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an understanding of the epigenetic mechanisms and protein dna interactions that affect gene expression is an important component of functional genomics research .
chromatin - immunoprecipitation ( chip ) has been used to study these interactions by allowing the isolation of genomic fragments targeted by an antibody or bound by a specific protein in vivo ( 14 ) .
the presence of a specific chromosomal target in the immunoprecipitated fragments can be determined by amplifying the region of interest with appropriately designed primers .
this approach is limiting in that it depends on prior knowledge of the expected gene targets , knowledge of the precise chromosomal location to which a protein binds near the gene , and requires separate pcr for each target .
global analysis of the dna products from a chip assay is possible by hybridizing the fragments against a microarray of chromosomal regions , an approach that has been called chip - chip ( 57 ) .
microarray analysis of chip samples requires the construction of arrays using probes against regulatory regions with which the protein might interact .
the first chip - chip experiments were performed in yeast , using intergenic regions as probes on the microarray ( 6 ) .
chip - chip experiments in humans have been conducted using array sequences derived from regions directly upstream of the genes and into the first exon ( 8,9 ) .
this approach assumes that the majority of protein dna interactions that affect rna transcription occur in regions near the transcription start site ( tss ) .
a more comprehensive approach has been to use arrays with a set of probes that represent all regions within a given locus ( 10 ) , chromosome ( 11 ) or genome ( 12,13 ) . an alternate approach to designing a microarray for chip - chip studies
cgis are genomic regions that are thought to have escaped the gradual loss of cpg dinucleotides that results from the transition of methylated cytosine to thymidine and is responsible for the cpg scarcity through most vertebrate genomes ( 14,15 ) .
it has been estimated that 60% of all human genes are associated with a cgi usually in the 5 end ( 16 ) , and 85% of cgis have been determined to be within 500 to + 1500 bp of a tss .
, a strong correlation has been noted between cgis and clusters of transcription factor binding sites ( 18 ) .
this association of cgis with promoter regions suggests that trans - acting factors bound to these sites prevent cytosine methylation and subsequent degradation ( 19 ) .
supporting this idea is the observation that de novo methylation of cgis can result in transcriptional repression and x - chromosome inactivation while demethylation with 5-azacytidine can remove gene repression ( 20 ) . the first large - scale effort to computationally identify cgis
was performed using genbank sequences ( 14 ) . with the completion of the human genome - sequencing project ,
bioinformatic approaches have been applied to identifying cgis across the human genome ( 21 ) and cgi tracks are available on the major publicly accessible , genome browsers ( ncbi , ucsc ) .
the criteria and algorithms used to determine the bounds of the islands vary , but generally it is accepted that cgis are 200 bp or greater in length with a g + c content > 50% and a cpg percentage that exceeds 60% of that expected in random sequence ( 1/16 ) ( 14 ) .
in contrast to bioinformatic identification of cgis , a physical cgi library was constructed using a two - step cloning strategy involving isolation of gc - rich chromosomal fragments based on their lack of methylation in vivo and followed by reselecting fragments that could be methylated in vitro ( 22 ) .
after analysis of 113 clones in this library , it was concluded that 77% of the clones were derived from cpg - rich regions . the first array constructed from this clone library
was utilized to determine the methylation status of cgis in breast cancer cells ( 23 ) .
these arrays have since been used effectively for analysis of e2f ( 5 ) and c - myc targets ( 24 ) using a chip - chip approach . we have recently constructed microarrays containing probes derived from the clones isolated from this library . to better understand the nature of the cgi library and to facilitate analysis of cgi arrays , we have sequenced 20 736 clones and conducted a large - scale characterization that includes identification of the genomic origins of each clone as well as other potential hybridization targets in the genome .
the cgi library had been prepared and described ( 25 ) . from this library ,
12 192 clones ( 12k set ) were obtained from the wellcome trust sanger institute ( cambridge , uk ) .
sequencing of these clones had been done previously at the sanger institute and this information is publicly available ( ) . a second set of 8544 clones ( 9k set ) , derived from the same library but screened with human cot1 dna to remove clones with repetitive elements , was obtained from t. h. huang ( ohio state university ) .
subsets of these clones had been used previously in the construction of several arrays ( 23,26 ) , including the cgi promoter arrays available through the uhn microarray centre in toronto ( ) .
the complete set of 20 736 clones was replicated and sent for sequencing to the genome sciences centre ( vancouver , bc ) .
blat ( blast - like alignment tool ) ( 27 ) was obtained from ucsc genome bioinformatics ( ) . the may 2004 build ( hg17 ) of the human genome was obtained from the ucsc genome bioinformatics site ( ) ( 28 ) and formatted for local blat alignments .
annotated cgis , refseq and known gene positions from this build were obtained from ucsc and used for analysis of clone alignments .
end reads from each clone were aligned to the genomic sequence using blat ( -fastmap option ) , masking out repetitive elements and low - complexity regions .
base - calls in the end read with a phred score < 20 were also masked .
alignments to the genome for each clone were constructed by combining the blat alignment from each end read when within 5000 bp of each other . in cases where only a single read was available ,
clone alignments were constructed from a single blat alignment , but marked as incomplete . in cases where sequence reads for a single clone aligned independent of each other , alignment information was stored and this was noted .
once all clone sequences had been aligned , genomic loci were defined by combining overlapping alignments resulting from redundant clones in the collection .
two sets of loci were generated from the human genomic sequence for comparison with the cgi library loci . to model the expected contents of the cgi library ,
in addition , a set of random loci was created by selection of 5000 random positions distributed across all 23 chromosomes proportional to chromosome length , and extracting a 1000 bp downstream from each position .
cgi library sequence and alignment data has been stored in a mysql database and made publicly available .
queryable web - based forms have been constructed ( ) to facilitate analysis of this library , as well as retrieval of information associated with specific probe identifiers on the cgi promoter arrays .
two samples of 100 ng of genomic dna , prepared from 2ftgh fibrosarcoma cells , were spiked with 2 ng arabidopsis dna control and random - primed with a final 50 l mixture of 1 sequenase buffer , 0.12 mm amino - allyl - dutp / dntp [ 0.12 mm datp / dgtp / dctp , 0.048 mm dttp ( invitrogen , 10297 - 018 ) and 0.072 mm aminoallyl - dutp ( sigma , a0410 ) ] and 1 l ( 13 u ) of sequenase t7 dna polymerase ( usb , 70775y / z ) for 4 h at 37c .
the resulting product was purified using the cyscribe gfx purification kit ( amersham biosciences , 27 - 9606 - 01 ) according to manufacturer 's directions .
recovered dna was reduced to a final volume of 8 l , and incubated with dmso reconstituted alexa 647 or alexa 555 dyes ( molecular probes , a32755 ) , in a final volume of 10 l for 1 h at room temperature .
both 100 ng samples were pooled and added to 85 l of hybridization cocktail [ 0.5 g/l calf thymus dna ( sigma , d8661 ) and 0.5 g/l yeast trna ( invitrogen , 15401 - 029 ) in dig easy hyb solution ( roche , 1603558 ) ] and hybridized to the array for 18 h. after washing ( 1 scc and 0.1% sds solution at 50c ) , the arrays were scanned using the genepix 4000a microarray scanner ( axon instruments ) at pmt voltages between 750 and 800 at 100% laser power .
background subtraction was done using an algorithm that fits a convolution of exponential and normal distributions to foreground intensities ( normexp ) with an offset of 50 for low - intensity shrinkage .
all algorithms were implemented in the limma package ( v1.8.21 ) ( 30 ) of the bioconductor library ( 31 ) for the r statistical package .
the cgi library had been prepared and described ( 25 ) . from this library ,
12 192 clones ( 12k set ) were obtained from the wellcome trust sanger institute ( cambridge , uk ) .
sequencing of these clones had been done previously at the sanger institute and this information is publicly available ( ) . a second set of 8544 clones ( 9k set ) , derived from the same library but screened with human cot1 dna to remove clones with repetitive elements , was obtained from t. h. huang ( ohio state university ) .
subsets of these clones had been used previously in the construction of several arrays ( 23,26 ) , including the cgi promoter arrays available through the uhn microarray centre in toronto ( ) .
the complete set of 20 736 clones was replicated and sent for sequencing to the genome sciences centre ( vancouver , bc ) .
blat ( blast - like alignment tool ) ( 27 ) was obtained from ucsc genome bioinformatics ( ) .
the may 2004 build ( hg17 ) of the human genome was obtained from the ucsc genome bioinformatics site ( ) ( 28 ) and formatted for local blat alignments .
annotated cgis , refseq and known gene positions from this build were obtained from ucsc and used for analysis of clone alignments .
end reads from each clone were aligned to the genomic sequence using blat ( -fastmap option ) , masking out repetitive elements and low - complexity regions .
base - calls in the end read with a phred score < 20 were also masked .
alignments to the genome for each clone were constructed by combining the blat alignment from each end read when within 5000 bp of each other . in cases where only a single read was available ,
clone alignments were constructed from a single blat alignment , but marked as incomplete . in cases where sequence reads for a single clone aligned independent of each other , alignment information was stored and this was noted .
once all clone sequences had been aligned , genomic loci were defined by combining overlapping alignments resulting from redundant clones in the collection .
two sets of loci were generated from the human genomic sequence for comparison with the cgi library loci . to model the expected contents of the cgi library ,
msei fragments containing complete or partial annotated cgis were identified . in addition , a set of random loci was created by selection of 5000 random positions distributed across all 23 chromosomes proportional to chromosome length , and extracting a 1000 bp downstream from each position .
cgi library sequence and alignment data has been stored in a mysql database and made publicly available .
queryable web - based forms have been constructed ( ) to facilitate analysis of this library , as well as retrieval of information associated with specific probe identifiers on the cgi promoter arrays .
two samples of 100 ng of genomic dna , prepared from 2ftgh fibrosarcoma cells , were spiked with 2 ng arabidopsis dna control and random - primed with a final 50 l mixture of 1 sequenase buffer , 0.12 mm amino - allyl - dutp / dntp [ 0.12 mm datp / dgtp / dctp , 0.048 mm dttp ( invitrogen , 10297 - 018 ) and 0.072 mm aminoallyl - dutp ( sigma , a0410 ) ] and 1 l ( 13 u ) of sequenase t7 dna polymerase ( usb , 70775y / z ) for 4 h at 37c .
the resulting product was purified using the cyscribe gfx purification kit ( amersham biosciences , 27 - 9606 - 01 ) according to manufacturer 's directions .
recovered dna was reduced to a final volume of 8 l , and incubated with dmso reconstituted alexa 647 or alexa 555 dyes ( molecular probes , a32755 ) , in a final volume of 10 l for 1 h at room temperature .
both 100 ng samples were pooled and added to 85 l of hybridization cocktail [ 0.5 g/l calf thymus dna ( sigma , d8661 ) and 0.5 g/l yeast trna ( invitrogen , 15401 - 029 ) in dig easy hyb solution ( roche , 1603558 ) ] and hybridized to the array for 18 h. after washing ( 1 scc and 0.1% sds solution at 50c ) , the arrays were scanned using the genepix 4000a microarray scanner ( axon instruments ) at pmt voltages between 750 and 800 at 100% laser power .
background subtraction was done using an algorithm that fits a convolution of exponential and normal distributions to foreground intensities ( normexp ) with an offset of 50 for low - intensity shrinkage .
data were normalized with a robust - spline algorithm ( 29 ) . all algorithms were implemented in the limma package ( v1.8.21 ) ( 30 ) of the bioconductor library ( 31 ) for the r statistical package .
sequence information was obtained for two sets of cgi clones . the 12k set ( 12 192 clones ) was a portion of the original clone selection that had been deposited at the wellcome trust sanger institute ( 22 ) . a second 9k set ( 8544 clones )
had been isolated from the same cgi library by the huang laboratory ( 32 ) .
vector - trimmed sequences that were longer than 50 bp and had a phred score > 20 were used for alignment to the genome .
a summary of the available sequence and alignment to the human genome is presented in table 1 for each set and for the combined set of 20 736 clones .
the 9k set was generally of better quality , with 95% having acceptable sequence versus 78% of the 12k set ( table 1 , cgi library sequence information ) .
combined , 85% of the clone sequences were considered suitable for alignment to the human genomic sequence .
the 9k set had a median sequence length of 499 bp with 2% of the reads
in contrast , 11% of the 12k set reads were < 100 bp in length , contributing to the shorter median sequence length of 307 bp .
a second round of sequencing performed on 768 blinded clones confirmed the sequence data obtained in the first round ( data not shown ) .
of the 17 645 clones with sequence , 14 901 ( 84% ) aligned to the human genome using blat ( table 1 , genomic alignment ) .
the proportion of clones aligning was higher for the 9k set ( 92% ) versus the 12k set ( 79% ) as expected due to the longer reads in this set .
overall , 90% of the clones with sequence aligned at a single position in the genome .
while many partial alignments were observed , the majority of sequences showed nearly full - length alignments to the genome .
the median sequence length for clones that did align was 480 bp versus only 217 bp for clones that did not align .
although it appears likely that read length affected the ability to align sequence , some sequences that did not align to genomic dna were as long as 962 bp .
alignments which overlapped each other on the genomic scaffold were combined to define 9595 distinct genomic loci ( table 1 , genomic loci ) .
interestingly , only 753 loci ( 8% ) were shared , indicating that the two sets were very distinct from each other .
the majority of loci ( 65% ) were defined by a single clone ( figure 1 , upper panel ) , although these accounted for only 30% of all clones which aligned ( figure 1 , lower panel ) . therefore
approximately 39% had a low degree of redundancy ( 25 per locus ) while 15% were highly redundant ( 11 + per locus ) .
this last group consists of 2958 clones defining only 118 loci and includes a single locus defined by 582 clones .
genomic loci ranged in size between 50 and 2589 bp , with a mean locus length of 511 bp .
many of the shorter loci result from partial sequence alignments and often these sequences align to a greater degree elsewhere in the genome .
the percentage of each sequence contributing to the total alignment length is shown in figure 2 ( upper panel ) .
most alignments < 200 bp tend to be due to partial alignments and this trend diminishes as the total aligned length increases . when the loci defined from these alignments are examined ( figure 2 , lower panel ) , shorter loci
are usually defined by partially aligning clones while loci 200 bp in size and greater generally result from nearly complete sequence alignments .
accordingly , we have not included loci < 200 bp in length in the subsequent analysis , reducing the number from 9595 to 7184 . to evaluate the overall quality of the loci defined by the physical cgi library , we calculated two metrics , the g + c content and cpg dinucleotide frequency , and compared this to the computationally annotated cgis . to make the appropriate comparison , we modeled in silico the library construction undertaken by cross et al .
( 22 ) by identifying msei sites in the genomic sequence and generating fragments between msei sites within or immediately flanking annotated cgis ( msei - cgis ) .
starting with the 27 801 computationally annotated cgis from hg17 , 42 450 msei - cgis were identified having an average length of 1106 bp ( range 551 , 939 bp ) . for additional comparison , a set of 5000 random loci , each 1000 bp in length and proportionally distributed across all 23 chromosomes ,
these three sets were then evaluated for the two above - mentioned metrics ( figure 3 ) .
both criteria were met ( cpg ratio > 0.6 , g + c content > 0.5 ) for 64% of the physical loci .
although this was less than the 87% observed in the msei - cgis , this stood in stark contrast to the 2% observed in the random loci .
annotated cgi will , by definition , exceed both criteria but 13% of the msei - cgis do not , and this can be directly attributed to the non - cgi - containing flanking sequences imposed by the msei positions . given this , it is likely that the 36% of the physical cgis not exceeding both criteria includes authentic cgi sequences .
we next determined the chromosomal distribution of the physical cgi loci and compared this to the distribution of msei - cgis ( figure 4 ) . in total , 63% of the physical cgi loci show overlap with an msei - cgi .
the proportional representation of both sets relative to chromosome size were similar ( figure 4 , right - hand side ) , suggesting that the clones isolated from the library are generally representative of annotated cgi .
a similar over - representation ( chromosomes 16,17 and 19 ) or under - representation ( chromosomes 13 , x , y ) of cgi on particular chromosomes was also observed in both sets .
given that cgis have been demonstrated to have a close relationship with the 5 upstream region of genes , we next examined the distance of each cgi library locus from the nearest tss .
distance from a tss to the annotated cgis as well as to the random loci was also calculated and these results are summarized in figure 5 . for the cgi - derived msei fragments , 47% are in the proximal promoter region ( + 200 bp to 1 kb ) , 41% of which directly overlap a tss .
an additional 12% are found in more distal promoter regions ( + 1 kb to 10 kb ) and 14% are found further into the gene sequence , > 1000 bp downstream from the tss .
less than 10% of the annotated cgis are found in regions far upstream of a tss ( 100k ) in contrast to the randomly generated loci which are predominantly in these regions .
the cgi library loci that overlapped a cgi - derived msei fragment showed a similar preferential localization around the tss and in promoter regions .
the cgi library loci that were not associated with the annotated cgi also showed a stronger association with tsss than observed with the random loci . although few were directly at a tss , 25% were within the distal promoter region , compared to only 10% of the random loci .
furthermore , 43% of the random loci were > 100 kb away from a tss compared to only 25% of this subset of the cgi library loci . to verify the overall ability to align these sequences to the human genome
, a 12k microarray constructed from the cgi clones was hybridized with dna fragments generated by sonication of isolated human genomic dna .
fluorescence measurements from non - human probes on this array were used as a measure of non - specific binding since the corresponding spike - in cdna was not included in the hybridization mixture ( figure 6a ) .
signal from only 396 of the 12 196 cgi probes were within 2 sd of the mean signal intensity of the non - human dna probes , suggesting that genomic dna bound specifically to > 97% of the cgi probes .
the array was hybridized with two independently labeled aliquots ( 100 ng each ) of sonicated genomic dna .
an ma plot of background - corrected and normalized log2 signal versus log2 differential expression is shown in figure 6b .
signal consistency is very strong between the two channels with > 99.5% of cgi probes showing
2-fold differential expression ( |m| < 1 ) and signal from the two channels showing a correlation ( pearson ) of 0.99 .
the mean log2 signal intensity for all probes was 10.9 1.9 ; in total 85% of cgi probes had signals within two orders of magnitude range . to facilitate examination of the cgi library clone alignments to the genome ,
a cgi library browser has been constructed and is available at ( figure 7 ) clone identifiers corresponding to spots on the 12k cgi microarray can be entered to obtain a graphical view of the genomic alignment , as well as the relative positions of upstream and downstream genes .
other features of the browser include the ability to view other clones aligning to the same locus and to identify clones aligning near a specified gene .
links have been created to map directly to the ucsc genome browser ( 33 ) to allow exploration of other genomic features near the locus of interest .
arrays constructed from a cgi library ( 22 ) have been applied successfully to study dna methylation status and to identify genomic fragments immunoprecipitated with antibodies against several target proteins ( 5,24,32,3436 ) .
the rationale for using a cgi array to analyze chip dna is based on the association between cgis and gene tsss ( 16,17 ) as well as the hypothesis that cpg conservation in these regions results from protein chromatin interactions which prevent methylation and subsequent cpg degeneration ( 19 ) .
the cgi library from which the probes for the array were selected was created by isolating genomic fragments that have a high g + c content , are rich in cpg dinucleotides , but are poorly methylated ( 22 ) .
the clones used as probes on earlier arrays were selected randomly from this library and therefore their sequence and identity was unknown .
in contrast to arrays constructed from defined genomic fragments , it had been necessary to identify probes of interest post - hybridization by sequencing the clone , aligning to known sequences , then identifying associated genes .
this study provides a comprehensive analysis of a set of 20 736 clones isolated from the cgi library validating the approach taken by cross et al .
( 22 ) in construction of the library as well as the use of this library for construction of a microarray suitable for chip - chip analysis .
initial attempts to align clone sequences to genomic dna utilized the sequence information which was publicly available for the 12k set ( ) . while sequence information was available for only 60% of the clones in this data set , nearly 85% of the clones had usable sequence following resequencing in this study .
furthermore , the average read length in the original data set was 215 bp compared to 414 bp in our sequences .
the higher quality of sequence data probably contributed to a higher percentage of successful alignments .
more importantly though , since discrepancies were observed between the two data sets ( data not shown ) and our sequence data is known to directly correspond to the clones used for array construction , this ensures an accurate annotation of the probes on the microarray . despite resequencing
, 15% of the clones did not have adequate sequence information to generate genomic alignments .
this included clones in which no sequence information was obtained as well as clones with < 50 bp of quality sequence ( phred score > 20 ) , which would generate short , less informative alignments .
this was not unexpected and may be due to lack of an insert in the clone , or to the presence of multiple clones in some wells , preventing proper sequencing of the inserts .
the first set of 12 192 clones ( 12k set ) was obtained directly from the sanger institute .
the second subset of 8544 clones ( 9k set ) had been previously isolated by huang et al .
the clones from this set have been used in the construction of various cgi arrays ( 5,24,32 ) .
the quality of the sequence information in the 9k set was superior to the 12k set .
fewer clones in the 9k set returned no sequence , and the read lengths were generally longer . this is probably due to the fact that this set had been prescreened with human cot-1 dna to remove sequences with a high degree of repeat elements .
consequently , a higher percentage of clones in the 9k set aligned to genomic sequence than in the 12k set .
cgi arrays constructed from the 12k set have been available through the uhn microarray centre ( ) since 2003 .
arrays constructed from the full set of 20 736 clones are now being produced .
we have used the blat software ( 27 ) to align sequences to genomic dna , both for its speed and because it is well - suited for identifying highly similar sequences .
some post - alignment processing was necessary to separate out partial alignments on a single chromosome that had been combined by blat 's stitching routine .
the 16% that did not align can be partially accounted for by their shorter sequence lengths although there did exist long sequences that did not align .
in addition , sequences that did not have long contiguous stretches of quality sequence may have not aligned due to masking of the poor quality bases .
use of other alignment algorithms may be more suitable for these sequences , but the results would include similar or homologous regions that would not necessarily represent either the genomic origin of a clone , nor an area that would hybridize against the clone on an array .
clone alignments were constructed after aligning each read separately , allowing the genomic sequence to act as a scaffold to determine the positioning of the two reads with respect to each other . in clones with non - overlapping ends ,
there were also instances of alignments of one read in the absence of a nearby alignment of the other read .
although these alignments may not represent the genomic origin of a particular clone , they do indicate a potential hybridization target in genomic dna against the clone when acting as a probe on a microarray .
such clones may have arisen by ligation of two or more distinct fragments into the same plasmid .
evidence suggesting this is observed in 2% of the clones ( 213 clones ) , where the end reads align to distinct genomic positions , often on different chromosome .
the absence of the other read 's contribution to the alignment is noted in the annotation available through the web browser .
the genomic scaffold was also used to identify redundancy in the clones derived from the cgi library .
cgi library loci were generated by combining overlapping alignments and identifying all clones contributing to each locus .
there is the possibility also that a locus might capture contiguous clones in cases where incompletely digested fragments were captured , but in the absence of this , contiguous clones will be represented by adjacent loci . in this way ,
9595 loci were defined , slightly less than one - half of the total number of clones available and slightly more than one - half of the clones with sequence .
this number is reduced to 7184 when eliminating shorter loci resulting from partial sequence alignments . in this way
, it was determined that only 30% of the clones were unique , the other 70% showing some degree of redundancy .
interestingly , the 12k and 9k sets show only a small degree of overlap which was not expected given the degree of redundancy in each set . of the 9595 loci ,
this finding suggests there may be value in continuing to isolate clones from the library .
approximately 60% of the loci that we have identified are within the msei restriction fragments associated with the annotated cgi .
this number corresponds well with the observation that 64% of the loci evaluate as a cgi based on g + c content and cpg dinucleotide frequency .
cgis by definition are regions of cpg conservation due to the absence of methylation and will therefore have high g + c content and a cpg frequency that approaches the expected random frequency of 1/16 . identification of cgi in promoter regions or in genomic sequence exploits algorithms that evaluate when these parameters exceed arbitrary values designed to minimize signal - to - noise . within the 40% of the loci that do not correspond to the annotated cgi - associated msei restriction fragments
alternately , some of these loci may represent noise in the original library due to capture of fragments not expected from the selection procedure .
the documented association of cgis with regions upstream of genes is based on bioinformatic analysis of the genome ( 14,17,21 ) and is dependent on the analysis of the nucleotide sequence alone .
we have approached this from a different direction , mapping the cgi library clones that were selected based on sequence characteristics and methylation status , to the genomic sequence .
approximately 60% of the cgi library loci in the subset that overlap annotated cpg sites are in the distal promoter region or closer to a tss .
this is nearly identical to the distribution of the annotated cgis , supporting the idea that cgis are associated with tsss .
in addition to collecting cgis near the tss , the cgi library also contains fragments representing cgis that are distant from a gene .
potentially these islands may represent regions to which transcriptional enhancers and repressors may bind outside the proximal or distal promoter regions .
alternately , they may be a part of promoter regions in proximity to an unknown tss and may ultimately play a role in identifying new genes ( 37 ) .
nearly 25% of the loci in the subset that does not overlap annotated cpg sites are within the distal promoter region or closer .
this is a significant enrichment relative to random loci in this region ( 10% ) .
furthermore , while > 40% of random loci are 100 kb or further from a tss , only 25% of this second subset is found here . despite not being associated with annotated cgis , this suggests that these loci are not simply due to non - specific collection of random fragments into the library .
possibly , the cpg content is too low to characterize these regions as cgis by the established criteria , but still high enough to have been effectively methylated and isolated during the procedure used to create the cgi library .
there have been a variety of approaches taken towards the design of microarrays suitable for analysis of chip dna .
proximal promoter arrays constructed using regions directly upstream of known tsss ( 8,9 ) can identify binding to transcriptional regulatory elements which exist close to the gene .
it has been well established though that regulatory factors can bind at more distal regions , and even within introns , exons or downstream of a gene ( 10,11,38 ) .
more comprehensive tiling arrays have been constructed targeting intergenic regions ( 6 ) , distinct loci ( 10,11 ) and the full genome ( 12,13 ) .
the cgi array approach is based primarily on the association of cgis with regulatory activity ( 16,17 ) , but in contrast to the proximal promoter arrays the location of cgis relative to the tss is not limited to 1000 bp upstream . while many of the loci identified in this study
are still within 10 kb of a tss , they also occur at far more distant regions .
although full genome tiling arrays obviously represent the most comprehensive tool for global methylation or protein chromatin interaction studies , technical issues such as costs , multiple array platforms and data analysis software , represent aspects which currently preclude widespread use of these arrays for a typical molecular biology laboratory . however , it is likely that all these array types represent complementary approaches to studying global epigenetic mechanisms and transcription factor binding sites .
the primary focus of this study is to annotate clones isolated from the cgi library and the cgi arrays which use these clones as probes .
while more extensive studies are in progress to further evaluate these arrays , we conducted a straight - forward experiment to examine probe hybridization to the 12k cgi array .
furthermore , the degree of binding observed after hybridization with equal amounts of genomic dna in both channels was highly consistent , with only 32 of 12 192 cgi probes showing differential hybridization .
the signal obtained from each probe varied over 2 orders of magnitude , probably due to differences in hybridization strength , dye incorporation and hybridization specificity between different dna fragments .
the data produced in this analysis is available online through the cpg island library browser ( ) . this has been designed to allow detailed examination of individual probes on the cgi array as well as providing a means to summarize information for a list of probes .
the alignments detailed in this browser include complete and near - complete alignments of clone sequences to genomic loci , as well as partial alignments that may require consideration when analyzing hybridization results .
this interactive tool will facilitate analysis of chip - chip experiments by allowing faster identification of targets .
in addition , redundant probes on the array have been identified and probes that align near a gene of interest as well as the spatial relationship of the alignment to the gene can be easily determined .
the cgi library is expected to contain only a subset of all genomic cgi , those that are unmethylated in whole blood genomic dna from which the library was constructed ( 22 ) .
the analysis presented here will contribute to the production of next generation cgi arrays by allowing removal of redundant probes , enrichment with unique clones , and the inclusion of probes designed against other regulatory regions which complement this cgi approach .
a clearer understanding of the nature of the cgi probes will also facilitate the design of coordinated expression arrays that can be used in conjunction with chip - chip studies with cgi arrays to study and define transcriptional networks .
furthermore , the development of high quality cgi arrays will contribute to more accurate analysis of global methylation status and the relationship of epigenetic mechanisms to these networks .
the majority of loci ( 6179/9552 ) are each defined by a single , non - redundant clone ( upper panel , left - most bar ) . fewer loci are represented by redundant clones with 1782 loci each defined by a pair of clones , 1240 by 35 clones , 235 by 610 clones and 118 loci each represented by 11 or more clones ( range 11582 clones ) . in the lower panel ,
the percentage of the sequence length aligning is plotted against the total number of bases aligned ( alignment length ) .
the shorter alignments generally result from partial sequence alignments and as the total length of the alignment increases , so does the % aligning .
loci < 100 bp in length are generated mostly from these partial alignments , while the longer loci ( 200 bp ) are derived from nearly complete sequence alignments .
cgi library loci ( right panel ) were evaluated for g + c content and cpg dinucleotide content ( expressed as a ratio of the expected frequency of 1/16 ) . for comparison ,
msei fragments containing annotated cgis ( left panel ) and random loci ( center panel ) were also evaluated .
dotted lines indicate the values frequently used for assessment of cgis ( g + c > 0.5 , cpg observed / expected > 0.6 ) . the percentage of loci in each quadrant is indicated .
a schematic diagram of the 23 human chromosomes is shown with giemsa staining patterns in grayscale . annotated cgis are indicated in green ( top of schematic diagram ) and the number identified on each chromosome is indicated to the right ( cgi ) .
the position of each mapped locus is indicated in red ( bottom of schematic diagram ) . the number identified on each chromosome
the first column is the number of loci with a position that overlaps an annotated cgi / msei .
the second column is the number of loci that do not overlap an annotated cgi / msei . to the far right
is indicated the proportional representation of the number of loci or annotated cgis relative to chromosome length .
loci were also identified on mitochondrial dna ( 14 loci ) as well as on undesignated chromosome sequence collected into ucsc random sequence files ( 94 loci ) .
the distance to the nearest annotated tss is shown for three sets of loci : the annotated cgi in the current build of the human genome ( hg17 , may 2004 ) ; the random loci ; and the loci derived from the cgi library .
the last set has been subdivided into loci which overlap an annotated cgi ( solid ) and those that do not ( speckled ) .
the percentage of loci in each set at various positions relative to the tss is shown .
percentage of loci overlapping an annotated tss , in the proximal promoter region ( + 200 to 1000 bp ) and in the distal promoter region ( + 1000 to 10000 bp ) .
( iii ) upstream between 10 and 100 kb upstream of a tss or > 100 kb upstream of a tss .
( a ) a representative block from the cgi array after hybridization with 100 ng sonicated human genomic dna .
the faint spots in the lower left are the non - human dna control probes .
arabidopsis controls were added to the hybridization and the arabidopsis probes are in the lower right .
( b ) ma plot of differential expression ( m ) versus mean signal intensity ( a ) in log2-space for all cgi probes on the array .
only 32 of 12 196 probes display > 2-fold differential expression ( |m| > 1 ) .
an online cgi library browser ( ) has been constructed to allow users of the cgi promoter microarrays as well as users interested in the cgi library to explore alignments of the clones to the human genome .
for each clone , alignments to the genome are listed and displayed diagrammatically , including the relative positions of annotated cgi and nearby genes .
cgi library sequence information , genomic alignment and genomic loci statistics is shown for the 12k clone set , the 9k clone set and the combined 21k set .
the number of clones sent for sequencing , the number with usable sequence . the number of clones with usable sequence that aligned to the human genome build .
the overlap between the two clone sets is indicated , along with the number of loci unique to each clone set .
also indicated are the number of loci generated by a single clone alignment and multiple clone alignments . | an effective tool for the global analysis of both dna methylation status and protein chromatin interactions is a microarray constructed with sequences containing regulatory elements .
one type of array suited for this purpose takes advantage of the strong association between cpg islands ( cgis ) and gene regulatory regions .
we have obtained 20 736 clones from a cgi library and used these to construct cgi arrays .
the utility of this library requires proper annotation and assessment of the clones , including cpg content , genomic origin and proximity to neighboring genes .
alignment of clone sequences to the human genome ( ucsc hg17 ) identified 9595 distinct genomic loci ; 64% were defined by a single clone while the remaining 36% were represented by multiple , redundant clones .
approximately 68% of the loci were located near a transcription start site .
the distribution of these loci covered all 23 chromosomes , with 63% overlapping a bioinformatically identified cgi .
the high representation of genomic cgi in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein chromatin interactions . a browsable database is available on - line to facilitate exploration of the cgis in this library and their association with annotated genes or promoter elements . | INTRODUCTION
MATERIALS AND METHODS
CGI library and sequence data
Sequence alignment
CGI array hybridization
RESULTS
DISCUSSION
Figures and Tables | global analysis of the dna products from a chip assay is possible by hybridizing the fragments against a microarray of chromosomal regions , an approach that has been called chip - chip ( 57 ) . with the completion of the human genome - sequencing project ,
bioinformatic approaches have been applied to identifying cgis across the human genome ( 21 ) and cgi tracks are available on the major publicly accessible , genome browsers ( ncbi , ucsc ) . after analysis of 113 clones in this library , it was concluded that 77% of the clones were derived from cpg - rich regions . to better understand the nature of the cgi library and to facilitate analysis of cgi arrays , we have sequenced 20 736 clones and conducted a large - scale characterization that includes identification of the genomic origins of each clone as well as other potential hybridization targets in the genome . once all clone sequences had been aligned , genomic loci were defined by combining overlapping alignments resulting from redundant clones in the collection . the may 2004 build ( hg17 ) of the human genome was obtained from the ucsc genome bioinformatics site ( ) ( 28 ) and formatted for local blat alignments . once all clone sequences had been aligned , genomic loci were defined by combining overlapping alignments resulting from redundant clones in the collection . a summary of the available sequence and alignment to the human genome is presented in table 1 for each set and for the combined set of 20 736 clones . of the 17 645 clones with sequence , 14 901 ( 84% ) aligned to the human genome using blat ( table 1 , genomic alignment ) . to evaluate the overall quality of the loci defined by the physical cgi library , we calculated two metrics , the g + c content and cpg dinucleotide frequency , and compared this to the computationally annotated cgis . furthermore , 43% of the random loci were > 100 kb away from a tss compared to only 25% of this subset of the cgi library loci . to verify the overall ability to align these sequences to the human genome
, a 12k microarray constructed from the cgi clones was hybridized with dna fragments generated by sonication of isolated human genomic dna . to facilitate examination of the cgi library clone alignments to the genome ,
a cgi library browser has been constructed and is available at ( figure 7 ) clone identifiers corresponding to spots on the 12k cgi microarray can be entered to obtain a graphical view of the genomic alignment , as well as the relative positions of upstream and downstream genes . arrays constructed from a cgi library ( 22 ) have been applied successfully to study dna methylation status and to identify genomic fragments immunoprecipitated with antibodies against several target proteins ( 5,24,32,3436 ) . the rationale for using a cgi array to analyze chip dna is based on the association between cgis and gene tsss ( 16,17 ) as well as the hypothesis that cpg conservation in these regions results from protein chromatin interactions which prevent methylation and subsequent cpg degeneration ( 19 ) . ( 22 ) in construction of the library as well as the use of this library for construction of a microarray suitable for chip - chip analysis . in this way ,
9595 loci were defined , slightly less than one - half of the total number of clones available and slightly more than one - half of the clones with sequence . within the 40% of the loci that do not correspond to the annotated cgi - associated msei restriction fragments
alternately , some of these loci may represent noise in the original library due to capture of fragments not expected from the selection procedure . we have approached this from a different direction , mapping the cgi library clones that were selected based on sequence characteristics and methylation status , to the genomic sequence . the alignments detailed in this browser include complete and near - complete alignments of clone sequences to genomic loci , as well as partial alignments that may require consideration when analyzing hybridization results . furthermore , the development of high quality cgi arrays will contribute to more accurate analysis of global methylation status and the relationship of epigenetic mechanisms to these networks . fewer loci are represented by redundant clones with 1782 loci each defined by a pair of clones , 1240 by 35 clones , 235 by 610 clones and 118 loci each represented by 11 or more clones ( range 11582 clones ) . the distance to the nearest annotated tss is shown for three sets of loci : the annotated cgi in the current build of the human genome ( hg17 , may 2004 ) ; the random loci ; and the loci derived from the cgi library . an online cgi library browser ( ) has been constructed to allow users of the cgi promoter microarrays as well as users interested in the cgi library to explore alignments of the clones to the human genome . | [
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] |
this article presents statistics on health care utilization , prices , expenses , employment , and work hours , as well as on national economic activity .
some of these statistics are based on sample surveys conducted monthly or quarterly by government agencies or private organizations , and are available 1 to 3 months after the completion of the period .
these statistics provide an early indication of changes occurring within the general economy and in the health care sector . the accompanying tables report selected quarterly statistics for 1991 through the first quarter of 1994 , and the calendar year aggregation of quarterly information for the past 3 years .
additional tables show the change from the same period 1 year earlier . for quarterly information
, this calculation permits analysis of quarterly data to focus on the direction and magnitude of changes , without interference introduced by seasonal fluctuations . in the national health accounts , indicators such as these play an important role in the estimation of the latest historical year of health care expenditures .
information that is more comprehensive tends to lag behind the close of a calendar year by 9 to 12 months or more .
therefore , we rely extensively on indicators such as these to anticipate and predict changes in health care sector expenditures for the most recent year .
other indicators help to identify specific reasons ( e.g. , increases in price inflation or declines in utilization ) for expenditure change . in the following sections
, we will identify important indicators of health care and national economic activity and their sources .
we will then describe what these indicators tell us about general economic and health sector activity during the most recent quarter .
since 1963 , the american hospital association ( aha ) , in cooperation with member hospitals , has been collecting data on the operation of community hospitals through its national hospital panel survey .
community hospitals , which comprised more than 80 percent of all hospital facilities in the united states in 1993 , include all non - federal , short - term general , and other special hospitals open to the public .
they exclude hospital units of institutions ; psychiatric facilities ; tuberculosis , other respiratory , and chronic disease hospitals ; institutions for the mentally retarded ; and alcohol and chemical dependency hospitals .
the sample is designed to produce estimates of community hospital indicators by bed size and region ( american hospital association , 1963 - 94 ) . in tables 1 and 2 ,
statistics covering expenses , utilization , beds , and personnel depict trends in the operation of community hospitals annually from 1991 and quarterly for selected quarters for 1991 through 1994 .
figures 1 and 2 show changes from the same quarter 1 year earlier for various measures of hospital operating expenses and utilization for 1985 to 1994 . for purposes of national health expenditures
( nhe ) , survey statistics on revenues ( not shown on table 1 ) are analyzed in estimating the growth in the largest component of health care costs community hospital expenditures .
this one segment of nhe accounted for 33 percent of all health spending in 1993 ( levit et al . , 1994 ) .
the survey also identifies important factors influencing expenditure growth patterns , such as changes in the number of beds in operation , number of admissions , length of stay , use of outpatient facilities , and number of surgeries .
the bureau of labor statistics ( bls ) collects monthly information on employment for all workers , and earnings and work hours for non - supervisory workers in a sample of 385,000 establishments .
data are collected through cooperative agreements with state agencies that also use this information to create state and local area statistics .
the survey is designed to collect industry - specific information on wage and salary jobs in non - agricultural industries .
it excludes statistics on self - employed persons and on those employed in the military ( u.s . department of labor , 1994a ) .
approximately 5 percent of the population hold more than one job at any one time .
( other surveys that are household - based , such as the current population survey [ cps ] , also record employment . in the cps , however , each person 's employment status is counted only once , as either employed , unemployed , or not in the labor force . ) once each year , monthly establishment - based employment statistics are adjusted to benchmarks created from annual establishment census information , resulting in revisions to previously published employment estimates .
tables 3 and 4 present statistics on employment , average hourly earnings , and average weekly hours in private ( non - government ) health service establishments .
similar statistics for the private non - agricultural sector , included on these tables , provide a basis for comparing the economy as a whole with the health sector in employment , earnings , and work hours .
figure 3 shows changes from the same quarter 1 year earlier in employment in the private non - agricultural sector and the health services industry for 1985 to 1994 .
table 5 summarizes business activity in the health sector and the overall economy by measuring change in the implied non - supervisory work hours and payroll . implied work hours
are the product of the number of non - supervisory employees and average weekly hours . implied non - supervisory payrolls
are calculated by multiplying implied work hours by average hourly earnings . for purposes of nhe , changes in work hours by industry combined with changes in prices ( discussed in a later section ) can be used to gauge the direction and magnitude of expenditure change in specific industries .
we use these composite indicators in the estimation of growth in physician and dental expenditures for the most recent period .
we study the historical relationship of changes in this indicator to changes in expenditures and estimate this relationship for the most recent period .
bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services .
tables 6 and 7 present information on the all urban consumer price index ( cpi ) that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states .
( the more restrictive wage earner cpi gauges prices faced by wage earners and clerical workers
. these workers account for 32 percent of the non - institutionalized population [ u.s .
the index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . for most items , the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products .
those shares or weights remain constant in all years , even though consumption patterns of the household may change over time .
cpis for health care goods and services depict price changes for out - of - pocket expenditures made directly by consumers .
the composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items .
for example , the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out of pocket by consumers ; the remaining 97 percent of the costs of hospital care paid by private health insurers , medicare , medicaid , and other payers are not weighted into the cpi for medical care .
in addition , some medical care sector indexes measure changes in list or charged prices , rather than in prices actually received by providers after discounts are deducted . in several health care areas , received or transaction prices are difficult to capture , although bls is making advances in this area . in the nhe ,
a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by hcfa to provide transaction price changes , are used as measures of inflation for the health industry .
the indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index ( letsch , 1993 ) . in 1979
, hcfa developed the medicare hospital input price index ( hospital market basket ) which was designed to measure the pure price changes associated with expenditure changes for hospital services . in the early 1980s ,
the skilled nursing facility ( snf ) and home health agency ( hha ) input price indexes , often referred to as market baskets , were developed to price a consistent set of goods and services over time .
also in the early 1980s , the original medicare hospital input price index was revised for use in updating payment rates for the prospective payment system ( pps ) .
all of these indexes have played an important role in helping to set medicare payment percent increases and in understanding the contribution of input price increases to growing health expenditures .
the input price indexes , or market baskets , are laspeyres or fixed - weight indexes that are constructed in two steps .
for example , for the pps hospital input price index , the base period is 1987 .
cost categories , such as food , fuel , and labor , are identified and their 1987 expenditure amounts determined . the proportion or share of total expenditures included in specific spending categories
its purpose is to measure the rate of price increase of the goods or services in that category .
the price proxy index for each spending category is multiplied by the expenditure weight for the category .
the sum of these products ( weights multiplied by the price index ) over all cost categories yields the composite input price index for any given time period , usually a fiscal year or a calendar year .
the percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider .
the input price indexes are estimated on a historical basis and forecasted out several years .
the hcfa - chosen price proxies are forecasted under contract with data resources , inc./mcgraw hill ( dri ) . following every calendar year quarter , in march , june , september , and december
, dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions .
some of the data in tables 8 through 13 are forecasted and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received .
the methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits .
a description of the current structure of the pps input price index was published in the september 4 , 1990 , federal register .
the most recent pps update for payment rates was published in the september 1 , 1993 , federal register .
the latest hha regulatory input price index was published in the july 8 , 1993 , federal register , and the latest snf input price index was published in the october 7 , 1992 , federal register .
periodically , the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased .
each revision allows for new base weights , a new base year , and changes to certain price variables used for price proxies .
each input price index is presented in two tables : the first table shows the quarterly levels for each price index , and the second is a percentage change table .
bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services .
tables 6 and 7 present information on the all urban consumer price index ( cpi ) that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states .
( the more restrictive wage earner cpi gauges prices faced by wage earners and clerical workers
. these workers account for 32 percent of the non - institutionalized population [ u.s .
the index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . for most items , the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products .
those shares or weights remain constant in all years , even though consumption patterns of the household may change over time .
cpis for health care goods and services depict price changes for out - of - pocket expenditures made directly by consumers .
the composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items .
for example , the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out of pocket by consumers ; the remaining 97 percent of the costs of hospital care paid by private health insurers , medicare , medicaid , and other payers are not weighted into the cpi for medical care .
in addition , some medical care sector indexes measure changes in list or charged prices , rather than in prices actually received by providers after discounts are deducted . in several health care areas , received or transaction prices are difficult to capture , although bls is making advances in this area . in the nhe ,
a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by hcfa to provide transaction price changes , are used as measures of inflation for the health industry .
the indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index ( letsch , 1993 ) .
in 1979 , hcfa developed the medicare hospital input price index ( hospital market basket ) which was designed to measure the pure price changes associated with expenditure changes for hospital services . in the early 1980s ,
the skilled nursing facility ( snf ) and home health agency ( hha ) input price indexes , often referred to as market baskets , were developed to price a consistent set of goods and services over time .
also in the early 1980s , the original medicare hospital input price index was revised for use in updating payment rates for the prospective payment system ( pps ) .
all of these indexes have played an important role in helping to set medicare payment percent increases and in understanding the contribution of input price increases to growing health expenditures .
the input price indexes , or market baskets , are laspeyres or fixed - weight indexes that are constructed in two steps .
for example , for the pps hospital input price index , the base period is 1987 .
cost categories , such as food , fuel , and labor , are identified and their 1987 expenditure amounts determined . the proportion or share of total expenditures included in specific spending categories
its purpose is to measure the rate of price increase of the goods or services in that category .
the price proxy index for each spending category is multiplied by the expenditure weight for the category .
the sum of these products ( weights multiplied by the price index ) over all cost categories yields the composite input price index for any given time period , usually a fiscal year or a calendar year .
the percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider .
the input price indexes are estimated on a historical basis and forecasted out several years .
the hcfa - chosen price proxies are forecasted under contract with data resources , inc./mcgraw hill ( dri ) . following every calendar year quarter ,
in march , june , september , and december , dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions . some of the data in tables 8 through 13
are forecasted and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received .
the methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits .
a description of the current structure of the pps input price index was published in the september 4 , 1990 , federal register .
the most recent pps update for payment rates was published in the september 1 , 1993 , federal register .
the latest hha regulatory input price index was published in the july 8 , 1993 , federal register , and the latest snf input price index was published in the october 7 , 1992 , federal register .
periodically , the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased .
each revision allows for new base weights , a new base year , and changes to certain price variables used for price proxies .
each input price index is presented in two tables : the first table shows the quarterly levels for each price index , and the second is a percentage change table .
national economic indicators provide a context for understanding health - specific indicators and how change in the health sector relates to change in the economy as a whole .
economy as the market value of goods and services produced within the geographic boundaries of the united states by u.s . or foreign citizens or companies .
real gdp removes the effects of price changes from the valuation of goods and services produced , so that the growth of real gdp reflects changes in the physical quantity of the output of the economy ( u.s . department of commerce , 1994 ) .
the growth in operating expenses of community hospitals continued to decelerate in the first quarter of 1994 .
measured over the same quarter a year earlier , total operating expenses increased 4.6 percent in the first quarter of 1994 , the lowest growth rate since the first quarter of 1985 .
the growth rate in total operating expenses has decelerated markedly in the last 2 years , as the 4.6-percent increase in the first quarter of 1994 is less than one - half of the 11.5-percent increase registered in the first quarter of 1992 . both labor and
non - labor expenses contributed to the continued deceleration in the growth of operating expenses .
labor expenses increased 4.5 percent in the first quarter , as measured over the same quarter a year earlier , while non - labor expenses increased 4.8 percent .
these increases compare with increases of 7.7 percent and 7.3 percent , respectively , in the first quarter of 1993 .
inpatient expenses followed the same trend as total operating expenses in the first quarter of 1994 , increasing 3.1 percent from the first quarter of 1993 ( figure 1 ) .
statistics on community hospital utilization indicate that in the first quarter of 1994 admissions increased slightly while the adult length of stay decreased ( figure 2 ) .
admissions in community hospitals increased 0.2 percent from the first quarter of 1993 to the first quarter of 1994 , while the adult length of stay decreased from 6.4 to 6.2 days .
taken together , these two factors led to a decrease in the number of inpatient days .
the number of inpatient days decreased 2.9 percent from the first quarter of 1993 to the first quarter of 1994 .
employment in the health care industry increased more rapidly than employment in the overall economy in the first quarter of 1994 .
however , the difference between the rate of growth in health care employment and the rate of growth in overall employment was smaller in the first quarter of 1994 than it has been since the beginning of the 1990 - 91 recession .
employment in health services grew 3.1 percent from first quarter 1993 to first quarter 1994 , increasing to a level of 8.9 million workers .
this was essentially the same growth as the 3.1 percent registered from the first quarter of 1992 to the first quarter of 1993 . in comparison ,
employment in the private non - agricultural sector grew 2.3 percent from the first quarter of 1993 to the first quarter of 1994 , an acceleration of 0.8 percentage point from the 1.5-percent growth registered from the first quarter of 1992 to the first quarter of 1993 ( figure 3 ) .
implied non - supervisory work hours and payrolls , developed from the bls establishment survey , are frequently cited as composite measures of economic activity . implied work hours are the product of the number of non - supervisory employees and average weekly hours . implied non - supervisory payrolls
are calculated by multiplying implied work hours by average hourly earnings . for private health service establishments ,
growth in both of these measures was approximately equal to the growth in the private non - agricultural sector in the first quarter of 1994 .
implied non - supervisory work hours in the health services industry grew 3.0 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 3.1 percent for the private non - agricultural sector .
implied non - supervisory payrolls in the health services industry grew 5.8 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 5.9 percent for the private non - agricultural sector . within the health services industry , private hospitals were responsible for most of the deceleration in these measures .
private hospitals continued to be the slowest - growing segment of the health care sector .
implied non - supervisory payrolls in private hospitals grew 2.8 percent from the first quarter of 1993 to the first quarter of 1994 , while implied non - supervisory work hours increased 0.4 percent in the same period .
consumer prices , as measured by the cpi for all urban consumers , increased 2.5 percent from the first quarter of 1993 to the first quarter of 1994 , a slight deceleration from the 3.2-percent increase registered from the first quarter of 1992 to the first quarter of 1993 . the increase in consumer prices for medical care , as measured by the cpi , was 5.0 percent from the first quarter of 1993 to the first quarter of 1994 , a deceleration from the 6.3-percent increase registered from the first quarter of 1992 to the first quarter of 1993 .
consumer prices for medical goods and services continued to increase more rapidly than consumer prices in the rest of the economy in the first quarter of 1994 .
however , price increases for medical goods and services are also decelerating more rapidly than price increases in the rest of the economy . in effect , the gap between increases in consumer prices for all items and consumer prices for medical care narrowed in first quarter 1994 .
this pattern of converging price increases follows a trend established in 1992 ( figure 4 ) .
the most significant changes in prices for medical goods and services in the first quarter of 1994 were in prescription drugs and outpatient hospital services .
consumer prices for prescription drugs , as measured by the cpi , increased 3.0 percent from the first quarter of 1993 to the first quarter of 1994 .
this followed an even larger deceleration in the preceding year , when price increases for prescription drugs increased 9.9 percent in the first quarter of 1992 , compared with 5.0 in the first quarter of 1993 . in effect , in the first quarter of 1994 , consumer prices for prescription drugs were increasing at only one - third the rate observed 2 years previously .
consumer price increases for outpatient hospital services , as measured by the cpi , also decelerated rapidly in the first quarter of 1994 .
consumer prices for outpatient hospital services increased 6.5 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 10.1 percent from the first quarter of 1992 to the first quarter of 1993 .
the current expansion continued , as the first quarter of 1994 marked the twelfth consecutive quarter of growth in the economy .
real gdp increased 3.7 percent from first quarter 1993 to first quarter 1994 , a slight acceleration in the rate of growth from the 3.2-percent increase recorded in the preceding 12 months .
inflation , measured by the change in the implicit price deflator for gdp , continued to moderate in the first quarter of 1994 , as the rate of increase in prices decelerated from the first quarter of 1993 to the first quarter of 1994 .
the implicit price deflator for gdp , a measure of aggregate price changes in the economy , increased 1.7 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 2.5 percent from the first quarter of 1992 to the first quarter of 1993 .
the growth in operating expenses of community hospitals continued to decelerate in the first quarter of 1994 .
measured over the same quarter a year earlier , total operating expenses increased 4.6 percent in the first quarter of 1994 , the lowest growth rate since the first quarter of 1985 .
the growth rate in total operating expenses has decelerated markedly in the last 2 years , as the 4.6-percent increase in the first quarter of 1994 is less than one - half of the 11.5-percent increase registered in the first quarter of 1992 .
both labor and non - labor expenses contributed to the continued deceleration in the growth of operating expenses .
labor expenses increased 4.5 percent in the first quarter , as measured over the same quarter a year earlier , while non - labor expenses increased 4.8 percent .
these increases compare with increases of 7.7 percent and 7.3 percent , respectively , in the first quarter of 1993 .
inpatient expenses followed the same trend as total operating expenses in the first quarter of 1994 , increasing 3.1 percent from the first quarter of 1993 ( figure 1 ) .
statistics on community hospital utilization indicate that in the first quarter of 1994 admissions increased slightly while the adult length of stay decreased ( figure 2 ) .
admissions in community hospitals increased 0.2 percent from the first quarter of 1993 to the first quarter of 1994 , while the adult length of stay decreased from 6.4 to 6.2 days .
taken together , these two factors led to a decrease in the number of inpatient days .
the number of inpatient days decreased 2.9 percent from the first quarter of 1993 to the first quarter of 1994 .
employment in the health care industry increased more rapidly than employment in the overall economy in the first quarter of 1994 .
however , the difference between the rate of growth in health care employment and the rate of growth in overall employment was smaller in the first quarter of 1994 than it has been since the beginning of the 1990 - 91 recession .
employment in health services grew 3.1 percent from first quarter 1993 to first quarter 1994 , increasing to a level of 8.9 million workers .
this was essentially the same growth as the 3.1 percent registered from the first quarter of 1992 to the first quarter of 1993 . in comparison ,
employment in the private non - agricultural sector grew 2.3 percent from the first quarter of 1993 to the first quarter of 1994 , an acceleration of 0.8 percentage point from the 1.5-percent growth registered from the first quarter of 1992 to the first quarter of 1993 ( figure 3 ) .
implied non - supervisory work hours and payrolls , developed from the bls establishment survey , are frequently cited as composite measures of economic activity . implied work hours are the product of the number of non - supervisory employees and average weekly hours .
implied non - supervisory payrolls are calculated by multiplying implied work hours by average hourly earnings . for private health service establishments ,
growth in both of these measures was approximately equal to the growth in the private non - agricultural sector in the first quarter of 1994 .
implied non - supervisory work hours in the health services industry grew 3.0 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 3.1 percent for the private non - agricultural sector .
implied non - supervisory payrolls in the health services industry grew 5.8 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 5.9 percent for the private non - agricultural sector . within the health services industry , private hospitals were responsible for most of the deceleration in these measures .
private hospitals continued to be the slowest - growing segment of the health care sector .
implied non - supervisory payrolls in private hospitals grew 2.8 percent from the first quarter of 1993 to the first quarter of 1994 , while implied non - supervisory work hours increased 0.4 percent in the same period .
consumer prices , as measured by the cpi for all urban consumers , increased 2.5 percent from the first quarter of 1993 to the first quarter of 1994 , a slight deceleration from the 3.2-percent increase registered from the first quarter of 1992 to the first quarter of 1993 . the increase in consumer prices for medical care , as measured by the cpi , was 5.0 percent from the first quarter of 1993 to the first quarter of 1994 , a deceleration from the 6.3-percent increase registered from the first quarter of 1992 to the first quarter of 1993 .
consumer prices for medical goods and services continued to increase more rapidly than consumer prices in the rest of the economy in the first quarter of 1994 .
however , price increases for medical goods and services are also decelerating more rapidly than price increases in the rest of the economy . in effect , the gap between increases in consumer prices for all items and consumer prices for medical care narrowed in first quarter 1994 .
this pattern of converging price increases follows a trend established in 1992 ( figure 4 ) .
the most significant changes in prices for medical goods and services in the first quarter of 1994 were in prescription drugs and outpatient hospital services .
consumer prices for prescription drugs , as measured by the cpi , increased 3.0 percent from the first quarter of 1993 to the first quarter of 1994 .
this followed an even larger deceleration in the preceding year , when price increases for prescription drugs increased 9.9 percent in the first quarter of 1992 , compared with 5.0 in the first quarter of 1993 . in effect , in the first quarter of 1994 , consumer prices for prescription drugs were increasing at only one - third the rate observed 2 years previously .
consumer price increases for outpatient hospital services , as measured by the cpi , also decelerated rapidly in the first quarter of 1994 .
consumer prices for outpatient hospital services increased 6.5 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 10.1 percent from the first quarter of 1992 to the first quarter of 1993 .
the current expansion continued , as the first quarter of 1994 marked the twelfth consecutive quarter of growth in the economy .
real gdp increased 3.7 percent from first quarter 1993 to first quarter 1994 , a slight acceleration in the rate of growth from the 3.2-percent increase recorded in the preceding 12 months .
inflation , measured by the change in the implicit price deflator for gdp , continued to moderate in the first quarter of 1994 , as the rate of increase in prices decelerated from the first quarter of 1993 to the first quarter of 1994 .
the implicit price deflator for gdp , a measure of aggregate price changes in the economy , increased 1.7 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 2.5 percent from the first quarter of 1992 to the first quarter of 1993 . | this regular feature of the journal includes a discussion of each of the following four topics : community hospital statistics ; employment , hours , and earnings in the private health sector ; health care prices ; and national economic indicators .
these statistics are valuable in their own right for understanding the relationship between the health care sector and the overall economy . in addition
, they allow us to anticipate the direction and magnitude of health care cost changes prior to the availability of more comprehensive data . | Introduction
Community Hospital Statistics
Private Health Sector: Employment, Hours, and Earnings
Prices
Consumer Prices
Background on Input Price Indexes
National Economic Indicators
First Quarter Indicators | this article presents statistics on health care utilization , prices , expenses , employment , and work hours , as well as on national economic activity . some of these statistics are based on sample surveys conducted monthly or quarterly by government agencies or private organizations , and are available 1 to 3 months after the completion of the period . these statistics provide an early indication of changes occurring within the general economy and in the health care sector . for quarterly information
, this calculation permits analysis of quarterly data to focus on the direction and magnitude of changes , without interference introduced by seasonal fluctuations . in the national health accounts , indicators such as these play an important role in the estimation of the latest historical year of health care expenditures . in the following sections
, we will identify important indicators of health care and national economic activity and their sources . for purposes of national health expenditures
( nhe ) , survey statistics on revenues ( not shown on table 1 ) are analyzed in estimating the growth in the largest component of health care costs community hospital expenditures . similar statistics for the private non - agricultural sector , included on these tables , provide a basis for comparing the economy as a whole with the health sector in employment , earnings , and work hours . figure 3 shows changes from the same quarter 1 year earlier in employment in the private non - agricultural sector and the health services industry for 1985 to 1994 . table 5 summarizes business activity in the health sector and the overall economy by measuring change in the implied non - supervisory work hours and payroll . for purposes of nhe , changes in work hours by industry combined with changes in prices ( discussed in a later section ) can be used to gauge the direction and magnitude of expenditure change in specific industries . in addition , some medical care sector indexes measure changes in list or charged prices , rather than in prices actually received by providers after discounts are deducted . in the nhe ,
a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by hcfa to provide transaction price changes , are used as measures of inflation for the health industry . in the nhe ,
a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by hcfa to provide transaction price changes , are used as measures of inflation for the health industry . the methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits . national economic indicators provide a context for understanding health - specific indicators and how change in the health sector relates to change in the economy as a whole . employment in the health care industry increased more rapidly than employment in the overall economy in the first quarter of 1994 . however , the difference between the rate of growth in health care employment and the rate of growth in overall employment was smaller in the first quarter of 1994 than it has been since the beginning of the 1990 - 91 recession . in comparison ,
employment in the private non - agricultural sector grew 2.3 percent from the first quarter of 1993 to the first quarter of 1994 , an acceleration of 0.8 percentage point from the 1.5-percent growth registered from the first quarter of 1992 to the first quarter of 1993 ( figure 3 ) . for private health service establishments ,
growth in both of these measures was approximately equal to the growth in the private non - agricultural sector in the first quarter of 1994 . implied non - supervisory work hours in the health services industry grew 3.0 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 3.1 percent for the private non - agricultural sector . implied non - supervisory payrolls in the health services industry grew 5.8 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 5.9 percent for the private non - agricultural sector . private hospitals continued to be the slowest - growing segment of the health care sector . employment in the health care industry increased more rapidly than employment in the overall economy in the first quarter of 1994 . however , the difference between the rate of growth in health care employment and the rate of growth in overall employment was smaller in the first quarter of 1994 than it has been since the beginning of the 1990 - 91 recession . for private health service establishments ,
growth in both of these measures was approximately equal to the growth in the private non - agricultural sector in the first quarter of 1994 . implied non - supervisory work hours in the health services industry grew 3.0 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 3.1 percent for the private non - agricultural sector . implied non - supervisory payrolls in the health services industry grew 5.8 percent from the first quarter of 1993 to the first quarter of 1994 , compared with an increase of 5.9 percent for the private non - agricultural sector . private hospitals continued to be the slowest - growing segment of the health care sector . | [
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] |
alzheimer s disease ( ad ) affects 5%15% of the population over 65 years , and about 20% of individuals over 80 years .
ad is characterized by a gradual onset of multiple cognitive deficits and is associated with continuing cognitive decline .
there is a high prevalence of psychotic symptoms ( paulsen et al 2000 ) and behavioral disturbances in ad ( devanand et al 1992 ; eustace et al 2002 ) .
a review of studies that assessed the prevalence of psychosis associated with ad showed a median prevalence rate of 41.1% ( range , 12.2%74.1% ) ( ropacki and jeste 2005 ) ; however , the patients followed up in the outpatient clinics showed lower rates ( bassiony et al 2000 ) .
psychotic symptoms have been linked to increased cognitive and functional decline ( chui et al 1994 ; lopez et al 1999 ) leading to caregiver distress ( everitt et al 1991 ; hopkins and libon 2005 ) and an increased likelihood of institutionalization ( lopez et al 1999 ) in patients with ad .
jeste and finkel ( 2000 ) have defined the diagnostic criteria for psychosis of ad ( table 1 ) . among the psychotropic medications , antipsychotics are the most studied treatment for psychosis , aggression , and agitation in elderly patients with dementia ( doody et al 2001 ; kindermann et al 2002 ) .
these studies have shown the efficacy of atypical antipsychotics in treating some of the behavioral symptoms that are associated with ad ( katz et al 1999 ; street et al 2000 ; tariot et al 2002 ; brodaty et al 2003 ; de deyn et al 1999 , 2004 ; zhong et al 2004 ) .
even though there is no psychotropic medication that is approved by the us food and drug administration ( fda ) for the treatment of psychosis of ad , a number of expert consensus statements recommend the use of atypical antipsychotic agents as a first - line pharmacologic approach to treatment ( alexopoulos et al 2004 ; ags / aagp ) . despite these findings
, only a few studies have prospectively evaluated the antipsychotic effects of this class of compounds in patients with ad using the criteria defined by jeste and finkel ( 2000 ) .
in contrast , most published studies with atypical agents have also included patients with vascular and mixed dementias , and those with behavioral disturbances , such as agitation .
safety and tolerability of a medication is of great concern while treating the elderly due to their increased sensitivity to adverse events ( aes ) , the increased likelihood of co - morbid health problems ( eg , cardiovascular disease ) , and the potential for some side effects to worsen the progression of dementia ( kindermann et al 2002 ) .
studies have shown that risperidone , olanzapine , and quetiapine can reduce psychotic symptoms and behavioral disturbances in patients with dementia ( katz et al 1999 ; defilippi and crismon 2000 ; street et al 2000 ; madhusoodanan et al 2001 ; tariot et al 2002 ; brodaty et al 2003 ; de deyn et al 1999 , 2004 ; zhong et al 2004 .
however , the side - effect profiles of individual atypical antipsychotics , especially the metabolic effects may adversely influence their overall effectiveness and acceptability ( jeste et al 1999 ; masand 2000 ) .
movement disorders , sedation , and orthostasis are associated with an increased risk of falls and subsequent fractures .
analyses of safety data from several studies have raised concerns about an increased risk of cerebrovascular adverse events ( cvaes ) , such as stroke , with the use of some atypical antipsychotics compared with placebo in elderly patients with dementia ( wooltorton 2002 , 2004 ) .
this has led fda to issue warnings on the use of atypical antipsychotics in the treatment of behavioral and psychological symptoms in elderly patients with dementia , particularly those with a history of cerebrovascular disease ( us fda 2003 ; health canada ; mhra ) .
more recently , the fda issued a separate warning regarding an increased mortality risk in elderly patients with dementia who were treated with atypical antipsychotics ( us fda 2005 ) .
we have dealt with these issues in detail in our recent review article ( madhusoodanan et al 2007 ) .
aripiprazole is a novel atypical antipsychotic that has shown rapid and sustained efficacy and tolerability in short - term and long - term controlled clinical trials and retrospective studies in patients with schizophrenia , schizoaffective disorder and bipolar disorder ( liebermann 2004 ; madhusoodanan et al 2004 ; sajatovic et al 2008 ) .
recently fda approved the use of aripiprazole as an adjunctive to antidepressants for the treatment of major depressive disorder in adults ( prescribing information 2007 ) .
aripiprazole is a partial d2-receptor agonist ( burris et al 2002 ) , 5-ht2a receptor antagonist ( byars et al 2002 ) , and a partial agonist at 5-ht1a receptors ( jordan et al 2002 ) .
in addition , aripiprazole has been shown to exhibit moderate affinity for the alpha1 , alpha2 and histamine h1 receptors and negligible affinity for muscarinic m1 receptors ( deleon et al 2004 ; prescribing information 2007 ) .
three 10-week , placebo - controlled , randomized , double - blind clinical studies have evaluated the efficacy and tolerability of aripiprazole for the treatment of psychosis related to ad ( table 2a ) ( streim et al 2004 ; de deyn et al 2005 ; mintzer et al 2007 ) .
of these , the studies conducted by de deyn et al and streim et al examined flexible dosing of aripiprazole ( 215 mg / d ) , and mintzer et al studied fixed doses of aripiprazole ( 2 mg / d , 5 mg / d , 10 mg / d ) .
patients in the streim et al and mintzer et al studies were either full time nursing home or assisted living facility residents , while patients in the de deyn et al study were living in assisted living facilities or adult communities , or with a caregiver .
the inclusion criteria were delusions / hallucinations lasting for 1 month or longer , mini mental state exam ( mmse ; fostein et al 1975 ) score of 624 , and score of more than 6 on delusions or hallucinations items of neuropsychiatric inventory ( npi ; cummings et al 1994 ) scale at baseline . in the de deyn
et al ( 2005 ) study , 208 outpatients ( mean age 81.5 years , range 5595 ) with ad - associated psychosis and mean mmse score of 14.35 ( aripiprazole ) and 14.13 ( placebo ) , were randomized to aripiprazole ( n = 106 ) or placebo ( n = 102 ) .
the initial aripiprazole dose of 2 mg / d was titrated upwards ( 5 , 10 , or 15 mg / d ) usually every 2 weeks according to efficacy and tolerability .
patients in this study were noninstitutionalized , in contrast to most studies with other atypicals published to date , which included institutionalized patients ( katz et al 1999 ; street et al 2000 ; tariot et al 2002 ; brodaty et al 2003 ; de deyn et al 1999 , 2004 ; zhong et al 2004 ) . in this study
, the degree of cognitive impairment ( mean mmse , 14.35 for aripiprazole , 14.13 for placebo ) was less compared with most of the studies with other atypicals ( mean mmse , 6.19.3 ) . in the streim et al ( 2004 ) study , 256 inpatients ( mean age 83 years , range 5996 ) with ad - associated psychosis and mean mmse score of 12.9 were randomized to aripiprazole ( n = 131 ) or placebo ( n = 125 ) .
the suggested titration for this study was : week 1 : 2 mg / d ; week 23 : 5mg / d ; week 45 : 10 mg / d ; week 610 : 15 mg / d .
in the mintzer et al ( 2007 ) study , 487 inpatients ( mean age 82.5 years , range 5697 ) with ad - associated psychosis and mean mmse score of 12.4 were randomized to fixed doses of aripiprazole , 2 mg / d ( n = 118 ) , 5 mg / d ( n = 122 ) , 10 mg / d ( n = 126 ) ( patients were started on 5 mg / d for 7 days and the dose was then increased to 10 mg / d ) , or placebo ( n= 121 ) . by week 6 , patients with clinical global impression of improvement ( cgi - i ; guy 1976 ) score of 4 were allowed open - label treatment with aripiprazole through week 10 . in total , 284 patients completed 10 weeks of double - blind therapy .
in the de deyn et al ( 2005 ) study , the caregiver - assessed npi psychosis subscale ( cummings et al 1994 ) score showed improvements in both groups ( aripiprazole 6.55 , placebo 5.52 ; p = 0.17 at end point ) , but the difference between aripiprazole and placebo was not statistically significant . in the streim
et al ( 2004 ) study , there was no statistically significant difference between aripiprazole and placebo on the npi psychosis subscale score at week 10 .
there were decreases in clinical global impression - severity of illness ( cgi - s ; guy 1976 ) scores in both aripiprazole and placebo groups , but the difference in the mean change in cgi - s in aripiprazole vs placebo was significant ( p < 0.05 ) only at week 8 . in the mintzer
et al ( 2007 ) study , there was a statistically significant difference between aripiprazole and placebo ( aripiprazole 10 mg / d , 6.87 .
p 0.013 ) on the neuropsychiatric inventory - nursing home ( npi - nh ) psychosis subscale ( guy 1976 ; cummings et al 1994 ; wood et al 2000 ) score at week 10 .
mean change in npi - nh psychosis subscale score was also significant for the aripiprazole 5 mg group vs placebo at week 8 ( p < 0.05 ) .
there was a statistically significant difference ( p < 0.05 ) in the response to treatment as defined by 50% reduction in the npi - nh psychosis subscale score at week 10 for aripiprazole ( 65% ) vs placebo ( 50% ) . in the de
deyn et al ( 2005 ) study , aripiprazole - treated patients showed significantly greater improvements from baseline in physician assessed brief psychiatric rating scale ( bprs ) ( overall and gorham 1962 ) psychosis and bprs core subscale scores at end point compared with placebo .
there were no statistically significant differences between aripiprazole and placebo in the mean change in bprs total score ( aripiprazole 8.53 , placebo 6.58 ; p value not significant ) , npi total ( aripiprazole 9.75 , placebo 11.20 ; p value not significant ) , cgi - s ( aripiprazole 0.69 , placebo 0.54 ; p value not significant ) , cgi - i ( aripiprazole 3.17 , placebo , 3.07 ; p value not significant ) .
patients in both the aripiprazole and placebo groups showed little change in cognitive function over the course of the study , with minimal mean changes ( < 1 point ) in mmse score observed from baseline to end point ( aripiprazole 0.81 , placebo 0.53 ; p 0.001 ) .
the investigators explained the small improvement in mmse scores seen in the placebo group as a possible response to the increased attention patients received during the study .
the minimal changes observed in both groups and high mean baseline scores , coupled with the wide variation in baseline mmse scores ( from 2 to 23 ) , suggested that these are of little clinical significance .
aripiprazole has negligible affinity for muscarinic m1 receptors , and hence has a low potential for cognitive impairment , and has shown improvements in cognitive function in a study in patients with schizophrenia or schizoaffective disorder ( cornblatt et al 2002 ) . in the streim et al ( 2004 ) study , there was a statistically significant difference ( p = 0.006 ) in the response to treatment as defined by 50% reduction in the npi total score at week 10 for aripiprazole ( 46% ) and placebo ( 28% ) .
there were statistically significant improvements in the npi - nh total scores ( aripiprazole 16.4 , placebo 10.0 ; p 0.009 ) , bprs total score ( aripiprazole , 7.7 placebo , 5.1 ; p 0.031 ) and cornell depression in dementia scale ( cdds ; alexopoulos et al 1988 ) total score ( aripiprazole 0.13 , placebo 1.98 ; p 0.006 ) at week 10 .
there were statistically significant ( p < 0.05 ) improvements with aripiprazole vs placebo group in cohen - mansfield agitation inventory ( cmai ; cohen - mansfield and billig 1986 ; cohen - mansfield 1995 ; miller et al 1995 ; weiner et al 2002 ) and cgi - i scores . in the mintzer et al ( 2007 )
study , there was a statistically significant improvement at week 10 with aripiprazole 10 mg / d vs placebo group in bprs total score ( aripiprazole 7.12 , placebo 4.17 ; p 0.03 ) and in the cgi - s ( aripiprazole 0.72 , placebo , 0.46 ; p 0.031 ) .
there was a significant reduction in the cmai scores with aripiprazole 5 mg / d and 10 mg / d vs placebo at weeks 6 through 10 .
there was a significant reduction in the bprs core score with aripiprazole 5 mg / d vs placebo at weeks 4 , 6 , and 8 , and with 10 mg / d vs placebo at weeks 4 through 10 .
there was a significant reduction in the npi - nh total score with aripiprazole 5 mg / d vs placebo at weeks 6 and 8 .
kostic et al ( 2005 ) analyzed the data from the two studies ( streim et al 2004 ; mintzer et al 2007 ) to investigate further the effect of aripiprazole on agitation as measured by reduction in npi - agitation item , cmai , and tension / hostility / uncooperativeness / excitement scores of bprs .
in the streim et al ( 2004 ) study , npi agitation / aggression , cmai , and bprs tension / hostility / uncooperativeness / excitement scores were significantly reduced at study endpoint ( p = 0.001 , p < 0.05 , p < 0.05 respectively ) . in the mintzer
et al ( 2007 ) study , aripiprazole 5 mg / d and 10 mg / d demonstrated significant reduction ( p < 0.05 ) of npi - nh agitation item and bprs tension / hostility / uncooperativeness / excitement subscore compared with placebo . a significant decrease from baseline on bprs tension / hostility / uncooperativeness / excitement subscore was also observed with aripiprazole 2 mg / d ( beginning at week 6 ) at study endpoint ( p 0.05 ) .
the effect of aripiprazole on agitation as measured by bprs tension / hostility / uncooperativeness / excitement subscore was seen earlier with higher doses ( beginning at week 2 with 10 mg / d and beginning at week 4 with 5
discontinuation due to lack of efficacy occurred in 3 patients on aripiprazole and 6 patients on placebo in the de deyn et al ( 2005 ) study , and in 1 patient on aripiprazole and 3 patients on placebo in the streim et al study ( 2004 ) .
discontinuation due to lack of response occurred in 13% of patients on aripiprazole vs 30% of patients on placebo , and time to discontinuation was significantly longer in the aripiprazole group vs the placebo group ( p = 0.022 ) in the streim et al ( 2004 ) study . in the mintzer
et al ( 2007 ) study , discontinuation due to lack of efficacy occurred in 3% of patients on 2 mg / d of aripiprazole , 2% of patients on 5 mg / d of aripiprazole , 2% of patients on 10 mg / d of aripiprazole , and 2% of patients on placebo .
discontinuation due to lack of response occurred in 21% of patients on 2 mg / d of aripiprazole , 16% of patients on 5
mg / d of aripiprazole , 13% of patients on 10 mg / d of aripiprazole , and 24% of patients on placebo .
thus the discontinuation due to lack of response was highest in the lowest - dose group and placebo group . in the de deyn et al ( 2005 )
study , aes were generally mild to moderate and included ( aripiprazole vs placebo ) : urinary tract infection ( 8% vs 12% ) , accidental injury ( 8% vs 5% ) , somnolence ( 8% vs 1% ) , and bronchitis ( 6% vs 3% ) .
there were no significant differences vs placebo in the extrapyramidal symptoms ( eps ) scores , or clinically significant electrocardiogram ( ecg ) abnormalities , vital signs , or weight . in the streim
et al ( 2004 ) study , aes that occurred at the rate of 10% in the aripiprazole group were accidental injury , somnolence , urinary tract infection , ecchymosis , asthenia , vomiting , and rash , and in the placebo group were accidental injury , urinary act infection , ecchymosis , and rash .
eps occurred in 5% of aripiprazole group vs 4% of placebo group , somnolence occurred in 14% of aripiprazole group vs 4% of placebo group , and accidental injuries occurred in 21% of aripiprazole group vs 30% of placebo group . in the mintzer
et al ( 2007 ) study , aes occurring at 10% incidence in any dosage group of aripiprazole were accidental injury , agitation , peripheral edema , extremity pain , somnolence , urinary incontinence , asthenia , vomiting , and skin ulcer , and in the placebo group , accidental injury , anorexia , ecchymosis , and agitation .
eps occurred in 8% of aripiprazole 2 mg / d group , 7% in aripiprazole 5 mg / d group , 7% in aripiprazole 10 mg / d group , and 6% in the placebo group .
somnolence occurred in 3% of aripiprazole 2 mg / d group , 10% in aripiprazole 5 mg / d group , 7% in aripiprazole 10 mg / d group , and 3% in the placebo group .
accidental injuries occurred in 30% of aripiprazole 2 mg / d group , 24% in aripiprazole 5 mg / d group , 20% in aripiprazole 10 mg / d group , and 19% in the placebo group .
analysis of the data on mortality in elderly patients with dementia - related psychosis ( breder et al 2005 ; pers comm jan 8 2008 , bristol - myers squibb and otsuka pharmaceutical co , ltd , data on file ) of the three trials ( streim et al 2004 ; de deyn et al 2005 ; minzter et al 2007 ) for deaths that occurred during or within 30 days of discontinuation from the placebo - controlled phase of the clinical trial showed that incidence of deaths was 3.5% in aripiprazole - treated patients and 1.7% in placebo - treated patients ( p= 0.139 ) .
deaths were mostly associated with cardiovascular or infectious conditions . in these three trials ( streim et al 2004 ; de deyn et al 2005 ;
minzter et al 2007 ) , there was increased incidence of cvaes ( eg , stroke , transient ischemic attack ) , including fatalities , in aripiprazole - treated patients . in the fixed - dose study of mintzer
et al ( 2007 ) there was a statistically significant dose - response relationship for cvaes in patients treated with aripiprazole . in an analysis ( pers comm jan 8 2008 , bristol - myers squibb and otsuka pharmaceutical co , ltd , data on file ) of the three 10-week , placebo - controlled studies of aripiprazole in elderly patients with psychosis associated with ad ( n = 938 , mean age 82.4 years , range 5699 years ) , the treatment - emergent aes that were reported at an incidence of 3% and at least twice that for placebo were lethargy ( placebo 2% , aripiprazole 5% ) , somnolence ( including sedation ) ( placebo 3% , aripiprazole 8% ) , and incontinence ( primarily , urinary incontinence ) ( placebo 1% , aripiprazole 5% ) , excessive salivation ( placebo 0% , aripiprazole 4% ) , and lightheadedness ( placebo 1% , aripiprazole 4% ) .
discontinuation due to aes occurred in 10 patients on aripiprazole vs 7 patients on placebo in the de deyn et al ( 2005 ) study , 14% on aripiprazole vs 9% patients on placebo in the streim et al ( 2004 ) study . in the mintzer
et al ( 2007 ) study , discontinuation occurred in 8% on aripiprazole 2 mg / d , 18% on aripiprazole 5 mg / d , 25% on aripiprazole 10 mg / d vs 13% patients on placebo .
the most common ae leading to discontinuation among the aripiprazole and placebo groups were asthenia and agitation , respectively .
discontinuations due to asthenia appeared to be dose - related , with the greatest number of patients experiencing asthenia in the aripiprazole 10 mg / day dose group ( placebo 1% , aripiprazole 2 mg / day 1% , aripiprazole 5 mg / day 2% , and aripiprazole 10 mg / day 4% ) .
in the de deyn et al ( 2005 ) study , the caregiver - assessed npi psychosis subscale ( cummings et al 1994 ) score showed improvements in both groups ( aripiprazole 6.55 , placebo 5.52 ; p = 0.17 at end point ) , but the difference between aripiprazole and placebo was not statistically significant . in the streim
et al ( 2004 ) study , there was no statistically significant difference between aripiprazole and placebo on the npi psychosis subscale score at week 10 .
there were decreases in clinical global impression - severity of illness ( cgi - s ; guy 1976 ) scores in both aripiprazole and placebo groups , but the difference in the mean change in cgi - s in aripiprazole vs placebo was significant ( p < 0.05 ) only at week 8 . in the mintzer
et al ( 2007 ) study , there was a statistically significant difference between aripiprazole and placebo ( aripiprazole 10 mg / d , 6.87 .
p 0.013 ) on the neuropsychiatric inventory - nursing home ( npi - nh ) psychosis subscale ( guy 1976 ; cummings et al 1994 ; wood et al 2000 ) score at week 10 .
mean change in npi - nh psychosis subscale score was also significant for the aripiprazole 5 mg group vs placebo at week 8 ( p < 0.05 ) .
there was a statistically significant difference ( p < 0.05 ) in the response to treatment as defined by 50% reduction in the npi - nh psychosis subscale score at week 10 for aripiprazole ( 65% ) vs placebo ( 50% ) . in the de
deyn et al ( 2005 ) study , aripiprazole - treated patients showed significantly greater improvements from baseline in physician assessed brief psychiatric rating scale ( bprs ) ( overall and gorham 1962 ) psychosis and bprs core subscale scores at end point compared with placebo .
there were no statistically significant differences between aripiprazole and placebo in the mean change in bprs total score ( aripiprazole 8.53 , placebo 6.58 ; p value not significant ) , npi total ( aripiprazole 9.75 , placebo 11.20 ; p value not significant ) , cgi - s ( aripiprazole 0.69 , placebo 0.54 ; p value not significant ) , cgi - i ( aripiprazole 3.17 , placebo , 3.07 ; p value not significant ) .
patients in both the aripiprazole and placebo groups showed little change in cognitive function over the course of the study , with minimal mean changes ( < 1 point ) in mmse score observed from baseline to end point ( aripiprazole 0.81 , placebo 0.53 ; p 0.001 ) .
the investigators explained the small improvement in mmse scores seen in the placebo group as a possible response to the increased attention patients received during the study .
the minimal changes observed in both groups and high mean baseline scores , coupled with the wide variation in baseline mmse scores ( from 2 to 23 ) , suggested that these are of little clinical significance .
aripiprazole has negligible affinity for muscarinic m1 receptors , and hence has a low potential for cognitive impairment , and has shown improvements in cognitive function in a study in patients with schizophrenia or schizoaffective disorder ( cornblatt et al 2002 ) . in the streim et al ( 2004 ) study , there was a statistically significant difference ( p = 0.006 ) in the response to treatment as defined by 50% reduction in the npi total score at week 10 for aripiprazole ( 46% ) and placebo ( 28% ) .
there were statistically significant improvements in the npi - nh total scores ( aripiprazole 16.4 , placebo 10.0 ; p 0.009 ) , bprs total score ( aripiprazole , 7.7 placebo , 5.1 ; p 0.031 ) and cornell depression in dementia scale ( cdds ; alexopoulos et al 1988 ) total score ( aripiprazole 0.13 , placebo 1.98 ; p 0.006 ) at week 10 .
there were statistically significant ( p < 0.05 ) improvements with aripiprazole vs placebo group in cohen - mansfield agitation inventory ( cmai ; cohen - mansfield and billig 1986 ; cohen - mansfield 1995 ; miller et al 1995 ; weiner et al 2002 ) and cgi - i scores . in the mintzer et al ( 2007 )
study , there was a statistically significant improvement at week 10 with aripiprazole 10 mg / d vs placebo group in bprs total score ( aripiprazole 7.12 , placebo 4.17 ; p 0.03 ) and in the cgi - s ( aripiprazole 0.72 , placebo , 0.46 ; p 0.031 ) .
there was a significant reduction in the cmai scores with aripiprazole 5 mg / d and 10 mg / d vs placebo at weeks 6 through 10 .
there was a significant reduction in the bprs core score with aripiprazole 5 mg / d vs placebo at weeks 4 , 6 , and 8 , and with 10 mg / d vs placebo at weeks 4 through 10 .
there was a significant reduction in the npi - nh total score with aripiprazole 5 mg / d vs placebo at weeks 6 and 8 .
kostic et al ( 2005 ) analyzed the data from the two studies ( streim et al 2004 ; mintzer et al 2007 ) to investigate further the effect of aripiprazole on agitation as measured by reduction in npi - agitation item , cmai , and tension / hostility / uncooperativeness / excitement scores of bprs .
in the streim et al ( 2004 ) study , npi agitation / aggression , cmai , and bprs tension / hostility / uncooperativeness / excitement scores were significantly reduced at study endpoint ( p = 0.001 , p < 0.05 , p < 0.05 respectively ) . in the mintzer
et al ( 2007 ) study , aripiprazole 5 mg / d and 10 mg / d demonstrated significant reduction ( p < 0.05 ) of npi - nh agitation item and bprs tension / hostility / uncooperativeness / excitement subscore compared with placebo . a significant decrease from baseline on bprs tension / hostility / uncooperativeness / excitement subscore was also observed with aripiprazole 2 mg / d ( beginning at week 6 ) at study endpoint ( p 0.05 ) .
the effect of aripiprazole on agitation as measured by bprs tension / hostility / uncooperativeness / excitement subscore was seen earlier with higher doses ( beginning at week 2 with 10 mg / d and beginning at week 4 with 5
discontinuation due to lack of efficacy occurred in 3 patients on aripiprazole and 6 patients on placebo in the de deyn et al ( 2005 ) study , and in 1 patient on aripiprazole and 3 patients on placebo in the streim et al study ( 2004 ) .
discontinuation due to lack of response occurred in 13% of patients on aripiprazole vs 30% of patients on placebo , and time to discontinuation was significantly longer in the aripiprazole group vs the placebo group ( p = 0.022 ) in the streim et al ( 2004 ) study . in the mintzer
et al ( 2007 ) study , discontinuation due to lack of efficacy occurred in 3% of patients on 2 mg / d of aripiprazole , 2% of patients on 5 mg / d of aripiprazole , 2% of patients on 10 mg / d of aripiprazole , and 2% of patients on placebo .
discontinuation due to lack of response occurred in 21% of patients on 2 mg / d of aripiprazole , 16% of patients on 5
mg / d of aripiprazole , 13% of patients on 10 mg / d of aripiprazole , and 24% of patients on placebo .
thus the discontinuation due to lack of response was highest in the lowest - dose group and placebo group .
in the de deyn et al ( 2005 ) study , the caregiver - assessed npi psychosis subscale ( cummings et al 1994 ) score showed improvements in both groups ( aripiprazole 6.55 , placebo 5.52 ; p = 0.17 at end point ) , but the difference between aripiprazole and placebo was not statistically significant . in the streim
et al ( 2004 ) study , there was no statistically significant difference between aripiprazole and placebo on the npi psychosis subscale score at week 10 .
there were decreases in clinical global impression - severity of illness ( cgi - s ; guy 1976 ) scores in both aripiprazole and placebo groups , but the difference in the mean change in cgi - s in aripiprazole vs placebo was significant ( p < 0.05 ) only at week 8 . in the mintzer
et al ( 2007 ) study , there was a statistically significant difference between aripiprazole and placebo ( aripiprazole 10 mg / d , 6.87 .
p 0.013 ) on the neuropsychiatric inventory - nursing home ( npi - nh ) psychosis subscale ( guy 1976 ; cummings et al 1994 ; wood et al 2000 ) score at week 10 .
mean change in npi - nh psychosis subscale score was also significant for the aripiprazole 5 mg group vs placebo at week 8 ( p < 0.05 ) .
there was a statistically significant difference ( p < 0.05 ) in the response to treatment as defined by 50% reduction in the npi - nh psychosis subscale score at week 10 for aripiprazole ( 65% ) vs placebo ( 50% ) .
in the de deyn et al ( 2005 ) study , aripiprazole - treated patients showed significantly greater improvements from baseline in physician assessed brief psychiatric rating scale ( bprs ) ( overall and gorham 1962 ) psychosis and bprs core subscale scores at end point compared with placebo .
there were no statistically significant differences between aripiprazole and placebo in the mean change in bprs total score ( aripiprazole 8.53 , placebo 6.58 ; p value not significant ) , npi total ( aripiprazole 9.75 , placebo 11.20 ; p value not significant ) , cgi - s ( aripiprazole 0.69 , placebo 0.54 ; p value not significant ) , cgi - i ( aripiprazole 3.17 , placebo , 3.07 ; p value not significant ) .
patients in both the aripiprazole and placebo groups showed little change in cognitive function over the course of the study , with minimal mean changes ( < 1 point ) in mmse score observed from baseline to end point ( aripiprazole 0.81 , placebo 0.53 ; p 0.001 ) .
the investigators explained the small improvement in mmse scores seen in the placebo group as a possible response to the increased attention patients received during the study .
the minimal changes observed in both groups and high mean baseline scores , coupled with the wide variation in baseline mmse scores ( from 2 to 23 ) , suggested that these are of little clinical significance .
aripiprazole has negligible affinity for muscarinic m1 receptors , and hence has a low potential for cognitive impairment , and has shown improvements in cognitive function in a study in patients with schizophrenia or schizoaffective disorder ( cornblatt et al 2002 ) . in the streim
et al ( 2004 ) study , there was a statistically significant difference ( p = 0.006 ) in the response to treatment as defined by 50% reduction in the npi total score at week 10 for aripiprazole ( 46% ) and placebo ( 28% ) .
there were statistically significant improvements in the npi - nh total scores ( aripiprazole 16.4 , placebo 10.0 ; p 0.009 ) , bprs total score ( aripiprazole , 7.7 placebo , 5.1 ; p 0.031 ) and cornell depression in dementia scale ( cdds ; alexopoulos et al 1988 ) total score ( aripiprazole 0.13 , placebo 1.98 ; p 0.006 ) at week 10 .
there were statistically significant ( p < 0.05 ) improvements with aripiprazole vs placebo group in cohen - mansfield agitation inventory ( cmai ; cohen - mansfield and billig 1986 ; cohen - mansfield 1995 ; miller et al 1995 ; weiner et al 2002 ) and cgi - i scores . in the mintzer
et al ( 2007 ) study , there was a statistically significant improvement at week 10 with aripiprazole 10 mg / d vs placebo group in bprs total score ( aripiprazole 7.12 , placebo 4.17 ; p 0.03 ) and in the cgi - s ( aripiprazole 0.72 , placebo , 0.46 ; p 0.031 ) .
there was a significant reduction in the cmai scores with aripiprazole 5 mg / d and 10 mg / d vs placebo at weeks 6 through 10 .
there was a significant reduction in the bprs core score with aripiprazole 5 mg / d vs placebo at weeks 4 , 6 , and 8 , and with 10 mg / d vs placebo at weeks 4 through 10 .
there was a significant reduction in the npi - nh total score with aripiprazole 5 mg / d vs placebo at weeks 6 and 8 .
kostic et al ( 2005 ) analyzed the data from the two studies ( streim et al 2004 ; mintzer et al 2007 ) to investigate further the effect of aripiprazole on agitation as measured by reduction in npi - agitation item , cmai , and tension / hostility / uncooperativeness / excitement scores of bprs . in the streim
et al ( 2004 ) study , npi agitation / aggression , cmai , and bprs tension / hostility / uncooperativeness / excitement scores were significantly reduced at study endpoint ( p = 0.001 , p < 0.05 , p < 0.05 respectively ) . in the mintzer
et al ( 2007 ) study , aripiprazole 5 mg / d and 10 mg / d demonstrated significant reduction ( p < 0.05 ) of npi - nh agitation item and bprs tension / hostility / uncooperativeness / excitement subscore compared with placebo . a significant decrease from baseline on bprs tension / hostility / uncooperativeness / excitement subscore was also observed with aripiprazole 2 mg / d ( beginning at week 6 ) at study endpoint ( p 0.05 ) .
the effect of aripiprazole on agitation as measured by bprs tension / hostility / uncooperativeness / excitement subscore was seen earlier with higher doses ( beginning at week 2 with 10 mg / d and beginning at week 4 with 5 mg / d ) compared with 2 mg / d .
discontinuation due to lack of efficacy occurred in 3 patients on aripiprazole and 6 patients on placebo in the de deyn et al ( 2005 ) study , and in 1 patient on aripiprazole and 3 patients on placebo in the streim et al study ( 2004 ) .
discontinuation due to lack of response occurred in 13% of patients on aripiprazole vs 30% of patients on placebo , and time to discontinuation was significantly longer in the aripiprazole group vs the placebo group ( p = 0.022 ) in the streim et al ( 2004 ) study . in the mintzer
et al ( 2007 ) study , discontinuation due to lack of efficacy occurred in 3% of patients on 2 mg / d of aripiprazole , 2% of patients on 5 mg / d of aripiprazole , 2% of patients on 10 mg / d of aripiprazole , and 2% of patients on placebo . discontinuation due to lack of response occurred in 21% of patients on 2 mg / d of aripiprazole , 16% of patients on 5 mg / d of aripiprazole , 13% of patients on 10 mg / d of aripiprazole , and 24% of patients on placebo .
thus the discontinuation due to lack of response was highest in the lowest - dose group and placebo group .
in the de deyn et al ( 2005 ) study , aes were generally mild to moderate and included ( aripiprazole vs placebo ) : urinary tract infection ( 8% vs 12% ) , accidental injury ( 8% vs 5% ) , somnolence ( 8% vs 1% ) , and bronchitis ( 6% vs 3% ) .
there were no significant differences vs placebo in the extrapyramidal symptoms ( eps ) scores , or clinically significant electrocardiogram ( ecg ) abnormalities , vital signs , or weight . in the streim
et al ( 2004 ) study , aes that occurred at the rate of 10% in the aripiprazole group were accidental injury , somnolence , urinary tract infection , ecchymosis , asthenia , vomiting , and rash , and in the placebo group were accidental injury , urinary act infection , ecchymosis , and rash .
eps occurred in 5% of aripiprazole group vs 4% of placebo group , somnolence occurred in 14% of aripiprazole group vs 4% of placebo group , and accidental injuries occurred in 21% of aripiprazole group vs 30% of placebo group . in the mintzer
et al ( 2007 ) study , aes occurring at 10% incidence in any dosage group of aripiprazole were accidental injury , agitation , peripheral edema , extremity pain , somnolence , urinary incontinence , asthenia , vomiting , and skin ulcer , and in the placebo group , accidental injury , anorexia , ecchymosis , and agitation .
eps occurred in 8% of aripiprazole 2 mg / d group , 7% in aripiprazole 5 mg / d group , 7% in aripiprazole 10 mg / d group , and 6% in the placebo group .
somnolence occurred in 3% of aripiprazole 2 mg / d group , 10% in aripiprazole 5 mg / d group , 7% in aripiprazole 10 mg / d group , and 3% in the placebo group .
accidental injuries occurred in 30% of aripiprazole 2 mg / d group , 24% in aripiprazole 5 mg / d group , 20% in aripiprazole 10 mg / d group , and 19% in the placebo group .
analysis of the data on mortality in elderly patients with dementia - related psychosis ( breder et al 2005 ; pers comm jan 8 2008 , bristol - myers squibb and otsuka pharmaceutical co , ltd , data on file ) of the three trials ( streim et al 2004 ; de deyn et al 2005 ; minzter et al 2007 ) for deaths that occurred during or within 30 days of discontinuation from the placebo - controlled phase of the clinical trial showed that incidence of deaths was 3.5% in aripiprazole - treated patients and 1.7% in placebo - treated patients ( p= 0.139 ) .
deaths were mostly associated with cardiovascular or infectious conditions . in these three trials ( streim et al 2004 ; de deyn et al 2005 ;
minzter et al 2007 ) , there was increased incidence of cvaes ( eg , stroke , transient ischemic attack ) , including fatalities , in aripiprazole - treated patients . in the fixed - dose study of mintzer et al ( 2007 ) there was a statistically significant dose - response relationship for cvaes in patients treated with aripiprazole . in an analysis ( pers comm jan 8 2008 , bristol - myers squibb and otsuka pharmaceutical co , ltd , data on file ) of the three 10-week , placebo - controlled studies of aripiprazole in elderly patients with psychosis associated with ad ( n = 938 , mean age 82.4 years , range 5699 years ) , the treatment - emergent aes that were reported at an incidence of 3% and at least twice that for placebo were lethargy ( placebo 2% , aripiprazole 5% ) , somnolence ( including sedation ) ( placebo 3% , aripiprazole 8% ) , and incontinence ( primarily , urinary incontinence ) ( placebo 1% , aripiprazole 5% ) , excessive salivation ( placebo 0% , aripiprazole 4% ) , and lightheadedness ( placebo 1% , aripiprazole 4% ) .
discontinuation due to aes occurred in 10 patients on aripiprazole vs 7 patients on placebo in the de deyn et al ( 2005 ) study , 14% on aripiprazole vs 9% patients on placebo in the streim et al ( 2004 ) study . in the mintzer
et al ( 2007 ) study , discontinuation occurred in 8% on aripiprazole 2 mg / d , 18% on aripiprazole 5 mg / d , 25% on aripiprazole 10 mg / d vs 13% patients on placebo .
the most common ae leading to discontinuation among the aripiprazole and placebo groups were asthenia and agitation , respectively .
discontinuations due to asthenia appeared to be dose - related , with the greatest number of patients experiencing asthenia in the aripiprazole 10 mg / day dose group ( placebo 1% , aripiprazole 2 mg / day 1% , aripiprazole 5 mg / day 2% , and aripiprazole 10 mg / day 4% ) .
the clinical antipsychotic trials of intervention effectiveness - alzheimer s disease ( catie - ad ) study ( schneider et al 2006 ) evaluated the overall effectiveness of three of the most widely used antipsychotic medications compared with placebo among patients in non - nursing home settings , who were experiencing delusions , hallucinations , aggression , or agitation .
the study followed the participants over 9 months and also included the involvement of caregivers . in this 42-site , double - blind , placebo - controlled trial ,
421 outpatients suffering from ad and having psychosis , aggression , or agitation were randomly assigned to receive olanzapine ( mean dose , 5.5 mg / d ) , quetiapine ( mean dose , 56.5 mg / d ) , risperidone ( mean dose , 1.0 mg / d ) , or placebo .
there were no significant differences among treatments for the main outcome measures , ie , time to discontinuation of treatment for any reason and number of patients with at least minimal improvement on the clinical global impression of change ( cgic ) ( schneider et al 1997 ) scale at 12 weeks .
the median time to discontinuation of treatment due to a lack of efficacy favored olanzapine and risperidone over quetiapine and placebo . the time to discontinuation of treatment due to adverse events or intolerability favored placebo .
this study showed that the adverse effects of atypical antipsychotics offset their benefits in the treatment of psychosis , aggression , or agitation in patients with ad .
the flexible dose studies of aripiprazole showed modest effect ( streim et al 2004 ; de deyn et al 2005 ) .
in these two studies , which assessed the efficacy of flexible doses of aripiprazole up to 15 mg / d in patients with ad , the change from baseline npi - nh psychosis subscale score was similar for the aripiprazole and placebo groups , with significant improvements compared with placebo in secondary measures of psychosis ( eg , bprs score subscale and psychosis subscale ) ( de deyn et al 2005 ) and other behavioral symptoms ( eg , cmai , cdds , cgi - i , npi - nh scores ) ( streim et al 2004 ) in patients suffering from psychosis associated with ad . the study assessing the efficacy of fixed doses of aripiprazole ( mintzer et al 2007 ) showed that aripiprazole 10 mg / d was efficacious and safe for psychosis associated with ad , significantly improving psychotic symptoms and agitation , and showed improvement in both primary and secondary outcome measures in that patient population . the safety and tolerability profile of antipsychotic treatment is of particular concern in elderly patients who are more susceptible to adverse effects such as eps , delirium due to anticholinergic adverse events , and orthostatic hypotension .
there is also a high prevalence of co - existing metabolic ( eg , high cholesterol , diabetes mellitus ) and cardiovascular illnesses ( eg , hypertension , ischemic heart disease ) in the elderly .
in addition , aes may be associated with falls and accidents resulting in fractures in the elderly .
hence the risk of such aes should be considered while choosing a medication and making treatment decisions .
it is therefore important to note that the incidence of eps - related aes in these studies was low across all treatment groups .
this concurs with the safety profile of aripiprazole in patients with schizophrenia and bipolar disorder in adults and in elderly patients ( marder et al 2003 ; madhusoodanan et al 2004 ; sajatovic et al 2008 ) .
however , not all studies with atypical antipsychotics in patients with ad have shown eps - related adverse events similar to placebo ( street et al 2000 ) .
high levels of somnolence may also be associated with falls in the elderly . in the two studies assessing the flexible doses of aripiprazole , occurrence of somnolence showed a > 5% difference between the aripiprazole and placebo groups . in the study assessing fixed doses of aripiprazole , the incidence of somnolence with aripiprazole was low ( 7% at the 10 mg / d dose ) and did not increase with dose .
for example , 36.5% with risperidone ( brodaty et al 2003 ) and 35.8% with olanzapine ( street et al 2000 ) .
there was no significant difference between placebo and aripiprazole in all the three studies for weight change , in contrast to studies with other atypical antipsychotics where significantly higher weight gain was observed with both olanzapine ( de deyn et al 2004 ) and risperidone ( brodaty et al 2003 ) vs placebo in patients with ad .
recently , cvaes have emerged as a significant issue with the use of atypical antipsychotics in elderly individuals ( wooltorton 2002 , 2004 ) .
however , some retrospective cohort studies have suggested that conventional and atypical antipsychotics do not differ significantly in their risk of cvaes ( herrmann et al 2004 ; finkel et al 2005 ) or mortality ( wang et al 2005 ) in older adults .
cvaes were reported for 7 aripiprazole - treated patients during the mintzer et al ( 2007 ) fixed dose study ( 1 in the 2 mg / d dose group , 2 in the 5 mg / d dose group , and 4 in the 10 mg / d dose group ) , compared with no patients in the placebo group .
the effect was statistically dose - dependent ( p = 0.03 , cochran mantel
haenszel [ cmh ] row means score test ) . all cvaes reported with aripiprazole occurred in subjects with a prior history of cva , stroke , or related risk factors
it is also important to note that the fda has issued a black box warning for increased mortality risk in elderly patients with dementia , treated with atypical antipsychotics ( us fda 2003 ) .
analyses of 17 placebo - controlled trials ( modal duration of 10 weeks ) in these patients revealed a risk of death in the drug - treated patients of 1.6 to 1.7 times to that seen in placebo - treated patients .
over the course of a typical 10-week controlled trial , the rate of death in drug - treated patients was about 4.5% , compared with a rate of about 2.6% in the placebo group .
although the causes of death were varied , most of the deaths appeared to be the result of either cardiovascular causes ( eg , heart failure , sudden death ) or infections ( eg , pneumonia ) .
aripiprazole should be used with caution in patients with known cardiovascular disease , cerebrovascular disease , or conditions that would predispose patients to hypotension .
the risks and benefits of arpipirazole in the treatment of psychosis of ad should be considered by the clinicians and discussed with the patients / caregivers ( schneider et al 2005 ) . a thorough evaluation of the medical and psychiatric status along with a comprehensive treatment approach including environmental manipulation
are required prior to considering use of atypical antipsychotic in patients with psychosis associated with ad .
the efficacy results in the study by mintzer et al ( 2007 ) show that aripiprazole 10 mg / d is an effective dose for these patients , although some patients achieved significant benefit with aripiprazole 5 mg / d . the dose effectiveness relationship is further supported by the rate of discontinuation due to lack of efficacy , which was greatest in the 2 mg / d dose group .
however , when selecting an appropriate dose , the discontinuation rate due to dose - related adverse events should also be considered .
the rates of discontinuation due to adverse events for the 5 and 10 mg / d doses ( 18% and 25% , respectively ) were higher than those reported in the flexible - dose aripiprazole studies in noninstitutionalized patients ( 9.4% ) ( de deyn et al 2005 ) and in institutionalized patients ( 14% ) ( streim et al 2004 ) . in both of the flexible - dose studies ,
patients were started with 2 mg / d and titrated gradually to higher aripiprazole doses .
this suggests that slow titration from a low initial dose may improve the tolerability and prove to be the most clinically appropriate strategy for starting aripiprazole treatment in this elderly patient population .
the control of psychotic symptoms is important for improving outcome in elderly patients with ad .
furthermore , treatment of the psychotic symptoms improves patient quality of life , decreases caregiver burden , decreases the need for institutionalization , and reduces overall healthcare costs .
there are no psychotropic medications approved by the fda for the treatment of psychosis of ad .
the limited evidence available for atypical antipsychotics suggests that they offer modest benefits over placebo for treating psychosis .
the studies evaluating aripiprazole suggest that it may also be beneficial in treatment of psychosis for patients with ad .
however , the benefits of relieving psychosis need to be balanced against the risk of neurologic , metabolic , and cardiovascular aes in each individual patient .
the safety and tolerability profile seen with aripiprazole suggests a low potential for negative impact on overall patient health and makes aripiprazole a reasonable treatment option . however , in the absence of head - to - head comparison studies between aripiprazole and other atypical antipsychotics , it is difficult to conclude whether there is any added value of the uses of aripiprazole .
further studies comparing the efficacy and tolerability of aripiprazole versus other atypical antipsychotics in psychosis of ad are needed to guide treatment choice among the atypical antipsychotics agents . | psychosis of alzheimer s disease ( ad ) is characterized by delusions or hallucinations and may be associated with agitation , negative symptoms or depression .
there are no psychotropic medications that are approved by the us fda for the treatment of psychosis of ad .
however , atypical antipsychotics have been widely used and recommended by geriatric experts in the management of psychosis of ad in view of the modest efficacy and relative safety until fda warnings were issued in 2005 and meta - analytic studies showed no significant difference to placebo .
the fda warnings on the cardiac , metabolic , cerebrovascular , and mortality risks have caused serious concerns for the use of atypical antipsychotic agents in elderly patients with dementia .
only a few studies have evaluated prospectively the effects of aripiprazole in psychosis associated with ad .
these studies show improvement in the symptoms of psychosis associated with ad with aripiprazole .
the safety and tolerability profile of aripiprazole suggests a low potential for negative impact on dementia and overall patient health .
further studies comparing the efficacy and tolerability of aripiprazole vs other atypical antipsychotics in dementia are needed . | Introduction
Results
Efficacy (
Primary outcome measures
Secondary outcome measures
Tolerability (
Discussion
Conclusion | these studies have shown the efficacy of atypical antipsychotics in treating some of the behavioral symptoms that are associated with ad ( katz et al 1999 ; street et al 2000 ; tariot et al 2002 ; brodaty et al 2003 ; de deyn et al 1999 , 2004 ; zhong et al 2004 ) . even though there is no psychotropic medication that is approved by the us food and drug administration ( fda ) for the treatment of psychosis of ad , a number of expert consensus statements recommend the use of atypical antipsychotic agents as a first - line pharmacologic approach to treatment ( alexopoulos et al 2004 ; ags / aagp ) . analyses of safety data from several studies have raised concerns about an increased risk of cerebrovascular adverse events ( cvaes ) , such as stroke , with the use of some atypical antipsychotics compared with placebo in elderly patients with dementia ( wooltorton 2002 , 2004 ) . this has led fda to issue warnings on the use of atypical antipsychotics in the treatment of behavioral and psychological symptoms in elderly patients with dementia , particularly those with a history of cerebrovascular disease ( us fda 2003 ; health canada ; mhra ) . three 10-week , placebo - controlled , randomized , double - blind clinical studies have evaluated the efficacy and tolerability of aripiprazole for the treatment of psychosis related to ad ( table 2a ) ( streim et al 2004 ; de deyn et al 2005 ; mintzer et al 2007 ) . in an analysis ( pers comm jan 8 2008 , bristol - myers squibb and otsuka pharmaceutical co , ltd , data on file ) of the three 10-week , placebo - controlled studies of aripiprazole in elderly patients with psychosis associated with ad ( n = 938 , mean age 82.4 years , range 5699 years ) , the treatment - emergent aes that were reported at an incidence of 3% and at least twice that for placebo were lethargy ( placebo 2% , aripiprazole 5% ) , somnolence ( including sedation ) ( placebo 3% , aripiprazole 8% ) , and incontinence ( primarily , urinary incontinence ) ( placebo 1% , aripiprazole 5% ) , excessive salivation ( placebo 0% , aripiprazole 4% ) , and lightheadedness ( placebo 1% , aripiprazole 4% ) . in an analysis ( pers comm jan 8 2008 , bristol - myers squibb and otsuka pharmaceutical co , ltd , data on file ) of the three 10-week , placebo - controlled studies of aripiprazole in elderly patients with psychosis associated with ad ( n = 938 , mean age 82.4 years , range 5699 years ) , the treatment - emergent aes that were reported at an incidence of 3% and at least twice that for placebo were lethargy ( placebo 2% , aripiprazole 5% ) , somnolence ( including sedation ) ( placebo 3% , aripiprazole 8% ) , and incontinence ( primarily , urinary incontinence ) ( placebo 1% , aripiprazole 5% ) , excessive salivation ( placebo 0% , aripiprazole 4% ) , and lightheadedness ( placebo 1% , aripiprazole 4% ) . this study showed that the adverse effects of atypical antipsychotics offset their benefits in the treatment of psychosis , aggression , or agitation in patients with ad . the study assessing the efficacy of fixed doses of aripiprazole ( mintzer et al 2007 ) showed that aripiprazole 10 mg / d was efficacious and safe for psychosis associated with ad , significantly improving psychotic symptoms and agitation , and showed improvement in both primary and secondary outcome measures in that patient population . all cvaes reported with aripiprazole occurred in subjects with a prior history of cva , stroke , or related risk factors
it is also important to note that the fda has issued a black box warning for increased mortality risk in elderly patients with dementia , treated with atypical antipsychotics ( us fda 2003 ) . a thorough evaluation of the medical and psychiatric status along with a comprehensive treatment approach including environmental manipulation
are required prior to considering use of atypical antipsychotic in patients with psychosis associated with ad . there are no psychotropic medications approved by the fda for the treatment of psychosis of ad . the safety and tolerability profile seen with aripiprazole suggests a low potential for negative impact on overall patient health and makes aripiprazole a reasonable treatment option . further studies comparing the efficacy and tolerability of aripiprazole versus other atypical antipsychotics in psychosis of ad are needed to guide treatment choice among the atypical antipsychotics agents . | [
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] |
diabetes is one of the major public health problems worldwide , affecting 24 million individuals in the u.s . alone
more than 70% of patients with type 2 diabetes die of cardiovascular causes ( 2 ) .
although a consistent association between glycemic control and cardiovascular disease has been noted in epidemiological studies ( 35 ) , randomized clinical trials ( rcts ) did not show a benefit ( 69 ) . in the uk prospective diabetes study ( ukpds ) , there was a 16% reduction ( p = 0.052 ) in cardiovascular events in the intensive glycemic control arm in the original rct period ( 6 ) .
after an additional 10 years of follow - up for the ukpds , those originally assigned randomly to intensive glycemic control had significant long - term reductions in incident myocardial infarction and in all - cause mortality despite a loss of glycemic differences with the control group ( 10 ) .
several other rcts , however , did not lead to a significant reduction in macrovascular complications in the intensive glycemic treatment among diabetic patients ( 79 ) .
rcts may have insufficient sample sizes or follow - up periods to detect moderate differences in risk , as well as potential differences among ethnic groups .
such inconsistent evidence has resulted in the american heart association ( aha ) , the american college of cardiology ( acc ) , and the american diabetes association ( ada ) providing a conservative class iib ( usefulness / efficacy is less well established by evidence / opinion ) recommendation with level of evidence a for the benefit of glycemic control on cardiovascular disease compared with microvascular complications , which has a superior level recommendation of class i ( abundant epidemiological studies and rcts to confirm the benefit ) ( 11 ) .
thus , there is still an urgent need for more observational data to support the case for causality given the lack of conclusive evidence from rcts .
in addition , most epidemiological studies only use a single baseline measurement of hba1c to predict risk of future coronary heart disease ( chd ) , which may produce potential bias .
moreover , very few studies have assessed the race - specific association of hba1c with chd risk .
the aim of the current study is to examine the race - specific association between different levels of hba1c at baseline and during follow - up and the risk of incident chd among african american and white diabetic patients in the louisiana state university hospital - based longitudinal study ( lsuhls ) .
the lsu health care services division ( lsuhcsd ) operates seven public hospitals and affiliated clinics in louisiana , which provide quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1219 ) .
overall , lsuhcsd facilities have served 1.6 million patients ( 35% of the louisiana population ) since 1997 .
administrative , anthropometric , laboratory ( test code , test collection date , test result values , and abnormal flag ) , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data ,
a cohort of diabetic patients was established by using the icd-9 ( code 250 ) through the lsuhls database between 1 january 1999 and 31 december 2009 . both inpatients and outpatients
internal diabetes disease management guidelines call for physician confirmation of diabetes diagnoses by applying the ada criteria : a fasting plasma glucose level 126 mg / dl , 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test , one or more classic symptoms , and a random plasma glucose level 200 mg / dl ( 20 ) .
the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . before diagnosis with diabetes
the agreement of diabetes diagnosis was 97% ; 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes using the ada diabetes diagnosis criteria ( 20 ) .
the current study included 30,102 diabetic patients ( 12,592 white and 17,510 african american ) who were 3094 years of age at baseline without a history of chd or stroke and with complete repeated data on all risk factor variables . in these diabetic patients ,
79% of patients qualify for free care ( by virtue of being low income and uninsured , any individual or family unit whose income is at or < 200% of federal poverty level ) , 5.1% of patients are self - pay ( uninsured , but incomes not low enough to qualify for free care ) , 5.1% of patients are covered by medicaid , 8.9% of patients have medicare , and 2.2% of patients are covered by commercial insurance .
the study and analysis plan were approved by the pennington biomedical research center and lsu health sciences center institutional review boards , lsu system .
we did not obtain informed consent from the participants involved in our study because we used anonymized data compiled from electronic medical records .
the patient characteristics , including age of diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , body weight , height , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , hba1c , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive , cholesterol - lowering , and antidiabetic drugs ) , within a half year after the diabetes diagnosis ( baseline ) and during follow - up after the diabetes diagnosis ( follow - up ) were extracted from the computerized hospitalization records .
the updated mean values of hba1c , ldl cholesterol , hdl cholesterol , triglycerides , bmi , blood pressure , and egfr over time were measured first at baseline and second as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up .
for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean value was from baseline to the year before this event occurred ( 21 ) .
follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals .
the diagnosis of chd was the primary end point of interest of the study and was defined according to the icd-9 : chd ( icd-9 codes 410414 ) . since 1997 ,
diagnoses of chd were made by the treating physicians based on a clinical assessment and examinations as considered relevant by the clinician in charge of treatments .
follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , death , or 31 may 2012 ( 17 ) .
the association between hba1c and the risk of chd was analyzed by using cox proportional hazards models .
hba1c was evaluated in the following two ways : 1 ) as seven categories ( hba1c < 6.0% [ 42 mmol / mol ] [ reference group ] , 6.06.9% [ 4252 mmol / mol ] , 7.07.9% [ 5363 mmol / mol ] , 8.08.9% [ 6474 mmol / mol ] , 9.09.9% [ 7585 mmol / mol ] , 10.010.9% [ 8696 mmol / mol ] , and 11.0% [ 97 mmol / mol ] ) and
different levels of hba1c were included in the models as dummy variables , and the significance of the trend over different categories of hba1c was tested in the same models by giving an ordinal numerical value for each dummy variable .
all analyses were adjusted for age and sex and further for smoking , income , type of insurance , bmi , systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , egfr , and use of antihypertensive drugs , diabetes medications , and cholesterol - lowering agents . when we analyzed the association between updated mean of hba1c and chd risk , we adjusted for updated means of bmi , ldl cholesterol , hdl cholesterol , triglycerides , systolic blood pressure , and egfr instead of the baselines of these variables .
we stratified the samples by race because there was a significant interaction between race and hba1c on chd risk . to avoid the potential bias due to severe diseases at baseline ,
additional analyses were performed excluding the subjects who were diagnosed with chd during the first 2 years of follow - up .
we used restricted cubic splines in cox models to test whether there is a dose - response or nonlinear association of hba1c as a continuous variable with chd risk .
all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss , inc . , chicago , il ) and sas for windows , version 9.3 ( sas institute , cary , nc ) .
the lsu health care services division ( lsuhcsd ) operates seven public hospitals and affiliated clinics in louisiana , which provide quality medical care to the residents of louisiana regardless of their income or insurance coverage ( 1219 ) .
overall , lsuhcsd facilities have served 1.6 million patients ( 35% of the louisiana population ) since 1997 .
administrative , anthropometric , laboratory ( test code , test collection date , test result values , and abnormal flag ) , clinical diagnosis , and medication data collected at these facilities are available in electronic form for both inpatients and outpatients from 1997 . using these data ,
a cohort of diabetic patients was established by using the icd-9 ( code 250 ) through the lsuhls database between 1 january 1999 and 31 december 2009 . both inpatients and outpatients
internal diabetes disease management guidelines call for physician confirmation of diabetes diagnoses by applying the ada criteria : a fasting plasma glucose level 126 mg / dl , 2-h glucose level 200 mg / dl after a 75-g 2-h oral glucose tolerance test , one or more classic symptoms , and a random plasma glucose level 200 mg / dl ( 20 ) .
the first record of diabetes diagnosis was used to establish the baseline for each patient in the present analyses due to the design of the cohort study . before diagnosis with diabetes
the agreement of diabetes diagnosis was 97% ; 20,919 of a sample of 21,566 hospital discharge diagnoses based on icd codes also had physician - confirmed diabetes using the ada diabetes diagnosis criteria ( 20 ) .
the current study included 30,102 diabetic patients ( 12,592 white and 17,510 african american ) who were 3094 years of age at baseline without a history of chd or stroke and with complete repeated data on all risk factor variables . in these diabetic patients ,
79% of patients qualify for free care ( by virtue of being low income and uninsured , any individual or family unit whose income is at or < 200% of federal poverty level ) , 5.1% of patients are self - pay ( uninsured , but incomes not low enough to qualify for free care ) , 5.1% of patients are covered by medicaid , 8.9% of patients have medicare , and 2.2% of patients are covered by commercial insurance .
the study and analysis plan were approved by the pennington biomedical research center and lsu health sciences center institutional review boards , lsu system .
we did not obtain informed consent from the participants involved in our study because we used anonymized data compiled from electronic medical records .
the patient characteristics , including age of diabetes diagnosis , sex , race / ethnicity , family income , smoking status , types of health insurance , body weight , height , bmi , blood pressure , total cholesterol , hdl cholesterol , ldl cholesterol , triglycerides , hba1c , estimated glomerular filtration rate ( egfr ) , and medication ( antihypertensive , cholesterol - lowering , and antidiabetic drugs ) , within a half year after the diabetes diagnosis ( baseline ) and during follow - up after the diabetes diagnosis ( follow - up ) were extracted from the computerized hospitalization records .
the updated mean values of hba1c , ldl cholesterol , hdl cholesterol , triglycerides , bmi , blood pressure , and egfr over time were measured first at baseline and second as an updated mean of annual measurements , calculated for each participant from baseline to each year of follow - up .
for example , at 1 year , the updated mean is the average of the baseline and 1-year values , and at 3 years , it is the average of baseline , 1-year , 2-year , and 3-year values . in the case of an event during follow - up , the period for estimating updated mean value was from baseline to the year before this event occurred ( 21 ) .
follow - up information was obtained from the lsuhls inpatient and outpatient database by using the unique number assigned to every patient who visits the lsuhcsd hospitals .
the diagnosis of chd was the primary end point of interest of the study and was defined according to the icd-9 : chd ( icd-9 codes 410414 ) . since 1997 ,
diagnoses of chd were made by the treating physicians based on a clinical assessment and examinations as considered relevant by the clinician in charge of treatments .
follow - up of each cohort member continued until the date of the diagnosis of chd , the date of the last visit if the subject stopped use of lsuhcsd hospitals , death , or 31 may 2012 ( 17 ) .
the association between hba1c and the risk of chd was analyzed by using cox proportional hazards models .
hba1c was evaluated in the following two ways : 1 ) as seven categories ( hba1c < 6.0% [ 42 mmol / mol ] [ reference group ] , 6.06.9% [ 4252 mmol / mol ] , 7.07.9% [ 5363 mmol / mol ] , 8.08.9% [ 6474 mmol / mol ] , 9.09.9% [ 7585 mmol / mol ] , 10.010.9% [ 8696 mmol / mol ] , and 11.0% [ 97 mmol / mol ] ) and
different levels of hba1c were included in the models as dummy variables , and the significance of the trend over different categories of hba1c was tested in the same models by giving an ordinal numerical value for each dummy variable .
all analyses were adjusted for age and sex and further for smoking , income , type of insurance , bmi , systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , egfr , and use of antihypertensive drugs , diabetes medications , and cholesterol - lowering agents . when we analyzed the association between updated mean of hba1c and chd risk , we adjusted for updated means of bmi , ldl cholesterol , hdl cholesterol , triglycerides , systolic blood pressure , and egfr instead of the baselines of these variables .
we stratified the samples by race because there was a significant interaction between race and hba1c on chd risk . to avoid the potential bias due to severe diseases at baseline ,
additional analyses were performed excluding the subjects who were diagnosed with chd during the first 2 years of follow - up .
we used restricted cubic splines in cox models to test whether there is a dose - response or nonlinear association of hba1c as a continuous variable with chd risk .
all statistical analyses were performed with pasw for windows , version 20.0 ( ibm spss , inc . , chicago , il ) and sas for windows , version 9.3 ( sas institute , cary , nc ) .
general characteristics of the study population are presented by race in table 1 . during a mean follow - up period of 6.0 years
a significantly increased risk of chd was observed among both african american and white diabetic patients with increasing baseline hba1c after adjustment for age and sex ( table 2 ) .
after further adjustment for other confounding factors ( smoking , income , type of insurance , bmi , systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , egfr , and use of antihypertensive drugs , diabetes medications , and cholesterol - lowering agents ) , this graded positive association remained significant among white ( p trend < 0.001 ) and african american ( p = 0.002 ) diabetic patients ( table 2 ) . when hba1c was considered as a continuous variable by using restricted cubic splines , a linear association of hba1c with chd risk was observed ( supplementary fig .
each one percentage increase in baseline hba1c was associated with a 2% ( 95% ci 1.011.04 ) increased risk of chd in african americans and a 6% ( 1.051.08 ) increased risk of chd in whites , and this association was significantly stronger among white diabetic patients than african american patients ( p = 0.001 ) .
baseline characteristics of african american and white patients with diabetes hr ( 95% ci ) of chd according to different levels of hba1c at baseline and during follow - up among african american and white patients with diabetes when we stratified by sex , age , smoking status , and family income , the graded positive association of baseline hba1c with chd risk did not change ( almost all p trend < 0.05 ) ( table 3 ) .
the graded positive association of hba1c with chd risk was also confirmed among diabetic patients using glucose - lowering agents and those who were not using ( all p trend < 0.001 ) ( table 3 ) .
hr ( 95% ci ) of chd according to different levels of hba1c at baseline among various subpopulations after excluding the subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 588 ) , the multivariable - adjusted hazard ratios ( hrs ) of chd associated with different levels of hba1c did not change ( data not shown ) .
when we performed an additional analysis by using an updated mean of hba1c during follow - up , we found almost the same graded positive associations between baseline hba1c levels and updated mean levels of hba1c with chd risk among both african american and white diabetic patients ( supplementary table 1 ) . during follow - up , each one percentage increase of hba1c was associated with a more obvious increase in chd risk in white ( hr 1.11 [ 95% ci 1.091.14 ] ) than in african american ( 1.05 [ 1.031.08 ] ) diabetic patients ( p < 0.001 ) .
moreover , we performed another analysis using age as the timescale and the results did not change ( supplementary table 2 ) .
our study found a graded positive association between hba1c at baseline and during follow - up with the risk of chd among both african american and white diabetic patients .
this graded positive association was more significant in white than african american patients with diabetes .
observational studies have confirmed the continuous and positive association between glycemic control and the risk of cardiovascular disease among diabetic patients ( 4,5 ) .
three large rcts ( 79 ) designed primarily to determine whether targeting different glucose levels can reduce the risk of cardiovascular events in patients with type 2 diabetes failed to confirm the benefit .
first , small sample sizes , short follow - up duration , and few chd cases in some rcts may limit the statistical power .
second , most epidemiological studies only assess a single baseline measurement of hba1c with chd risk , which may produce potential bias .
the recent analysis of 10 years of posttrial follow - up of the ukpds showed continued reductions for myocardial infarction and death from all causes despite an early loss of glycemic differences ( 10 ) .
the scientific evidence from rcts was not sufficient to generate strong recommendations for clinical practice .
thus , consensus groups ( aha , acc , and ada ) have provided a conservative endorsement ( class iib recommendation , level of evidence a ) for the cardiovascular benefits of glycemic control ( 11 ) . in the absence of conclusive evidence from rcts
, observational epidemiological studies might provide useful information to clarify the relationship between glycemia and chd risk . in the current study with 30,102 participants with diabetes and 7,258 incident chd cases during a mean follow - up of 6.0 years , we found a graded positive association by various hba1c intervals of clinical relevance or by using hba1c as a continuous variable at baseline and during follow - up with chd risk among both african american and white diabetic patients .
each one percentage increase in baseline and follow - up hba1c was associated with a 2 and 5% increased risk of chd in african american and 6 and 11% in white diabetic patients .
each one percentage increase of hba1c was associated with a greater increase in chd risk in white versus african american diabetic patients .
this magnitude of chd risk increase especially in african americans is lower than that reported from the atherosclerosis risk in communities ( aric ) study ( 4 ) of a relative risk for chd of 1.14 ( 95% ci 1.071.21 ) and a recent meta - analysis of cohort studies of a relative risk of 1.15 ( 1.01.20 ) ( 22 ) with 1% increase of hba1c .
the hazard rates for chd in african american patients were consistently lower than white patients for nearly all levels of hba1c .
the higher mean values of hba1c at baseline and during follow - up among african american diabetic patients than white patients might result in the absolute lower hazard rates for chd associated with each one percentage increase in hba1c among african american diabetic patients than white patients .
in addition , the differences in hazard rates might be related to the recently recognized differences in hba1c levels between african american and white patients who have the same levels of blood glucose ( 23 ) .
the mechanism underlying this racial difference in hba1c level due to biological differences or to other sociobehavioral differences , including disparities in access to health and prevention care , has not been established .
in addition , we found that this graded positive association was present in patients with diabetes with and without glucose - lowering agent treatment , and in patients in different age , sex , and smoking status groups .
several plausible biological mechanisms have been proposed to explain a possible direct relationship between chronically elevated blood glucose levels and chd ( 24 ) .
glucose can react with many different proteins , creating advanced glycation end products , which contribute to long - term complications in diabetes as well as to endothelial dysfunction , changes in arterial distensibility , plaque formation , and atherosclerosis ( 25,26 ) .
but the pathophysiology may not only be linked directly to hyperglycemia but also to diabetic dyslipidemia , hypertension , and inflammation , which can accelerate vascular injury and cardiovascular disease risk . in the steno-2 study ,
a multifactorial intervention showed an 50% reduction in the risk of cardiovascular and microvascular events among diabetic patients ( 27 ) .
there are several strengths to our study , including the large sample size , high proportion of african americans , long follow - up time , and use of administrative databases to avoid differential recall bias .
we have used both baseline hba1c levels and updated mean values of hba1c during follow - up in the analyses , which can avoid potential bias from a single baseline measurement .
in addition , participants in this study use the same public health care system , which minimizes the influence from the accessibility of health care , particularly in comparing african americans and whites .
one limitation of our study is that our analysis was not performed on a representative sample of the population , which limits the generalizability of this study ; however , lsuhcsd hospitals are public hospitals and cover > 1.6 million patients , most of whom are low - income people in louisiana .
the results of the current study will have wide applicability for the population with low income and without health insurance in the u.s .
second , the validity of myocardial infarction diagnoses in our study has not been confirmed by specialists . but the method we used ( hospital discharge register ) to diagnose major nonfatal chd has been widely used in american and european cohort studies , such as the kaiser permanente medical care program ( 28,29 ) , the aric study ( 4 ) , the framingham study ( 30,31 ) , and the national finrisk survey ( 32 ) . the validity of the diagnoses of myocardial infarction by using the hospital discharge register in these cohort studies is available ( agreement 8398% ) ( 29,33 ) .
third , even though our analyses adjusted for an extensive set of confounding factors , residual confounding due to measurement error in the assessment of confounding factors , unmeasured factors such as heart rate , physical activity , education , dietary factors , and cognitive function for all patients , can not be excluded . based on the limitations above , our findings may need to be further confirmed by other studies .
in summary , our study demonstrates that there is a graded association between hba1c at baseline and during follow - up with the risk of chd among both african american and white diabetic patients .
our study provides epidemiological support for glucose lowering as a strategy to reduce chd in a large sample size of both white and african american diabetes patients with low socioeconomic status . | objectiveclinical trials to date have not provided definitive evidence regarding the effects of glucose lowering with coronary heart disease ( chd ) risk among diabetic patients.research design and methodswe prospectively investigated the association of hba1c at baseline and during follow - up with chd risk among 17,510 african american and 12,592 white patients with type 2 diabetes.resultsduring a mean follow - up of 6.0 years , 7,258 incident chd cases were identified .
the multivariable - adjusted hazard ratios of chd associated with different levels of hba1c at baseline ( < 6.0 [ reference group ] , 6.06.9 , 7.07.9 , 8.08.9 , 9.09.9 , 10.010.9 , and 11.0% ) were 1.00 , 1.07 ( 95% ci 0.971.18 ) , 1.16 ( 1.041.31 ) , 1.15 ( 1.011.32 ) , 1.26 ( 1.091.45 ) , 1.27 ( 1.091.48 ) , and 1.24 ( 1.101.40 ) ( p trend = 0.002 ) for african americans and 1.00 , 1.04 ( 0.941.14 ) , 1.15 ( 1.031.28 ) , 1.29 ( 1.131.46 ) , 1.41 ( 1.221.62 ) , 1.34 ( 1.141.57 ) , and 1.44 ( 1.261.65 ) ( p trend < 0.001 ) for white patients , respectively .
the graded association of hba1c during follow - up with chd risk was observed among both african american and white diabetic patients ( all p trend < 0.001 ) .
each one percentage increase of hba1c was associated with a greater increase in chd risk in white versus african american diabetic patients . when stratified by sex , age , smoking status , use of glucose - lowering agents , and income , this graded association of hba1c with chd was still present.conclusionsthe current study in a low - income population suggests a graded positive association between hba1c at baseline and during follow - up with the risk of chd among both african american and white diabetic patients with low socioeconomic status . | Introduction
Research Design and Methods
Study Population
Baseline and Follow-up Measurements
Prospective Follow-up
Statistical Analyses
Results
Conclusions
Supplementary Material | the aim of the current study is to examine the race - specific association between different levels of hba1c at baseline and during follow - up and the risk of incident chd among african american and white diabetic patients in the louisiana state university hospital - based longitudinal study ( lsuhls ) . hba1c was evaluated in the following two ways : 1 ) as seven categories ( hba1c < 6.0% [ 42 mmol / mol ] [ reference group ] , 6.06.9% [ 4252 mmol / mol ] , 7.07.9% [ 5363 mmol / mol ] , 8.08.9% [ 6474 mmol / mol ] , 9.09.9% [ 7585 mmol / mol ] , 10.010.9% [ 8696 mmol / mol ] , and 11.0% [ 97 mmol / mol ] ) and
different levels of hba1c were included in the models as dummy variables , and the significance of the trend over different categories of hba1c was tested in the same models by giving an ordinal numerical value for each dummy variable . hba1c was evaluated in the following two ways : 1 ) as seven categories ( hba1c < 6.0% [ 42 mmol / mol ] [ reference group ] , 6.06.9% [ 4252 mmol / mol ] , 7.07.9% [ 5363 mmol / mol ] , 8.08.9% [ 6474 mmol / mol ] , 9.09.9% [ 7585 mmol / mol ] , 10.010.9% [ 8696 mmol / mol ] , and 11.0% [ 97 mmol / mol ] ) and
different levels of hba1c were included in the models as dummy variables , and the significance of the trend over different categories of hba1c was tested in the same models by giving an ordinal numerical value for each dummy variable . after further adjustment for other confounding factors ( smoking , income , type of insurance , bmi , systolic blood pressure , ldl cholesterol , hdl cholesterol , triglycerides , egfr , and use of antihypertensive drugs , diabetes medications , and cholesterol - lowering agents ) , this graded positive association remained significant among white ( p trend < 0.001 ) and african american ( p = 0.002 ) diabetic patients ( table 2 ) . each one percentage increase in baseline hba1c was associated with a 2% ( 95% ci 1.011.04 ) increased risk of chd in african americans and a 6% ( 1.051.08 ) increased risk of chd in whites , and this association was significantly stronger among white diabetic patients than african american patients ( p = 0.001 ) . baseline characteristics of african american and white patients with diabetes hr ( 95% ci ) of chd according to different levels of hba1c at baseline and during follow - up among african american and white patients with diabetes when we stratified by sex , age , smoking status , and family income , the graded positive association of baseline hba1c with chd risk did not change ( almost all p trend < 0.05 ) ( table 3 ) . the graded positive association of hba1c with chd risk was also confirmed among diabetic patients using glucose - lowering agents and those who were not using ( all p trend < 0.001 ) ( table 3 ) . hr ( 95% ci ) of chd according to different levels of hba1c at baseline among various subpopulations after excluding the subjects who were diagnosed with chd during the first 2 years of follow - up ( n = 588 ) , the multivariable - adjusted hazard ratios ( hrs ) of chd associated with different levels of hba1c did not change ( data not shown ) . during follow - up , each one percentage increase of hba1c was associated with a more obvious increase in chd risk in white ( hr 1.11 [ 95% ci 1.091.14 ] ) than in african american ( 1.05 [ 1.031.08 ] ) diabetic patients ( p < 0.001 ) . our study found a graded positive association between hba1c at baseline and during follow - up with the risk of chd among both african american and white diabetic patients . in the current study with 30,102 participants with diabetes and 7,258 incident chd cases during a mean follow - up of 6.0 years , we found a graded positive association by various hba1c intervals of clinical relevance or by using hba1c as a continuous variable at baseline and during follow - up with chd risk among both african american and white diabetic patients . each one percentage increase of hba1c was associated with a greater increase in chd risk in white versus african american diabetic patients . in summary , our study demonstrates that there is a graded association between hba1c at baseline and during follow - up with the risk of chd among both african american and white diabetic patients . | [
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1 ) is comprised of subsystems for two - photon near - infrared ( nir ) excitation , visible fluorescence detection and wavefront measurement , and continuous wave ( cw ) visible excitation . in the two - photon subsystem ,
pulsed light from a ti : sapphire laser ( coherent , chameleon ultra ii ) , intensity controlled by a pockels cell ( conoptics , 350 - 80-la-02 ) , is expanded to a 1/e diameter of 8 mm before being reflecting at 8 from the normal off of a nir - responsive spatial light modulator ( slm nir , boulder nonlinear systems , hsp256 - 1064 ) .
the slm is used to apply the corrective pattern needed to retain a diffraction - limited two - photon excitation ( tpe ) focus in the specimen . a pair of nir achromatic relay lenses ( focal lengths f1 = 150 mm and f2 = 125 mm ) operating in a 2f1 + 2f2 configuration are then used to image the slm onto the 5 mm mirror of a galvanometer ( y galvo , cambridge technology , 6215h ) .
another pair of f1 = f2 = 85 mm relay lenses then image the slm onto a second 5 mm galvo mirror ( x galvo , cambridge technology , 6215h ) .
a final pair of f1 = 89 mm and f2 = 350 mm relay lenses creates a magnified image of the slm at the rear pupil plane of the imaging objective ( nikon , cfi apo lwd 25xw , 1.1 na and 2 mm wd ) .
mutual conjugation of the slm , both galvos , and the objective rear pupil insures that the corrective phase pattern from the slm is stationary at the objective rear pupil , even as the galvos scan the focused nir light laterally across the specimen .
the visible excitation subsystem begins when four cw lasers ( = 440 nm , 50 mw , crystalaser ; = 488 nm , 200 mw , coherent sapphire 488 lp ; = 514 nm , 300 mw , mpb communications , model 2ru - vfl - p-300 - 514-r ; and = 561 nm , 200 mw , coherent sapphire 561 lp ) are expanded to a common a 1/e diameter of 2 mm and combined into a single co - linear beam using dichroic beamsplitters ( semrock , lasermux family ) .
an acousto - optic tunable filter ( aotf , aa opto - electronic , aotfnc-400.650-tn ) selects one or more wavelengths and controls the power of each .
the linearly polarized output of the aotf is expanded to a 1/e diameter of 10 mm , inserted into the microscope beam path using a dichroic beamsplitter ( d2 in supplementary fig .
1 , semrock di01-r442/510 - 25x36 or di01-r488/561 - 25x36 ) , and reflected from a spatial light modulator responsive to visible light ( slm vis , boulder nonlinear systems , hsp256 - 0532 ) .
the slm is used to apply the corrective pattern needed to retain both a diffraction - limited visible excitation focus in the specimen , and a diffraction - limited focus of the fluorescence emission at a pinhole ( 50 m , thorlabs , p50s ) that provides filtering for the confocal imaging mode .
this pinhole is manually removed when imaging in the two - photon mode . after passing through a polarizing beam splitter ( pbs , thorlabs , pbs251 ) , a pair of f1 = 150 mm and f2 = 125 mm relay lenses image slm vis onto y galvo .
consequently , slm vis , both galvos , and the objective rear pupil are also mutually conjugate , and corrective phase pattern from slm vis is stationary at the objective rear pupil , even as the galvos scan the focused visible light laterally across the specimen . in the fluorescence detection subsystem , the focused fluorescence emission generated by either tpe or visible light
is collected by the objective and initially follows the reverse path of the nir and visible excitation beams .
after y galvo , however , another dichroic beamsplitter ( d1 , semrock ff705-di01 - 25x36 ) diverts the emission through the relay lens pair that conjugates y galvo to slm vis .
half of this unpolarized light passes through pbs , reflects off slm vis , and is focused by an f = 300 mm lens before a photomultiplier tube ( pmt , hamamatsu , h7422 - 40 or r10467u-40 ) . the signal from this detector forms the image in either the confocal or the tpe imaging mode ,
the other half of the fluorescence is reflected by pbs and is sent to the shack - hartmann ( sh ) wavefront sensor , positioned such that the lenslet array ( 10 10 lenses , 0.5 mm pitch , f = 46.7 mm , edmund optics , 64 - 483 ) is conjugate to the objective rear pupil and the two galvos . as a result ,
the detected light is de - scanned by the galvos , and a stationary wavefront is presented at the sensor , even as the focused excitation scans laterally across the specimen .
displacements of the foci on the sh camera ( andor ixon3 897 emccd ) then solely represent the local wavefront gradients , as desired .
the choice of pbs to split the fluorescence signal equally between the sh sensor and the imaging pmt is an obvious one , given that slm vis requires linearly polarized light to modulate the phase properly .
however , while this configuration is perhaps the simplest for an slm - based system , the 50% signal loss at the pmt is a substantial price to pay . on the other hand ,
the question of the optimum split ratio is a complex one , as a number of factors influence how much signal the sh sensor requires to accurately measure the displacement of each lenslet - defined focal spot . increasing the number of lenslets increases the complexity of the aberration that can be measured , but divides the sh signal among more elements .
decreasing the size of each ao corrective subvolume provides more local measurement of the aberration , but decreases the total integrated signal collected for each such measurement .
finally , increased imaging depth generally leads to greater aberration and thus more dramatic improvement after ao correction , but also results in more scattered background and less ballistic ( focused ) light at the sh sensor , requiring more signal to accurately measure the focal spot displacements . in short , while the 50/50 ratio of the pbs configuration represents a simple compromise that works well for the specimens studied here , other configurations , including a system with a variable split ratio , can be envisioned for other biological systems . before measuring and correcting sample - induced aberrations ,
we measure these system aberrations by the phase retrieval method , since it provides an independent means to determine the correction necessary to recover an ideal diffraction - limited focus for an ideal , non - aberrating point object . to correct the aberrations in the visible light path
, the pinhole near the pmt is removed , and a 3d image of an isolated , 200 nm diameter fluorescent bead on a glass slide is obtained by scanning the visible focus in a series of xy planes , and stepping the sample in z to different planes with a piezoelectric flexure stage ( physik instrumente , p-622.2cd ) . the sampling interval must be smaller than the nyquist limit :
nx , y=/(4na),nz=/[2n(1 - 1-(na / n)2 ) ] in each direction , and the field of view must be large enough that aberrated images of the bead ( see below ) are not cropped at the edges .
the 3d image is then inspected , particularly for axial asymmetry indicative of spherical aberration , and the correction collar on the objective is adjusted .
this process of 3d imaging and collar correction is repeated until the spherical aberration is minimized .
next , the bead is moved to the z plane of best focus , and a series of seven 2d images are taken while applying seven different zernike polynomial phase patterns of 2 p - p amplitude on slm vis : flat phase ; positive defocus ; negative defocus ; positive x astigmatism ; negative x astigmatism ; positive y astigmatism ; and negative y astigmatism . from these images , the wavefront correction for system aberration in the visible excitation path is retrieved using the gerchberg - saxton algorithm . thereafter
, this pattern is applied to slm vis , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to correct for aberrations in the nir light path ,
a ccd camera ( avt , guppy f-146 ) is placed at the intermediate image plane located at the focus of the first relay lens after x galvo .
seven 2d images of this focus are taken while applying the seven zernike polynomial phase patterns listed above to slm nir , and the wavefront correction for system aberration in this portion of the nir excitation path is retrieved using the gerchberg - saxton algorithm .
thereafter , this pattern is applied to slm nir , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to calibrate the sh sensor , the visible and nir wavefront corrections for system aberration
are applied to slm vis and slm nir , respectively . a 2d image of a field of fluorescent beads
is then taken in the two - photon imaging mode while integrating the signal at the sh camera .
the resulting sh image consists of an array of foci , matching the elements of the lenslet array .
the centroids of these foci are determined to sub - pixel precision , and serve as the calibration reference .
thereafter , the displacements of these centroids from their reference positions indicate the local gradient of the sample - induced wavefront error , from which the wavefront itself can be calculated using a generalized matrix inversion method .
note that all of the wavefronts presented in this work represent the corrections for sample - induced aberrations , i.e. , after compensation for system aberrations . because aberrations can vary rapidly as a function of position within biological samples
, we image large volumes by dividing them into smaller sub - volumes , and determine an averaged ao correction unique to each sub - volume .
stacked , closed - loop ultrasonic piezomotor stages ( physik instrumente , m-663.465 ) are used initially for x - y positioning of the sample to the focal point of the objective , as well as for lateral translation between sub - volumes . a closed loop ball - screw driven stage ( physik instrumente , m-110.2dg ) provides similar functions in z. within each sub - volume , x galvo and y galvo scan the focus laterally , while a piezo flexure stage ( physik instrumente , p-622.2cd ) steps between scan planes to build a 3d image of the sub - volume . at each voxel ,
the fluorescence photons reaching pmt generate current spikes which are first amplified ( femto messtechnik gmbh , dlpca-200 ) and then integrated over the pixel dwell time in a custom , fast - resetting analog integrator .
the integrator output is digitized by an fpga - based reconfigurable i / o board ( national instruments , pcie-7852r ) just prior to integrator reset from the same board at the end of the dwell period . in the two - photon imaging mode , ao correction occurs simultaneously with image acquisition . the sh camera exposure time is chosen to be just long enough to yield an snr sufficient to accurately measure the gradient of the wavefront .
calculation of the wavefront from this gradient occurs concurrently with the next sh exposure , and the resulting correction of sample - induced aberration is added immediately to the individual system corrections at slm vis and slm nir .
currently , the fastest closed - loop update time for new ao corrections is 14 ms , limited in bright samples by the read - out speed of the emccd - based sh camera . in the future
in the confocal mode , ao correction occurs sequentially : in each corrective sub - volume , the visible excitation is first blocked with the aotf , the nir light is passed by the pockels cell , and a fraction of the sub - volume ( often a single plane ) is scanned by the tpe focus while the resulting de - scanned fluorescence is collected in a single exposure at the sh camera .
after the wavefront correction is calculated and added to the system corrections at slm vis and slm nir , the nir light is blocked , and the visible light is passed in order to image the entire sub - volume . in either imaging mode , the aberration - corrected 3d point spread function ( psf ) of the microscope
is first determined by imaging an isolated 200 nm diameter fluorescent bead on a glass slide with system corrections applied to both slms . for regions of the sample where ao correction recovers near diffraction - limited resolution
, these measured psfs can be used to deconvolve the 3d imaging data via the lucy - richardson algorithm in matlab .
this provides a sharper 3d representation of the imaging volume that depicts the sample and the relative amplitudes of its spatial frequencies more accurately .
volume renderings of the data are created in amira ( fei visualization sciences group ) . for data sets with intensities covering a large dynamic range , a gamma function
the imaging conditions and visualization parameters for all figures and videos are listed ( supplementary table 1 , 2 ) .
the emccd which serves as the sh camera is set to internal triggering mode and serves as the master timing source -- timing pulses from the fire out ttl output of the emccd are read by the fpga card in a control computer ( pc ) to synchronize all operations ( supplementary figs .
analog outputs from the fpga card provide user - defined waveforms that control x galvo , y galvo , and the z sample piezo during imaging .
these are conditioned by individual scaling amplifiers ( srs , sim983 , and sim900 mainframe ) to match their 16-bit resolution to the control range of each device .
additional analog outputs to the pockels cell and the aotf control the intensity and blanking of the nir and two selected wavelengths of visible light .
a digital output synchronizes the integrator to the scanning , while an analog input digitizes the integrated signal at each voxel .
all other hardware , including slm nir , slm vis , the coarse ball screw driven z stage , the x and y ultrasonic piezo stages , and a filter wheel are each directly controlled by cards in the pc .
the pc itself consists of a rack - mounted chassis with motherboard ( supermicro , superserver sys-7046gt - trf ) , dual microprocessors ( intel , hexa - core xeon x5680 3.33 ghz 12 mb ) , 48 gb of ram , and a 1 tb hard drive running under 64-bit windows 7 pro .
wild - type and transgenic lines were maintained according to institutional animal care and use protocols .
the following lines were used : roy ; gmc604et ; gmc930tg , which expresses yfp in the membranes of a sparse set of neurons ( fig . 1 , supplementary fig . 7 and supplementary video 1 ) ; tg(-actin : mgfp ) , which expresses gfp in the membranes of all cells ( fig .
2a and supplementary video 3 ) ; tg(4.9sox10:egfp ) , which expresses egfp cytosolically in a subset of oligodendrocytes in the brain ( fig .
tg(4.9sox10:egfp ) crossed with tg(-8.4neurog1:nrfp ) , which expresses rfp in neuronal nuclei ( fig .
3 and supplementary video 4 ) ; s1101t - gal4 x uas - memcerulean , uas - centrin2-yfp , which expresses yfp - tagged centrin2 in a broad subset of neurons , including in the retina ( supplementary fig . 5a
e ) ; and huc - gal4 x uas - mitocfp / memyfp , which expresses cfp in the mitochondria and yfp in the plasma membrane of a broad subset of neurons ( supplementary figs .
embryos were transferred at 12h or 24 h post - fertilization to e3 solution ( 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl2 , 0.33 mm mgso4 ) containing n - phenylthiourea ( sigma ) to inhibit pigmentation . for imaging embryos
were anesthetized using tricaine ( sigma ) in e3 solution and mounted in 0.7% low - melting agarose ( sigma - aldrich a4018 ) as described by godinho .
1 ) is comprised of subsystems for two - photon near - infrared ( nir ) excitation , visible fluorescence detection and wavefront measurement , and continuous wave ( cw ) visible excitation . in the two - photon subsystem ,
pulsed light from a ti : sapphire laser ( coherent , chameleon ultra ii ) , intensity controlled by a pockels cell ( conoptics , 350 - 80-la-02 ) , is expanded to a 1/e diameter of 8 mm before being reflecting at 8 from the normal off of a nir - responsive spatial light modulator ( slm nir , boulder nonlinear systems , hsp256 - 1064 ) .
the slm is used to apply the corrective pattern needed to retain a diffraction - limited two - photon excitation ( tpe ) focus in the specimen . a pair of nir achromatic relay lenses ( focal lengths f1 = 150 mm and f2 = 125 mm ) operating in a 2f1 + 2f2 configuration are then used to image the slm onto the 5 mm mirror of a galvanometer ( y galvo , cambridge technology , 6215h ) .
another pair of f1 = f2 = 85 mm relay lenses then image the slm onto a second 5 mm galvo mirror ( x galvo , cambridge technology , 6215h ) .
a final pair of f1 = 89 mm and f2 = 350 mm relay lenses creates a magnified image of the slm at the rear pupil plane of the imaging objective ( nikon , cfi apo lwd 25xw , 1.1 na and 2 mm wd ) .
mutual conjugation of the slm , both galvos , and the objective rear pupil insures that the corrective phase pattern from the slm is stationary at the objective rear pupil , even as the galvos scan the focused nir light laterally across the specimen .
the visible excitation subsystem begins when four cw lasers ( = 440 nm , 50 mw , crystalaser ; = 488 nm , 200 mw , coherent sapphire 488 lp ; = 514 nm , 300 mw , mpb communications , model 2ru - vfl - p-300 - 514-r ; and = 561 nm , 200 mw , coherent sapphire 561 lp ) are expanded to a common a 1/e diameter of 2 mm and combined into a single co - linear beam using dichroic beamsplitters ( semrock , lasermux family ) .
an acousto - optic tunable filter ( aotf , aa opto - electronic , aotfnc-400.650-tn ) selects one or more wavelengths and controls the power of each .
the linearly polarized output of the aotf is expanded to a 1/e diameter of 10 mm , inserted into the microscope beam path using a dichroic beamsplitter ( d2 in supplementary fig .
1 , semrock di01-r442/510 - 25x36 or di01-r488/561 - 25x36 ) , and reflected from a spatial light modulator responsive to visible light ( slm vis , boulder nonlinear systems , hsp256 - 0532 ) .
the slm is used to apply the corrective pattern needed to retain both a diffraction - limited visible excitation focus in the specimen , and a diffraction - limited focus of the fluorescence emission at a pinhole ( 50 m , thorlabs , p50s ) that provides filtering for the confocal imaging mode .
this pinhole is manually removed when imaging in the two - photon mode . after passing through a polarizing beam splitter ( pbs , thorlabs , pbs251 ) , a pair of f1 = 150 mm and f2 = 125 mm relay lenses image slm vis onto y galvo .
consequently , slm vis , both galvos , and the objective rear pupil are also mutually conjugate , and corrective phase pattern from slm vis is stationary at the objective rear pupil , even as the galvos scan the focused visible light laterally across the specimen . in the fluorescence detection subsystem , the focused fluorescence emission generated by either tpe or visible light
is collected by the objective and initially follows the reverse path of the nir and visible excitation beams .
after y galvo , however , another dichroic beamsplitter ( d1 , semrock ff705-di01 - 25x36 ) diverts the emission through the relay lens pair that conjugates y galvo to slm vis .
half of this unpolarized light passes through pbs , reflects off slm vis , and is focused by an f = 300 mm lens before a photomultiplier tube ( pmt , hamamatsu , h7422 - 40 or r10467u-40 ) . the signal from this detector forms the image in either the confocal or the tpe imaging mode ,
the other half of the fluorescence is reflected by pbs and is sent to the shack - hartmann ( sh ) wavefront sensor , positioned such that the lenslet array ( 10 10 lenses , 0.5 mm pitch , f = 46.7 mm , edmund optics , 64 - 483 ) is conjugate to the objective rear pupil and the two galvos . as a result ,
the detected light is de - scanned by the galvos , and a stationary wavefront is presented at the sensor , even as the focused excitation scans laterally across the specimen .
displacements of the foci on the sh camera ( andor ixon3 897 emccd ) then solely represent the local wavefront gradients , as desired .
the choice of pbs to split the fluorescence signal equally between the sh sensor and the imaging pmt is an obvious one , given that slm vis requires linearly polarized light to modulate the phase properly .
however , while this configuration is perhaps the simplest for an slm - based system , the 50% signal loss at the pmt is a substantial price to pay . on the other hand ,
the question of the optimum split ratio is a complex one , as a number of factors influence how much signal the sh sensor requires to accurately measure the displacement of each lenslet - defined focal spot . increasing the number of lenslets increases the complexity of the aberration that can be measured , but divides the sh signal among more elements .
decreasing the size of each ao corrective subvolume provides more local measurement of the aberration , but decreases the total integrated signal collected for each such measurement .
finally , increased imaging depth generally leads to greater aberration and thus more dramatic improvement after ao correction , but also results in more scattered background and less ballistic ( focused ) light at the sh sensor , requiring more signal to accurately measure the focal spot displacements . in short , while the 50/50 ratio of the pbs configuration represents a simple compromise that works well for the specimens studied here , other configurations , including a system with a variable split ratio , can be envisioned for other biological systems .
before measuring and correcting sample - induced aberrations , the microscope must be calibrated to compensate for its own aberrations .
we measure these system aberrations by the phase retrieval method , since it provides an independent means to determine the correction necessary to recover an ideal diffraction - limited focus for an ideal , non - aberrating point object . to correct the aberrations in the visible light path ,
the pinhole near the pmt is removed , and a 3d image of an isolated , 200 nm diameter fluorescent bead on a glass slide is obtained by scanning the visible focus in a series of xy planes , and stepping the sample in z to different planes with a piezoelectric flexure stage ( physik instrumente , p-622.2cd ) . the sampling interval must be smaller than the nyquist limit :
nx , y=/(4na),nz=/[2n(1 - 1-(na / n)2 ) ] in each direction , and the field of view must be large enough that aberrated images of the bead ( see below ) are not cropped at the edges .
the 3d image is then inspected , particularly for axial asymmetry indicative of spherical aberration , and the correction collar on the objective is adjusted .
this process of 3d imaging and collar correction is repeated until the spherical aberration is minimized .
next , the bead is moved to the z plane of best focus , and a series of seven 2d images are taken while applying seven different zernike polynomial phase patterns of 2 p - p amplitude on slm vis : flat phase ; positive defocus ; negative defocus ; positive x astigmatism ; negative x astigmatism ; positive y astigmatism ; and negative y astigmatism . from these images , the wavefront correction for system aberration in the visible excitation path is retrieved using the gerchberg - saxton algorithm . thereafter
, this pattern is applied to slm vis , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to correct for aberrations in the nir light path ,
a ccd camera ( avt , guppy f-146 ) is placed at the intermediate image plane located at the focus of the first relay lens after x galvo .
seven 2d images of this focus are taken while applying the seven zernike polynomial phase patterns listed above to slm nir , and the wavefront correction for system aberration in this portion of the nir excitation path is retrieved using the gerchberg - saxton algorithm .
thereafter , this pattern is applied to slm nir , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . to calibrate the sh sensor , the visible and nir wavefront corrections for system aberration
are applied to slm vis and slm nir , respectively . a 2d image of a field of fluorescent beads
is then taken in the two - photon imaging mode while integrating the signal at the sh camera .
the resulting sh image consists of an array of foci , matching the elements of the lenslet array .
the centroids of these foci are determined to sub - pixel precision , and serve as the calibration reference .
thereafter , the displacements of these centroids from their reference positions indicate the local gradient of the sample - induced wavefront error , from which the wavefront itself can be calculated using a generalized matrix inversion method .
note that all of the wavefronts presented in this work represent the corrections for sample - induced aberrations , i.e. , after compensation for system aberrations .
because aberrations can vary rapidly as a function of position within biological samples , we image large volumes by dividing them into smaller sub - volumes , and determine an averaged ao correction unique to each sub - volume .
stacked , closed - loop ultrasonic piezomotor stages ( physik instrumente , m-663.465 ) are used initially for x - y positioning of the sample to the focal point of the objective , as well as for lateral translation between sub - volumes . a closed loop ball - screw driven stage ( physik instrumente , m-110.2dg ) provides similar functions in z. within each sub - volume , x galvo and y galvo scan the focus laterally , while a piezo flexure stage ( physik instrumente , p-622.2cd ) steps between scan planes to build a 3d image of the sub - volume . at each voxel ,
the fluorescence photons reaching pmt generate current spikes which are first amplified ( femto messtechnik gmbh , dlpca-200 ) and then integrated over the pixel dwell time in a custom , fast - resetting analog integrator .
the integrator output is digitized by an fpga - based reconfigurable i / o board ( national instruments , pcie-7852r ) just prior to integrator reset from the same board at the end of the dwell period . in the two - photon imaging mode , ao correction occurs simultaneously with image acquisition .
the sh camera exposure time is chosen to be just long enough to yield an snr sufficient to accurately measure the gradient of the wavefront .
calculation of the wavefront from this gradient occurs concurrently with the next sh exposure , and the resulting correction of sample - induced aberration is added immediately to the individual system corrections at slm vis and slm nir .
currently , the fastest closed - loop update time for new ao corrections is 14 ms , limited in bright samples by the read - out speed of the emccd - based sh camera . in the future , a scmos camera may permit faster correction . in the confocal mode ,
ao correction occurs sequentially : in each corrective sub - volume , the visible excitation is first blocked with the aotf , the nir light is passed by the pockels cell , and a fraction of the sub - volume ( often a single plane ) is scanned by the tpe focus while the resulting de - scanned fluorescence is collected in a single exposure at the sh camera .
after the wavefront correction is calculated and added to the system corrections at slm vis and slm nir , the nir light is blocked , and the visible light is passed in order to image the entire sub - volume . in either imaging mode , the aberration - corrected 3d point spread function ( psf ) of the microscope is first determined by imaging an isolated 200 nm diameter fluorescent bead on a glass slide with system corrections applied to both slms . for regions of the sample where ao correction recovers near diffraction - limited resolution , these measured psfs can be used to deconvolve the 3d imaging data via the lucy - richardson algorithm in matlab .
this provides a sharper 3d representation of the imaging volume that depicts the sample and the relative amplitudes of its spatial frequencies more accurately .
volume renderings of the data are created in amira ( fei visualization sciences group ) . for data sets with intensities covering a large dynamic range , a gamma function
the imaging conditions and visualization parameters for all figures and videos are listed ( supplementary table 1 , 2 ) .
the emccd which serves as the sh camera is set to internal triggering mode and serves as the master timing source -- timing pulses from the fire out ttl output of the emccd are read by the fpga card in a control computer ( pc ) to synchronize all operations ( supplementary figs .
analog outputs from the fpga card provide user - defined waveforms that control x galvo , y galvo , and the z sample piezo during imaging .
these are conditioned by individual scaling amplifiers ( srs , sim983 , and sim900 mainframe ) to match their 16-bit resolution to the control range of each device .
additional analog outputs to the pockels cell and the aotf control the intensity and blanking of the nir and two selected wavelengths of visible light .
a digital output synchronizes the integrator to the scanning , while an analog input digitizes the integrated signal at each voxel .
all other hardware , including slm nir , slm vis , the coarse ball screw driven z stage , the x and y ultrasonic piezo stages , and a filter wheel are each directly controlled by cards in the pc .
the pc itself consists of a rack - mounted chassis with motherboard ( supermicro , superserver sys-7046gt - trf ) , dual microprocessors ( intel , hexa - core xeon x5680 3.33 ghz 12 mb ) , 48 gb of ram , and a 1 tb hard drive running under 64-bit windows 7 pro .
wild - type and transgenic lines were maintained according to institutional animal care and use protocols .
the following lines were used : roy ; gmc604et ; gmc930tg , which expresses yfp in the membranes of a sparse set of neurons ( fig . 1 , supplementary fig . 7 and supplementary video 1 ) ; tg(-actin : mgfp ) , which expresses gfp in the membranes of all cells ( fig .
2a and supplementary video 3 ) ; tg(4.9sox10:egfp ) , which expresses egfp cytosolically in a subset of oligodendrocytes in the brain ( fig .
tg(4.9sox10:egfp ) crossed with tg(-8.4neurog1:nrfp ) , which expresses rfp in neuronal nuclei ( fig . 3 and supplementary video 4 ) ; s1101t - gal4 x uas - memcerulean , uas - centrin2-yfp , which expresses yfp - tagged centrin2 in a broad subset of neurons , including in the retina ( supplementary fig . 5a
e ) ; and huc - gal4 x uas - mitocfp / memyfp , which expresses cfp in the mitochondria and yfp in the plasma membrane of a broad subset of neurons ( supplementary figs .
embryos were transferred at 12h or 24 h post - fertilization to e3 solution ( 5 mm nacl , 0.17 mm kcl , 0.33 mm cacl2 , 0.33 mm mgso4 ) containing n - phenylthiourea ( sigma ) to inhibit pigmentation . for imaging embryos
were anesthetized using tricaine ( sigma ) in e3 solution and mounted in 0.7% low - melting agarose ( sigma - aldrich a4018 ) as described by godinho . | using a de - scanned , laser - induced guide star and direct wavefront sensing , we demonstrate adaptive correction of complex optical aberrations at high numerical aperture and a 14 ms update rate .
this permits us to compensate for the rapid spatial variation in aberration often encountered in biological specimens , and recover diffraction - limited imaging over large ( > 240 m)3 volumes .
we applied this to image fine neuronal processes and subcellular dynamics within the zebrafish brain . | ONLINE METHODS
Scanning adaptive optical microscope using a de-scanned nonlinear guide star
System Calibration
Image Acquisition, Wavefront Sensing, and Adaptive Optical Correction
Control electronics and timing
Zebrafish care and preparation
Supplementary Material | the slm is used to apply the corrective pattern needed to retain both a diffraction - limited visible excitation focus in the specimen , and a diffraction - limited focus of the fluorescence emission at a pinhole ( 50 m , thorlabs , p50s ) that provides filtering for the confocal imaging mode . as a result ,
the detected light is de - scanned by the galvos , and a stationary wavefront is presented at the sensor , even as the focused excitation scans laterally across the specimen . before measuring and correcting sample - induced aberrations ,
we measure these system aberrations by the phase retrieval method , since it provides an independent means to determine the correction necessary to recover an ideal diffraction - limited focus for an ideal , non - aberrating point object . to correct the aberrations in the visible light path
, the pinhole near the pmt is removed , and a 3d image of an isolated , 200 nm diameter fluorescent bead on a glass slide is obtained by scanning the visible focus in a series of xy planes , and stepping the sample in z to different planes with a piezoelectric flexure stage ( physik instrumente , p-622.2cd ) . thereafter , this pattern is applied to slm nir , and the wavefront correction for sample - induced aberrations is added to it to provide complete correction during normal operation . calculation of the wavefront from this gradient occurs concurrently with the next sh exposure , and the resulting correction of sample - induced aberration is added immediately to the individual system corrections at slm vis and slm nir . in the future
in the confocal mode , ao correction occurs sequentially : in each corrective sub - volume , the visible excitation is first blocked with the aotf , the nir light is passed by the pockels cell , and a fraction of the sub - volume ( often a single plane ) is scanned by the tpe focus while the resulting de - scanned fluorescence is collected in a single exposure at the sh camera . for regions of the sample where ao correction recovers near diffraction - limited resolution
, these measured psfs can be used to deconvolve the 3d imaging data via the lucy - richardson algorithm in matlab . all other hardware , including slm nir , slm vis , the coarse ball screw driven z stage , the x and y ultrasonic piezo stages , and a filter wheel are each directly controlled by cards in the pc . the pc itself consists of a rack - mounted chassis with motherboard ( supermicro , superserver sys-7046gt - trf ) , dual microprocessors ( intel , hexa - core xeon x5680 3.33 ghz 12 mb ) , 48 gb of ram , and a 1 tb hard drive running under 64-bit windows 7 pro . the slm is used to apply the corrective pattern needed to retain both a diffraction - limited visible excitation focus in the specimen , and a diffraction - limited focus of the fluorescence emission at a pinhole ( 50 m , thorlabs , p50s ) that provides filtering for the confocal imaging mode . as a result ,
the detected light is de - scanned by the galvos , and a stationary wavefront is presented at the sensor , even as the focused excitation scans laterally across the specimen . before measuring and correcting sample - induced aberrations , the microscope must be calibrated to compensate for its own aberrations . calculation of the wavefront from this gradient occurs concurrently with the next sh exposure , and the resulting correction of sample - induced aberration is added immediately to the individual system corrections at slm vis and slm nir . in the confocal mode ,
ao correction occurs sequentially : in each corrective sub - volume , the visible excitation is first blocked with the aotf , the nir light is passed by the pockels cell , and a fraction of the sub - volume ( often a single plane ) is scanned by the tpe focus while the resulting de - scanned fluorescence is collected in a single exposure at the sh camera . for regions of the sample where ao correction recovers near diffraction - limited resolution , these measured psfs can be used to deconvolve the 3d imaging data via the lucy - richardson algorithm in matlab . all other hardware , including slm nir , slm vis , the coarse ball screw driven z stage , the x and y ultrasonic piezo stages , and a filter wheel are each directly controlled by cards in the pc . the pc itself consists of a rack - mounted chassis with motherboard ( supermicro , superserver sys-7046gt - trf ) , dual microprocessors ( intel , hexa - core xeon x5680 3.33 ghz 12 mb ) , 48 gb of ram , and a 1 tb hard drive running under 64-bit windows 7 pro . | [
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post - traumatic stress disorder ( ptsd ) is characterized by various altered emotional responses as a result of trauma exposure ( e.g. , combat , assault , and disasters ) .
patients with ptsd not only experience intense negative emotional reactions when reminded of their trauma but also report exaggerated arousal ( poor sleep , restlessness , hypervigilance ) , anhedonia , social withdrawal , and decreased emotional expressivity , referred to as emotional numbing . characterizing the neural basis of these diverse , distorted emotional responses poses a major challenge to contemporary psychiatric research .
functional neuroimaging techniques have focused primarily on the study of brain function related to fear perception and response , and have consistently implicated aberrant amygdala reactivity to fear - relevant probes and other abnormalities in a broad aberrant amygdala - linked circuitry involving the medial prefrontal cortex ( mpfc ) , insula , anterior cingulate cortex ( acc ) , and hippocampus ( rauch and shin , 1997 ; pitman et al . , 2001 ; nemeroff et al . , 2006 ; rauch et al . , 2006 ; etkin and wager , 2007 ; liberzon and sripada , 2008 ; shin , 2009 ; shin and liberzon , 2010 ) .
together these interconnected regions form a disrupted functional network thought to be responsible for impaired regulation of fear responses , enhanced attention to threat - related stimuli , and biased memory for adverse events ( shin , 2009 ) .
anxiety disorders , such as ptsd , are believed to manifest from dysfunction in a complex integrated functional network , largely , between cortical and limbic regions ( gilboa et al .
, 2008 ; bluhm et al . , 2009 ; shaw et al . , 2009 ; daniels et al . , 2010 ) .
some studies have begun to examine these brain circuits and region - to - region interactions , by measuring the extent to which activity in one region is correlated with activity in another during a particular task .
although these dysfunctional networks have been implicated in mediating several characteristics of ptsd , such as , hyperarousal , abnormal reactivity to emotional stimuli , and avoidance of emotionally distressing memories ( nemeroff et al . , 2006 ; shin and liberzon , 2010 ) , little is known about how these regions may interact dynamically within individual subjects .
some clues exist from anatomical and functional studies that these brain regions may indeed form a network responsible for emotion processing .
tracer studies in non - human primates ( amaral and price , 1984 ; saunders et al . , 1988 ; barbas and de olmos , 1990 ; stefanacci et al . , 1996 ; ghashghaei and barbas , 2002 ; ghashghaei et al . , 2007
; freese and amaral , 2009 ) and , more recently , diffusion tensor imaging studies in humans ( croxson et al . , 2005 ; johansen - berg et al . , 2008 ;
2009 ) have identified robust bidirectional projections between the amygdala and the mpfc , rostral acc ( racc ) , insula , and hippocampus .
consistent with known anatomical connections , several studies that have examined functional connectivity of the amygdala have found significant co - activation and/or functionally correlated activation of the amygdala and the mpfc , insula , hippocampus , and racc ( phan et al . , 2002 ; wager et al .
, 2007a ; kober et al . , 2008 ; etkin et al . , 2009 ;
it is well established that negatively valenced emotional stimuli activate the amygdala , which mediates subjective and attentional - vigilance aspects of threat processing ( liberzon et al . , 1999
liberzon and phan , 2003 ; taylor et al . , 2003 ; wager et al . , 2003 ;
etkin and wager , 2007 ; kober et al . , 2008 ; liberzon and sripada , 2008 ; etkin , 2009 ; shin and liberzon , 2010 ) .
similarly , insula activity also increases in response to emotionally aversive stimuli that evoke visceral or somatic sensations ( simmons et al . , 2004 ) .
increased amygdala and insula activation during fear conditioning have been shown to be reliably associated with one another ( etkin and wager , 2007 ) .
amygdala activity is decreased in response to suppression of negative affect via reappraisal and during inhibition of conditioned fear responses as a result of increased activation in the mpfc and racc , which exert top - down inhibitory influences on amygdala reactivity to fear and threat ( ochsner et al . , 2002 ; taylor et al . , 2003 ; phelps et al . , 2004 ; etkin et al . , 2006 , 2011 ; urry et al . , 2006 ;
the magnitude of task - dependent functional coupling between the amygdala and mpfc / racc has been shown to be negatively correlated with intensity of subjective reports of negative affect ( banks et al . , 2007 ) .
increased functional connectivity between the amygdala and the hippocampus has been attributed to the persistence of memories for emotionally arousing events ( hamann et al .
, 1999 ; kilpatrick and cahill , 2003 ; phelps , 2004 ; ritchey et al .
, 2008 ; murty et al . , 2011 ) . specifically , the hippocampus forms episodic representations of the emotional significance and interpretation of events , and influences amygdala activity when emotional stimuli are encountered ( phelps , 2004 )
. these lines of convergent evidence suggests that how the amygdala interacts with other regions may mediate the control , or lack thereof , of fear perception and emotional arousal in humans .
dysfunctions within discrete areas that form an amygdala paralimbic / frontal network have been implicated in mediating several characteristics of ptsd , such as , hyperarousal , abnormal reactivity to emotional stimuli , and avoidance of emotionally distressing memories ( nemeroff et al . , 2006 ; shin and liberzon , 2010 ) . in particular , many studies have shown amygdala hyperactivity in ptsd in response to trauma - related imagery ( shin et al .
, 1997 , 2004a ) , combat - related sounds or smells ( liberzon et al . , 1999 ; pissiota et al . , 2002 ; vermetten et al . , 2007 ) , trauma - related photographs or words ( hendler et al . , 2003 ; driessen et al . , 2004 ;
, 2000 ; shin et al . , 2005 ; williams et al . , 2006 ; bryant et al . , 2008 ) . exaggerated amygdala reactivity observed in ptsd has been posited to be a result of insufficient top - down regulation from the mpfc and acc , consequently leading to hyperarousal and deficits in extinction as well as the inability to suppress enhanced fear perception or exaggerated fear responses to trauma - related stimuli ( rauch and shin , 1997 ; rauch et al .
, 1998 ; pitman et al . , 2001 ; liberzon and phan , 2003 ) ; for example , shin et al .
( 2004a , 2005 ) have observed that exaggerated amygdala reactivity is negatively correlated with responses in the dorsal and ventral mpfc across individuals with ptsd .
however , gilboa et al . ( 2004 ) found little evidence for failure of inhibition of acc over the amygdala in individuals with ptsd related to civilian trauma during symptom provocation and in fact found that amygdala activity significantly influenced acc activity .
insula hyperactivity has been observed in ptsd patients and given its role in the experience ( e.g. , somatic sensation ) of negative emotions and structural connectivity to amygdala ( augustine , 1996 ; aggleton and saunders , 2000 ; freese and amaral , 2009 ) , the insula may be working in concert with aberrant amygdala responses ( bremner et al . , 2003 , 2005 ;
, 2007 ; vermetten et al . , 2007 ; lindauer et al . , 2008 ; simmons et al . , 2008 ; werner et al . , 2009 ; whalley et al
although less commonly implicated , abnormal hippocampal function , and diminished hippocampal volumes in ptsd patients have been associated with deficits in contextual processing , as well as memory impairments , and neuroendocrine dysregulation ( bremner et al . , 1999 , 2003 ; bonne et al . , 2001 ;
shin et al . , 2004a , b , 2006 ; werner et al . , 2009 ) .
recently these functional connectivity techniques have been applied to the study of corticolimbic circuitry abnormalities at baseline or at rest ( resting - state functional connectivity ) .
studies of functional interconnectivity of brain regions derived from resting - state scans provides insight into the relationship of spontaneous brain activity between brain regions without being confounded by task influences on activation and has even been shown to reflect structural connectivity between brain regions ( greicius et al . , 2009 ;
resting - state functional connectivity of the amygdala has revealed patterns of connectivity consistent with task - based connectivity patterns ( stein et al .
moreover , resting - state functional connectivity has been a useful tool for identifying abnormalities in the functional organization of brain systems in several anxiety and mood disorders ( greicius , 2008 ) .
however , little is known about what abnormalities , if any , in amygdala connectivity exist at rest in ptsd .
therefore the primary aim of the present experiment was to investigate aberrant amygdala functional connectivity patterns in returning operation enduring freedom / operation iraqi freedom ( oef / oif ) veterans with combat - related ptsd ( ptsd group ) and combat - exposed oef / oif veterans without ptsd [ combat - exposed control ( cec ) group ] during resting - state .
we hypothesized that amygdala connectivity to the acc , mpfc , insula , and hippocampus would differentiate the ptsd group from the cec group .
if observed , such findings would extend our understanding of the pathophysiology of ptsd by identifying a disturbed network that exists outside of the presence of an overt threat / danger or in the absence of stimulus or task - induced negative emotional processing .
thirty - four , right - handed , male veterans returning from oef / oif with documented exposure to combat - related trauma participated in this study . based on the dsm - iv ( apa , 1994 ) ,
17 participants met criteria for current ptsd ( ptsd group ; age : 30.12 7.70 years ; caucasian = 16 ; hispanic or latino = 1 ) and the other 17 participants were combat - exposed matched controls without ptsd ( cec group ; age : 33.71 9.12 ; caucasian = 16 ; asian = 1 ) .
psychiatric diagnoses were established via the structured clinical interview for dsm - iv ( first et al . , 1996 ) .
additional standardized clinical instruments including the clinician administered ptsd scale ( caps ; blake et al . , 1995 ) , the ptsd checklist : military ( pcl - m ; blanchard et al . , 1996 ) , the combat exposure scale ( ces ; keane et al . , 1989 ) , the hamilton depression inventory ( ham - d ; williams , 1988 ) , and the beck depression inventory ( bdi - ii ; beck et al . , 1996 ) were administered to quantitatively characterize ptsd symptoms , severity of trauma exposure , and depression .
relative to the cec group , the ptsd group had significantly higher scores on the caps , pcl - m , and ham - d and bdi - ii . of note
, the groups did not differ in severity of trauma exposure . some of the ptsd patients had current psychiatric co - morbidity ( n = 2 with current major depressive disorder ; n = 2 with current alcohol abuse ) or had a past co - morbidity more than 6 months ago ( n = 1 had major depressive disorder ; n = 4 had alcohol abuse , one of whom also had past opioid abuse ; n = 1 had alcohol dependence in full sustained remission ) at the time of scanning .
in addition , some ptsd patients had a history of psychotropic medication usage ( n = 8 had taken an selective serotonin reuptake inhibitor , one of whom had also taken a norepinephrine dopamine reuptake inhibitor ; n = 1 had taken a tri - cyclic antidepressant ; n = 2 had taken a serotonin antagonist - reuptake inhibitor ) , but none of the ptsd patients were currently taking any psychotropic medications at the time of scanning .
all participants were free of any clinically significant medical or neurologic condition that would affect brain blood flow / metabolism or function and/or task performance .
none of the subjects had a positive urine toxicology screen at the time of scanning .
all participants gave written informed consent after explanation of the experimental protocol , as approved by the va ann arbor healthcare system and university of michigan institutional review boards .
ptsd , post - traumatic stress disorder ; cec , combat - exposed controls ; caps , clinician administered ptsd scale ; pcl - m , ptsd checklist : military ; bdi - ii , beck depression inventory ; ham - d , hamilton depression inventory ; ces , combat exposure scale .
all participants underwent an 8-min resting - state fmri scan in which they were instructed to fixate on a white crosshair that was centrally projected against a black background and let their mind wander without falling asleep .
fmri scanning was performed on a 3 t ge signa system ( general electric ; milwaukee , wi , usa ) using a standard radiofrequency coil at the university of michigan functional mri laboratory .
whole - brain functional images ( i.e. , blood oxygenated level - dependent , bold ) were collected from 43 axial , 3-mm - thick slices using a t2 * -sensitive gradient echo reverse spiral acquisition sequence ( repetition time , 2000 ms ; echo time , 30 ms ; 64 64 matrix ; 220 mm field of view ; flip angle , 90 ) , optimized to minimize susceptibility artifacts ( signal loss ) at the medial temporal lobe ( including the amygdala ; stenger et al . , 2000 ) .
cardiac and respiratory cycles were recorded with mri vendor supplied pulse - oximeter and respiratory belt for physiological corrections on resting - state data .
a t1-weighted anatomical image was collected in the same planes as the functional data , but with higher in - plane resolution ( 1 mm , t1-overlay ) to aid in later co - registration .
a high resolution , t1-weighted volumetric anatomical scan ( t1-spgr ; three - dimensional spoiled gradient echo ) was also acquired for precise anatomical localization and normalization .
data from 32 participants ( cec = 17 ; ptsd = 15 ) met criteria for high quality and scan stability with minimum motion correction and were subsequently included in fmri analyses ( <3 mm displacement in any one direction ; two ptsd patients were excluded for poor data quality due to excessive head movement ) .
functional data were processed and analyzed using statistical parametric mapping software ( spm8 ; wellcome trust centre for neuroimaging , london ) using similar methods previously published from our lab ( jelsone - swain et al . , 2010 ) .
images were corrected for physiological signal fluctuations using a custom code written in matlab ( mathworks , natick , ma , usa ; noll et al . , 1991 ) .
slice timing and movement correction was done to the time - series data using spm8 .
each participant s t1-overlay was co - registered to the time - series data and the t1-spgr was then co - registered to the co - registered t1-overlay image .
the co - registered t1-spgr was then segmented into gray matter , white matter , and cerebrospinal fluid ( csf ) and normalized to montreal neurological institute ( mni ) space using vbm8 toolbox of spm8 and the resulting normalization matrix was applied to the time - series data .
these normalized time - series data were subsequently re - sampled to 2 mm voxels and smoothed with an 8-mm gaussian kernel to minimize noise and effects due to residual differences in functional and gyral anatomy during inter - subject averaging .
then the resulting white matter and csf segments were further defined using a custom algorithm previously described ( welsh et al . , 2007 ) .
each voxel s time - series was detrended to correct for linear drift over time .
nine nuisance covariates ( time - series predictors for global signal , white matter , csf , and the six movement parameters , including the first derivative , obtained during realignment to account for motion - related effects in bold ) were sequentially regressed from the time - series .
the resulting time - series were then band - passed filtered between the frequencies of 0.01 and 0.10-hz to limit the analysis to resting - state frequencies of interest . to determine amygdala connectivity during resting - state , seed regions in the left and right amygdala
were defined by an anatomically based amygdala mask in each hemisphere ( from mask of region of interest analysis software , marina ; tzourio - mazoyer et al . , 2002 ; walter et al . , 2003 ) .
we then extracted the averaged time course from these seed regions in each participant s data and calculated correlation coefficients between these average time courses and all other voxels of the brain resulting in an r - image for amygdala connectivity .
the resulting correlation coefficients were then transformed into z - scores using a fisher r - to - z transformation and the resulting z images were analyzed at the second level in a random - effects statistical model .
two - tailed independent samples t tests were used to identify areas of the brain that exhibited activity that covaried with the amygdala differentially during resting - state between the two groups ( ptsd > cec ; cec > ptsd ) .
significant activations were identified with a whole - brain voxel - wise threshold of p < 0.005 with a minimum cluster extent of > 387 contiguous voxels ( 3096 mm ) , to correct for multiple comparisons at a corrected p < 0.05 calculated using monte - carlo simulations ( afni 3dclustsim ) .
previous studies interested in differences in brain connectivity between patients with ptsd and trauma - exposed controls without ptsd have used similar significance thresholding approaches to balance type i and ii error rates ( yin et al .
, 2011a , b ) . to clarify the signal direction , variance , and specificity of differences in strength of connectivity between the cec and ptsd groups during resting - state , we extracted individual subject s z - score values from activated voxels that fell within an anatomically based mask for each a priori region from the between - group contrast ( ptsd > cec ; tzourio - mazoyer et al . , 2002 ; walter et al . , 2003 ) .
of note , we did not conduct statistical tests on these measures , as they were defined from significant activations resulting from whole - brain maps of group differences in connectivity .
thirty - four , right - handed , male veterans returning from oef / oif with documented exposure to combat - related trauma participated in this study . based on the dsm - iv ( apa , 1994 ) ,
17 participants met criteria for current ptsd ( ptsd group ; age : 30.12 7.70 years ; caucasian = 16 ; hispanic or latino = 1 ) and the other 17 participants were combat - exposed matched controls without ptsd ( cec group ; age : 33.71 9.12 ; caucasian = 16 ; asian = 1 ) .
psychiatric diagnoses were established via the structured clinical interview for dsm - iv ( first et al . , 1996 ) .
additional standardized clinical instruments including the clinician administered ptsd scale ( caps ; blake et al . , 1995 ) , the ptsd checklist : military ( pcl - m ; blanchard et al . , 1996 ) , the combat exposure scale ( ces ; keane et al . , 1989 ) , the hamilton depression inventory ( ham - d ; williams , 1988 ) , and the beck depression inventory ( bdi - ii ; beck et al . , 1996 ) were administered to quantitatively characterize ptsd symptoms , severity of trauma exposure , and depression .
relative to the cec group , the ptsd group had significantly higher scores on the caps , pcl - m , and ham - d and bdi - ii . of note
, the groups did not differ in severity of trauma exposure . some of the ptsd patients had current psychiatric co - morbidity ( n = 2 with current major depressive disorder ; n = 2 with current alcohol abuse ) or had a past co - morbidity more than 6 months ago ( n = 1 had major depressive disorder ; n = 4 had alcohol abuse , one of whom also had past opioid abuse ; n = 1 had alcohol dependence in full sustained remission ) at the time of scanning .
in addition , some ptsd patients had a history of psychotropic medication usage ( n = 8 had taken an selective serotonin reuptake inhibitor , one of whom had also taken a norepinephrine dopamine reuptake inhibitor ; n = 1 had taken a tri - cyclic antidepressant ; n = 2 had taken a serotonin antagonist - reuptake inhibitor ) , but none of the ptsd patients were currently taking any psychotropic medications at the time of scanning .
all participants were free of any clinically significant medical or neurologic condition that would affect brain blood flow / metabolism or function and/or task performance .
none of the subjects had a positive urine toxicology screen at the time of scanning .
all participants gave written informed consent after explanation of the experimental protocol , as approved by the va ann arbor healthcare system and university of michigan institutional review boards .
ptsd , post - traumatic stress disorder ; cec , combat - exposed controls ; caps , clinician administered ptsd scale ; pcl - m , ptsd checklist : military ; bdi - ii , beck depression inventory ; ham - d , hamilton depression inventory ; ces , combat exposure scale .
all participants underwent an 8-min resting - state fmri scan in which they were instructed to fixate on a white crosshair that was centrally projected against a black background and let their mind wander without falling asleep .
fmri scanning was performed on a 3 t ge signa system ( general electric ; milwaukee , wi , usa ) using a standard radiofrequency coil at the university of michigan functional mri laboratory .
whole - brain functional images ( i.e. , blood oxygenated level - dependent , bold ) were collected from 43 axial , 3-mm - thick slices using a t2 * -sensitive gradient echo reverse spiral acquisition sequence ( repetition time , 2000 ms ; echo time , 30 ms ; 64 64 matrix ; 220 mm field of view ; flip angle , 90 ) , optimized to minimize susceptibility artifacts ( signal loss ) at the medial temporal lobe ( including the amygdala ; stenger et al . , 2000 ) .
cardiac and respiratory cycles were recorded with mri vendor supplied pulse - oximeter and respiratory belt for physiological corrections on resting - state data .
a t1-weighted anatomical image was collected in the same planes as the functional data , but with higher in - plane resolution ( 1 mm , t1-overlay ) to aid in later co - registration . a high resolution , t1-weighted volumetric anatomical scan ( t1-spgr ; three - dimensional spoiled gradient echo ) was also acquired for precise anatomical localization and normalization .
data from 32 participants ( cec = 17 ; ptsd = 15 ) met criteria for high quality and scan stability with minimum motion correction and were subsequently included in fmri analyses ( <3 mm displacement in any one direction ; two ptsd patients were excluded for poor data quality due to excessive head movement ) .
functional data were processed and analyzed using statistical parametric mapping software ( spm8 ; wellcome trust centre for neuroimaging , london ) using similar methods previously published from our lab ( jelsone - swain et al . , 2010 ) .
images were corrected for physiological signal fluctuations using a custom code written in matlab ( mathworks , natick , ma , usa ; noll et al . , 1991 ) .
slice timing and movement correction was done to the time - series data using spm8 .
each participant s t1-overlay was co - registered to the time - series data and the t1-spgr was then co - registered to the co - registered t1-overlay image .
the co - registered t1-spgr was then segmented into gray matter , white matter , and cerebrospinal fluid ( csf ) and normalized to montreal neurological institute ( mni ) space using vbm8 toolbox of spm8 and the resulting normalization matrix was applied to the time - series data .
these normalized time - series data were subsequently re - sampled to 2 mm voxels and smoothed with an 8-mm gaussian kernel to minimize noise and effects due to residual differences in functional and gyral anatomy during inter - subject averaging . then the resulting white matter and csf segments
were further defined using a custom algorithm previously described ( welsh et al . , 2007 ) .
each voxel s time - series was detrended to correct for linear drift over time .
nine nuisance covariates ( time - series predictors for global signal , white matter , csf , and the six movement parameters , including the first derivative , obtained during realignment to account for motion - related effects in bold ) were sequentially regressed from the time - series .
the resulting time - series were then band - passed filtered between the frequencies of 0.01 and 0.10-hz to limit the analysis to resting - state frequencies of interest . to determine amygdala connectivity during resting - state , seed regions in the left and right amygdala
were defined by an anatomically based amygdala mask in each hemisphere ( from mask of region of interest analysis software , marina ; tzourio - mazoyer et al .
we then extracted the averaged time course from these seed regions in each participant s data and calculated correlation coefficients between these average time courses and all other voxels of the brain resulting in an r - image for amygdala connectivity .
the resulting correlation coefficients were then transformed into z - scores using a fisher r - to - z transformation and the resulting z images were analyzed at the second level in a random - effects statistical model .
two - tailed independent samples t tests were used to identify areas of the brain that exhibited activity that covaried with the amygdala differentially during resting - state between the two groups ( ptsd > cec ; cec > ptsd ) .
significant activations were identified with a whole - brain voxel - wise threshold of p < 0.005 with a minimum cluster extent of > 387 contiguous voxels ( 3096 mm ) , to correct for multiple comparisons at a corrected p < 0.05 calculated using monte - carlo simulations ( afni 3dclustsim ) .
previous studies interested in differences in brain connectivity between patients with ptsd and trauma - exposed controls without ptsd have used similar significance thresholding approaches to balance type i and ii error rates ( yin et al .
to clarify the signal direction , variance , and specificity of differences in strength of connectivity between the cec and ptsd groups during resting - state , we extracted individual subject s z - score values from activated voxels that fell within an anatomically based mask for each a priori region from the between - group contrast ( ptsd > cec ; tzourio - mazoyer et al .
, 2002 ; walter et al . , 2003 ) . of note , we did not conduct statistical tests on these measures , as they were defined from significant activations resulting from whole - brain maps of group differences in connectivity .
across the entire brain , we observed a discretely localized difference in amygdala connectivity pattern between groups . from the right amygdala anatomical seed region , we observed that ptsd patients exhibited stronger connectivity with the insula than cec subjects ( mni peak : [ 38 , 18 , 2 ] , z - score = 4.29 , volume = 440 voxels ; figure 1 ) ; this pattern was not detected from the left amygdala seed .
follow - up roi analyses on the extracted z - scores of the strength of connectivity from the insula revealed that both groups exhibited positive amygdala insula coupling , however , the extent of connectivity between the amygdala and insula was greater in the ptsd group than the cec group ( figure 1 ) . to explore the clinical relevance of the observed amygdala
insula connectivity abnormalities , we performed correlational analyses between the extracted values of the strength of connectivity and ptsd symptom severity measures ( caps , bdi - ii , pcl - m , and ham - d ) but did not observe any significant correlations ( all ps > 0.05 , corrected for multiple comparisons ) . of note
, we did not observe group differences in any other a priori areas that we predicted , such as the acc , mpfc , and hippocampus in relation to amygdala connectivity at rest . between - group
whole - brain voxel - wise statistical t map overlaid on a canonical brain rendering ( left , mni coronal , y - plane = 14 ; right , mni sagittal , x - plane = 38 [ right ] ) showing stronger amygdala connectivity to the insula during rest in the ptsd group ( ptsd < cec ) . connectivity target is displayed at whole - brain voxel - wise p <
0.005 , uncorrected ; color bar represents statistical t - scores ; bar graph shows mean extracted z - scores ( sem ) within each group from activated voxels that fell within an anatomically based insula mask , showing stronger connectivity in ptsd patients ( > cec ) .
ptsd , post - traumatic stress disorder ; cec , combat - exposed control ; l , left .
this is the first study to our knowledge that examines intrinsic amygdala functional connectivity patterns during rest in returning oef / oif veterans with combat - related ptsd compared to a group of oef / oif veterans with combat exposure , but without ptsd .
we found stronger amygdala insula resting - state functional connectivity in the ptsd group compared to the cec group . of note ,
this connectivity pattern was from the right amygdala seed and was not shown from the left amygdala . although we did not make an a priori prediction about lateralization of amygdala resting - state connectivity several findings of amygdala hyperactivity in ptsd and correlations between ptsd symptom severity and amygdala activity have been right - sided ( rauch et al . , 1996 , 2000 ;
pissiota et al . , 2002 ; fredrikson and furmark , 2003 ; driessen et al . , 2004 ; shin et al . , 2004a ) .
contrary to our original hypothesis we did not observe any significant differences in amygdala connectivity to any other a priori regions ( mpfc , acc , hippocampus ) in the ptsd group compared to the cec group at rest .
this is a notable negative finding and requires replication ; however , we acknowledge that the absence of differences in amygdala frontal or amygdala hippocampal connectivity between groups could have resulted from : ( 1 ) our stringent , whole - brain correction for multiple comparisons to detect significance coupled with a small sample size may have led to false negatives and/or more subtle connectivity abnormalities ; and/or ( 2 ) the resting - state task may be insensitive to detecting amygdala prefrontal and amygdala hippocampal connectivity abnormalities , which may require engagement by an overt task . both the amygdala and insula
have been separately implicated in the pathophysiology of anxiety and ptsd ( rauch et al . , 2000 ;
pissiota et al . , 2002 ; shin et al . , 2004a ; protopopescu et al . , 2005 ; nemeroff et al .
, 2006 ; hopper et al . , 2007 ; carrion et al . , 2008 ; shin and liberzon , 2010 ) .
the amygdala plays an important role in the subjective and attentional - vigilance aspects of threat processing , and thus abnormalities in amygdala activity may be associated with hyperarousal and hypervigilance to threat in ptsd ( etkin , 2009 ) .
moreover , several studies have shown that the amygdala is hyperresponsive to both trauma - related ( rauch et al . , 1996 ;
shin et al . , 1997 , 2004a ; liberzon et al . , 1999 ;
, 2002 ; hendler et al . , 2003 ; driessen et al . , 2004 ;
, 2005 ; vermetten et al . , 2007 ) and unrelated stimuli in ptsd ( rauch et al . , 2000 ; armony et al . , 2005 ;
; williams et al . , 2006 ) , amygdala activation is positively correlated with ptsd symptom severity ( rauch et al . , 1996 ;
shin et al . , 2004a ; armony et al . , 2005 ; protopopescu et al . ,
fredrikson and furmark , 2003 ) , and symptom reduction after treatment is associated with decreased amygdala activation ( felmingham et al . , 2007 ) .
likewise , ptsd patients display exaggerated insula activation during script - driven imagery ( lanius et al .
2008 ) , fear conditioning and extinction ( bremner et al . , 2005 ) , the anticipation of negative images ( simmons et al . , 2006 ) , the retrieval of emotional or neutral stimuli ( bremner et al . , 2003 ; werner et al .
, 2009 ; whalley et al . , 2009 ) , and aversive smells and painful stimuli ( vermetten et al . , 2007 ) and is also positively correlated with ptsd symptom severity ( osuch et al . , 2001 ; hopper et al . , 2007 ;
the insula controls evaluative , experiential , and expressive aspects of internal emotional states via visceral and somatic changes ( e.g. , autonomic flight - or - fight responses ) evoked during presentations of aversive stimuli ( phan et al . , 2002 ; anderson et al . , 2003 ;
critchley et al . , 2004 ; simmons et al . , 2004 ; paulus and stein , 2006 ) and it has been posited that the insula relays interoceptive information to the amygdala to help guide behavioral responses ( augustine , 1996 ; craig , 2002 ; simmons et al . , 2004 ; paulus and stein , 2006 ) .
in fact , the insula provides some of the strongest cortical connections to the major output division of the amygdala responsible for generating fear responses to symptom - provoking stimuli ( augustine , 1996 ; aggleton and saunders , 2000 ; freese and amaral , 2009 ) and abnormalities in these structures has been suggested to underlie exaggerated fear responses and the persistence of traumatic memories ( shin and liberzon , 2010 ) , as well as anxiety proneness ( paulus and stein , 2006 ; simmons et al .
furthermore , evidence from a recent study suggests that a functional network between the amygdala and insula mediates anxious anticipation of negative events and anxious individuals display exaggerated activity within this network during anticipation of aversive stimuli ( carlson et al . , 2010 ) .
individuals with ptsd display excess anticipation of negative events and because of this are preoccupied with studying their environment for possible threats ( i.e. , hypervigilance ) and increased amygdala
insula functional coupling may be a mechanism supporting hypervigilance in patients with ptsd . besides the present study , others have investigated baseline connectivity patterns in patients with ptsd and observed abnormalities in functional connectivity within the default - mode network when compared to healthy controls ( although sometimes inconsistent ; bluhm et al . , 2009 ; daniels et al .
our study extends these findings to resting - state amygdala coupling within a corticolimbic network known to be dysfunctional during trauma - related anxiety provocation , emotionally based tasks , and evocative stimuli in ptsd patients . however , our study has some important limitations .
first , our study only included males and therefore can not be generalized to females .
second , the resting - state analysis of changes in amygdala insula connectivity do not allow for inferences about directionality or causality , which await task - based path or dynamic causal analyses .
in addition , we have interpreted our resting - state findings based on previous functional and structural imaging studies , however , research with converging methods ( i.e. , task - dependent and -independent fmri , diffusion tensor imaging ) are much needed to link connectivity at rest with brain structure and function .
lastly , the cross - sectional nature of our measurement does not allow us to ascertain whether enhanced amygdala
insula resting - state connectivity was present before the traumatic experience and if so , makes it a potential vulnerability maker for ptsd .
despite these limitations , our findings demonstrate that alterations in these connectivity patterns in a network involved in emotional processing and regulation may be relevant to a brain model of ptsd that involves baseline abnormalities in amygdala insula functional connectivity that exist even without task induction .
these findings suggest that the aberrant amygdala and insula activation to fear - evocative probes previously characterized in ptsd may be driven by an underlying enhanced connectivity between amygdala , a region known for perceiving threat and generating fear responses , and the insula , a region known for processing the meaning and prediction of aversive bodily states .
insula connectivity may reflect an exaggerated , pervasive state of arousal that exists outside the presence of an overt , actual threat / danger .
extends our understanding of the pathophysiology of ptsd , and the current findings prompt further investigation in this emerging area of neuroimaging research .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | post - traumatic stress disorder ( ptsd ) is often characterized by aberrant amygdala activation and functional abnormalities in corticolimbic circuitry , as elucidated by functional neuroimaging .
these activation studies have primarily relied on tasks designed to induce region - specific , and task - dependent brain responses in limbic ( e.g. , amygdala ) and paralimbic brain areas through the use of aversive evocative probes .
it remains unknown if these corticolimbic circuit abnormalities exist at baseline or at rest , in the absence of fear / anxiety - related provocation and outside the context of task demands .
therefore the primary aim of the present experiment was to investigate aberrant amygdala functional connectivity patterns in combat - related ptsd patients during resting - state .
seventeen operation enduring freedom / operation iraqi freedom ( oef / oif ) veterans with combat - related ptsd ( ptsd group ) and 17 combat - exposed oef / oif veterans without ptsd [ combat - exposed control ( cec ) group ] underwent an 8-min resting - state functional magnetic resonance imaging scan . using an anatomically derived amygdala
seed region we observed stronger functional coupling between the amygdala and insula in the ptsd group compared to the cec group , but did not find group differences in amygdala prefrontal connectivity .
these findings suggest that the aberrant amygdala and insula activation to fear - evocative probes previously characterized in ptsd may be driven by an underlying enhanced connectivity between the amygdala , a region known for perceiving threat and generating fear responses , and the insula , a region known for processing the meaning and prediction of aversive bodily states .
this enhanced amygdala
insula connectivity may reflect an exaggerated , pervasive state of arousal that exists outside the presence of an overt actual threat / danger .
studying amygdala functional connectivity at rest
extends our understanding of the pathophysiology of ptsd . | Introduction
Materials and Methods
Participants
Functional imaging acquisition
Functional imaging analysis
Results
Discussion
Conflict of Interest Statement | post - traumatic stress disorder ( ptsd ) is characterized by various altered emotional responses as a result of trauma exposure ( e.g. recently these functional connectivity techniques have been applied to the study of corticolimbic circuitry abnormalities at baseline or at rest ( resting - state functional connectivity ) . therefore the primary aim of the present experiment was to investigate aberrant amygdala functional connectivity patterns in returning operation enduring freedom / operation iraqi freedom ( oef / oif ) veterans with combat - related ptsd ( ptsd group ) and combat - exposed oef / oif veterans without ptsd [ combat - exposed control ( cec ) group ] during resting - state . if observed , such findings would extend our understanding of the pathophysiology of ptsd by identifying a disturbed network that exists outside of the presence of an overt threat / danger or in the absence of stimulus or task - induced negative emotional processing . based on the dsm - iv ( apa , 1994 ) ,
17 participants met criteria for current ptsd ( ptsd group ; age : 30.12 7.70 years ; caucasian = 16 ; hispanic or latino = 1 ) and the other 17 participants were combat - exposed matched controls without ptsd ( cec group ; age : 33.71 9.12 ; caucasian = 16 ; asian = 1 ) . to clarify the signal direction , variance , and specificity of differences in strength of connectivity between the cec and ptsd groups during resting - state , we extracted individual subject s z - score values from activated voxels that fell within an anatomically based mask for each a priori region from the between - group contrast ( ptsd > cec ; tzourio - mazoyer et al . based on the dsm - iv ( apa , 1994 ) ,
17 participants met criteria for current ptsd ( ptsd group ; age : 30.12 7.70 years ; caucasian = 16 ; hispanic or latino = 1 ) and the other 17 participants were combat - exposed matched controls without ptsd ( cec group ; age : 33.71 9.12 ; caucasian = 16 ; asian = 1 ) . to clarify the signal direction , variance , and specificity of differences in strength of connectivity between the cec and ptsd groups during resting - state , we extracted individual subject s z - score values from activated voxels that fell within an anatomically based mask for each a priori region from the between - group contrast ( ptsd > cec ; tzourio - mazoyer et al . follow - up roi analyses on the extracted z - scores of the strength of connectivity from the insula revealed that both groups exhibited positive amygdala insula coupling , however , the extent of connectivity between the amygdala and insula was greater in the ptsd group than the cec group ( figure 1 ) . this is the first study to our knowledge that examines intrinsic amygdala functional connectivity patterns during rest in returning oef / oif veterans with combat - related ptsd compared to a group of oef / oif veterans with combat exposure , but without ptsd . we found stronger amygdala insula resting - state functional connectivity in the ptsd group compared to the cec group . contrary to our original hypothesis we did not observe any significant differences in amygdala connectivity to any other a priori regions ( mpfc , acc , hippocampus ) in the ptsd group compared to the cec group at rest . this is a notable negative finding and requires replication ; however , we acknowledge that the absence of differences in amygdala frontal or amygdala hippocampal connectivity between groups could have resulted from : ( 1 ) our stringent , whole - brain correction for multiple comparisons to detect significance coupled with a small sample size may have led to false negatives and/or more subtle connectivity abnormalities ; and/or ( 2 ) the resting - state task may be insensitive to detecting amygdala prefrontal and amygdala hippocampal connectivity abnormalities , which may require engagement by an overt task . in fact , the insula provides some of the strongest cortical connections to the major output division of the amygdala responsible for generating fear responses to symptom - provoking stimuli ( augustine , 1996 ; aggleton and saunders , 2000 ; freese and amaral , 2009 ) and abnormalities in these structures has been suggested to underlie exaggerated fear responses and the persistence of traumatic memories ( shin and liberzon , 2010 ) , as well as anxiety proneness ( paulus and stein , 2006 ; simmons et al . these findings suggest that the aberrant amygdala and insula activation to fear - evocative probes previously characterized in ptsd may be driven by an underlying enhanced connectivity between amygdala , a region known for perceiving threat and generating fear responses , and the insula , a region known for processing the meaning and prediction of aversive bodily states . insula connectivity may reflect an exaggerated , pervasive state of arousal that exists outside the presence of an overt , actual threat / danger . | [
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] |
the e2f transcription factor is best known for its ability to regulate cell cycle progression by coordinating a large group of genes involved in g1-to - s phase transition . e2f
regulated genes include cell cycle regulators , such as cyclin eand cdc25a , and genes that encode essential components of the dna replication machinery . in agreement with the idea that e2f is an important regulator of cell proliferation , studies in several experimental systems
have shown that the ectopic expression of e2f is sufficient to drive quiescent cells into s phase , while the inhibition of e2f - dependent transcription blocks cell proliferation [ 24 ] .
e2f cell cycle activity is controlled through the temporally regulated physical association of pocket proteins .
the hypophosphorylated retinoblastoma protein prb represses e2f activity by shielding the e2f transactivation domain leading to restriction of cell cycle progression . during the course of cell cycle progression through the restriction point ,
prb becomes phosphorylated by the activity of cyclindependent kinases ( cdks ) resulting in the release of e2f , thereby causing its activation ( fig .
it is currently believed that most , and perhaps all , human cancers contain mutations that comprise prb function and that this contributes to their ability to proliferate in an environment that would cause normal cells to arrest . during transition of cells from g1 to s phase
, the activity of e2f is controlled by prb , which binds e2f / dp , leading to transcriptional repression of e2f - responsive promoters in go / g1 . in late g1 prb
is phosphorylated by cyclin - dependent kinases ( cdk ) , which results in the release of e2f .
free e2f transactivates growth promoting genes such as cyclin e or cdc25a , resulting in s phase entry and cell cycle progression .
e2f is a family composed of at least eight members that can be divided into distinct subgroups on the basis of their structural and functional similarities ( reviewed in ref . ) .
e2f1 , e2f2 and e2f3a are transcriptional activators , whereas e2f3b , e2f4 , e2f5 , e2f6 and e2f7 act as repressors of transcription .
the most recently identified e2f family member , e2f8 , resembles the organization of e2f7 in the presence of two separate dna - binding domains . like e2f7
all e2f proteins except e2f7 and e2f8 function in heterodimeric complexes with dp family ( dp1-dp4 ) proteins . from mutant mouse models it became clear that different e2f and dp subunits perform distinct , perhaps overlapping functions in the control of cell cycle progression , as well as unique roles during development , tissue homeostasis and apoptosis . for example , e2f3 is important for the control of cellular proliferation , while e2f4 and e2f5 are essential for g1 control and cell cycle exit as well as for normal mouse development .
previous studies suggest a role for e2f7 in facilitating cell cycle arrest in both g1 and g2 phases .
in contrast , e2f4 can associate with all pocket proteins , while e2f5 solely binds p130 .
e2f6 as well as the newest family members e2f7 and e2f8 lack a transactivation domain and do not interact with any pocket protein .
recent genomic studies have found that the function of the e2f transcriptional program extends further than the g1/s transition .
chromatin immunoprecipitation experiments [ 1013 ] , expression profiling studies [ 1417 ] and a recently performed proteomic analysis showed that e2f proteins bind to and modulate expression of a large fraction of genes with diverse functions , suggesting a widespread role for e2f in the human genome .
currently , the best - characterized activity , especially at the hands of e2f1 , in addition to its traditional role in cell cycle progression is its ability to induce apoptosis [ 1922 ] . in some setting , another member of the e2f family , e2f3 , also induces apoptosis .
however , this e2f3-induced apoptosis was found to be e2f1-dependent and is largely attributed to the ability of e2f3 to transactivate the e2f1 gene , indicating that accumulation of crucial levels of e2f1 activity , and not total e2f activity , is essential for the induction of apoptosis .
a large number of studies clearly support the role of e2f1 as a tumour suppressor rather than an onco - gene [ 2426 ] . under deregulated conditions the activity of e2f1
is linked to events that determine cell fate through the induction of apoptosis , thus protecting the organism against oncogenic transformation .
mice , for example , lacking e2f1 ( e2f1 ) exhibit defects in apoptosis together with an increased incidence of tumour development [ 24 , 25 ] , and the level of apoptosis seen in rbmice is suppressed by e2f1 deficiency .
different mechanisms have been attributed to the ability of e2f1 to cause apoptosis , which can occur through both p53-dependent and independent path - ways ( fig .
, p53 accumulates following e2f1 expression through activation of the cdkn2a transcript p14arf , which in turn interacts with mdm2 , thereby preventing mdm2 from targeting p53 for ubiquitination and sub - sequent degradation . lately
, additional arf - independent pathways have been described , and some reports even imply a negative feedback by arf since over - expression of arf inhibits e2f1-dependent apoptosis , and targets e2f1 for proteasomal degradation .
e2f1-induced apoptosis in the absence of arf was shown to correlate with p53 phosphorylation at residues that are also phosphory - lated in response to dna damage [ 29 , 31 ] .
moreover , induction of both apoptosis and p53 phosphorylation by e2f1 are abolished by the atm and atr protein kinase inhibitor caffeine , supporting that e2f1 uses the atm signalling pathway to induce p53 and chk2 phosphorylation and thereby apoptosis [ 32 , 33 ] .
correspondingly , it was reported that the e2f1 transcription factor elevates atm promoter activity by direct binding , which is accompanied by enhanced p53 phosphorylation .
in addition , e2f1 interacts directly with p53 via the cyclin a binding domain of e2f1 , and this interaction enhances p53 apoptotic activity in response to dna damage
e2f1 promotes apoptosis via the tumour suppressor p53 and independent of p53 . in the p53-dependent pathway ,
e2f1 activates arf , which in turn stabilizes p53 by alleviating its proteosome degradation through mdm2 .
arf negatively regulates e2f1 in a feedback loop mechanism . in response to dna damage e2f1
is stabilized through phosphorylation by atm / atr and chk2 kinases ( chk2 also stabilizes p53 by phosphorylation ) .
in addition , e2f1 interacts directly with p53 via the cyclin a binding domain , thereby inducing p53-mediated apoptosis . alternatively ,
p53-independent apoptosis by e2f1 occurs via direct up - regulation of genes including p73 and apaf-1 .
the assembly of apaf1 with cytochrome c released from the mitochondria leads to formation of the apoptosome that catalyzes caspase-9 , and successive initiation of proapoptotic effector caspases including caspase-3 .
e2f1-induced apoptosis occurs also independent of p53 in tissue culture and transgenic mice [ 3638 ] , and prb has been shown to protect p53-null cells from apoptosis in an e2f1-binding dependent manner .
apoptotic targets of e2f1 in the absence of p53 include the p53 homolog protein p73 [ 40 , 41 ] and apoptosis protease - activating factor 1 ( apaf1 ) both of which are transcriptionally regulated by e2f1 .
this initiates the assembly of apaf1 with cytochrome c followed by procaspase-9 activation and successive initiation of proapoptotic effector caspases including caspase-3 , -6 and -7 .
mapping studies revealed that the apoptotic ability of e2f1 requires the dna - binding domain but not its transactivation function , since mutants of e2f1 that lack the transactivation domain are still able to induce cell death [ 36 , 37 , 43 ] .
from these experiments , it was supposed that proapoptotic e2f1 target genes are activated by removal of e2f1/rb repression rather than direct transactivation . moreover , bell et al . recently reported dna - binding independent cell death from a minimal proapoptotic region of e2f1 that is consequently unable to transactivate , repress or derepress e2f target genes .
however , since this activity is also present in e2f2 and e2f3 proteins , it might therefore define a distinct mechanism of death by e2f proteins . because normal cells proliferate without suffering e2f1-induced apoptosis , its proapoptotic potential
integration of external signals appears to play an important role in determining the sensitivity to e2f1-induced apoptosis .
for example , cell survival signals via the pi3/akt and the egfr / ras / raf pathway have been shown to promote e2f1-driven cell proliferation by suppressing e2f1-induced apoptosis [ 4547 ] .
in addition , dna damage signals have been suggested to specifically activate e2f1-dependent transcription of proapoptotic genes [ 4850 ] .
these changes stabilize e2f1 , increase its transactivation potential and allow it to preferentially bind the promoters of some proapoptotic genes .
overall , the final decision of whether e2f1 leads to cell proliferation or apoptosis might thus depend on the genetic status or molecular back - ground of the cell .
this means that e2f1 would function strictly as a growth promoter if pathways that mediate e2f1-induced apoptosis are disabled .
many of the drugs cause dna damage and the inflicted dna damage activates apoptotic programs that lead to cell death . forced expression of e2f1 has been shown to sensitize tumour cells to the proapoptotic signals generated by chemotherapeutic drugs or ionizing radiation [ 5156 ] .
in addition , endogenous e2f1 is up - regulated after dna damage in a manner analogous to that of p53 , suggesting its direct role in responsiveness to conventional genotoxic stress signals . in response to dna damage , the e2f1 protein
is stabilized through distinct mechanisms , including direct phosphorylation by the ataxia - telangiectasia mutated ( atm ) kinase , the atm and rad3-related ( atr ) kinase and the chk2 kinase [ 32 , 48 , 58 ] , and also by acetylation through p300/creb - binding protein - associated factor ( p / caf ) .
activation of the atm dna damage response pathway is commonly observed in a variety of early - stage tumours , suggesting that this checkpoint response functions to suppress the development of cancer .
has demonstrated that e2f1 is a critical determinant of the cellular response in cancer cells to genotoxic stress .
taking advantage of drosophila ddp and de2f1;de2f2 mutant animals , it was shown that the role of de2f1 within individual developing wing discs exposed to -irradiation is completely context - dependent .
de2f1/ddp appears to protect non - proliferating cells and sensitizes proliferating cells to -irradiation - induced apoptosis .
the loss of the prb - dependent cell cycle checkpoint might allow cancer cells to enter s phase and initiate apoptosis under conditions where normal cells would undergo a g1 arrest and initiate dna repair .
in addition , e2f can increase the effectiveness of chemotherapeutic agents that are most active in s phase cells and/or that require the presence of a particular cell cycle dependent target for the induction of cell death [ 60 , 61 ] .
the frequent deregulation of e2f1 in human tumours , taken together with its apoptotic potential and its stabilization after damage suggest that e2f1 may play an important role in the enhanced sensitivity of tumour cells to dna damage - induced cell death . in agreement with this , increased levels of proapoptotic target genes of e2f1 such as adenoviral e1a and p73 have been invoked as a potential basis for selective killing of cancer cells , including p53-defective tumour cells , by dna - damaging agents relative to normal cells [ 6264 ] .
in the past few years , a large number of novel e2f1-regulated target genes or gene networks functioning in the apoptotic program have been identified . from these studies
it becomes evidently clear that e2f1 is a key regulator of the apoptotic machinery by being involved in many aspects of programmed cell death , both by activating proapoptotic genes and through the inhibition of antiapoptotic survival signals .
e2f1 induces the expression of the proapoptotic bcl-2 homology 3 ( bh3)-only proteins puma , noxa , bim , hrk / dp5 and bik in cancer cells through a direct transcriptional mechanism [ 65 , 66 ] .
in fact , e2f1 binds to the promoters of these genes and transactivates them by a p53-independent mechanism .
bh3-only proteins are members of the bcl-2 protein family and are required for the execution of apoptotic cell death by integrating diverse apoptotic stimuli into a common death pathway governed by other multidomain bcl-2 family members . while some bh3-only proteins , such as bid , may chaperone the activation of bax and bak at the mitochondrial membrane , most others antagonize the functions of the anti - apoptotic bcl-2 family members .
reversely , reducing the expression of noxa and puma by rna interference diminished apoptosis induced by e2f1 . like e2f1 itself ,
some of these proteins , for example , bik and puma are also up - regulated in response to chemotherapeutic agents [ 65 , 66 ] .
consistent with their responsiveness to e2f1 , it has been shown that the dna damage - induced elevation of puma and bik is abolished by suppression of e2f1 activity , indicating that both pathways contribute to the apoptotic response to damaging agents .
increased expression of proapoptotic bcl-2 genes including bad , bak and bid after e2f1 activation in p53-deficient tumour cells was also reported in a gene array analysis by stanelle et al .. in the same study , kiaa0767 , termed d(eath)-i(nducing)-p(rotein ) , was identified as a relevant apoptotic target of e2f1 .
the dip protein is localized in the mitochondria and mediates e2f1-induced apoptosis independently of p53 in a partially caspase - independent manner . in addition
, transcriptional activation of the second mitochondrial activator of caspases or direct inhibitor - of - apoptosis - binding protein with low pi ( smac / diablo ) was reported as a mechanism by which e2f1 promotes p53-independent apoptosis via the mitochondria .
these proteases are synthesized as inactive proenzymes and processed to an active state during apoptotic cell death .
initiator caspases ( caspase-2 , -8 and -9 ) trigger a cascade that result in the activation of effector caspases ( caspase-3 and -7 ) , which in turn produce the characteristic morpho - logical changes associated with apoptosis . enforced e2f1 expression has been shown to result in the accumulation of caspase-2 , -3 , -7 , -8 and -9 through a direct transcriptional mechanism .
e2f1 can facilitate caspase activation through p53-dependent signals , resulting in mitochondrial release of cytochrome c , while simultaneously increasing caspase expression through direct caspase promoter binding that is independent of p53 . by up - regulation of caspases ,
particularly caspase-8 , e2f1 might sensitize cells to death - inducing ligands such as tumour necrosis factor ( tnf ) .
another novel death effector protein of e2f1 is siva , which was identified to be directly up - regulated by e2f1 and p53 via their corresponding consensus sites in the siva promoter .
siva is a proapoptotic protein containing a death domain that is expressed on a broad scope of tissues , including cells of the immune system and the cns as well as tumour cells . as a potential mechanism for apoptosis induction by siva
, its interaction with members of the tnf - receptor family and antiapoptotic bcl-2 family members has been considered .
these studies suggest that siva may function through multiple mechanisms possibly dependent on the cell type and apoptotic stimulus .
in addition to the findings by fortin et al . indicating that the siva gene exhibits a striking induction during p53-mediated neuronal apoptosis , up - regulation of siva was also observed in p53-deficient hepatoma cells in response to cisplatinum - induced apoptosis , suggesting that it is also regulated in a p53-independent manner , perhaps by e2f1 .
some clarity has been shed on the requirement of the ask1-p38 mapk pathway for e2f1-induced apoptosis .
the ginsberg group showed that e2f1 modulates the activity of the p38 mapk path - way through up - regulation of p38 mapk phosphorylation .
this involves transcriptional induction of the ask1 ( apoptosis signal - regulating kinase 1 ) gene ( also known as map3k5 ) , a member of the mitogen - activated protein kinase family that phosphorylates p38 mkks .
dna microarray analysis revealed map3k5 up - regulation following e2f1 over - expression in p53-positive u-2os osteosarcoma and in p53-deficient melanoma cells .
for example , map3k5 has been shown to mediate rapamycin - induced apoptosis via phosphorylation of c - jun in p53-negative mouse embryonic fibroblasts , whereas suppression of kinase activity and ask1-jnk signalling through the interaction of ask1 with p21cip1 in p53 wild - type cells leads to apoptosis inhibition .
the relevance of the ask1-p38 connection for e2f1-induced apoptosis is also evidently clear from data demonstrating that e2f1 regulates the activity of the p38 signalling inhibitor wip1 .
further insight into the mechanism by which e2f1 directs p53 activity towards apoptosis comes from studies showing that e2f1 up - regulates the expression of proapoptotic cofactors of p53 , jmy , tp53inp1 and the apoptosis - stimulating proteins of p53 ( aspp ) family members aspp1 and aspp2 by a direct transcriptional mechanism [ 80 , 81 ] . tp53inp1 was shown to mediate phosphorylation of p53 on serine 46 .
both aspp proteins modulate the cellular apoptotic threshold by direct interaction with p53 , thereby enhancing the dna binding and trans - activation function of p53 on the promoters of proapoptotic genes in vivo .
herein , aspp specifically renders p53-guided apoptosis by stimulation of the p53-regulated promoters bax and pig-3 .
while these studies provide additional pathways by which e2f1 cooperates with p53 to induce apoptosis , the aspp promoters can also be transactivated by e2f1 in a p53-deficient context as shown by chip and reporter gene assays [ 80 , 83 ] .
since aspp1 and aspp2 are specific activators of the apoptotic function of all p53 family members , e2f1 may use this pathway to increase the apoptotic function of p63 and p73 independently of p53 .
this ambivalence seems to be a common concept for death proteins since it has also been observed for other apoptosis factors such as siva and map3k5 .
siva seems to link the e2f / arf / p53- and the p53-independent apoptotic pathway . in a functional so - called technical knockout approach using the saos-2 er - e2f1 cell line , where e2f1 activity can be conditionally induced on 4-hydroxytamoxifen treatment , several other potential death targets of e2f1 were identified such as galectin-1 ( or lgals1 ) .
e2f1-induced apoptosis was significantly abolished when galectin-1 anti - sense cdna was introduced into p53-negative cells .
galectin-1 has been shown to play a key role in tumour - immune escape by killing antitumour effector t cells .
it sensitizes , for example , human - resting t cells to fas ( cd95)/caspase-8mediated cell death .
e2f1 was earlier shown to be essential for t - cell apoptosis by a process called t - cell receptor ( tcr ) activation - induced cell death ( aicd ) .
lissy et al . demonstrated that t cells were protected from tcr - mediated apoptosis by disruption of the e2f1 gene or p73 .
together these findings support a function for e2f1 in balancing life and death of immune effector cells .
beside direct activation of various apoptosis - related genes described earlier , a second major mechanism by which e2f1 sensitizes cells to apoptosis is via inhibition of antiapoptotic signalling .
one example is the inhibition of nf-b that functions in most cases as a survival signal .
activation of tnfr in response to tnf results in the stimulation of nf-b via traf2 , which contributes to the inhibition of cell death .
importantly , e2f1 expression sensitizes cells to apoptosis by down - regulation of traf2 protein levels , thereby inhibiting antiapoptotic nf-b signalling .
the general principle of blocking survival factors by e2f1 to induce apoptosis is supported by the data from two other studies [ 89 , 90 ] .
they reported that over - expression of e2f1 suppresses the expression of the apoptotic antagonists bcl-2 and its family member mcl-1 which leads to apoptosis . in both cases ,
the e2f1-induced decrease in bcl-2 and mcl-1 levels occurs independently of the arf / p53 pathway .
this scenario renders cells susceptible to apoptosis by altering the balance between anti- and proapoptotic bcl-2 family members in favour of those that induce cell death . together with the induction of bh3-only proteins and other proapoptotic bcl-2 family members by e2f1
there is increasing evidence that execution of the apoptotic program is achieved by an intense cross - talk between the mitochondria and the endoplasmic reticulum ( er ) ( reviewed in ref . ) .
a number of molecules involved in mitochondrial apoptosis are also involved in er stress - induced cell death , suggesting that the er might regulate apoptosis by sensitizing the mitochondria by a number of extrinsic and intrinsic stimuli , as well as by inducing apoptosis .
bcl-2 family members , for example , localize to the er membrane , thereby disrupting er homeostasis by altering er membrane permeability as a result of mitochondrial dysfunction .
unfolded protein response mediated cell survival or cell death is regulated by the balance between the er chaperones grp78 and gadd153 .
e2f1 can down - regulate the expression of grp78 ( also known as bip ) , which normally protects cells from death induced by disturbance of er homeostasis [ 94 , 95 ] .
moreover , expression of another er chaperon , the 94 kd glucose - regulated protein ( grp94 ) , shown to be significantly up - regulated in human esophageal cancers , is also inhibited by e2f1 . from these data
it is evident that e2f1-mediated apoptosis involves both mitochondrial and er - related death pathways .
e2f1 sensitizes cells to apoptosis by both direct activation of proapoptotic genes and through inhibition of antiapoptotic survival genes , thereby engaging different death signalling pathways via the mito - chondria ( intrinsic ) , death receptors ( extrinsic ) and the endoplasmic reticulum .
since apoptosis programs can be manipulated to produce massive changes in cell death , the genes and proteins controlling apoptosis are potential drug targets . as indicated earlier , acquired apoptotic defects during cancer progression involve three types of molecular changes : ( i ) inactivation of proapoptotic effectors , for example , p53 or apaf-1 ; ( ii ) activation of antiapoptotic factors , for example , bcl-2 and ( iii ) reinforcement of survival signals .
thus , anti - cancer studies set out to either boost cell death or to impede antiapoptotic and proliferative pathways .
two observations suggest that such strategies are feasible . first , most antiapoptotic mutations act relatively upstream in the program implying that tumour cells generally retain the machinery and latent potential for apoptosis .
second , tumour - specific alterations in apoptotic programs provide opportunities to target cell death in a selective manner .
restoration of the apoptotic pathway , for example , by re - introduction of p53 or activation of apaf-1 has been shown to increase the sensitivity of neoplastic cells to dna - damaging agents .
the inef - fectiveness of a p53-based gene therapeutic strategy in certain conditions is , however , problematic ( e.g. mutant p53 in tumour cells trans - dominantly impairs the function of wild - type p53 ) . here , genes ( such as proapoptotic e2f1 targets ) are particularly useful that compensate or bypass cell death defects regardless of the p53 status . in the past few years , pre - clinical experiments mainly using e2f1 itself as anti - cancer therapeutic have been initiated . the effect of e2f1 over - expression on tumour growth has been evaluated in several types of human cancer in vitro and in vivo including glioma , melanoma , esophageal cancer , breast- and ovarian carcinoma , head and neck squamous cell cancer , gastric cancer ; pancreatic carcinoma and nonsmall - cell lung cancer [ 54 , 100108 ] .
these studies clearly indicate that apoptosis induction by adenoviral expressed e2f1 results in growth suppression and increased responsiveness of tumour cells to chemotherapy with relative sparing of normal tissues
. this selectivity might be due to the fact that normal cells contain wild - type prb , which likely can bind to and antagonize the activity of exogenous e2f1 .
furthermore , transcription from e2f1-responsive promoters is higher in cancer cells than in normal cells .
however , targeting e2f1 itself is a cumbersome mission possibly feasible for some but not suitable for all therapeutic purposes because of its dual role in cell cycle progression and apoptosis . regarding this issue
, it has been shown that a transcriptionally inactive form of e2f1 is able to induce apoptosis without activating proliferation in human coronary vascular smooth muscle cells ( vsmc ) .
therefore , this strategy has widespread potential for preventing hyperproliferation in artherosclerosis , hypertension and restenosis after injury .
in addition to e2f1 , its downsteam target p73 was proven to be a valuable candidate for cancer therapy in tumour cells .
infection with an adenoviral vector encoding the beta isoform of p73 resulted in potent cytotoxicity based on a combination of cell cycle arrest and apoptosis induction [ 64 , 109 , 110 ] .
similar to e2f1 , p73 can efficiently induce apoptosis and enhanced chemosensitivity of tumour cells primarily resistant to wild - type p53 .
although this strategy warrants further consideration , the antitumoural effect exhibited by over - expression of p73 , in contrast to its homolog p53 , is not restricted to tumour cells .
although present data provide compelling evidence that effectors of e2f1-induced apoptosis can significantly kill cancer cells , their role as a target for cancer treatment is only beginning to emerge .
so far , in clinical studies antiapoptotic e2f1 regulated genes were used to hamper tumour growth instead of using approaches for introducing proapoptotic molecules .
antiapoptotic bcl-2 , for example , has been targeted extensively by antisense oligonucleotide approaches which interfere with bcl-2 activity , resulting in less bcl-2 expression and elevated levels of apoptosis . in the phase i ii study ,
bcl-2 antisense oligonucleotide ( g3139 ) treatment of chemoresistant melanoma patients led to minor to complete therapeutic response in 6 of 14 cases when the antisense therapy was combined with dacarbazine administration . in a phase ii study , bcl-2 antisense oligonucleotide treatment of patients with multiple myeloma led to therapeutic responses in 55% of cases when combined with chemotherapy .
these data underline that inhibition of antiapoptotic bcl-2 is surely a promising treatment for otherwise resistant tumour entities . | abstractdefects in apoptotic programs contribute to a number of human diseases , ranging from neurodegenerative disorders to malignancy , and treatment failure .
the genetic basis for apoptosis implies that cell death can be disrupted by mutations , raising the intriguing possibility that cell numbers can be regulated by factors that influence cell survival .
it is well documented that the e2f1 transcription factor is a key regulator of apoptotic programs .
e2f1-induced cell death occurs via multiple pathways , some of which involve the tumour suppressor p53 , and autonomous of p53 .
this has led to the opinion that e2f1 functions as a tumour surveillance factor , detecting aberrant proliferation and engaging apoptotic pathways to protect the organism from developing tumours .
frequently , novel players are discovered that expand the interpretation of apoptosis control by e2f1 .
this information will help to produce new strategies to exploit e2f1-induced apoptosis for therapeutic benefit . | Introduction
Molecular mechanisms of E2F1-induced apoptosis
E2F1 sensitizes tumour cells to DNA damage
E2F1 guided death pathways in cancer
Activation of proapoptotic genes
Inhibition of survival signalling pathways
E2F1 apoptosis and new therapeutic strategies | however , this e2f3-induced apoptosis was found to be e2f1-dependent and is largely attributed to the ability of e2f3 to transactivate the e2f1 gene , indicating that accumulation of crucial levels of e2f1 activity , and not total e2f activity , is essential for the induction of apoptosis . a large number of studies clearly support the role of e2f1 as a tumour suppressor rather than an onco - gene [ 2426 ] . mice , for example , lacking e2f1 ( e2f1 ) exhibit defects in apoptosis together with an increased incidence of tumour development [ 24 , 25 ] , and the level of apoptosis seen in rbmice is suppressed by e2f1 deficiency . correspondingly , it was reported that the e2f1 transcription factor elevates atm promoter activity by direct binding , which is accompanied by enhanced p53 phosphorylation . in addition , e2f1 interacts directly with p53 via the cyclin a binding domain of e2f1 , and this interaction enhances p53 apoptotic activity in response to dna damage
e2f1 promotes apoptosis via the tumour suppressor p53 and independent of p53 . e2f1-induced apoptosis occurs also independent of p53 in tissue culture and transgenic mice [ 3638 ] , and prb has been shown to protect p53-null cells from apoptosis in an e2f1-binding dependent manner . apoptotic targets of e2f1 in the absence of p53 include the p53 homolog protein p73 [ 40 , 41 ] and apoptosis protease - activating factor 1 ( apaf1 ) both of which are transcriptionally regulated by e2f1 . mapping studies revealed that the apoptotic ability of e2f1 requires the dna - binding domain but not its transactivation function , since mutants of e2f1 that lack the transactivation domain are still able to induce cell death [ 36 , 37 , 43 ] . this means that e2f1 would function strictly as a growth promoter if pathways that mediate e2f1-induced apoptosis are disabled . from these studies
it becomes evidently clear that e2f1 is a key regulator of the apoptotic machinery by being involved in many aspects of programmed cell death , both by activating proapoptotic genes and through the inhibition of antiapoptotic survival signals . like e2f1 itself ,
some of these proteins , for example , bik and puma are also up - regulated in response to chemotherapeutic agents [ 65 , 66 ] . consistent with their responsiveness to e2f1 , it has been shown that the dna damage - induced elevation of puma and bik is abolished by suppression of e2f1 activity , indicating that both pathways contribute to the apoptotic response to damaging agents . the dip protein is localized in the mitochondria and mediates e2f1-induced apoptosis independently of p53 in a partially caspase - independent manner . another novel death effector protein of e2f1 is siva , which was identified to be directly up - regulated by e2f1 and p53 via their corresponding consensus sites in the siva promoter . indicating that the siva gene exhibits a striking induction during p53-mediated neuronal apoptosis , up - regulation of siva was also observed in p53-deficient hepatoma cells in response to cisplatinum - induced apoptosis , suggesting that it is also regulated in a p53-independent manner , perhaps by e2f1 . the relevance of the ask1-p38 connection for e2f1-induced apoptosis is also evidently clear from data demonstrating that e2f1 regulates the activity of the p38 signalling inhibitor wip1 . further insight into the mechanism by which e2f1 directs p53 activity towards apoptosis comes from studies showing that e2f1 up - regulates the expression of proapoptotic cofactors of p53 , jmy , tp53inp1 and the apoptosis - stimulating proteins of p53 ( aspp ) family members aspp1 and aspp2 by a direct transcriptional mechanism [ 80 , 81 ] . both aspp proteins modulate the cellular apoptotic threshold by direct interaction with p53 , thereby enhancing the dna binding and trans - activation function of p53 on the promoters of proapoptotic genes in vivo . a number of molecules involved in mitochondrial apoptosis are also involved in er stress - induced cell death , suggesting that the er might regulate apoptosis by sensitizing the mitochondria by a number of extrinsic and intrinsic stimuli , as well as by inducing apoptosis . unfolded protein response mediated cell survival or cell death is regulated by the balance between the er chaperones grp78 and gadd153 . since apoptosis programs can be manipulated to produce massive changes in cell death , the genes and proteins controlling apoptosis are potential drug targets . second , tumour - specific alterations in apoptotic programs provide opportunities to target cell death in a selective manner . | [
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antiretroviral therapy ( art ) has transformed the lives of millions of persons infected with hiv by gradually increasing their cd4 + t - cell counts over a decade .
however , cd4 + t - cell reconstitution with art is characterized by biphasic pattern of t - cell subsets characterized by an initial phase of expansion and redistribution of cd4 + memory t - cells , followed by the second phase where reconstitution of cd4 + naive t - cells occurs with a reduction of t - cell immune activation .
unfortunately , approximately 20% of patients may experience immune nonresponse even after suppressive art , and this is more commonly seen in late presenters ( patients with baseline cd4 + t - cell count < 350 cells / mm at the time of art initiation ) .
such population remains at a higher risk for the development of aids and non - aids events few years after having initiated art .
therefore , information on the dynamics of cd4 + t - cell reconstitution in the late presenters remains an important issue to address .
elevation of immune activation , increase of memory t - cells with late differentiation , has been reported . however , limited data exist on long - term outcome of patients initiating art late in the course of infection .
some groups have defined late presenter generally as patients initiating art below 350 cell / mm cd4 + t - cells .
data on late presenters and cd4 + t - cell reconstitution are limited owing to short - term study duration and limitation of the analyses to total cd4 t - cells but not their subsets .
it remains elusive whether baseline characteristics and t - cell subset profiles could predict long - term treatment responses . to this end , we analyzed data from our outpatient cohort with late presenter participants treated with art for over 8 years .
we determined the long - term t - cell subset trajectory after art initiation and attempted to identify prognostic factors of complete immune reconstitution .
a prospective study from october 2002 to september 2013 was conducted at the clinics of the department of infectious disease in peking union medical college hospital ( pumch ) , a large tertiary care hospital in beijing , china .
hiv diagnosis was determined by standard serum enzyme - linked immunosorbent assays and confirmed by western blot analyses .
patients were considered for enrollment in this study if they were treated with art for at least 8 years with regular follow - ups in the absence of treatment interruptions .
prospective blood samples were collected during the patients routine visits , and the patient characteristics were retrieved from the medical records .
all patients gave written informed consent , and the study was approved by the institutional review board of pumch .
patients received art continuously since the inclusion ; however , the drug combinations changed over the years due to new drug availability and tolerance .
control groups were established for comparison and characterized by 51 healthy volunteers recruited from the chinese blood donor corps .
patients were routinely followed up every 3 months for the first 6 months , and then every 6 months for at least 8 years . at each visit , a clinical assessment was recorded , and samples were collected for laboratory assessment , including t - cell subsets and plasma hiv rna quantification .
patients were classified into two groups : one group including patients with baseline cd4 + t - cell count > 200 cells / mm , and the other group with cd4 + t - cell count 200 cells / mm .
for immunofluorescent surface staining and flow cytometric analysis , three - color flow cytometry ( beckman - coulter , brea , ca , usa ) was performed using peripheral blood mononuclear cells .
immune subsets were defined using the following markers : naive cd4 + t - cells defined by cd4 + , cd45ra+ , and cd62l+ ; memory cd4 + t - cells defined by cd4 + , cd45ro+ , and cd45ra- ; functional cd4 + t - cells defined by cd28 + and cd4 + ; activated cd8 + cells defined by cd8 + , cd38 + , or hla - dr+ .
all monoclonal antibodies were purchased from beckman - coulter and immunotech ( brea , ca , usa ) . for viral load ( vl ) measurements ,
plasma was separated from whole blood by centrifugation within 4 h of collection and stored at 80c until tested .
the cobas ampliprep / cobas taqman real - time rt - pcr assay ( roche , ca , usa ) was performed according to the manufacturer 's instructions .
whitney u - test and were described as medians with interquartile ranges ( iqrs ) unless otherwise stated .
multivariate models by generalized estimating equations were utilized to analyze the following covariates : age at art initiation , gender , transmission route , development of clinical aids prior to art , time from diagnosis of hiv infection to treatment initiation , baseline vl , nadir pre - art cd4 + t - cell counts , duration of art , and delayed viral suppression .
clinical aids was defined as the presence of a clinical disease ( not cd4 + t - cell count ) meeting the 1993 centers for disease control aids case definition . since there is no consensus definition of normalized cd4/cd8 ratio , we used the criteria from our institution , which are at least two cd4/cd8 ratios over 0.95 .
we used a poisson regression model with a robust error variance to calculate relative risks of reaching complete immune reconstitution ( cd4 + t - cells 500 cells / mm ) . in the multivariate poisson regression model , we entered all factors except for age with a p < 0.20 in univariate analysis , and entered age as a continuous factor , since age might affect naive cd4 + t - cell percentage .
we used cox regression analysis to model the time from art initiation to the development of complete immune reconstitution , which was defined as the midpoint between the last cd4 + t - cell < 500 cells / mm and the first cd4 + t - cells 500 cells / mm
. we also used receiver operating characteristic ( roc ) curves to determine the diagnostic potency of different indices at baseline . sensitivity and specificity
were calculated to evaluate diagnostic performance for the complete immune reconstitution ( cd4 + t - cells 500 cells / mm ) at 8-year art .
a prospective study from october 2002 to september 2013 was conducted at the clinics of the department of infectious disease in peking union medical college hospital ( pumch ) , a large tertiary care hospital in beijing , china .
hiv diagnosis was determined by standard serum enzyme - linked immunosorbent assays and confirmed by western blot analyses .
patients were considered for enrollment in this study if they were treated with art for at least 8 years with regular follow - ups in the absence of treatment interruptions .
prospective blood samples were collected during the patients routine visits , and the patient characteristics were retrieved from the medical records .
all patients gave written informed consent , and the study was approved by the institutional review board of pumch .
patients received art continuously since the inclusion ; however , the drug combinations changed over the years due to new drug availability and tolerance .
control groups were established for comparison and characterized by 51 healthy volunteers recruited from the chinese blood donor corps .
patients were routinely followed up every 3 months for the first 6 months , and then every 6 months for at least 8 years . at each visit , a clinical assessment was recorded , and samples were collected for laboratory assessment , including t - cell subsets and plasma hiv rna quantification .
patients were classified into two groups : one group including patients with baseline cd4 + t - cell count > 200 cells / mm , and the other group with cd4 + t - cell count 200 cells / mm .
for immunofluorescent surface staining and flow cytometric analysis , three - color flow cytometry ( beckman - coulter , brea , ca , usa ) was performed using peripheral blood mononuclear cells .
immune subsets were defined using the following markers : naive cd4 + t - cells defined by cd4 + , cd45ra+ , and cd62l+ ; memory cd4 + t - cells defined by cd4 + , cd45ro+ , and cd45ra- ; functional cd4 + t - cells defined by cd28 + and cd4 + ; activated cd8 + cells defined by cd8 + , cd38 + , or hla - dr+ .
all monoclonal antibodies were purchased from beckman - coulter and immunotech ( brea , ca , usa ) . for viral load ( vl ) measurements ,
plasma was separated from whole blood by centrifugation within 4 h of collection and stored at 80c until tested .
the cobas ampliprep / cobas taqman real - time rt - pcr assay ( roche , ca , usa ) was performed according to the manufacturer 's instructions .
whitney u - test and were described as medians with interquartile ranges ( iqrs ) unless otherwise stated .
multivariate models by generalized estimating equations were utilized to analyze the following covariates : age at art initiation , gender , transmission route , development of clinical aids prior to art , time from diagnosis of hiv infection to treatment initiation , baseline vl , nadir pre - art cd4 + t - cell counts , duration of art , and delayed viral suppression .
clinical aids was defined as the presence of a clinical disease ( not cd4 + t - cell count ) meeting the 1993 centers for disease control aids case definition . since there is no consensus definition of normalized cd4/cd8 ratio , we used the criteria from our institution , which are at least two cd4/cd8 ratios over 0.95 .
we used a poisson regression model with a robust error variance to calculate relative risks of reaching complete immune reconstitution ( cd4 + t - cells 500 cells / mm ) . in the multivariate poisson regression model , we entered all factors except for age with a p < 0.20 in univariate analysis , and entered age as a continuous factor , since age might affect naive cd4 + t - cell percentage .
we used cox regression analysis to model the time from art initiation to the development of complete immune reconstitution , which was defined as the midpoint between the last cd4 + t - cell < 500 cells / mm and the first cd4 + t - cells 500 cells / mm .
we also used receiver operating characteristic ( roc ) curves to determine the diagnostic potency of different indices at baseline .
sensitivity and specificity were calculated to evaluate diagnostic performance for the complete immune reconstitution ( cd4 + t - cells 500 cells / mm ) at 8-year art .
these patients had been diagnosed with hiv for a median of 0.1 year ( iqr : 00.8 years ) before art initiation and had a median duration of art of 10.2 years ( iqr : 9.510.6 years ) . the majority of patients were infected via blood transfusion .
fourteen patients ( 45.2% ) had experienced aids - defining events , and eight patients ( 25.8% ) had a history of infection with hepatitis b or hepatitis c virus .
all patients were selected as late presenters based on cd4 + t - cell counts < 350 cells / mm .
baseline median cd4 + t - cell count was 70 ( iqr : 12223 ) cells / mm and median vl was 4.7 ( iqr : 4.35.3 ) lg copies / ml . of 31 patients , 30 had baseline memory and naive cell profiles available . in 22 patients with cd4 + t - cell count 200 cells / mm , naive cd4 + t - cell percentage was also lower ( 6.6% , iqr 4.112.3% ) than that in 9 patients with cd4 + t - cells over 200 cells / mm ( 27.5% , iqr 26.041.4% , p < 0.001 ) .
characteristics of the participants iqr : interquartile range ; hbsag : hepatitis b surface antigen ; hcv : hepatitis c virus . during 8 years of treatment
, 24 patients achieved virologic suppression within half a year of treatment initiation , whereas seven patients had virologic failure or rebound , and were switched to second - line regimens ( tenofovir + lamivudine + ritonavir - boosted lopinavir ) .
the 24 patients who demonstrated stable viral suppression were receiving zidovudine , stavudine , didanosine , and lamivudine - based regimens , which were first - line art at the time . in comparison with
our hiv - infected patients had lower cd4 + t - cell and naive cd4 + t - cell counts and proportions , as well as higher proportions and lower levels of memory cd4 + t - cells during 8-year art [ figures 1 and 2 , supplementary figure s1a and s1b ] . at year 8
, there were 13 patients ( 41.9% ) with cd4 + t - cells over 500 cells / mm .
the group with baseline cd4 + t - cell counts over 200 cells / mm had a higher rate of complete immune reconstitution than that with baseline cd4 + t - cell counts 200 cells / mm ( 77.8% vs. 27.3% , p = 0.017 ) .
most patients with baseline cd4 + t - cell counts 200 cells / mm did not exhibit complete immune reconstitution after 8 years of treatment [ supplementary figure s2 ] .
medians and interquartile ranges ( error bars ) of cd4 + t - cell percentage and count ( a and b ) , memory cd4 + t - cell count ( c ) and naive cd4 + t - cell count ( d ) during eight - year art .
( e and f ) demonstrate medians and interquartile ranges of cd8+cd38 + t - cell percentage and cd8+hla - dr+ t - cell percentage during 8-year art , respectively .
( g ) immune activation after 8-year art , grouped by cd4 + t - cell count at year 8 . the shaded bands in ( a - f ) reflect the reference ranges in hiv - negative population .
the slopes in ( a - f ) were calculated by a linear model regression .
the wilcoxon nonparametric test was used to compare slopes in different phases . * p < 0.05 , and ns denotes not significant .
total , memory , and naive cd4 + t - cell as well as cd8+cd38+/cd8 + percentage during 8-year follow - up in each patient .
patients were categorized by their baseline total cd4 + t - cell count ( < 50 , 50100 , 100200 , and 200350 cells / mm ) .
medians and interquartile ranges of memory cd4 + t cell percentage ( a ) and naive cd4 + t cell percentage ( b ) during 8 year art . the shaded band reflects the reference ranges in hiv negative population , * p < 0.05 .
medians and interquartile ranges ( error bars ) of cd4 + t cell in subgroups .
no clinical aids events , baseline cd4 + t - cell > 200 cells / mm , and high naive cd4 + t - cell percentage were associated with complete immune reconstitution .
there was a biphasic reconstitution of cd4 + t - cells : a rapid increase during the first 6 months followed by a more gradual increase over the subsequent 8 years [ figure 1a and 1b ] .
memory and naive cell recovery followed similar patterns [ figure 1c and 1d and supplementary figure s1a and s1b ] . in multivariate analysis , clinical aids events and lower baseline vl were associated with a less robust cd4 + t - cell recovery [ supplementary table s1 ] .
multivariate analysis of demographic and clinical characteristics associated with cd4 + t - cell subsets response vl : viral load ; cart : combination antiretroviral therapy ; ci : confidence interval .
naive cd4 + t - cell percentage after 8 years of art differed significantly between patients with complete immune reconstitution ( median percentage 31.1% , iqr 21.041.3% ) and those without complete immune reconstitution ( 20.1% , iqr 8.929.0% , p = 0.028 ) .
older age , baseline cd4 + t - cell 200 cells / mm , and lower baseline vl were associated with poorer recovery of naive cd4 + t - cells [ supplementary table s1 ] .
in addition , most patients did not exhibit normalized cd4/cd8 ratios after 8 years of treatment [ supplementary figure s3a - s3c ] .
baseline cd4 + t - cell count over 200 cells / mm was associated with better cd4/cd8 recovery since in this group , 66.7% ( 6/9 ) of patients achieved cd4/cd8 ratio exceeding 0.95 between year 7 and year 8 , in comparison with 9.1% ( 2/22 ) in patients with baseline cd4 + t - cell count 200 cells / mm ( p = 0.003 , by fisher 's exact test ) .
we also correlated baseline naive cd4 + t - cell percentage with normalization of cd4/cd8 ratio by adding it to univariate cox proportional model and discovered that the hazard ratio was 1.09 per 1% increase in baseline naive cd4 + t - cell percentage ( 95% confidence interval [ ci ] 1.041.15 , p = 0.001 ) .
medians and interquartile ranges ( error bars ) of cd4/cd8 ratio in the whole group ( a ) and in subgroups ( b ) .
the hiv - infected patients had higher levels of cd8+cd38 + and hla - dr+ percentage than the normal ranges at baseline [ figure 1e and 1f ] .
we also observed a biphasic recovery for immune activation : a rapid decrease during the first 6 months was followed by a plateau throughout 8 years [ figure 1e and 1f ] .
of note , the percentage of cd8+cd38 + cells from day 0 to year 2 was significantly higher than the controls , similar to the controls from year 2 to year 4 , and significantly lower than the controls from year 4 to year 8 [ figure 1e ] . during 8-year art ,
the greatest decreases of the percentage of cd8+cd38 + cells were noted within the first 6 months after art initiation . in multivariate regression analysis , male sex , viral hepatitis , and higher vl
were associated with a more robust response of the percentage of cd8+cd38 + cells during 8-year art [ supplementary table s2 ] .
we also summarized cd8+cd38+/cd8 + percentage during 8 years of follow - up in each patient [ figure 2 ] .
multivariate analysis of demographic and clinical characteristics associated with cd8 + t - cell subsets response hla - dr : human leukocyte antigen - d related ; vl : viral load ; cart : combination antiretroviral therapy ; ci : confidence interval . we next needed to determine whether immune activation might be associated with immune reconstitution after long - term therapy .
after 8 years of art , immune activation levels were comparable between patients who achieved complete immune reconstitution and patients who did not [ figure 1 g ] . to determine factors associated with complete immune reconstitution in individuals demonstrating cd4 + t - cell count 500 cells / mm after 8-year treatment , we first determined relative risks for each factor .
the percentage of naive cd4 + t - cell at baseline was strongly associated with complete immune reconstitution , with a relative risk ( rr ) of 1.08 ( per 1% increase in naive cd4 + t - cell percentage , 95% ci 1.021.44 ; p = 0.006 ) independent of baseline cd4 + count [ table 2 ] .
we then used roc curves to estimate the prognostic values of baseline naive cd4 + t - cell percentage .
the auc of the roc curve was 0.907 ( 95% ci : 0.7921.000 , p <
0.001 ) for baseline naive cd4 + t - cell percentage [ figure 3 ] .
we also compared roc curves of potential prognostic factors such as baseline total cd4 + t - cell count or naive cd4 + t - cell count , and baseline naive cd4 + t - cell percentage , and discovered that baseline naive cd4 + t - cell percentage predicts immune reconstitution better than baseline total cd4 + t - cell count or naive cd4 + t - cell count [ figure 3 ] .
relative risks of demographic and clinical characteristics associated with cd4 + t cell > 500 cells/l ( n = 30 ) in the multivariate model , we only included factors with p<0.20 in univariate model .
vl : viral load ; cart : combination antiretroviral therapy ; rr : relative risk ; ci : confidence interval .
areas under the curves were included in the parentheses . based on roc curve analysis ,
the cutoff value for the diagnosis of complete immune reconstitution at 8-year art was 12.4% for baseline naive cd4 + t - cells , with a sensitivity of 84.6% ( 95% ci : 53.797.3% ) , a specificity of 88.2% ( 95% ci : 62.297.9% ) , and an accuracy of 86.7% .
the positive predictive value was 84.6% ( 95% ci : 53.797.3% ) and negative predictive value was 88.2% ( 95% ci : 62.297.9% ) .
we then substituted baseline naive cd4 + t - cell percentage ( continuous values ) with categorical values ( < 12.4% vs. over 12.4% ) into the regression model and discovered that baseline naive cd4 + percentage over 12.4% had rr : 10.2 ( 95% ci : 2.836.5 , p < 0.001 ) in multivariate analysis .
these patients had been diagnosed with hiv for a median of 0.1 year ( iqr : 00.8 years ) before art initiation and had a median duration of art of 10.2 years ( iqr : 9.510.6 years ) . the majority of patients were infected via blood transfusion .
fourteen patients ( 45.2% ) had experienced aids - defining events , and eight patients ( 25.8% ) had a history of infection with hepatitis b or hepatitis c virus .
all patients were selected as late presenters based on cd4 + t - cell counts < 350 cells / mm .
baseline median cd4 + t - cell count was 70 ( iqr : 12223 ) cells / mm and median vl was 4.7 ( iqr : 4.35.3 ) lg copies / ml . of 31 patients , 30 had baseline memory and naive cell profiles available . in 22 patients with cd4 + t - cell count 200 cells / mm , naive cd4 + t - cell percentage was also lower ( 6.6% , iqr 4.112.3% ) than that in 9 patients with cd4 + t - cells over 200 cells / mm ( 27.5% , iqr 26.041.4% , p < 0.001 ) .
characteristics of the participants iqr : interquartile range ; hbsag : hepatitis b surface antigen ; hcv : hepatitis c virus .
during 8 years of treatment , 24 patients achieved virologic suppression within half a year of treatment initiation , whereas seven patients had virologic failure or rebound , and were switched to second - line regimens ( tenofovir + lamivudine + ritonavir - boosted lopinavir ) .
the 24 patients who demonstrated stable viral suppression were receiving zidovudine , stavudine , didanosine , and lamivudine - based regimens , which were first - line art at the time .
in comparison with the reference ranges , our hiv - infected patients had lower cd4 + t - cell and naive cd4 + t - cell counts and proportions , as well as higher proportions and lower levels of memory cd4 + t - cells during 8-year art [ figures 1 and 2 , supplementary figure s1a and s1b ] . at year 8 , there were 13 patients ( 41.9% ) with cd4 + t - cells over 500 cells / mm .
the group with baseline cd4 + t - cell counts over 200 cells / mm had a higher rate of complete immune reconstitution than that with baseline cd4 + t - cell counts 200 cells / mm ( 77.8% vs. 27.3% , p = 0.017 ) .
most patients with baseline cd4 + t - cell counts 200 cells / mm did not exhibit complete immune reconstitution after 8 years of treatment [ supplementary figure s2 ] .
medians and interquartile ranges ( error bars ) of cd4 + t - cell percentage and count ( a and b ) , memory cd4 + t - cell count ( c ) and naive cd4 + t - cell count ( d ) during eight - year art .
( e and f ) demonstrate medians and interquartile ranges of cd8+cd38 + t - cell percentage and cd8+hla - dr+ t - cell percentage during 8-year art , respectively .
( g ) immune activation after 8-year art , grouped by cd4 + t - cell count at year 8 . the shaded bands in ( a - f ) reflect the reference ranges in hiv - negative population .
the slopes in ( a - f ) were calculated by a linear model regression .
the wilcoxon nonparametric test was used to compare slopes in different phases . * p < 0.05 , and ns denotes not significant .
total , memory , and naive cd4 + t - cell as well as cd8+cd38+/cd8 + percentage during 8-year follow - up in each patient .
patients were categorized by their baseline total cd4 + t - cell count ( < 50 , 50100 , 100200 , and 200350 cells / mm ) .
medians and interquartile ranges of memory cd4 + t cell percentage ( a ) and naive cd4 + t cell percentage ( b ) during 8 year art . the shaded band reflects the reference ranges in hiv negative population , * p < 0.05 .
medians and interquartile ranges ( error bars ) of cd4 + t cell in subgroups .
no clinical aids events , baseline cd4 + t - cell > 200 cells / mm , and high naive cd4 + t - cell percentage were associated with complete immune reconstitution .
there was a biphasic reconstitution of cd4 + t - cells : a rapid increase during the first 6 months followed by a more gradual increase over the subsequent 8 years [ figure 1a and 1b ] .
memory and naive cell recovery followed similar patterns [ figure 1c and 1d and supplementary figure s1a and s1b ] . in multivariate analysis , clinical aids events and lower baseline vl were associated with a less robust cd4 + t - cell recovery [ supplementary table s1 ] .
multivariate analysis of demographic and clinical characteristics associated with cd4 + t - cell subsets response vl : viral load ; cart : combination antiretroviral therapy ; ci : confidence interval .
naive cd4 + t - cell percentage after 8 years of art differed significantly between patients with complete immune reconstitution ( median percentage 31.1% , iqr 21.041.3% ) and those without complete immune reconstitution ( 20.1% , iqr 8.929.0% , p = 0.028 ) .
older age , baseline cd4 + t - cell 200 cells / mm , and lower baseline vl were associated with poorer recovery of naive cd4 + t - cells [ supplementary table s1 ] . in addition , most patients did not exhibit normalized cd4/cd8 ratios after 8 years of treatment [ supplementary figure s3a - s3c ] .
baseline cd4 + t - cell count over 200 cells / mm was associated with better cd4/cd8 recovery since in this group , 66.7% ( 6/9 ) of patients achieved cd4/cd8 ratio exceeding 0.95 between year 7 and year 8 , in comparison with 9.1% ( 2/22 ) in patients with baseline cd4 + t - cell count 200 cells / mm ( p = 0.003 , by fisher 's exact test ) .
we also correlated baseline naive cd4 + t - cell percentage with normalization of cd4/cd8 ratio by adding it to univariate cox proportional model and discovered that the hazard ratio was 1.09 per 1% increase in baseline naive cd4 + t - cell percentage ( 95% confidence interval [ ci ] 1.041.15 , p = 0.001 ) .
medians and interquartile ranges ( error bars ) of cd4/cd8 ratio in the whole group ( a ) and in subgroups ( b ) . kaplan meier graph of patients achieving normalized cd4/cd8 ratio ( c ) .
the hiv - infected patients had higher levels of cd8+cd38 + and hla - dr+ percentage than the normal ranges at baseline [ figure 1e and 1f ] .
we also observed a biphasic recovery for immune activation : a rapid decrease during the first 6 months was followed by a plateau throughout 8 years [ figure 1e and 1f ] . of note ,
the percentage of cd8+cd38 + cells from day 0 to year 2 was significantly higher than the controls , similar to the controls from year 2 to year 4 , and significantly lower than the controls from year 4 to year 8 [ figure 1e ] . during 8-year art ,
the greatest decreases of the percentage of cd8+cd38 + cells were noted within the first 6 months after art initiation . in multivariate regression analysis , male sex , viral hepatitis , and higher vl
were associated with a more robust response of the percentage of cd8+cd38 + cells during 8-year art [ supplementary table s2 ] .
we also summarized cd8+cd38+/cd8 + percentage during 8 years of follow - up in each patient [ figure 2 ] .
multivariate analysis of demographic and clinical characteristics associated with cd8 + t - cell subsets response hla - dr : human leukocyte antigen - d related ; vl : viral load ; cart : combination antiretroviral therapy ; ci : confidence interval .
we next needed to determine whether immune activation might be associated with immune reconstitution after long - term therapy .
after 8 years of art , immune activation levels were comparable between patients who achieved complete immune reconstitution and patients who did not [ figure 1 g ] .
to determine factors associated with complete immune reconstitution in individuals demonstrating cd4 + t - cell count 500 cells / mm after 8-year treatment , we first determined relative risks for each factor .
the percentage of naive cd4 + t - cell at baseline was strongly associated with complete immune reconstitution , with a relative risk ( rr ) of 1.08 ( per 1% increase in naive cd4 + t - cell percentage , 95% ci 1.021.44 ; p = 0.006 ) independent of baseline cd4 + count [ table 2 ] .
we then used roc curves to estimate the prognostic values of baseline naive cd4 + t - cell percentage .
the auc of the roc curve was 0.907 ( 95% ci : 0.7921.000 , p < 0.001 ) for baseline naive cd4 + t - cell percentage [ figure 3 ] .
we also compared roc curves of potential prognostic factors such as baseline total cd4 + t - cell count or naive cd4 + t - cell count , and baseline naive cd4 + t - cell percentage , and discovered that baseline naive cd4 + t - cell percentage predicts immune reconstitution better than baseline total cd4 + t - cell count or naive cd4 + t - cell count [ figure 3 ] .
relative risks of demographic and clinical characteristics associated with cd4 + t cell > 500 cells/l ( n = 30 ) in the multivariate model , we only included factors with p<0.20 in univariate model .
vl : viral load ; cart : combination antiretroviral therapy ; rr : relative risk ; ci : confidence interval .
areas under the curves were included in the parentheses . based on roc curve analysis ,
the cutoff value for the diagnosis of complete immune reconstitution at 8-year art was 12.4% for baseline naive cd4 + t - cells , with a sensitivity of 84.6% ( 95% ci : 53.797.3% ) , a specificity of 88.2% ( 95% ci : 62.297.9% ) , and an accuracy of 86.7% .
the positive predictive value was 84.6% ( 95% ci : 53.797.3% ) and negative predictive value was 88.2% ( 95% ci : 62.297.9% ) .
we then substituted baseline naive cd4 + t - cell percentage ( continuous values ) with categorical values ( < 12.4% vs. over 12.4% ) into the regression model and discovered that baseline naive cd4 + percentage over 12.4% had rr : 10.2 ( 95% ci : 2.836.5 , p < 0.001 ) in multivariate analysis .
this study described t - cell dynamics during 8-year treatment and identified prognostic factors for immune reconstitution in late presenters .
we showed that in late presenters , cd4 + t - cells did not reach the normal range after long - term treatment .
in fact , the percentage of cd8+cd38 + , as a marker of immune activation , continued to decrease and reached a level even lower than the normal range after 8 years of art .
more importantly , it was shown at baseline that the percentage of naive cd4 + t - cell , rather than cd4 + t - cell count , could be a better prognostic factor for immune reconstitution in long - term art .
although some successfully treated hiv - infected patients have normalized their cd4 + t - cell count , cd4 + t - cells and naive cd4 + t - cells remained below reference ranges throughout 8 years , which is consistent with previous studies .
, in patients who initiated art with cd4 + t - cell counts < 350 cells / mm , the cd4 + t - cell count is less likely to return to reference range after 3-year art . in our study , all patients initiated art with cd4 + t - cell counts < 350 cells / mm , and cd4 + t - cell counts could not reach reference range even after 8-year art in most patients .
these results also justify the need to initiate art when cd4 + t - cell count is higher than 350 cells / mm , in a hope to achieve better immunologic responses .
furthermore , cd4/cd8 ratio was not normalized in most patients with baseline cd4 + t - cell count < 200 cells / mm after 8 years of treatment .
this index is particularly associated with mortality , immunosenescent phenotype , and non - aids morbidity and mortality . of note , younger age was associated with higher naive cd4 + t - cell count during long - term art [ 1 year younger is associated with three more naive cd4 + t - cells / mm , supplementary table s1 ] , which could be due to better thymic output in young patients . in terms of immune activation ,
expression of the t - cell activation marker ( cd38 + ) on cd8 + t - cells returned to normal levels a few years after initiation of art and even reached a level lower than normal range after 4 years of treatment .
some long - term studies demonstrated normalization in activated cd8 + t - cells in hiv - infected patients after long - term art while other short - term studies showed persistent residual activation in cd8 + compartments .
all of these studies are consistent with our findings that normalization of immune activation might be achieved after at least 4 years of art .
surprisingly , after long - term treatment , immune activation was not associated with cd4 + recovery [ figure 1 g ] , suggesting that immune activation after long - term art might not be the culprit behind limited immune reconstitution . on the other hand ,
cd38 is one of the many immune activation markers and may not recapitulate the whole picture of immune activation during long - term therapy .
monocyte activation , as measured by the expression of cd16 or by soluble cd14 , interleukin 6 , kynurenine to tryptophan ratio , etc . , may remain elevated even after treatment ; therefore , measurement of t - cell activation may not fully depict the immune activation picture in long - term art . on the other hand , could low naive cd4 + t - cell levels be responsible for the limited immune reconstitution during long - term art need to be determined .
we found baseline naive cd4 + t - cell count as a robust diagnostic approach for complete immune reconstitution at 8-year art .
based on our study , baseline naive percentage over 12.4 may be a good predictive index for complete immune reconstitution in long - term treatment .
to our surprise , baseline total cd4 + t - cell count seemed to be a less reliable predictor for complete immune reconstitution in comparison with baseline naive cell percentage , as is demonstrated in table 2 and figure 3 .
these findings suggest that depletion of naive cd4 + t - cells before art initiation could lead to failure in immune recovery . a previous study by schacker et al .
demonstrated that higher pretreatment naive cd4 + t - cell percentage was associated with better immune reconstitution during 2-year art in patients with baseline cd4 + t - cell count between 200 and 500 cells / mm .
this study supports our findings ; however , it did not show this correlation in long - term treatment . in comparison ,
our study extended the duration of observation to 8 years and gave a cutoff value for prediction , making baseline naive cd4 + percentage not only a predictor for short - term immune reconstitution but also a long - term predictor .
of note , since our patients are late presenters , whether this prognostic factor could be applied to patients with baseline cd4 + count over 350 cells / mm remains to be determined .
we would like to synthesize our findings and previous reports to depict the relationship between immune reconstitution and immune activation . before art initiation
, hiv infection and immune activation may lead to cd4 + t - cell depletion , lymphoid tissue fibrosis , and decreased thymic output , and all of these factors may lead to a decrease in naive cd4 + t - cell reservoir .
importantly , lymphoid tissue fibrosis might also cause irreversible disruption of lymphoid structure , leading to a permanent decrease in naive cell reservoir . at the early stage of art
, hiv replication can be suppressed , making redistribution of memory cd4 + t - cells and dampening of immune activation possible .
after this early stage , naive cd4 + t - cell expansion becomes the driving force behind cd4 + t - cell recovery .
should the naive reservoir have been jeopardized due to destruction of the lymphoid microenvironment during the pretreatment stage , patients may suffer from incomplete immune reconstitution .
this incomplete immune reconstitution may occur despite the fact that immune activation has been quenched after art use .
this led us to determine that baseline naive cd4 + t - cells rather than immune activation levels could predict immune reconstitution in long - term art . in line with previous studies
, we also suggest that early initiation of art might be warranted to rescue lymphoid tissue from fibrosis and to conserve the naive cell reservoir .
we also suggest that early initiation of anti - inflammatory agents might preserve naive cd4 + t - cell reservoir . on the other hand ,
thymopoiesis is also associated with effective cd4 + t - cell recovery by recovering naive cd4 + t - cells , and previous studies with recombinant human interleukin 7 ( rhil-7 ) demonstrate that rhil-7 boosts thymic output and promotes naive and central memory cd4 + t - cells in a dose - dependent manner .
we followed patients for 8 years , and such long period of time enabled us to observe continuous increase in cd4 + t - cell and decrease in cd8 + cell activation .
in addition , it is becoming rare to have late presenters in the developed countries , whereas such presentation is common in less - developed areas ; therefore , predicting the immune reconstitution for late presenters is of great importance for the initiation of art .
our study has some limitations as follows : ( 1 ) our sample size is still limited and further long - term study with a larger sample size is warranted to evaluate t - cell dynamics during long - term treatment .
( 2 ) t - cell comparison and reconstitution in lymphoid tissue may not be reflected in circulating cells .
( 3 ) we did not measure the stem - cell like central memory cd4 + t - cell , which may also have predictive values and may also reflect the size of reservoir .
( 4 ) in terms of immune activation markers , we did not obtain the proportions of cd38 and hla - dr coexpression during our follow - up ( cd4+cd38+hla - dr+ and cd8cd38+hla - dr+ cells ) . in conclusion , our results demonstrated the persistence of t - cell imbalance throughout 8 years of art .
baseline percentage of naive cd4 + t - cells could predict complete immune reconstitution at 8-year art , and an optimal cutoff value was estimated at 12.4% . promoting thymus function by immunotherapeutic agents such as rhil-7 may be promising in the recovery of cd4 + t - cell count in late presenters .
supplementary information is linked to the online version of the paper on the chinese medical journal website .
this study was supported by the national natural science foundation of china ( no . 81071372 ) , the national key technologies r and d program for the 11 five - year plan ( no .
2008zx10001 - 006 ) , the national key technologies r and d program for the 12 five - year plan ( no .
2012zx10001003 - 001 ) , and the key clinical program of the ministry of health ( 20102012 ) .
this study was supported by the national natural science foundation of china ( no . 81071372 ) , the national key technologies r and d program for the 11 five - year plan ( no .
2008zx10001 - 006 ) , the national key technologies r and d program for the 12 five - year plan ( no . 2012zx10001003 - 001 ) , and the key clinical program of the ministry of health ( 20102012 ) . | background : among hiv - infected patients initiating antiretroviral therapy ( art ) , early changes in cd4 + t - cell subsets are well described .
however , hiv - infected late presenters initiating treatment present with a suboptimal cd4 + t - cell reconstitution and remain at a higher risk for aids and non - aids events .
therefore , factors associated with cd4 + t - cell reconstitution need to be determined in this population , which will allow designing effective immunotherapeutic strategies.methods:thirty-one adult patients with baseline cd4 + t - cell count < 350 cells / mm3 exhibiting viral suppression after art initiation were followed in the hiv / aids research center of peking union medical college hospital in beijing , china , from october 2002 to september 2013 .
changes in t - cell subsets and associated determinants were measured.results:median baseline cd4 + t - cell count was 70 cells / mm3 .
we found a biphasic reconstitution of t - cell subsets and immune activation : a rapid change during the first 6 months followed by a more gradual change over the subsequent 8 years .
baseline cd4 + t - cell count > 200 cells / mm3 in comparison to cd4 + t - cell count 200 cells / mm3 was associated with more complete immune reconstitution ( 77.8% vs. 27.3% respectively ; p = 0.017 ) and normalized cd4/cd8 ratio .
we showed that the baseline percentage of naive cd4 + t - cell was a predictive marker for complete immune reconstitution ( area under receiver operating characteristic curve 0.907 ) , and 12.4% as cutoff value had a sensitivity of 84.6% and a specificity of 88.2%.conclusions : baseline naive cd4 + t - cell percentage may serve as a predictive marker for optimal immune reconstitution during long - term therapy .
such study findings suggest that increasing thymic output should represent an avenue to improve patients who are diagnosed late in the course of infection . | I
M
Patients
T-cell subsets and viral load
Statistical analyses
R
Baseline characteristics
Virologic suppression
Incomplete recovery of CD4+ T-cell subsets
Higher immune activation levels at baseline and normalization of activated CD8+ T-cells
Correlation of immune reconstitution and immune activation after 8 years of antiretroviral therapy
The predictive factors for complete immune reconstitution after 8 years of antiretroviral therapy
D
Financial support and sponsorship
Conflicts of interest | unfortunately , approximately 20% of patients may experience immune nonresponse even after suppressive art , and this is more commonly seen in late presenters ( patients with baseline cd4 + t - cell count < 350 cells / mm at the time of art initiation ) . patients were classified into two groups : one group including patients with baseline cd4 + t - cell count > 200 cells / mm , and the other group with cd4 + t - cell count 200 cells / mm . patients were classified into two groups : one group including patients with baseline cd4 + t - cell count > 200 cells / mm , and the other group with cd4 + t - cell count 200 cells / mm . the group with baseline cd4 + t - cell counts over 200 cells / mm had a higher rate of complete immune reconstitution than that with baseline cd4 + t - cell counts 200 cells / mm ( 77.8% vs. 27.3% , p = 0.017 ) . no clinical aids events , baseline cd4 + t - cell > 200 cells / mm , and high naive cd4 + t - cell percentage were associated with complete immune reconstitution . there was a biphasic reconstitution of cd4 + t - cells : a rapid increase during the first 6 months followed by a more gradual increase over the subsequent 8 years [ figure 1a and 1b ] . baseline cd4 + t - cell count over 200 cells / mm was associated with better cd4/cd8 recovery since in this group , 66.7% ( 6/9 ) of patients achieved cd4/cd8 ratio exceeding 0.95 between year 7 and year 8 , in comparison with 9.1% ( 2/22 ) in patients with baseline cd4 + t - cell count 200 cells / mm ( p = 0.003 , by fisher 's exact test ) . the percentage of naive cd4 + t - cell at baseline was strongly associated with complete immune reconstitution , with a relative risk ( rr ) of 1.08 ( per 1% increase in naive cd4 + t - cell percentage , 95% ci 1.021.44 ; p = 0.006 ) independent of baseline cd4 + count [ table 2 ] . based on roc curve analysis ,
the cutoff value for the diagnosis of complete immune reconstitution at 8-year art was 12.4% for baseline naive cd4 + t - cells , with a sensitivity of 84.6% ( 95% ci : 53.797.3% ) , a specificity of 88.2% ( 95% ci : 62.297.9% ) , and an accuracy of 86.7% . the group with baseline cd4 + t - cell counts over 200 cells / mm had a higher rate of complete immune reconstitution than that with baseline cd4 + t - cell counts 200 cells / mm ( 77.8% vs. 27.3% , p = 0.017 ) . no clinical aids events , baseline cd4 + t - cell > 200 cells / mm , and high naive cd4 + t - cell percentage were associated with complete immune reconstitution . there was a biphasic reconstitution of cd4 + t - cells : a rapid increase during the first 6 months followed by a more gradual increase over the subsequent 8 years [ figure 1a and 1b ] . naive cd4 + t - cell percentage after 8 years of art differed significantly between patients with complete immune reconstitution ( median percentage 31.1% , iqr 21.041.3% ) and those without complete immune reconstitution ( 20.1% , iqr 8.929.0% , p = 0.028 ) . baseline cd4 + t - cell count over 200 cells / mm was associated with better cd4/cd8 recovery since in this group , 66.7% ( 6/9 ) of patients achieved cd4/cd8 ratio exceeding 0.95 between year 7 and year 8 , in comparison with 9.1% ( 2/22 ) in patients with baseline cd4 + t - cell count 200 cells / mm ( p = 0.003 , by fisher 's exact test ) . the percentage of naive cd4 + t - cell at baseline was strongly associated with complete immune reconstitution , with a relative risk ( rr ) of 1.08 ( per 1% increase in naive cd4 + t - cell percentage , 95% ci 1.021.44 ; p = 0.006 ) independent of baseline cd4 + count [ table 2 ] . based on roc curve analysis ,
the cutoff value for the diagnosis of complete immune reconstitution at 8-year art was 12.4% for baseline naive cd4 + t - cells , with a sensitivity of 84.6% ( 95% ci : 53.797.3% ) , a specificity of 88.2% ( 95% ci : 62.297.9% ) , and an accuracy of 86.7% . demonstrated that higher pretreatment naive cd4 + t - cell percentage was associated with better immune reconstitution during 2-year art in patients with baseline cd4 + t - cell count between 200 and 500 cells / mm . | [
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the increasing use of liquid chromatography / mass spectrometry ( lc / ms ) instrumentation for proteomics studies at a large scale stimulates the development and improvement of data analysis tools .
the precise retrieval of biological information from a large lc / ms dataset critically depends on algorithms for data interpretation , which remains a current bottleneck in the rapid advance of proteomics technology .
the quantitation of differentially regulated proteins represents a major type of proteomics application in biological studies .
protein quantitation with lc / ms data includes three conceptually different methods , that is , spectral counting , differential stable isotope labeling , and label - free lc / ms measurements by using extracted ion chromatographic intensities . due to the increased time and complexity of sample preparation in stable isotope labeling , cost of labeling reagents and requirement of higher starting sample amount , however , researchers are increasingly using label - free proteomics for faster and simpler protein quantitation .
multiple algorithms and software solutions for label - free proteomics data analysis have been developed .
these algorithms and software solutions provide quantitation of protein differential abundances but do not always provide a statistical significance assessment of differential abundances .
algorithms for statistical significance analysis in label - free proteomics with spectral counting were investigated [ 4 , 5 ] . in label - free quantitation with extracted ion chromatographic intensities , there are still needs to improve approaches for assessing statistical significance , especially for low - replicate datasets .
most proteomics studies infer proteins with 2 identified peptides as reliable protein identifications and usually disregard proteins with a single - peptide hit as unreliable for quantitation . this two - peptide
rule was recently challenged with the evidence that it reduced protein identifications more in a target database than in a decoy database , and thus increased false discovery rates in protein identification .
indeed , it was shown that proteins with a single - peptide hit could represent 30% of the proteins identified with 2
ms2 spectrum matches at p < .01 . because those single - peptide proteins had 2 ms2 spectrum matches ( p < .01 ) in multiple lc / ms analyses under the same condition , they had an adequate level of statistical confidence to be included for quantitation .
but the inclusion of single - peptide proteins in a differential quantitative proteomics analysis raises two issues .
the first is that a conventional statistical test such as a t - test can not be applied toward these single - peptide proteins when the t - test relies on multiple quantified peptides as replicates to calculate the t - statistic for the protein relative abundance .
the second is that many single - peptide proteins are at a lower abundance and thus noisy .
more stringent thresholds are needed to control the false discovery rate when these single - peptide proteins are included for the selection of differentially regulated proteins .
pavelka et al . applied a power law global error model ( plgem ) and the signal - to - noise ratio ( stn ) statistic to select differentially regulated proteins based on a spectral counting quantitation method .
the plgem - stn statistic utilized a resampling approach to estimate the null distribution from replicates of a sample .
after the error model was calculated from a pool of resampling statistics that constituted the null distribution , a set of stn thresholds were applied at a specified confidence level toward samples with any level of replicates .
the plgem - stn method is attractive in that it could be applied toward samples with no replicates if several replicates for one sample are provided to estimate the null distribution .
the plgem - stn method , however , has not been demonstrated for label - free quantitation with extracted ion chromatographic intensities . in this paper ,
the plgem - stn statistic was applied toward a lc / ms dataset obtained with a high - resolution mass spectrometer .
the peptide and protein abundances were quantified with a label - free approach based on extracted ion chromatographic intensities .
the false discovery rate was estimated at different confidence levels of the plgem - stn statistics .
the plgem - stn statistic alone did not provide a desired level of false discovery rate control .
insufficient stringency in false discovery rate control was similar to the situation when a t - test statistic was used alone . with the combination of a t - test and the rule of minimum number of permuted significant pairings ( mpsp ) ,
however , the false discovery rate was significantly reduced in that study . in this study ,
the combination of mpsp and plgem - stn was tested for controlling the false discovery rate in order to extend the selection of differentially regulated proteins to those with lower fold - changes and to those with single - peptide hits .
the combination of mpsp and fold - change thresholds was also compared with the plgem - stn - mpsp approach .
the mycobacterium smegmatis ( msm ) strain mc 155 was obtained from the american type culture collection ( atcc ; rockville , md ) and cultured in 7h9 media .
a ph 5.0 and a ph 7.0 msm culture were grown in triplicate in unlabeled media and harvested as described previously [ 6 , 9 ] .
a cell pellet was collected from a 30-ml culture aliquot for each culture replicate in a log phase . a [ n]-labeled msm culture
was also grown for use as a control to determine false positive rates in protein quantitation .
hereafter , the stressed ph 5 culture is named as s , the reference ph 7 culture as r , and the control culture as c. as described previously , the medium for growing n labeled cells consisted of ( g / l ) 99at% ( nh4)2so4 : 0.5 ; glucose : 2 ; tween 80 : 0.5 ; citric acid : 0.094 ; biotin : 0.0005 ; pyridoxine : 0.001 ; nacl : 0.1 ; na2hpo4 : 2.5 ; kh2po4 : 1 ; mgso4 6h2o : 0.1 ; cuso4 5h2o : 0.001 ; znso4 6h2o : 0.002 ; cacl2 2h2o : 0.0007 ; ferric ammonium citrate : 0.04 ; ph 5.0 .
the single n labeled cell culture was grown at 50 ml in a loosely capped 250-ml nephelo culture flask under shaking at 37c .
thirty milliliter of the n labeled reference culture was collected at od 1.1 in the late - log phase .
preparation of proteins from the cell pellets of cultures s , r , and c was described previously [ 6 , 10 ] .
the s triplicates were pooled to generate protein sample sp and the r triplicates were pooled to generate protein sample rp .
in addition , the s triplicates sa , sb , and sc were also individually processed .
these five protein samples ; that is , sp , rp , sa , sb , and sc were , respectively , mixed with an equal amount of proteins from the [ n]-labeled c culture . after mixing with the labeled proteins from culture c , the five protein samples were separated on a 1d - sds / page gel , divided into five fractions , and processed for in - gel digestion and peptide extraction for lc / ms analysis as described in [ 9 , 10 ] . for the pooled samples sp and
rp , all five fractions were analyzed by lc / ms . for sa , sb , and sc ,
the peptide extract from each gel fraction was constituted in ~25 l 5% formic acid and was analyzed in duplicate injections with a nanolc / ltq - ftms system ( thermo finnigan ; san jose , ca ) . in each lc / ms injection ,
5 l of peptide extract solution was separated on a 150-mm 75-m c18 reverse phase column with 5% to 35% acetonitrile ( v / v ) gradient in 0.1% trifluoroacetic acid over 60 minutes .
the ltq - ftms was operated in a data - dependent acquisition mode with up to 10 ms / ms spectra acquired following each ms scan .
the bioworks ( thermo finnigan ; san jose , ca ) software was on a stand - alone workstation and utilized sequest as the search engine .
the raw data files were searched against an ncbi msm database in two separate bioworks searches .
the precursor ion tolerance was set at 1.5 da to include the peptides , which precursor ions had one c isotope .
only the peptide charge states ( pcss ) with p < .01 were accepted for subsequent quantitation .
lists of pcss selected at p < .01 were exported from bioworks into excel spreadsheets . the excel spreadsheets containing the accepted pcss , along with raw data files , were processed for quantitation as previously described [ 1012 ] .
the lc / ms raw data associated with this paper can be downloaded from http://proteomecommons.org/ tranche ( see supplementary material available online at doi:10.1155/2010/731582 ) .
protein abundances were quantified with a label - free approach as described in [ 6 , 9 ] .
the abundance of a protein was calculated as the sum of the extracted ion chromatographic intensities of the pcss detected for that protein .
the unlabeled protein samples were named as sa , sb , sc , sp , and rp . the [ n]-labeled protein sample from culture c had five sample preparation replicates because it was mixed with each of the five unlabeled proteins samples .
accordingly , each sample preparation replicate of the culture c protein sample was named by adding the prefix c before the unlabeled protein sample with which it was run together .
for example , the labeled sample that was mixed with sp was named csp , and so forth . thus , the labeled c culture protein sample had five replicates that were named as csa , csb , csc , csp , and crp , respectively . because each sample was analyzed in duplicate lc / ms injections , the lc / ms injections were named by adding the subscript 1 or 2 to each protein sample ( see table 1 )
. therefore , the lc / ms analysis of the five protein samples led to 20 quantitation categories ( table 1 ) .
here , a quantitation category referred to one lc / ms injection of a protein sample in unlabeled or labeled form . because each protein sample contained the unlabeled proteins from culture s or r , and the labeled proteins from control culture c , one lc / ms injection generated four quantitation categories with two belonging to the unlabeled protein sample and two to the labeled protein sample .
the five unlabeled protein samples ( sa , sb , sc , sp , and rp ) , the five sample preparation replicates of the labeled control protein sample ( csa , csb , csc , csp , and crp ) , and the 20 quantitation categories arising from the duplicate analysis of these samples are summarized in table 1 .
the complete analysis of the five gel fractions for sp and rp resulted in the quantitation of 5134 pcss and 1032 proteins ( see tables 1 and 2 in supplementary material available online at doi:10.1155/2010/731582 ) . in the label - free quantitation approach employed here
, the abundance of a pcs ( apcs ) was represented by the extracted ion chromatographic intensity of the pcs , and the abundance of a protein ( apro ) was represented by the sum of the extracted ion chromatographic intensities of the pcss that belonged to the protein . because the sample fractionation efficiency might dictate the approach to normalize the samples , the fractionation resolution was examined by plotting a histogram for the percentage of the detected pcss versus the number of gel fractions in which they were present ( figure 2 ) .
the result shows that 82.8% of the pcss were present in a single gel fraction and 96.3% were present in 2 gel fractions .
the selection of the single - band pcss was for the purpose of normalizing pcs abundances in different fractions . in each fraction , the pcs abundances were normalized in the following two steps . in the first normalization step , the pcs abundances were normalized by the median extracted ion chromatographic intensity sought from the single - band pcss .
then , the median - normalized pcss intensities were multiplied by the total intensity of the same fraction averaged over all of the samples .
in these two steps of normalization , the first median - normalization step improves the comparability of pcss in each fraction across different samples .
the second normalization step retained the relative fraction intensity information across the five fractions , so that the apcs values correlated more adequately to their protein abundances in the samples .
it is critical to perform the second step of normalization because it preserves the information about the abundance of a protein in a sample .
the information about the abundance of a protein in the samples will be indispensable to perform the power law global error and signal - to - noise statistic modeling as described later .
after pcs normalization , the protein abundance was calculated by summing the apcs values of that protein in each sample [ 14 , 15 ] .
one was to extend the selection of differentially regulated proteins to those that had single - peptide hits .
the other was to select differentially regulated proteins at smaller fold - changes and at a false discovery rate .05 .
the approaches to achieve this two - fold purpose were investigated under a scenario where the number of sample replicates was too small to apply other typical statistics such as a t - test .
more importantly , a conventional t - test alone might not provide the necessary specificity in the label - free quantitation of differentially regulated proteins .
therefore , in a prior test , it was found necessary to insert an additional measure , such as the mpsp rule .
the biological sample model used in the study was the proteome response of an acid stressed msm culture ( s ) in reference to a neutral ph culture ( r ) .
the proteins from a [ n]-labeled control culture ( c ) was used as an internal standard to mix with the proteins from the unlabeled cultures ( figure 1 ) . because the proteins from the control culture were analyzed repeatedly with two other unlabeled samples , the repeated analyses of the labeled control provided
replicates to construct a null distribution in which no true differentially regulated proteins were present .
such an error model could not be derived from the pair of unlabeled protein samples sp and rp that did not have protein sample replicates . with the null distribution provided by the labeled control sample ,
different approaches were experimented with to select differentially regulated proteins by using the combination of mpsp , plgem - stn , and fold - change .
differentially regulated proteins were selected from the unlabeled sample pair sp and rp . the other three samples sa , sb , and sc were used to evaluate the source of variability but not for the selection of differentially regulated proteins . the naming of these samples and their lc / ms runs is delineated in table 1 .
. an observed differential abundance of a pcs or protein between samples arose not only from the difference in biological samples but also from measurement noise that included the variability among lc / ms injection replicates , sample preparation replicates , biological replicates , or the data processing method . to assist in the assessment of the source of variability in the label - free quantitation of the lc / ms data ,
the 3rd of the five fractions of an sds / page gel lane was processed for lc / ms analysis for the protein samples sa , sb , sc , sp , and rp with duplicate injections for each sample ( table 1 ) .
the five samples with two lc / ms injections per sample resulted in 10 lc / ms runs .
these 10 lc / ms runs of the 3rd fraction allowed the quantitation of 349 proteins for the 3rd fraction . because a protein was quantified in both the unlabeled form ( for culture s or r ) and the labeled form ( for culture c ) , there were 20 quantitation categories for each protein .
thus , these 349 proteins and the 20 quantitation categories formed a 349 20 matrix .
the 349 20 matrix was examined by a clustering analysis to obtain an overview of the correlation among the protein samples and lc / ms injections with the purpose to reveal the major source of variability .
the naming of the 20 quantitation categories was shown in table 1 . from the clustering tree of the 20 quantitation categories shown in figure 4
, it could be seen that the distance between each pair of duplicate lc / ms injections was the shortest compared to those between any other sample pairings .
the closest distance of the duplicate lc / ms injections for a sample indicated that the variability between lc / ms injections was the smallest , which also excluded that the label - free data analysis methodology would introduce a significant variability . in figure 4 , it was also apparent that the unlabeled and labeled quantitation categories were separated into two distinct branches represented by nodes i and ii , respectively .
the separation of the unlabeled and labeled quantitation categories into the two distinct clusters indicated that the difference between cultures c and s or c and r was larger than the difference between s and r. from the tree branch under node ii , it could be seen that the distance between the unlabeled protein samples sp and rp was larger than the distance among the s culture replicates ; that is , sa , sb , and sc .
the result indicated that the difference between cultures s and r exceeded the difference among the s culture replicates , suggesting that the variability in biological sample replicates was less than the actual difference between the biological samples treated with different conditions .
therefore , the clustering result in figure 4 indicated that the variability increased in the order of lc / ms injections < sample preparation replicates ( under node i ) ~biological replicates ( under node iii ) < biological samples ( between nodes iii and iv ) . because these differences were evaluated based on the proteomic quantitation data , a variability observed among biological replicates also included the variability introduced during sample preparation for lc / ms analysis .
the similarity between the variability observed among the sample preparation replicates and the variability observed among the biological replicates suggested that the variability among biological replicates was not larger than the variability among sample preparation replicates .
this subsection describes the multiple steps leading to the extended selection of differentially regulated proteins from all quantified proteins including those with only a single - peptide hit .
therefore , it is desirable to have a procedure to select regulated proteins from all of the proteins including those with a single - peptide hit to maximize the potential of the global protein expression profiling .
the major steps to establish the criteria for extended selection of differentially regulated proteins are summarized in table 2 , and are described in detail in the following .
based on the evaluation with the clustering analysis ( figure 4 ) , the variability among sample preparation replicates appeared to be comparable with the variability among biological replicates .
samples sp and rp represented the average of triplicate biological replicates for cultures s and r , respectively , because each of them was the pooled sample of three biological replicates .
therefore , the [ n]-labeled control sample replicates ( table 1 ) were adequate to represent a null distribution in which there was no differentially regulated protein .
the determined measurement noise was then used to estimate the false discovery rate for the selected differentially regulated proteins between samples sp and rp .
the null distribution provided a reference for setting thresholds to maximize the selection of differentially regulated proteins ( positives ) while minimizing false positives . in figure 5 , such a null distribution was illustrated with the scatter plot represented by the pink dots . to investigate the relationship between measurement variability and protein abundance apro , relative standard deviation ( rstd )
was plotted against the mean apro value for each protein in the unlabeled protein samples ( blue trace ) or the labeled control protein samples ( pink trace ) ( figure 5 ) .
the rstd - apro trace in pink reflected the local noise of the null distribution .
the local noise of the null distribution was mainly due to the variability that was introduced during sample preparation ( figure 4 ) .
the rstd - apro trace in pink clearly suggested that the apro measurement noise had a reciprocal dependence on the apro amplitude .
the rstd - apro trace in blue reflected both sample preparation variability and biological sample difference between cultures s and r. thus , the blue trace had higher rstd values than the pink trace throughout the apro range . because of the reciprocal dependence of apro rstd on the apro value , a universal 3-fold - change cutoff missed some positives at higher apro values where a < 3-fold change was already significantly different from the local noise . missed positives at higher apro values could be observed in figure 5 by examining the spread of the two scatter plots in the high apro ranges . at apro > 1000 ,
the rstd was a few times smaller than that at apro of ~100 . from the figure
, it could be seen that it was possible to detect a < 2-fold change for the proteins with apro > 1000 . to the contrary , at apro < 10 , a 3-fold change threshold was not sufficient to eliminate many false positives
therefore , a criterion adaptive to the dependence of apro noise on apro values would uncover more differentially regulated proteins .
this extended selection of differentially regulated proteins could be achieved by penalizing proteins with higher apro values less than proteins with lower apro values .
such an adaptive criterion , however , requires a systematic modeling of the noise to establish the thresholds according to local variability .
the issue of the dependence of variability on mean gene expression level was addressed for gene differential expression studies with dna microarray .
for example , pavelka et al . proposed a power law global error model ( plgem ) in combination with the signal - to - noise - ratio ( stn ) test statistic for the identification of differentially expressed genes in microarray data .
pavelka et al . further applied the approach to spectral count - based quantitative proteomics data .
the plgem - stn statistic , however , has not been demonstrated for label - free proteomics data based on the quantitation of peptide and protein extracted ion chromatographic intensities . in this study ,
the plgem - stn statistic was experimented with for the selection of differentially regulated proteins quantified with label - free proteomics based on protein extracted ion chromatographic intensities .
the plgem - stn analysis was performed in four major steps for the dataset shown in figure 5(see scheme s1 in supplementary material available online at doi:10.1155/2010/731582).there were two reasons for the choice of the plgem - stn method .
first , the plgem - stn method allowed statistical analyses of the proteins quantified with a single pcs because the plgem - stn statistic did not rely on multiple pcss of a protein like a t - test . because single - peptide proteins constituted
a third of the quantified proteins ( figure 6 ) , being able to quantify these single - peptide proteins was important to maximize the potential value of the data .
second , the plgem - stn method took into account the dependence of apro noise on apro levels .
a threshold adjustable to the local dependence of apro noise on apro levels allowed the selection of differentially regulated proteins with a smaller fold - change threshold at a higher apro level .
therefore , the plgem - stn method potentially could select more differentially regulated proteins by applying a smaller fold - change threshold in the higher apro range where the variability was smaller .
table 3 shows the result of the plgem - stn analysis for the unlabeled samples sp and rp and the labeled sample replicates csp and crp .
csp and crp were the labeled control samples analyzed concurrently with sp and rp , respectively .
the differentially regulated proteins found between sp and rp were positives , and those found between csp and crp were false positives . because each protein sample was analyzed with duplicate lc / ms injections , permutation of the four lc / ms injections for a sample pair resulted in four permuted sample pairings .
these four permuted sample pairings were numbered as i to iv in table 3 . in each column for a permuted sample pairing in table 3 , the numbers of false positives and positives and the false discovery rate were listed .
the false positives were determined as the differentially regulated proteins for the sample pair csp / crp .
the positives were determined as the differentially expressed proteins for the sample pair sp / rp . for the labeled protein sample pair csp / crp , the four permuted sample pairings were csp,1/crp,1 , csp,1/crp,2 , csp,2/crp,1 , and csp,2/crp,2 .
for the unlabeled sample pair sp / rp , the four permuted sample pairings were sp,1/rp,1 , sp,1/rp,2 , sp,2/rp,1 , and sp,2/rp,2 .
the naming of the lc / ms injections noted in the permuted sample pairings is shown in table 1 . in table 3 ,
the positives and false positives were selected with the plgem - stn method at the confidence level of 0.01 and 0.002 , respectively .
the results indicate that the numbers of positives or false positives were not the same among the four permuted sample pairings . to estimate an average false discovery rate ,
the numbers of positives and false positives were respectively averaged among the four permuted sample pairings .
the false discovery rate was then calculated as the ratio of the average number of false positives divided by the average number of positives .
the false discovery rate was determined at two different plgem - stn confidence levels ( table 3 ) . with a receiver operating characteristic analysis ,
the plgem - stn approach is examined over a broader confidence level range ( figure 7 ) and will be compared with another approach that is to be described below .
initially , the plgem approach was carried out by comparing the duplicate lc / ms injections from the two samples r and s without permutation pairings .
but the false discovery rate stayed high unless the sensitivity was severely compromised to reduce the false discovery rate .
for example , at a confidence level of 0.0001 , only 16 differentially regulated proteins were selected at 6% false discovery rate ( data not shown ) . with all of the permutation pairs and a combination of plgem and mpsp ,
44 differentially regulated proteins were selected at a false discovery rate of 5% ( table 3 ) .
therefore , utilizing all possible permutation pairs with a combination of plgem and mpsp results in a higher sensitivity to uncover differentially regulated proteins . because of the variable numbers of positives and false positives among the four permuted sample pairings , it was necessary to determine a consensus list of differentially regulated proteins from the four permuted sample pairings .
previously , the rule of mpsp was applied to determine the consensus list of differentially regulated proteins from four permuted sample pairings .
the mpsp rule required that only those proteins that were found differentially regulated in a certain number of permuted sample pairings were counted as positives ( for sp / rp ) or false positives ( for csp / crp ) . when a sample pair such as sp / rp had no sample replicates but had duplicate lc / ms injections , mpsp was found to be optimum at four . setting mpsp at four meant that a differentially regulated protein had to be found differentially regulated in all of the four permuted sample pairings .
the application of the mpsp rule towards the plgen - stn results decreased both false positives and positives ( table 3 ) .
but the false discovery rate was also decreased relative to that when only the plgem - stn statistic was applied . from table 3
, it could be seen that the number of true positives , which was estimated from the difference between the numbers of positives and false positives , remained about the same . therefore , the combination of the mpsp rule with the plegm - stn method reduced the false discovery rate by 2 - 3 times without compromising the sensitivity .
as summarized in figure 7 , the receiver operating characteristic analysis clearly shows that the plgem - stn - mpsp approach significantly reduces false positives to improve the specificity without significantly affecting the sensitivity .
compared to the use of the plgem - stn statistic alone , the combination of plgem - stn and mpsp performs better in controlling false discovery rates without compromising the sensitivity to select differentially regulated proteins .
the use of mpsp with fold - change criteria was also examined ( table 4 ) . with fold - change criteria alone ,
the false discovery rate did not drop below 46% at 2- to 4-fold changes ( table 4 ) or even at a 5-fold change ( see figure s3 supplementary material available online at doi:10.1155/2010/731582 ) . with the combination of mpsp and the fold - change criteria ,
the false discovery rate was reduced from 46% to 21% at 2- and 3-fold changes . at a 4-fold change ,
compared to the combination of plgem - stn and mpsp , however , the combination of fold - change and mpsp reduced more true positives at the similar false discovery rate of 4%-5% .
the reduced sensitivity was due to the increase in the fold - change threshold . with the 4-fold - change - mpsp and the plgem - stn - mpsp approaches , 26 and 44 proteins were respectively selected as differentially regulated at a false discovery rate of 4% or 5% ( tables 3 and 4 ) . among these 26 and 44 proteins , there were 55 unique proteins(see table s1 in supplementary material available online at doi:10.1155/2010/731582).these 55 unique proteins included all of the 20 high - confidence differentially regulated proteins identified previously with an empirical fold - change and abundance level cutoff approach .
only 15 proteins were common between the two sets of differentially regulated proteins selected with the 4-fold - change - mpsp and the plgem - stn - mpsp approaches ( figure 8(a ) ) .
the 4-fold - change - mpsp approach selected more single - pcs proteins than the plgem - stn - mpsp approach ( figure 8(b ) ) .
the plgem - stn - mpsp approach selected proteins with a fold - change as low as 1.8-fold ( figure 8(c ) ) . however , these differentially regulated proteins selected with plgem - stn - mpsp had a protein abundance higher than most of the differentially regulated proteins selected with the 4-fold - change - mpsp approach ( figure 8(d ) ) .
despite the relative complexity in label - free proteomics data analysis and the demand of more stringently controlled lc / ms experimental conditions , there are strong motivations stemming from biological and experimental perspectives to use the label - free approach , as discussed below .
as shown in figure 4 , the unlabeled and labeled quantitation categories are separated into two distinct clusters .
one includes the quantitation categories from the labeled control culture c ( under node i ) .
the other includes the quantitation categories from the two unlabeled cultures s and r ( under node ii ) .
thus , there was a larger difference between the labeled ( c ) and either of the two unlabeled samples ( s or r ) than between the two unlabeled cultures ( s and r ) .
the number of differentially regulated proteins between the labeled culture and either of the unlabeled culture was about three times as many as that between the two unlabeled cultures . compared to the difference between the two unlabeled cultures , the difference between the labeled culture and either of the unlabeled cultures was larger .
this larger difference was probably because the labeled culture was cultured in a synthetic minimal medium while the two unlabeled cultures were grown in a commercial 7h9 broth that was richer in ingredients .
another factor was that the acidic growth condition was a relatively mild stress so that not many proteins were differentially regulated .
the apparent difference in proteome profile for cells cultured in different media is actually a strong motivation for this study . in microbiological works , it is not always convenient to make a [ n]-labeled medium with complex ingredients required to cultivate bacteria under more physiologically relevant conditions .
even some of the stable - isotope - labeled media are technically feasible to make , they often bear a costly price tag . for microbiological works , one might not want to be restricted by the type of medium that can be used because of the stable isotope labeling limitation .
for example , some mycobacteria are difficult to cultivate on simple synthetic media and prefer complex media . thus , unlabeled media are always convenient choices if the down - stream proteomic analysis is established to proceed with the quantitation . for such reasons ,
the focus of this study was on the comparison of protein expression profiles between the two unlabeled cultures s and r. the labeled control culture c was used as an internal standard to estimate false discovery rates . the label - free quantitation scheme presented in this study incorporated a labeled internal control to provide replicates for noise modeling without a requirement of other unlabeled sample replicates .
the inclusion of a labeled internal control facilitates the control of false discovery rates .
internal standards are commonly used to improve reliability of quantitative proteomics such as to aid in removing outlier data and to detect fluctuation in instrument performance .
compared to other synthetic peptide internal standards [ 18 , 19 ] , the [ n]-labeled control culture c provides more comprehensive peptide internal standards . for most of the peptides ,
the extracted ion chromatographic intensities can be matched among the three protein samples originated from the two unlabeled ( s and r ) cultures and the labeled ( c ) culture .
the c protein sample was mixed and run together with either s or r protein sample , so that the reliability of the internal standards was improved . for constructing the null distribution for the error model in plgem - stn , it would be ideal to have the labeled internal standard identical to an unlabeled sample in protein composition .
as mentioned above , however , that requirement could restrict the culturing conditions available for biological experiments .
thus , it is acceptable and sometimes necessary to use a labeled protein mixture sample as internal standard , even though the internal standard sample might be somewhat different from the unlabeled samples in protein abundance profiles .
nevertheless , the null distribution is only utilized to establish the relation between the signal - to - noise ratio and the peptide abundance in the plgem - stn method .
there is no requirement of direct one - to - one comparison between the labeled and unlabeled version of a protein during this process .
therefore , the difference in proteome composition between the labeled internal standard sample c and the two unlabeled samples s and r is not expected to affect the modeling parameters derived from the null distribution constructed from the labeled c sample .
one could choose to run multiple replicates of an unlabeled sample and use the replicates to construct the null distribution [ 4 , 6 ] .
the lc / ms data used in this work was acquired with a high - resolution mass spectrometer that resolved peptide peaks from a complex sample mixture to allow the determination of the extracted ion chromatographic intensities of peptides and proteins .
repeated lc / ms injections showed the highest reproducibility among several other types of replicates ( figure 4 ) , indicating that the major variability of the label - free quantitation did not lie within the lc / ms separation and the data analysis method .
rather , sample preparation replicates represented a major source of the variability . with a labeled control sample to run concurrently with each of the unlabeled samples , replicates for the labeled control sample were obtained .
the replicates of the control sample provided data to model the noise in the label - free quantitation with extracted ion chromatographic intensities ( figure 5 ) .
we performed a two - step normalization procedure in which the information about the abundance of a peptide or protein in a sample was preserved ( figure 3 ) .
the preservation of the information about the abundance of a peptide or protein in the samples is critical for performing the plgem - stn analysis .
in addition , because protein extracted ion chromatographic intensity was represented by the sum of the pcs extracted ion chromatographic intensities belonging to that protein , the summation weighed the low - intensity pcss less than the high - intensity pcss
. such a summation of pcs extracted ion chromatographic intensities probably suppressed noise from lower - intensity pcss .
when a protein abundance ratio is calculated as the average of pcs abundance ratios without weighing , the noise from a lower - intensity pcs would be amplified .
we have avoided this potential issue by summing the pcs intensities to represent protein abundances before calculating protein abundance ratios .
single - peptide proteins made up about 35% of the quantified proteins ( figure 6 ) .
selection of differentially regulated proteins from these single - peptide proteins required a significance assessment method that did not rely on multiple - peptide detection to calculate a statistic about the confidence of a protein differential abundance .
the use of a statistic that does not rely on the detection of multiple peptides is especially useful when the sample replicates are too low to use a typical statistical test such as a t - test .
plgem - stn was a method that fits this criterion . however , plgem - stn alone was not strict enough to control the false discovery rate without further diminishing the number of positives ( figure 7 ) .
the lack of stringency by using the plgem - stn method alone was similar to that by using the t - test alone .
in that prior study , the lack of specificity with a t - test alone was overcome by introducing the rule mpsp .
the mpsp rule simply required that a protein be selected as differentially regulated only when it was repeatedly found so in certain number of permuted sample pairings .
the mpsp rule was introduced to deal with datasets with small replicates where other more sophisticated statistical tests could not be applied .
although the mpsp rule was originally used in combination with a t - test statistic and a fold - change threshold , this study shows that it can be used in combination with other types of statistical tests such as the plgem - stn method ( figure 7 ) .
the combination of the mpsp rule allowed the selection of differentially regulated proteins at a false discovery rate < 5% , which would have been impossible for a fold - change method , at least for the data used in this study ( see figure s3 supplementary material available online at doi:10.1155/2010/731582 ) .
the mpsp rule significantly reduced false positives while keeping the number of true positives relatively constant , thus effectively improving the statistical confidence of the selected differentially regulated proteins by lowering the false discovery rate ( table 4 ) .
the results from this and the prior study suggest that mpsp is a rule that can be used in combination with different types of statistics to select differentially regulated proteins . the label - free quantitation simplified cell culturing and sample preparation .
another useful aspect of the label - free quantitation is that peptide cross - reference could be used to increase the number of proteins quantified in all of the samples run under the same condition .
introduced the concept of accurate mass and elution time peptide tag for global protein quantitation using high resolution mass spectrometry .
one advantage of this method over using the spectral counting method is that the large number of identifications that occur in a lc / ms injection can be used as the basis for improved quantitation of another lc / ms injection [ 13 , 21 , 22 ] .
the accurate mass and elution time peptide tag approach uses the extracted ion chromatographic intensities as the quantitative measurement of peptides and proteins .
the linear response of peptide extracted ion chromatographic intensities to protein quantities was demonstrated [ 2325 ] .
this method was thus used to improve the comparability of proteins quantified between samples , among lc / ms injections , and for different isotopic forms of a protein .
the quantitation of 349 proteins from a single gel fraction for several samples clearly demonstrated the power of the peptide cross - reference feature in extracted ion chromatographic intensity - based label - free quantitative proteomics .
one drawback of extracted ion chromatographic intensity - based label - free quantitative proteomics is that the success of an analysis critically depends upon the reproducibility of lc / ms runs that have to be maintained across multiple samples .
the reproducibility of lc / ms runs across multiple samples is a prerequisite to reliable peptide cross - reference . with the advancement in lc
/ ms instrumentation and the availability of improved lc / ms chromatogram alignment methods [ 26 , 27 ] , the reproducibility of lc / ms runs is unlikely to remain an obstacle for the increasing use of label - free quantitative proteomics .
a label - free quantitative proteomics scheme was demonstrated to select differentially regulated proteins with single - peptide hits and with < 2-fold changes at a 5% false discovery rate .
the label - free quantitation scheme incorporated a labeled internal control into multiple unlabeled samples to facilitate error modeling when there were no replicates for the unlabeled samples .
the error modeling allowed the use of the plgem - stn statistic to facilitate the selection of differentially regulated proteins with single - peptide hits .
the plgem - stn statistic also facilitated the selection of differentially regulated proteins at different fold - change thresholds according to the local abundance level of the proteins .
while the plgem - stn statistic uncovered more differentially regulated proteins at higher abundance with smaller fold - changes , the plgem error modeling of local variance versus abundance overpenalized the proteins with lower abundance . with a constant fold - change threshold , however ,
thus , the results from this study showed that the plgem - stn and a constant fold - change threshold were complementary to each other and could be used simultaneously .
but , neither the plgem - stn nor the 4-fold - change criterion alone was stringent enough for selecting differentially regulated proteins at a 5% false discovery rate .
mpsp was introduced and shown to be a rule that could decrease false discovery rates when being used in combination with the plgem - stn statistic or the 4-fold - change threshold .
the mpsp rule played a critical role in extending the selection of differentially regulated proteins to those with a single - peptide hit or with a lower fold - change in label - free proteomics when sample replicates were limited .
although the approaches were demonstrated for a representative replicate - limited scenario , they potentially can also be applicable to a situation where more sample replicates are available . | when sample replicates are limited in a label - free proteomics experiment , selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single - peptide hit or a small fold - change .
this paper aims to address this issue .
an important component of the approach employed here is to utilize the rule of minimum number of permuted significant pairings ( mpsp ) to reduce false positives .
the mpsp rule generates permuted sample pairings from limited analytical replicates and simply requires that a differentially regulated protein can be selected only when it is found significant in designated number of permuted sample pairings .
both a power law global error model with a signal - to - noise ratio statistic ( plgem - stn ) and a constant fold - change threshold were initially used to select differentially regulated proteins . but both methods were found not stringent enough to control the false discovery rate to 5% in this study . on the other hand , the combination of the mpsp rule with either of these two methods significantly reduces false positives with little effect on the sensitivity to select differentially regulated proteins including those with a single - peptide hit or with a < 2-fold change . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion | more stringent thresholds are needed to control the false discovery rate when these single - peptide proteins are included for the selection of differentially regulated proteins . applied a power law global error model ( plgem ) and the signal - to - noise ratio ( stn ) statistic to select differentially regulated proteins based on a spectral counting quantitation method . with the combination of a t - test and the rule of minimum number of permuted significant pairings ( mpsp ) ,
however , the false discovery rate was significantly reduced in that study . in this study ,
the combination of mpsp and plgem - stn was tested for controlling the false discovery rate in order to extend the selection of differentially regulated proteins to those with lower fold - changes and to those with single - peptide hits . the other was to select differentially regulated proteins at smaller fold - changes and at a false discovery rate .05 . with the null distribution provided by the labeled control sample ,
different approaches were experimented with to select differentially regulated proteins by using the combination of mpsp , plgem - stn , and fold - change . this subsection describes the multiple steps leading to the extended selection of differentially regulated proteins from all quantified proteins including those with only a single - peptide hit . therefore , it is desirable to have a procedure to select regulated proteins from all of the proteins including those with a single - peptide hit to maximize the potential of the global protein expression profiling . proposed a power law global error model ( plgem ) in combination with the signal - to - noise - ratio ( stn ) test statistic for the identification of differentially expressed genes in microarray data . in this study ,
the plgem - stn statistic was experimented with for the selection of differentially regulated proteins quantified with label - free proteomics based on protein extracted ion chromatographic intensities . the mpsp rule required that only those proteins that were found differentially regulated in a certain number of permuted sample pairings were counted as positives ( for sp / rp ) or false positives ( for csp / crp ) . therefore , the combination of the mpsp rule with the plegm - stn method reduced the false discovery rate by 2 - 3 times without compromising the sensitivity . compared to the use of the plgem - stn statistic alone , the combination of plgem - stn and mpsp performs better in controlling false discovery rates without compromising the sensitivity to select differentially regulated proteins . at a 4-fold change ,
compared to the combination of plgem - stn and mpsp , however , the combination of fold - change and mpsp reduced more true positives at the similar false discovery rate of 4%-5% . nevertheless , the null distribution is only utilized to establish the relation between the signal - to - noise ratio and the peptide abundance in the plgem - stn method . however , plgem - stn alone was not strict enough to control the false discovery rate without further diminishing the number of positives ( figure 7 ) . the mpsp rule simply required that a protein be selected as differentially regulated only when it was repeatedly found so in certain number of permuted sample pairings . although the mpsp rule was originally used in combination with a t - test statistic and a fold - change threshold , this study shows that it can be used in combination with other types of statistical tests such as the plgem - stn method ( figure 7 ) . the combination of the mpsp rule allowed the selection of differentially regulated proteins at a false discovery rate < 5% , which would have been impossible for a fold - change method , at least for the data used in this study ( see figure s3 supplementary material available online at doi:10.1155/2010/731582 ) . the mpsp rule significantly reduced false positives while keeping the number of true positives relatively constant , thus effectively improving the statistical confidence of the selected differentially regulated proteins by lowering the false discovery rate ( table 4 ) . a label - free quantitative proteomics scheme was demonstrated to select differentially regulated proteins with single - peptide hits and with < 2-fold changes at a 5% false discovery rate . the error modeling allowed the use of the plgem - stn statistic to facilitate the selection of differentially regulated proteins with single - peptide hits . with a constant fold - change threshold , however ,
thus , the results from this study showed that the plgem - stn and a constant fold - change threshold were complementary to each other and could be used simultaneously . but , neither the plgem - stn nor the 4-fold - change criterion alone was stringent enough for selecting differentially regulated proteins at a 5% false discovery rate . the mpsp rule played a critical role in extending the selection of differentially regulated proteins to those with a single - peptide hit or with a lower fold - change in label - free proteomics when sample replicates were limited . | [
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] |
, chemotherapy has become the first - line therapy because of its beneficial local and systemic effects . with the advent of preoperative chemotherapy ,
however , response to chemotherapy regimen is not uniform . to date , histological assessment of tumor necrosis on the resected tumor specimens has been the gold standard method of assessing response to preoperative chemotherapy . with the effect of chemotherapy
, tumor demonstrates large area of necrosis marked with sparsely fibrillar and calcified stromal cells , tumor bone with empty turtle shells of tumor osteoid , with few viable osteocytes in the lacunae or empty lacunae . with modern therapy ,
percentage of tumor necrosis is also the most important prognostic factor for both disease - free survival and recurrence .
good responders to chemotherapy have better disease - free survival rates and can be treated with limb salvage surgery with low risk of recurrence . but poor responders should be treated aggressively with radical surgery and a changed postoperative chemotherapy regimen .
early information regarding tumor response to initial chemotherapy cycles will help tailoring subsequent treatment to achieve better control of the tumor .
many imaging techniques like conventional radiography , computed tomography ( ct ) scan , angiography , bone scintigraphy , magnetic resonance imaging ( mri ) scan , and positron emission tomography ( pet ) scan have been evaluated for predicting tumor response preoperatively .
mr imaging in various forms like static , contrast - enhanced , diffusion - weighted , dynamic contrast - enhanced with or without pharmacokinetic modeling of bone sarcomas has been applied to the study of musculoskeletal tumors with encouraging results .
dynamic contrast - enhanced mri studies the kinetics of distribution of paramagnetic contrast in the micro vessels and the interstitial space of tissues .
it detects impaired viability by detecting disappearance of tumor vascularity using the slope value of signal intensity
compared to dynamic mri , in literature , there has not been common consensus about reliability of volumetric assessment by static mri .
though few studies have reported its significant role in predicting histological response , many studies have given an equivocal report .
although increase in tumor volume correlates well with the poor histological response , reduced or stable tumor volume does not guarantee a good response .
hence , we undertook this exercise to investigate the role of both volumetric and dynamic studies , by static and dynamic mri scan , respectively , in assessment of the tumor response to preoperative chemotherapy .
this was a prospective case study done at our hospital between september 2008 and august 2010 , after obtaining approval from institute review board .
twenty - five patients of bone sarcoma with a mean age of 21.8 years ( range 12 - 40 years ) were included in this study after taking informed consent from them or their parents ( for patients of age < 18 years ) .
11 patients were excluded from the study , which included six patients of ewing 's sarcoma with widespread disease , not amenable to surgical treatment .
they were primarily treated with chemotherapy and radiotherapy . among the other five patients who were excluded from the study , two patients had parosteal variety of osteosarcoma
, one patient had already received one cycle of chemotherapy , one patient had metallic implant , and one patient could not complete three cycles due to the development of ulcer over the tumor mass . finally , 14 patients formed the study group ; 10 were male and 4 were female patients .
the tumors were located in femur in nine patients , tibia in three patients , humerus in one patient , and scapula in one patient .
this study included 1 intracompartmental and 13 extracompartmental tumor , as per enneking 's criteria .
the treatment protocol for each patient was decided in tumor board meeting , by a team of specialists which included orthopedic surgeon , medical oncologist , radiotherapist , and pathologist .
patients were started on chemotherapy consisting of three drugs cisplatin [ 100 mg / m body surface area ( bsa ) in three divided doses over 3 days ] , adriamycin ( 50 mg / m bsa single dose ) , and ifosfamide ( 1.5 g / m bsa daily for 3 days ) .
we did not add methotrexate to the chemotherapy regimen due to lack of monitoring facility .
after inclusion , static mri followed by dynamic study was performed in all 14 patients .
mri study was performed with 1.5 t magnet ( siemens , tim technology , avanto , germany ) .
static mri was performed first in coronal , sagittal , and axial planes . both t1-weighted image ( spin echo , tr / te = 500 ms/12 ms , number of excitation 1 ) and t2-weighted images ( fast spin echo , tr / te = 3500 - 4000 ms/120 ms , 180 flip angle ,
120 khz band width , 15 turbo factor , number of excitations 2 ) were obtained with slice thickness 5 mm , interslice gap 0.5 mm , 340 mm field of view ( fov ) , and 512 256 image matrixes .
immediately after the first sequence , bolus injection of gadolinium diethylene ( 0.1 mmol / kg ) was administered intravenously within a period of 15 s. it was followed by five fast low angle shot ( flash ) sequences without pause , for a total of 6 min ( acquisition time of 72 s per sequence ) .
acquisition parameters for flash sequences included echo delay time of 1.45 ms , t1w1 ( tr / te = 4.27/1.45 ) , 0.9 0.8 0.9 mm voxel size , 358 448 image matrixes , 6 flip angle , 340 mm fov , 45 - 65 number of slices with 0.9 mm slice thickness , and use of surface coil and spine matrix coil .
tumor height , width , and depth were measured from the axial , sagittal , and coronal images in the static mri .
tumor volume ratio ( tumor volume after treatment divided by tumor volume before treatment ) was calculated .
changes in tumor volume were classified as increased ( ratio > 1.05 ) , stable ( ratio between 0.95 and 1.05 ) , or decreased ( ratio < 0.95 ) . decreased or stable tumor volume indicated good response to chemotherapy . in a software program for dynamic analysis ,
due to heterogeneity in enhancement , three circular regions of interest of around 1 cm diameter were selected in the areas of maximum enhancement .
signal intensity was measured and plotted against time in a graph . in all three regions of interest ,
slope of the curve ( percentage increase in signal intensity per minute over the baseline value ) was calculated using the equation
slope ( % /minute ) = ( simax siprior ) 100/(siprior tmax ) .
siprior is the baseline signal intensity before contrast , simax is signal intensity at tmax , and tmax is the time point at which si / siprior increased by at least 3% from image to image .
average of three slope values was considered as the final pre - chemotherapy dynamic mri slope value .
again , in post - chemotherapy dynamic mr image , three regions of similar size were selected in exactly the same areas as visualized in pre - chemotherapy image and the slope was calculated .
slope values obtained before chemotherapy were compared with the values after chemotherapy and the difference was calculated . more than 60% reduction in slope value after chemotherapy was considered as good response .
after completion of preoperative chemotherapy , patients were taken for surgery with a mean time interval of 25 days between chemotherapy and surgery .
twelve patients were operated with limb salvage surgery [ nine tumor resection , extracorporeal irradiation and reconstruction ( ecir ) ; two tumor resection and reconstruction with custom - made megaprosthesis ( cmp ) ; and one resection of scapula ] .
amputation was performed in two patients ( one transfemoral amputation and one forequarter amputation ) .
after surgical resection of the tumor , the specimens were sent to pathology laboratory and were evaluated histologically .
each specimen was placed in formalin and then sliced coronally or axially at its largest cross section .
five - millimeter - thick slice was prepared which was cut into multiple tissue bits of 5 - 10 mm length and 5 - 10 mm breadth .
the tissue bits were decalcified and processed further using xylene , alcohol , and formalin .
after embedding , 3.5 - 4 mm thin sections were cut , mounted , stained with hematoxylin and eosin stain , and then viewed under microscope for necrosis and viable tumor cells .
percentage area of necrosis was calculated in each slide and summed up to give percentage necrosis of the whole tumor .
tumor necrosis was scored according to huvos grading ( grade i , less than 50% necrosis ; grade ii , 50 - 90% necrosis ; grade iii , more than 90% necrosis with some foci of viable tumor cells ; and grade iv , 100% necrosis with no viable tumor cells ) .
at least 90% necrosis ( grades iii and iv ) was considered as good response to preoperative chemotherapy .
based on this histological response of the tumor , patients were divided into two groups , i.e. good responders and poor responders .
those with > 90% necrosis ( grades iii and iv ) were considered as good responders and those with < 90% necrosis were considered as poor responders .
the overall degree of relationship between the change in radiological parameters and histological necrosis was assessed by means of pearson 's correlation analysis using correlation coefficient , rp .
the criterion for statistical significance with a two - tailed test was chosen at a p value of less than 0.05 .
both the parameters were individually correlated with the histological response . using the cut - off criteria for positive response ,
statistical analysis was done using spss software for windows ( version 16.0 , spss inc ) .
twenty - five patients of bone sarcoma with a mean age of 21.8 years ( range 12 - 40 years ) were included in this study after taking informed consent from them or their parents ( for patients of age < 18 years ) .
11 patients were excluded from the study , which included six patients of ewing 's sarcoma with widespread disease , not amenable to surgical treatment .
they were primarily treated with chemotherapy and radiotherapy . among the other five patients who were excluded from the study , two patients had parosteal variety of osteosarcoma
, one patient had already received one cycle of chemotherapy , one patient had metallic implant , and one patient could not complete three cycles due to the development of ulcer over the tumor mass . finally , 14 patients formed the study group ; 10 were male and 4 were female patients .
the tumors were located in femur in nine patients , tibia in three patients , humerus in one patient , and scapula in one patient .
this study included 1 intracompartmental and 13 extracompartmental tumor , as per enneking 's criteria .
the treatment protocol for each patient was decided in tumor board meeting , by a team of specialists which included orthopedic surgeon , medical oncologist , radiotherapist , and pathologist .
patients were started on chemotherapy consisting of three drugs cisplatin [ 100 mg / m body surface area ( bsa ) in three divided doses over 3 days ] , adriamycin ( 50 mg / m bsa single dose ) , and ifosfamide ( 1.5 g / m bsa daily for 3 days ) .
we did not add methotrexate to the chemotherapy regimen due to lack of monitoring facility .
after inclusion , static mri followed by dynamic study was performed in all 14 patients .
mri study was performed with 1.5 t magnet ( siemens , tim technology , avanto , germany ) .
static mri was performed first in coronal , sagittal , and axial planes . both t1-weighted image ( spin echo , tr / te = 500 ms/12 ms , number of excitation 1 ) and t2-weighted images ( fast spin echo , tr / te = 3500 - 4000 ms/120 ms , 180 flip angle , 120 khz band width , 15 turbo factor , number of excitations 2 ) were obtained with slice thickness 5 mm , interslice gap 0.5 mm , 340 mm field of view ( fov ) , and 512 256 image matrixes .
immediately after the first sequence , bolus injection of gadolinium diethylene ( 0.1 mmol / kg ) was administered intravenously within a period of 15 s. it was followed by five fast low angle shot ( flash ) sequences without pause , for a total of 6 min ( acquisition time of 72 s per sequence ) .
acquisition parameters for flash sequences included echo delay time of 1.45 ms , t1w1 ( tr / te = 4.27/1.45 ) , 0.9 0.8 0.9 mm voxel size , 358 448 image matrixes , 6 flip angle , 340 mm fov , 45 - 65 number of slices with 0.9 mm slice thickness , and use of surface coil and spine matrix coil .
tumor height , width , and depth were measured from the axial , sagittal , and coronal images in the static mri . then , tumor volume was calculated using the formula for ellipsoid mass
tumor volume ratio ( tumor volume after treatment divided by tumor volume before treatment ) was calculated .
changes in tumor volume were classified as increased ( ratio > 1.05 ) , stable ( ratio between 0.95 and 1.05 ) , or decreased ( ratio < 0.95 ) . decreased or stable tumor volume indicated good response to chemotherapy . in a software program for dynamic analysis , all dynamic sequences were loaded .
imaging plane was selected representing largest area of the tumor . due to heterogeneity in enhancement ,
three circular regions of interest of around 1 cm diameter were selected in the areas of maximum enhancement .
in all three regions of interest , slope of the curve ( percentage increase in signal intensity per minute over the baseline value ) was calculated using the equation
siprior is the baseline signal intensity before contrast , simax is signal intensity at tmax , and tmax is the time point at which si / siprior increased by at least 3% from image to image .
average of three slope values was considered as the final pre - chemotherapy dynamic mri slope value .
again , in post - chemotherapy dynamic mr image , three regions of similar size were selected in exactly the same areas as visualized in pre - chemotherapy image and the slope was calculated .
slope values obtained before chemotherapy were compared with the values after chemotherapy and the difference was calculated . more than 60% reduction in slope value after chemotherapy was considered as good response .
after completion of preoperative chemotherapy , patients were taken for surgery with a mean time interval of 25 days between chemotherapy and surgery .
twelve patients were operated with limb salvage surgery [ nine tumor resection , extracorporeal irradiation and reconstruction ( ecir ) ; two tumor resection and reconstruction with custom - made megaprosthesis ( cmp ) ; and one resection of scapula ] .
amputation was performed in two patients ( one transfemoral amputation and one forequarter amputation ) .
after surgical resection of the tumor , the specimens were sent to pathology laboratory and were evaluated histologically .
each specimen was placed in formalin and then sliced coronally or axially at its largest cross section .
five - millimeter - thick slice was prepared which was cut into multiple tissue bits of 5 - 10 mm length and 5 - 10 mm breadth .
the tissue bits were decalcified and processed further using xylene , alcohol , and formalin .
after embedding , 3.5 - 4 mm thin sections were cut , mounted , stained with hematoxylin and eosin stain , and then viewed under microscope for necrosis and viable tumor cells .
percentage area of necrosis was calculated in each slide and summed up to give percentage necrosis of the whole tumor .
tumor necrosis was scored according to huvos grading ( grade i , less than 50% necrosis ; grade ii , 50 - 90% necrosis ; grade iii , more than 90% necrosis with some foci of viable tumor cells ; and grade iv , 100% necrosis with no viable tumor cells ) .
at least 90% necrosis ( grades iii and iv ) was considered as good response to preoperative chemotherapy .
based on this histological response of the tumor , patients were divided into two groups , i.e. good responders and poor responders .
those with > 90% necrosis ( grades iii and iv ) were considered as good responders and those with < 90% necrosis were considered as poor responders .
the overall degree of relationship between the change in radiological parameters and histological necrosis was assessed by means of pearson 's correlation analysis using correlation coefficient , rp .
the criterion for statistical significance with a two - tailed test was chosen at a p value of less than 0.05 .
both the parameters were individually correlated with the histological response . using the cut - off criteria for positive response ,
statistical analysis was done using spss software for windows ( version 16.0 , spss inc ) .
the necrotic foci showed cellular degeneration with sparsely fibrillar , calcified stromal cells and empty tumor osteoid .
considering necrosis of the tumor , according to huvos grading , nine patients were poor responders ( six grade i and three grade ii ) and five were good responders ( three grade iii and two grade iv ) . in two cases of poor response
pre - chemotherapy tumor volume of all patients ranged from 70.73 to 4244.42 cm ( mean 852.8 cm ) . following chemotherapy ,
increase in tumor volume ( tumor volume ratio > 1.05 ) was seen in six poor responders ( five huvos grade i and one grade ii necrosis ) and one good responder ( grade iii necrosis ) .
reduction of volume ( tumor volume ratio < 0.95 ) was seen in four good responders ( two grade iii and two grade iv necrosis ) and three poor responders ( one grade i and two grade ii necrosis ) .
none of the patients showed stable tumor volume . among the five good responders , four patients showed 9.39 - 39.20% ( 59.8 - 761.85 cm ) decrease in volume and one patient showed 32.88% ( 207.15 cm ) increase in volume . among the nine poor responders , the tumor volume increased by 6.47 - 76.62% ( 12.66 - 774.35 cm ) in six patients , while three patients experienced reduction by 9.91 - 36.13% ( 25.92 - 112.76 cm ) .
no statistically significant correlation was found between change in tumor volume and histological response ( rp = 0.496 , p = 0.071 ) as depicted in figure 1 .
predictive value of decreased tumor volume for a good response was 57.14% ( four of seven patients ) , and predictive value of increased tumor volume for a poor response was 85.7% ( six of seven patients ) .
we observed changes in all three dimensions of the tumor mass including longitudinal dimension ( height of the tumor ) in post - chemotherapy static mr imaging .
correlation of tumor volume change with histological necrosis the mean dynamic mri slope value for all patients before chemotherapy was 41.53% per minute ( range 15.33 - 67.09% per minute ) .
following preoperative chemotherapy , there was fall in slope values in 12 patients and increase in 2 patients .
post - chemotherapy mean slope value for five good responders was 15.52% per minute ( range 11.27 - 22.7% ) and for nine poor responders , it was 26.66% per minute ( range 10.41 - 40.29% ) .
there was reduction in slope value of an average of 67.55% of the pre - chemotherapy value in good responders and of 20.21% in poor responders .
one responding patient showed less than 60% reduction in slope ( 55.97% ) and one poor responder showed more than 60% reduction in slope ( 61.46% ) .
these two patients were classified incorrectly , using criteria of 60% reduction in slope following chemotherapy .
figures 2a , b and 3a , b show dynamic mr images and signal intensity
time curves in a case with 50% necrosis , respectively , and figures 4a , b , and 5a ,
( a and b ) : ( a ) pre - chemotherapy dynamic mr image of osteosarcoma of distal femur in a 15-year - old girl showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 67.09% ( b ) post - chemotherapy signal intensity time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 34.13% ( 49.13% reduction in slope value following chemotherapy ) ( a ) pre - chemotherapy dynamic mr image of telangiectatic osteosarcoma of proximal tibia in a 15-year - old boy showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 36.7% ( b ) post - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 21.9% ( 40.3% reduction in slope value following chemotherapy ) significant correlation was found between percentage change in slope and tumor necrosis ( rp = 0.894 , p = 0.001 ) as depicted in figure 6 .
more than 60% reduction in slope was predictive of good response [ positive predictive value ( ppv ) = 80% ] , while less than 60% reduction was predictive of poor response [ negative predictive value ( npv ) = 88.89% ] . while pre - chemotherapy slope value did not correlate well with necrosis ( rp = 0.384 , p = 0.175 ) , post - chemotherapy slope value showed significant negative correlation with tumor necrosis ( rp = 0.706 , p = 0.004 ) .
correlation of change in dynamic mri slope with histological necrosis table 1 shows the changes in tumor volume and dynamic mri slope , and tumor necrosis in response to preoperative chemotherapy in patients in different huvos grades .
changes in static and dynamic mri parameters ( tumor volume and dynamic mri slope ) and histological necrosis in response to preoperative chemotherapy
the necrotic foci showed cellular degeneration with sparsely fibrillar , calcified stromal cells and empty tumor osteoid .
considering necrosis of the tumor , according to huvos grading , nine patients were poor responders ( six grade i and three grade ii ) and five were good responders ( three grade iii and two grade iv ) . in two cases of poor response
pre - chemotherapy tumor volume of all patients ranged from 70.73 to 4244.42 cm ( mean 852.8 cm ) . following chemotherapy ,
increase in tumor volume ( tumor volume ratio > 1.05 ) was seen in six poor responders ( five huvos grade i and one grade ii necrosis ) and one good responder ( grade iii necrosis ) .
reduction of volume ( tumor volume ratio < 0.95 ) was seen in four good responders ( two grade iii and two grade iv necrosis ) and three poor responders ( one grade i and two grade ii necrosis ) .
, four patients showed 9.39 - 39.20% ( 59.8 - 761.85 cm ) decrease in volume and one patient showed 32.88% ( 207.15 cm ) increase in volume . among the nine poor responders , the tumor volume increased by 6.47 - 76.62% ( 12.66 - 774.35 cm ) in six patients , while three patients experienced reduction by 9.91 - 36.13% ( 25.92 - 112.76 cm ) .
no statistically significant correlation was found between change in tumor volume and histological response ( rp = 0.496 , p = 0.071 ) as depicted in figure 1 .
predictive value of decreased tumor volume for a good response was 57.14% ( four of seven patients ) , and predictive value of increased tumor volume for a poor response was 85.7% ( six of seven patients ) .
we observed changes in all three dimensions of the tumor mass including longitudinal dimension ( height of the tumor ) in post - chemotherapy static mr imaging .
the mean dynamic mri slope value for all patients before chemotherapy was 41.53% per minute ( range 15.33 - 67.09% per minute ) . following preoperative chemotherapy , there was fall in slope values in 12 patients and increase in 2 patients .
post - chemotherapy mean slope value for five good responders was 15.52% per minute ( range 11.27 - 22.7% ) and for nine poor responders , it was 26.66% per minute ( range 10.41 - 40.29% ) .
there was reduction in slope value of an average of 67.55% of the pre - chemotherapy value in good responders and of 20.21% in poor responders .
one responding patient showed less than 60% reduction in slope ( 55.97% ) and one poor responder showed more than 60% reduction in slope ( 61.46% ) .
these two patients were classified incorrectly , using criteria of 60% reduction in slope following chemotherapy .
figures 2a , b and 3a , b show dynamic mr images and signal intensity time curves in a case with 50% necrosis , respectively , and figures 4a , b , and 5a , b show the same in a case with no necrosis . ( a and b ) : ( a ) pre - chemotherapy dynamic mr image of osteosarcoma of distal femur in a 15-year - old girl showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 67.09% ( b ) post - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 34.13% ( 49.13% reduction in slope value following chemotherapy ) ( a ) pre - chemotherapy dynamic mr image of telangiectatic osteosarcoma of proximal tibia in a 15-year - old boy showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 36.7% ( b ) post - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 21.9% ( 40.3% reduction in slope value following chemotherapy ) significant correlation was found between percentage change in slope and tumor necrosis ( rp = 0.894 , p = 0.001 ) as depicted in figure 6 .
more than 60% reduction in slope was predictive of good response [ positive predictive value ( ppv ) = 80% ] , while less than 60% reduction was predictive of poor response [ negative predictive value ( npv ) = 88.89% ] . while pre - chemotherapy slope value did not correlate well with necrosis ( rp = 0.384 , p = 0.175 ) , post - chemotherapy slope value showed significant negative correlation with tumor necrosis ( rp = 0.706 , p = 0.004 ) .
correlation of change in dynamic mri slope with histological necrosis table 1 shows the changes in tumor volume and dynamic mri slope , and tumor necrosis in response to preoperative chemotherapy in patients in different huvos grades .
changes in static and dynamic mri parameters ( tumor volume and dynamic mri slope ) and histological necrosis in response to preoperative chemotherapy
reduction in the tumor volume occurs due to necrosis , decrease in the volume of normal supporting stroma , and resolution of inflammatory edema .
many studies have been done in the past to find out the usefulness of tumor volume measured by ct or mri to predict histological response .
as ct does not provide good contrast to show the distinction between extraosseous tumor and adjacent normal tissue , volume measurement may be inaccurate with ct .
few studies have found significant correlation between change in tumor volume assessed by mri and histological necrosis .
although , in our study , change in tumor volume showed trend toward correlation with necrosis ( rp = 0.497 ) , this correlation was not found to be significant ( p = 0.071 ) .
these findings are supported by other studies which also failed to find a significant correlation .
although changes in volume do not accurately reflect the extent of necrosis , increase in volume is associated with poor histological response and decrease in volume indicates good response .
we also found increased tumor volume as a better predictor of poor response ( npv = 85.7% ) but decreased tumor volume as a poor predictor of good response to chemotherapy ( ppv = 57.14% ) .
all our patients , except one , had extracompartmental tumor , i.e. , tumor spreading beyond well delineated surgical compartment of its origin .
review of literature shows that most of the changes occur in vascularity or in size of the extraosseous component of the tumor .
did not find any change in the intramedullary extent of the tumor , i.e. the longitudinal dimension .
in contrast , in our study , we found changes occurring in all three dimensions of the tumor , including the intraosseous length .
in dynamic mri , the pharmacokinetics of the intravenously injected gadolinium is studied . in this , the slope of signal intensity time curve indirectly reflects the change in vascularity and , hence , the viability of the tumor .
slope value reflects the increase in signal intensity per unit time in a specific area of tumor , i.e. rate of uptake in the vascularized portion of the tumor .
it shows steeper slope in the vascularized portion and gradual slope in the area of necrosis .
dynamic study by contrast - enhanced mri detects impaired viability by detecting the disappearance of tumor vascularity with the effect of chemotherapy , indirectly by measuring reduction in slope values over consecutive mri .
it helps differentiating regions of necrosis , viable tumor , muscle , and blood vessels , as they display distinct signal intensity
it also helps differentiating extraosseous tumor , muscle infiltrated by the tumor , edematous muscle , and normal muscle .
recently , in a study by guo et al . , they found significant correlation between histological response and the pharmacokinetic parameters of dynamic contrast -enhanced mri .
they also found its efficacy in prediction of event -free and overall survival of osteosarcoma patients .
tumor viability is also indicated by rapid upward slope ( greater than 30% per minute ) , early enhancement of areas within 6 s after arrival of the bolus in a feeding artery , and early wash in and wash out of the contrast agent .
more gradual slopes are observed in areas of necrosis , cystic areas , edema , peritumoral inflammation , and paucicellular cartilaginous and myxoid regions .
dynamic mri has the advantage of correctly determining the area of viable tumor by showing different enhancement pattern and change in slope values .
the prediction of histological response by dynamic mr imaging has proved to be very significant in our study .
this is in accordance with the findings of other studies which establish its role in assessing necrosis in malignant bone tumor .
the cut - off criteria of > 60% reduction in the slope of erlemann et al
. could predict good histological response in 80% of our patients , while correctly predicting poor response in 88.89% of patients .
slope value of less than 40% per minute after full course of neoadjuvant chemotherapy indicated good response to it .
but in our study , all the patients showed less than 40% post - chemotherapy slope value , except one poor responder who showed more than 40% slope value .
procedural errors in performing contrast mri were minimized by consistency of the method of contrast injection , using the portal at peripheral site .
others factors like chemotherapy - induced cardiomyopathy and infection that could lead to relative change in uptake of contrast by tumor did not develop in any patient .
the present study proves the efficacy of dynamic mri as an accurate , quantitative , and noninvasive method of assessing response of malignant bone tumors to preoperative chemotherapy .
it has the advantage of accurately localizing the sites of viable residual tumor following therapy .
this study also indicates that although tumor volume change provides some information about the responding nature of the tumor , the volumetric assessment should not be trusted as an accurate method of evaluating tumor response to preoperative chemotherapy . | objectives : preoperative chemotherapy plays a key role in management of bone sarcomas . postoperative evaluation of histological necrosis has been the gold standard method of assessing response to preoperative chemotherapy .
this study was done to evaluate the efficacy of static and dynamic magnetic resonance imaging ( mri ) for assessing response preoperatively.materials and methods : our study included 14 patients ( 12 osteosarcomas and 2 malignant fibrous histiocytomas ) with mean age of 21.8 years , treated with preoperative chemotherapy followed by surgery .
they were evaluated with static and dynamic mri twice , before starting chemotherapy and again prior to surgery .
change in tumor volume and slope of signal intensity - time curve were calculated and correlated with percentage of histological necrosis using pearson correlation test.results:the change in dynamic mri slope was significant ( p = 0.001 ) .
also , 60% reduction in slope of the curve proved to be an indicator of good histological response [ positive predictive value ( ppv ) = 80% ] .
change in tumor volume failed to show significant correlation ( p = 0.071 ) .
although it showed high negative predictive value ( npv = 85.7% ) , ppv was too low ( ppv = 57.14%).conclusions : dynamic mri correctly predicts histological necrosis after administration of preoperative chemotherapy to bone sarcomas .
hence , it can be used as a preoperative indicator of response to neoadjuvant chemotherapy . on the other hand , volumetric assessment by static mri
is not an effective predictor of histological necrosis .
this study proves the superiority of dynamic contrast - enhanced study over volumetric study by mri . | Introduction
Materials and Methods
Patients
MRI evaluation
Histological evaluation
Statistical analysis
Results
Histological evaluation of tumor necrosis
Tumor volume change
Dynamic MRI slope
Discussion
Conclusion | to date , histological assessment of tumor necrosis on the resected tumor specimens has been the gold standard method of assessing response to preoperative chemotherapy . figures 2a , b and 3a , b show dynamic mr images and signal intensity
time curves in a case with 50% necrosis , respectively , and figures 4a , b , and 5a ,
( a and b ) : ( a ) pre - chemotherapy dynamic mr image of osteosarcoma of distal femur in a 15-year - old girl showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 67.09% ( b ) post - chemotherapy signal intensity time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 34.13% ( 49.13% reduction in slope value following chemotherapy ) ( a ) pre - chemotherapy dynamic mr image of telangiectatic osteosarcoma of proximal tibia in a 15-year - old boy showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 36.7% ( b ) post - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 21.9% ( 40.3% reduction in slope value following chemotherapy ) significant correlation was found between percentage change in slope and tumor necrosis ( rp = 0.894 , p = 0.001 ) as depicted in figure 6 . more than 60% reduction in slope was predictive of good response [ positive predictive value ( ppv ) = 80% ] , while less than 60% reduction was predictive of poor response [ negative predictive value ( npv ) = 88.89% ] . correlation of change in dynamic mri slope with histological necrosis table 1 shows the changes in tumor volume and dynamic mri slope , and tumor necrosis in response to preoperative chemotherapy in patients in different huvos grades . ( a and b ) : ( a ) pre - chemotherapy dynamic mr image of osteosarcoma of distal femur in a 15-year - old girl showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 67.09% ( b ) post - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 34.13% ( 49.13% reduction in slope value following chemotherapy ) ( a ) pre - chemotherapy dynamic mr image of telangiectatic osteosarcoma of proximal tibia in a 15-year - old boy showing greater enhancement ( b ) post - chemotherapy dynamic mr image showing reduced enhancement following chemotherapy ( a ) pre - chemotherapy signal intensity time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 36.7% ( b ) post - chemotherapy signal intensity
time curve [ signal intensity on the x - axis and time ( minute ) on the y - axis ] showing dynamic mri slope value of 21.9% ( 40.3% reduction in slope value following chemotherapy ) significant correlation was found between percentage change in slope and tumor necrosis ( rp = 0.894 , p = 0.001 ) as depicted in figure 6 . more than 60% reduction in slope was predictive of good response [ positive predictive value ( ppv ) = 80% ] , while less than 60% reduction was predictive of poor response [ negative predictive value ( npv ) = 88.89% ] . correlation of change in dynamic mri slope with histological necrosis table 1 shows the changes in tumor volume and dynamic mri slope , and tumor necrosis in response to preoperative chemotherapy in patients in different huvos grades . although , in our study , change in tumor volume showed trend toward correlation with necrosis ( rp = 0.497 ) , this correlation was not found to be significant ( p = 0.071 ) . we also found increased tumor volume as a better predictor of poor response ( npv = 85.7% ) but decreased tumor volume as a poor predictor of good response to chemotherapy ( ppv = 57.14% ) . the present study proves the efficacy of dynamic mri as an accurate , quantitative , and noninvasive method of assessing response of malignant bone tumors to preoperative chemotherapy . | [
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in ancient times , medical practitioners treated patients through shamanism or used materials that were available in their area , such as plants , animals or alcohol . however , these practitioners may not have utilized therapeutic knowledge , and patients may have not received satisfactory medical care . over time , by trial and error , therapeutic strategies using readily available materials were established , as demonstrated by evidence unearthed from ancient tombs ( e.g. , the mawangdui tomb ) , including the wushier bing fang ( ) . over time , medical information accumulated , and medical practitioners used many suitable medicinal plants or animals for the treatment of diseases , including ginseng .
ginseng is a medicinal plant that has been used in medical practices for more than 2,000 years .
the shennong bencao jing ( shennong 's herbal classic , ) , one of the first books specializing in herbal medicine , was edited in the 1st century ad . in this text , ginseng was first recognized as a medicinal herb by medical practitioners ( fig .
1 ) . in addition , in the shang - han lun ( translated as the treatise on diseases caused by cold factors , ) , zhong jing zhang ( ) described the use of ginseng in herbal prescriptions . however , because ginseng has been cultivated since the 15th century , the type of ginseng that appeared in the shennong bencao jing may have been different from that which is currently in use . in previous centuries , wild ginseng would have been collected from mountainous areas ; thus , this plant is commonly referred to as mountain ginseng .
, lugano - bioggio , switzerland , ginseng has been widely used as an active ingredient in modern medicine . for example
, the german commission e approved ginseng as a tonic for invigoration and fortification to treat fatigue , debility or declining sexual capacity .
ginseng has several beneficial properties , including providing the nourishing effects , anti - fatigue , or immune - enhancing effects , as mentioned in the traditional descriptions of the shennong bencao jing .
therefore , the characteristics of ginseng described in historical documents , such as the shennong bencao jing , the bencao gangmu ( , which was written by shizhen li [ ] during the ming dynasty ) , and the hyangyak - jibseongbang ( ) or the donguibogam ( , which was published during the chosun dynasty ) , should be examined .
the role of ginseng in traditional prescriptions should also be analyzed to expand the use of ginseng in modern medicine .
in addition , investigations on the experimental evidence in pharmacological studies of ginseng - based traditional prescriptions would yield information on how traditional knowledge or historical accounts described in the aforementioned documents can be interpreted in contemporary medical terms .
panax ginseng is a traditional medicinal plant that has been used therapeutically for millennia in east asia . in korea ,
the name panax means all healing , which describes the traditional belief that ginseng can heal all aspects of the body .
the most common ginsengs are ginseng ( p. ginseng meyer ) , chinese ginseng ( p. notoginseng [ burk . ]
fh chen ) , and american ginseng ( p. quinquefolium l. ) . among the ginseng varieties ,
therefore , p. ginseng is cultivated in limited areas , including korea , the manchuria region of china ( the region of dongbei ) , and the maritime province of siberia in russia . although it is not known whether historical documents described cultivated or wild ginseng , in the present review , we discuss historical accounts concerning p. ginseng , the role of p. ginseng in traditional prescriptions , and experimental evidence from pharmacological and clinical studies of traditional prescriptions containing p. ginseng ( table 1 ) .
summary of the pharmacological activities of ginseng - containing prescriptions described in the shang - han lun in either animal or human studies
the pharmacological functions of ginseng , which was considered a high - grade herb , were first described in the shennong bencao jing .
ginseng can nourish or tonify 5 vital organs of the body ( the spleen , lung , heart , kidney , and liver ) , has sedative properties , is used for palpitations to restore a normal pulse , dispels pathogenic factors , improves visual acuity and mental activity , and enhances longevity with long - term intake ( fig .
the shennong bencao jing was written in the 1st century by an unknown author , and the mingyi bielu ( ) , which described 365 different types of herbs , was written by hongjing tao ( , ad 456 - 536 ) .
hongjing tao also published a book titled the bencaojing jizhu ( ) , in which 730 kinds of herbs were discussed . in the mingyi bielu ,
additional properties of ginseng were described , including curing internal coldness , pain in the chest or abdomen , sensations of fullness in the chest , vomiting , and diarrhea .
ginseng can also be used to relieve thirst and feelings of solidness , to enhance cognitive function , and to improve blood circulation ( fig .
these pharmacological properties of ginseng were also described in the bencao gangmu , which is the most complete and comprehensive pre - modern herbal textbook .
in addition , shizhen li ( , 1518 - 1593 ) discussed several symptoms treatable with ginseng , including general weakness , spontaneous sweating and fever , vertigo and headache , regurgitation and vomiting , alternating fever and chills , chronic diarrhea , increased urination or stranguria , fatigue , externally contracted wind or hot attack , cramps , vomiting blood ( hematemesis ) , bleeding from the rectum , bloody urinary leakage , abnormal uterine bleeding , and discomfort before or after parturition ( fig .
several published documents have also referred to the descriptions in the shennong bencao jing , mingyi bielu , and bencao gangmu .
the hyangyak - jibseongbang was published during the chosun dynasty and referred to the pharmacological activities of ginseng described in the mingyi bielu .
the donguibogam , which was added to the world heritage list in 2009 by unesco , also described the activities of ginseng , including those described in the shennong bencao jing ( i.e. , the use of ginseng to cure general weakness , acute vomiting with diarrhea [ cholera ] , hiccups and vomiting , atrophic lung disease and phlegm ) ( fig .
therefore , p. ginseng was likely considered a medicinal herbal for the treatment of general weakness , acute vomiting with diarrhea , anxiety or mental health .
it is unclear whether ancient medical practitioners such as shizhen li ( ) , zhong jing zhang ( ) , or jun heo ( ) knew the functions of ginseng .
however , the pharmacological activities listed in historical documents are also supported by recent experimental and clinical studies ( vide infra ) .
although the pharmacological activities of ginseng were described in the shennong bencao jing , illustrations of the variety of ginseng used at that time were not provided .
the bencao gangmu described ginseng used in the baekje dynasty , and , in this case , an illustration was provided ( fig .
the baekje dynasty ( , 18 bc - ad 660 ) governed the western part of the korean peninsula and actively traded with china , suggesting that the korean peninsula might have been suitable for the cultivation of ginseng and that korean ginseng may have been used in herbal prescriptions prior to the ming dynasty .
however , the illustration of ginseng in the bencao gangmu differs slightly from that of the bencao baiyao ( ) , which was written by wang ang ( ) in 1694 during the qing dynasty .
wang ang edited the bencao baiyao and included herbs from the shennong bencao jing and the bencao gangmu . in the bencao gangmu ,
the shape of the ginseng root was described as resembling the human body or the extremities of the human body .
therefore , although the depiction of ginseng differs slightly among historical documents , the description provided in the bencao gangmu may have referred to p. ginseng .
among the 113 prescriptions ( 18.6% ) described in the shang - han lun , which was written by zhong jing zhang ( 150 - 219 ad ) , 21 prescriptions contained ginseng , including banhasasim - tang ( , ban - xia xie - xin tang in chinese or hangeshashin - to in japanese ) and sosiho - tang ( , xiao - chai - hu tang in chinese or sho - saiko - to in japanese ) . the shang- han lun covers specific symptoms of disorders and their corresponding treatments and is designated as one of the four fundamental texts of traditional chinese medicine .
in addition , among the 3,944 prescriptions ( 16.6% ) in the donguibogam , which was written by jun heo , 653 prescriptions containing ginseng as an ingredient .
several traditional prescriptions were prepared from ginseng , which was used for its restorative , tonic , nootropic , and anti - aging properties , or for dispelling pathogenic factors , as described in the shennong bencao jing . among ginseng - containing prescriptions , the classic and basic prescription is sagunja - tang ( , sijunzi tang in chinese or shikunshi - to in japanese ) .
sagunjatang was prescribed for conditions such as hypodynamia , lassitude and anorexia . as a main ingredient in sagunjatang , ginseng exerts various pharmacological activities , such as organ tonification and restorative activities , as described in the shennong bencao jing and bencao gangmu .
these pharmacological activities were also accomplished by sipjeondaebo - tang ( , shiquan - da - bu - tang in chinese or juzen - taiho - to in japanese ) , which was composed of 10 types of herbs , including four herbs ( ginseng radix , atractylodis rhizoma alba , poria sclerotium , glycyrrhizae radix et rhizoma ) from sagunja - tang , four herbs ( angelicae gigantis radix , cnidii rhizoma , paeoniae radix , rehmanniae radix preparata ) from samul - tang ( , si - wu - tang in chinese , and shimotsu - to in japanese ) and additional herbal materials , such as astragali radix ( astragalus membranaceus ) and cinnamomi cortex ( cinnamomum cassia ) .
sipjeondaebo - tang was prescribed to patients who suffered from weakness or spontaneous sweating , which were described as being treatable with ginseng in the bencao gangmu .
in addition , doksam - tang ( , dushen tang in chinese or dokujin - to in japanese ) , which was only composed of ginseng , was used for seriously ill patients , as depicted in the shi yao shen shu ( ) , which was written by ke - jiu ge ( ) in 1348 during the yuan dynasty . in the shennong bencao jing ,
ginseng was said to dispel pathogenic factors . in the donguibogam , talmyung - san ( identical to doksam - tang )
recently , an et al . reported the effects of ginseng formulae on stable chronic obstructive pulmonary disease , which represents a pattern of qi deficiency involving either the lung or the spleen .
for seriously ill patients , ginseng can also be used with aconiti root , which is found in sambu - tang ( , shen fu tang in chinese ) , as described in the fu ren da quan liang fang ( ) , which was written by zi- ming chen ( ) during the song dynasty . in the aforementioned formulae ,
ginseng is the main active ingredient and exerts restorative , tonic or nourishing effects on hypodynamia and ameliorates the effects of serious illness , as described in the shennong bencao jing and the bencao gangmu .
although jeongji - whan prescription ( , dingzhi wan in chinese ) is listed in the yixue xinwu ( ) , which was written by goupeng cheng ( ) in 1732 , this prescription has not been certified as a herbal prescription in japan . however , in the mid-1990s , the nishiyama group in japan reported the effects of dx-9386 , which possesses ingredients that are identical to those of jeongji - whan , on brain function .
the jeongjiwhan prescription was described as being active against anxiety , palpitation , and amnesia , and ginseng was used as the main ingredient .
kami - guibi - tang ( , jia wei gu pi tang in chinese or kami - kihi - to in japanese ) , or guibi - tang ( , gu pi tang in chinese or kihi - to in japanese ) , was described in classical literature as being effective for the treatment of insomnia , anemia , amnesia , depression , and neurosis .
suggested that guibi - tang is an attractive candidate as an anti - dementia drug .
thus , although the improvement in brain function was not solely dependent on ginseng , these prescriptions made use of the effects of ginseng on anxiety , amnesia , and palpitation , which were described in the shennong bencao jing . in particular , the mingyi bielu stated that ginseng relieves thirst ( sogal in korean , xiao ke in chinese or shoukatsu in japanese , ) , and the bencao gangmu referred to the description of ginseng in the mingyi bielu .
as described in historical documents , the symptoms of thirst are similar to those of diabetes .
baekho - ga - insamtang ( , byakko - ka - ninjin - to in japanese or bai hu jia ren shen tang in chinese ) , which was a shang - han lun formula , relieves thirst caused by internal fever .
in addition to the aforementioned pharmacological properties , ginseng can also be used to alleviate diarrhea and vomiting .
insam - tang ( , ninjin - to in japanese or ren shen tang in chinese ) , which is composed of ginseng radix , glycyrrhizae radix , atractyloides rhizome , and zingiberis rhizome , has been used for the treatment of diarrhea or anorexia and tonifies the qi of the spleen . in banhasasim - tang , ginseng is used as a coingredient . even when included as co - ingredient in banhasasim - tang , ginseng enhances qi and contributes to the alleviation of diarrhea or intestinal catarrh .
recently , taiwanese doctors investigated the most common prescriptions from the shang - han lun .
banhasasimtang and sosiho - tang ( sho - saiko - to in japanese and xiao chai hu tang in chinese ) were prescribed at 10.24% and 9.11% in the shan - han lun formulae , respectively .
additionally , in the shennong bencao jing , ginseng is listed as an anti - aging factor .
kyungok - ko , a sticky extract composed of ginseng radix , rehmnania juice , hoelen ( poria sclerotium ) , and honey , was described as having anti - aging activities when administered for an extended period of time .
thus , various pharmacological activities of ginseng were described along with those of other active ingredients . in doksam - tang ,
the ginseng content is equal to 37.5 g , and ginseng is the active ingredient in this prescription . according to the german commission e , crude preparations of 1 to 2 g of dried root powder of ginseng
a decoction can be prepared by simmering 3 to 9 g of dried ginseng root in 720 to 960 ml of water . in decoction
, ginseng is included at a dosage of 3 to 9 g as the main ingredient in most prescriptions , except for doksamtang . in some formulations , such as kami - guibi - tang or guibi - tang ,
thus , ginseng has been used at dosages ranging from 3 to 37.5 g. in chemical drugs , the amount of intake rarely exceeds 5 to 10 times the usual dose .
however , p. ginseng is well tolerated , and its adverse effects are mild and reversible .
over the last several decades , traditional chinese medicine has become a significant form of complementary and alternative medicine used by patients in europe and north america .
in addition to traditional chinese medicine , traditional japanese medicine ( kampo ) and traditional korean medicine ( hanbang ) , which are referred to as traditional herbal medicine in this review , have been accepted as a form of complementary and alternative medicine by patients . in western medicine ,
randomized clinical trials are generally accepted as the most reliable approach to testing the efficacy of medicines and treatments ; however , in traditional herbal medicine , clinical evidence was observed and recorded descriptively in the literature .
although the theoretical frameworks of western medicine and traditional herbal medicine are different , scientific studies on the efficacy of traditional herbal medicine using randomized clinical trial techniques are becoming more popular .
such evidence - based medicine integrates the best evidence from research with clinical expertise and patient values . using clinical trials or basic scientific research ,
the efficacy of traditional prescriptions for specific diseases can be determined , which has the potential to expand the use of traditional formulae .
western research models are used in japan to study traditional medicine , and the approach is based on conventional western disease nosology and conventional immunology . to date , most traditional herbal medicine experiments have been performed in vivo on animals or humans by japanese scientists , and the results have been published in peer - reviewed international journals . sipjeondaebo - tang is the most commonly investigated herbal medicine by scientists and clinicians .
traditionally , sipjeondaebo - tang , a decoction with 10 medicinal herbs , has been used to treat patients with anemia , anorexia , or fatigue .
the pharmacological activities of sipjeondaebotang affect the systemic immune functions of t and b cells , macrophages and nk cells and cells of the hematopoietic and intestinal immune system .
evaluated the effects of sipjeondaebo - tang in patients receiving hemodialysis with erythropoietin - resistant anemia .
their results showed that sipjeondaebotang improved erythropoietin - resistant anemia in endstage renal disease patients , and similar findings were observed in the perioperative period .
in addition , sipjeondaebo - tang exerted a protective influence against intractable and recurrent infections in immature immune systems .
thus , the effects of sipjeondaebo - tang on the immune system have been confirmed by clinical trials and animal experiments , providing experimental evidence in support of the traditional use of sipjeondaebo - tang .
bojungikki - tang ( , bu - zhong - yi - qi - tang in chinese or hochu - ekki - to in japanese ) has been widely used in china , japan , and korea for chronic fatigue syndrome and is composed of 10 species of medicinal herbs . in particular , in bojungikki - tang , ginseng radix and astragali radix are used in higher proportions .
several clinical trials were conducted to examine whether this formulation alleviates cancer - related fatigue and improves immunological capacity in elderly patients .
the results of these trials suggested that bojungikki - tang could help to improve age- or disease - related impairment in immune function .
animal studies also showed that bojungikki - tang exhibits immunopharmacological activity against microbial infections , and positive results were obtained in a murine model of chronic fatigue syndrome .
although the main active ingredient in bojungikki - tang has not been elucidated , ginseng radix and astragali radix could contribute to the effects of bojungikki - tang .
insamyangyoung - tang ( , ren - shen - yang - rong - tang in chinese or ninjin - youeito in japanese ) is another prescription for the modulation of physiological immunity .
several ginseng - containing prescriptions , including daegeonjung - tang ( , da jian zhong tang in chinese or dai - kenchu - to in japanese ) , insam - tang ( , ninjin - to in japanese or ren shen tang in chinese ) , and banhasasim - tang , have been used to treat gastrointestinal problems , such as gastric atony , vomiting and anorexia .
substantial effort has been made to elucidate the pharmacological activities of these formulations . in a human study ,
daegeonjung - tang increased gastrointestinal motility and improved ileal function by increasing motilin and vasoactive intestinal peptide levels in the plasma .
postoperative ileus is an adverse consequence of abdominal surgery and leads to prolonged periods of hospitalization and increased healthcare costs .
itoh et al . reported the effectiveness of daegeonjungtang on postoperative ileus , and this prescription has attracted attention for the treatment of postoperative ileus in japan .
insam - tang , which has been used for the treatment of gastroenteritis , gastric atony , gastrectasis , vomiting , and anorexia , is also useful for the treatment of postoperative ileus .
because ginseng is not the main ingredient of either of these prescriptions , the active compound of insam - tang may be 6-gingerol or 6-shogaol from zingiberis rhizome .
recently , banhasasim - tang was reported to be effective against irinotecan - induced diarrhea .
cholinergic hypofunction and inflammatory responses induced by amyloid- protein are the main causes of this disease .
consequently , acetylcholinesterase inhibitors are clinically used for the treatment of alzheimer s disease , and anti - oxidative or anti - inflammatory agents may also be applicable because a redox imbalance is observed in the brains of alzheimer s disease sufferers .
in addition , degenerative changes in the forebrain cholinergic system are reportedly linked to oxidative stress .
ginseng is a well - known , anti - aging herb , and cognitive functions decrease gradually with age .
saengmaek - san ( , shengmaisan in chinese ) , which is composed of 3 different kinds of herbs ( ginseng radix , ophiopogon rhizome , and schisandrae fructus ) , has been used to treat symptoms related to cardiovascular diseases , such as heart failure and stroke .
recently , saengmaek - san , which possesses anti - oxidative properties , was reported to ameliorate scopolamine - induced cognitive dysfunctions via acetylcholinesterase inhibition .
in addition , dx-9386 , which consists of ginseng radix , polygalae radix , acorus radix and hoelen and is identical to jeongji - whan , has anti - aging and memory - ameliorating effects .
guibi - tang was described in historical documents as an effective prescription for psychotic problems such as insomnia , amnesia , depression , and neurosis .
recently , the administration of guibi - tang for 3 consecutive days was shown to ameliorate spatial and object - recognition memories in amyloid protein - injected mice , suggesting that this formulation is an attractive candidate for anti - dementia drugs .
baekho - ga- insam - tang is a herbal medicine used for the relief of diuresis , thirst and dermal pruritus , which are associated with diabetes .
this result has been confirmed by experiments performed on , kka mice , which are genetic animal models of diabetes mellitus .
the administration of baekho - ga - insam - tang relieves diuresis , thirst , and dermal pruritus by increasing kidney aquaporin 2 and skin aquaporin expression .
in addition , baekhoga- insam - tang enhances salivary secretion by increasing aquaporin 5 in a rat thirst model , suggesting that this formulation could be useful for xerostomia .
thus , the functions and efficacies of the aforementioned ginseng - containing prescriptions have been confirmed by modern experimental pharmacology .
we summarized the results of in vivo animal or human studies on ginseng - containing prescriptions in table 1 , which includes the prescriptions mentioned in the present review .
there are 13 species of ginseng in the araliaceae family , including 1 ) p. ginseng meyer , 2 ) p. quinquefolius l. , 3 ) p. japonicus l. , 4 ) p. notoginseng ( burk . )
fh chen , 5 ) p. pseudoginseng wallich , 6 ) p. vietnamensis ha et grushv , 7 ) p.
p. stipuleanatus ht tsai & km feng , 11 ) p. wangianus sun , 12 ) p. zingiberensis cy wu & km feng , and 13 ) p. major ting .
the most commonly used panax species include plants 1 ) to 6 ) in the list above .
p. vietnamensis was found in the mountainous regions of central vietnam in the 1970s , and p. pseudoginseng has been used as a folk medicine in the himalayas . since the pioneering work of dr .
reported the vasodilatory effect of p. ginseng obtained from keumsan , korea , and in 1971 , lee and huemer reported the antitumor activity of ginseng obtained from kangwhado , korea .
the shibata group in japan has studied the chemistry of the saponins and sapogenins of the white ginseng , p. ginseng , which is cultivated in japan .
notoginseng was first employed as a medicine not much earlier than the time of shizhen li s first report in the bencao gangmu .
therefore , this species of ginseng was not mentioned in texts on chinese medicine produced before this period .
thus , although other species of ginseng are also active , p. ginseng is the most popular and active species .
the therapeutic potency of ginseng is dependent on its geographical locality , dosage , and processing .
several studies have been conducted to compare the effects of different species of ginseng . for wound healing and hypoglycemic effects ,
p. ginseng is superior to p. quinquefolius , whereas p. quinquefolius displays better anticancer effects .
similarly , p. ginseng ( ginseng in korea ) has the highest therapeutic potency for the treatment of diabetes .
p. quinquefolius ( american ginseng ) is a medium - potency - grade ginseng , and p. japonicus ( japanese ginseng ) is considered a lowpotency- grade ginseng . in the future , a comprehensive and objective evaluation of ginseng should be conducted using evidence - based medicine to further elucidate the intriguing properties of ginseng in terms of treatment of various diseases shown in recent review articles .
in the future , a comprehensive and objective evaluation of ginseng should be conducted using evidence - based medicine to further elucidate the intriguing properties of ginseng in terms of treatment of various diseases shown in recent review articles .
since the description of ginseng from the korean peninsula was first published in the bencao gangmu , p. ginseng has become the most commonly used therapeutic ginseng .
modern chemical and biological drugs are suitable for the treatment of diseases with specific causes and pathologies but are not suitable for diseases with multiple factors , such as diabetes , alzheimer s disease , and chronic fatigue . based on historical accounts and recent experimental or clinical studies , ginseng and ginseng - containing herbal prescriptions are useful for the treatment of chronic diseases . | panax ginseng meyer has been widely used as a tonic in traditional korean , chinese , and japanese herbal medicines and in western herbal preparations for thousands of years . in the past , ginseng was very rare and was considered to have mysterious powers . today , the efficacy of drugs must be tested through well - designed clinical trials or meta - analyses , and ginseng is no exception . in the present review , we discuss the functions of ginseng described in historical documents and describe how these functions are taken into account in herbal prescriptions .
we also discuss the findings of experimental pharmacological research on the functions of ginseng in ginseng - containing prescriptions and how these prescriptions have been applied in modern therapeutic interventions .
the present review on the functions of ginseng in traditional prescriptions helps to demystify ginseng and , as a result , may contribute to expanding the use of ginseng or ginseng - containing prescriptions . | INTRODUCTION
A RETROSPECTIVE ON GINSENG IN HISTORICAL DOCUMENTS
THE PHARMACOLOGICAL ACTIVITIES OF PANAX GINSENG IN TRADITIONAL PRESCRIPTIONS
THE USE OF GINSENG-CONTAINING PRESCRIPTIONS IN MODERN PHARMACOLOGICAL EXPERIMENTS
THE VARIETY OF SPECIES IN THE PLANT GENUS PANAX
PERSPECTIVES
CONCLUSION | in addition , in the shang - han lun ( translated as the treatise on diseases caused by cold factors , ) , zhong jing zhang ( ) described the use of ginseng in herbal prescriptions . , lugano - bioggio , switzerland , ginseng has been widely used as an active ingredient in modern medicine . therefore , the characteristics of ginseng described in historical documents , such as the shennong bencao jing , the bencao gangmu ( , which was written by shizhen li [ ] during the ming dynasty ) , and the hyangyak - jibseongbang ( ) or the donguibogam ( , which was published during the chosun dynasty ) , should be examined . the role of ginseng in traditional prescriptions should also be analyzed to expand the use of ginseng in modern medicine . in addition , investigations on the experimental evidence in pharmacological studies of ginseng - based traditional prescriptions would yield information on how traditional knowledge or historical accounts described in the aforementioned documents can be interpreted in contemporary medical terms . although it is not known whether historical documents described cultivated or wild ginseng , in the present review , we discuss historical accounts concerning p. ginseng , the role of p. ginseng in traditional prescriptions , and experimental evidence from pharmacological and clinical studies of traditional prescriptions containing p. ginseng ( table 1 ) . summary of the pharmacological activities of ginseng - containing prescriptions described in the shang - han lun in either animal or human studies
the pharmacological functions of ginseng , which was considered a high - grade herb , were first described in the shennong bencao jing . the baekje dynasty ( , 18 bc - ad 660 ) governed the western part of the korean peninsula and actively traded with china , suggesting that the korean peninsula might have been suitable for the cultivation of ginseng and that korean ginseng may have been used in herbal prescriptions prior to the ming dynasty . several traditional prescriptions were prepared from ginseng , which was used for its restorative , tonic , nootropic , and anti - aging properties , or for dispelling pathogenic factors , as described in the shennong bencao jing . as a main ingredient in sagunjatang , ginseng exerts various pharmacological activities , such as organ tonification and restorative activities , as described in the shennong bencao jing and bencao gangmu . in the aforementioned formulae ,
ginseng is the main active ingredient and exerts restorative , tonic or nourishing effects on hypodynamia and ameliorates the effects of serious illness , as described in the shennong bencao jing and the bencao gangmu . thus , although the improvement in brain function was not solely dependent on ginseng , these prescriptions made use of the effects of ginseng on anxiety , amnesia , and palpitation , which were described in the shennong bencao jing . in particular , the mingyi bielu stated that ginseng relieves thirst ( sogal in korean , xiao ke in chinese or shoukatsu in japanese , ) , and the bencao gangmu referred to the description of ginseng in the mingyi bielu . in western medicine ,
randomized clinical trials are generally accepted as the most reliable approach to testing the efficacy of medicines and treatments ; however , in traditional herbal medicine , clinical evidence was observed and recorded descriptively in the literature . using clinical trials or basic scientific research ,
the efficacy of traditional prescriptions for specific diseases can be determined , which has the potential to expand the use of traditional formulae . thus , the effects of sipjeondaebo - tang on the immune system have been confirmed by clinical trials and animal experiments , providing experimental evidence in support of the traditional use of sipjeondaebo - tang . several ginseng - containing prescriptions , including daegeonjung - tang ( , da jian zhong tang in chinese or dai - kenchu - to in japanese ) , insam - tang ( , ninjin - to in japanese or ren shen tang in chinese ) , and banhasasim - tang , have been used to treat gastrointestinal problems , such as gastric atony , vomiting and anorexia . thus , the functions and efficacies of the aforementioned ginseng - containing prescriptions have been confirmed by modern experimental pharmacology . we summarized the results of in vivo animal or human studies on ginseng - containing prescriptions in table 1 , which includes the prescriptions mentioned in the present review . p. vietnamensis was found in the mountainous regions of central vietnam in the 1970s , and p. pseudoginseng has been used as a folk medicine in the himalayas . based on historical accounts and recent experimental or clinical studies , ginseng and ginseng - containing herbal prescriptions are useful for the treatment of chronic diseases . | [
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] |
thyroidectomy has gained popularity for the treatment of thyroid disease over the past few decades due to markedly improved cosmetic satisfaction .
compared to conventional open thyroidectomy , the bilateral axillo - breast approach ( baba ) robotic or endoscopic thyroidectomy offers superior aesthetic results , as no scar is left on the anterior neck area .
however , large flap dissection of the anterior chest and cervical area and significant tension in the tissue flap due to insufflation with co2 is required to obtain a satisfactory view during the surgery .
it has been reported that patients who undergo endoscopic or robotic surgery on the neck or thyroid suffer from moderate to severe neck , anterior chest , and axillary pain following surgery .
in addition , about 70% of patients complain of anterior chest discomfort within 4 days after endoscopic thyroidectomy , and hypoesthesia with discomfort to the anterior chest area may persist for 3 months following surgery .
postoperative pain is associated with reduced quality of recovery , delayed mobilization , prolonged hospitalization , and the development of chronic pain syndrome .
acute pain control following baba robotic or endoscopic thyroidectomy is essential for quality of recovery and to prevent chronic pain .
opioids are the first - line postoperative analgesia ; however , the high incidence of postoperative nausea and vomiting ( ponv ) limits their use following thyroidectomy .
several trials with nonopioid analgesics have been carried out for the management of acute pain following baba robotic or endoscopic thyroidectomy . in one study ,
perioperative administration of intravenous paracetamol was effective for pain management in patients with robot - assisted thyroidectomy performed via the transaxillary approach ; however , this required infusion over 24 hours . in another study ,
pregabalin was administered from the day before surgery to 2 days postoperatively in patients who had undergone robot - assisted thyroidectomy ; this was effective for reducing early postoperative pain , but in some cases led to dizziness and sedation .
ryu et al investigated the effects of spraying 0.25% levobupivacaine on the dissected area , and reported reduced pain in the anterior chest region for 2 days postoperatively following baba robotic thyroidectomy .
the use of nonsteroidal antiinflammatory drugs ( nsaids ) as a systemic analgesic should be avoided due to the increased risk of bleeding complications , which may lead to fatal airway obstruction following thyroidectomy ; however , the clinical impact of nsaids on postoperative bleeding remains unclear .
ketamine is a phencyclidine derivative , and was first developed as a general anesthetic in the 1960s .
it also acts as a noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist , which reduces acute postoperative pain and prevents the development of chronic postoperative pain .
it may be administered perioperatively for postoperative pain as an adjuvant to systemic opioids , with fewer adverse effects ; however , the effects of perioperative ketamine infusion on postoperative pain following baba robotic or endoscopic thyroidectomy have not been investigated .
this randomized , double - blinded , placebo - controlled study investigated the effects of perioperative ketamine infusion on acute postoperative pain and recovery profiles in patients who underwent baba robotic or endoscopic thyroidectomy .
the institutional review board of seoul national university bundang hospital ( b-15.9/314 - 009 ) approved this prospective , randomized , and double - blinded controlled trail and the protocol was registered at http://cris.nih.go.kr ( registration number kct0001887 ) . written informed consent
was obtained from all patients . a total of 58 patients , american society of anesthesiologists ( asa ) physical status i or ii , 19 to 69 years of age , scheduled for elective baba robotic or endoscopic thyroidectomy from november 2015 to june 2016 were enrolled for this study .
inclusion criteria are thyroid mass smaller than 4 cm in benign cases and smaller than 2 cm in potentially malignant cases without evidence of extracapsular soft tissue invasion or metastasis .
exclusion criteria are patients with a history of chronic pain , allergy or hypersensitivity to ketamine , chronic use of analgesics or opioids for > 2 weeks before the surgery , previous history of surgery on chest or neck area and those who were not able to understand a numerical rating scale ( nrs ) were excluded .
patients were transferred to the operating room after midazolam premedication ( 0.03 mg / kg ) .
on arrival at the operating room , patients were monitored with standard monitoring including electrocardiography , noninvasive arterial pressure , and pulse oximetry .
total intravenous anesthesia with propofol and remifentanil was used for the induction and maintenance of anesthesia using an orchestra infusion pump system ( fresenius vial , brezins , france ) to maintain bispectral index monitoring ( a-2000 bis monitor ; aspect medical systems , inc . ,
rocuronium bromide 0.6 mg / kg was injected to facilitate muscle relaxation and additional doses of rocuronium bromide 0.15 mg / kg was administered during operation to maintain 1 to 2 twitches on train - of - four stimulation of ulnar nerve .
the ventilator was set to maintain end - tidal co2 between 35 and 40 mm hg with oxygen and medical air ( fio2 0.5 ) .
after surgery , neuromuscular blockade was reversed with neostigmine 0.04 mg / kg and glycopyrrolate 0.01 mg / kg .
patients were extubated after they were fully recovered and transferred to the postanesthesia care unit ( pacu ) .
a single experienced surgeon ( jyc ) performed all the operations to maintain a uniform application of surgical stimulus .
the operating procedures for endoscopic or robotic ( with the aid of the da vinci robot system [ intuitive surgical , inc .
, mountain view , ca ] ) assisted thyroidectomy using baba approaches have been described previously .
patients were placed in a supine position with a slight neck extension using a pillow under the shoulders .
skin flaps were outlined and 200 ml of 1:200,000 diluted epinephrine was injected along the upper chest and subplatysmal space for vasoconstriction .
then , 2 superomedial circumareolar marginal incisions ( right , 1.2 cm ; left , 0.8 cm ) and 2 axillary skin incisions ( 0.8 cm each ) were made for the instrumental ports .
flap dissections were initially carried out using endoscopic instruments and extended to the thyroid cartilage superiorly , 3 cm below the clavicle inferiorly , and laterally from just beyond the lateral border of one sternocleidomastoid muscle to the other .
the surgical space was insufflated with co2 gas ( pressure of 56 mm hg ) via the 12 mm camera port .
after docking of robot or endoscopic instruments , the midline was divided using monopolar electrocautery until the thyroid was visualized .
the cricothyroid membrane , the isthmus , and the central group of lymph nodes were identified and then thyroid isthmus was routinely divided .
the remainder of the thyroidectomy was performed with identification of the middle and inferior thyroid pedicles , the recurrent laryngeal nerve , and the superior and inferior parathyroid glands .
after unilateral thyroidectomy , the thyroid specimen was removed through the left axillary incision using an endoplastic bag .
after thyroidectomy , antiadhesive solution ( guardix - sg , 6 g ; hanmi pharmaceutical , seoul , korea ) was sprayed onto the whole flap .
randomization was performed before the induction of anesthesia by an anesthesiologist who is only in charge of the randomization .
a computer generated random number table ( random allocation software version 1.0 ) with block size 4 was used . from a table of random numbers
, patients were allocated to control group ( n = 29 ) or ketamine group ( n = 29 ) .
patients , anesthesiologists responsible for the patients and outcome assessors were blinded to the group assignment .
study solutions ( normal saline for control group or ketamine 1 mg / kg bolus , 60 g / kg / h continuous infusion for ketamine group ) were infused right after the induction of anesthesia by the anesthesiologist as identical syringes ( 50 ml ) to ensure blinding until the end of surgery ( skin closure ) .
postoperative pain in each area ( neck , axilla , and anterior chest ) was evaluated by the patients on a 101-point nrs ( 0 = no pain , 100 = the worst imaginable pain ) at 1 , 6 , 24 , and 48 hours after surgery .
patients were evaluated every 15 minutes using the modified aldrete scoring system until ready for discharge from the pacu and the criterion used for patient discharge from pacu was the achievement of a modified aldrete score of 9 . in the pacu ,
first - line rescue analgesic was fentanyl 50 g and second - line was intravenously ketorolac tromethamin 30 mg if nrs is more than 30 .
after that , intravenous ketorolac tromethamin 15 mg was used as the rescue analgesics in the ward if nrs was more than 30 or the patient wanted the analgesic drug .
in addition , postoperative adverse effects such as ponv , shivering , headache , dizziness , or drowsiness were also recorded during the study period .
sample size calculation was based on mean pain scores of 6 ( 3.5 ) 1 hour after robot - assisted thyroidectomy using g power 3.0 .
we considered that a decrease of 2 in the mean pain scores would be clinically significant .
twenty - six patients per group were required ( an alpha value of 0.05 and power of 80% ) and a total of 58 patients were finally decided to be needed considering 10% drop - out rate .
student t test was used to compare normally distributed variables ( tumor size , resected thyroid weight , and discharge time ) , and the mann
chi - square test or fisher exact test was used for analysis of other categorical outcomes such as the incidences of rescue analgesics and adverse effects .
a full analysis set was used for the analysis of data . all values presented are mean ( standard deviation ) , median ( interquartile range ) , or number of patients ( % ) .
the institutional review board of seoul national university bundang hospital ( b-15.9/314 - 009 ) approved this prospective , randomized , and double - blinded controlled trail and the protocol was registered at http://cris.nih.go.kr ( registration number kct0001887 ) . written informed consent
was obtained from all patients . a total of 58 patients , american society of anesthesiologists ( asa ) physical status i or ii , 19 to 69 years of age , scheduled for elective baba robotic or endoscopic thyroidectomy from november 2015 to june 2016 were enrolled for this study .
inclusion criteria are thyroid mass smaller than 4 cm in benign cases and smaller than 2 cm in potentially malignant cases without evidence of extracapsular soft tissue invasion or metastasis .
exclusion criteria are patients with a history of chronic pain , allergy or hypersensitivity to ketamine , chronic use of analgesics or opioids for > 2 weeks before the surgery , previous history of surgery on chest or neck area and those who were not able to understand a numerical rating scale ( nrs ) were excluded .
patients were transferred to the operating room after midazolam premedication ( 0.03 mg / kg ) .
on arrival at the operating room , patients were monitored with standard monitoring including electrocardiography , noninvasive arterial pressure , and pulse oximetry .
total intravenous anesthesia with propofol and remifentanil was used for the induction and maintenance of anesthesia using an orchestra infusion pump system ( fresenius vial , brezins , france ) to maintain bispectral index monitoring ( a-2000 bis monitor ; aspect medical systems , inc . ,
rocuronium bromide 0.6 mg / kg was injected to facilitate muscle relaxation and additional doses of rocuronium bromide 0.15 mg / kg was administered during operation to maintain 1 to 2 twitches on train - of - four stimulation of ulnar nerve .
the ventilator was set to maintain end - tidal co2 between 35 and 40 mm hg with oxygen and medical air ( fio2 0.5 ) .
after surgery , neuromuscular blockade was reversed with neostigmine 0.04 mg / kg and glycopyrrolate 0.01 mg / kg .
patients were extubated after they were fully recovered and transferred to the postanesthesia care unit ( pacu ) .
a single experienced surgeon ( jyc ) performed all the operations to maintain a uniform application of surgical stimulus .
the operating procedures for endoscopic or robotic ( with the aid of the da vinci robot system [ intuitive surgical , inc .
, mountain view , ca ] ) assisted thyroidectomy using baba approaches have been described previously .
patients were placed in a supine position with a slight neck extension using a pillow under the shoulders .
skin flaps were outlined and 200 ml of 1:200,000 diluted epinephrine was injected along the upper chest and subplatysmal space for vasoconstriction .
then , 2 superomedial circumareolar marginal incisions ( right , 1.2 cm ; left , 0.8 cm ) and 2 axillary skin incisions ( 0.8 cm each ) were made for the instrumental ports .
flap dissections were initially carried out using endoscopic instruments and extended to the thyroid cartilage superiorly , 3 cm below the clavicle inferiorly , and laterally from just beyond the lateral border of one sternocleidomastoid muscle to the other .
the surgical space was insufflated with co2 gas ( pressure of 56 mm hg ) via the 12 mm camera port .
after docking of robot or endoscopic instruments , the midline was divided using monopolar electrocautery until the thyroid was visualized .
the cricothyroid membrane , the isthmus , and the central group of lymph nodes were identified and then thyroid isthmus was routinely divided .
the remainder of the thyroidectomy was performed with identification of the middle and inferior thyroid pedicles , the recurrent laryngeal nerve , and the superior and inferior parathyroid glands .
after unilateral thyroidectomy , the thyroid specimen was removed through the left axillary incision using an endoplastic bag .
after thyroidectomy , antiadhesive solution ( guardix - sg , 6 g ; hanmi pharmaceutical , seoul , korea ) was sprayed onto the whole flap .
randomization was performed before the induction of anesthesia by an anesthesiologist who is only in charge of the randomization .
a computer generated random number table ( random allocation software version 1.0 ) with block size 4 was used . from a table of random numbers
, patients were allocated to control group ( n = 29 ) or ketamine group ( n = 29 ) .
patients , anesthesiologists responsible for the patients and outcome assessors were blinded to the group assignment .
study solutions ( normal saline for control group or ketamine 1 mg / kg bolus , 60 g / kg / h continuous infusion for ketamine group ) were infused right after the induction of anesthesia by the anesthesiologist as identical syringes ( 50 ml ) to ensure blinding until the end of surgery ( skin closure ) .
postoperative pain in each area ( neck , axilla , and anterior chest ) was evaluated by the patients on a 101-point nrs ( 0 = no pain , 100 = the worst imaginable pain ) at 1 , 6 , 24 , and 48 hours after surgery .
patients were evaluated every 15 minutes using the modified aldrete scoring system until ready for discharge from the pacu and the criterion used for patient discharge from pacu was the achievement of a modified aldrete score of 9 . in the pacu ,
first - line rescue analgesic was fentanyl 50 g and second - line was intravenously ketorolac tromethamin 30 mg if nrs is more than 30 .
after that , intravenous ketorolac tromethamin 15 mg was used as the rescue analgesics in the ward if nrs was more than 30 or the patient wanted the analgesic drug .
ketamine related adverse events such as sedation or diplopia were recorded if it occurred . in addition , postoperative adverse effects such as ponv , shivering , headache , dizziness , or drowsiness were also recorded during the study period .
sample size calculation was based on mean pain scores of 6 ( 3.5 ) 1 hour after robot - assisted thyroidectomy using g power 3.0 .
we considered that a decrease of 2 in the mean pain scores would be clinically significant .
twenty - six patients per group were required ( an alpha value of 0.05 and power of 80% ) and a total of 58 patients were finally decided to be needed considering 10% drop - out rate .
student t test was used to compare normally distributed variables ( tumor size , resected thyroid weight , and discharge time ) , and the mann
chi - square test or fisher exact test was used for analysis of other categorical outcomes such as the incidences of rescue analgesics and adverse effects .
a full analysis set was used for the analysis of data . all values presented are mean ( standard deviation ) , median ( interquartile range ) , or number of patients ( % ) .
sixty - seven patients were screened for eligibility and 58 patients were randomized for the study .
of the 58 patients enrolled in the study , 1 patient was eliminated from the data collection and a total of 57 patients were included in the final analysis .
one patient from ketamine was excluded after randomization for the patient controlled analgesia instead of rescue analgesics ( fig .
fifty - eight patients were randomized and 1 patient was excluded from final analysis due to patient controlled analgesia instead of rescue analgesics .
patient , surgery , and anesthetic characteristics were similar between the 2 groups ( table 1 ) .
postoperative pathology of the patients showed 45 patients with papillary carcinoma , 1 with follicular carcinoma , 5 with nodular hyperplasia , and 4 with follicular adenoma .
there were no significant differences in tumor size or pathologic type between the 2 groups .
postoperative pain scores were evaluated in each area of the surgery ( neck , axilla , and chest ) using 101 nrs at postoperative 1 , 6 , 24 , and 48 hours .
there are statistically significant differences in pain scores between control and ketamine group during postoperative 48 hours although pain scores at postoperative 1 hour were not different between the 2 groups .
pain scores of the ketamine group were significantly lower than those of control group in the neck area at 6 hours ( 30 vs 50 ; p = 0.017 ) , 24 hours ( 20 vs 30 ; p < 0.001 ) , and 48 hours ( 10 vs 20 ; p = 0.005 ) postoperatively ( fig .
2 ) . patients in ketamine group showed lower pain scores in the axilla area than those in control group at postoperative 24 hours ( 15 vs 30 ; p = 0.024 ) and 48 hours ( 10 vs 20 ; p = 0.005 ) ( fig .
2 ) . pain in the chest area of the ketamine group was lower than control group at postoperative 48 hours ( 10 vs 20 ; p = 0.007 ) ( fig .
box plot with median ( solid line ) , interquartile range ( box ) , and values within 1.5 interquartile range from each side of the box ( whiskers ) .
vnrs = verbal numerical rating scale ; p < 0.05 compared with control groups .
there were no statistically significant differences in the need of rescue analgesics between the 2 groups ( table 2 ) .
the total numbers of rescue analgesic administration were smaller in ketamine group than in control group during the study period though they could not reach statistically significant differences ( 1.5 vs 2 ; p = 0.051 ) ( table 2 ) .
postoperative adverse events including ponv , headache , dizziness , and shivering were recorded during the study period and there were no significant differences in postoperative complications between the 2 groups ( table 3 ) .
we set out this prospective , randomized , and controlled trial to determine whether intraoperative ketamine infusion would decrease postoperative pain following baba robotic or endoscopic thyroidectomy .
the principal finding was that low - dose ketamine infusion during anesthesia significantly reduced postoperative pain , with no increased incidence of adverse events . during baba robotic or endoscopic thyroidectomy ,
a subplatysmal skin flap is dissected from the axilla to the anterior neck , and forcefully lifted to enable visualization of the surgical space . to expose the thyroid ,
the dissected area is wider during robotic or endoscopic thyroidectomy than with conventional open thyroidectomy , which explains the significant postoperative pain following robotic or endoscopic thyroidectomy .
baba leads to additional operative trauma along the route to the thyroid ; therefore , this technique may be expected to result in greater postoperative pain compared to conventional open thyroidectomy . in this study , a subanesthetic dose of ketamine was infused intravenously .
the postoperative pain scores in the neck , axilla , and chest area were significantly lower in the ketamine group than in the control group during the first 48 hours postoperatively .
pain scores at 1 hour postoperatively did not differ between the 2 groups in all areas ; however , this can be explained by considering the residual anesthetic effect .
ketamine is a noncompetitive nmda receptor antagonist related to pain sensitivity , and has been administered during various kinds of surgery for postoperative pain control .
preemptive analgesia can prevent the development and establishment of the central processing of noxious stimulation ( i.e. , surgical incision ) during the pain pathway . in this study , a subanesthetic dose of ketamine ( 1 mg / kg bolus , 60 g / kg / h continuous infusion ) was infused immediately following induction of anesthesia ( before the surgical incision ) , and was retained until the end of the surgery .
the optimal dose and infusion timing of ketamine for postoperative pain control has not yet been established ; however , most trials have used ketamine in subanesthetic doses during the perioperative period .
reviews and meta - analyses that have evaluated the preemptive analgesic effects of ketamine for postoperative pain have concluded that subanesthetic doses of ketamine ( 0.150.5 mg / kg bolus , 60120 g / kg / h continuous infusion ) result in preemptive analgesic effects that can reduce postoperative opioid usage with no adverse effects .
there were no statistically significant differences in the requirement for rescue analgesics between the 2 groups , although the total number of administrations of rescue analgesics was smaller in the ketamine group than in the control group .
rescue analgesics were used in preference to opioid - based patient control analgesia , because the most distressing obstacle during postoperative pain management for thyroidectomy patients is ponv due to the routine use of opioids . moreover
, the incidence of ponv following thyroidectomy appears to be higher than with other surgeries .
no major complications were associated with ketamine infusion , consistent with the results of a review on the use of ketamine for postoperative analgesia . the recovery time ( discharge time from pacu ) and incidence of postoperative adverse effects did not differ between the 2 groups .
two patients in the control group and 7 in the ketamine group exhibited ponv , although this difference was not statistically significant .
no trials have provided extractable data regarding the effects of ketamine on ponv , because ponv has not been the primary outcome of pain control studies .
intraoperative ketamine infusion has not been shown to reduce ponv , and may even have a negative effect on the severity of nausea following lumbar spinal surgery . however , a reduction in ponv has been reported with ketamine usage because of the reduced opioid dosage it enables . in this study
, there were no statistically significant differences in the requirement for rescue analgesics , including opioids and nsaids .
a major limitation of the present study is that the effects of ketamine infusion on acute postoperative pain were evaluated for only 2 days postoperatively , with no assessment of chronic pain .
acute pain control may be predictive of the development of long - term sensory disturbances or discomfort , and these chronic symptoms may persist for up to 3 months following robot or endoscopy - assisted thyroidectomy .
tae et al reported that postoperative anterior chest pain scores were higher following robotic thyroidectomy than after conventional open thyroidectomy for the first week postoperatively but that there was no difference between the 2 groups after 1 or 3 months .
however , song et al found that anterior chest discomfort and sensory disturbance were greater and required longer recovery times following robotic thyroidectomy than did conventional open thyroidectomy .
therefore , ketamine may reduce the development of chronic postoperative pain via nmda receptor blockade , which reduces wind - up and central sensitization .
this study addressed the efficacy and safety profiles of intraoperative ketamine infusion during for baba robotic or endoscopic thyroidectomy .
ketamine in subanesthetic dose ( 1 mg / kg bolus , 60 g / kg / h continuous infusion ) may be a useful adjunct in that it reduced postoperative pain scores during postoperative 48 hours after baba robotic or endoscopic thyroidectomy without adverse effect .
further studies on optimal dose of ketamine and the effect of intraoperative ketamine infusion on chronic postsurgical pain with long - term follow - up are needed . | abstractbackground : robotic or endoscopic thyroidectomy using bilateral axillo - breast approach ( baba ) is frequently performed for excellent cosmesis .
however , postoperative pain is remained as concerns due to the extent tissue dissection and tension during the operation .
ketamine is a noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist that reduces acute postoperative pain .
we evaluated the effects of intraoperative ketamine infusion on postoperative pain control and recovery profiles following baba robotic or endoscopic thyroidectomy.methods:fifty-eight adult patients scheduled for baba robotic or endoscopic thyroidectomy were randomized into a control group ( n = 29 ) and ketamine group ( n = 29 ) .
following induction of anesthesia , patients in each group were infused with the same volume of saline or ketamine solution ( 1 mg / kg bolus , 60 g / kg / h continuous infusion ) .
total intravenous anesthesia with propofol and remifentanil was used to induce and maintain anesthesia .
pain scores ( 101-point numerical rating scale , 0 = no pain , 100 = the worst imaginable pain ) , the consumption of rescue analgesics , and other postoperative adverse effects were assessed at 1 , 6 , 24 , and 48 hours postoperatively.results:patients in the ketamine group reported lower pain scores than those in the control group at 6 hours ( 30 [ 30 ] vs 50 [ 30 ] ; p = 0.017 ) , 24 hours ( 20 [ 10 ] vs 30 [ 20 ] ; p < 0.001 ) , and 48 hours ( 10 [ 10 ] vs 20 [ 15 ] ; p <
0.001 ) in neck area .
no statistically significant differences were found between the 2 groups in terms of the requirements for rescue analgesics or the occurrence of adverse events.conclusion:intravenous ketamine infusion during anesthesia resulted in lower postoperative pain scores following baba robotic or endoscopic thyroidectomy , with no increase in adverse events . | Introduction
Methods
Protocol and patients
Anesthesia
Surgery
Randomization and intervention
Outcomes
Statistical analysis
Results
Discussion
Conclusion | compared to conventional open thyroidectomy , the bilateral axillo - breast approach ( baba ) robotic or endoscopic thyroidectomy offers superior aesthetic results , as no scar is left on the anterior neck area . it also acts as a noncompetitive n - methyl - d - aspartate ( nmda ) receptor antagonist , which reduces acute postoperative pain and prevents the development of chronic postoperative pain . it may be administered perioperatively for postoperative pain as an adjuvant to systemic opioids , with fewer adverse effects ; however , the effects of perioperative ketamine infusion on postoperative pain following baba robotic or endoscopic thyroidectomy have not been investigated . this randomized , double - blinded , placebo - controlled study investigated the effects of perioperative ketamine infusion on acute postoperative pain and recovery profiles in patients who underwent baba robotic or endoscopic thyroidectomy . study solutions ( normal saline for control group or ketamine 1 mg / kg bolus , 60 g / kg / h continuous infusion for ketamine group ) were infused right after the induction of anesthesia by the anesthesiologist as identical syringes ( 50 ml ) to ensure blinding until the end of surgery ( skin closure ) . postoperative pain in each area ( neck , axilla , and anterior chest ) was evaluated by the patients on a 101-point nrs ( 0 = no pain , 100 = the worst imaginable pain ) at 1 , 6 , 24 , and 48 hours after surgery . study solutions ( normal saline for control group or ketamine 1 mg / kg bolus , 60 g / kg / h continuous infusion for ketamine group ) were infused right after the induction of anesthesia by the anesthesiologist as identical syringes ( 50 ml ) to ensure blinding until the end of surgery ( skin closure ) . postoperative pain in each area ( neck , axilla , and anterior chest ) was evaluated by the patients on a 101-point nrs ( 0 = no pain , 100 = the worst imaginable pain ) at 1 , 6 , 24 , and 48 hours after surgery . postoperative pain scores were evaluated in each area of the surgery ( neck , axilla , and chest ) using 101 nrs at postoperative 1 , 6 , 24 , and 48 hours . there are statistically significant differences in pain scores between control and ketamine group during postoperative 48 hours although pain scores at postoperative 1 hour were not different between the 2 groups . pain scores of the ketamine group were significantly lower than those of control group in the neck area at 6 hours ( 30 vs 50 ; p = 0.017 ) , 24 hours ( 20 vs 30 ; p < 0.001 ) , and 48 hours ( 10 vs 20 ; p = 0.005 ) postoperatively ( fig . patients in ketamine group showed lower pain scores in the axilla area than those in control group at postoperative 24 hours ( 15 vs 30 ; p = 0.024 ) and 48 hours ( 10 vs 20 ; p = 0.005 ) ( fig . pain in the chest area of the ketamine group was lower than control group at postoperative 48 hours ( 10 vs 20 ; p = 0.007 ) ( fig . the total numbers of rescue analgesic administration were smaller in ketamine group than in control group during the study period though they could not reach statistically significant differences ( 1.5 vs 2 ; p = 0.051 ) ( table 2 ) . the postoperative pain scores in the neck , axilla , and chest area were significantly lower in the ketamine group than in the control group during the first 48 hours postoperatively . in this study , a subanesthetic dose of ketamine ( 1 mg / kg bolus , 60 g / kg / h continuous infusion ) was infused immediately following induction of anesthesia ( before the surgical incision ) , and was retained until the end of the surgery . reviews and meta - analyses that have evaluated the preemptive analgesic effects of ketamine for postoperative pain have concluded that subanesthetic doses of ketamine ( 0.150.5 mg / kg bolus , 60120 g / kg / h continuous infusion ) result in preemptive analgesic effects that can reduce postoperative opioid usage with no adverse effects . there were no statistically significant differences in the requirement for rescue analgesics between the 2 groups , although the total number of administrations of rescue analgesics was smaller in the ketamine group than in the control group . a major limitation of the present study is that the effects of ketamine infusion on acute postoperative pain were evaluated for only 2 days postoperatively , with no assessment of chronic pain . ketamine in subanesthetic dose ( 1 mg / kg bolus , 60 g / kg / h continuous infusion ) may be a useful adjunct in that it reduced postoperative pain scores during postoperative 48 hours after baba robotic or endoscopic thyroidectomy without adverse effect . | [
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] |
multiple sclerosis ( ms ) is a progressive , degenerative disease of the central nervous system , characterized by multiple symptoms , including spasticity ; problems with walking , balance , and coordination ; fatigue , depression , and mood disorders ; bladder , bowel , and sexual dysfunction ; and cognitive impairment . as such
, ms is the leading cause of disability among nonelderly individuals in industrialized countries.1 in germany , it is estimated that at least 120,000 people have ms,2 with the total annual cost of ms in germany estimated to be 40,000 per person with ms.3,4 ms pathogenesis is characterized by inflammatory and neurodegenerative mechanisms,5 which manifest as relapses6,7 and disability progression,8 respectively .
the most common form of ms is relapsing remitting ms ( rrms ) , whereby an unpredictable course of relapses is followed by a period of remission and recovery.9 ms relapses have been shown to profoundly affect an individual s quality of life , as occurrences interfere with the ability to perform daily activities , are linked to emotional distress and depression , and impact family , friends , and caregivers.10 thus , the societal , economic , familial , and personal burden of ms relapses is enormous .
people with ms who experience relapses are often treated with corticosteroids and admitted to the hospital.11 ms relapses contribute significantly to the costs associated with the disease,12 which increase with disease severity.1214 treatments that can slow or prevent the progression of ms have the potential to significantly improve the quality of life for people with ms and reduce the economic impact of the disease.14,15 natalizumab , a selective adhesion - molecule inhibitor , has been shown to reduce annualized relapse rates by 68% versus placebo among people with rrms16,17 and by 81% versus placebo among people with highly active rrms.18 in the european union , natalizumab is indicated in adults with highly active rrms who failed to respond to an adequate course of interferon beta or glatiramer acetate , and in adults with rapidly evolving severe rrms ( with 2 relapses in 1 year and 1 gadolinium - enhancing lesion or a significant increase in t2 lesion load compared with a previous recent magnetic resonance imaging assessment).19 in germany , ~90% of the population ( ~70 million individuals ) is insured as part of the statutory health insurance ( shi ) program,20,21 and the prevalence of ms among people insured by the shi in 2010 was estimated to range from 120,000 to 200,000.22 the objective of this study was to evaluate ms - related and all - cause health care resource utilization ( hru ) and costs among german people with ms during the 12 months before and 12 months after initiation of natalizumab in a real - world setting .
in addition , hru and costs were evaluated after stratifying people with ms by whether or not they had used a disease - modifying therapy ( dmt ) during the pre - index period .
although several studies have evaluated the economic burden of ms in germany,3,4,15 to our knowledge this is the first study to evaluate ms - related hru and costs among natalizumab - treated people with ms based on their previous dmt exposure .
the current analysis was conducted using the health risk institute ( hri ) research database , which contains anonymized , patient - level inpatient , outpatient , sick leave , and pharmacy claims data from about 80 health insurance companies , representing a high proportion of th130 health insurance companies that comprise the shi in germany20 and providing good external validity.23 for 20082013 , the hri research database contained individual , de - identified claims data for about 4 million people , with a patient sample that is representative of the german population for age and gender.24,25 the database is fully compliant with all data protection regulations in germany and has been certified . as the hri research database includes
verified accounting data of the participating insurance companies , these claims data are regularly audited by the insurance companies for reimbursement purposes and are prepared in accordance with german social law ( paragraphs 287 sgb v and 75 sgb x ) .
this study utilized an existing data set in line with all data protection regulations , and patients were not identified for the purpose of this study .
this study is a retrospective database study based on fully anonymized claims data , and claims data are recorded for accounting purposes and not for clinical research . no electronic medical records or other clinical parameters were used . as a result , no ethical approval or consent from an ethics committee or review board was required for this study .
the study sample included patients aged 18 years who initiated treatment with natalizumab ( index date [ anatomical therapeutic chemical { atc } code l04aa23 ] between january 1 , 2009 , and december 31 , 2012 ) .
all patients had a primary or secondary diagnosis of ms ( international statistical classification of diseases and related health problems , 10th revision , german modification [ icd-10-gm ] codes g35.xx ) in the inpatient sector in the pre - index period or a verified diagnosis of ms in the outpatient sector during any of the four quarters before the index quarter .
at least 24 months of continuous eligibility was required , including a 12-month pre - index period and a 12-month post - index period .
furthermore , patients had to have 1 natalizumab prescription in the fourth quarter of the post - index period to indicate continuous natalizumab use .
thus , the entire study covered the time interval from january 1 , 2008 , to december 31 , 2013 ( figure 1 ) .
outcomes were evaluated in the overall sample of patients who initiated treatment with natalizumab and in natalizumab initiators based on the prior use of other dmts .
patients were categorized as having used ( prior dmt use ) or not used ( no prior dmt use ) another dmt during the 12-month pre - index period .
the deyo - charlson comorbidity index ( dci),26,27 a summary measure of comorbidity burden , is reported as both a continuous and categorical measure .
individual dci comorbidities are reported as binary outcomes ( i.e. , present or not present ) .
ms - related and all - cause hospitalizations were defined as having a primary ms - related icd-10-gm code ( g35.xx ) or any icd-10-gm code , respectively . for the total population ,
the total number of inpatient stays and total length of stay were each summed across all admissions for the pre- and post - index period .
the use of corticosteroids ( intravenous [ iv ] and oral ) was determined if a patient had 1 prescription claim for dexamethasone , methylprednisolone , prednisolone , prednisone , or adrenocorticotropic hormone .
similarly , immunosuppressant use was recorded affirmatively if a patient had 1 claim for an immunosuppressant . per the german association of neurologists guidelines for diagnosis and treatment of ms,28 drugs for ms symptom relief
include antidementia agents , antidepressants , antiepileptics , muscle relaxants ( i.e. , baclofen , botulinum toxin , dantrolene , tizanidine , tolperisone , and tetrazepam ) , antispasmodics , and select medications to manage fatigue ( i.e. , amantadine and modafinil ) , tremor ( i.e. , propranolol ) , and sexual dysfunction ( i.e. , sildenafil , tadalafil , and tibolone ) .
use of ms symptom relief and all ms - related prescription medications was identified using the atc classification system .
health care related costs ( in euros [ ] ) were calculated separately for the pre- and post - index periods on a per - patient basis and converted to 2013 using the general rate of inflation for germany.29 ms - related inpatient costs were defined as the total costs for patient hospitalizations with a primary icd-10-gm code for ms , and all - cause costs were defined as the total costs for admissions for any reason .
total costs for corticosteroids ( iv , oral , and self - injectable ) and immunosuppressants were summed across all relevant prescription claims .
total costs for ms - related outcomes were calculated as the sum of all costs for ms - related hospitalizations , corticosteroids , and immunosuppressants .
costs were collected in the hri database and the reported costs were those for which the shis pay . in the inpatient sector , a german diagnosis - related group system was used .
additional end points included the percentage of patients having any sick or disability days ( sick leave > 6 weeks ) , the number of ms - related and all - cause sick days , and all - cause disability days .
results for baseline demographic and clinical characteristics , hru and costs , and sick days and disability days are summarized descriptively .
continuous variables are presented as means ( with standard deviations [ sds ] ) or medians ( with ranges ) , depending on the distributions of the data .
statistical comparisons of baseline demographic and clinical characteristics between those with and without prior dmt use were performed using nonpaired t - tests for means and mann whitney u tests for medians ; chi - square and fisher s exact tests were used for comparisons of proportions .
statistical comparisons of the changes from the pre- to post - index period for hru , costs , sick days , and disability days were performed using one - sample t - tests for means and wilcoxon signed - rank tests for medians ; mcnemar chi - square tests were used for comparisons of categorical data .
statistical comparisons of the changes from the pre- to post - index period between patients with and without prior dmt use were performed using two - sample t - tests for means and wilcoxon rank - sum tests for medians ; mantel haenszel chi - square tests were used for categorical data .
the current analysis was conducted using the health risk institute ( hri ) research database , which contains anonymized , patient - level inpatient , outpatient , sick leave , and pharmacy claims data from about 80 health insurance companies , representing a high proportion of th130 health insurance companies that comprise the shi in germany20 and providing good external validity.23 for 20082013 , the hri research database contained individual , de - identified claims data for about 4 million people , with a patient sample that is representative of the german population for age and gender.24,25 the database is fully compliant with all data protection regulations in germany and has been certified . as the hri research database includes
verified accounting data of the participating insurance companies , these claims data are regularly audited by the insurance companies for reimbursement purposes and are prepared in accordance with german social law ( paragraphs 287 sgb v and 75 sgb x ) .
this study utilized an existing data set in line with all data protection regulations , and patients were not identified for the purpose of this study .
this study is a retrospective database study based on fully anonymized claims data , and claims data are recorded for accounting purposes and not for clinical research . no electronic medical records or other clinical parameters were used . as a result , no ethical approval or consent from an ethics committee or review board was required for this study .
the study sample included patients aged 18 years who initiated treatment with natalizumab ( index date [ anatomical therapeutic chemical { atc } code l04aa23 ] between january 1 , 2009 , and december 31 , 2012 ) .
all patients had a primary or secondary diagnosis of ms ( international statistical classification of diseases and related health problems , 10th revision , german modification [ icd-10-gm ] codes g35.xx ) in the inpatient sector in the pre - index period or a verified diagnosis of ms in the outpatient sector during any of the four quarters before the index quarter .
at least 24 months of continuous eligibility was required , including a 12-month pre - index period and a 12-month post - index period .
furthermore , patients had to have 1 natalizumab prescription in the fourth quarter of the post - index period to indicate continuous natalizumab use .
thus , the entire study covered the time interval from january 1 , 2008 , to december 31 , 2013 ( figure 1 ) .
outcomes were evaluated in the overall sample of patients who initiated treatment with natalizumab and in natalizumab initiators based on the prior use of other dmts .
patients were categorized as having used ( prior dmt use ) or not used ( no prior dmt use ) another dmt during the 12-month pre - index period .
the deyo - charlson comorbidity index ( dci),26,27 a summary measure of comorbidity burden , is reported as both a continuous and categorical measure .
individual dci comorbidities are reported as binary outcomes ( i.e. , present or not present ) .
ms - related and all - cause hospitalizations were defined as having a primary ms - related icd-10-gm code ( g35.xx ) or any icd-10-gm code , respectively . for the total population , the total number of inpatient stays and total length of stay were each summed across all admissions for the pre- and post - index period .
the use of corticosteroids ( intravenous [ iv ] and oral ) was determined if a patient had 1 prescription claim for dexamethasone , methylprednisolone , prednisolone , prednisone , or adrenocorticotropic hormone .
similarly , immunosuppressant use was recorded affirmatively if a patient had 1 claim for an immunosuppressant . per the german association of neurologists guidelines for diagnosis and treatment of ms,28 drugs for ms symptom relief
include antidementia agents , antidepressants , antiepileptics , muscle relaxants ( i.e. , baclofen , botulinum toxin , dantrolene , tizanidine , tolperisone , and tetrazepam ) , antispasmodics , and select medications to manage fatigue ( i.e. , amantadine and modafinil ) , tremor ( i.e. , propranolol ) , and sexual dysfunction ( i.e. , sildenafil , tadalafil , and tibolone ) .
use of ms symptom relief and all ms - related prescription medications was identified using the atc classification system .
health care related costs ( in euros [ ] ) were calculated separately for the pre- and post - index periods on a per - patient basis and converted to 2013 using the general rate of inflation for germany.29 ms - related inpatient costs were defined as the total costs for patient hospitalizations with a primary icd-10-gm code for ms , and all - cause costs were defined as the total costs for admissions for any reason .
total costs for corticosteroids ( iv , oral , and self - injectable ) and immunosuppressants were summed across all relevant prescription claims .
total costs for ms - related outcomes were calculated as the sum of all costs for ms - related hospitalizations , corticosteroids , and immunosuppressants .
costs were collected in the hri database and the reported costs were those for which the shis pay . in the inpatient sector , a german diagnosis - related group system was used .
additional end points included the percentage of patients having any sick or disability days ( sick leave > 6 weeks ) , the number of ms - related and all - cause sick days , and all - cause disability days .
results for baseline demographic and clinical characteristics , hru and costs , and sick days and disability days are summarized descriptively .
continuous variables are presented as means ( with standard deviations [ sds ] ) or medians ( with ranges ) , depending on the distributions of the data .
statistical comparisons of baseline demographic and clinical characteristics between those with and without prior dmt use were performed using nonpaired t - tests for means and mann whitney u tests for medians ; chi - square and fisher s exact tests were used for comparisons of proportions .
statistical comparisons of the changes from the pre- to post - index period for hru , costs , sick days , and disability days were performed using one - sample t - tests for means and wilcoxon signed - rank tests for medians ; mcnemar chi - square tests were used for comparisons of categorical data .
statistical comparisons of the changes from the pre- to post - index period between patients with and without prior dmt use were performed using two - sample t - tests for means and wilcoxon rank - sum tests for medians ; mantel haenszel chi - square tests were used for categorical data .
the final sample included 193 patients who initiated treatment with natalizumab between 2009 and 2012 and met all selection criteria ( figure 2 ) .
the majority of patients ( 75% ; n=145 ) had used another dmt during the pre - index period .
the mean ( sd ) age was 37.1 ( 10.2 ) years , and 65% of the sample was female .
overall , hemiplegia or paraplegia ( 20.7% ) and chronic pulmonary disease ( 19.7% ) were the most prevalent conditions contributing to the dci . with the exception of mean dci , which was significantly higher among patients with prior dmt use ,
baseline demographic and clinical characteristics were comparable regardless of prior dmt use ( table 1 ) .
ms - related and all - cause inpatient hru and expenditures were reduced after initiating natalizumab compared with the pre - index period . in the overall sample ,
the proportion of patients with ms - related hospitalizations decreased significantly on natalizumab therapy , from 49.7% to 14.0% ( p<0.001 ; figure 3a ) , with a corresponding decrease in the per - patient mean number of ms - related hospitalizations from 1.1 to 0.2 ( p<0.001 ; table 2 ) .
the mean number of ms - related inpatient days among those with hospital stays decreased by 61.3% , from 7.0 days ( n=96 ) in the pre - index period to 2.7 ( n=27 ) on natalizumab therapy ( p<0.001 ; table 2 ) . in the overall sample , the proportion of patients with ms - related hospitalizations decreased by 71.8% while on natalizumab therapy ( p<0.001 ; figure 3a ) .
similarly significant reductions were observed for patients with ( 70.2% , p<0.001 ) and without ( 77.3% , p<0.001 ) prior dmt use . the mean number of ms - related hospitalizations also decreased significantly in both groups , from 1.1 to 0.2 in patients with prior dmt use ( p<0.001 ) and from 0.9 to 0.1 in patients without prior dmt use ( p<0.001 ; table s1 ) . among all ms patients , the mean duration of hospital stays
decreased by 52.8% in patients with prior dmt use ( from 7.2 to 3.4 days ; p<0.001 ) and by 90.5% in patients without prior dmt use ( from 6.3 to 0.6 days ; p<0.001 ) .
mean annual per - patient ms - related inpatient costs decreased by 78.3% , from 3,759 ( median , 706 ; range , 060,583 ) in the pre - index period to 815 ( median , 0 ; range , 025,934 ) in the post - index period in the overall sample , and by 76.6% and 83.9% in patients with and without prior dmt use , respectively ( all p<0.001 ; figure 3b ) .
in the overall sample , the proportion of patients hospitalized for any cause decreased by 52.4% while on natalizumab therapy ( p<0.001 ; figure 4a ) .
the mean number of all - cause hospitalizations was 1.3 in the pre - index period and 0.4 in the post - index period ( p<0.001 ) , and the mean duration of these stays decreased by 48.2% , from 9.6 to 5.0 days ( p<0.001 ; table 2 ) .
similar reductions after natalizumab initiation were observed in the subgroups of patients with and without prior dmt use ( figure 4a ; table s1 ) .
mean annual per - patient all - cause inpatient costs decreased by 64.0% , from 4,610 ( median , 2,365 ; range , 060,583 ) in the pre - index period to 1,660 ( median , 0 ; range , 033,719 ) in the post - index period in the overall sample and by 58.9% and 78.7% in patients with and without prior dmt use , respectively ( all p<0.001 ; figure 4b ) .
in the first year of treatment with natalizumab , statistically significant reductions from the pre - index period in the proportions of patients using corticosteroids were observed among all patients ( 62.3% reduction ) and among those with ( 63.8% reduction ) and without ( 56.6% reduction ) prior dmt use ( all p<0.001 ; figure 5a ) .
similarly , a significant decrease was observed in the mean number of corticosteroid prescriptions for all patients ( from 2.4 to 0.6 ; p<0.001 ) .
patients who had received prior dmts showed a larger change in corticosteroid use ( from 2.6 to 0.6 ) than those who did not receive prior dmts ( from 2.1 to 0.9 ; all p<0.001 ; table 2 ; table s1 ) .
mean expenditures associated with corticosteroid use also significantly declined in each patient group ( decreased by 78.9% , 79.0% , and 78.2% for patients overall , with prior dmt use , and without prior dmt use , respectively ; all p<0.001 ; figure 5b ) .
after initiation of natalizumab , the percentage of patients with ms - related and all - cause sick days decreased from the pre - index period by 27.6% ( p<0.001 ) and 14.0% ( p=0.011 ) , respectively ( figure 6a , b ) .
the percentages of patients with sick days also decreased in patients with and without prior dmt use ; however , only the changes in the former group were statistically significant ( figure 6a , b ) .
in contrast , changes in the mean number of ms - related and all - cause sick days were statistically significant for all patients regardless of previous dmt use ( table 2 ; table s1 ) . the mean number of ms - related sick days per patient was reduced by 8.0 days ( p<0.001 ) for patients overall , by 7.7 days ( p=0.003 ) for those with prior dmt use , and by 8.9 days ( p=0.012 ) for those without prior dmt use .
similarly , the mean number of all - cause sick days was reduced by 8.3 days ( p<0.001 ) , 9.0 days ( p=0.001 ) , and 6.2 days ( p=0.026 ) , respectively . both for patients with and without prior dmt use , changes in the proportion of patients with all - cause disability days and in the mean number of all - cause disability days ( table 2 ; table s1 ) were not statistically significant .
the final sample included 193 patients who initiated treatment with natalizumab between 2009 and 2012 and met all selection criteria ( figure 2 ) .
the majority of patients ( 75% ; n=145 ) had used another dmt during the pre - index period .
the mean ( sd ) age was 37.1 ( 10.2 ) years , and 65% of the sample was female .
overall , hemiplegia or paraplegia ( 20.7% ) and chronic pulmonary disease ( 19.7% ) were the most prevalent conditions contributing to the dci . with the exception of mean dci , which was significantly higher among patients with prior dmt use ,
baseline demographic and clinical characteristics were comparable regardless of prior dmt use ( table 1 ) .
ms - related and all - cause inpatient hru and expenditures were reduced after initiating natalizumab compared with the pre - index period . in the overall sample ,
the proportion of patients with ms - related hospitalizations decreased significantly on natalizumab therapy , from 49.7% to 14.0% ( p<0.001 ; figure 3a ) , with a corresponding decrease in the per - patient mean number of ms - related hospitalizations from 1.1 to 0.2 ( p<0.001 ; table 2 ) .
the mean number of ms - related inpatient days among those with hospital stays decreased by 61.3% , from 7.0 days ( n=96 ) in the pre - index period to 2.7 ( n=27 ) on natalizumab therapy ( p<0.001 ; table 2 ) . in the overall sample , the proportion of patients with ms - related hospitalizations decreased by 71.8% while on natalizumab therapy ( p<0.001 ; figure 3a ) .
similarly significant reductions were observed for patients with ( 70.2% , p<0.001 ) and without ( 77.3% , p<0.001 ) prior dmt use .
the mean number of ms - related hospitalizations also decreased significantly in both groups , from 1.1 to 0.2 in patients with prior dmt use ( p<0.001 ) and from 0.9 to 0.1 in patients without prior dmt use ( p<0.001 ; table s1 ) . among all ms patients , the mean duration of hospital stays
decreased by 52.8% in patients with prior dmt use ( from 7.2 to 3.4 days ; p<0.001 ) and by 90.5% in patients without prior dmt use ( from 6.3 to 0.6 days ; p<0.001 ) .
mean annual per - patient ms - related inpatient costs decreased by 78.3% , from 3,759 ( median , 706 ; range , 060,583 ) in the pre - index period to 815 ( median , 0 ; range , 025,934 ) in the post - index period in the overall sample , and by 76.6% and 83.9% in patients with and without prior dmt use , respectively ( all p<0.001 ; figure 3b ) .
in the overall sample , the proportion of patients hospitalized for any cause decreased by 52.4% while on natalizumab therapy ( p<0.001 ; figure 4a ) .
the mean number of all - cause hospitalizations was 1.3 in the pre - index period and 0.4 in the post - index period ( p<0.001 ) , and the mean duration of these stays decreased by 48.2% , from 9.6 to 5.0 days ( p<0.001 ; table 2 ) .
similar reductions after natalizumab initiation were observed in the subgroups of patients with and without prior dmt use ( figure 4a ; table s1 ) .
mean annual per - patient all - cause inpatient costs decreased by 64.0% , from 4,610 ( median , 2,365 ; range , 060,583 ) in the pre - index period to 1,660 ( median , 0 ; range , 033,719 ) in the post - index period in the overall sample and by 58.9% and 78.7% in patients with and without prior dmt use , respectively ( all p<0.001 ; figure 4b ) .
in the first year of treatment with natalizumab , statistically significant reductions from the pre - index period in the proportions of patients using corticosteroids were observed among all patients ( 62.3% reduction ) and among those with ( 63.8% reduction ) and without ( 56.6% reduction ) prior dmt use ( all p<0.001 ; figure 5a ) .
similarly , a significant decrease was observed in the mean number of corticosteroid prescriptions for all patients ( from 2.4 to 0.6 ; p<0.001 ) .
patients who had received prior dmts showed a larger change in corticosteroid use ( from 2.6 to 0.6 ) than those who did not receive prior dmts ( from 2.1 to 0.9 ; all p<0.001 ; table 2 ; table s1 ) .
mean expenditures associated with corticosteroid use also significantly declined in each patient group ( decreased by 78.9% , 79.0% , and 78.2% for patients overall , with prior dmt use , and without prior dmt use , respectively ; all p<0.001 ; figure 5b ) .
after initiation of natalizumab , the percentage of patients with ms - related and all - cause sick days decreased from the pre - index period by 27.6% ( p<0.001 ) and 14.0% ( p=0.011 ) , respectively ( figure 6a , b ) .
the percentages of patients with sick days also decreased in patients with and without prior dmt use ; however , only the changes in the former group were statistically significant ( figure 6a , b ) .
in contrast , changes in the mean number of ms - related and all - cause sick days were statistically significant for all patients regardless of previous dmt use ( table 2 ; table s1 ) .
the mean number of ms - related sick days per patient was reduced by 8.0 days ( p<0.001 ) for patients overall , by 7.7 days ( p=0.003 ) for those with prior dmt use , and by 8.9 days ( p=0.012 ) for those without prior dmt use .
similarly , the mean number of all - cause sick days was reduced by 8.3 days ( p<0.001 ) , 9.0 days ( p=0.001 ) , and 6.2 days ( p=0.026 ) , respectively . both for patients with and without prior dmt use , changes in the proportion of patients with all - cause disability days and in the mean number of all - cause disability days ( table 2 ; table s1 ) were not statistically significant .
natalizumab has been available in germany since 2006.30 among german people with ms initiating natalizumab treatment between january 2009 and december 2012 in real - world settings , this study demonstrated significant reductions at 12 months in ms - related and all - cause hospitalizations and corticosteroid use .
administrative medical and pharmacy claims data from the german hri research database showed that , from the pre- to the post - index period , the proportion of people with ms hospitalized for ms - related and all - cause events decreased by 72% and 52% , respectively , and the percentage of people with ms using corticosteroids was reduced by 62% . for ms - related and all - cause hospitalizations ,
per - person costs were reduced by nearly 3,000 ; for corticosteroid use , per - person costs were reduced by a mean of approximately 300 . in this study , 75% of people with ms
the reductions in ms - related and all - cause hospitalizations and costs and ms - related and all - cause sick days were larger among people with ms without prior dmt use .
in contrast , reductions in corticosteroid use and disability days were comparable between people with ms with and without prior dmt use .
these findings are supported by studies in other countries of people with ms starting or switching to natalizumab.3133 in a similarly designed us - based analysis , significant reductions were observed in ms - related and all - cause hospitalizations , corticosteroid use , and associated costs after starting treatment with natalizumab.31 seventy percent of people with ms had used pre - index dmt ; for ms - related hospitalizations and costs , reductions were larger in the cohort of people with ms who had not used pre - index dmt.31 in an italian observational study of people with rrms who had continued disease activity after interferon beta or glatiramer acetate therapy , a significantly larger percentage of those who switched their therapy to natalizumab showed no evidence of disease activity compared with those who switched to another dmt after 24 months.32 in other studies , people with ms with breakthrough disease after the first - line therapy showed larger reductions in disease activity after switching from another dmt to natalizumab.17,33,34 in a 2006 german cost - of - illness analysis , the mean total annual cost of ms per person was estimated at 40,000 , two - fifths of which was related to decreased productivity owing to sick leave and early retirement.3 decreased numbers of ms - related sick days ( 8.0 days ) and all - cause sick days ( 8.3 days ) in the first year following natalizumab treatment initiation in this study could correlate with a significant decline in the indirect costs of ms . in combination with the declines in direct medical costs also observed here , the addition of natalizumab to a treatment regimen for a person with ms could have a positive impact on the economic burden of ms in germany .
while decreases in the number of disability - related days were observed , these changes did not reach statistical significance . this may be due to the short 12-month followup period and/or the small number of disabled people with ms .
in addition , dmt treatment may mitigate the number of people with ms who become disabled as 70% of the population is pretreated .
these reductions and cost offsets suggest that natalizumab may be a cost - effective treatment option for ms in germany .
an analysis of shi claims data showed that the prevalence of ms in bavaria had increased by 42% from 2005 to 2009 , corresponding to an overall projected prevalence of 142,856 people with ms in germany in 2009.30 in addition , the percentage of people with ms receiving 1 dmt increased from 46% in 2005 to 51% in 2009 .
thus , given the increasing prevalence of ms and the increasing proportion of people with ms receiving multiple dmts in germany , the findings from the current study suggest that natalizumab may positively impact the high economic burden associated with hru among german people with ms.4 as this is a descriptive study , these results should be interpreted in the context of certain limitations .
first , although the hri research database is generally representative of the german population , the relatively small sample size of this study restricts the extent to which the findings are generalizable to the german population of people with ms , at large .
furthermore , the study results may not be generalizable to pregnant women , as they were excluded from this analysis .
it was not possible to evaluate clinical measures of effectiveness , including disability progression , as typically assessed by changes in expanded disability status scale scores , clinically identified relapses , and severity of the relapses , as this information is not available from the hri research database .
in addition , the time period of the analysis does not allow for a complete record of a person s history with ms , including disease duration , prior treatment , or relapse , all of which may differ over time .
also , to have a comparable patient sample , only patients staying on natalizumab therapy were included .
these analyses were limited by the 1-year pre- and post - index periods , and future analyses can examine 2-year pre- and post - index periods , provided that sample sizes are large enough .
these analyses were also limited to hru ( i.e. , hospitalizations , corticosteroid use , and sick days ) related to ms outcomes and did not include any dmt costs , such as the cost of natalizumab or dmt costs in the pre - index period .
in addition , corticosteroid use may be underestimated in this study because its use in the inpatient setting could not be included in this analysis .
corticosteroid use was evaluated as a dichotomous outcome , and corticosteroid dosing changes around natalizumab were not evaluated .
finally , in german claims data , outpatient visits are not coded with a diagnosis code , and emergency room ( er ) visits can not be assessed ; therefore , ms - specific outpatient visits and er visits were not included in this analysis .
further research is warranted , including a more detailed analysis to evaluate study outcomes among people with ms by disease severity and to evaluate hospitalizations that may be preventable , thereby reducing hru - related costs .
in a real - world setting in germany , people with ms who initiated treatment with natalizumab demonstrated significant reductions in ms - related and all - cause inpatient stays and corticosteroid use with associated costs .
natalizumab was also associated with significant decreases from the pre - index period in the percentage of people with ms - related and all - cause sick days and in the mean number of ms - related and all - cause sick days .
these findings suggest that the benefits of natalizumab , as demonstrated in clinical trials , translate into real - world reductions in ms - related and all - cause hospitalizations and corticosteroid use .
in addition , people with ms without dmt use had a greater magnitude of reduction in both ms - related and all - cause inpatient utilization and costs following natalizumab initiation than people with ms with prior dmt use , further highlighting the need to start treatment early .
health care resource utilization among the subgroups of patients with and without prior dmt use before and after initiating treatment with natalizumab abbreviations : dmt , disease - modifying therapy ; ms , multiple sclerosis ; sd , standard deviation . | backgroundmultiple sclerosis ( ms ) , a progressive neurodegenerative disease , greatly impacts the quality of life and economic status of people affected by this disease . in germany , the total annual cost of ms is estimated at 40,000 per person with ms .
natalizumab has shown to slow ms disease progression , reduce relapses , and improve the quality of life of people with ms.objectiveto evaluate ms - related and all - cause health care resource utilization and costs among german ms patients during the 12 months before and after initiation of natalizumab in a real - world setting.methodsthe current analysis was conducted using the health risk institute research database .
identified patients were aged 18 years with 1 diagnosis of ms and had initiated natalizumab therapy ( index ) , with 12-month pre and post index - period data .
patients were stratified by prior disease - modifying therapy ( dmt ) usage or no dmt usage in the pre - index period .
outcome measures included corticosteroid use and number of sick / disability days , inpatient stays , and outpatient visits .
health care costs were calculated separately for pre- and post - index periods on a per - patient basis and adjusted for inflation.resultsin a final sample of 193 natalizumab - treated patients , per - patient ms - related corticosteroid use was reduced by 62.3% , ms - related sick days by 27.6% , and inpatient costs by 78.3% from the pre- to post - index period .
furthermore , the proportion of patients with ms - related hospitalizations decreased from 49.7% to 14.0% ( p<0.001 ) ; this reduction was seen for patients with and without prior dmt use.conclusionsin a real - world setting in germany , initiation of natalizumab treatment in people with ms significantly reduced ms - related hospitalizations , corticosteroid use , sick days , and associated costs . | Introduction
Methods
Database
Case selection
Patient groups
Outcome measures
Statistical analyses
Results
Patient disposition and characteristics
Inpatient hospitalizations and expenditures
Corticosteroid use
Sick days and disability days
Discussion
Conclusions
Supplementary material | people with ms who experience relapses are often treated with corticosteroids and admitted to the hospital.11 ms relapses contribute significantly to the costs associated with the disease,12 which increase with disease severity.1214 treatments that can slow or prevent the progression of ms have the potential to significantly improve the quality of life for people with ms and reduce the economic impact of the disease.14,15 natalizumab , a selective adhesion - molecule inhibitor , has been shown to reduce annualized relapse rates by 68% versus placebo among people with rrms16,17 and by 81% versus placebo among people with highly active rrms.18 in the european union , natalizumab is indicated in adults with highly active rrms who failed to respond to an adequate course of interferon beta or glatiramer acetate , and in adults with rapidly evolving severe rrms ( with 2 relapses in 1 year and 1 gadolinium - enhancing lesion or a significant increase in t2 lesion load compared with a previous recent magnetic resonance imaging assessment).19 in germany , ~90% of the population ( ~70 million individuals ) is insured as part of the statutory health insurance ( shi ) program,20,21 and the prevalence of ms among people insured by the shi in 2010 was estimated to range from 120,000 to 200,000.22 the objective of this study was to evaluate ms - related and all - cause health care resource utilization ( hru ) and costs among german people with ms during the 12 months before and 12 months after initiation of natalizumab in a real - world setting . health care related costs ( in euros [ ] ) were calculated separately for the pre- and post - index periods on a per - patient basis and converted to 2013 using the general rate of inflation for germany.29 ms - related inpatient costs were defined as the total costs for patient hospitalizations with a primary icd-10-gm code for ms , and all - cause costs were defined as the total costs for admissions for any reason . health care related costs ( in euros [ ] ) were calculated separately for the pre- and post - index periods on a per - patient basis and converted to 2013 using the general rate of inflation for germany.29 ms - related inpatient costs were defined as the total costs for patient hospitalizations with a primary icd-10-gm code for ms , and all - cause costs were defined as the total costs for admissions for any reason . after initiation of natalizumab , the percentage of patients with ms - related and all - cause sick days decreased from the pre - index period by 27.6% ( p<0.001 ) and 14.0% ( p=0.011 ) , respectively ( figure 6a , b ) . in the overall sample ,
the proportion of patients with ms - related hospitalizations decreased significantly on natalizumab therapy , from 49.7% to 14.0% ( p<0.001 ; figure 3a ) , with a corresponding decrease in the per - patient mean number of ms - related hospitalizations from 1.1 to 0.2 ( p<0.001 ; table 2 ) . after initiation of natalizumab , the percentage of patients with ms - related and all - cause sick days decreased from the pre - index period by 27.6% ( p<0.001 ) and 14.0% ( p=0.011 ) , respectively ( figure 6a , b ) . these findings are supported by studies in other countries of people with ms starting or switching to natalizumab.3133 in a similarly designed us - based analysis , significant reductions were observed in ms - related and all - cause hospitalizations , corticosteroid use , and associated costs after starting treatment with natalizumab.31 seventy percent of people with ms had used pre - index dmt ; for ms - related hospitalizations and costs , reductions were larger in the cohort of people with ms who had not used pre - index dmt.31 in an italian observational study of people with rrms who had continued disease activity after interferon beta or glatiramer acetate therapy , a significantly larger percentage of those who switched their therapy to natalizumab showed no evidence of disease activity compared with those who switched to another dmt after 24 months.32 in other studies , people with ms with breakthrough disease after the first - line therapy showed larger reductions in disease activity after switching from another dmt to natalizumab.17,33,34 in a 2006 german cost - of - illness analysis , the mean total annual cost of ms per person was estimated at 40,000 , two - fifths of which was related to decreased productivity owing to sick leave and early retirement.3 decreased numbers of ms - related sick days ( 8.0 days ) and all - cause sick days ( 8.3 days ) in the first year following natalizumab treatment initiation in this study could correlate with a significant decline in the indirect costs of ms . | [
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transient ischemic attack ( tia ) has long time been one of the most important risk factors for severe ischemic stroke , the incidence of which is dramatically high during the early stage of ischemic stroke [ 13 ] .
thus , advanced evaluation and management of tia is imperative . with the development of new neuroimaging modalities , such as cta , mra , and dsa ,
increasing imaging records on tia can be found , especially with the application of mra , which can be used to improve the determination of patients with high risk of having recurrent stroke or tia .
application of neuroimaging modalities have revealed that stenosis or occlusion of a large proximal intracranial or extracranial symptomatic artery were significantly associated with recurrent stroke / tia [ 48 ] .
most previous studies concentrated on the imaging results of brain tissues and vascular lesions , and the degree of artery stenosis and plaques , but few researchers have paid attention to the difference of vascular characteristics between tia and infarction patients .
the mechanism underlying occurrence of tia in vertebral artery lesions , such as hypoplasia should be studied . in the current study , we present a heuristic model of determination of tia patients by utilizing mra , ce - mra , and dwi . the contributing factors of tia attack are explained in terms of the entire circulatory system .
thirty - four healthy subjects ( 18 males and 16 females , average age 55.2413.84 ) with no history of hypertension , hyperlipoidemia , diabetes mellitus , heart or cerebrovascular disease , or abnormal ct / mri results were randomly selected as the control group ( group a ) in the present study .
forty tia patients ( 30 males and 16 females , average age 60.5310.39 ) were enrolled from september 2009 to august 2012 in the rizhao city people s hospital ( group b ) .
the tia patients were diagnosed based on the criteria of easton et al . and had transient ischemic symptoms with a mean attack time of 0.9320.665 h ( range , 1 min to 9 h ) .
eight patients in group b had tia of the internal carotid artery ( ica ) system and 32 patients had tia of the vertebral artery system .
group c consisted of 36 cerebral infarction patients ( 21 males and 15 females , average age 56.588.21 ) .
patients were diagnosed as having cerebral infarction when infarct size was larger than 2 cm as illustrated by ct or mri .
there was no significant difference in age or sex between the 3 groups ( =4.267 , p=0.118 ) .
the ethics committee of rizhao city people s hospital approved the relating screening , inspection , and data collection of the patients , and all subjects signed a written informed consent form .
mra and dwi detection was conducted on all subjects using a 1.5-t mr scanner ( twin - speed infinity with excite i , ge , usa ) following standard procedure : intracranial 3d tof ( time of flight ) ( tr / te=32/3.3 ms , fov=1818 cm , flip angle=20 , section thickness=1.5 mm , matrix=288128 , band width=15.6 khz , total acquisition time=5 min ) and a cervical 3d ce - mra ( tr / te=6 ms / minimum , fov=2428 cm , flip angle=45 , section thickness=1.8 mm , slab thickness=65 mm , matrix=256256 , total acquisition time=55 s , from aorta to circle of willis ) with intravenous administration of gd - dtpa ( 2 ml / s of 0.1 mmol / kg gd , 20 ml saline flash ) .
dwi was also performed using 3 sensitive gradients for brain tissue ( tr / te=10000/107 ms , matrix=128128 , fov = 2424 cm , section thickness=6 mm with a gap of 1.5 mm , b value=0 s / mm and 1000 s / mm , acquisition time=40 s ) .
mra range was from the aortic arch to top of skull , and dwi was from the skull base to top of skull .
the inner diameter of vertebral artery was measured at the level between c6 and c2 .
vertebral artery hypoplasia ( vah ) was defined as that the diameter of vertebral artery ( va ) was less than 23 mm by 3d tof mra .
all patients were evaluated by the same 2 senior vascular radiology physicians . according to the standard of the north american symptomatic carotid endarterectomy trial ( nascet )
, lesions were classified as mild ( 029% ) , moderate ( 3069% ) , severe ( 7099% ) , and occlusion ( 100% ) .
arterial stenosis was graded by comparing the diameter of the maximally stenosed artery with the diameter of the more proximal normal segment of the same vessel .
least significant difference ( lsd ) and chi - square analyses were performed with a significant level of 0.05 using spss version 16.0 ( ibm , armonk , ny , usa ) .
thirty - four healthy subjects ( 18 males and 16 females , average age 55.2413.84 ) with no history of hypertension , hyperlipoidemia , diabetes mellitus , heart or cerebrovascular disease , or abnormal ct / mri results were randomly selected as the control group ( group a ) in the present study .
forty tia patients ( 30 males and 16 females , average age 60.5310.39 ) were enrolled from september 2009 to august 2012 in the rizhao city people s hospital ( group b ) .
the tia patients were diagnosed based on the criteria of easton et al . and had transient ischemic symptoms with a mean attack time of 0.9320.665 h ( range , 1 min to 9 h ) .
eight patients in group b had tia of the internal carotid artery ( ica ) system and 32 patients had tia of the vertebral artery system .
group c consisted of 36 cerebral infarction patients ( 21 males and 15 females , average age 56.588.21 ) .
patients were diagnosed as having cerebral infarction when infarct size was larger than 2 cm as illustrated by ct or mri .
there was no significant difference in age or sex between the 3 groups ( =4.267 , p=0.118 ) .
the ethics committee of rizhao city people s hospital approved the relating screening , inspection , and data collection of the patients , and all subjects signed a written informed consent form .
mra and dwi detection was conducted on all subjects using a 1.5-t mr scanner ( twin - speed infinity with excite i , ge , usa ) following standard procedure : intracranial 3d tof ( time of flight ) ( tr / te=32/3.3 ms , fov=1818 cm , flip angle=20 , section thickness=1.5 mm , matrix=288128 , band width=15.6 khz , total acquisition time=5 min ) and a cervical 3d ce - mra ( tr / te=6 ms / minimum , fov=2428 cm , flip angle=45 , section thickness=1.8 mm , slab thickness=65 mm , matrix=256256 , total acquisition time=55 s , from aorta to circle of willis ) with intravenous administration of gd - dtpa ( 2 ml / s of 0.1 mmol / kg gd , 20 ml saline flash ) . dwi was also performed using 3 sensitive gradients for brain tissue ( tr / te=10000/107 ms , matrix=128128 , fov = 2424 cm , section thickness=6 mm with a gap of 1.5 mm , b value=0 s / mm and 1000 s / mm , acquisition time=40 s ) .
mra range was from the aortic arch to top of skull , and dwi was from the skull base to top of skull .
the inner diameter of vertebral artery was measured at the level between c6 and c2 .
vertebral artery hypoplasia ( vah ) was defined as that the diameter of vertebral artery ( va ) was less than 23 mm by 3d tof mra .
according to the standard of the north american symptomatic carotid endarterectomy trial ( nascet ) , lesions were classified as mild ( 029% ) , moderate ( 3069% ) , severe ( 7099% ) , and occlusion ( 100% ) .
arterial stenosis was graded by comparing the diameter of the maximally stenosed artery with the diameter of the more proximal normal segment of the same vessel .
least significant difference ( lsd ) and chi - square analyses were performed with a significant level of 0.05 using spss version 16.0 ( ibm , armonk , ny , usa ) .
both observers came to agreement in interpreting 3d tof , dwi , and ce - mra images for more than 97% of patients .
if there was any disagreement between the 2 readers , the results were evaluated in a joint session .
the number and degree of stenoses of cranial and cervical arteries are provided in table 1 .
seventy - six arteries in 35 tia patients ( 87.5% ) had different severities of artery tortuosity and stenosis , of which 43 affected arteries ( 56.58% ) were intracranial lesions , 24 ( 31.58% ) were extracranial lesions , and 9 ( 11.84% ) were extracranial - intracranial lesions .
the number of the lesions in group a was significantly less than in group b and group c. the major types of lesions were tortuosity and mild stenosis ( 16 arteries ) , which were common in the middle - aged population .
severe stenoses and occlusions were more frequently in group b and group c. chi - square test showed that the difference between the 2 groups was significant ( =18.02 , p=0.006 ) .
after partition , there was significant difference between the 3 groups ( =13.524 , p=0.004 ; =10.407 , p=0.015 ) but no significant difference was detected between group b and group c ( =5.351 , p=0.148 ) .
twenty - one brain infarction patients were followed up ; we found there were 13 patients ( 61.9% ) had suffered from transient ischemia symptom in the last 2 years .
multiple artery lesions were more common in group b and group c compared with group a ( =25.921 , p<0.001 ; =29.179 , p<0.001 ) , but no significant difference was found between group b and group c ( =1.194 , p=0.755 ) .
the 8 patients with tia of the internal carotid artery ( ica ) system had no maldevelopment of the vertebral artery but 13 patients with tia of the vertebral artery system ( 40.63% ) were found to have unilateral maldevelopment .
moreover , 6 cases ( 17.65% ) in healthy controls were found to have maldevelopment of the vertebral artery ( table 2 ) .
the hvas had a mean inner diameter of 1.720.89 mm and the mean diameter of the opposite side vertebral arteries was 3.900 .
arthrosclerosis was common in the hva patients in group b , characterized by low signal intensity , irregularity of the artery wall , stenosis , and occlusion .
the opposite side always showed dilatation of the lumen and tortuosity , but 6 ( 46.15% ) of them had focal stenosis ( figure 1 ) .
eight patients in group b had high signal intensity in dwi images , 4 of which were attacked by tia of the ica system .
the focus was 12 cm in area , most of them were solitary but a few were multiple .
one had focus in the brain stem , but no obvious lesion was found in the va system .
12 were found to have severe stenosis and occlusion . compared with the 32 patients ( 17 arteries ) who had no high dwi signal intensity , it was significantly different ( =8.869 , p=0.003 ) .
one patient with tia of the ica system showed large area of abnormal signal intensity on apparent diffusion coefficient ( adc ) image in the right temporal and occipital lobe ( figure 2 . ) .
the adc value was 27.26% lower than in the contralateral area . however , no abnormal adc value was found in other patients .
both observers came to agreement in interpreting 3d tof , dwi , and ce - mra images for more than 97% of patients .
if there was any disagreement between the 2 readers , the results were evaluated in a joint session .
the number and degree of stenoses of cranial and cervical arteries are provided in table 1 .
seventy - six arteries in 35 tia patients ( 87.5% ) had different severities of artery tortuosity and stenosis , of which 43 affected arteries ( 56.58% ) were intracranial lesions , 24 ( 31.58% ) were extracranial lesions , and 9 ( 11.84% ) were extracranial - intracranial lesions .
the number of the lesions in group a was significantly less than in group b and group c. the major types of lesions were tortuosity and mild stenosis ( 16 arteries ) , which were common in the middle - aged population .
severe stenoses and occlusions were more frequently in group b and group c. chi - square test showed that the difference between the 2 groups was significant ( =18.02 , p=0.006 ) .
after partition , there was significant difference between the 3 groups ( =13.524 , p=0.004 ; =10.407 , p=0.015 ) but no significant difference was detected between group b and group c ( =5.351 , p=0.148 ) .
twenty - one brain infarction patients were followed up ; we found there were 13 patients ( 61.9% ) had suffered from transient ischemia symptom in the last 2 years .
multiple artery lesions were more common in group b and group c compared with group a ( =25.921 , p<0.001 ; =29.179 , p<0.001 ) , but no significant difference was found between group b and group c ( =1.194 , p=0.755 ) .
the 8 patients with tia of the internal carotid artery ( ica ) system had no maldevelopment of the vertebral artery but 13 patients with tia of the vertebral artery system ( 40.63% ) were found to have unilateral maldevelopment .
moreover , 6 cases ( 17.65% ) in healthy controls were found to have maldevelopment of the vertebral artery ( table 2 ) .
the hvas had a mean inner diameter of 1.720.89 mm and the mean diameter of the opposite side vertebral arteries was 3.900 .
arthrosclerosis was common in the hva patients in group b , characterized by low signal intensity , irregularity of the artery wall , stenosis , and occlusion .
the opposite side always showed dilatation of the lumen and tortuosity , but 6 ( 46.15% ) of them had focal stenosis ( figure 1 ) .
eight patients in group b had high signal intensity in dwi images , 4 of which were attacked by tia of the ica system .
the focus was 12 cm in area , most of them were solitary but a few were multiple .
one had focus in the brain stem , but no obvious lesion was found in the va system .
compared with the 32 patients ( 17 arteries ) who had no high dwi signal intensity , it was significantly different ( =8.869 , p=0.003 ) .
one patient with tia of the ica system showed large area of abnormal signal intensity on apparent diffusion coefficient ( adc ) image in the right temporal and occipital lobe ( figure 2 . ) .
it is reported that more than 90% tia patients had lesions in brain - feeding arteries , which was confirmed by our study ( 87.5% ) . however , 31.58% of tia patients in our study had lesions at the extracranial part of the cervical arteries and the result was inconsistent with previous studies .
this high percentage of lesions at cervical arteries might be due to the improved living standard of chinese or the higher sensitivity of the ce - mra in the diagnosis of cervical disease .
the number and degree of stenoses of cranial and cervical arteries was not significantly different between group b and group c in our study .
no large infarcted area was found as in multiple artery stenosis and occlusions in most tia patients , but these lesions might have the same pathological basis , with large area of the infarcted lesion .
brain infarction may happen at any time because of the shedding of the existing embolism or the impairment of the compensating path .
the recurrence of transient ischemia symptoms suggests that artery stenosis and thrombus might already exist and the severe stenosis and irregular filling defect might be one of the most important risk factors for severe ischemia stroke , which makes it necessary for tia patients to have accurate assessment of artery lesions as early as possible .
according to george s study , 5.7% of patients have left - side va maldevelopment , 8.8% have right - side va maldevelopment , and 0.6% have it on both sides .
almost all the va maldevelopment patients have non - homogenous signal intensity and irregularity of the artery wall , causing most maldevelopment va patients to have atherosclerosis , and the contralateral va compensates by enlarging .
self - compensation always occurs when 1 side of the va is compromised . with the compensation of the contralateral va being enhanced and more dominant , the contralateral va has a high incidence rate of maldevelopment va , in which case , even small atherosclerosis will cause decompensation and lead to ischemic brain artery disease . in the present study ,
a much higher percentage of unilateral va maldevelopment was detected compared with previous ones ( 40.63% ) and 6 of the 13 ( 46.15% ) tia patients who had unilateral va maldevelopment suffered from contralateral va stenosis .
although other compensatory mechanisms of va insufficiency , such as circle of willis , were also important , the maldevelopment of posterior communicating arteries was more frequent .
we have thought that unilateral va maldevelopment could be a predisposing factor for vertebrobasilar tia because this vulnerable compensatory mechanism would promote the incidence rate of the disease .
it is quite important to determine the situation and give an accurate assessment for normal people and asymptomatic patients .
high dwi signal intensity is commonly detected in tia patients , which involves the swelling of neurons and possibility of infarction . in the present study ,
22.5% ( 9/40 ) of tia patients had high dwi signal intensity or adc value abnormality and the patients with high dwi signal intensity had more severe artery stenosis than those with negative dwi detection .
in addition , only 12.5% of vb tia patients had high dwi signal intensity , suggesting that the incidence of infarction of tia patients is very low , which might be caused by the perfect compensatory mechanism .
kamal et al . analyzed the images of tia patients when attack occurred within 6 h and demonstrated that the affected area that seemed normal had lower adc value ( 9% to 26% ) than the contralateral side .
however , the change was only detected clearly on adc images , while it was negative with dwi detection . in the present study , adc value abnormality with negative dwi finding
was found in only 1 patient , which was obviously inconsistent with the result of kamal s study .
the nerve system damages could be quantitatively evaluated by the measurement of adc value , and it would be of great help for the diagnosis of this slight but measurable abnormality during the evaluation of brain damage in tia patients .
there were several limitations of our study : 1 ) the sample size was small ; 2 ) because of limitations on obtaining research sequences in acute tia patients , we covered only 1 imaging modality ( mri ) ; 3 ) the nature of the lesions was unrevealed , generally , and stenosis is usually attributed to intracranial atherosclerosis but can also result from angiospasm .
it is reported that more than 90% tia patients had lesions in brain - feeding arteries , which was confirmed by our study ( 87.5% ) . however , 31.58% of tia patients in our study had lesions at the extracranial part of the cervical arteries and the result was inconsistent with previous studies .
this high percentage of lesions at cervical arteries might be due to the improved living standard of chinese or the higher sensitivity of the ce - mra in the diagnosis of cervical disease .
the number and degree of stenoses of cranial and cervical arteries was not significantly different between group b and group c in our study .
no large infarcted area was found as in multiple artery stenosis and occlusions in most tia patients , but these lesions might have the same pathological basis , with large area of the infarcted lesion .
brain infarction may happen at any time because of the shedding of the existing embolism or the impairment of the compensating path .
the recurrence of transient ischemia symptoms suggests that artery stenosis and thrombus might already exist and the severe stenosis and irregular filling defect might be one of the most important risk factors for severe ischemia stroke , which makes it necessary for tia patients to have accurate assessment of artery lesions as early as possible .
va maldevelopment is a congenital variation , most of which is unilateral . according to george s study ,
5.7% of patients have left - side va maldevelopment , 8.8% have right - side va maldevelopment , and 0.6% have it on both sides .
almost all the va maldevelopment patients have non - homogenous signal intensity and irregularity of the artery wall , causing most maldevelopment va patients to have atherosclerosis , and the contralateral va compensates by enlarging .
self - compensation always occurs when 1 side of the va is compromised . with the compensation of the contralateral va being enhanced and more dominant , the contralateral va has a high incidence rate of maldevelopment va , in which case , even small atherosclerosis will cause decompensation and lead to ischemic brain artery disease . in the present study ,
a much higher percentage of unilateral va maldevelopment was detected compared with previous ones ( 40.63% ) and 6 of the 13 ( 46.15% ) tia patients who had unilateral va maldevelopment suffered from contralateral va stenosis .
although other compensatory mechanisms of va insufficiency , such as circle of willis , were also important , the maldevelopment of posterior communicating arteries was more frequent .
we have thought that unilateral va maldevelopment could be a predisposing factor for vertebrobasilar tia because this vulnerable compensatory mechanism would promote the incidence rate of the disease .
it is quite important to determine the situation and give an accurate assessment for normal people and asymptomatic patients .
high dwi signal intensity is commonly detected in tia patients , which involves the swelling of neurons and possibility of infarction . in the present study , 22.5% ( 9/40 ) of tia patients had high dwi signal intensity or adc value abnormality and the patients with high dwi signal intensity had more severe artery stenosis than those with negative dwi detection .
in addition , only 12.5% of vb tia patients had high dwi signal intensity , suggesting that the incidence of infarction of tia patients is very low , which might be caused by the perfect compensatory mechanism .
kamal et al . analyzed the images of tia patients when attack occurred within 6 h and demonstrated that the affected area that seemed normal had lower adc value ( 9% to 26% ) than the contralateral side .
however , the change was only detected clearly on adc images , while it was negative with dwi detection . in the present study , adc value abnormality with negative dwi finding
was found in only 1 patient , which was obviously inconsistent with the result of kamal s study .
the nerve system damages could be quantitatively evaluated by the measurement of adc value , and it would be of great help for the diagnosis of this slight but measurable abnormality during the evaluation of brain damage in tia patients .
there were several limitations of our study : 1 ) the sample size was small ; 2 ) because of limitations on obtaining research sequences in acute tia patients , we covered only 1 imaging modality ( mri ) ; 3 ) the nature of the lesions was unrevealed , generally , and stenosis is usually attributed to intracranial atherosclerosis but can also result from angiospasm .
the severe stenosis and filling defect of cranial and cervical arteries of tia patients might be one of the most important risk factors of severe stroke .
the patients with unilateral va malformation were prone to have atherosclerosis , which might cause decompensation and be a predisposing factor for tia.the fresh ischemia foci on dwi were correlated with severe artery lesions .
it would help to reveal the pathologic mechanism of tia patients to detect the variation of adc value .
all these findings suggest that imaging of cranial and cervical arteries should be included in the initial evaluation of tia to prevent severe stroke . | backgroundmagnetic resonance angiography ( mra ) and diffusion - weighted imaging ( dwi ) have been widely used in the prediction of ischemic stroke ; however , the differences of the 2 methods in detection the artery lesion differences between transient ischemic attack ( tia ) and infarction patients have been long neglected .
we performed the present study to investigate the differences between vessel characteristics detected by mra and dwi in acute stroke and tia patients.material/methodswe classified 110 subjects into 2 groups and all the patients underwent both mra and dwi .
the degree of stenosis of cranial and cervical arteries , the distribution of the stenosis , the development and changes of the vessels , and the dwi scanning results of the brain tissue were all analyzed.resultswe detected a significant difference in the number and the degree of stenosis of cranial and cervical arteries among the 3 groups ( p=0.006 ) . compared with health controls ,
patients with tia and cerebral infraction had much more severe stenosis and occlusive arteries ( p<0.05 ) .
however , no significant difference was detected between tia and cerebral infraction patients ( p=0.148 ) .
moreover , a higher rate of unilateral vertebral artery dysplasia was found in the vertebrobasilar tia patients .
higher lesion signals were also observed by dwi in tia patients of internal carotid artery system ( 4/8 , 50%).conclusionsvessel characteristics were not significantly different between tia and infarction patients .
unilateral vertebral artery hypoplasia was a predisposing factor for vertebrobasilar tia and ischemic focus in dwi detection was always caused by severe artery lesions . | Background
Material and Methods
Patients
MRA and DWI detection
Statistical analysis
Results
The stenosis degree and lesion distribution
Maldevelopment of vertebral artery
The results of DWI
Discussion
The degree and distribution of stenosis in TIA patients
The meaning of unilateral VA maldevelopment for TIA patients
DWI findings of TIA patients
Conclusions | transient ischemic attack ( tia ) has long time been one of the most important risk factors for severe ischemic stroke , the incidence of which is dramatically high during the early stage of ischemic stroke [ 13 ] . most previous studies concentrated on the imaging results of brain tissues and vascular lesions , and the degree of artery stenosis and plaques , but few researchers have paid attention to the difference of vascular characteristics between tia and infarction patients . there was no significant difference in age or sex between the 3 groups ( =4.267 , p=0.118 ) . the number and degree of stenoses of cranial and cervical arteries are provided in table 1 . after partition , there was significant difference between the 3 groups ( =13.524 , p=0.004 ; =10.407 , p=0.015 ) but no significant difference was detected between group b and group c ( =5.351 , p=0.148 ) . the 8 patients with tia of the internal carotid artery ( ica ) system had no maldevelopment of the vertebral artery but 13 patients with tia of the vertebral artery system ( 40.63% ) were found to have unilateral maldevelopment . the number and degree of stenoses of cranial and cervical arteries are provided in table 1 . after partition , there was significant difference between the 3 groups ( =13.524 , p=0.004 ; =10.407 , p=0.015 ) but no significant difference was detected between group b and group c ( =5.351 , p=0.148 ) . the 8 patients with tia of the internal carotid artery ( ica ) system had no maldevelopment of the vertebral artery but 13 patients with tia of the vertebral artery system ( 40.63% ) were found to have unilateral maldevelopment . the number and degree of stenoses of cranial and cervical arteries was not significantly different between group b and group c in our study . the recurrence of transient ischemia symptoms suggests that artery stenosis and thrombus might already exist and the severe stenosis and irregular filling defect might be one of the most important risk factors for severe ischemia stroke , which makes it necessary for tia patients to have accurate assessment of artery lesions as early as possible . almost all the va maldevelopment patients have non - homogenous signal intensity and irregularity of the artery wall , causing most maldevelopment va patients to have atherosclerosis , and the contralateral va compensates by enlarging . in the present study ,
a much higher percentage of unilateral va maldevelopment was detected compared with previous ones ( 40.63% ) and 6 of the 13 ( 46.15% ) tia patients who had unilateral va maldevelopment suffered from contralateral va stenosis . we have thought that unilateral va maldevelopment could be a predisposing factor for vertebrobasilar tia because this vulnerable compensatory mechanism would promote the incidence rate of the disease . in the present study ,
22.5% ( 9/40 ) of tia patients had high dwi signal intensity or adc value abnormality and the patients with high dwi signal intensity had more severe artery stenosis than those with negative dwi detection . the number and degree of stenoses of cranial and cervical arteries was not significantly different between group b and group c in our study . the recurrence of transient ischemia symptoms suggests that artery stenosis and thrombus might already exist and the severe stenosis and irregular filling defect might be one of the most important risk factors for severe ischemia stroke , which makes it necessary for tia patients to have accurate assessment of artery lesions as early as possible . almost all the va maldevelopment patients have non - homogenous signal intensity and irregularity of the artery wall , causing most maldevelopment va patients to have atherosclerosis , and the contralateral va compensates by enlarging . in the present study ,
a much higher percentage of unilateral va maldevelopment was detected compared with previous ones ( 40.63% ) and 6 of the 13 ( 46.15% ) tia patients who had unilateral va maldevelopment suffered from contralateral va stenosis . we have thought that unilateral va maldevelopment could be a predisposing factor for vertebrobasilar tia because this vulnerable compensatory mechanism would promote the incidence rate of the disease . in the present study , 22.5% ( 9/40 ) of tia patients had high dwi signal intensity or adc value abnormality and the patients with high dwi signal intensity had more severe artery stenosis than those with negative dwi detection . the severe stenosis and filling defect of cranial and cervical arteries of tia patients might be one of the most important risk factors of severe stroke . the patients with unilateral va malformation were prone to have atherosclerosis , which might cause decompensation and be a predisposing factor for tia.the fresh ischemia foci on dwi were correlated with severe artery lesions . | [
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our present description of the fundamental laws of nature is based on two disconnected pieces : quantum mechanics and general relativity . on the quantum - mechanics side our most significant successes
were obtained applying relativistic quantum field theory , which turns out to be the appropriate formalization of ( special- ) relativistic quantum mechanics .
this theory neglects gravitational effects and is formulated in a flat / minkowskian spacetime background .
interesting results ( but , so far , with little experimental support ) can be obtained by reformulating this theory in certain curved spacetime backgrounds , but there is no rigorous generalization allowing for the dynamics of gravitational fields .
the only known way for having a manageable formulation of some gravitational effects within quantum field theory is to adopt the perspective of effective field theory [ 144 , 276 ] , which allows lagrangians that are not renormalizable . at leading order ,
this effective theory just gives us back einstein s general relativity ( gr ) , but beyond leading order it predicts corrections proportional to powers of \documentclass[12pt]{minimal }
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\begin{document}${e^2}/e_p^2$\end{document } , where e is the characteristic energy scale of the process under consideration ( typically , the center - of - mass energy for a scattering experiment ) and ep is the planck scale ( ep 10 ev ) .
the effective - field - theory description evidently breaks down at energies e on the order of the planck scale , leaving unanswered [ 144 , 276 ] most of the core issues concerning the interplay between gravity and quantum mechanics .
most importantly , the experiments that have formed our trust in quantum mechanics are nearly exclusively experiments in which gravitational effects are negligible at the presently - achievable levels of experimental sensitivity ( some of the rare instances where the outcome of a quantum - mechanical measurement is affected by gravitational effects , such as the one reported in ref .
our trust in gr has emerged in experimental studies and observations in which gravitational interactions can not be neglected , such as the motion of planets around the sun .
planets are composed of a huge number of fundamental particles , and the additive nature of energy ( playing in such contexts roughly the role of gravitational charge ) is such that the energy of a planet is very large , in spite of the fact that each composing fundamental particle carries only a small amount of energy . as a result , for planets
moreover , a planet satisfies the conditions under which quantum theory is in the classical limit : in the description of the orbits of the planets the quantum properties of the composing particles can be safely neglected .
gr and relativistic quantum mechanics do have some shared tools , such as the notion of spacetime , but they handle these entities in profoundly different manners .
the differences are indeed so profound that it might be natural to expect only one or the other language to be successful , but instead they have both been extremely successful .
this is possible because of the type of experiments in which they have been tested so far , with two sharply separated classes of experiments , allowing complementary approximations . while somewhat puzzling from a philosopher s perspective , all this would not on its own amount to a scientific problem . in the experiments we are presently able to perform and at the level of sensitivities we are presently able to achieve there is no problem .
but a scientific problem , which may well deserve to be called a quantum - gravity problem , is found if we consider , for example , the structure of the scattering experiments done in particle - physics laboratories .
there are no surprises in the analysis of processes with an in state with two particles each with an energy of 10 ev .
relativistic quantum mechanics makes definite predictions for the ( distributions / probabilities of ) results of this type of measurement procedure , and our experiments fully confirm the validity of these predictions .
we are presently unable to redo the same experiments having as in state two particles with energy of 10 ev ( i.e. , energy higher than the planck scale ) , but , nonetheless , if one factors out gravity , relativistic quantum mechanics makes a definite prediction for these conceivable ( but presently undoable ) experiments . however , for collisions of particles of 10 ev energy , the gravitational interactions predicted by gr are very strong and gravity should not be negligible . on the other hand , the quantum properties predicted for the particles by relativistic quantum mechanics ( for example the fuzziness of their trajectories ) can not be neglected , contrary to the desires of the classical mechanics of our present description of gravity .
one could naively attempt to apply both theories simultaneously , but it is well established that such attempts do not produce anything meaningful ( for example by encountering uncontrollable divergences ) . as mentioned above ,
a framework where these issues can be raised in very precise manner is the one of effective quantum field theory , and the break down of the effective quantum field theory of gravitation at the planck scale signals the challenges that are here concerning me .
picture is one ( not necessarily the best , but a sufficiently clear ) way to introduce a quantum - gravity problem . but is the conceivable measurement procedure i just discussed truly sufficient to introduce a scientific problem ?
one ingredient appears to be missing : the measurement procedure is conceivable but presently we are unable to perform it .
moreover , one could argue that mankind might never be able to perform the measurement procedure i just discussed .
there appears to be no need to elaborate predictions for the outcomes of that measurement procedure .
however , it is easy to see that the measurement procedure i just discussed contains the elements of a true scientific problem .
one relevant point can be made considering the experimental / observational evidence we are gathering about the
this evidence strongly supports the idea that in the early universe particles with energies comparable to the planck energy scale ep were abundant , and that these particles played a key role in those early stages of evolution of the universe .
( controlled repeatable experiments ) , but it does represent a context in which proposals for the quantum - gravity / planck - scale realm could be tested .
different scenarios for the physical theory that applies in the quantum - gravity realm could be compared on the basis of their description of the early universe .
the detailed analysis of a given physical theory for the quantum - gravity realm could allow us to establish some characteristic predictions for the early universe and for some manifestations in our observations ( cosmology ) of those early stages of evolution of the universe .
the theory would be testable on the basis of those predictions for our present observations .
therefore , these early - universe considerations provide an opportunity for comparison between the predictions of a quantum - gravity theory and measurement results . and it might not be necessary to resort to cosmology : the fact that ( in setting up the quantum - gravity problem ) we have established some objective limitations of our present theories implies that some qualitatively new effects will be predicted by the theory that applies to the quantum - gravity realm .
these effects should dominate in that realm ( in particular , they will profoundly affect the results of measurements done on particles with planck - scale energy ) , but they should always be present . for processes involving particles with energy e much smaller than ep the implications of a typical quantum - gravity theory will be rather marginal but not altogether absent .
the magnitude of the associated effects should be suppressed by some small overall coefficients , probably given by powers of the ratio e / ep ; small but different from zero .
therefore , we do have a genuine quantum - gravity problem , and this problem has been studied for more than 70 years . unfortunately , most of this research has been conducted assuming that no guidance could be obtained from experiments .
but , if there is to be a science of the quantum - gravity problem , this problem must be treated just like any other scientific problem , seeking desperately the guidance of experimental facts , and letting those facts take the lead in the development of new concepts .
clearly , physicists must hope this also works for the quantum - gravity problem , or else abandon it to the appetites of philosophers .
it is unfortunately true that there is a certain level of risk that experiments might never give us any clear lead toward quantum gravity , especially if we are correct in expecting that the magnitude of the characteristic effects of the new theory should be set by the tiny planck length ( p 1/ep
but even if the new effects were really so small we could still try to uncover experimentally some manifestations of quantum gravity .
this is hard , and there is no guarantee of success , but we must try . as i shall stress again in later parts of this first section ,
let me note here that some degree of optimism could be inspired by considering , for example , the prediction of proton decay within certain modern grand unified theories of particle physics .
the decay probability for a proton in those theories is really very small , suppressed by the fourth power of the ratio between the mass of the proton and the grand unification scale ( a scale that is only some three orders of magnitude smaller than the planck scale ) , but meaningful tests of scenarios for proton decay in grand unified theories have been devised .
while the possibility of a quantum gravity phenomenology could be considered , on the basis of these arguments , even in the early days of quantum - gravity research , a sizable effort has finally matured only since the second half of the 1990s . in particular , only over this recent period do we have the first cases of phenomenological programs that truly affect the directions taken by more formal work in quantum gravity . and , especially in relation to this healthy two - way cross - influence between formal theory and phenomenology , a prominent role has been played by proposals testing features that could be manifestations of spacetime quantization .
the expectation that the fundamental description of spacetime should not be given by a classical geometry is shared by a large majority of quantum - gravity researchers . and , as a result ,
the phenomenology inspired by this expectation has had influence on a sizable part of the recent quantum - gravity literature .
my goal here is primarily the one of reviewing the main results and proposals produced by this emerging area of phenomenology centered on the possibility of spacetime quantization .
the notion of quantum - gravity research can have a different meaning for different researchers .
this is due both to the many sides of the quantum - gravity problem and the fact that most researchers arrive to the study of quantum gravity from earlier interests in other areas of physics research .
because of its nature the quantum - gravity problem has a different appearance , for example , to a particle physicist and to a relativist .
in particular , this affects the perception of the implications of the double role of gravitational fields : unlike all other fields studied in fundamental physics the gravitational field is not just used to describe gravitational interactions but also characterizes the structure of spacetime itself .
the structure of einstein s theory of gravitational phenomena tells us both of the geometry of spacetime , which should be described in terms of smooth riemannian manifolds , and of the implications of einstein s equations for dynamics .
but in most approaches these two sides of gravity are not handled on the same footing .
particularly from the perspective of a particle physicist it makes sense to focus on contexts amenable to treatment assuming some given riemannian - manifold spacetime background and gravitons as mediators of perturbative gravitational interactions .
other researchers , typically not coming from a particle - physics background , are instead primarily interested in speculations for how to replace riemannian manifolds in the description of the structure of spacetime , and contemplating a regime describing perturbative gravitational interactions is not one of their main concerns . because of my objectives , it is appropriate for me to locate early on in this review the quantum - spacetime issues within the broader spectrum of quantum - gravity research .
we do expect that there is a regime of physics where quantum gravity does not simply amount to small corrections to our currently adopted theories , but rather our current theories should be there completely inapplicable .
an example of this is the class of hypothetical situations discussed in my opening remarks : if we consider a collision with impact parameter on the order of the planck length between two particles , which exchange in the collision an energy on the order of the planck scale , then our current theories do not even give us a reliable first approximation of the outcome .
such collisions would create a concentration of energy comparable to the planck scale in a region of planck - length size .
and we have no previous experience with systems concentrated in a planck - length region with rest energy1 on the order of the planck scale . in such cases ,
the pillars of our current description of the laws of physics come in very explicit conflict .
on one side , we have quantum mechanics , with its characteristic property that a rest energy m can only be localized within a region the size of the compton wavelength \documentclass[12pt]{minimal }
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\begin{document}$${r_c}\sim { \hbar \over m}.$$\end{document } on the other side , gr assigns to any localized ( point - like ) amount m of rest energy a region of size its schwarzschild radius \documentclass[12pt]{minimal }
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\begin{document}$${r_s}\sim { g_n}m\sim { { \ell _ p^2 m } \over \hbar},$$\end{document } where gn denotes newton s constant and p denotes the planck length ( \documentclass[12pt]{minimal }
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\begin{document}${\ell _ p } \equiv \sqrt { \hbar g }
\sim { 10^{- 35}}$\end{document } , in units with speed - of - light scale set to unity , c = 1 ) .
if m /p ( rest energy on the order of the planck scale ) , the compton and schwarzschild radii are of the same order of magnitude and quantum mechanics can not ignore gravitation but at the same time gravitation can not ignore quantum mechanics .
evidently we can get nowhere attempting to investigate this issue by just combining2 somehow the standard model of particle physics and the general - relativistic classical description of gravitational phenomena .
another context of similar conceptual content can be imagined if we take for granted ( which we can do only as working assumptions ) the existence of hawking radiation .
we could then start with an isolated macroscopic black hole and attempt to describe its whole future evolution .
as long as the black hole remains macroscopic , but loses weight through hawking radiation , we can imagine to be able to devise a reliable first approximation .
but when the black hole reaches planck - length size ( and planck - scale rest energy ) we are again left without any even approximate answers . the description of these types of quantum - black - hole regimes
( description that i shall use rather generically , including for example the regime characteristic of the very early universe ) is evidently an example of cases such that we could only have a satisfactory picture by understanding how both of the two roles of the gravitational fields need to be revised ( how spacetime structure should be then described and how the gravitational - interaction aspects of gravitation should be then described ) .
providing a description of such a quantum - black - hole regime is probably the most fascinating challenge for quantum - gravity research , but evidently it is not a promising avenue for actually discovering quantum - gravity effects experimentally .
as i shall mention , somewhat incidentally in a few points of this review , this expectation would change if , surprisingly , gravitation turns out to be much stronger than we presently expect , so that at least in some contexts its strength is not characterized by the planck scale . but this review adopts the conservative view that quantum - gravity effects are at least roughly as small as we expect , and , therefore , characterized roughly by the planck scale . and if that is the case , it is hard to even imagine a future in which we gain access to a quantum - black - hole regime .
a key assumption of this review is that quantum gravity will manifest itself experimentally in the shape of small corrections to contexts , which we are able to describe in first approximation within our current theories . for particle physicists ( and , therefore , for at least part of the legitimate overall perspective on the quantum - gravity problem ) the most natural opportunities in which quantum gravity could introduce small corrections are in contexts involving the gravitational - interaction aspects of quantum gravity . rather than attempting to give general definitions let me offer a clear example .
these are studies of long - range corrections to the newtonian limit of gravitation , where gravity does look like a newton - force interaction . by focusing on long - range features
but there are still issues of considerable interest , at least conceptually , that quantum gravity should address in that regime .
it is natural to expect that the description of gravity in terms of a newton - force interaction would also show traces of the new laws that quantum gravity will bring about .
this possibility can be investigated coherently ( but without any guarantee of a reliable answer ) with effective - field - theory techniques applied to the nonrenormalizable theory of quantum gravity obtained by linearizing the einstein - hilbert theory before quantization .
it essentially turns into an exercise of exploring the properties that such an effective theory attributes to gravitons . and one does derive a correction to newton s potential with behavior [ 210 , 27 , 329 , 120 , 324 , 449 ] \documentclass[12pt]{minimal }
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\begin{document}$$\delta { v_{{\rm{newton}}}}\sim { { \hbar { g^2}m } \over { { r^3}}}\sim { { \ell _ p^2 } \over { { r^2}}}{v_{{\rm{newton}}}},$$\end{document } where m is the mass of the source of the gravitational potential and on the right - hand side i highlighted the fact that this correction would come in suppressed with respect to the standard leading newtonian term by a factor given by the square of the ratio of the planck length versus the distance scale at which the potential is probed .
this illustrates the sort of effects one may look for within schemes centered on a background minkowski spacetime and properties of the graviton . in this specific case
the effect is unmanageably small,3 but in principle one could look for other effects of this sort that might be observably large in some applications .
having given some examples of the ways in which quantum gravity might change our description of gravitational interactions , let me now turn to the complementary type of issues that are in focus when one studies the idea of spacetime quantization .
the nature of the quantum - gravity problem tells us in many ways that the ultimate description of spacetime structure is not going to be in terms of a smooth classical geometry .
we do not have at present enough information to deduce how our formalization of spacetime should change , but it must change .
the collection of arguments in support of this expectation ( see , e.g. , refs . [ 406 , 532 , 269 , 44 , 332 , 442 , 211 , 20 , 432 , 50 , 249 , 489 ] ) is impressive and relies both on aspects of the quantum - gravity problem and on analyses of proposed approaches to the solution of the quantum - gravity problem . surely , some very dramatic manifestations of spacetime quantization should be expected in what i labeled as the quantum - black - hole regime .
but , as already stressed above , it is hard to even imagine managing to derive evidence of spacetime quantization from experimental access to that regime .
it is easy to see that our best chance for uncovering non - classical properties of spacetime is to focus on the implications of spacetime quantization for the minkowski limit ( or perhaps the de sitter limit ) of quantum gravity . our data on contexts we presently describe as involving particle propagation in a background minkowski spacetime is abundant and of high quality .
if the fundamental formalization of spacetime is not in terms of a smooth classical geometry then we should find some traces of spacetime quantization also in those well - studied contexts .
the effects are likely to be very small , but the quality of the data available to us in this quantum - spacetime regime is very high , occasionally high enough to probe spacetime structure with planck - scale sensitivity .
i do not elaborate further on it here since it will take full shape in the following .
it is an interesting aspect of how the quantum - gravity community is fragmented to observe that it is sometimes difficult to explain to a relativist how graviton - exchange studies could be seen as part of quantum - gravity research and it is difficult to explain to particle physicists how studies of particles not interacting gravitationally in a quantum - spacetime can play a role in quantum - gravity research .
let me also discuss one more aspect of the interplay between quantum mechanics and gravitation that is of interest from a quantum - gravity perspective , even though at first sight it does not look like quantum gravity at all .
these are studies of quantum mechanics in a curved background spacetime , without assuming spacetime is quantized and without including any graviton - like contribution to the interactions .
no aspect of gravity is quantized in such studies , but they concern a regime that must be present as a limiting case of quantum gravity , and , therefore , by studying this regime we are establishing constraints on how quantum gravity might look . on the conceptual side
perhaps the most significant example of how quantum mechanics in curved spacetime backgrounds can provide important hints toward quantum gravity is provided by studies of black - hole thermodynamics . and
it is a regime of physics where we do have some experimental access mainly through studies of the quantum properties of particles in cases when the geometry of spacetime near the surface of the earth ( essentially gravity of earth , the acceleration g ) does matter .
while my focus here is on quantum - spacetime studies , it will occasionally be useful for me to adopt the perspective of quantum mechanics in curved classical background spacetimes . in order to fully expose the change of perspective , which matured over the last decade , it is useful to first discuss briefly some earlier analyses that made contact with experiments / observations and are relevant for the understanding of the interplay between gr and quantum mechanics .
in particular , the renowned chandrasekhar limit [ 164 , 165 ] , which describes the maximum mass of a white - dwarf star , was obtained introducing some quantum - mechanical properties of particles ( essentially pauli s exclusion principle ) within an analysis of gravitational phenomena .
a fully rigorous derivation of the chandrasekhar limit would require quantum gravity , but not all of it : it would suffice to master one special limit of quantum gravity , the classical - gravity limit , in which one takes into account the quantum properties of matter fields ( particles ) in the presence of rather strong spacetime curvature ( treated , however , classically ) . by testing experimentally the chandrasekhar - limit formula ,
one is , therefore , to some extent probing ( the classical - gravity limit of ) quantum gravity . also relevant to the classical - gravity limit of quantum gravity
are the relatively more recent studies of the implications of the earth s gravitational field in matter - interferometry experiments .
experiments investigating these effects have been conducted since the mid 1970s and are often called cow experiments from the initials of colella , overhauser and werner who performed the first such experiment .
the main target of these studies is the form of the schrdinger equation in the presence of the earth s gravitational field , which could be naturally conjectured to be of the form4
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\begin{document}$$\left [ { - \left({{1 \over { 2{m_i } } } } \right){{\vec \nabla}^2 } + { m_g}\phi ( \vec r ) } \right]\psi ( t,\vec r ) = i{{\partial \psi ( t,\vec r ) } \over { \partial t}}$$\end{document } for the description of the dynamics of matter ( with wave function \documentclass[12pt]{minimal }
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\begin{document}$\psi ( t,\vec r)$\end{document } in the presence of the earth s gravitational potential \documentclass[12pt]{minimal }
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\begin{document}$\phi ( \vec r)$\end{document}. the cow experiments exploit the fact that the earth s gravitational potential puts together the contributions of a very large number of particles ( all the particles composing the earth ) and , as a result , in spite of its per - particle weakness , the overall gravitational field is large enough to introduce observable effects .
while the basic message is that a gravity - improved schrdinger equation of the form ( 1 ) is indeed essentially applicable , some interesting discussions have been generated by these cow experiments , particularly as a result of the data reported by one such experiment ( data whose reliability is still being debated ) , which some authors have interpreted as a possible manifestation of a violation of the equivalence principle . in the same category of studies relevant for the classical - gravity limit of quantum gravity
i should mention some proposals put forward mainly by anandan ( see , e.g. , ref .
[ 77 , 76 ] ) , already in the mid 1980s , and some very recent remarkable studies that test how the gravitational field affects the structure of quantum states , such as the study reported in ref . that i shall discuss in some detail later in this review .
evidently , the study of the classical limit provides only a limited window on quantum gravity , and surely can not provide any insight on the possibility of short - distance spacetime quantization , on which i shall focus here . a list of early examples of studies raising at least the issue that spacetime structure might one day be probed with planck - scale sensitivity should start with the arguments reported by mead in 1965 .
there , mead contemplated the broadening of spectral lines possibly resulting from adopting the planck length as the value of the minimum possible uncertainty in position measurements .
then , in works published in the 1980s and early 1990s , there were a few phenomenological studies , adopting the planck scale as target and focusing essentially on the possibility that quantum - mechanical coherence might be spoiled by quantum - gravity effects .
one example is provided by the studies of planck - scale - induced cpt - symmetry violation and violations of ordinary quantum mechanics reported in refs .
[ 219 , 220 ] and references therein ( also see , for aspects concerning mainly the cpt - symmetry aspects , refs . [ 298 , 108 ] ) , which are particularly relevant for the analysis of data on the neutral - kaon system .
a quantization of spacetime is encoded in the non - critical - string - theory formalism adopted in refs .
[ 219 , 220 ] , but only to the extent that one can view as such the novel description of time there adopted .
[ 452 , 453 , 454 ] , which considered violations of ordinary quantum mechanics of a type describable in terms of the primary - state - diffusion formalism , with results that could be relevant for atom interferometry . also in refs .
[ 452 , 453 , 454 ] the main quantum - spacetime feature is found in the description of time . from a broader quantum - gravity - problem perspective
i should also mention the possibility of violations of cpt and lorentz symmetry within string theory analyzed in refs .
these studies , like most phenomelogy - relevant studies inspired by string theory ( see related comments later in this review ) , do not involve any spacetime quantization and do not necessarily imply that the magnitude of the effects is set by a planckian scale .
but they should nonetheless be prominently listed among the early proposals assuming that some of the theories used in quantum - gravity research might be testable with currently - available experimental techniques .
in particular , some of these studies , perhaps most notably the ones in ref . and ref . , were providing first preliminary evidence of the fact that it might be possible to investigate experimentally the structure of spacetime at the planck scale , which is expected to be the main key to the understanding of the quantum - gravity realm , and should involve spacetime quantization .
but , in spite of their objective significance , these studies did not manage to have an impact on the overall development of quantum - gravity research .
for example , all mainstream quantum - gravity reviews up to the mid 1990s still only mentioned the experiments issue in the form of some brief folkloristic statements , such as the only way to test planck scale effects is to build a particle accelerator all around our galaxy .
the fact that up to the mid 1990s the possibility of a quantum - spacetime phenomenology was mostly ignored , resulted in large part from a common phenomenon of human inertia
that affects some scientific communities , but some role was also played by a meaningful technical observation : the studies available up to that point relied on models with the magnitude of the effect set by a free dimensionless parameter , and at best the sensitivity of the experiment was at a level such that one could argue setting the value of the dimensionless parameter as a ratio between the planck length and one of the characteristic length scales of the relevant physical context .
it is true that this kind of dimensional - analysis reasoning does not amount to really establishing that the relevant candidate quantum - gravity effect is being probed with planck - scale sensitivity , and this resulted in a perception that such studies , while deserving some interest , could not be described objectively as probes of the quantum - gravity realm .
for some theorists a certain level of uneasiness also originated from the fact that the formalisms adopted in studies such as the ones in ref . and ref .
still , it did turn out that those earlier attempts to investigate the quantum - gravity problem experimentally were setting the stage for a wider acceptance of quantum - spacetime phenomenology .
the situation started to evolve rather rapidly when in the span of just a few years , between 1997 and 2000 , several analyses were produced describing different physical contexts in which effects introduced genuinely at the planck sale could be tested .
it started with some analyses of observations of gamma - ray bursts at sub - mev energies [ 66 , 247 , 491 ] , then came some analyses of large laser - light interferometers [ 51 , 54 , 53 , 433 ] , quickly followed by the first discussions of planck scale effects relevant for the analysis of ultra - high - energy cosmic rays [ 327 , 38 , 73 ] and the first analyses relevant for observations of tev gamma rays from blazars [ 38 , 73 , 463 ] ( also see ref . [ 331 , 119 ] ) . in particular , the fact that some of these analyses ( as i discuss in detail later ) considered planck - scale effects amounting to departures from classical lorentz symmetry played a key role in their ability to have an impact on a significant portion of the overall quantum - gravity - research effort .
classical lorentz symmetry is a manifestation of the smooth ( classical ) light - cone structure of minkowski spacetime , and it has long been understood that by introducing new quantum features
( e.g. , discreteness or noncommutativity of the spacetime coordinates ) in spacetime structure , as some aspects of the quantum - gravity problem might invite us to do , lorentz symmetry may be affected . and the idea of having some departure from lorentz symmetry does not necessarily require violations of ordinary quantum mechanics .
moreover , by offering an opportunity to test quantum - gravity theories at a pure kinematical level , these lorentz - symmetry - test proposals provided a path toward testability that appeared to be accessible even to the most ambitious theories that are being considered as candidates for the solution of the quantum gravity problem .
some of these theories are so complex that one can not expect ( at least not through the work of only a few generations of physicists ) to extract all of their physical predictions , but the kinematics of the minkowski limit may well be within our reach .
an example of this type is provided by loop quantum gravity ( lqg ) [ 476 , 96 , 502 , 524 , 93 ] , where one is presently unable to even formulate many desirable physics questions , but at least some ( however tentative ) progress has been made [ 247 , 33 , 523 , 75 , 128 ] in the exploration of the kinematics of the minkowski limit . from a pure - phenomenology perspective , the late-1990s transition is particularly significant , as i shall discuss in greater detail later , in as much as it marks a sharp transition toward falsifiability .
some of the late-1990s phenomenology proposals concern effects that one can imagine honestly deriving in a given quantum - gravity theory . instead the effects described in studies such as the ones reported in ref . and ref . were not really derived from proposed models but rather they were inspired by some paths toward the solution of the quantum - gravity problem ( the relevant formalisms were not really manageable to the point of allowing a rigorous derivation of the nature and size of the effects under study , but some intuition for the nature and size of the effects was developed combining our limited understanding of the formalisms and some heuristics ) .
such a line of reasoning is certainly valuable , and can inspire some meaningful new physics experimental searches , but if the results of the experiments are negative the theoretical ideas that motivated them are not falsified : when the link from theory to experiments is weak ( contaminated by heuristic arguments ) it is not possible to follow the link in the opposite direction ( use negative experimental results to falsify the theory ) . through further developments of the work that started in the late 1990s we are now getting close to taking quantum - spacetime phenomenology from the mere realm of searches of quantum - spacetime effects ( which are striking if they are successful but
have limited impact if they fail ) to the one of falsification tests of some theoretical ideas .
this is a point that i am planning to convey strongly with some key parts of this review , together with another sign of maturity of this phenomenology : the ability to discriminate between different ( but similar ) planck - scale physics scenarios . in order for a phenomenology
to even get started one must find some instances in which the new - physics effects can be distinguished from the effects predicted by current theories , but a more mature phenomenology should also be able to discriminate between similar ( but somewhat different ) new - physics scenarios . together with some ( however slow ) progress toward establishing the ability to falsify models and discriminate between models , the phenomenology work of this past decade
has also shown that the handful of examples of planck - scale sensitivities that generated excitement between 1997 and 2000 were not a one - time lucky streak :
the list of examples of experimental / observational contexts in which sensitivity to some effects introduced genuinely at the planck scale is established ( or found to be realistically within reach ) has continued to grow at a steady pace , as the content of this review will indicate , and the number of research groups joining the quantum - spacetime - phenomenology effort is also growing rapidly . and it is not uncommon for recent quantum - gravity reviews [ 91 , 475 , 501 , 151 ] , even when the primary focus is on developments on the mathematics side , to discuss in some detail ( and acknowledge the significance of ) the work done in quantum - gravity phenomenology . so far ,
. it may be useful to now provide a simple example of analysis that illustrates some of the concepts i have discussed and renders more explicit the fact that some of the sensitivity levels now available experimentally do correspond to effects introduced genuinely at the planck scale .
these objectives motivate me to invite the reader to contemplate the possibility of a discretization of spacetime on a lattice with \documentclass[12pt]{minimal }
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\begin{document}$e_p^{- 1}$\end{document } lattice spacing and a free particle propagating on such a spacetime .
it is well established that in these hypotheses there are \documentclass[12pt]{minimal }
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\begin{document}$e_p^{- 2}$\end{document } corrections to the energy - momentum on - shell relation , which in general are of the type5
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\begin{document}$${m^2 } \simeq { e^2 } - { \vec p^2 } + \sum\limits_{\{{m_\mu}\ } } { { \eta _ { { m_0},{m_1},{m_2},{m_3}}}\left({{{{e^{{m_0}}}p_1^{{m_1}}p_2^{{m_2}}p_3^{{m_3 } } } \over { e_p^2 } } } \right ) } + o\left({{{{e^6 } } \over { e_p^4 } } } \right),$$\end{document } where the non - negative integers { m } are such that m0 + m1 + m2 + m3 = 4 , and the parameters \documentclass[12pt]{minimal }
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\begin{document}${\eta _ { { m_0},{m_1},{m_2},{m_3}}}$\end{document},m1,m2,m3 , which for \documentclass[12pt]{minimal }
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\begin{document}$e_p^{- 1}$\end{document } lattice spacing typically turn out to be of order 1 ( when non - zero ) , reflect the specifics of the chosen discretization .
i should stress that the idea of a rigid - lattice description of spacetime is not really one of the most advanced for quantum - gravity research ( but see the recent related study in ref . ) .
moreover , while it is easy to describe a free particle on such a lattice , the more realistic case of interacting fields is very different , and its implications for the form of the on - shell relation are expected to be significantly more complex than assumed in eq .
in particular , if described within effective field theory , the implications for interacting theories of such a lattice description of spacetime include departures from special - relativistic on - shellness for which there is no planck - scale suppression , and are therefore unacceptable .
this is due to loop corrections , through a mechanism of the type discussed in refs .
[ 455 , 182 , 515 , 190 ] ( on which i shall return later ) , and assumes one is naturally unwilling to contemplate extreme fine - tuning .
i feel it is nonetheless very significant that the , however , unrealistic case of a free particle propagating in a lattice with planck - scale lattice spacing leads to features of the type shown in eq .
( 2 ) have magnitude set by nothing else but a feature of planck - scale magnitude introduced in spacetime structure .
so , in spite of the idealizations involved , the smallness of the effects discussed in this section is plausibly representative of the type of magnitude that quantum - spacetime effects could have , even though any realistic model of the standard model of particle physics in a quantum spacetime , should evidently remove those idealizations .
one finds that in most contexts corrections to the energy - momentum relation of the type in eq .
, for the analysis of center - of - mass collisions between particles of energy 1 tev ( such as the ones studied at the lhc ) these correction terms affect the analysis at the level of 1 part in 10 . however ( at least if such a modified dispersion relation is part of a framework with standard laws of energy - momentum conservation ) , one easily finds [ 327 , 38 , 463 , 73 ] significant implications for the cosmic - ray spectrum .
( named after greisen - zatsepin - kuzmin ) , which is a key expected feature of the cosmic - ray spectrum , and is essentially given by the threshold energy for cosmicray protons to produce pions in collisions with cosmic microwave background radiation ( cmbr ) photons . in the evaluation of the threshold energy for p + cmbr p + , the \documentclass[12pt]{minimal }
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\begin{document}$1/e_p^2$\end{document } correction terms of ( 2 ) can be very significant . as i shall discuss in greater detail in section 3.5 , whereas the classical - spacetime prediction for the gzk cutoff is around 510 ev , a much higher value of the cutoff is naturally obtained [ 327 , 38 , 463 , 73 ] in frameworks with the structure of eq .
( 2 ) turn into corresponding correction terms for the threshold - energy formula , and the significance of these corrections can be roughly estimated with \documentclass[12pt]{minimal }
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\begin{document}$\eta { e^4}/(\epsilon e_p^2)$\end{document } , where e is the energy of the cosmic - ray proton and is the energy of the cmbr photon , to be compared to m/16 , where m here is the proton mass , which roughly gives the gzk scale . adopting the typical quantum - gravity estimate6
|| 1 it turns out that in the gzk regime the ratio e / m is large enough to compensate for
the smallness of the ratio e / ep , so that a term of the type \documentclass[12pt]{minimal }
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\begin{document}${e^4}/(\epsilon e_p^2)$\end{document } is not negligible with respect to m/. this observation is one of the core ingredients of the quantum - spacetime phenomenology that has been done [ 327 , 38 , 463 , 73 ] analyzing gzk - scale cosmic rays . another key ingredient of those analyses is the quality of cosmicray data , which has improved very significantly over these last few years , especially as a result of observations performed at the pierre auger observatory . let me here use this cosmic - ray context also as an opportunity to discuss explicitly a first example of the type of amplifier that is inevitably needed in quantum - gravity phenomenology .
it is easy to figure out [ 52 , 73 ] that the large ordinary - physics number that acts as amplifier of the planck - scale effect in this case is provided by the ratio between a cosmic - ray proton ultra - high energy , which can be of order 10 ev , and the mass ( rest energy ) of the proton .
this is clearly shown by the comparison i made between an estimate of planck - scale corrections of order \documentclass[12pt]{minimal }
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\begin{document}${e^4}/(\epsilon e_p^2)$\end{document } and an estimate of the uncorrected result of order m / e . evidently , e / m is the amplifier of the planck - scale corrections , which also implies that these planck - scale modifications of the photopion - production threshold formula go very quickly from being significant to being completely negligible , as the proton energy is decreased .
a cosmic - ray proton with energy e on the order of 10 ev is so highly boosted that e / mp 10 , and this leads to \documentclass[12pt]{minimal }
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\begin{document}${e^4}/(\epsilon e_p^2 ) \sim { m^2}/\epsilon$\end{document } in my estimates , but at accelerator - accessible proton energies ( and proton boosts with respect to its rest frame ) the correction is completely negligible . according to traditional quantum - gravity arguments , which focus only on the role played by the ratio e / ep , one should assume that this analysis could be successful only when e / ep 1 ; clearly instead this analysis is successful already at energies of order 10 ev ( i.e. , some 8 orders of magnitude below the planck scale ) . and
this is not surprising since the relevant planck - scale effect is an effect of lorentz symmetry violation , so that large boosts ( i.e. , in this context , large values of e / mp ) can act as powerful amplifiers of the effect , even when the energies are not planckian . there are a strikingly large number of arguments pointing to the planck scale as the characteristic scale of quantum - gravity effects .
although clearly these arguments are not all independent , their overall weight must certainly be judged as substantial .
i shall not review them here since they can easily be found in several quantum - gravity reviews , and there are even some dedicated review papers ( see , e.g. , ref . ) .
faithful to the perspective of this review , i do want to stress one argument in favor of the planck scale as the quantum - gravity / quantum - spacetime scale , which is often overlooked , but is in my opinion particularly significant , especially since it is based ( however indirectly ) on experimental facts .
these are the well - known experimental facts pointing to a unification of the coupling
while gravity usually is not involved in arguments that provide support for unification of the nongravitational couplings , it is striking from a quantum - gravity perspective that , even just using the little information we presently have ( mostly at scales below the tev scale ) , our present best extrapolation of the available data on the running of these coupling constants rather robustly indicates that there will indeed be a unification and that this unification will occur at a scale that is not very far from the planck scale . in spite of the fact that we are not in a position to exclude that it is just a quantitative accident , this correspondence between ( otherwise completely unrelated ) scales
must presently be treated as the clearest hint of new physics that is available to us . as hinted in figure 1 , the present ( admittedly preliminary ) status of our understanding of this
unification puzzle might even suggest that there could be a single stage of full unification of all forces , including gravity .
however , according to the arguments that are presently fashionable among theoretical physicists , it would seem that the unification of nongravitational coupling constants should occur sizably above the scale of ( 10 ev ) ( presently preferred is a value close to ( 210 ev ) ) and at such relatively large distance scales gravity should still be too weak to matter , since it is indeed naively expected that gravity should be able to compete with the other forces only starting at scales as short as the planck length , of ( 10 ev ) .
constants of the standard model of particle physics , and also shows a naive description ( which , however , we are so far unable to improve upon ) of the strength of gravitational interactions , obtained by dividing the newton constant by the square of the length scale characteristic of the process .
constants of the standard model of particle physics , and also shows a naive description ( which , however , we are so far unable to improve upon ) of the strength of gravitational interactions , obtained by dividing the newton constant by the square of the length scale characteristic of the process . even setting aside this coupling - unification argument
, there are other compelling reasons for attributing to the planck scale the role of characteristic sale of quantum - gravity effects .
in particular , if one adopts the perspective of the effective - quantum - field - theory description of gravitational phenomena the case for the planck scale can be made rather precisely . a particularly compelling argument in this respect
is found in ref . which focuses on the loss of unitarity within the effective - quantum - field - theory description of gravitational phenomena .
unitarity has been a successful criterion for determining the scale at which other effective quantum field theories break down , such as the fermi theory of weak interactions . and it does turn out that the scale at which unitarity is violated for the effective - quantum - field - theory description of gravitational phenomena is within an order of magnitude of the planck scale .
for example , some authors ( see , e.g. , ref . ) consider it to be likely that the effective newton constant is also affected by some sort of renormalization - group running , and , if this is the case , then the prospects of all these arguments would change significantly . for the length scale of spacetime quantization , qst , naively assumed to be given by \documentclass[12pt]{minimal }
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\begin{document}$\sqrt { { g_n}(\infty)}$\end{document } , where gn ( ) is the measured value of the newton constant ( characteristic of gravity at large distances ) , any running of gravity would imply an estimate7 of the type \documentclass[12pt]{minimal }
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\begin{document}${\ell _ { { \rm{qst } } } } \sim \sqrt { { g_n}({\ell _ { { \rm{qst}}}})}$\end{document}. in relation to estimates of the scale of spacetime quantization these considerations should invite us to consider the planck length , 10 m only as a crude , very preliminary estimate . throughout this review
i shall tentatively take into account this issue by assuming that the scale where nonclassical properties of spacetime emerge should be somewhere between 10 m and 10 m , hoping that three orders of magnitude of prudence from above and below should suffice .
it is striking that these considerations also allow one to be more optimistic with respect to the ( already intrinsically appealing ) hypothesis of a single stage of unification of all forces , possibly even at distance scales as
large as ( 10 ev ) 10 m. and i find that , in relation to this issue , the recent ( mini-)burst of interest in the role of gravity in unification is particularly exciting .
a convincing case is being built concerning the possibility that gravity might affect the running of the standard - model coupling constants , and this too could have significant effects for the estimate of the unification scale ( see , e.g. , refs .
[ 473 , 529 ] and references therein ) . and in turn there is a rather robust argument ( see , e.g. , refs .
[ 146 , 147 ] and references therein ) suggesting that the other fields might significantly affect the strength of gravity .
my personal perspective on the overall balance of this limited insight that is available to us is summarized by the attitude i adopted for this review in relation to the expectations for the value of the quantum - spacetime scale .
unsurprisingly , i give top priority for this to the only ( and however faint ) indication we have from experiments : the values measured for coupling constants at presently accessible ultra - large distance scales appear to be arranged in such a way as to produce a unification of nongravitational forces at a much smaller length scale , which happens to be not distant from where we would naively expect gravity to come into the picture .
this in some sense tells us that our naive estimate of where gravity becomes strong ( and spacetime turns nonclassical ) can not be too far off the mark .
but at the same time imposes upon us at least a certain level of prudence : we can not assume that the quantum - spacetime scale is exactly the planck length , but we have some encouragement for assuming that it is within a few orders of magnitude of the planck length . in closing this long aside on the quantum - gravity / quantum - spacetime scale ,
let me stress that even prudently assuming a few orders of uncertainty above and below the planck length is not necessarily safe .
it is in my opinion the most natural working assumption in light of the information presently available to us , but we should be fully aware of the fact that our naive estimates might be off by more than a few orders of magnitude . following the line of reasoning adopted here
this would take the shape of a solution for \documentclass[12pt]{minimal }
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\begin{document}${\ell _ { { \rm{qst } } } } \sim \sqrt { { g_n}({\ell _ { { \rm{qst}}}})}$\end{document } that unexpectedly turned out to be wildly different form the planck length . the outlook of the analysis of the unification of forces appears to discourage such speculations , but we must be open to the possibility that the story here summarized in figure 1 might just be a cruel numerical accident ( more on this toward the end of this review , when i briefly consider the large extra dimensions scenario ) . having commented on the first
ingredient for the search of experiments relevant for quantum spacetime and quantum gravity , which is the estimate of the characteristic scale of this new physics , let me next comment on a few other ingredients , starting with some intuition for the type of quantum - spacetime effects that one might plausibly look for , and what that requires . as stressed earlier in this section , we can not place much hope of experimental breakthroughs in the full quantum - black - hole regime .
our best chances are for studies of contexts amenable to a description in terms of the properties of particles in a background quantum spacetime . and , as also already stressed , these effects will be minute , with magnitude governed by some power of the ratio between the planck length and the wavelength of the particles involved .
the presence of these suppression factors on the one hand reduces sharply our chances of actually discovering quantum - spacetime effects , but on the other hand simplifies the problem of figuring out what are the most promising experimental contexts , since these experimental contexts must enjoy very special properties that would not easily go unnoticed . for laboratory experiments ,
even an optimistic estimate of these suppression factors leads to a suppression of order 10 , which one obtains by assuming ( probably already using some optimism ) that at least some quantum - gravity effects are only linearly suppressed by the planck length and taking as particle wavelength the shorter wavelengths we are able to produce ( 10 m ) . in astrophysics ( which , however , limits one to
observations rather than experiments ) particles of shorter wavelength are being studied , but even for the highest energy cosmic rays , with energy of 10 ev and , therefore , wavelengths of 10 m , a suppression of the type lp/ would take values of order 10 .
it is mostly as a result of this type of consideration that traditional quantum - gravity reviews considered the possibility of experimental studies with unmitigated pessimism .
however , the presence of these large suppression factors surely can not suffice for drawing any conclusions .
even just looking within the subject of particle physics we know that certain types of small effects can be studied , as illustrated by the example of the remarkable limits obtained on proton instability .
the prediction of proton decay within certain grand unified theories of particle physics is really a small effect , suppressed by the fourth power of the ratio between the mass of the proton and grand - unification scale , which is only three orders of magnitude smaller than the planck scale . in spite of this horrifying suppression , of order [ mproton / egut ] 10 , with a simple idea we have managed to acquire full sensitivity to the new effect : the proton lifetime predicted by grand unified theories is of order 10 s and quite a few generations of physicists
should invest their entire lifetimes staring at a single proton before its decay , but by managing to keep under observation a large number of protons ( think for example of a situation in which 10 protons are monitored ) our sensitivity to proton decay is dramatically increased . in that context
the number of protons is the ( ordinary - physics ) dimensionless quantity that works as amplifier of the new - physics effect . outside of particle physics
more success stories of this type are easily found : think for example of the brownian - motion studies conducted a century ago . within the 1905 einstein description one
uses brownian - motion measurements on macroscopic scales as evidence for the atomic structure of matter . for the brownian - motion case
the needed amplifier is provided by the fact that a very large number of microscopic processes intervenes in each single macroscopic effect that is being measured .
it is hard but clearly not impossible to find experimental contexts in which there is effectively a large amplification of some small effects of interest . and
this is the strategy that is adopted in the attempts to gain access to the planck - scale realm .
something else that characterizes the work attitude of the community , whose results i am here reviewing , is the expectation that the solution of the quantum - gravity problem will require a significant change of theory paradigm .
members of this community find in the structure of the quantum - gravity problem sufficient elements for expecting that the transition from our current theories to a successful theory of quantum gravity should be no less ( probably more ) significant then the transition from classical mechanics to quantum mechanics , the prototypical example of a change of theory paradigm .
this marks a strong difference in intuition and methodology with respect to other areas of quantum - gravity research , which do not assume the need of a paradigm change .
if the string theory program turned out to be successful then quantum gravity should take the shape of just one more ( particularly complex but nonetheless consequential ) step in the exploitation of the current theory paradigm , the one that took us all the way from qed to the standard model of particle physics .
this difference of intuitions even affects the nature of the sort of questions the different communities ask .
the expectation of those not preparing for a change of theory paradigm is that one day some brilliant mind will wake up with the correct full quantum - gravity theory , with a single big conceptual jump .
what is expected is a single big conceptual step leading to a theory that describes potentially everything we know so far.8 something of the sort of the discovery of qcd : a full theory even though some of its answers to our questions are not immediately manifest once the theory is written out ( see , e.g. , confinement ) .
the expectation of those who are instead preparing for a change of theory paradigm is that we will get to a mature formulation of quantum gravity only at the end of a multi - step journey , with each step being of rather humble nature .
the change of theory paradigm in going from classical mechanics to quantum mechanics was of such magnitude that we could not possibly get it right in one single jump .
imagine someone , however brilliant , looking at black - body radiation and proposing a solution based on observables described as self - adjoint operators on hilbert spaces and all that .
planck s description of black - body radiation was very far from being a full formalization of quantum mechanics , and was even internally unsatisfactory , with a very limited class of contexts and regimes where it could be applied .
it was a theory of very few things , but it was a necessary step toward quantum mechanics .
a similar role in the gradual emergence of quantum mechanics was played by other theories of limited scope , such as einstein s description of the photoelectric effect , bohr s description of atoms , and the successful proposal by de broglie that wave - particle duality should be applied also to matter .
so , while those not preparing for a change of paradigm look for theories of everything , we are looking for theories of very few things , like planck , einstein , bohr , de broglie and other great contributors to the ultimate advent of quantum mechanics .
let me here add that even when exploiting a successful theory paradigm , often the next level of exploitation still requires us taking some clumsy steps based on theories of few things .
consider fermi s description of weak interactions in terms of four - fermion - vertex processes .
fermi s theory can be applied to a limited class of phenomena and only in a relatively narrow regime , and it is even a theory that is not satisfactory from the perspective of internal logical consistency . yet fermi s theory was an important and necessary step toward richer and more satisfactory descriptions of weak interactions .
the difference in methodologies is also connected with some practical considerations , connected with the fact that the formalisms presently being considered as solutions for the quantum - gravity problem are so complex that very little is understood of their truly physical implications .
some theories of few things can even be inspired by a given theory of everything : since it is de facto impossible to compare to data present full candidates for quantum gravity one ends up comparing to data the predictions of an associated test theory , a model that is inspired by some features we do understand ( usually not more than qualitatively or semi - quantitatively ) of the original theory , but casts them within a simple framework that is well suited for comparison to experiments ( but for which there is no actual guarantee of full compatibility with the original theory ) .
so , in the eyes of some workers these test theories of few things are needed to bridge the gap between the experimental data and our present understanding of the relevant formalisms . in the eyes of others
the test theories of few things are just attempts to bridge the gap between the experimental data available to us and our limited understanding of the quantum - gravity problem . essentially in working in quantum - spacetime phenomenology one must first develop some intuition for some candidate quantum - spacetime effects . and this can come either from analyzing the structure of the formalisms that are being considered in the search of a solution to the quantum - gravity problem or from analyzing the structure of the quantum - gravity problem .
once a class of effects is deemed of interest some test theories of these candidate effects must be developed so that they can be used as guidance for experimental searches . from the perspective of a phenomenologist , some carefully tailored test theories can also be valuable as some sort of common language to be used in assessing the progresses made in improving the sensitivity of experiments , a language that must be suitable both for experimentalists and for those working on the development of quantum - gravity theories .
the possibility to contemplate such quantum - gravity theories of not everything is facilitated by the fact that the quantum - gravity problem can be described in terms of several subproblems , each challenging us perhaps as much as some full open problems of other areas of physics . to mention just a few of these subproblems
let me notice that : ( i ) it appears likely that the solution of this problem requires a nonclassical description of spacetime geometry , ( ii ) quantum gravity might have to be profoundly different ( from an information - theory perspective ) from previous fundamental - physics theories , as suggested by certain analyses of the evolution of pure states in a black - hole background , ( iii ) the perturbative expansions that are often needed for the analysis of experimental data might require the development of new techniques , since it appears that the ones that rely on perturbative renormalizability might be unavailable , and ( iv ) we must find some way to reconcile general - relativistic background independence with the apparent need of quantum mechanics to be formulated in a given background spacetime .
for each of these aspects of the quantum - gravity problem we can , in principle , attempt to devise formalisms , intended as descriptions of those regimes of the quantum - gravity realm that are dominantly characterized by the corresponding features . in providing my description of the present status of quantum - spacetime phenomenology
, i shall adopt as my default mode that of characterizing the sensitivities that are within reach for certain classes of experiments / observations , with only a few cases where i discuss both sensitivities and available experimental limits .
the analysis of sensitivities was the traditional exercise a decade ago , in the early days of modern quantum - spacetime phenomenology , since the key objective then was to establish that sensitivity to effects introduced genuinely at the planck scale is achievable . in light of the observation i already reported in section 1.5 ( and
several other observations reported later in this review ) , the case for existence of quantum - spacetime phenomenology is at this point well settled .
we are now entering a more mature phase in which we start having the first examples of candidate quantum - spacetime effects for which the development of suitable test theories is approaching a level of maturity such that placing experimental bounds ( limits ) on the parameters of these test theories does deserve intrinsic interest .
however , at the time of writing , the transition from sensitivities to limits is not yet complete .
the cases where i will offer comments on available experimental limits are cases for which ( in my opinion ) this transition has been made satisfactorily .
but in several areas of quantum - spacetime phenomenology it is still common practice to discuss experimental bounds on the basis of a single little - understood experimental result ( often a single observation in astrophysics ) and most of the test theories are not yet developed to the point that we can attach much significance to placing limits on their parameters .
this is a key issue , and throughout this review i will find opportunities to discuss in more detail my concerns and offer some remarks that are relevant for completing the needed transition from sensitivities to limits .
i do plan to regularly update this review , and with each update readers should find the emphasis gradually going more and more from sensitivities to experimental limits . after having clarified that the default mode of this review provides descriptions of sensitivities ( with occasional characterizations of experimental bounds ) , i should comment on the types of theory and phenomenology that are the main focus of this review .
i have prepared other reviews on these and related topics [ 52 , 62 ] with a broader perspective but much more limited depth . here
my main focus is to analyze and review in some detail the healthy interface between pure theory and phenomenology of quantum spacetime .
i shall mostly describe the phenomenology proposals , but the selection of which proposals should be included is primarily based on their proven ability to motivate developments on the pure - theory side and to react to ( take into account adaptively of ) the indications that then emerge from these pure - theory studies
. this will be the default mode of my selection of topics , with some exceptions allowed in cases where i find that there are promising opportunities for such a healthy interface to mature over the next few years .
the net result of these goals of the review produces a certain bias toward proposals for quantum spacetime , which originated from ( or were inspired by ) the study of lqg and/or the study of planck - scale spacetime noncommutativity .
these are the two areas of pure - theory research in which , so far , the desirable two - way interface has most concretely materialized : pure - theory specialists have redirected part of their work toward the topics that phenomenologists have highlighted as most promising for phenomenology ; and the work of quantum - spacetime phenomenologists has been in turn influenced by the results then obtained on the pure - theory side .
in addition to a relatively long list of proposals inspired by lqg and/or by planck - scale space - time noncommutativity , i shall also comment on a few proposals inspired by other approaches to spacetime quantization ( e.g. , causal sets and noncritical string theory ) . from a broader quantum - gravity - problem perspective one should also consider critical string theory , which actually remains the most studied candidate for quantum gravity .
however , i focus here on quantum - spacetime effects and effects whose natural characteristic scale is the planck scale , whereas the phenomenology proposals so far inspired by the critical - string - theory research program do not revolve around quantum properties of spacetime and often the characteristic scale of the effects is not naturally the planck scale .
i shall observe in section 2.1.1 that the analysis of critical string theory actually has provided encouragement for the idea that it could also be a model of spacetime quantization , but the relevant aspects of critical string theory are still poorly understood and have not produced phenomenological proposals of the sort i am here reviewing .
i do believe that it is likely that in a not - so - distant future some new opportunities for quantum - spacetime phenomenology will arise from this avenue .
the main objective of the next section 2 is to motivate a list of candidate quantum - spacetime effects , on the basis of the structure of the quantum - gravity problem and/or of results obtained in certain theories that are being considered as relevant to the understanding of the quantum - gravity problem .
the rest of this review attempts to describe the status of searches of these candidate quantum - spacetime effects .
choosing what structure to give to sections 3 , 4 , 5 and 6 was the main challenge faced by my work on this review .
the option that finally prevailed attempts to assign each phenomenological proposal to a certain area of quantum - spacetime phenomenology . these should be viewed only as tentative assignments , or at least assignments based on a perception of what could be the primary targets of a given phenomenological proposal .
and there are some visible limitations : some readers could legitimately argue that a certain subsection that i placed in one of the sections should instead find a more fitting setting in another section . indeed ,
as i was working on this review , there were a few subsections that kept switching from one section to another .
if used wisely , i feel that the structure i gave is still preferable to some of the alternatives that could have been considered .
for example , even such a tentative structure of organization is probably going to be more easy to use than a long unstructured list of all the many phenomenological proposals i am considering . and the option of organizing phenomenological proposals on the basis of the theories that motivate them , rather than roughly on the basis of their primary area of relevance in phenomenology , would have been against the whole spirit of this review .
section 3 focuses on effects that amount to planck - scale departures from lorentz / poincar symmetry , which is the type of effects on which the most energetic quantum - spacetime phenomenology effort has been so far directed .
the content of section 3 has some overlap with that describes the status of modern tests of lorentz symmetry , and , therefore , is in part also devoted to cases in which such tests are motivated by quantum - spacetime research .
however , my perspective will be rather different , focused on the quantum - spacetime - motivated searches and also using the example of lorentz / poincar - symmetry tests to comment on the level of maturity reached by quantum - spacetime phenomenology in relation to the falsification of ( test ) theories and to the discrimination between different but similar theories . and whereas from the broader viewpoint of probing the robustness of lorentz symmetry one should consider as significant any proposal capable of improving the bounds established within a given parametrization of departures from lorentz symmetry , i shall focus on the demands of planck - scale sensitivity , as required by the objectives of research on planck - scale quantization of spacetime , that is my main focus here . in section 4 ,
i describe the status of other areas of quantum - spacetime phenomenology in which the planck - scale also characterizes the onset of ultraviolet effects , but not of the types that require departures from lorentz / poincar symmetry .
while the primary objectives of this review are the ultraviolet effects linked with the planck - scale structure of spacetime , in section 5 i briefly consider the possibility of ultraviolet / infrared ( uv / ir ) mixing . in
such uv / ir - mixing scenarios the role of the planck scale would be in governing the uv side , and possibly then combining with other scales when ir effects are considered .
sections 3 , 4 , and 5 concern proposals of ( only a few ) controlled laboratory experiments and ( several ) observations in astrophysics .
these are the contexts in which currently one finds more mature proposals , particularly concerning the robustness of claims of planck - scale sensitivity of some relevant data analyses .
however , observations in cosmology should also provide some very valuable opportunities , and there are some quantum - spacetime - cosmology proposals , to which i devote section 6 , that can already be used to expose the great potential reach of this type of analyses . while different proposals of quantum - spacetime phenomenology often involve different formalizations and completely different experimental techniques ,
there is a common setup of all proposals described in sections 3 , 4 , 5 , and 6 .
this main strategy of quantum - spacetime phenomenology is summarized in section 7 , also pondering what might be some of its limitations .
before getting to the main task of this review , which concerns phenomenology proposals , it is useful to summarize briefly the motivation for studying certain candidate quantum - spacetime effects .
the possible sources of motivation come either from analyses of the structure of the quantum - gravity problem or from what is emerging in the development of some theories that have been proposed as candidate solutions of the quantum - gravity problem . as already stressed ,
my main focus here is on effects that can be linked to spacetime quantization at ( about ) the planck scale , and particularly the ones that were involved in the two - way interface that materialized over this last decade between phenomenologists and theorists working on the lqg approach and spacetime noncommutativity . in the first part of this section ,
i offer a few comments on some of the approaches being pursued in the study of the quantum - gravity problem , mostly focusing on whether or not they support a quantum - spacetime picture and the role played by the planck scale .
this part focuses primarily on lqg and spacetime noncommutativity , but i also comment briefly on critical string theory and other approaches .
then in the second part of this section i list some key candidates as phenomena that could characterize the quantum - spacetime realm .
this list is only very tentative but it seems to me we can not do any better than this at the present time .
analogous situations in other areas of physics are usually such that there are a few new theories that have started to earn our trust by successfully describing some otherwise unexplained data , and then often we let those theories guide us toward new effects that should be looked for .
the theories that are under consideration for the solution of the quantum - gravity problem , and for a quantum ( non - classical ) description of spacetime , can not yet claim any success in the experimental realm
. moreover , even if nonetheless we wanted to use them as guidance for experiments , the complexity of these theories proves to be a formidable obstruction . in most cases , especially concerning testable predictions , the best we can presently do with these theories is analyze their general structure and use this as a source of intuition for the proposal of a few candidate effects .
similarly , when we motivate the search of certain quantum - spacetime features on the basis of our present understanding of the quantum - gravity problem we are in no way assured that they should still find support in future better insight on the nature of this problem , but it is the best we can do at the present time .
the most studied approach to the quantum - gravity problem is a version of string theory that adopts supersymmetry and works in a
if this main - stream perspective turned out to be correct it would be bad news for quantum - spacetime phenomenologists , since the theory is formulated in classical minkowski background spacetime .
it would be bad news for phenomenology in general because ( critical , supersymmetric ) string theory is a particularly soft modification of current theories , and the new effects that can be accommodated by the theory are untestably small , if all the new features are indeed introduced ( as traditionally assumed ) at a string scale roughly given by the planck scale .
string theory is the natural attempt from a particle - physics perspective , but other perspectives on the quantum - gravity problem remain unimpressed , particularly considering that most results of string theory still only apply in a fixed background minkowski spacetime . and
it is interesting to notice how the most careful analyses performed even adopting a string - theory perspective end up finding that the case for applicability to the quantum - gravity problem is still rather weak ( see , e.g. , ref . ) .
this not withstanding there has been in recent years a more vigorous effort of development of a string - inspired phenomenology , with inspiration found in mechanisms that are , however , outside the traditional formulation of string theory .
this string - inspired phenomenology does not involve spacetime quantization and often does not refer explicitly to the planck scale , so i shall not discuss it in detail in this review ( although there will be scattered opportunities , at points of this review , where it becomes indirectly relevant ) .
extra dimensions [ 80 , 375 , 552 , 84 , 85 , 480 ] . the existence of extra dimensions can be conceived even outside string theory , but it is noteworthy that in string theory the criticality criterion actually requires extra dimensions .
if the extra dimensions , as traditionally assumed , have finite size on the order of the planck length , then one ends up having associated planck - scale effects for the low - energy realm , where our experiments and observations take place .
this would be a classic exercise for quantum - gravity phenomenology but it appears that the planck - scale suppression of these extra - dimension effects is so strong that they really could not ever be seen / tested .
the recent interest in the large extra dimensions scenario originates from the observation that dimensions of size much larger than the planck length ( but still microscopic ) , while not particularly natural from a string - theory perspective , may well be allowed in string theory [ 80 , 375 , 552 , 84 , 85 , 480 ] . and
for some choices of number and sizes of extra dimensions a rich phenomenology is produced .
most other phenomenological proposals inspired by string theory essentially make use of the fact that , at least as seen by a traditional particle physicist , string theory makes room for several new fields .
the new effects are indeed of types that are naturally described by introducing new fields in a classical spacetime background , rather than quantum - spacetime features , and the magnitude of these effects is not naturally governed by the planck scale.9 in spite of these profound differences there are some points of contact between the planck - scale quantum - spacetime phenomenology , which i am here concerned with , and this string phenomenology . in a quantum spacetime it is necessary to reexamine the issue of spacetime symmetries , and certain specific scenarios for the fate of lorentz symmetry come into focus . from a different perspective and in a technically different way
one also finds reasons to scrutinize lorentz symmetry in string phenomenology : it is plausible that some string - theory tensor fields ( most likely some of the new fields introduced by the theory ) could acquire a nonzero vacuum expectation value , in which case evidently one would have a spontaneous breakdown of lorentz symmetry .
i shall also comment on the possibility that spacetime quantization might affect the equivalence principle .
again , from a different perspective and in a technically different way , one also finds reasons to scrutinize the equivalence principle in string phenomenology . and
again it is typically due to the extra fields introduced in string theory : most notably some scenarios involving the dilaton , a scalar partner to the graviton predicted by string theory , produce violations of the equivalence principle ( see , e.g. , ref . ) .
i should stress here , because of the scope of this review , that the idea of a quantum space - time is not completely foreign to string theory .
it is presently appearing at an undigested and/or indirect level of analysis , but it is plausible that future evolutions of the string - theory program might have a primitive / fundamental role for spacetime quantization .
so far the most studied connection with quantum - spacetime ideas comes from a mechanism analogous to the emergence of noncommutativity of position coordinates in the landau model ( see , e.g. , ref . )
that is found to be applicable to the description of strings in the presence of a constant neveu - schwarz two - form ( b ) field [ 213 , 516 ] .
( effective descriptions applicable only in certain specific regimes ) do not amount to genuine nonclassicality of spacetime .
still , these recent string - theory results do create a point of contact between research ( and particularly phenomenology ) on fundamental spacetime noncommutativity and string theory , with the peculiarity that from the string - theory perspective one would not necessarily focus ( and typically there is no focus ) on the case of noncommutativity introduced at about the planck scale , since it is instead given in terms of the free specification of the field b. for the hope of a possible future reformulation of string theory in some way that would accommodate a primitive role for spacetime nonclassicality my impression is that the key opportunities should be seen in results suggesting that there are fundamental limitations for the localization of a spacetime event in string theory [ 532 , 269 , 44 , 332 ] .
the significance of these results on limitations of localizability in string theory probably has not been appreciated sufficiently .
only a few authors have emphasized the possible significance of these results , but i would argue that finding such limitations in a theory originally formulated in a classical spacetime background may well provide the starting point for reformulating the theory completely , perhaps codifying spacetime quantization at a primitive level . the most studied theory framework providing a quantum description of spacetime is lqg [ 476 , 96 , 502 , 524 , 93 ] .
the intuition of many phenomenologists who have looked at ( or actually worked on ) lqg is that this theory should predict quite a few testable effects , some of which may well be testable with existing technologies .
however , the complexity of the formalism has proven so far to be unmanageable from the point of view of obtaining crisp physical predictions . among the many challenges i should at least mention the much debated classical - limit problem , which obstructs the way toward a definite set of predictions for the quasi - minkowski ( or quasi - desitter , or quasi - frw ) regime , which is where most of the opportunities for phenomenology can be found . however , one may attempt to infer from the general structure of the theory motivation for the study of some candidate lqg effects . and ,
as i shall stress in several parts of this review , this type of attitude has generated a healthy interface between phenomenologists and lqg theorists .
most of the relevant proposals are ignited indeed by the quantum properties of spacetime in lqg , which appear to be primarily codified in a discretization of the area and volume observables [ 477 , 95 , 476 ] in particular , several studies ( see later in this review ) have argued that the type of discretization of spacetime observables usually attributed to lqg could be responsible10 for planck - scale departures from lorentz symmetry .
in addition to a large effort focused on the fate of lorentz symmetry , there has also been a rather large effort focused on early - universe cosmology inspired by lqg . among the appealing features of this cosmology work i should at least mention singularity avoidance . for the lqg approach
, there might be no alternative to avoiding the big - bang singularity , since indeed , at least as presently understood , lqg describes spacetime has a fundamentally discrete structure governed by difference ( rather than differential ) equations .
this discreteness is expected to become a dominant characteristic of the framework for processes involving comparably small ( planckian ) length scales , and in particular it should inevitably give rise to a totally unconventional picture of the earliest stages of evolution of the universe .
attempts at developing a setup for a quantitative description of these early - universe features have been put forward in refs . [ 125 , 94 , 126 , 92 ] and references therein , but one must inevitably resort to rather drastic approximations , since a full lqg analysis is not possible at present . for other areas of phenomenology discussed in this review
the influence of lqg has been less direct , but it appears safe to assume that it will inevitably grow in the coming years . to give a particularly striking example , let me mention the many proposals here discussed that concern spacetime fuzziness .
it is evident that lqg gives a fuzzy picture of spacetime ( in the sense discussed more precisely in later parts of this review ) , and it would be of important guidance for the phenomenologists to have definite predictions for these features
. even just a semiheuristic derivation of such effects is beyond the reach of our present understanding of lqg , but it will come .
the idea of having a nonclassical fundamental description of spacetime is central to the study of spacetime noncommutativity .
the formalization that is most applied in the study of the quantum - gravity / quantum - spacetime problem is mainly based on the formalism of quantum - groups and essentially assumes that the quantum properties of spacetime should be at least to some extent analogous to the quantum properties of phase space in ordinary quantum mechanics .
ordinary quantum mechanics introduces some limitations for procedures intending to obtain a combined determination of both position and momentum , and this is formalized in terms of noncommutativity of the position and momentum observables . with spacetime noncommutativity
one essentially assumes that spacetime coordinates should not commute [ 211 , 391 , 374 , 384 , 70 , 98 ] among themselves , producing some limitations for the combined determination of more than one coordinate of a spacetime point / event .
this has been the formalization of spacetime noncommutativity for which the two - way interface between theory and phenomenology , which is at center stage in this review , has been most significant .
looking ahead at the future of quantum - spacetime phenomenology , it appears legitimate to hope that another , perhaps even more compelling , candidate concept of noncommutative geometry , the one championed by connes [ 185 , 184 ] , may provide guidance . at present the most studied applications of this notion of noncommutative geometry
are focused on giving a fully geometric description of the standard model of particle physics , with the noncommutativity of geometry used to codify known properties of particle physics in geometric fashion , while keeping spacetime as a classical geometry .
going back to the quantum - group - based description of spacetime noncommutativity i should stress that , so far , the most significant developments have concerned attempts to describe the minkowski limit of the quantum - gravity problem , i.e. , a noncommutative version of minkowski spacetime ( spacetimes that reproduce classical minkowski spacetime in the limit in which the noncommutativity parameters are taken to 0 ) .
some related work has also been directed toward quantum versions of de sitter spacetime , but very little about spacetime dynamics and only at barely an exploratory level .
but at the present time this situation could be described by stating that most work on spacetime noncommutatvity is considering only one half of the quantum - gravity problem , the quantum - spacetime aspects ( neglecting the gravity aspects ) .
because of the double role of the gravitational field , which in some ways is just like another ( e.g. , electromagnatic ) field given in spacetime but it is also the field that describes the structure of spacetime , in quantum - gravity research the idea that this classical field be replaced by a nonclassical one ends up amounting to two concepts : some sort of quantization of gravitational interactions ( which might be mediated by a graviton ) and some sort of quantization of spacetime structure . at present one might say that only within the lqg approach are we truly exploring both aspects of the problem .
string theory , as long as it is formulated in a classical ( background ) spacetime , focuses in a sense on the quantization of the gravitational interaction , and sets aside ( or will address in the future ) the possible quantization of spacetime .
spacetime noncommutativity is an avenue for exploring the implications of the other side , the quantization of spacetime geometry .
the description of ( minkowski - limit ) spacetime in terms of ( quantum - group - based ) space - time noncommutativity has proven particularly valuable in providing intuition for the fate of ( minkowski - limit / poincar ) spacetime symmetries at the planck scale .
also parity transformations appear to be affected by at least some schemes of spacetime noncommutativity and this in turn provides motivation for testing cpt symmetry .
unfortunately , spacetime fuzziness , which is the primary intuition that leads most researchers to noncommutativity , frustratingly remains only vaguely characterized in current research on non - commutative spacetimes ; certainly not characterized with the sharpness needed for phenomenology .
the challenge of reviewing and offering a perspective on quantum - spacetime phenomenology is already overwhelming . and according to the perspective of this phenomenological approach the central challenge of quantum - gravity research is to find the first experimental manifestations of the quantum - gravity realm .
the different formalisms proposed for the study of the quantum - gravity problem can be very valuable for this objective , but only in as much as they provide intuition for the type of new effects that might characterize the quantum - gravity realm . in practice , at least for the next few decades , what will be compared to data will be simple test theories inspired by our understanding of the quantum - gravity problem or by the intuition obtained in the study of formal theories of quantum gravity .
the possibility of comparing a full quantum - gravity theory directly to experiments appears to be for a still distant future , as a result of the complexity of these theories ( which prevents us from deriving testable predictions ) .
i have invested a few pages on string theory , lqg and spacetime noncommutativity for different reasons . providing some reasonably detailed comments on string theory
was encouraged , in spite of the lack of a fundamental role for spacetime quantization , by its prominent role in the quantum - gravity literature . and , as stressed above , lqg and spacetime noncommutativity are particularly relevant for this review because the scenarios of spacetime quantization these approaches consider / derive have been a particularly influential source of intuition for proposals in quantum - spacetime phenomenology .
moreover , it is within the lqg and spacetime - noncommutativity communities that we have , so far , witnessed the most significant examples of the healthy two - way cross - influence between formal theory and phenomenology .
i shall not offer comparably detailed comments on any other quantum - gravity formalism , but there are a few that i should mention because of the significance of their role in quantum - spacetime phenomenology .
approach championed by ellis , mavromatos and nanopoulos [ 221 , 223 , 65 , 399 ] .
this is a variant of the string - theory approach that ( unlike the main - stream critical - string - theory approach ) adopts the choice of working in
noncritical number of spacetime dimensions , and describes time in a novel way . as will be evident in several points of this review , ellis , mavromatos , nanopoulos and collaborators have developed noncritical liouville string theory from a perspective that admirably keeps phenomenology always at center stage , and this has been a key influence on several quantum - spacetime - phenomenology research lines .
another approach for which there is by now a rather sizable research program aimed at phenomenological consequences is the one based on discrete causal sets [ 131 , 470 ] .
this is an approach of spacetime discretization that exploits the fact that a lorentzian metric determines both a geometry and a causal structure and also determines the metric up to a conformal factor .
one can then take the causal structure as primary , and start with a finite set of points with a causal ordering , recovering the conformal factor by counting points .
still , on the subject of approaches in which a role is played by spacetime discretization i should also bring to the attention of my readers the recent developments in the study of causal dynamical triangulations [ 45 , 371 , 46 , 47 , 372 , 49 ] . through causal dynamical triangulations
one gives an explicit , nonperturbative and background - independent , realization of the formal gravitational path integral on a given differential manifold . and some of the results obtained within this approach already provide elements of valuable intuition for quantum - spacetime phenomenology , as exemplified by the results providing first evidence for a scale - dependent spectral dimension of spacetime , varying from four at large scales to two at scales on the order of the planck length .
these running spectral dimensions could have very significant applications in phenomenology , and early signs that this might indeed be the case can be found in the debate reported in refs .
[ 424 , 505 , 425 ] concerning the implication for primordial gravity waves . also particularly important for quantum - spacetime phenomenology
this is an attempt at the nonperturbative construction of a predictive quantum field theory of the metric tensor centered on the availability of a non - gaussian renormalization - group fixed point [ 544 , 466 , 212 ] .
there are a few perspectives from which this asymptotic - safety program is influencing part of the research on quantum - spacetime phenomenology .
as an example of phenomenology work that was directly inspired by asymptotic safety , i should mention the expectation that quantum - gravity effects might also be important in a large - distance regime , with possible relevance for phenomenology .
i shall comment on this later in this review , also in relation to the idea of uv / ir mixing as a possibility that appears to be plausible even within other perspectives on quantum gravity and quantum spacetime .
that ultimately the description of spacetime in a quantum gravity with asymptotic safety will be a quantum - spacetime description .
also significant for quantum - spacetime phenomenology is the whole idea of running gravitational couplings , which is central to asymptotic safety .
as mentioned we tentatively assume that quantum - spacetime effects originate at the planck scale , but the planck scale is computed in terms of ( the ir value of ) newton s constant and might give us a misleading intuition for the characteristic scales of spacetime quantization .
there are also some perspectives on the quantum - gravity problem that at present i do not see as direct opportunities for quantum - spacetime phenomenology , but certainly are playing the role of intuition builders for the phenomenologists , affecting the perception of the quantum - gravity problem that guides some of the relevant research . among these
i should mention the rather large literature on the emergent gravity paradigm ( see , e.g. , refs .
[ 103 , 538 , 443 , 513 , 555 , 499 , 297 ] ) .
this literature actually contains a variety of possible way through which gravity could be described not as a fundamental aspect of the laws of nature , but rather as an emergent feature .
a simple analogy here is with pion - mediated strong interactions , which emerge from the quantum chromodynamics of quarks and gluons at low energies .
and i should mention as another potential intuition builder for the phenomenologists a class of studies that in various ways place dissipation in connection with aspects of the quantum - gravity problem ( see , e.g. , refs .
the theory proposals i briefly considered in section 2.1.4 ( all still lacking any experimental success ) can only serve the purpose of inspiring some test theories suitable for comparison to data . in this section ,
i will briefly motivate a partial list of possible classes of effects that could characterize the quantum - gravity / quantum - spacetime realm . and indeed in compiling such a list , one ends up using both intuition based on the general structure of the quantum - gravity problem and intuition based on what has been so far understood of theories that predict or assume spacetime quantization .
both the analysis of the general structure of the quantum - gravity problem and the analysis of proposed approaches to the solution of the quantum - gravity problem provide a rather broad collection of intuitions for what might be the correct quantization of spacetime ( see , e.g. , refs .
[ 406 , 532 , 269 , 44 , 332 , 442 , 211 , 20 , 432 , 50 , 249 , 489 ] ) , and in turn this variety of scenarios produces a rather broad collection of hypothesis concerning possible experimental manifestations of spacetime quantization . from a quantum - spacetime perspective
it is natural to expect that some opportunities for phenomenology might come from tests of spacetime symmetries .
it is relatively easy to test spacetime symmetries very sensitively , and it is natural to expect that introducing new ( quantum ) features in spacetime structure would affect the symmetries . let us consider in particular the minkowski limit , the one described by the classical minkowski spacetime in current theories : there is a duality one - to - one relation between the classical minkowski spacetime and the classical ( lie- ) algebra of poincar symmetry .
poincar transformations are smooth arbitrary - magnitude classical transformations and it is , therefore , natural to subject them to scrutiny11 if the classical minkowski spacetime is replaced by a quantized / discretized version .
the most active quantum - spacetime - phenomenology research area is indeed the one considering possible planck - scale departures from poincar / lorentz symmetries .
one possibility that has been considered in detail is the one of some symmetry - breaking mechanism affecting poincar / lorentz symmetry .
an alternative , which i advocated a few years ago [ 58 , 55 ] , is the one of a spacetime quantization that deforms but does not break some spacetime symmetries . besides the analysis of the general structure of the quantum - gravity problem , encouragement for these poincar / lorentz - symmetry studies is also found within some of the most popular proposals for spacetime quantization . as mentioned ,
according to the present understanding of lqg , the fundamental description of spacetime involves some intrinsic discretization [ 476 , 502 ] , and , although very little of robust is presently known about the minkowski limit of the theory , several indirect arguments suggest that this discretization should induce departures from classical poincar symmetry . while most of the lqg literature on the fate of poincar symmetries argues for symmetry violation ( see , e.g. , refs .
[ 247 , 33 ] ) , there are some candidate mechanisms ( see , e.g. , refs .
[ 75 , 237 , 503 ] ) that appear to provide opportunities for a deformation of symmetries in lqg . a growing number of quantum - gravity researchers are also studying noncommutative versions of minkowski spacetime , which are promising candidates as quantum - gravity theories of not everything , i.e. , opportunities to get insight on some , but definitely not all , aspects of the quantum - gravity problem . for the most studied examples , canonical noncommutativity , 3\documentclass[12pt]{minimal }
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\begin{document}$$[{x_m},t ] = { i \over \kappa}{x_m},\quad \;[{x_m},{x_l } ] = 0,$$\end{document } the issues relevant for the fate of poincar symmetry are very much in focus , and departures from poincar symmetry appear to be inevitable.12 arguments suggesting that cpt violation might arise in the quantum - gravity realm have a long tradition [ 279 , 445 , 540 , 446 , 42 , 222 , 298 , 345 , 117 ] ( and also see , e.g. , the more recent refs .
[ 21 , 423 , 330 ] ) . and , in light of the scope of this review , i should stress that specifically the idea of spacetime quantization invites one to place cpt symmetry under scrutiny . indeed ,
locality ( in addition to unitarity and lorentz invariance ) is a crucial ingredient for ensuring cpt invariance , and a common feature of all the proposals for spacetime quantization is the presence of limitations to locality , at least intended as limitations to the localizability of a spacetime event .
unfortunately , a proper analysis of cpt symmetry requires a level of understanding of the formalism that is often beyond our present reach in the study of formalizations of the concept of quantum spacetime . in lqg
one should have a good control of the minkowski ( classical- ) limit , and of the description of charged particles in that limit , and this is still beyond what can presently be done within lqg .
similar remarks apply to spacetime noncommutativity , although in that case some indirect arguments relevant for cpt symmetry can be meaningfully structured . for example , in ref .
it is observed that certain spacetime noncommutativity scenarios appear to require a deformation of p ( parity ) transformations , which would result in a corresponding deformation of cpt transformations . in the mentioned quantum - spacetime picture based on noncritical liouville string theory
[ 221 , 224 ] , evidence of violations of cpt symmetry has been reported , and later in this review i shall comment on the exciting phenomenology that was inspired by these results .
it is well established that the availability of a classical spacetime background has been instrumental to the successful tests of quantum mechanics so far performed .
the applicability of quantum mechanics to a broader class of contexts remains an open experimental question . if indeed space - time is quantized there might be some associated departures from quantum mechanics .
and this quantum - spacetime intuition fits well with a rather popular intuition for the broader context of quantum - gravity research , as discussed for example in refs .
some of the test theories used to model spacetime quantization have been found to provide motivation for departures from quantum mechanics in the form of decoherence , loss of quantum coherence [ 432 , 50 , 246 ] . a description of decoherence has been inspired by the mentioned noncritical liouville string theory [ 221 , 224 ] , and is essentially the core feature of the formalism advocated by percival and collaborators [ 452 , 453 , 454 ] .
the possibility of modifications of the heisenberg principle and of the de broglie relation has also been much studied in accordance with the intuition that some aspects of quantum mechanics might need to be adapted to spacetime quantization .
although the details of the mechanism that produces such modifications vary significantly from one picture of spacetime quantization to another [ 322 , 22 , 122 ] , one can develop an intuition of rather general applicability by noticing that the form of the de broglie relation in ordinary quantum mechanics reflects the properties of the classical geometry of spacetime that is there assumed . more precisely , the de broglie relation reflects the properties of the differential calculus on the spacetime manifold , since ordinary quantum mechanics describes the momentum observable in terms of a derivative operator ( assuming the heisenberg principle holds ) , which , acting on wave functions with wavelength , leads to the de broglie relation p = h/. in a nonclassical ( quantum ) spacetime one must adopt new forms of differential calculus [ 500 , 390 ] , and as a result the description of the momentum observable and its relation to the wavelength of a wave must be reformulated [ 322 , 22 , 122 , 63 ] . while the possibility of spacetime quantization provides a particularly direct logical line toward modifications of laws of quantum mechanics , one should consider such modifications as natural for the whole quantum - gravity problem ( even when studied without assuming spacetime quantization ) .
for example , in string theory , assuming the availability of a classical spacetime background , one finds some evidence of modification of the heisenberg principle ( the generalized uncertainty principle discussed , e.g. , in refs . [ 532 , 269 , 44 , 332 , 551 ] ) . a description that is often used to give some intuition for the effects induced by spacetime quantization is wheeler s spacetime foam , even though it does not amount to an operative definition .
most authors see it as motivation to look for formalizations of spacetime in which the distance between two events can not be sharply determined , and the metric is correspondingly fuzzy . as i shall discuss in section 4 , a few attempts to operatively characterize the concept of spacetime foam and to introduce corresponding test theories have been recently developed
. and a rather rich phenomenology is maturing from these proposals , often centered both on spacetime fuzziness per se and associated decoherence .
unfortunately , very little guidance can be obtained from the most studied quantum - spacetime pictures . in lqg
remarkably even with spacetime noncommutativity , an idea that was mainly motivated by the spacetime - foam intuition of a nonclassical spacetime , we are presently unable to describe , for example , the fuzziness that would intervene in operating an interferometer with the type of crisp physical characterization needed for phenomenology . the possibility of violations of the equivalence principle has not been extensively studied from a quantum - spacetime perspective , in spite of the fact that spacetime quantization does provide some motivation for placing under scrutiny at least some implications of the equivalence principle .
this is at least suggested by the observation that locality is a key ingredient of the present formulation of the equivalence principle : the equivalence principle ensures that ( under appropriate conditions ) two point particles would go on the same geodesic independent of their mass .
but it is well established that this is not applicable to extended bodies , and presumably also not applicable to
delocalized point particles ( point particles whose position is affected by uncontrolled uncertainties )
. presumably also the description of particles in a spacetime that is nonclassical ( quantized ) , and , therefore , sets absolute limitations on the identification of a spacetime point , would require departures from some aspects of the equivalence principle .
relatively few studies have been devoted to violations of the equivalence principle from a quantum - spacetime perspective .
examples are the study reported in ref . , which obtained violations of the equivalence principle from quantum - spacetime - induced decoherence , the study based on noncritical liouville string theory reported in ref . , and the study based on metric fluctuations reported in ref .
also the broader quantum - gravity literature ( even without spacetime quantization ) provides motivation for scrutinizing the equivalence principle .
in particular , a strong phenomenology centered on violations of the equivalence principle was proposed in the string - theory - inspired studies reported in refs . [ 521 , 195 , 196 , 194 , 193 , 192 ] and references therein , which actually provide a description of violations of the equivalence principle13 at a level that might soon be within our experimental reach . also relevant to this review
is the possibility that violations of the equivalence principle might be a by - product of violations of lorentz symmetry . in particular , this is suggested by the analysis in ref . , where the gravitational couplings of matter are studied in the presence of lorentz violation .
the largest area of quantum - spacetime - phenomenology research concerns the fate of lorentz ( /poincar ) symmetry at the planck scale , focusing on the idea that the conjectured new effects might become manifest at low energies ( the particle energies accessible to us , which are much below the planck scale ) in the form of uv corrections
, correction terms with powers of energy in the numerator and powers of the planck scale in the denominator . among the possible effects that might signal departures from lorentz / poincar symmetry
, the interest has been predominantly directed toward the study of the form of the energy - momentum ( dispersion ) relation .
this was due both to the ( relative ) robustness of associated theory results in quantum - spacetime research and to the availability of very valuable opportunities of related data analyses .
indeed , as several examples in this section will show , over the last decade there were very significant improvements of the sensitivity of lorentz- and poincar - symmetry tests . before discussing some actual phenomenologic al analyses ,
, which i have already referred to but should be discussed a bit more carefully . and
i shall also give a rather broad perspective on the quantum - spacetime implications for the set up of test theories suitable for the study of the fate of lorentz / poincar symmetry at the planck scale .
in our current conceptual framework poincar symmetry emerges in situations that allow the adoption of a minkowski metric throughout .
it is not inconceivable that quantum gravity might admit a limit in which one can assume throughout a ( expectation value of the ) metric of minkowski type , but some planck - scale features of the fundamental description of spacetime ( such as spacetime discreteness and/or spacetime noncommutativity ) are still not completely negligible
. this nontrivial minkowski limit would be such that essentially the role of the planck scale in the description of gravitational phenomena can be ignored ( so that indeed one can make reference to a fixed minkowski metric ) , but the possible role of the planck scale in spacetime structure / kinematics is still significant .
this intuition inspires the work on quantum - minkowski spacetimes , and the analysis of the symmetries of these quantum spacetimes .
it is not obvious that the correct quantum gravity should admit such a nontrivial minkowski limit . with the little we presently know about the quantum - gravity problem we must be open to the possibility that the minkowski limit could actually be trivial , i.e. , that whenever the role of the planck scale in the description of gravitational phenomena can be neglected ( and the metric is minkowskian at least on average ) one should also neglect the role of the planck scale in spacetime structure .
but the hypothesis of a nontrivial minkowski limit is worth exploring : it is a plausible hypothesis and it would be extremely valuable for us if quantum gravity did admit such a limit , since it might open a wide range of opportunities for accessible experimental verification , as i shall stress in what follows .
when i mention a result on the theory side concerning the fate of poincar symmetry at the planck scale clearly it must be the case that the authors have considered ( or attempted to consider ) the minkowski limit of their preferred formalism .
it is fair to state that each quantum - gravity research line can be connected with one of three perspectives on the problem : the particle - physics perspective , the gr perspective and the condensed - matter perspective . from a particle - physics perspective
it is natural to attempt to reproduce as much as possible the successes of the standard model of particle physics .
one is tempted to see gravity simply as one more gauge interaction . from this particle - physics perspective
a natural solution of the quantum - gravity problem should have its core features described in terms of graviton - like exchange in a background classical spacetime .
indeed this structure is found in string theory , the most developed among the quantum - gravity approaches that originate from a particle - physics perspective .
the particle - physics perspective provides no a priori reasons to renounce poincar symmetry , since minkowski classical spacetime is an admissible background spacetime , and in classical minkowski there can not be any a priori obstruction for classical poincar symmetry .
still , a breakdown of lorentz symmetry , in the sense of spontaneous symmetry breaking , is possible , and this possibility has been studied extensively over the last few years , especially in string theory ( see , e.g. , ref .
complementary to the particle - physics perspective is the gr perspective , whose core characteristic is the intuition that one should firmly reject the possibility of relying on a background spacetime [ 476 , 502 ] . according to gr the evolution of particles and the structure of spacetime are self - consistently connected : rather than specify a spacetime arena ( a spacetime background ) beforehand , the dynamical equations determine at once both the spacetime structure and the evolution of particles .
although less publicized , there is also growing awareness of the fact that , in addition to the concept of background independence , the development of gr relied heavily on the careful consideration of the in - principle limitations that measurement procedures can encounter.14 in light of the various arguments suggesting that , whenever both quantum mechanics and gr are taken into account , there should be an in - principle planck - scale limitation to the localization of a spacetime point ( an event ) , the gr perspective invites one to renounce any direct reference to a classical spacetime [ 211 , 20 , 432 , 50 , 249 ] . indeed , this requirement that spacetime be described as fundamentally nonclassical ( fundamentally quantum ) , so that the measurability limitations be reflected by a corresponding measurability - limited formalization of spacetime , is another element of intuition that is guiding quantum - gravity research from the gr perspective .
this naturally leads one to consider discretized spacetimes , as in the lqg approach or noncommutative spacetimes .
results obtained over the last few years indicate that this gr perspective naturally leads , through the emergence of spacetime discreteness and/or noncommutativity , to some departures from classical poincar symmetry .
lqg and some other discretized - spacetime quantum - gravity approaches appear to require a description of the familiar ( classical , continuous ) poincar symmetry as an approximate symmetry , with departures governed by the planck scale .
and in the study of noncommutative spacetimes some planck - scale departures from poincar symmetry appear to be inevitable .
the third possibility is a condensed - matter perspective on the quantum - gravity problem ( see , e.g. , refs .
[ 537 , 358 , 166 ] ) , in which spacetime itself is seen as a sort of emerging critical - point entity .
condensed - matter theories are used to describe the degrees of freedom that are measured in the laboratory as collective excitations within a theoretical framework , whose primary description is given in terms of much different , and often practically inaccessible , fundamental degrees of freedom .
close to a critical point some symmetries arise for the collective - excitation theory , which do not carry the significance of fundamental symmetries , and are , in fact , lost as soon as the theory is probed away from the critical point .
notably , some familiar systems are known to exhibit special - relativistic invariance in certain limits , even though , at a more fundamental level , they are described in terms of a nonrelativistic theory .
so , from the condensed - matter perspective on the quantum - gravity problem it is natural to see the familiar classical continuous poincar symmetry only as an approximate symmetry .
further encouragement for the idea of an emerging spacetime ( though not necessarily invoking the condensed - matter perspective ) comes from the realization [ 304 , 533 , 444 ] that the einstein equations can be viewed as an equation of state , so in some sense thermodynamics implies gr and the associated microscopic theory might not look much like gravity .
if the fate of poincar symmetry at the planck scale is nontrivial , the simplest possibility is the one of broken poincar symmetry , in the same sense that other symmetries are broken in physics .
as mentioned , an example of a suitable mechanism is provided by the possibility that a tensor field might have a vacuum expectation value . an alternative possibility , that in recent years has attracted the interest of a growing number of researchers within the quantum - spacetime and the quantum - gravity communities , is the one of deformed ( rather than broken ) spacetime symmetries , in the sense of the doubly - special - relativity ( dsr ) proposal i put forward a few years ago .
still , because of the purposes of this review , i must take into account that the development of phenomenologically - viable dsr models is still in its infancy .
[ 56 , 493 , 202 , 292 ] ) have highlighted the challenges for the description of spacetime and in particular spacetime locality that inevitably arise when contemplating a dsr scenario .
i am confident that some of the most recent dsr studies , particularly those centered on the analysis of the relative locality [ 71 , 504 , 88 , 67 ] , contain the core ideas that in due time will allow us to fully establish a robust dsr picture of spacetime , but i nonetheless feel that we are still far from the possibility of developing a robust dsr phenomenology .
interested readers have available a rather sizable dsr literature ( see , e.g. , ref .
[ 58 , 55 , 349 , 140 , 386 , 387 , 354 , 388 , 352 , 353 , 350 , 26 , 200 , 493 , 465 , 291 , 314 , 366 ] and references therein ) , but for the purposes of this review i shall limit my consideration of dsr ideas on phenomenology to a single one of the ( many ) relevant issues , which is an observation that concerns the compatibility between modifications of the energy - momentum dispersion relation and modifications of the law of conservation of energy - momentum .
my main task in this section is to illustrate the differences ( in relation to this compatibility issue ) between the broken - symmetry hypothesis and the dsr - deformed - symmetry hypothesis .
the dsr scenario was proposed as a sort of alternative perspective on the results on planck - scale departures from lorentz symmetry that had been reported in numerous articles [ 66 , 247 , 327 , 38 , 73 , 463 , 33 ] between 1997 and 2000 .
these studies were advocating a planck - scale modification of the energy - momentum dispersion relation , usually of the form \documentclass[12pt]{minimal }
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\begin{document}${e^2 } = { p^2 } + { m^2 } + \eta l_p^n{p^2}{e^n } + o(l_p^{n + 1}{e^{n + 3}})$\end{document } , on the basis of preliminary findings in the analysis of several formalisms in use for planck - scale physics .
the complexity of the formalisms is such that very little else was known about their physical consequences , but the evidence of a modification of the dispersion relation was becoming robust . in all of the relevant papers
it was assumed that such modifications of the dispersion relation would amount to a breakdown of lorentz symmetry , with associated emergence of a preferred class of inertial observers ( usually identified with the natural observer of the cosmic microwave background radiation ) . however , it then turned out to be possible to avoid this preferred - frame expectation , following a line of analysis in many ways analogous to the one familiar from the developments that led to the emergence of special relativity ( sr ) , now more than a century ago . in galileian relativity
there is no observer - independent scale , and in fact the energy - momentum relation is written as e = p/(2 m ) . as experimental evidence in favor of maxwell s equations started to grow , the fact that those equations involve a fundamental velocity scale appeared to require the introduction of a preferred class of inertial observers .
but in the end we discovered that the situation was not demanding the introduction of a preferred frame , but rather a modification of the laws of transformation between inertial observers .
einstein s sr introduced the first observer - independent relativistic scale ( the velocity scale c ) , its dispersion relation takes the form e = cp + cm ( in which c plays a crucial role in relation to dimensional analysis ) , and the presence of c in maxwell s equations is now understood as a manifestation of the necessity to deform the galilei transformations .
research in quantum gravity is increasingly providing reasons for interest in planck - scale modifications of the dispersion relation , and , while it was customary to assume that this would amount to the introduction of a preferred class of inertial frames ( a quantum - gravity ether ) , the proper description of these new structures might require yet again a modification of the laws of transformation between inertial observers .
the new transformation laws would have to be characterized by two scales ( c and ) rather than the single one ( c ) of ordinary sr . while the dsr idea came to be 0proposed in the context of studies of modifications of the dispersion relation
, one could have other uses for the second relativistic scale , as stressed in parts of the dsr literature [ 58 , 55 , 349 , 140 , 386 , 387 , 354 , 388 , 352 , 353 , 350 , 26 , 200 , 493 , 465 , 291 , 314 , 366 ] . instead of promoting to the status of relativistic invariant a modified dispersion relation , one can have dsr scenarios with undeformed dispersion relations but , for example , with an observer - independent bound on the accuracy achievable in the measurement of distances .
however , as announced , within the confines of this quantum - spacetime - phenomenology review i shall only make use of one dsr argument , that applies to cases in which indeed the dispersion relation is modified .
this concerns the fact that in the presence of observer - independent modifications of the dispersion relation ( dsr-)relativistic invariance imposes the presence of associated modifications of the law of energy - momentum conservation .
[ 58 , 63 ] , but it is here sufficient to illustrate it in a specific example . let us then consider a dispersion relation whose leading - order deformation ( by a length scale ) is given by 5\documentclass[12pt]{minimal }
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\begin{document}$${e^2 } \simeq { \vec p^2 } + { m^2 } + \lambda { \vec p^2}e.$$\end{document } this dispersion relation is clearly an invariant of classical space rotations , and of deformed boost transformations generated by [ 58 , 63 ] 6\documentclass[12pt]{minimal }
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\begin{document}$${\mathcal{b}_j } \simeq i{p_j}{\partial \over { \partial e } } + i\left({e + { \lambda \over 2}{{\vec p}^2 } + \lambda { e^2 } } \right){\partial \over { \partial { p_j } } } - i\lambda { p_j}\left({{p_k}{\partial \over { \partial { p_k } } } } \right).$$\end{document } the issue concerning energy - momentum conservation arises because both the dispersion relation and the law of energy - momentum conservation must be ( dsr-)relativistic . and the boosts ( 6 ) , which enforce relativistically the modification of the dispersion relation , are incompatible with the standard form of energy - momentum conservation .
for example , for processes with two incoming particles , a and b , and two outgoing particles , c and d , the requirements ea + eb ec ed = 0 and pa + pb pc pd = 0 are not observer - independent laws according to ( 6 ) .
an example of a modification of energy - momentum conservation that is compatible with ( 6 ) is 7\documentclass[12pt]{minimal }
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\begin{document}$${e_a } + { e_b } + \lambda { p_a}{p_b } \simeq { e_c } + { e_d } + \lambda { p_c}{p_d},$$\end{document }
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\begin{document}$${p_a } + { p_b } + \lambda ( { e_a}{p_b } + { e_b}{p_a } ) \simeq { p_c } + { p_d } + \lambda ( { e_c}{p_d } + { e_d}{p_c}).$$\end{document } and analogous formulas can be given for any process with n incoming particles and m outgoing particles . in particular , in the case of a two - body particle decay a b + c the laws 9\documentclass[12pt]{minimal }
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\begin{document}$${e_a } \simeq { e_b } + { e_c } + \lambda { p_b}{p_c},$$\end{document }
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\begin{document}$${p_a } \simeq { p_b } + { p_c } + \lambda ( { e_b}{p_c } + { e_c}{p_b})$$\end{document } provide an acceptable ( observer - independent , covariant according to ( 6 ) ) possibility .
this observation provides a general motivation for contemplating modifications of the law of energy - momentum conservation in frameworks with modified dispersion relations .
and i shall often test the potential impact on the phenomenology of introducing such modifications of the conservation of energy - momentum by using as examples dsr - inspired laws of the type ( 7 ) , ( 8) , ( 9 ) , ( 10 ) .
i shall do this without necessarily advocating a dsr interpretation : knowing whether or not the outcome of tests of modifications of the dispersion relation depends on the possibility of also having a modification of the momentum - conservation laws is of intrinsic interest , with or without the dsr intuition .
but i must stress that when the relativistic symmetries are broken ( rather than deformed in the dsr sense ) there is no a priori reason to modify the law of energy - momentum conservation , even when the dispersion relation is modified .
indeed most authors adopting modified dispersion relations within a broken - symmetry scenario keep the law of energy - momentum conservation undeformed . on the other hand
the dsr research program has still not reached the maturity for providing a fully satisfactory interpretation of the nonlinearities in the conservation laws . for some time the main challenge came ( in addition to the mentioned interpretational challenges connected with spacetime locality ) from arguments suggesting that one might well replace a given nonlinear setup for a dsr model with one obtained by redefining nonlinearly the coordinatization of momentum space ( see , e.g. , ref . ) . when contemplating such changes of coordinatization of momentum space many interpretational challenges appeared to arise . in my opinion ,
also in this direction the recent dsr literature has made significant progress , by casting the nonlinearities for momentum - space properties in terms of geometric entities , such as the metric and the affine connection on momentum space ( see , e.g. , ref . ) .
this novel geometric interpretation is offering several opportunities for addressing the interpretational challenges , but the process is still far from complete .
so far the main focus of poincar - symmetry tests planned from a quantum - spacetime - phenomenology perspective has been on the form of the energy - momentum dispersion relation . indeed , certain analyses of formalisms provide encouragement for the possibility that the minkowski limit of quantum gravity might indeed be characterized by modified dispersion relations . however , the complexity of the formalisms that motivate the study of planck - scale modifications of the dispersion relation is such that one has only partial information on the form of the correction terms and actually one does not even establish robustly the presence of modifications of the dispersion relation .
still , in some cases , most notably within some lqg studies and some studies of noncommutative space - times , the theoretical evidence in favor of modifications of the dispersion relations appears to be rather robust .
this is exactly the type of situation that i mentioned earlier in this review as part of a preliminary characterization of the peculiar type of test theories that must at present be used in quantum - spacetime phenomenology .
it is not possible to compare to data the predictions for departures from poincar symmetry of lqg and/or noncommutative geometry because these theories do not yet provide a sufficiently rich description of the structures needed for actually doing phenomenology with modified dispersion relations .
what we can compare to data are some simple models inspired by the little we believe we understand of the relevant issues within the theories that provide motivation for this phenomenology .
and the development of such models requires a delicate balancing act . if we only provide them with the structures we do understand of the original theories they will be as sterile as the original theories .
so , we must add some structure , make some assumptions , but do so with prudence , limiting as much as possible the risk of assuming properties that could turn out not to be verified once we understand the relevant formalisms better . as this description should suggest ,
there has been a proliferation of models adopted by different authors , each reflecting a different intuition on what could or could not be assumed .
correspondingly , in order to make a serious overall assessment of the experimental limits so far established with quantum - spacetime phenomenology of modified dispersion relations , one should consider a huge zoo of parameters .
even the parameters of the same parametrization of modifications of the dispersion relation when analyzed using different assumptions about other aspects of the model should really be treated as different / independent sets of parameters .
i shall be satisfied with considering some illustrative examples of models , chosen in such a way as to represent possibilities that are qualitatively very different , and representative of the breadth of possibilities that are under consideration .
these examples of models will then be used in some relevant parts of this review as language for the description of the sensitivity to planck - scale effects that is within the reach of certain experimental analyses . before describing actual test theories
, i should at least discuss the most significant among the issues that must be considered in setting up any such test theory with modified dispersion relation .
this concerns the choice of whether or not to assume that the test theory should be a standard low - energy effective quantum field theory .
a significant portion of the quantum - gravity and quantum - spacetime community is rather skeptical of the results obtained using low - energy effective field theory in analyses relevant to the planck - scale regime .
one of the key reasons for this skepticism is the description given by effective field theory of the cosmological constant .
the cosmological constant is the most significant experimental fact of evident gravitational relevance that could be within the reach of effective field theory . and current approaches to deriving the cosmological constant within effective field theory produce results , which are some 120 orders of magnitude greater than allowed by observations.15 however , just like there are several researchers who are skeptical about any results obtained using low - energy effective field theory in analyses relevant for the quantum - gravity / quantum - spacetime regime , there are also quite a few researchers who feel that it should be ok to assume a description in terms of effective field theory for all low - energy ( sub - planckian ) manifestations of the quantum - gravity / quantum - spacetime regime . adopting a strict phenomenologist viewpoint ,
perhaps the most important observation is that for several of the effects discussed in this section on uv corrections to lorentz symmetry , and for some of the effects discussed in later sections , studies based on effective quantum field theory can only be performed with a rather strongly pragmatic attitude .
one would like to confine the new effects to unexplored high - energy regimes , by adjusting bare parameters accordingly , but , as i shall stress again later , quantum corrections produce [ 455 , 182 , 515 , 190 ] effects that are nonetheless significant at accessible low energies , unless one allows for rather severe fine - tuning . on the other hand
, we do not have enough clues concerning setups alternative to quantum - field theory that could be used .
for example , as i discuss in detail later , some attempts are centered on density - matrix formalisms that go beyond quantum mechanics , but those are ( however legitimate ) mere speculations at the present time .
nonetheless several of the phenomenologists involved , myself included , feel that in such a situation phenomenology can not be stopped by the theory impasse , even at the risk of later discovering that the whole ( or a sizable part of ) the phenomenological effort was not on sound conceptual bases .
but i stress that even when contemplating the possibility of physics outside the domain of effective quantum field theory , one inevitably must at least come to terms with the success of effective field theory in reproducing a vast class of experimental data . in this respect , at least for studies of planck - scale departures from classical - spacetime relativistic symmetries i find particularly intriguing a potential order - of - limits issue .
the effective - field - theory description might be applicable only in reference frames in which the process of interest is essentially occurring in its center of mass ( no planck - large boost with respect to the center - of - mass frame ) .
the field theoretic description could emerge in a sort of low - boost limit
the regime of low boosts with respect to the center - of - mass frame is often indistinguishable from the low - energy limit .
for example , from a planck - scale perspective , our laboratory experiments ( even the ones conducted at , e.g. , cern , desy , slac , ) are both low boost ( with respect to the center - of - mass frame ) and low energy .
however , some contexts that are of interest in quantum - gravity phenomenology , such as the collisions between ultra - high - energy cosmic - ray protons and cmbr photons , are situations where all the energies of the particles are still tiny with respect to the planck energy scale , but the boost with respect to the center - of - mass frame could be considered to be
large from a planck - scale perspective : the lorentz factor with respect to the proton rest frame is much greater than the ratio between the planck scale and the proton mass 11\documentclass[12pt]{minimal }
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\begin{document}$$\gamma = e/{m_{{\rm{proton } } } } \gg { e_p}/e.$$\end{document } another interesting scenario concerning the nature of the limit through which quantum - spacetime physics should reproduce ordinary physics is suggested by results on field theories in noncommutative spacetimes .
one can observe that a spacetime characterized by an uncertainty relation of the type 12\documentclass[12pt]{minimal }
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\begin{document}$$\delta x\delta y \geq \theta ( x , y)$$\end{document } never really behaves as a classical spacetime , not even at very low energies .
in fact , according to this type of uncertainty relation , a low - energy process involving soft momentum exchange in the x direction ( large x ) should somehow be connected to the exchange of a hard momentum in the y direction ( y /x ) , and this feature can not faithfully be captured by our ordinary field - theory formalisms . for the canonical noncommutative spacetimes
one does obtain a plausible - looking field theory , but the results actually show that it is not possible to rely on an ordinary effective low - energy quantum - field - theory description because of the presence of uv / ir mixing [ 213 , 397 ] ( a mechanism such that the high - energy sector of the theory does not decouple from the low - energy sector , which in turn very severely affects the prospects of analyses based on an ordinary effective low - energy quantum - field - theory description ) . for other ( non - canonical ) noncommutative spacetimes we are still struggling in the search for a satisfactory formulation of a quantum field theory [ 335 , 64 ] , and
it is at this point legitimate to worry that such a formulation of dynamics in those spacetimes does not exist . and the assumption of availability of an ordinary effective low - energy quantum - field - theory description has also been challenged by some perspectives on the lqg approach . for example , the arguments presented in ref . suggest that in several contexts in which one would naively expect a low - energy field theory description lqg might instead require a density - matrix description with features going beyond the reach of effective quantum field theory . in order to be applicable to a significant ensemble of experimental contexts ,
a test theory should specify much more than the form of the dispersion relation . in light of the type of data that we expect to have access to ( see later , e.g. , sections 3.4 , 3.5 , and 3.8 ) , besides
the choice of working within or without low - energy effective quantum field theory , there are at least three other issues that the formulation of such a test theory should clearly address :
( i)is the modification of the dispersion relation universal ? or
should one instead allow different modification parameters for different particles?(ii)in the presence of a modified dispersion relation between the energy e and the momentum p of a particle , should we still assume the validity of the relation = de / dp between the speed of a particle and its dispersion relation?(iii)in the presence of a modified dispersion relation , should we still assume the validity of the standard law of energy - momentum conservation ? is the modification of the dispersion relation
in the presence of a modified dispersion relation between the energy e and the momentum p of a particle , should we still assume the validity of the relation = de / dp between the speed of a particle and its dispersion relation ? in the presence of a modified dispersion relation ,
should we still assume the validity of the standard law of energy - momentum conservation ? unfortunately on these three key points , the quantum - spacetime pictures that are providing motivation for the study of planck - scale modifications of the dispersion relation are not giving us much guidance yet .
for example , in lqg , while we do have some ( however fragile and indirect ) evidence that the dispersion relation should be modified , we do not yet have a clear indication concerning whether the law of energy - momentum conservation should also be modified and we also can not yet establish whether the relation = de / dp should be preserved .
similarly , in the analysis of noncommutative spacetimes we are close to establishing rather robustly the presence of modifications of the dispersion relation , but other aspects of the relevant theories have not yet been clarified . while most of the literature for canonical noncommutative spacetimes assumes [ 213 , 397 ] that the law of energy - momentum conservation should not be modified , most of the literature on -minkowski spacetime argues in favor of a modification of the law of energy - momentum conservation .
there is also still no consensus on the relation between speed and dispersion , and particularly in the -minkowski literature some departures from the = de / dp relation are actively considered [ 336 , 414 , 199 , 351 ] . and at least for canonical noncommutative spacetimes the possibility of a nonuniversal dispersion relation is considered extensively [ 213 , 397 ] . concerning the relation = de / dp it may be useful to stress that it can be obtained assuming that a hamiltonian description is still available , = dx / dt [ x , h(p ) ] , and that the heisenberg uncertainty principle still holds exactly ( [ x , p ] = 1 x /p ) .
the possibility of modifications of the hamiltonian description is an aspect of the debate on planck - scale dynamics that was in part discussed in section 3.2.1 .
and concerning the heisenberg uncertainty principle i have already mentioned some arguments that invite us to contemplate modifications . with so many possible alternative ingredients to mix one can of
as mentioned , i intend to focus on some illustrative examples of test theories for my characterization of achievable experimental sensitivities .
my first example is a test theory of very limited scope , since it is conceived to only describe pure - kinematics effects
. this will strongly restrict the class of experiments that can be analyzed in terms of this test theory , but the advantage is that the limits obtained on the parameters of this test theory will have rather wide applicability ( they will apply to any quantum - spacetime theory with that form of kinematics , independent of the description of dynamics ) . the first element of this test theory , introduced from a quantum - spacetime - phenomenology perspective in refs .
universal ( same for all particles ) dispersion relation of the form 13\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { \vec p^2 } + \eta { \vec p^2}\left({{{{e^n } } \over { e_p^n } } } \right),$$\end{document } with real of order 1 and integer n ( > 0 ) .
this formula is compatible with some of the results obtained in the lqg approach and reflects some results obtained for theories in -minkowski noncommutative spacetime .
already in the first studies that proposed a phenomenology based on ( 13 ) it was assumed that even at the planck scale the familiar description of group velocity
, obtained from the dispersion relation according to = de / dp , would hold . and
in other early phenomenology works [ 327 , 38 , 73 , 463 ] based on ( 13 ) it was assumed that the law of energy - momentum conservation should not be modified at the planck scale , so that , for example , ina a + b c + d particle - physics process one would have 14\documentclass[12pt]{minimal }
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\begin{document}$${e_a } + { e_b } = { e_c } + { e_d},$$\end{document }
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\begin{document}$${\vec p_a } + { \vec p_b } = { \vec p_c } + { \vec p_d}.$$\end{document } in the following , i will refer to this test theory as the pkv0 test theory , where pk reflects its pure - kinematics nature , v reflects its lorentz - symmetry violation content , and 0 reflects the fact that it combines the dispersion relation ( 13 ) with what appears to be the most elementary set of assumptions concerning other key aspects of the physics : universality of the dispersion relation , = de / dp , and the unmodified law of energy - momentum conservation .
for example , the undeformed conservation of energy - momentum is relativistically incompatible with the deformation of the dispersion relation ( so , in particular , the pkv0 test theory requires a preferred frame ) .
modifications of the law of energy - momentum conservation would be required in a dsr picture , and may be considered even in other scenarios.16 evidently , the universality of the effect can and should be challenged . and
there are indeed ( as i shall stress again later in this review ) several proposals of test theories with different magnitudes of the effects for different particles [ 395 , 308 ] .
let me just mention , in closing this section , a case that is particularly challenging for phenomenology : the case of the variant of the pkv0 test theory allowing for nonuniversality such that the effects are restricted only to photons [ 227 , 74 ] , thereby limiting significantly the class of observations / experiments that could test the scenario ( see , however , ref . ) . the restriction to pure kinematics has the merit to allow us to establish constraints that are applicable to a relatively large class of quantum - spacetime scenarios ( different formulations of dynamics
would still be subject to the relevant constraints ) , but it also severely restricts the type of experimental contexts that can be considered , since it is only in rare instances ( and only to some extent ) that one can qualify an analysis as purely kinematical .
therefore , the desire to be able to analyze a wider class of experimental contexts is , therefore , providing motivation for the development of test theories more ambitious than the pkv0 test theory , with at least some elements of dynamics .
this is rather reasonable , as long as one proceeds with awareness of the fact that , in light of the situation on the theory side , for test theories adopting a given description of dynamics there is a risk that we may eventually find out that none of the quantum - gravity approaches that are being pursued are reflected in the test theory . when planning to devise a test theory that includes the possibility to describe dynamics , the first natural candidate ( not withstanding the concerns reviewed in section 3.2.1 ) is the framework of low - energy effective quantum field theory . in this section
i want to discuss a test theory that is indeed based on low - energy effective field theory , and has emerged primarily17 from the analysis reported by myers and pospelov in ref . .
, this test theory explores the possibility of a linear - in - lp modification of the dispersion relation 16\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { \vec p^2 } + \eta { \vec p^2}{l_p}e,$$\end{document } i.e. , the case n = 1 of eq . ( 13 ) .
perhaps the most notable outcome of the exercise of introducing such a dispersion relation within an effective low - energy field - theory setup is the observation that for the case of electromagnetic radiation , assuming essentially only that the effects are characterized mainly by an external four - vector , one arrives at a single possible correction term for the lagrangian density : 17\documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{l } = - { 1 \over 4}{f_{\mu \nu}}{f^{\mu \nu } } + { 1 \over { 2{e_p}}}{\eta ^\alpha}{f_{\alpha \delta}}{n^\sigma}{\partial _ \sigma}({n_\beta}{\varepsilon ^{\beta
this is also a framework for broken lorentz symmetry , since the ( dimensionless ) components of n take different values in different reference frames , transforming as the components of a four - vector . and
a full - scope phenomenology for this proposal should explore the four - dimensional parameter space , n , taking into account the characteristic frame dependence of the parameters n. as i discuss in later parts of this section , there is already a rather sizable literature on this phenomenology , but still mainly focused on what turns out to be the simplest possibility for the myers - pospelov framework , which relies on the assumption that one is in a reference frame where n only has a time component , n = ( n0 , 0,0,0 ) .
then , upon introducing the convenient notation ( n0 ) , one can rewrite ( 17 ) as 18\documentclass[12pt]{minimal }
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\begin{document}$${\mathcal l } = - { 1 \over 4}{f_{\mu \nu}}{f^{\mu \nu } } + { \xi \over { 2{e_p}}}{\varepsilon ^{jkl}}{f_{0j}}{\partial _ 0}{f_{kl}},$$\end{document } and in particular one can exploit the simplifications provided by spatial isotropy . and a key feature that arises is birefringence : within this setup it turns out that when right - circular polarized photons satisfy the dispersion relation e p + p , then necessarily left - circular polarized photons satisfy the opposite sign dispersion relation e p p . in the same spirit
one can add spin-1/2 particles to the model , but for them the structure of the framework does not introduce constraints on the parameters , and in particular there can be two independent parameters + and to characterize the modification of the dispersion relation for fermions of different helicity : 19\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { \vec p^2 } + { \eta _ + } { \vec p^2}\left({{e \over { { e_p } } } } \right),$$\end{document } in the positive - helicity case , and 20\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { \vec p^2 } + { \eta _ -}{\vec p^2}\left({{e \over { { e_p } } } } \right),$$\end{document } in the negative - helicity case .
the formalism is compatible with the possibility of introducing further independent parameters for each additional fermion in the theory ( so that , e.g. , protons would have different values of + and with respect to electrons ) .
and there is no constraint on the relation between + and , but the consistency of the framework requires that for particle - antiparticle pairs , the deformation should have opposite signs on opposite helicities , so that , for example , \documentclass[12pt]{minimal }
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\begin{document}$\eta _ + ^{({\rm{electron } } ) } = - \eta _ - ^{({\rm{positron}})}\;{\rm{and}}\;\eta _ - ^{({\rm{electron } } ) } = - \eta
_ + ^{({\rm{positron}})}$\end{document}. in some investigations one might prefer to look at particularly meaningful portions of this large parameter space .
for example , one might consider the possibility that the deformation for all spin-1/2 particles be characterized by only two parameters , the same two parameters for all particle - antipartic le pairs ( leaving open , however , some possible sign ambiguities to accommodate the possibility to choose between , for example , \documentclass[12pt]{minimal }
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\begin{document}$\eta _
+ ^{({\rm{muon } } ) } = \eta _ + ^{({\rm{electron } } ) } = - \eta _ - ^{({\rm{positron}})}\;{\rm{and}}\;\eta _ + ^{({\rm{muon } } ) } = \eta _ + ^{({\rm{positron } } ) } = - \eta _ - ^{({\rm{electron}})}$\end{document}. in the following i will refer to this test theory as the ftv0 test theory , where ft reflects its adoption of a low - energy effective field theory description , v reflects its lorentz - symmetry violation content , and 0 reflects the minimalistic assumption of universality for spin-1/2 particles . before starting my characterization of experimental sensitivities in terms of the parameters of some test theories
i find it appropriate to add a few remarks warning about some difficulties that are inevitably encountered .
for the pure - kinematics test theories , some key difficulties originate from the fact that sometimes an effect due to the modification of dynamics can take a form that is not easily distinguished from a pure - kinematics effect .
and other times one deals with an analysis of effects that appear to be exclusively sensitive to kinematics but then at the stage of converting experimental results into bounds on parameters some level of dependence on dynamics arises .
an example of this latter possibility will be provided by my description of particle - decay thresholds in test theories that violate lorentz symmetry .
the derivation of the equations that characterize the threshold requires only the knowledge of the laws of kinematics . and if , according to the kinematics of a given test theory , a certain particle at a certain energy can not decay , then observation of the decay allows one to set robust pure - kinematics limits on the parameters .
but if the test theory predicts that a certain particle at a certain energy can decay then by not finding such decays we are not in a position to truly establish pure - kinematics limits on the parameters of the test theory .
if the decay is kinematically allowed but not seen , it is possible that the laws of dynamics prevent it from occurring ( small decay amplitude ) . by adopting a low - energy quantum field theory this type of limitations
is removed , but other issues must be taken into account , particularly in association with the fact that the ftv0 quantum field theory is not renormalizable .
quantum - field - theory - based descriptions of planck - scale departures from lorentz symmetry can only be developed with a rather strongly pragmatic attitude .
in particular , for the ftv0 test theory , with its planck - scale suppressed effects at tree level , some authors ( notably refs .
[ 455 , 182 , 515 , 190 ] ) have argued that the loop expansion could effectively generate additional terms of modification of the dispersion relation that are unsuppressed by the cut - off scale of the ( nonrenormalizable ) field theory .
the parameters of the field theory can be fine - tuned to eliminate unwanted large effects , but the needed level of fine tuning is usually rather unpleasant . while certainly undesirable , this severe fine - tuning problem should not discourage us from considering the ftv0 test theory , at least not at this early stage of the development of the relevant phenomenology .
actually some of the most successful theories used in fundamental physics are affected by severe fine tuning .
it is not uncommon to eventually discover that the fine tuning is only apparent , and some hidden symmetry is actually naturally setting up the hierarchy of parameters . in particular
, it is already established that supersymmetry can tame the fine - tuning issue [ 268 , 130 ] .
if one extends supersymmetric quantum electrodynamics by adding interactions with external vector and tensor backgrounds that violate lorentz symmetry at the planck scale , then exact supersymmetry requires that such interactions correspond to operators of dimension five or higher , so that no fine - tuning is needed in order to suppress the unwanted operators of dimension lower than five .
supersymmetry can only be an approximate symmetry of the physical world , and the effects of the scale of soft - supersymmetry - breaking masses controls the renormalization - group evolution of dimension five lorentz - violating operators and their mixing with dimension three lorentz - violating operators [ 268 , 130 ] .
it has also been established that if lorentz violation occurs in the gravitational sector , then the violations of lorentz symmetry induced on the matter sector do not require severe fine - tuning .
in particular , this has been investigated by coupling the standard model of particle physics to a hoava - lifshitz description of gravitational phenomena .
the study of planck - scale departures from lorentz symmetry may find some encouragement in perspectives based on renormalization theory , at least in as much as it has been shown [ 79 , 78 , 289 , 507 ] that some field theories modified by lorentz - violating terms are actually rather well behaved in the uv .
the first example of planck - scale sensitivity that i discuss is the case of a process that is kinematically forbidden in the presence of exact lorentz symmetry , but becomes kinematically allowed in the presence of certain departures from lorentz symmetry .
305 , 59 , 334 , 115 ] ) that when lorentz symmetry is broken at the planck scale , there can be significant implications for certain decay processes .
at the qualitative level , the most significant novelty would be the possibility for massless particles to decay . and certain observations in astrophysics , which allow us to establish that photons of energies up to 10 ev are stable ,
can then be used [ 305 , 59 , 334 , 115 ] to set limits on schemes for departures from lorentz symmetry . for my purposes
let us start from the perspective of the pkv0 test theory , and therefore adopt the dispersion relation ( 13 ) and unmodified energy - momentum conservation .
one easily finds a relation between the energy e of the incoming photon , the opening angle between the outgoing electron - positron pair , and the energy e+ of the outgoing positron ( the energy of the outgoing electron is simply given by e e+ ) . setting n = 1 in ( 13 )
one finds that , for the region of phase space with me
e
ep , this relation takes the form 21\documentclass[12pt]{minimal }
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\begin{document}$$\cos ( \theta ) \simeq { { { e _ + } ( { e_\gamma } - { e _ + } ) + m_e^2 - \eta { e_\gamma}{e _ + } ( { e_\gamma } - { e _ + } ) /{e_p } } \over { { e _ + } ( { e_\gamma } - { e _ + } ) } } , $ $ \end{document } where me is the electron mass . the fact that for = 0 eq .
( 21 ) would require cos( ) > 1 reflects the fact that , if lorentz symmetry is preserved , the process ee is kinematically forbidden . for
< 0 the process is still forbidden , but for positive high - energy photons can decay into an electron - positron pair .
in fact , for \documentclass[12pt]{minimal }
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\begin{document}${e_\gamma } \gg { ( m_e^2{e_p}/\vert\eta \vert)^{1/3}}$\end{document } one finds that there is a region of phase space where cos( ) < 1 , i.e. , there is a physical phase space available for the decay .
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\begin{document}${(m_e^2{e_p})^{1/3}}$\end{document } 10 ev is not too high for testing , since , as mentioned , in astrophysics we see photons of energies up to 10 ev that are stable ( they clearly travel safely some large astrophysical distances ) .
the level of sensitivity that is within reach of these studies therefore goes at least down to values of ( positive ) of order 1 and somewhat smaller than 1 .
this is what one describes as planck - scale sensitivity in the quantum - spacetime phenomenology literature : having set the dimensionful deformation parameter to the planck - scale value , the coefficient of the term that can be tested is of order 1 or smaller .
however , specifically for the case of the photon - stability analysis it is rather challenging to transform this planck - scale sensitivity into actual experimental limits . within pkv0 kinematics , for n = 1 and positive of order 1 , it would have been natural to expect that photons with 10 ev energy are unstable .
but the fact that the decay of 10 ev photons is allowed by pkv0 kinematics of does not guarantee that these photons should rapidly decay .
it depends on the relevant probability amplitude , whose evaluation goes beyond the reach of kinematics .
still , it is likely that these observations are very significant for theories that are compatible with pkv0 kinematics . for a theory that is compatible with pkv0 kinematics ( with positive )
this evidence of stability of photons imposes the identification of a dynamical mechanism that essentially prevents photon decay .
if one finds no such mechanism , the theory is ruled out ( or at least its parameters are severely constrained ) , but in principle one could look endlessy for such a mechanism . a balanced approach to this issue must take into account that quantum - spacetime physics may well modify both kinematics and the strength ( and nature ) of interactions at a certain scale , and it might in principle do this in ways that can not be accommodated within the confines of effective quantum field theory , but one should take notice of the fact that , even in some new ( to - be - discovered ) framework outside effective quantum field theory , it is unlikely that there will be very large conspiracies between the modifications of kinematics and the modifications of the strength of interaction . in principle , models based on pure kinematics are immune from certain bounds on parameters that are also derived also using descriptions of the interactions , and it is conceivable that in the correct theory the actual bound would be somewhat shifted from the value derived within effective quantum field theory . but in order to contemplate large differences in the bounds one would need to advocate very large and ad hoc modifications of the strength of interactions , large enough to compensate for the often dramatic implications of the modifications of kinematics .
the challenge then is to find satisfactory criteria for confining speculations about variations of the strengths of interaction only within a certain plausible range . to my knowledge
a completely analogous calculation can be done within the ftv0 test theory , and there one can easily arrive at the conclusion that the ftv0 description of dynamics should not significantly suppress the photon - decay process . however , as mentioned , consistency with the effective - field - theory setup requires that the two polarizations of the photon acquire opposite - sign modifications of the dispersion relation .
we observe in astrophysics some photons of energies up to 10 ev that are stable over large distances , but as far as we know those photons could be all right - circular polarized ( or all left - circular polarized ) .
this evidence of stability of photons , therefore , is only applicable to the portion of the ftv0 parameter space in which both polarizations should be unstable ( a subset of the region with |+| > || and || > || ) . so far i have discussed photon stability assuming that only the dispersion relation is modified . if the modification of the dispersion relation is instead combined with a modification of the law of energy - momentum conservation the results can change very significantly . in order to expose these changes in rather striking fashion let me consider the example of dsr - inspired laws of energy - momentum conservation for the case of ee : 22\documentclass[12pt]{minimal }
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\begin{document}$${e_\gamma } \simeq { e _ + } + { e _ - } - \eta { \vec p _ + } \cdot { \vec p _ -},$$\end{document }
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\begin{document}$${\vec p_\gamma } \simeq { \vec p _ + } + { \vec p _ - } - \eta { e _ + } { \vec p _ - } - \eta { e _ -}{\vec p _ + } .$$\end{document } using these in place of ordinary conservation of energy - momentum , one ends up with a result for cos( ) that is still of the form ( a + b)/a but now with a = 2e+(e e+ ) + ee+(e e+ ) and \documentclass[12pt]{minimal }
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\begin{document}$b = 2m_e^2$\end{document } : 24\documentclass[12pt]{minimal }
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\begin{document}$$\cos ( \theta ) \simeq { { 2{e _ + } ( { e_\gamma } - { e _ + } ) + \eta { e_\gamma}{e _ + } ( { e_\gamma } - { e _ + } ) + 2m_e^2 } \over { 2{e _ + } ( { e_\gamma } - { e _ + } ) + \eta { e_\gamma}{e _ + } ( { e_\gamma } - { e _ + } ) } } .$$\end{document } evidently , this formula always gives cos( ) > 1 , so there are combinations of modifications of the dispersion relation and modifications of energy - momentum conservation such that ee is still forbidden .
if the modification of the dispersion relation and the modification of the law of energy - momentum conservation are not matched exactly to get this result , then one can have the possibility of photon decay , but in some cases it can be further suppressed ( in addition to the planck - scale suppression ) by the partial compensation between the two modifications .
the fact that the matching between modification of the dispersion relation and modification of the law of energy - momentum conservation that produces a stable photon is obtained using a dsr - inspired setup is not surprising .
the relativistic properties of the framework are clearly at stake in this derivation . a threshold - energy requirement for particle decay ( such as the \documentclass[12pt]{minimal }
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\begin{document}${e_\gamma } \gg { ( m_e^2{e_p}/\vert\eta \vert)^{1/3}}$\end{document } mentioned above ) can not be introduced as an observer - independent law , and is therefore incompatible with any relativistic ( even dsr - relativistic ) formulation of the laws of physics .
in fact , different observers assign different values to the energy of a particle and , therefore , in the presence of a threshold - energy requirement for particle decay a given particle should be allowed to decay , according to some observers while being totally stable for others . another opportunity to investigate quantum - spacetime - inspired planck - scale departures from lorentz symmetry
is provided by certain types of energy thresholds for particle - production processes that are relevant in astrophysics .
this is a very powerful tool for quantum - spacetime phenomenology [ 327 , 38 , 73 , 463 , 512 , 364 , 307 , 494 ] , and , in fact , at the beginning of this review , i chose the evaluation of the threshold energy for photopion production , p + cmber p + , as the basis for illustrating how the sensitivity levels that are within our reach can be placed in rather natural connection with effects introduced at the planck scale .
i discuss the photopion production threshold analysis in more detail in section 3.5 . here , i consider instead the electron - positron pair production process , ee . the threshold for ee is relevant for studies of the opacity of our universe to photons . in particular , according to the conventional ( classical - spacetime ) description , the ir diffuse extra - galactic background should give rise to the strong absorption of tev photons ( here understood as photons with energy 1 tev < e < 30 tev ) , but this prediction must be reassessed in the presence of violations of lorentz symmetry . to show that this is the case ,
let me start once again from the perspective of the pkv0 test theory , and analyze a collision between a soft photon of energy and a high - energy photon of energy e , which might produce an electron - positron pair . using the dispersion relation ( 13 ) ( for n = 1 ) and the ( unmodified ) law of energy - momentum conservation
, one finds that for given soft - photon energy e , the process ee is allowed only if e is greater than a certain threshold energy eth that depends on and \documentclass[12pt]{minimal }
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\begin{document}$m_e^2$\end{document } , as implicitly codified in the formula ( valid for me eth ep ) 25\documentclass[12pt]{minimal }
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\begin{document}$${e_{th}}\epsilon + \eta { { e_{th}^3 } \over { 8{e_p } } } \simeq m_e^2.$$\end{document } the special - relativistic result \documentclass[12pt]{minimal }
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\begin{document}${e_{th } } = m_e^2/\epsilon$\end{document } corresponds to the 0 limit of ( 25 ) . for || 1
the planck - scale correction can be safely neglected as long as \documentclass[12pt]{minimal }
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\begin{document}$\epsilon \gg { ( m_e^4/{e_p})^{1/3}}$\end{document}. but eventually , for sufficiently small values of ( and correspondingly large values of eth ) the planck - scale correction can not be ignored .
this provides an opportunity for a pure - kinematics test : if a 10 tev photon collides with a photon of 0.03 ev and produces an electron - positron pair the case n = 1 , 1 for the pkv0 test theory is ruled out . a 10 tev photon and
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\begin{document}${e_{th } } = m_e^2/\epsilon$\end{document } ) , but they can not produce an electron - positron pair according to pkv0 kinematics with n = 1 and 1 . for positive
while negative increases the energy requirement for electron - positron pair production , positive decreases the energy requirement for electron - positron pair production . in some cases , where one would expect electron - positron pair production to be forbidden
but once a process is allowed there is no guarantee that it will actually occur , not without some information on the description of dynamics ( that allows us to evaluate cross sections ) . as in the case of photon decay
, one must conclude that a pure - kinematics framework can be falsified when it predicts that a process can not occur ( if instead the process is seen ) but in principle it can not be falsified when it predicts that a process is allowed . here too , one should gradually develop balanced criteria taking into account the remarks i offer in section 3.3.1 concerning the plausibility ( or lack thereof ) of conspiracies between modifications of kinematics and modifications of the strengths of interaction . concerning the level of sensitivity that we can expect to achieve in this case one can robustly claim that planck - scale sensitivity is within our reach .
this , as anticipated above , is best seen considering the tev photons emitted by some blazars , for which ( as they travel toward our earth detectors ) the photons of the ir diffuse extragalactic background are potential targets for electron - positron pair production . in estimating the sensitivity achievable with this type of analyses it is necessary to take into account the fact that , besides the form of the threshold condition , there are at least three other factors that play a role in establishing the level of absorption of tev photons emitted by a given blazar : our knowledge of the type of signal emitted by the blazar ( at the source ) , the distance of the blazar , and most importantly the density of the ir diffuse extragalactic background .
the availability of observations of the relevant type has increased very significantly over these past few years .
markarian 501 ( at a redshift of z = 0.034 ) and the blazar h1426 + 428 ( at a redshift of z = 0.129 ) robust observations up to the 20-tev range have been reported [ 15 , 16 ] , and for the blazar
markarian 421 ( at a redshift of z = 0.031 ) observations of photons of energy up to 45 tev has been reported , although a more robust signal is seen once again up to the 20-tev range [ 355 , 17 ] .
the key obstruction for translating these observations into an estimate of the effectiveness of pair - production absorption comes from the fact that measurements of the density of the ir diffuse extragalactic background are very difficult , and as a result our experimental information on this density is still affected by large uncertainties [ 235 , 536 , 111 , 278 ] . the observations do show convincingly that some absorption is occurring [ 15 , 16 , 438 , 355 , 17 ] .
i should stress the fact that the analysis of the combined x - ray / tev - gamma - ray spectrum for the markarian 421 blazar , as discussed in ref .
the x - ray part of the spectrum allows one to predict the tev - gamma - ray part of the spectrum in a way that is rather insensitive to our poor knowledge of the source .
this in turn allows us to establish in a source - independent way that some absorption is occurring .
for the associated quantum - spacetime - phenomenology analysis , the fact that some absorption is occurring does not allow us to infer much : the analysis will become more and more effective as the quantitative characterization of the effectiveness of absorption becomes more and more precise ( as measured by the amount of deviation from the level of absorption expected within a classical - spacetime analysis that would still be compatible with the observations ) .
and we are not yet ready to make any definite statement about this absorption levels .
this is not only a result of our rather poor knowledge of the ir diffuse extragalactic background , but it is also due to the status of the observations , which still presents us with some apparent puzzles .
for example , it is not yet fully understood why , as observed by some [ 15 , 355 , 17 , 536 ] , there is a difference between the absorption - induced cutoff energy found in data concerning markarian 421 , \documentclass[12pt]{minimal }
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\begin{document}$e_{{\rm{mk}}421}^{{\rm{cutoff } } } \simeq 3.6\;{\rm{tev}}$\end{document } , and the corresponding cutoff estimate obtained from markarian-501 data , \documentclass[12pt]{minimal }
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\begin{document}$e_{{\rm{mk}}501}^{{\rm{cutoff } } } \simeq 6.2\;{\rm{tev}}$\end{document}. and the observation of tev -rays emitted by the blazar h1426 + 428 , which is significantly more distant than markarian 421 and markarian 501 , does show a level of absorption that is higher than the ones inferred for markarian 421 and markarian 501 , but ( at least assuming a certain description of the ir diffuse extragalactic background ) the h1426 + 428 tev luminosity seems to exceed the level anticipated from the current models of tev blazars by far .
clearly , the situation requires further clarification , but it seems reasonable to expect that within a few years we should fully establish facts such as
-rays with energies up to 20 tev are absorbed by the ir diffuse extragalactic background.18 this would imply that at least some photons with energy smaller than 200 mev can create an electron - positron pair in collisions with a 20 tev -ray . in turn
this would imply for the pkv0 test theory , with n = 1 , that necessarily 50 ( i.e. , either is positive or is negative with absolute value smaller than 50 ) .
this means that this strategy of analysis will soon take us robustly to sensitivities that are less than a factor of a 100 away from planck - scale sensitivities , and it is natural to expect that further refinements of these measurements will eventually take us to planck - scale sensitivity and beyond .
the line of reasoning needed to establish whether this planck - scale sensitivity could apply to pure - kinematics frameworks is somewhat subtle .
one could simplistically state that when we see a process that is forbidden by a certain set of laws of kinematics then those laws are falsified .
however , in principle this statement is correct only when we have full knowledge of the process , including a full determination of the momenta of the incoming particles . in the case of the absorption of multi - tev gamma rays from blazars
it is natural to assume that this absorption be due to interactions with ir photons , but we are not in a position to exclude that the absorption be due to higher - energy background photons .
therefore , we should contemplate the possibility that the pkv0 kinematics be implemented within a framework in which the description of dynamics is such to introduce a large - enough modification of cross sections to allow absorption of multi - tev blazar gamma rays by background photons of energy higher than 200 mev .
as mentioned above repeatedly , i advocate a balanced perspective on these sorts of issues , which should not extend all the way to assuming wild conspiracies centered on very large changes in cross sections , even when testing a pure - kinematics framework .
but , as long as a consensus on criteria for such a balanced approach is not established , it is difficult to attribute a quantitative confidence level to experimental bounds on a pure - kinematics framework through mere observation of some absorption of multi - tev blazar gamma rays .
the concerns are not applicable to test theories that do provide a description of dynamics , such as the ftv0 test theory , with its effective - field - theory setup . however , for the ftv0 test theory one must take into account the fact that the modification of the dispersion relation carries the opposite sign to the two polarizations of the photon and might have an helicity dependence in the case of electrons and positrons .
so , in the case of the ftv0 test theory , as long as observations only provide evidence of some absorption of tev gamma rays ( without much to say about the level of agreement with the amount of absorption expected in the classical - spacetime picture ) , and are , therefore , consistent with the hypothesis that only one of the polarizations of the photon is being absorbed , only rather weak limits can be established . for the derivation of threshold anomalies combining a modification of the law of energy - momentum conservation with the modification of the dispersion relation can lead to results that are very different from the case in which only the modifications of the dispersion relations are assumed .
this is a feature already stressed in the case of the analysis of photon stability . in order to establish it also for threshold anomalies
let me consider an example of the dsr - inspired modified law of energy - momentum conservation .
i assume that the modification of the law of energy - momentum conservation for the case of ee takes the form 26\documentclass[12pt]{minimal }
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\begin{document}$$e + \epsilon - { \eta \over { { e_p}}}\vec p \cdot \vec p \simeq { e _ + } + { e _ - } - { \eta \over { { e_p}}}{\vec p _ + } \cdot { \vec p _ -},$$\end{document }
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\begin{document}$$\vec p + \vec p + { \eta \over { { e_p}}}e\vec p + { \eta \over { { e_p}}}\epsilon \vec p \simeq { \vec p _ + } + { \vec p _ - } + { \eta \over { { e_p}}}{e _ + } { \vec p _ - } + { \eta \over { { e_p}}}{e _ -}{\vec p _ + } , $ $ \end{document } where i denote with \documentclass[12pt]{minimal }
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\begin{document}$\vec p$\end{document } the momentum of the photon of energy e and i denote with \documentclass[12pt]{minimal }
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\begin{document}$\vec p$\end{document } the momentum of the photon of energy . using these ( 26 ) , ( 27 ) and the
n = 1 dispersion relation , one obtains ( keeping only terms that are meaningful for me eth ep ) 28\documentclass[12pt]{minimal }
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\begin{document}$${e_{th } } \simeq { { m_e^2 } \over \epsilon},$$\end{document } i.e. , one ends up with the same result as in the special - relativistic case .
this shows very emphatically that modifications of the law of energy - momentum conservation can compensate for the effects on threshold derivation produced by modified dispersion relations
. the cancellation should typically be only partial , but in cases in which the two modifications are matched exactly
the fact that a dsr - inspired modification of the law of conservation of energy - momentum produces this exact matching admits a tentative interpretation that the interested reader can find in refs . [ 58 , 63 ] . in the preceding section 3.4
, i discussed the implications of possible planck - scale effects for the process ee , but this is not the only process in which planck - scale effects can be important . in particular , there has been strong interest [ 327 , 38 , 73 , 463 , 305 , 59 , 115 , 35 , 431 ] in the analysis of the photopion production
process , p p. as already stressed in section 1.5 , interest in the photopion - production process originates from its role in our description of the high - energy portion of the cosmic - ray spectrum .
gzk cutoff feature of that spectrum is linked directly to the value of the minimum ( threshold ) energy required for cosmic - ray protons to produce pions in collisions with cmbr photons [ 267 , 558 ] ( see , e.g. , refs . [ 240 , 348 ] ) . the argument suggesting that planck - scale modifications of the dispersion relation may significantly affect the estimate of this threshold energy is completely analogous to that discussed in preceding section 3.4 for ee .
however , the derivation is somewhat more tedious : in the case of ee the calculations are simplified by the fact that both outgoing particles have mass me and both incoming particles are massless , whereas for the threshold conditions for the photopion - production process one needs to handle the kinematics for a head - on collision between a soft photon of energy and a high - energy particle of mass mp and momentum \documentclass[12pt]{minimal }
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\begin{document}${\vec k_p}$\end{document } producing two ( outgoing ) particles with masses mp , m and momenta \documentclass[12pt]{minimal }
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\begin{document}$\vec k_p{\prime},{\vec k_\pi}$\end{document}. the threshold can then be conveniently characterized as a relationship describing the minimum value , denoted by kp , th , that the spatial momentum of the incoming particle of mass mp must have in order for the process to be allowed for given value of the photon energy : 29\documentclass[12pt]{minimal }
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\begin{document}$${k_{p , th } } \simeq { { { { ( { m_p } + { m_\pi})}^2 } - m_p^2 } \over { 4\epsilon } } + \eta { { k_{p , th}^{2 + n } } \over { 4\epsilon e_p^n}}\left({{{m_p^{1 +
n } + m_\pi ^{1 + n } } \over { { { ( { m_p } + { m_\pi})}^{1 + n } } } } - 1 } \right)$$\end{document } ( dropping terms that are further suppressed by the smallness of \documentclass[12pt]{minimal }
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\begin{document}$e_p^{- 1}$\end{document } and/or the smallness of or mp , ) . notice that whereas in discussing the pair - production threshold relevant for observations of tev gamma rays i had immediately specialized ( 13 ) to the case n = 1 , here i am contemplating values of n that are even greater than 1 .
one could also admit n > 1 for the pair - production threshold analysis , but it would be a mere academic exercise , since it is easy to verify that in that case planck - scale sensitivity is within reach only for n not significantly greater than 1 . instead ( as i briefly stressed already in section 1.5 ) the role of the photopion - production threshold in cosmic - ray analysis is such that even for the case of values of n as high as 2 ( i.e. , even for the case of effects suppressed quadratically by the planck scale ) planck - scale sensitivity is not unrealistic . in fact , using for mp and m the values of the masses of the proton and the pion and for a typical cmbr - photon energy one finds that for negative of order 1 ( effects introduced at the planck scale ) the shift of the threshold codified in ( 29 ) is gigantic for n = 1 and still observably large [ 38 , 73 ] for n = 2 . for negative
the planck - scale correction shifts the photopion - production threshold to higher values with respect to the standard classical - spacetime prediction , which estimates the photopion - production threshold scale to be of about 5 10 ev . assuming19 that the observed cosmic rays of highest energies are protons , when the spectrum reaches the photopion - production threshold one should first encounter a pileup of cosmic rays with energies just in the neighborhood of the threshold scale , and then above the threshold the spectrum should be severely depleted .
the pileup results from the fact that protons with above - threshold energy tend to lose energy through photopion production and slow down until their energy is comparable to the threshold energy .
the depletion above the threshold is the counterpart of this pileup ( protons emitted at the source with energy above the threshold tend to reach us , if they come to us from far enough away , with energy comparable to the threshold energy ) .
the availability in this cosmic - ray context of planck - scale sensitivities for values of n all the way up to n = 2 was fully established by the year 2000 [ 38 , 73 ] .
the debate then quickly focused on establishing what exactly the observations were telling us about the photopion - production threshold .
the fact that the agasa cosmic - ray observatory was reporting evidence of a behavior of the spectrum that was of the type expected in this planck - scale picture generated a lot of interest . however , more recent cosmic - ray observations , most notably the ones reported by the pierre auger observatory [ 448 , 8 ] , appear to show no evidence of unexpected behavior .
there is even some evidence ( see , however , the updated ref . )
suggesting that to the highest - energy observed cosmic rays , one can associate some relatively nearby sources , and that all this is occurring at scales that could fit within the standard picture of the photopion - production threshold , without planck scale effects . these results reported by the pierre auger observatory are already somewhat beyond the preliminary status , and we should soon have at our disposal very robust cosmic - ray data , which should be easily converted into actual experimental bounds on the parameters of planck - scale test theories . among the key ingredients that are still missing i should assign priority to the mentioned issue of correlation of cosmic - ray observations with the large scale distribution of matter in the nearby universe and the issue of the composition of cosmic rays ( protons versus heavy nuclei ) .
the rapidly - evolving [ 5 , 11 ] picture of correlations with matter in the nearby universe focuses on cosmic - ray events with energy 5.7 10 ev , while the growing evidence of a significant heavy - nuclei component at high energies is limited so far at energies of 4 10 ev . and this state of affairs , as notably stressed in ref . , limits our insight on several issues relevant for the understanding of the origin of cosmic rays and the related issues for tests of lorentz symmetry , since it leaves open several options for the nature and distance of the sources above and below 5 10 ev . postponing more definite claims on the situation on the experimental side ,
let me stress , however , that there is indeed a lot at stake in these studies for the hypothesis of quantum - spacetime - induced planck - scale departures from lorentz symmetry .
even for pure - kinematics test theories this type of data analysis is rather strongly relevant .
for example , the kinematics of the pkv0 test theory forbids ( for negative of order 1 and n 2 ) photopion production when the incoming proton energy is in the neighborhood of 5 10 ev and the incoming photon has typical cmbr energies . for reasons already stressed ( for other contexts ) , in order to establish a robust experimental limit on pure - kinematics scenarios using the role of the photopion - production threshold in the cosmic - ray spectrum , it would be necessary to also exclude that other background photons ( not necessarily cmbr photons ) be responsible for the observed cutoff.20 it appears likely that such a level of understanding of the cosmic - ray spectrum will be achieved in the not - so - distant future .
for the ftv0 test theory , since it goes beyond pure kinematics , one is not subject to similar concerns .
however , the fact that it admits the possibility of different effects for the two helicities of the incoming proton , complicates and renders less sharp this type of cosmic - ray analyses .
it does lead to intriguing hypotheses : for example , exploiting the possibility of helicity dependence of the planck scale effect for protons , one can rather naturally end up with a scenario that predicts a pileup / cutoff structure somewhat similar to the one of the standard classical - spacetime analysis , but softer , as a result of the fact that only roughly half of the protons would be allowed to lose energy by photopion production .
for the photopion - production threshold one finds exactly the same mechanism , which i discussed in some detail for the pair - production threshold , of possible compensation between the effects produced by modified dispersion relations and the effects produced by modified laws of energy - momentum conservation .
so , the analysis of frameworks where both the dispersion relation and the energy - momentum conservation law are modified , as typical in dsr scenarios , should take into account that added element of complexity .
also relevant to the analysis of cosmic - ray observations is another aspect of the possible implications of quantum - spacetime - motivated planck - scale departures from lorentz symmetry : the possibility of a suppression of pion decay at ultrahigh energies .
while in some cases departures from lorentz symmetry allow the decay of otherwise stable particles ( as in the case of ee , discussed above , for appropriate choice of values of parameters ) , it is indeed also possible for departures from lorentz symmetry to either introduce a threshold value of the energy of the particle , above which a certain decay channel for that particle is totally forbidden [ 179 , 81 ] , or introduce some sort of suppression of the decay probability that increases with energy and becomes particularly effective above a certain threshold value of the energy of the decaying particle [ 59 , 115 , 244 ]
. this may be relevant [ 81 , 59 ] for the description of the air showers produced by cosmic rays , whose structure depends rather sensitively on certain decay probabilities , particularly the one for the decay
. the possibility of suppression at ultrahigh energies of the decay has been considered from the quantum - gravity - phenomenology perspective primarily adopting pkv0-type frameworks [ 59 , 115 ] . using the kinematics of the pkv0 test theory one
easily arrives at the following relationship between the opening angle of the directions of the momenta of the outgoing photons , the energy of the pion ( e ) and the energies ( e and e = e e ) of the outgoing photons : 30\documentclass[12pt]{minimal }
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\begin{document}$$\cos ( \phi ) = { { 2e{e{\prime } } - m_\pi ^2 + 3\eta { e_\pi}e{e{\prime}}/{e_p } } \over { 2e{e{\prime } } + \eta { e_\pi}e{e{\prime}}/{e_p}}}.$$\end{document } this relation shows that , for positive , at high energies the phase space available to the decay is anomalously reduced : for a given value of e certain values of e that would normally be accessible to the decay are no longer accessible ( they would require cos > 1 ) .
this anomaly starts to be noticeable at pion energies of order \documentclass[12pt]{minimal }
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\begin{document}${(m_\pi ^2/{l_p})^{1/3 } } \sim { 10^{15}}$\end{document } ev , but only very gradually ( at first only a small portion of the available phase space is excluded ) .
this is rather intriguing since there is a report of experimental evidence of anomalies for the structure of the air showers produced by cosmic rays , particularly their longitudinal development . and it has been argued in ref . that these unexpected features of the longitudinal development of air showers could be explained in terms of a severely reduced decay probability for pions of energies of 10 ev and higher .
this is still to be considered a very preliminary observation , not only because of the need to acquire data of better quality on the development of air showers , but also because of the role that our limited control of nonperturbative qcd has in setting our expectations for what air - shower development should look like without new physics .
it is becoming rather urgent to reassess this issue in light of recent data on cosmic rays and cosmic - ray shower development .
such an exercise has not been made for a few years now , and for the mentioned auger data , with the associated debate on the composition of cosmic rays , the analysis of shower development ( and , therefore , of the hypothesis of some suppression of pion decay ) is acquiring increasing significance [ 509 , 6 , 36 , 549 ] .
as for the other cases in which i discuss effects of modifications of the dispersion relation for kinematics of particle reactions , for this pion - decay argument scenarios hosting both a modified dispersion relation and modifications of the law of conservation of energy - momentum , as typical in dsr scenarios , can lead to a compensation of the correction terms .
the quantum - spacetime - phenomenology analyses i have reviewed so far have played a particularly significant role in the rapid growth of the field of quantum - spacetime phenomenology over the last decade .
this is particularly true for the analyses of the pair - production threshold for gamma rays and of the photopion - production threshold for cosmic rays , in which the data relevant for the planck - scale effect under study can be perceived as providing some encouragement for new physics .
one can legitimately argue [ 463 , 302 ] that the observed level of absorption of tev gamma rays is low enough to justify speculations about new physics
( even though , as mentioned , there are conventional - physics descriptions of the relevant data ) .
the opportunities for planck scale physics to play a role in the neighborhood of the gzk scale of the cosmic - ray spectrum are becoming slimmer , as stressed in section 3.5 , but still it has been an important sign of maturity for quantum - spacetime phenomenology to play its part in the debate that for a while was generated by the preliminary and tentative indications of an anomaly around the gzk cutoff .
it is interesting how the hypothesis of a pion - stability threshold , another planck - scale - motivated hypothesis , also plays a role in the assessment of the present status of studies of ultra - high - energy cosmic rays .
i am giving disproportionate attention to the particle - interaction analyses described in sections 3.4 , 3.5 , 3.6 because they are the most discussed and clearest evidence in support of the claim that quantum - spacetime planck - scale phenomenology does have the ability to discover its target new physics , so much so that some ( however tentative ) experimental puzzles have been considered and are being considered from the quantum - spacetime perspective .
but it is of important to also consider the implications of quantum - spacetime - inspired planck - scale departures from lorentz symmetry , and particularly planck - scale modifications of the dispersion relation , for all possible particle - physics processes . and a very valuable type of particle - physics processes to be considered are the ones that are forbidden in a standard special - relativistic setup but could be allowed in the presence of planck - scale departures from lorentz symmetry .
anomalous processes , and in the analysis of some of them one does find opportunities for planck - scale sensitivity , as already discussed for the case of the process ee in section 3.3 . for a comprehensive list ( and more detailed discussion ) of other analyses of anomalous processes , which are relevant for the whole subject of the study of possible departures from lorentz symmetry ( within or without quantum spacetime ) , readers can rely on refs .
i will just briefly mention one more significant example of an anomalous process that is relevant from a quantum - spacetime - phenomenology perspective : the vacuum cerenkov process , e e , which in certain scenarios [ 395 , 308 , 41 ] with broken lorentz symmetry is allowed above a threshold value of electron energy .
this is analyzed in close analogy with the discussion in section 3.3 for the process ee ( which is another example of anomalous particle interaction ) .
since we have no evidence at present of vacuum - cerenkov processes , the relevant analyses are of the type that sets limits on the parameters of some test theories . clearly , this observational evidence against vacuum - cerenkov processes is also relevant for pure - kinematics test theories , but in ways that it is difficult to quantify , because of the dependence on the strength of the interactions ( an aspect of dynamics ) .
so , here too , one should contemplate the implications of these findings from the perspective of the remarks offered in section 3.3.1 concerning the plausibility ( or lack thereof ) of conspiracies between modifications of kinematics and modifications of the strengths of interaction . within the ftv0 test theory one
can rigorously analyze the vacuum - cerenkov process , and there actually , if one arranges for opposite - sign dispersion - relation correction terms for the two helicities of the electron , one can in principle have helicity - changing e e at any energy ( no threshold ) , but estimates performed [ 395 , 308 ] within the ftv0 test theory show that the rate is extremely small at low energies . above the threshold for helicity - preserving e e the ftv0 rates are substantial , and this in particular would allow an analysis with planck - scale sensitivity that relies on observations of 50-tev gamma rays from the crab nebula .
the argument is based on several assumptions ( but all apparently robust ) and its effectiveness is somewhat limited by the combination of parameters allowed by ftv0 setup and by the fact that for these 50-tev gamma rays we observe from the crab nebula we can only reasonably guess a part of the properties of the emitting particles . according to the most commonly adopted model the relevant gamma rays are emitted by the crab nebula as a result of inverse compton processes , and from this one infers [ 395 , 308 , 40 ] that for electrons of energies up to 50 tev the vacuum cerenkov process is still ineffective , which in turn allows one to exclude certain corresponding regions of the ftv0 parameter space .
analyses of thresholds for particle - physics processes , discussed in the previous sections 3.4 , 3.5 , 3.6 , and 3.7 , played a particularly important role in the development of quantum - spacetime phenomenology over the last decade , because the relevant studies were already at planck - scale sensivity . in june 2008 , with the launch of the fermi ( /glast ) space telescope [ 436 , 201 , 440 , 3 , 4 , 413 ] we gained access to planck - scale effects also for in - vacuo dispersion as well .
these studies deserve particular interest because they have broad applicability to quantum - spacetime test theories of the fate of lorentz / poincar symmetry at the planck scale . in the previous sections 3.4 ,
3.5 , 3.6 , and 3.7 , i stressed how the analyses of thresholds for particle - physics processes provided information that is rather strongly model dependent , and dependent on the specific choices of parameters within a given model .
the type of insight gained through in - vacuo - dispersion studies is instead significantly more robust .
a wavelength dependence of the speed of photons is obtained [ 66 , 497 ] from a modified dispersion relation , if one assumes the velocity to still be described by = de / dp . in particular , from the dispersion relation of the pkv0 test theory one obtains ( at intermediate energies ,
m < e ep ) a velocity law of the form 31\documentclass[12pt]{minimal }
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\begin{document}$$v \simeq 1 - { { { m^2 } } \over { 2{e^2 } } } + \eta { { n + 1 } \over 2}{{{e^n } } \over { e_p^n}}.$$\end{document } arguments and semi - heuristic derivations in support of this type of speed law for massless particles have been reported21 both in the spacetime - noncommutativity literature ( see , e.g. , refs . [ 70 , 191 ] ) and in the lqg literature ( see , e.g. , refs . [ 247 , 33 , 523 ] ) . on the basis of the speed law ( 31 )
one would find that two simultaneously - emitted photons should reach the detector at different times if they carry different energy . and this time - of - arrival - difference effect can be significant [ 66 , 491 , 459 , 539 , 232 ] in the analysis of short - duration gamma - ray bursts that reach us from cosmological distances . for a gamma - ray burst , it is not uncommon22 that the time traveled before reaching our earth detectors be of order t 10 s. microbursts within a burst can have very short duration , as short as 10 s , and this should suggest that the photons that compose such a microburst are all emitted at the same time , up to an uncertainty of 10 s. some of the photons in these bursts have energies that extend even above 10 gev , and for two photons with energy difference of order e 10 gev a e / ep speed difference over a time of travel of 10 s would lead to a difference in times of arrival of order \documentclass[12pt]{minimal }
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\begin{document}$\delta t\sim\eta t\delta { e \over { { e_p}}}\sim\eta \cdot 1$\end{document } which is not negligible23 with respect to the typical variability time scales one expects for the astrophysics of gamma - ray bursts .
indeed , it is rather clear [ 74 , 264 ] that the studies of gamma - ray bursts conducted by the fermi telescope provide us access to testing planck - scale effects , in the linear - modification ( n = 1 ) scenario .
( 31 ) since for redshifts of 1 and higher , spacetime curvature / expansion is a very tangible effect . and
most results in quantum - spacetime research hinting at modifications of the dispersion relation , and possible associated energy / momentum dependence of the speed of massless particles , were derived working essentially in the flat - spacetime / minkowski limit : it is obvious that analogous effects would also be present when spacetime expansion is switched on , but it is not obvious how formulas should be generalized to that case . in particular , the formula ( 31 ) is essentially unique for ultrarelativistic particles in the flat - spacetime limit : we are only interested in leading - order formulas and the difference between ( e / ep ) and \documentclass[12pt]{minimal }
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\begin{document}${p^2}{e^{n - 2}}/e_p^n$\end{document } is negligible for ultrarelativistic particles ( with p m ) .
how spacetime expansion renders these considerations more subtle is visible already in the case of de sitter expansion .
adopting conformal coordinates in de sitter spacetime , with metric ds = dt a(t ) dx ( and a(t ) = e ) we have for ultrarelativistic particles ( with p m ) the velocity formula 32\documentclass[12pt]{minimal }
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\begin{document}$$v \simeq { a^{- 1}}(t ) - { { { m^2 } } \over { 2{p^2}}}a(t),$$\end{document } so already in the undeformed case the coordinate velocity ( from which physical time delays will be derived ) depends not only on momentum but also on the scale factor a(t ) .
it is not obvious how one should describe leading - order planck - scale corrections to this , going as some power of momentum .
it is natural to make the ansatz 33\documentclass[12pt]{minimal }
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\begin{document}$$v \simeq { a^{- 1}}(t ) - { { { m^2 } } \over { 2{p^2}}}a(t ) + \eta { { n + 1 } \over 2}{{{p^n } } \over { e_p^n}}{a^k}(t),$$\end{document } with the integer k being at this point one more phenomenological parameter to be determined experimentally .
arguments on value of the integer k would be most natural were reported in refs . [ 228 , 474 , 303 , 229 ] , ultimately leading to a consensus [ 303 , 229 ] converging on describing k = n as the most natural choice .
i shall not dwell much on this : let me just confirm that i would also give priority to the case k = n , but doing this in such a way as not to by - pass the obvious fact that the value of k would have to be determined experimentally ( and nature might well have chosen a value for k different from n ) . assuming that indeed k = n one would expect for simultaneously emitted massless particles in a universe parametrized by the cosmological parameters m , , h0 ( evaluated today ) a momentum - dependent difference in times of arrival at a telescope given by 34\documentclass[12pt]{minimal }
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\begin{document}$$\delta t \simeq \eta { { n + 1 } \over { 2{h_0}}}{{{p^n } } \over { e_p^n}}\int\nolimits_0^z { d{z{\prime}}{{{{(1 + { z{\prime}})}^n } } \over { \sqrt { { \omega _ m}{{(1 + { z{\prime}})}^3 } + { \omega _ \lambda}}}},}$$\end{document } where p is the momentum of the particle when detected at the telescope .
actually , planck - scale sensitivity to in - vacuo disperson can also be provided by observations of tev flares from certain active galactic nuclei , at redshifts much smaller than 1 ( cases in which spacetime expansion is not really tangible ) . in particular , studies of tev flares from mk 501 and pks 2155 - 304 performed by the magic and hess
observatories have established [ 218 , 29 , 226 , 18 , 10 , 129 ] bounds on the scale of dispersion , for the linear - effects ( n = 1 ) scenario , at about 1/10 of the planck scale .
but the present best constraints on quantum - spacetime - induced in - vacuo dispersion are derived from observations of gamma - ray bursts reported by the fermi telescope .
there are , so far , four fermi - detected gamma - ray bursts that are particularly significant for the hypothesis of in - vacuo dispersion : grb 090816c , grb 090510 , grb 090902b , grb 090926a .
the data for each one of these bursts has the strength of constraining the scale of in - vacuo dispersion , for the linear - effects ( n = 1 ) scenario , at better than 1/10 of the planck scale .
in particular , grb 090510 was a truly phenomenal short burst and the structure of its observation allows us to conservatively establish that the scale of in - vacuo dispersion , for the linear - effects ( n = 1 ) scenario , is higher than 1.2 times the planck scale .
the simplest way to do such analyses is to take one high - energy photon observed from the burst and take as reference its delay t with respect to the burst trigger : if one could exclude conspiracies such that the specific photon was emitted before the trigger ( we can not really exclude it , but we would consider that as very unlikely , at least with present knowledge ) evidently t would have to be bigger than any delay caused by the quantum - spacetime effects .
this , in turn , allows us , for the case of grb 090510 , to establish the limit at 1.2 times the planck scale . and , interestingly , even more sophisticated techniques of analysis , using not a single photon but the whole structure of the high - energy observation of grb 090510 , also encourage the adoption of a limit at 1.2 times the planck scale .
it has also been noticed that if one takes at face value the presence of high - energy photon bunches observed for grb 090510 , as evidence that these photons were emitted nearly simultaneously at the source and they are being detected nearly simultaneously , then the bound inferred could be even two orders of magnitude above the planck scale .
i feel that at least the limit at 1.2 times the planck scale is reasonably safe / conservative .
but it is obvious that here we would feel more comfortable with a wider collection of gamma - ray bursts usable for our analyses .
this would allow us to balance , using high statistics , the challenges for such studies of in - vacuo dispersion that ( as for other types of studies based on observations in astrophysics discussed earlier ) originate from the fact that we only have tentative models of the source of the signal .
in particular , the engine mechanisms causing the bursts of gamma rays also introduce correlations at the source between the energy of the emitted photons and the time of their emission .
this was in part expected by some astrophysicists , and fermi data allows one to infer it at levels even beyond expectations [ 3 , 4 , 527 , 376 , 187 , 256 ] . on a single observation of gamma - ray - burst events
such at - the - source correlations are , in principle , indistinguishable from the effect we expect from in - vacuo dispersion , which indeed is a correlation between times of arrival and energies of the photons . and another challenge i should mention originates from the necessity of understanding at least partly the precursors of a gamma - ray burst , another feature that was already expected and to some extent known , but recently came to be known as a more significant effect than expected [ 4 , 530 ] .
so , we will reach a satisfactory comfort level with our bounds on in - vacuo dispersion only with
high statistics , a relatively large collection of gamma - ray bursts usable for our analyses .
high statistics always helps , but in this case it will also provide a qualitatively new handle for the data analysis : a relatively large collection of high - energy gamma - ray bursts , inevitably distributed over different values of redshift , would help our analyses also because comparison of bursts at different redshifts can be exploited to achieve results that are essentially free from uncertainties originating from our lack of knowledge of the sources .
this is due to the fact that the structure of in - vacuo dispersion is such that the effect should grow in predictable manner with redshift , whereas we can exclude that the exact same dependence on redshift ( if any ) could characterize the correlations at the source between the energy of the emitted photons and the time of their emission . in this respect
we might be experiencing a case of tremendous bad luck : as mentioned we really still only have four gamma - ray bursts to work with , grb 090816c , grb 090510 , grb 090902b , grb 090926a , but on the basis of how fermi observations had been going for the first 13 months of operation we were led to hope that by this time ( end of 2012 ) , after 50 months of operation of fermi , we might have had as many as 15 such bursts and perhaps 4 or 5 bursts of outstanding interest for in - vacuo dispersion , comparable to grb 090510 .
these four bursts we keep using from the fermi data set were observed during the first 13 months of operation ( in particular grb 090510 was observed during the 10th month of operation ) and we got from fermi nothing else of any use over the last 37 months . if our luck turns around we should be able to claim for quantum - spacetime phenomenology a first small but tangible success : ruling out at least the specific hypothesis of planck - scale in - vacuo dispersion , at least specifically for the case of linear - effects ( n= 1 ) .
this being said about the opportunities and challenges facing the phenomenology of in - vacuo dispersion , let me , in closing this section , offer a few additional remarks on the broader picture . from a quantum - spacetime - phenomenology perspective it is noteworthy that , while in the analyses discussed in the previous sections 3.4 , 3.5 , 3.6 , and 3.7 , the amplifier of the planck - scale effect was provided by a large boost , in this in - vacuo - dispersion case the amplification is due primarily to the long propagation times , which essentially render the analysis sensitive to the accumulation of very many minute planck - scale effects . for propagation times that are realistic in controlled earth experiments , in which one perhaps could manage to study the propagation of photons of tev energies , over distances of 10 m ,
the in - vacuo dispersion would still induce , even for n = 1 , only time delays of order 10 s. in - vacuo - dispersion analyses of gamma - ray bursts are also extremely popular within the quantum - spacetime - phenomenology community because of the very limited number of assumptions on which they rely .
one comes very close to having a direct test of a planck - scale modification of the dispersion relation . in comparing the pkv0 and the ftv0 test theories
, one could exploit the fact that whereas for the pkv0 test theory the planck - scale - induced time - of - arrival difference would affect a multi - photon microburst by producing a difference in the average arrival time of the signal in different energy channels , within the ftv0 test theory , for an ideally unpolarized signal , one would expect a dependence of the time - spread of a microburst that grows with energy , but no effect for the average arrival time in different energy channels .
this originates from the polarization dependence imposed by the structure of the ftv0 test theory : for low - energy channels the whole effect will be small , but in the highest - energy channels , the fact that the two polarizations travel at different speed will manifest itself as spreading in time of the signal , without any net average - time - of - arrival effect for an ideally unpolarized signal . since there is evidence that at least some gamma - ray bursts are somewhat far from being ideally unpolarized ( see evidence of polarization reported , e.g. , in refs . [ 359 , 556 , 528 ] ) , one could also exploit a powerful correlation : within the ftv0 test theory one expects to find some bursts with sizeable energy - dependent average - time - of - arrival differences between energy channels ( for bursts with some predominant polarization ) , and some bursts ( the ones with no net polarization ) with much less average - time - of - ar11rival differences between energy channels but a sizeable difference in time spreading in the different channels .
polarization - sensitive observations of gamma - ray bursts would allow one to look directly for the polarization dependence predicted by the ftv0 test theory .
clearly , these in - vacuo dispersion studies using gamma rays in the gev - tev range provide us at present with the cleanest opportunity to look for planck - scale modifications of the dispersion relation .
unfortunately , while they do provide us comfortably with planck - scale sensitivity to linear ( n = 1 ) modifications of the dispersion relation , they are unable to probe significantly the case of quadratic ( n = 2 ) modifications . and , while , as stressed , these studies apply to a wide range of quantum - spacetime scenarios with modified dispersion relations , mostly as a result of their insensitivity to the whole issue of description of dynamical aspects of a quantum - spacetime theory , one should be aware of the fact that it might be inappropriate to characterize these studies as tests that must necessarily apply to all quantum - spacetime pictures with modified dispersion relations .
most notably , the assumption of obtaining the velocity law from the dispersion relation through the formula = de / dp may or may not be valid in a given quantum - spacetime picture .
validity of the formula = de / dp essentially requires that the theory is still
, at least in the sense that the velocity along the x axis is obtained from the commutator with a hamiltonian ( x [ x , h ] ) , and that the heisenberg commutator preserves its standard form ( [ x , px ] so that x /px ) .
especially this second point is rather significant since heuristic arguments of the type also used to motivate modified dispersion relations suggest [ 22 , 122 , 323 , 415 , 243 , 408 ] that the heisenberg commutator might have to be modified in the quantum - spacetime realm .
observations of gamma rays in the gev - tev range could provide us with a very sharp picture of planck - scale - induced dispersion , if it happens to be a linear ( n = 1 ) effect , but , as stressed above , one would need observations of similar quality for photons of significantly higher energies in order to gain access to scenarios with quadratic ( n = 2 ) effects of planck - scale - induced dispersion .
the prospect of observing photons with energies up to 10 ev at ground observatories [ 471 , 74 ] is very exciting , and should be pursued very forcefully , but it represents an opportunity whose viability still remains to be fully established . and in any case we expect photons of such high energies to be absorbed rather efficiently by background soft photons ( e.g. , cmbr photons ) so that we could not observe them from very distant sources .
one possibility that could be considered is the one of 1987a - type supernovae ; however such supernovae are typically seen at distances not greater than some 10 light years . and
the fact that neutrinos from 1987a - type supernovae can be definitely observed up to energies of at least tens of tev s is not enough to compensate for the smallness of the distances ( as compared to typical gamma - ray - burst distances ) . as a result , using 1987a - type supernovae one might have serious difficulties even to achieve planck - scale sensitivity for linear ( n = 1 ) modifications of the dispersion relation , and going beyond linear order clearly is not possible . the most advanced plans for in - vacuo - dispersion studies with sensitivity up to quadratic ( n = 2 ) planck - scale modifications of the dispersion relation actually exploit [ 230 , 168 , 61 , 301 ] ( also see , for a similar argument within a somewhat different framework , ref . ) once again the extraordinary properties of gamma - ray bursters , but their neutrino emissions rather than their production of photons . indeed , according to current models [ 411 , 543 ] , gamma - ray bursters should also emit a substantial amount of high - energy neutrinos .
some neutrino observatories should soon observe neutrinos with energies between 10 and 10 ev , and one could either ( as it appears to be more feasible ) compare the times of arrival of these neutrinos emitted by gamma - ray bursters to the corresponding times of arrival of low - energy photons or compare the times of arrivals of different - energy neutrinos ( which , however , might require larger statistics than it seems natural to expect ) . in assessing the significance of these foreseeable studies of neutrino propagation within different test theories , one should again take into account issues revolving around the possibility of anomalous reactions
. in particular , in spite of the weakness of their interactions with other particles , within an effective - field - theory setup neutrinos can be affected by cherenkov - like processes at levels that are experimentally significant , though not if the scale of modification of the dispersion relation is as high as the planck scale .
the recent overall analysis of modified dispersion for neutrinos in quantum field theory given in ref .
shows that for the linear ( n = 1 ) case we are presently able to establish constraints at levels of about 10 times the planck scale ( and even further from the planck scale for the quadratic case , n = 2 ) .
it is well established [ 179 , 141 , 225 , 83 , 421 , 169 ] that flavor - dependent modifications to the energy - momentum dispersion relations for neutrinos may lead to neutrino oscillations even if neutrinos are massless .
this point is not directly relevant for the three test theories i have chosen to use as frameworks of reference for this review .
the pkv0 test theory adopts universality of the modification of the dispersion relation , and also the ftv0 test theory describes flavor - independent effects ( its effects are nonuniversal only in relation to polarization / helicity ) .
still , i should mention this possibility both because clearly flavor - dependent effects may well attract gradually more interest from quantum - spacetime phenomenologists ( some valuable analyses have already been produced ; see , e.g. , refs .
[ 395 , 308 ] and references therein ) , and because even for researchers focusing on flavor - independent effects , it is important to be familiar with constraints that may be set on flavor - dependent scenarios ( those constraints , in a certain sense , provide motivation for the adoption of flavor independence ) .
most studies of neutrino oscillations induced by violations of lorentz symmetry were actually not motivated by quantum - gravity / quantum - spacetime research ( they were part of the general lorentz - symmetry - test research area ) and assumed that the flavor - dependent violations would take the form of a flavor - dependent speed - of - light scale , which essentially corresponds to the adoption of a dispersion relation of the type ( 13 ) , but with n = 0 , and flavor - dependent values of . a few studies have considered the case24
n = 1 with flavor - dependent , which is instead mainly of interest from a quantum - spacetime perspective,25 and found [ 141 , 225 , 421 ] that for n = 1 from eq .
( 13 ) one naturally ends up with oscillations lengths that depend quadratically on the inverse of the energies of the particles ( l e ) , whereas in the case n = 0 ( flavor - dependent speed - of - light scale ) such a strong dependence on the inverse of the energies is not possible . in principle , this opens an opportunity for the discovery of manifestations of the flavor - dependent n = 1 case through studies of neutrino oscillations [ 141 , 421 ] ; however , at present there is no evidence of a role for these effects in neutrino oscillations and , therefore , the relevant data analyses produce bounds [ 141 , 421 ] on flavor dependence of the dispersion relation . in a part of the next section ( 4.6 ) , i shall comment again on neutrino oscillations , but in relation to the possible role of quantum - spacetime - induced decoherence ( rather than lorentz - symmetry violations ) .
another opportunity to set limits on test theories with planck - scale modified dispersion relations is provided by the study of the implications of modified dispersion relations for synchrotron radiation [ 306 , 62 , 309 , 378 , 231 , 420 , 39 ] . an important point for these analyses [ 306 , 309 , 378 ]
is the observation that in the conventional ( lorentz - invariant ) description of synchrotron radiation one can estimate the characteristic energy ec of the radiation through a semi - heuristic derivation leading to the formula 35\documentclass[12pt]{minimal }
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\begin{document}$${e_c } \simeq { 1 \over { r \cdot \delta \cdot [ { v_\gamma } - { v_e}]}},$$\end{document } where e is the speed of the electron , is the speed of the photon , is the angle of outgoing radiation , and r is the radius of curvature of the trajectory of the electron . assuming that the only planck - scale modification in this formula should come from the velocity law ( described using = de / dp in terms of the modified dispersion relation )
, one finds that in some instances the characteristic energy of synchrotron radiation may be significantly modified by the presence of planck - scale modifications of the dispersion relation .
this originates from the fact that , for example , according to ( 31 ) , for n = 1 and < 0 , an electron can not have a speed that exceeds the value \documentclass[12pt]{minimal }
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\begin{document}$v_e^{\max } \simeq 1 - ( 3/2){(\vert \eta \vert { m_e}/{e_p})^{2/3}}$\end{document } , whereas in sr e can take values arbitrarily close to 1 . as an opportunity to test such a modification of the value of the synchrotron - radiation characteristic energy one can attempt to use data on photons emitted by the crab nebula .
this must be done with caution since the observational information on synchrotron radiation being emitted by the crab nebula is rather indirect : some of the photons we observe from the crab nebula are attributed to sychrotron processes , but only on the basis of a ( rather successful ) model , and the value of the relevant magnetic fields is also not directly measured .
but the level of planck - scale sensitivity that could be within the reach of this type of analysis is truly impressive : assuming that indeed the observational situation has been properly interpreted , and relying on the mentioned assumption that the only modification to be taken into account is the one of the velocity law , one could [ 306 , 378 ] set limits on the parameter of the pkv0 test theory that go several orders of magnitude beyond || 1 , for negative and n = 1 , and even for quadratic ( n = 2 ) planck - scale modifications the analysis would fall just short of reaching planck - scale sensitivity ( only a few orders of magnitude away from || 1 sensitivity for n = 2 ) .
however , the assumptions of this type of analysis , particularly the assumption that nothing changes but the velocity law , can not even be investigated within pure - kinematics test theories , such as the pkv0 test theory .
synchrotron radiation is due to the acceleration of the relevant charged particles and , therefore , implicit in the derivation of the formula ( 35 ) is a subtle role for dynamics . from a quantum - field - theory perspective
, the process of synchrotron - radiation emission can be described in terms of compton scattering of the electrons with the virtual photons of the magnetic field , and its analysis is , therefore , rather sensitive even to details of the description of dynamics in a given theory .
indeed , essentially this synchrotron - radiation phenomenology has focused on the ftv0 test theory and its generalizations , so that one can rely on the familiar formalism of quantum field theory .
making reasonably prudent assumptions on the correct model of the source one can establish valuable ( sub - planckian ! ) experimental bounds on the parameters of the ftv0 test theory . as i stressed already a few times earlier in this review , the ftv0 test theory , as a result of a rigidity of the adopted effective - field - theory framework , necessarily predicts birefringence , by assigning different speeds to different photon polarizations .
birefringence is a pure - kinematics effect , so it can also be included in straightforward generalizations of the pkv0 test theory , if one assigns a different dispersion relation to different photon polarizations and then assumes that the speed is obtained from the dispersion relation via the standard = de / dp relation .
i have already discussed some ways in which birefringence may affect other tests of dispersion - inducing ( energy - dependent ) modifications of the dispersion relation , as in the example of searches of time - of - arrival / energy correlations for observations of gamma - ray bursts .
the applications i already discussed use the fact that for large enough travel times birefringence essentially splits a group of simultaneously - emitted photons with roughly the same energy and without characteristic polarization into two temporally and spatially separated groups of photons , with different circular polarization ( one group being delayed with respect to the other as a result of the polarization - dependent speed of propagation ) . another feature that can be exploited
is the fact that even for travel times that are somewhat shorter than the ones achieving a separation into two groups of photons , the same type of birefringence can already effectively erase [ 261 , 262 ] any linear polarization that might have been there to begin with , when the signal was emitted .
this observation can be used in turn to argue that for given magnitude of the birefringence effects and given values of the distance from the source it should be impossible to observe linearly polarized light , since the polarization should have been erased along the way . using observations of polarized light from distant radio galaxies [ 395 , 261 , 262 , 158 , 342 , 495 ]
one can comfortably achieve planck - scale sensitivity ( for n = 1 linear modifications of the dispersion relation ) to birefringence effects following this strategy .
in particular , the analysis reported in ref . [ 261 , 262 ] leads to a limit of || < 2 10 on the parameter of the ftv0 test theory . and more recent studies of this type allowed even more stringent bounds to be established(see refs .
interestingly , even for this strategy based on the effect of removal of linear polarization , gamma - ray bursts could in principle provide formidable opportunities .
and there was a report of observation of polarized mev gamma rays in the prompt emission of the gamma - ray burst grb 021206 , which would have allowed very powerful bounds on energy - dependent birefringence to be established
. however , ref . has been challenged ( see , e.g. , ref . [ 481 , 124 ] ) .
still , experimental studies of polarization for gamma - ray bursts continue to be a very active area of research ( see , e.g. , refs .
[ 359 , 556 , 528 ] ) , and it is likely that this will gradually become the main avenue for constraining quantum - spacetime - induced birefringence . over this past decade
there has been growing awareness of the fact that data analyses with good sensitivity to effects introduced genuinely at the planck scale are not impossible , as once thought .
it is at this point well known , even outside the quantum - gravity / quantum - spacetime community , that planck - scale sensitivity is achieved in certain ( however rare ) astrophysics studies
. it would be very very valuable if we could establish the availability of analogous tests in controlled laboratory setups , but this is evidently more difficult , and opportunities are rare and of limited reach .
still , i feel it is important to keep this goal as a top priority , so in this section i mention a couple of illustrative examples , which can at least show that laboratory tests are possible . considering these objectives it makes sense to focus again on quantum - spacetime - motivated planck - scale modifications of the dispersion relation , so that the estimates of sensitivity levels achievable in a controlled laboratory setup
one possibility is to use laser - light interferometry to look for in - vacuo - dispersion effects . in ref .
two examples of interferometric setups were discussed in some detail , with the common feature of making use of a frequency doubler , so that part of the beam would be for a portion of its journey through the interferometer at double the reference frequency of the laser beam feeding the interferometer .
the setups must be such that the interference pattern is sensitive to the fact that , as a result of in - vacuo dispersion , there is a nonlinear relation between the phase advancement of a beam at frequency and a beam at frequency 2. for my purposes here it suffices to discuss briefly one such interferometric setup .
specifically , let me give a brief description of a setup in which the frequency ( or energy ) is the parameter characterizing the splitting of the photon state , so the splitting is in energy space ( rather than the more familiar splitting in configuration space , in which two parts of the beam actually follow geometrically different paths ) .
second harmonic generator so that if a wave reaches the frequency doubler with frequency then , after passing through the frequency doubler , the outgoing wave in general consists of two components , one at frequency and the other at frequency 2. if two such frequency doublers are placed along the path of the beam at the end , one has a beam with several components , two of which have frequency 2 : the transmission of the component that left the first frequency doubler as a 2 wave , and another component that is the result of frequency doubling of that part of the beam that went through the first frequency doubler without change in the frequency .
therefore , the final 2 beam represents an interferometer in energy space . as shown in detail in ref .
the intensity of this 2 beam takes a form of type 36\documentclass[12pt]{minimal }
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\begin{document}$${i^{(2\omega ) } } = { i_a } + { i_b}\cos \ , ( \alpha + ( { k{\prime } } - 2k)l),$$\end{document } where l is the distance between the two frequency doublers , ia and ib are l - independent ( they depend on the amplitude of the original wave and the effectiveness of the frequency doublers ) , the phase is also l - independent and is obtained combining several contributions to the phase ( both a contribution from the propagation of the wave and a contribution introduced by the frequency doublers ) , k is the wave number corresponding to the frequency through the dispersion relation , and k is the wave number corresponding to the frequency 2 through the dispersion relation ( since the dispersion relation is planck - scale modified one expects departures from the special - relativistic result k = 2k ) . since the intensity only depends on the distance l between the frequency doublers through the planck - scale correction to the phase , ( k 2k ) l , by exploiting a setup that allows one to vary l , one should rather easily disentangle the planck - scale effect .
and one finds that the accuracy achievable with modern interferometers is sufficient to achieve planck - scale sensitivity ( e.g. , sensitivity to || 1 in the pkv0 test theory with n = 1 ) .
it is rather optimistic to assume that the accuracy achieved in standard interferometers would also be achievable with this peculiar setup , particularly since it would require the optics aspects of the setup ( such as lenses ) to work with that high accuracy simultaneously with two beams of different wavelength .
moreover , it would require some very smart techniques to vary the distance between the frequency doublers without interfering with the effectiveness of the optics aspects of the setup .
so , in practice we would not presently be capable of using such setups to set planck - scale - sensitive limits on in - vacuo dispersion , but the fact that the residual obstructions are of rather mundane technological nature encourages us to think that in the not - so - distant future tests of planck - scale in - vacuo dispersion in controlled laboratory experiments will be possible .
besides in - vacuo dispersion , another aspect of the physics of planck - scale modified dispersion relations that we should soon be able to test in controlled laboratory experiments is the one concerning anomalous thresholds , at least in the case of the ee process that i already considered from an astrophysics perspective in section 3.4 .
it is not so far from our present technical capabilities to set up collisions between 10 tev photons and 0.03 ev photons , thereby reproducing essentially the situation of the analysis of blazars that i discussed in section 3.4 . and notice that with respect to the analysis of observations of blazars , such controlled laboratory studies would give much more powerful indications .
in particular , for the analysis of observations of blazars discussed in section 3.4 , a key limitation on our ability to translate the data into experimental bounds on parameters of a pure - kinematics framework was due to the fact that ( even assuming we are indeed seeing absorption of multitev photons ) the astrophysics context does not allow us to firmly establish whether the absorption is indeed due to the ir component of the intergalactic background radiation ( as expected ) or instead is due to a higher - energy component of the background ( in which case the absorption would instead be compatible with some corresponding planck - scale pictures ) .
if collisions between 10 tev and 0.03 ev photons in the lab do produce pairs , since we would in that case have total control of the properties of the particles in the in state of the process , we would then have firm pure - kinematics bounds on the parameters of certain corresponding planck scale test theories ( such as the pkv0 test theory ) .
these laboratory studies of planck - scale - modified dispersion relations could also be adapted to the ftv0 test theory , by simply introducing some handles on the polarization of the photons that are placed under observation ( also see refs . [ 254 , 255 ] ) , with sensitivity not far from planck - scale sentivity in controlled laboratory experiments .
readers for which this review is the first introduction to the world of quantum - spacetime phenomenology might be surprised that this long section , with an ambitious title announcing related tests of lorentz symmetry , was so heavily biased toward probing the form of the energy - momentum dispersion relation .
other aspects of the implications of lorentz ( and poincar ) symmetry did intervene , such as the law of energy - momentum conservation and its deformations ( and the form of the interaction vertices and their deformations ) , and are in part probed through the data analyses reviewed , but the feature that clearly is at center stage is the structure of the dispersion relation .
the reason for this is rather simple : researchers that recognize themselves as quantum - spacetime phenomenologists will consider a certain data analysis as part of the field if that analysis concerns an effect that can be robustly linked to quantum properties of spacetime ( rather than , for example , some classical - field background ) and if the analysis exposes the availability of planck - scale sensitivities , in the sense i described above . at least according to the results obtained so far ,
the aspect of lorentz / poincar symmetry that is most robustly challenged by the idea of a quantum spacetime is the form of the dispersion relation , and this is also an aspect of lorentz / poincar symmetry for which the last decade of work on this phenomenology robustly exposed opportunities for planck - scale sensitivities . for the type of modifications of the dispersion relation that i considered in this section we have at present rather robust evidence of their applicability in certain noncommutative pictures of spacetime ,
where the noncommutativity is very clearly introduced at the planck scale . and several independent ( although all semi - heuristic ) arguments suggest that the same general type of modified dispersion relations should apply to the minkowski limit of lqg , a framework where a certain type of discretization of spacetime structure is introduced genuinely at the planck scale
. unfortunately , these two frameworks are so complex that one does not manage to analyze spacetime symmetries much beyond building a case ( and not a waterproof case ) for modified dispersion relations
. a broader range of lorentz - symmetry tests could be valuable for quantum - spacetime research , but without the support of a derivation it is very hard to argue that the relevant effects are being probed with sensitivities that are significant from a quantum - spacetime / planck - scale perspective .
think , for example , of a framework , such as the one adopted in ref .
, in which the form of the dispersion relation is modified , but not in an energy - dependent way : one still has dispersion relations of the type \documentclass[12pt]{minimal }
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\begin{document}${e^2 } = c_\ # ^2{p^2 } + m_\ # ^2$\end{document } , but with a different value of the velocity scale c # for different particles .
this is not necessarily a picture beyond the realm of possibilities one would consider from a quantum - spacetime perspective , but there is no known quantum - spacetime picture that has provided direct support for it . and
it is also essentially impossible to estimate what accuracy must be achieved in measurements of cproton celectron , in order to reach planck - scale sensitivity .
some authors qualify as planckian magnitude of this type of effect , the case in which the dimensionless parameter has value on the order of the ratio of the mass of the particles involved in the process versus the planck scale ( as in cproton celectron ( mproton melectron)/ep ) but this arbitrary criterion clearly does not amount to establishing genuine planck - scale sensitivity , at least as long as we do not have a derivation starting with spacetime quantization at the planck scale that actually finds such magnitudes of these sorts of effects . still , it is true that the general structure of the quantum - gravity problem and the structure of some of the quantum spacetimes that are being considered for the minkowski limit of quantum gravity might host a rather wide range of departures from classical lorentz symmetry . correspondingly , a broad range of lorentz - symmetry tests could be considered of potential interest .
i shall not review here this broader lorentz - symmetry - tests literature , since it is not specific to quantum - spacetime research ( these are tests that could be done and in large part were done even before the development of research on lorentz symmetries from within the quantum - spacetime literature ) and it has already been reviewed very effectively in ref . .
let me just stress that for these broad searches of departures from lorentz symmetry one needs test theories with many parameters .
formalisms that are well suited to a systematic program of such searches are already at an advanced stage of development [ 180 , 181 , 340 , 343 , 123 , 356 , 357 ] ( also see ref . ) , and in particular the standard - model - extension framework [ 180 , 181 , 340 , 343 ] has reached a high level of adoption of preference for theorists and experimentalists as the language in which to characterize the results of systematic multi - parameter lorentz - symmetry - test data analyses .
the standard model extension was originally conceived as a generalization of the standard model of particle - physics interactions restricted to power - counting - renormalizable correction terms , and as such it was of limited interest for the bulk of the quantum - spacetime / quantum - gravity community : since quantum gravity is not a ( perturbatively ) renormalizable theory , many quantum - spacetime researchers would be unimpressed with lorentz - symmetry tests restricted to powercounting - renormalizable correction terms .
however , over these last few years most theorists involved in studies of the standard model extension have started to add correction terms that are not powercounting renormalizable.26 a good entry point for the literature on limits on the parameters of the standard model extension is provided by refs .
[ 395 , 123 , 346 ] . from a quantum - gravity - phenomenology perspective
it is useful to contemplate the differences between alternative strategies for setting up a completely general
in particular , it has been stressed ( see , e.g. , refs . [ 356 , 357 ] ) that violations of lorentz symmetry can be introduced directly at the level of the dynamical equations , without assuming ( as done in the standard model extension ) the availability of a lagrangian generating the dynamical equations .
this is more general than the lagrangian approach : for example , the generalized maxwell equation discussed in ref .
[ 356 , 357 ] predicts effects that go beyond the standard model extension . and charge conservation , which automatically comes from the lagrangian approach , can be violated in models generalizing the field equations [ 356 , 357 ] .
the comparison of the standard - model - extension approach and of the approach based on generalizations introduced directly at the level of the dynamical equations illustrates how different philosophies lead to different strategies for setting up a completely general systematic investigation of possible departures from lorentz symmetry . by removing the assumption of the availability of a lagrangian ,
still , no general approach can be absolutely general : in principle one could always consider removing an extra layer of assumptions . as the topics
i have reviewed in this section illustrate , from a quantum - spacetime - phenomenology perspective it is not necessarily appropriate to seek the most general parametrizations .
on the contrary , we would like to single out some particularly promising candidate quantum - spacetime effects ( as in the case of modified dispersion relations ) and focus our efforts accordingly .
tests of lorentz symmetry , and particularly of the form of the dispersion relation , probably make up something on the order of half of the whole quantum - spacetime - phenomenology literature .
the other half is spread over a few other , evidently less developed , research lines .
nonetheless , for some of these other research lines the literature has reached some nonnegligible maturity , and even those that are at preliminary stages of development could be precious potential opportunities for quantum - spacetime research . evidently , the most challenging part of this review work concerns these other components of quantum - spacetime - phenomenology research , since it is harder to summarize and organize intelligibly the results and scopes of research programs that are still in early stages of development .
but it is also the part of this review that could be most valuable , since there is already some work [ 52 , 308 , 485 , 294 ] attempting to summarize and review , although more concisely than done here in section 3 , the results obtained by the quantum - spacetime phenomenology of planck - scale modified dispersion relations . in reporting on the work done in these other quantum - spacetime - phenomenology reseach lines
, i shall use as one of the guiding concepts the one of assessing whether a given research program concerns uv quantum - spacetime effects or ir quantum - spacetime effects .
the typical situation for a uv quantum - spacetime effect is that it takes the form of correction terms that grow with the energy of the particles , and whose significance is therefore increasingly high as the energy of the particles increases . for
any given standard - physics ( no - quantum - spacetime ) prediction a0 , this will take the general form 37\documentclass[12pt]{minimal }
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\begin{document}$${a_0 } \rightarrow { a_0}\left({1 + { \eta _ \#}{{{e^n } } \over { e_p^n } } } \right)\quad \;({\rm{with}}\;{\rm{context}}/{\rm{theory}}\;{\rm{specific}}\;{\rm{numerical}}\;{\rm{factor}}{\eta _ \#})$$\end{document } this is the type of quantum - spacetime effect that one traditionally one to be inevitably produced by any form of spacetime quantization , and is the focus of this section .
the possibility of ir quantum - spacetime effects , effects that are due to planck - scale spacetime quantization but are significant in some deep - ir regime , came to the attention of the community only rather recently , emerging mainly from work on ir / uv mixing in quantum spacetime , and i shall focus on it in the next section 5 .
in this review , as natural for phenomenology , i am primarily looking at quantum - spacetime effects from the perspective of the type of pre - quantum - spacetime laws that they affect ( so we have departures from classical spacetime symmetries , violations of the quantum - mechanical coherence , and so on ) . and
our experimental opportunities are such that the main focus is on how spacetime quantization could affect particle propagation ( and , for a restricted sample of phenomenological opportunities , interactions among particles ) . for this section on other uv quantum - spacetime effects a significant role ( particularly noticeable in sections 4.2 , 4.3 , and 4.5 will be played by the idea that quantum - spacetime effects may introduce an additional irreducible contribution to the fuzziness of the worldlines of particles .
this should be contrasted to the content of section 3 , which focuses mainly on phenomenological proposals involving mechanisms for systematic departures from the currently - adopted laws of propagation of ( and interaction among ) particles . in most cases
such systematic effects amount to departures from the predictions of lorentz symmetry ( such as a systematic dependence on the velocity of a massless particle on its energy , which would produce a systematic difference between the arrival times of high - energy and low - energy photons that are simultaneously emitted ) .
if it ends up being the case that the correct quantum - spacetime picture does not provide us any such systematic effects , then we will be left with non - systematic effects , i.e. , fuzziness . in looking for such effects
we can be guided by the intuition that spacetime quantization might act as an environment inducing apparently random fluctuations in certain observables .
for example , by distance fuzziness one does not describe an effect that would systematically gives rise to larger ( or smaller ) distance - measurement results , but rather one describes a sort of new uncertainty principle for distance measurements .
this distinction between systematic and nonsystematic effects can easily be characterized for any given observable \documentclass[12pt]{minimal }
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\begin{document}$\hat x$\end{document } for which the pre - quantum - spacetime theoretical prediction can be described in terms of a prediction
x and , possibly , a fundamental ( ordinarily quantum mechanical ) uncertainty x . the effects of spacetime quantization in general could lead to a new prediction x and a new uncertainty x. one would attribute to quantum spacetime the effects 38\documentclass[12pt]{minimal }
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\begin{document}$${(\delta x)_{{\rm{qg } } } } \equiv { x{\prime } } - x$$\end{document } and 39\documentclass[12pt]{minimal }
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\begin{document}$${(\delta x)_{{\rm{qg } } } } \equiv \delta { x{\prime } } - \delta x.$$\end{document } one can speak of purely systematic quantum - spacetime effects when ( x)qg 0 and ( x)qg = 0 , while the opposite case , ( x)qg = 0 and ( x)qg > 0 , can be qualified as purely non - systematic .
it is likely that for many observables both types of quantum - spacetime effect may be present simultaneously , but it is natural that at least the first stages of development of a quantum - spacetime phenomenology on an observable \documentclass[12pt]{minimal }
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\begin{document}$\hat x$\end{document } be focused on one or the other special case ( ( x)qg = 0 or ( x)qg = 0 ) . clearly , the discusions of effects given in section 3 were all with ( x)qg = 0 , while for most of the proposals discussed in this section the main focus will be on the effects characterized by ( x)qg = 0 .
the scenarios for spacetime fuzziness that are most studied from a quantum - spacetime perspective are intuitively linked to the notion of spacetime foam , championed by wheeler and studied extensively in the quantum - gravity literature , more or less directly , for several decades ( see , e.g. , refs .
[ 547 , 203 , 178 , 281 , 150 , 250 , 553 ] ) . from a modern perspective one is attempting to characterize the physics of matter particles as effectively occurring in an environment of short - distance quantum - gravitational degrees of freedom .
and one may expect that for propagating particles with wavelength much larger than the planck length , when it may be appropriate to integrate out these short - distance quantum - gravitational degrees of freedom , the main residual effect of short - distance gravity would indeed be an additional contribution to the fuzziness of worldlines .
while in full - fledged quantum - spacetime theories , such a lqg , such analyses are still beyond our reach , one can find partial encouragement for this intuition in recent progress on the understanding of quantum gravity in 3d ( 2 + 1-dimensional ) spacetime .
[ 75 , 237 , 412 , 152 , 259 , 238 , 313 , 441 ] establish that for 3d quantum gravity ( exploiting the much lower complexity than for the 4d case ) we are able to perform the task needed for studies of spacetime foam : we can actually integrate out gravity , reabsorbing its effects into novel properties for a gravity - free propagation of particles . and foaminess is formalized in the fact that this procedure integrating out gravity leaves us with a theory of free particles in a noncommutative spacetime , refs .
[ 75 , 237 , 259 , 238 ] , specifically a spacetime with lie - algebra noncommutativity
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\begin{document}$$[{x_\alpha},{x_\beta } ] = i\kappa _ { \alpha \beta}^\gamma { x_\gamma}$$\end{document } ( in particular the choice of \documentclass[12pt]{minimal }
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\begin{document}$\kappa _ { \alpha \beta}^\gamma$\end{document } as the levi - civita tensor is the one suggested by the direct derivation given in ref . ) .
in other words , upon integrating out the gravitational degrees of freedom , the quantum dynamics of matter fields coupled to 3d gravity is effectively described by matter fields in a noncommutative spacetime , a fuzzy / foamy spacetime . while the only direct / deductive derivations of such results are for 3d quantum gravity , it is natural to take that as a starting point for the study of real 4d quantum gravity , whereas analogous results are still unavailable . and a sizable literature has been devoted to the search of possible experimental manifestations of spacetime foam .
i start with spacetime - foam test theories , whose structure renders them well suited for interferometric tests . the first challenge for a phenomenology investigating the possibility of spacetime foam originates in the fact that wheeler s spacetime foam intuition , while carrying strong conceptual appeal , can not on its own be used for phenomenology , since it is not characterized in terms of observable properties .
the phenomenology then is based on test theories inspired by the spacetime - foam intuition .
a physical / operative definition of at least one aspect of spacetime foam is given in refs .
[ 51 , 54 , 53 , 433 ] and is well suited for a phenomenology based on interferometry27 .
according to this definition the fuzziness / foaminess of a spacetime is established[51 , 54 , 53 , 433 ] on the basis of an analysis of strain noise28 in interferometers set up in that spacetime . in achieving their remarkable accuracy , modern interferometers must deal with several classical - physics strain noise sources ( e.g. , thermal and seismic effects induce fluctuations in the relative positions of the test masses ) . and importantly strain noise sources associated with effects due to ordinary quantum mechanics are also significant for modern interferometers ( the combined minimization of photon shot noise and radiation pressure noise leads to a noise source that originates from ordinary quantum mechanics )
. one can give an operative definition [ 51 , 53 ] of fuzzy / foamy spacetime in terms of a corresponding additional source of strain noise . a theory in which the concept of distance is fundamentally fuzzy in this operative sense
would be such that the read - out of an interferometer would still be noisy ( because of quantum - spacetime effects ) even in the idealized limit in which all classical - physics and ordinary - quantum - mechanics noise sources are completely eliminated / subtracted . before even facing the task of developing test theories for spacetime foaminess in interferometry
it is best to first check whether there is any chance of using realistic interferometric setups to uncover effects as small as expected if introduced at the planck scale .
a first encouraging indication comes from identifying the presence of a huge amplifier in modern interferometers : a well - known quality of these modern interferometers is their ability to detect gravity waves of amplitude 10 m by carefully monitoring distances of order 10 m , and this should provide opportunities for an amplifier that is of order 10 .
this also means that our modern interferometers have outstanding control over noise sources , which is ideal for the task at hand , involving scenarios for how quantum - spacetime effects may contribute an additional source of noise in such interferometers .
clearly , the noise we could conceivably see emerging from spacetime quantization should be modeled in terms of some random vibrations .
for example there is in general no spendable notion of amplitude of random vibrations . the most fruitful way to characterize them ,
also for the purposes of comparing their intensity to other non - random sources of vibration that might affect the same system , is by using the power spectral density .
let me introduce some notation , which will prove useful when i move on to discuss crude models of quantum - spacetime - induced noise .
for this i simple - mindedly consider the readout of an interferometer as h(t ) , given by the position x(t ) of a mirror divided by a reference length scale l ( h(t ) = x(t)/l ) , and adjust the reference frame so that on average x(t ) vanishes , x = 0 .
given some rules for fluctuations of this readout one can indeed be interested in its power spectral density ( ) , in principle computable via 41\documentclass[12pt]{minimal }
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\begin{document}$$\sigma ( \omega ) = \int\nolimits_{- \infty}^\infty { d\tau \;{\mu _ { [ h(t)h(t + \tau)]}}\;{e^{- 2\pi i\omega \tau}},}$$\end{document } where [h(t)h(t+ ) ] depends only on and is the value expected on average for h(t)h(t + ) in the presence of the vibration / fluctuation process of interest in the analysis .
having characterized the noise source in terms of its power spectral density we can then easily compute some primary characteristics , such as its root mean square deviation h , which for cases of zero - mean noise , such as the one i am considering , will be given by the expectation of h. this can be expressed in terms of the power spectral density as follows 42\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _
h^2 = { \mu _ { { h^2 } } } = \int\nolimits_{- \infty}^\infty { d\omega \sigma ( \omega)}.$$\end{document } in experimental practice , for a frequency - band limited signal ( fmax ) and a finite observation time ( tobs ) , this relation will take the shape 43\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _
\simeq \int\nolimits_{1/{t_{{\rm{obs}}}}}^{{f_{\max } } } { d\omega \sigma ( \omega)}.$$\end{document } in modern interferometers such as ligo [ 9 , 1 ] and virgo [ 157 , 12 ] the power spectral density of the noise is controlled at a level of ( ) 10 hz at observation frequencies of about 100 hz , and in turn this ( also considering the length of the arms of these modern interferometers ) implies [ 9 , 1 , 157 , 12 ] that for a gravity wave with 100 hz frequency the detection threshold is indeed around 10 m. the challenge here for quantum - spacetime phenomenologists is to characterize the relevant quantum - spacetime effects in terms of a novel contribution ( ) to the power spectral density of the noise . if at some point experimentalists manage to bring the total noise ( ) , for some range of observation frequencies , below the level predicted by a certain quantum - spacetime test theory , then that test theory will be ruled out .
is there any hope for a reasonable quantum - spacetime test theory to predict noise at a level comparable to the ones that are within the reach of modern interferometry ?
well , this is the type of question that one can only properly address in the context of models , but it may be valuable to first use dimensional analysis , assuming the most optimistic behavior of the quantum - spacetime effects , and check if the relevant order of magnitude is at all providing any encouragement for the painful ( if at all doable ) analysis of the relevant issues in quantum - spacetime models . to get what is likely to be the most optimistic ( and certainly the simplest , but not necessarily the most realistic ) planck - scale estimate of the effect ,
let us assume that quantum - spacetime noise is white noise , ( ) = 0 ( frequency independent ) , so that it is fully specified by a single dimensionful number setting the level of this white noise . and
since carries units of hz one easily notices a tempting simple naive estimate in terms of the planck length and the speed - of - light29 scale : 0 lp / c , which , since lp / c 10 hz , encouragingly happens to be just at the mentioned level of sensitivity of ligo - virgo - type interferometers .
this provides some initial encouragement for a phenomenology based on interferometric noise , though only within the limitations of a very crude and naive estimate .
my next task is going beyond assuming for simplicity that the quantum - spacetime noise be white and beyond adopting a naive dimensional - analysis estimate of what could constitute a planck - scale level of such a noise .
the ultimate objective here would be to analyze an interferometer in the framework of a compelling quantum - spacetime theory , but this is beyond our capabilities at present .
however , we can start things off by identifying some semi - heuristic pictures ( the basis for a test theory ) with effects introduced genuinely at the planck scale that turn out to produce strain noise at the level accessible with modern interferometers .
having in mind this objective , let us take as a starting point for a first naive picture of spacetime fuzziness the popular arguments suggesting that the planck scale should also set some absolute limitation on the measurability of distances . and
let us ( optimistically ) assume that this translates to the fact that any experiment in which a distance l plays a key role ( meaning that one is either measuring l itself or the observable quantity under study depends strongly on l ) is affected by a mean square deviation \documentclass[12pt]{minimal }
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\begin{document}$\sigma _ l^2$\end{document}. it turns out to be useful [ 51 , 53 ] to consider this \documentclass[12pt]{minimal }
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\begin{document}$\sigma _ l^2$\end{document } as a possible stepping stone toward the strain - noise power - spectrum estimate . and a particularly striking picture arises by assuming that the distances l between the test masses of an interferometer be affected by planck - length fluctuations of random - walk type occurring at a rate of one per planck time ( 10 s ) , so that [ 51 , 53 ] 44\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _ l^2 \simeq { l_p}t \simeq { l_p}l\quad [ { \rm{random}}\;{\rm{walk}}\;{\rm{case}}],$$\end{document } where t is the time scale over which the experiment monitors the distance l , assuming the use of ultrarelativistic particles ( t l ) .
it is noteworthy that \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \simeq { l_p}t$\end{document } can be motivated independently ( without having in mind the idea of such effective spacetime fluctuations ) on the basis of some aspects of the quantum - gravity problem . and the study of certain quantum - spacetime pictures that have been of interest to the quantum - gravity community , such as the -minkowski noncommutative spacetime of eq .
( 4 ) , provide some support for this random - walk picture : from [ xj , t ] = ilpxj one could guess roughly a law of the form \documentclass[12pt]{minimal }
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\begin{document}$\sigma _ x^2 \sim \delta x\delta t \sim { l_p}x$\end{document}. some arguments inspired by the holography paradigm for quantum gravity [ 433 , 430 , 170 ] suggest even weaker effects , characterized by 45\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _
l^2 \simeq l_p^{4/3}{l^{2/3}}\quad [ { \rm{holography } } - { \rm{inspired}}\;{\rm{case}}].$$\end{document } interestingly , this ansatz \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \simeq l_p^{4/3}{l^{2/3}}$\end{document } had been independently proposed in the quantum - gravity literature on the basis of a perspective on the quantum - gravity problem ( see ref . [ 432 , 319 , 209 ] ) , which originally in no way involved spacetime fuzziness . probably the most conservative ( and pessimistic ) expectation for spacetime fuzziness one can find in the quantum - spacetime literature is the one omitting any opportunity for amplification by the involvement of a long observation time ( see , e.g. , parts of refs . [ 249 , 293 ] ) 46\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _
l^2 \simeq l_p^2\quad [ { \rm{weakest}}\;{\rm{case}}].$$\end{document } the random - walk case is the most typical textbook study case for random noise .
its power spectral density goes like , so one should have 47\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _ { \rm{l}}^{[{\rm{qg}};{\rm{rw } } ] } \simeq { { { l_p } } \over { { \omega ^2}}},$$\end{document } which gives 48\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _ h^2\sim { { \sigma _ l^2 } \over { { l^2 } } } \simeq { 1 \over { { l^2}}}\int\nolimits_{1/{t_{{\rm{obs}}}}}^{{f_{\max } } } { d\omega { { { l_p } } \over { { \omega ^2 } } } \simeq { { { l_p}{t_{{\rm{obs } } } } } \over { { l^2}}}}$$\end{document } ( so , for l tobs one indeed finds \documentclass[12pt]{minimal }
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\begin{document}$\sigma _ l^2 \sim { l_p}l$\end{document } ) .
( 45 ) a power spectral density going as / , so one should have 49\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _ l^{[{\rm{qg}};{\rm{holo } } ] } \simeq { { l_p^{4/3 } } \over { { \omega ^{5/3}}}},$$\end{document } which indeed gives \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \simeq l_p^{4/3}t_{{\rm{obs}}}^{2/3 } \simeq l_p^{4/3}{l^{2/3}}$\end{document}. and , finally , for the \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
( 46 ) , a rough but valuable approximate description of the power spectral density goes like , so one should have 50\documentclass[12pt]{minimal }
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\begin{document}$$\sigma _ l^{[{\rm{qg}};{\rm{weak } } ] } \simeq { { l_p^2 } \over \omega},$$\end{document } which indeed gives \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \simeq l_p^2$\end{document}. it is tempting to obtain from these estimates of the quantum - spacetime - induced distance uncertainty an estimate for the quantum - spacetime - induced strain noise , by simply dividing by the square of the length of the arms of the interferometer , \documentclass[12pt]{minimal }
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\begin{document}${\sigma ^{[{\rm{qg } } ] } } = \sigma _ l^{[{\rm{qg}}]}/{l^2}$\end{document}. this would be the way to proceed if we were converting distance noise into strain noise , but really here we are obtaining a rough estimate of strain noise from an estimate of distance uncertainty , and i shall therefore proceed in some sense sub judice ( see in particular my comments below concerning the large number of photons collectively used for producing the accurate measurements of a modern interferometer ) . assuming that indeed \documentclass[12pt]{minimal }
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\begin{document}${\sigma ^{[{\rm{qg } } ] } }
= \sigma _ l^{[{\rm{qg}}]}/{l^2}$\end{document } , and taking as reference value an observation frequency of 100 hz , one would get for the three cases i discussed the following estimates of strain noise at 100 hz , for arm lengths of a few kilometers : 51\documentclass[12pt]{minimal }
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\begin{document}$${\sigma ^{[{\rm{qg}};{\rm{weak}}]}}\sim { 10^{- 78}}{\rm{h}}{{\rm{z}}^{- 1}},\quad { \sigma ^{[{\rm{qg}};{\rm{holo}}]}}\sim { 10^{- 52}}{\rm{h}}{{\rm{z}}^{- 1}},\quad { \sigma ^{[{\rm{qg}};{\rm{rw}}]}}\sim { 10^{-
38}}{\rm{h}}{{\rm{z}}^{- 1}}.$$\end{document } these estimates are rather naive but it is nonetheless interesting to compare them to the levels of noise control achieved experimentally .
as mentioned , around 100 hz both ligo and virgo achieve noise control at the level of strain noise of 10 hz , so estimates like and would be safe , but the estimate must be excluded : the estimate would assign more noise of quantum - spacetime origin than the total noise that ligo and virgo managed to control ( which would include the hypothetical quantum - spacetime - induced noise ) . in spite of the crudeness of the derivations i discussed so far ,
this does give a rather worthy input for those who fancy the random - walk picture , as i shall stress in section 4.2.4 .
before i get to that issue , let me stress that there is a possible source of confusion for terminology ( and content ) in the literature . in the quantum - gravity literature
there has been some discussion for several years of holography - inspired noise in the sense of my eq .
more recently , a different mechanism for quantum - spacetime - induced noise , also labeled as holography inspired , was proposed in a series of papers by hogan [ 287 , 286 , 288 ] .
i do not think it is particularly important at the present time to establish which ( if either ) of the two proposals is more directly inspired by holography .
[ 433 , 430 , 170 ] is a rather mature proposal , centered on eq .
( 49 ) and meaningful at least as a quantum - spacetime test theory in the sense i just described .
instead it is probably fair to describe the alternative version of holographic noise more recently proposed in ref .
[ 287 , 286 , 288 ] as a young proposal still looking for some maturity : it does not amount to any however - wild variant of the description of interferometric noise i summarized here , and actually it is claimed to be immune not only to the sort of interferometric noise i discussed in this section but also to all other effects that have been typically associated with spacetime quantization in the literature . it would be a quantum - spacetime picture whose effects can only be detected in an experiment that coherently compares transverse positions over an extended spacetime volume to extremely high precision , and with high time resolution or bandwidth .
evidently some work is still needed on the conceptual aspects ( as a rigorous theory of spacetime quantization ) and on the phenomenological aspects ( as a computably predictive and broadly applicable test theory of spacetime quantization ) of this proposal .
only time will tell if this present lack of maturity is due to intrinsic unsurmountable limitations of the proposal or is simply a result of the fact that the proposal was made only rather recently ( so there was not much time for this maturity to be reached ) .
i should note that at some point , in spite of its lack of maturity , this proposal started to attract some pronounced interest in relation to reports by the geo600 interferometer of unexplained excess noise : it had been claimed that hogan s version of holographic noise could match exactly the anomaly that was being reported by geo600 .
however , it appears that experimenters at geo600 have recently achieved a better understanding of their noise sources , and no unexplained contribution is at this point reported ( this is at least implicit in ref . and
( at some point known among specialists as the mystery noise ) is over . at the present time the state of the art of phenomenologically - spendable descriptions of planck - scale - induced strain noise does not go much beyond the simple - minded estimates i just described in relation to eqs . ( 47 ) , ( 49 ) , and ( 50 ) . but some lessons were nonetheless learned , as usually happens even with the most humble phenomenology . and these lessons do point toward some directions worthy of exploration in the future . in this section
i highlight some of these lessons and possible future developments . among the few steps of simple derivation , which i described in section 4.2.3 ,
evidently much scrutiny should be particularly directed toward the assumption \documentclass[12pt]{minimal }
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\begin{document}${\sigma ^{[{\rm{qg } } ] } } = \sigma _ l^{[{\rm{qg}}]}/{l^2}$\end{document } : i motivated some candidate forms for \documentclass[12pt]{minimal }
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\begin{document}$\sigma _ l^{[{\rm{qg}}]}$\end{document } using essentially the sort of arguments that usually allow us to establish uncertainty principles for single particles , such as the ones taking as a starting point a postulated noncommutativity of single - particle coordinates ; however , the strain noise relevant for our interferometers is not at all a single - particle feature .
let me use the example of random - walk fuzziness for illustrating how the relationship between single - particle quantum - spacetime arguments and interferometric strain noise could be more subtle than assumed in \documentclass[12pt]{minimal }
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\begin{document}${\sigma ^{[{\rm{qg } } ] } } = \sigma _
i specialize the more general idea of random - walk quantum - spacetime fuzziness in the sense of assuming that each single photon in an interferometer experiences a random - walk path : a random planck - length fluctuation per planck - time would affect the path of each photon of the beam .
this would imply in particular that as a photon goes from one mirror of the interferometer to the other , over a distance l , it reaches its destination with an uncertainty corresponding to \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \sim { l_p}t \sim { l_p}l$\end{document}. however , the interferometer ( and this is key to its outstanding sensitivity ) does not depend on determining the position of each single photon in the beam : on the contrary the key observable is the average position of the photons composing the beam , which may be viewed as the putative position of the mirror
if l is now viewed as the distance between positions of mirrors defined in this way , rather than as the distance of propagation of an individual photon , then evidently the result is an estimate \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \sim { l_p}t \sim { l_p}l/{n_\gamma}$\end{document } , where n is the ( very large ! ) number of photons contributing to each such determination of the position of the mirror .
while the noise levels produced by a random - walk ansatz assuming \documentclass[12pt]{minimal }
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\begin{document}${\sigma ^{[{\rm{qg } } ] } } = \sigma _ l^{[{\rm{qg}}]}/{l^2}$\end{document } are , as stressed in section 4.2.3 , already ruled out by the achievements of ligo and virgo , this single - particle picture of a random - walk scenario , which evidently leads us to assume 52\documentclass[12pt]{minimal }
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\begin{document}$${\sigma ^{[{\rm{qg } } ] } } = { { \sigma _ l^{[{\rm{qg } } ] } } \over { { n_\gamma}{l^2}}}$$\end{document } is still safely compatible with the noise results of ligo and virgo , thanks to the large n suppression .
a similar n suppression could naturally be expected for the holographic noise scenario of eq .
as discussed in the previous section 4.2.3 , that holographic noise scenario would be safe from ligo / virgo bounds even without the n suppression .
( in some sense that holographic noise scenario would turn into unpleasantly too safe from ligo / virgo , i.e. , probably beyond the reach of any foreseeable interferometric experiment , if it were to take into account the plausible n suppression ) . concerning the scenario for weak quantum - spacetime - induced fuzziness , the one of eq .
( 50 ) , contemplating the possibility of an n suppression is of mere academic interest : those noise levels are so low , even without the possible additional n suppression , that we should exclude their testability for the foreseeable future . but for random - walk noise and for the holographic - noise scenario of eq .
( 49 ) this issue of a possible n suppression needs to be investigated and understood .
this is probably not for the ligo / virgo season : ligo and virgo have not found any excess noise so far , and at this point it is unlikely they will ever find it . but a completely new drawing board for phenomenology would materialize with the advent of lisa : lisa will operate at lower observational frequencies than ligo / virgo - type interferometers , which is important from the quantum - spacetime perspective since both random - walk noise , as described by eq . (
( 49 ) predict effects that increase at lower observational frequencies.30 the outcome of such lisa quantum - spacetime - noise studies may then depend on issues such as the possible n suppression .
i should also stress that the analysis of these opportunities for quantum - spacetime phenomenology from experiments operating at low observational frequencies , is perhaps the most significant and most robust conceptual achievement of the sort of phenomenology of spacetime foam that i am discussing in this section .
when these pictures were first proposed it was seen by many as a total surprise that one could contemplate planck - scale effects at frequencies of observation of only 100 hz . the naive argument goes something like planck - scale - induced noise must be studied at planck frequency , and the planck frequency is ep/ 10 hz . however , in analyzing actual pictures of quantum - spacetime fuzziness , even the simple - minded ones described above , one becomes familiar with well - known facts establishing ( and we should expect this lesson to apply even to more sophisticated picture of quantum - spacetime - induced fuzziness ) that discrete fluctuation mechanisms tend to produce very significant effects at low observational frequencies , with typical behaviors of the type , even when their charateristic time scale is ultrashort . since the phenomenology of the implications of spacetime foam for interferometry is at an early stage of development , at the present time it may be premature to enter into detailed discussions of what type of interferometry might be best suited for uncovering quantum - spacetime / planck - scale effects . accordingly , in section 4.2 i focus by default on the simplest case of interferometric studies , the one using a laser - light beam .
however , in recent times atom interferometry has reached equally astonishing levels of sensitivity and for several interferometric measurements it is presently the best choice .
laser - light interferometry is still preferred for certain well - established techniques of interferometric studies of spacetime observables , as in the case of searches for gravity waves , and the observations i reported above for the phenomenology of strain noise induced by quantum - spacetime effects appear to be closely linked to the issues encountered in the search for gravity waves .
however , it seems plausible that soon there will be some atom - interferometry setups that are competitive for gravity - wave searches ( see , e.g. , refs . [ 526 , 208 ] ) .
this in turn might imply that searches of quantum - spacetime - induced strain noise could rely on atom interferometry .
the alternative between light and matter interferometry might prove valuable at later more mature stages of this phenomenology .
it is likely that different test theories will give different indications in this respect , so that atom interferometry might provide the tightest constraints on some spacetime - foam test theories , whereas laser - light interferometry might provide the best constraints on other spacetime - foam test theories .
a key aspect of the description of planck - scale effects for atom interferometry to be addressed by the test theories ( and hopefully , some day , by some fully - developed quantum - spacetime / quantum - gravity theories ) is the role played by the mass of the atoms . with respect to laser - light interferometry , the case of
atom interferometry challenges us with at least two more variables to be controlled at the theory level , which are the mass of the atoms and their compositeness
. how do these two aspects of atom interferometry interface with the quantum - spacetime features that are of interest here ?
do they effectively turn out to introduce suppressions of the relevant effects or on the contrary could they be exploited to see the effects ?
for none of the quantum - spacetime models that are presently studied have we reached a level of understanding of physical implications robust enough for us to answer confidently these questions .
perhaps we should also worry about ( or exploit ) another feature that is in principle tunable in atom interferometry , which is the velocity of the particles in the beam .
interferometric studies of spacetime - foam are another rare example of tests of quantum - spacetime effects that can be conducted in a controlled laboratory setup ( also see section 3.13 ) . astrophysics may turn into the most powerful arena for this type of study . indeed
, the studies i discussed in the previous section 4.2 , which started toward the end of the 1990s , inspired a few years later some follow - up studies from the astrophysics side .
as should be expected , the main opportunities come from observations of waves that have propagated over very large distances , thereby possibly accumulating a significant collective effect of the fuzziness encountered along the way to our detectors .
an implication of distance fuzziness that one should naturally consider for waves propagating over large distances is the possibility of time spreading of the signal : if at the source the signal only lasted a certain very short time , but the photons that compose the signal travel a large distance l , affected by uncertainty \documentclass[12pt]{minimal }
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\begin{document}$\sigma _ l^2$\end{document } , before reaching our detectors the observed spread of times of arrival might carry little trace of the original time spread at the source and be instead a manifestation of the quantum - gravity - induced l . if the distance l is affected by a quantum - spacetime uncertainty l then different photons composing the signal will effectively travel distances that are not all exactly given by l but actually differ from l and from each other up to an amount l .
again , it is of particular interest to test laws of the type discussed in the previous section 4.2 , but it appears that these effects would be unobservably small even in the case that provides the strongest effects , which is the random - walk ansatz \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
l^2 \simeq { l_p}l \simeq { l_p}t$\end{document } ( assuming ultrarelativistic particles , for which l is at least roughly equal to the time duration t of the journey ) . to see this
let me consider once again gamma - ray bursts , which often travel for times on the order of 10 s before reaching earth detectors and are sometimes characterized by time structures ( microbursts within the burst ) that have durations as short as 10 s. values of \documentclass[12pt]{minimal }
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l^2 \sim { c^2}{10^{- 27}}{{\rm{s}}^2}$\end{document } could be noticeable in the analysis of such bursts .
however , the estimate \documentclass[12pt]{minimal }
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\begin{document}$\sigma _
i shall comment in section 4.8 on an alternative formulation of the phenomenology of quantum - spacetime - induced worldline fuzziness , the one in ref .
inspired by the causal - set approach ( the approach on which section 4.8 focuses ) . as an alternative way to model spacetime fuzziness , there has been some interest [ 431 , 72 , 37 ] in the possibility that there might be effects resembling the ones discussed in section 3 , which are systematic deviations from the predictions of poincar symmetry , but are nonsystematic in the sense discussed at the beginning of this section .
the possibility of fuzziness of particle worldlines governed by e / ep , mentioned in the previous section 4.3.1 , is an example of such nonsystematic violations of poincar symmetry .
these speculations are not on firm ground on the theory side , in the sense that there is not much in support of this among available results on actual analysis of formalizations of spacetime quantization .
but it is legitimate to expect that this might be due to our limited abilities in mastering these complex formalisms .
, if spacetime geometry is fuzzy then it may be inevitable for the operative procedures that give sense to the notion of energy and momentum of a particle to also be fuzzy .
[ 74 , 57 , 72 ] , scenarios such that spacetime fuzziness effectively produces an uncertainty in the velocity of particles of order e / ep
. this would give rise to a magnitude of these nonsystematic effects comparable to the one discussed in section 3.8 for the corresponding systematic effects .
after a journey of 10 s the acquired fuzziness of arrival times could be within the reach of suitably arranged gamma - ray - burst studies . however , there is no significant effort to report here on establishing bounds following this strategy .
there are instead some studies of this phenomenological picture [ 431 , 72 , 37 ] , which take as a starting point the possibility , discussed in sections 3.4 and 3.5 , of modifications of the dispersion relation leading to modifications of the threshold requirements for certain particle - production processes , such as the case of two incoming photons producing an outgoing electron - positron pair
[ 431 , 72 , 37 ] considered the possibility of a non - systematic quantum - spacetime - induced deformation of the dispersion relation , specifically the case in which the classical relation e = p + m still holds on average , but for a given particle with large momentum \documentclass[12pt]{minimal }
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\begin{document}$$\vert \vec p\vert + { { { m^2 } } \over { 2\vert \vec p\vert } } - { { \vert \eta \vert } \over 2}{{{{\vec p}^2 } } \over { { e_p } } } \leq e \leq \vert \vec p\vert + { { { m^2 } } \over { 2\vert \vec p\vert } } + { { \vert \eta \vert } \over 2}{{{{\vec p}^2 } } \over { { e_p}}},$$\end{document } with some ( possibly gaussian ) probability distribution . a quantum - spacetime theory with this feature
should be characterized by a fundamental value of , but each given particle would satisfy a dispersion relation of the type 54\documentclass[12pt]{minimal }
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\begin{document}$$e \simeq \vert \vec p\vert + { { { m^2 } } \over { 2\vert \vec p\vert } } + { { \tilde \eta } \over 2}{{{{\vec p}^2 } } \over { { e_p}}},$$\end{document } with || || . in analyses such as the one discussed in section 3.4 ( for observations of gamma rays from blazars )
one would then consider electron - positron pair production in a head - on photon - photon collision assuming that one of the photons is very hard while the other is very soft . to leading order , for the soft photon
only , the energy is significant ( for an already small the actual value of will not matter in leading order ) .
so , the soft photon can , in leading order , be treated as satisfying a classical dispersion relation . in a quantum - spacetime theory predicting such non - systematic effects ,
the hard photon would be characterized both by its energy e and its value of . in order to establish whether a collision between two such photons can produce an electron - positron pair , one should establish whether , for some admissible values of + and ( the values of pertaining to the outgoing positron and the electron respectively ) , the conditions for energy - momentum conservation can be satisfied . the process will be allowed if 55\documentclass[12pt]{minimal }
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\begin{document}$$e \geq { { { m^2 } } \over \epsilon } - { { \tilde \eta } \over 4}{{{e^3 } } \over { \epsilon { e_p } } } + { { { { \tilde \eta } _ + } + { { \tilde \eta } _ - } } \over { 16}}{{{e^3 } } \over { \epsilon { e_p}}}.$$\end{document } since , + and are bound by the range || to || , the process is only allowed , independent of the value of , if the condition 56\documentclass[12pt]{minimal }
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\begin{document}$$e \geq { { { m^2 } } \over \epsilon } - { { 3\vert \eta \vert } \over 8}{{{e^3 } } \over { \epsilon { e_p}}}$$\end{document } is satisfied .
clearly , in this sense the threshold is inevitably decreased by the non - systematic effect .
however , there is only a tiny chance that a given photon would have = || , since this is the limiting case of the range allowed by the nonsystematic effect , and unless = || , the process will still not be allowed even if 57\documentclass[12pt]{minimal }
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\begin{document}$$e \simeq { { { m^2 } } \over \epsilon } - { { 3\vert \eta \vert } \over 8}{{{e^3 } } \over { \varepsilon { e_p}}}.$$\end{document } moreover , even assuming = || , the energy value described by ( 57 ) will only be sufficient to create an electron positron pair with + = || and = || , which again are isolated points at the extremes of the relevant probability distributions . therefore the process becomes possible at the energy level described by ( 57 ) but it remains extremely unlikely , strongly suppressed by the small probability that the values of , + and would satisfy the kinematical requirements . with reasoning of this type , one can easily develop an intuition for the dependence on the energy e , for fixed value of ( and treating , + and as totally unknown ) , of the likelihood that the pair - production process can occur : ( i ) when ( 56 ) is not satisfied the process is not allowed ; ( ii ) as the value of e is increased above the value described by ( 57 ) , pair production becomes less and less suppressed by the relevant probability distributions for , + and , but some suppression remains up to the value of e that satisfies 58\documentclass[12pt]{minimal }
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\begin{document}$$e \simeq { { { m^2 } } \over \epsilon } + { { 3\vert \eta \vert } \over 8}{{{e^3 } } \over { \epsilon { e_p}}};$$\end{document } ( iii ) finally for energies e higher than the one described by ( 58 ) , the process is kinematically allowed for all values of , + and , and , therefore , the likelihood of the process is just the same as in the classical - spacetime theory .
one should next consider that for a hard photon traveling toward our earth detectors from a distant astrophysical source there are many opportunities to collide with soft photons with energy suitable for pair production to occur ( the mean free path is much shorter than the distance between the source and the earth ) .
thus , one expects [ 72 , 37 ] that even a small probability of producing an electron - positron pair in a single collision would be sufficient to lead to the disappearance of the hard photon before reaching our detectors .
the probability is small in a single collision with a soft background photon , but the fact that there are , during the long journey , many such pair - production opportunities renders it likely that in one of the many collisions the hard photon would indeed disappear into an electron - positron pair .
therefore , for this specific scheme of non - systematic effects it appears that a characteristic prediction is that the detection of such hard photons from distant astrophysical sources should start being significantly suppressed already at the energy level described by ( 57 ) , which is below the threshold corresponding to the classical - spacetime kinematics .
it is interesting [ 57 , 72 , 74 ] to contemplate in this case the possibility that systematic and nonsystematic effects may both be present .
it is not unnatural to arrange the framework in such a way that the systematic effects tend to give higher values of the threshold energy , but then the nonsystematic effects would allow ( with however small probability ) configurations below threshold to produce the electron - positron pair . and for very large propagation distances
available ) the nonsystematic effect can essentially erase the systematic effect ( no noticeable upward shift of the threshold ) .
i illustrated the implications of nonsystematic effects within a given scenario and specifically for the case of observations of gamma rays from blazars .
one can implement the non - systematic effects in some alternative ways and the study of the observational implications can consider other contexts . in this respect
i should bring to the attention of my readers the studies of non - systematic effects for ultra - high - energy cosmic rays reported in refs .
[ 37 , 310 , 106 ] . combinations of systematic and nonsystematic effects can also be relevant [ 57 , 74 ] for studies of the correlations between times of arrival and energy of simultaneously - emitted particles . for
that type of study both the systematic and the nonsystematic effects could leave an observable trace in the data , codified in the mean arrival time and the standard deviation of arrival times found in different energy channels .
the two examples of studies in astrophysics of quantum - spacetime - induced distance fuzziness i discuss in sections 4.3.1 and 4.3.2 have only been moderately popular .
i have left as last the most intensely studied opportunity in astrophysics for quantum - spacetime - induced distance fuzziness .
it is interesting that these studies were started by ref . , which cleverly combined some aspects of refs .
[ 51 , 54 , 53 , 433 ] , providing the main concepts for the proposal summarized in section 4.2 , and some aspects of ref . , providing the main concepts for the proposal summarized in section 3.8 .
ref . was interested in the same phenomenology of distance fuzziness introduced and analyzed for controlled interferometers in refs .
[ 51 , 54 , 53 , 433 ] , but looked for opportunities to perform analogous tests using the whole universe as laboratory , in the sense first introduced in ref . .
gradually over the last decade this became a rather active research area , as illustrated by the studies reported in refs .
[ 367 , 434 , 189 , 464 , 363 , 171 , 167 , 402 , 514 , 403 , 404 , 520 , 456 , 405 ] .
the phenomenological idea is powerfully simple : effects of quantum - spacetime - induced space - time fuzziness had been shown [ 51 , 54 , 53 , 433 ] to be potentially relevant for ligo / virgo - like ( and lisa - like ) intereferometers , exploiting not only the distance - monitoring accuracy of those interferometers , but also the fact that such accuracy on distance monitoring is achieved for rather large terrestrial distances .
essentially the universe gives much larger distances for us to monitor , and although we can monitor them with accuracy inferior to the one of a ligo / virgo - like intereferometer , on balance , the astrophysics route may also be advantageous also for studies of quantum - spacetime - induced spacetime fuzziness . as for refs .
[ 51 , 54 , 53 , 433 ] , reviewed in section 4.2 , the core intuition here is that the quantum - spacetime contribution to the fuzziness of a particle s worldline might grow with propagation distance . and collecting the scenarios summarized in eqs .
( 47 ) , ( 49 ) , and ( 50 ) , one arrives at a one - parameter family of phenomenological anstze for the characterization of this dependence of fuzziness on distance 59\documentclass[12pt]{minimal }
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an assumption shared by most explorations [ 367 , 434 , 189 , 464 , 363 , 171 , 167 , 402 , 514 , 403 , 404 , 520 , 456 , 405 ] of this phenomenological avenue is that from eq .
( 59 ) , that there would also follow an associated uncertainty in the specification of momenta 60\documentclass[12pt]{minimal }
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\begin{document}$$\delta e\sim l_p^\alpha \;{e^{1 + \alpha}},\quad \;\;\delta p\sim l_p^\alpha \;{p^{1 + \alpha}}.$$\end{document } i must stress that this ( however plausible ) deduction of the heuristic arguments has not been confirmed in any explicit model of spacetime quantization . and
it plays a crucial role in most astrophysics tests of distance fuzziness : from eq .
( 60 ) it is easy to see that it follows that ( assuming a classical - wave description is still admissible when such effects are nonnegligible ) there should be a mismatch between uncertainty in the group velocity and in the phase velocity of a classical wave , and this in turn proves to be a very powerful tool for the phenomenology . during a propagation time t = l/g ( g being the group velocity ) the phase of a wave advances by = 2(p/g)(l/ ) ( where p is the phase velocity and is the wavelength ) .
there are two schools of intuition concerning how quantitatively spacetime fuzziness should scramble the phase of a wave . according to ref . and
followers the effect should go as 61\documentclass[12pt]{minimal }
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\begin{document}$$\delta \phi \simeq { { { e^\alpha } } \over { e_p^\alpha}}{l \over \lambda } = { { { e^{1 + \alpha } } } \over { e_p^\alpha}}l,$$\end{document } whereas according to ref .
[ 434 , 171 ] and followers , the effect should grow more slowly with the distance of propagation , going like 62\documentclass[12pt]{minimal }
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\begin{document}$$\delta \phi \simeq { e \over { e_p^\alpha}}{l^{1 - \alpha}}.$$\end{document } as first observed in ref .
, the alternative formulas ( 61 ) and ( 62 ) should be improved to account for redshift . for the case of eq .
( 62 ) ref . proposes the following 63\documentclass[12pt]{minimal }
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\begin{document}$$\delta \phi \sim { e \over { e_p^\alpha}}{{1 - \alpha } \over { { h_0}{q_0}}}\int\nolimits_0^\infty { dz(1 + z){l^{- \alpha}}\left({1 - { { 1 - { q_0 } } \over { \sqrt { 1 + 2z{q_0 } } } } } \right)},$$\end{document } where q0 is the decelaration parameter , \documentclass[12pt]{minimal }
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\begin{document}${q_0 } = { \omega _ 0}/2 - \lambda/(3h_0 ^ 2)$\end{document } and l is the luminosity distance , \documentclass[12pt]{minimal }
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\begin{document}$l = [ z{q_0 } + ( 1 - { q_0})(1 - \sqrt { 1 + 2z{q_0}})]/({h_0}q_0 ^ 2)$\end{document } ( , h0 and 0 here denote , as usual , respectively the cosmological constant , the hubble parameter and the matter fraction ) . evidently , this phenomenology still has a few too many quantitative details subject to further scrutiny and a few too many alternative scenarios .
this is the result of the fact that work on actual formalizations of spacetime quantization , while encouraging the general intuitive picture , has been unable to provide detailed guidance . and the heuristic arguments based on these preliminary studies have been unable to narrow the range of possibilities . but pursuing this path further appears to be an exciting opportunity for quantum - spacetime phenomenology , and we should
( 61 ) , ( 62 ) , and ( 63 ) , several authors ( see , e.g. , refs . [ 171 , 514 , 520 ] ) have concluded that a phenomenology based on blurring of the images of distant sources can provide planck - scale sensitivity for a rather broad range of possible phenomenological test theories and for values of significantly greater than 1/2 , possibly going all the way up to values of close to 1 . in ref .
one even finds a preliminary data analysis suggesting that for observations of quasars there might be a trend towards lower observed strehl ratios with increasing redshift , which would provide encouragement for the hope of discovering quantum - spacetime - induced image blurring .
the main opportunities appear to be provided by observations of distant quasars [ 171 , 514 , 520 ] , whose dimensions are small and are rather abundantly observed at high redshift .
investigations of spacetime symmetries and distance fuzziness are evidently relevant for some of the core features of the idea of spacetime quantization .
my next task concerns cpt - symmetry tests , and the possibility that indirectly some scenarios for the quantization of spacetime might affect cpt symmetry . a complication , but also an opportunity , for quantum - spacetime - motivated tests of cpt symmetries comes from the fact that cpt symmetry should be and is tested independently of the quantum - spacetime motivation .
from this perspective the situation is somewhat analogous to that discussed earlier concerning quantum - spacetime - motivated tests of lorentz symmetry
. the quantum spacetime literature can provide special motivation for probing cpt symmetry in certain specific ways , but there is already plenty of motivation , even without quantum - spacetime research , for testing cpt symmetry as broadly as possible [ 389 , 439 , 234 ] .
also , in this case , the standard model extension provides a much appreciated and widely adopted formalization , finding a good balance between the desire of searching for violations of cpt symmetry ( and/or , as mentioned , violations of lorentz symmetry ) within the confines of quantum field theory but allowing for both effects that have been discussed from the quantum - spacetime perspective and effects for which so far there is no quantum - spacetime motivation .
i shall focus here on the hypothesis of quantum - spacetime - induced and planck - scale - magnitude cpt violation effects , so i shall not review the broad subject of cpt violation within the standard model extension , for which readers can find valuable reviews and perspectives in refs .
[ 345 , 117 , 180 , 339 , 299 , 341 , 346 ] ( see also parts of ref . ) .
another issue that should always be kept in mind in relation to cpt symmetry is the fact that it can be derived as a theorem for local quantum field theories with lorentz invariance . in approaches based on local field theory ,
it is natural to perform combined studies of cpt and lorentz symmetry.31 however , the notion of spacetime quantization at the planck scale involves some aspects of nonlocality ( at least the notion of points that coincide with accuracy better than the planck length is typically abandoned ) and in most quantum - spacetime studies of the fate of cpt symmetry the expectation is that these aspects of non - locality may be primarily responsible for the conjectured violations of cpt symmetry .
i shall not attempt to summarize here the results on violations of cpt symmetry arising from spacetime quantization not introduced at the planck scale ( but rather at some much lower scale ) , for which readers can find valuable starting points to the related literature in refs .
consistent with the scope of this review , i shall focus exclusively on scenarios for violations of cpt symmetry based on nonclassicality ( quantization ) of spacetime introduced at the planck scale . as a result of some technical challenges , mentioned in section 2.2.2 , this literature can only rely on preliminary theory results , but does suggest convincingly that planck - scale sensitivity to quantum - spacetime - induced violations of cpt symmetry is within our reach . in the case of the test of cpt symmetry it is easier for me to start by discussing the availability of planck - scale sensitivity , postponing briefly some comments on test theories based on the idea of spacetime quantization .
there is a sizable literature establishing that cpt symmetry can be tested with planck - scale sensitivity in the neutral - kaon and the neutral - b systems ( see , e.g. , refs .
it turns out that in these neutral - meson systems there are plenty of opportunities for planck - scale departures from cpt symmetry to be amplified .
in particular , the neutral - kaon system hosts the peculiarly small mass difference between long - lived and short - lived kaons |ml
710 and other small numbers naturally show up in the analysis of the system , such as the ratio |l s|/ml , s 1.4 10 . and
for certain types of departures from cpt symmetry the inverse of one of these small numbers amplifies the small cpt - violation effect [ 219 , 220 , 298 , 108 ] .
in particular , this mechanism turns out to provide sufficient amplification for planck - scale effects , inducing a difference of order \documentclass[12pt]{minimal }
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\begin{document}$m_{{k^0}}^2/{e_p}$\end{document } between the terms on the diagonal of the \documentclass[12pt]{minimal }
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\begin{document}${k^0},{\bar k^0}$\end{document } mass matrix ( exact classical cpt symmetry would require the terms on the diagonal to be identical )
. it should be noticed that \documentclass[12pt]{minimal }
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\begin{document}${m_{{k^0}}}/{e_p } \sim { 10^{- 19}}$\end{document } , which is not overwhelmingly smaller than |ml ms|/ml , s 710 .
a much studied quantum - spacetime description of violations of cpt symmetry is centered on the mentioned liouville - strings approach [ 221 , 220 ] , particularly with its description of spacetime foam and its non - classical description of time , involving a non - trivial role for the liouville field .
this model is , in particular , the reference for the analysis of planck - scale limits on quantum - spacetime - induced cpt violation reported by the cplear collaboration on the basis of studies of neutral kaons ( also see the related results reported using neutral - kaon data gathered at the particle - physics laboratory in frascati [ 13 , 534 , 205 ] ) .
interestingly , the liouville - string model hosts both departures from cpt symmetry and decoherence , and i find it most convenient to discuss it in the later part of this section devoted to decoherence studies .
let me highlight a recent development that is in part inspired by these liouville - string studies .
[ 112 , 113 ] ) that quantum - spacetime scenarios with violations of cpt symmetry might also require some corresponding modifications of the recipe for obtaining multiparticle states from single - particle states for identical particles .
this may apply in particular to the neutral - kaon \documentclass[12pt]{minimal }
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\begin{document}${k_0 } - { \bar k_0}$\end{document } system , since standard cpt transformations take k0 into \documentclass[12pt]{minimal }
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\begin{document}${\bar k_0}$\end{document } but violations of cpt symmetry are likely to also induce a modification of the link between k0 and \documentclass[12pt]{minimal }
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\begin{document}${\bar k_0}$\end{document}. refs .
[ 112 , 113 ] proposed a phenomenology inspired by this argument and based on the following parametrization of the state |i > initially produced by a -meson decay : 64\documentclass[12pt]{minimal }
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\begin{document}$$\vert i > \propto ( ( \vert { k_s}(p),{k_l}(- p ) > - \vert { k_l}(p),{k_s}(- p ) > ) + \omega ( \vert { k_s}(p),{k_s}(- p ) > - \vert { k_l}(p),{k_l}(- p ) > ) ) , $ $ \end{document } where the complex parameter essentially characterizes the level of contamination of the state |i > by the ( otherwise unexpected ) c - even component |ks(p ) , ks( p ) >
stringent constraints on can be placed by performing measurements of the chain of processes kk xy , in which first the meson decays into a pair of neutral kaons and then one of the kaons decays at time t1 into a final state x , while the other kaon decays at time t2 into a final state y. by following this strategy the kloe experiment [ 534 , 522 ] at dane is setting [ 204 , 205 ] experimental limits on at the level 10 ( |re()| < 10 , |im()| < 10 ) .
it is not easy at present to establish robustly what level of sensitivity to could really amount to planck - scale sensitivity , but it is noteworthy that there are semi - quantitative / semi - heuristic estimates based on a certain intuition for spacetime foam suggesting [ 112 , 113 , 398 ] that sensitivities in the neighborhood of 10 , 10 could already be significant .
another formalism for spacetime quantization at the planck scale where violations of cpt symmetry have been discussed to some extent is
a first hint that this might be appropriate comes from the fact that the -minkowski formalism is one of those providing support for the possibility of modifications of the dispersion relation of the form \documentclass[12pt]{minimal }
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\begin{document}${m^2 } \simeq { e^2 } - { \bar p^2 } + \lambda e{\bar p^2}/2$\end{document } , with on the order of the planck length .
it may be relevant for the relation between particles and antiparticles ( for which cpt symmetry is a crucial player ) that for the values of e allowed by the dispersion relation for given \documentclass[12pt]{minimal }
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\begin{document}$\vert \vec p\vert$\end{document } one does not recover the ordinary result ( with its traditional two solutions of equal magnitude and opposite sign ) ; instead , one finds that the two solutions e+ , e are given by 65\documentclass[12pt]{minimal }
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\begin{document}$${e _ \pm } \simeq - { \lambda \over 2}{\vec p^2 } \pm \sqrt { { m^2 } + { { \vec p}^2}}.$$\end{document } the fact that the solutions e+ and e are not exactly opposite may suggest that one should make room for a mismatch m of the terms on the diagonal of the \documentclass[12pt]{minimal }
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\begin{document}${k^0},{\bar k^0}$\end{document } mass matrix , of order 66\documentclass[12pt]{minimal }
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\begin{document}$$\vert \delta m\vert \;\sim { { \vert { e _ + } \vert - \vert { e _ -}\vert } \over { \vert { e _ + } \vert + \vert { e _
-}\vert}}2 m \simeq \lambda { { { { \vec p}^2}m } \over { \sqrt { { m^2 } + { { \vec p}^2}}}}.$$\end{document } the most significant feature of this description of m is its momentum dependence , and , for given || , |m| is an increasing function of \documentclass[12pt]{minimal }
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\begin{document}$\vert \vec p\vert$\end{document } , quadratic in the non - relativistic limit and linear in the ultra - relativistic limit .
therefore , among experiments achieving comparable m sensitivity the ones studying more energetic kaons are going to lead to more stringent bounds on . considering that , as mentioned , neutral - kaon experiments at factories are now sensitive at the level m 10 gev , one infers a sensitivity to this type of candidate quantum - gravity effect that , for kaons of momenta of about 110 mev ( at the resonance ) , corresponds to a sensitivity to values of || around 10 m , i.e. , not far ( just 3 orders of magnitude away ) from the planck scale .
because of the premium on high momenta of this scenario , better limits could be set using experiments with high - momentum kaons fermilab s e731 [ 554 , 450 ] .
and studies with neutral b mesons of relatively high momenta could also be valuable from this perspective .
however , we are at a very early stage of understanding of the fate of cpt symmetry in these spacetimes with quantization at the planck scale .
specifically , for the case of -minkowski space - time , analyses such as the one in ref . suggest that cpt symmetry is deformed rather than broken / lost . indeed , in -minkowski the anomalies one
can presently preliminarily see for cpt symmetry are all linked to the peculiarity of p - parity transformations .
it appears that in -minkowski p - parity transformations for momenta should not take a momentum \documentclass[12pt]{minimal }
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\begin{document}$\ominus \vec p$\end{document } denotes the antipode operation : \documentclass[12pt]{minimal }
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\begin{document}$\ominus \vec p \equiv - \vec p{e^{- \lambda { p_0}}}$\end{document } ( where denotes again the -minkowski noncommutativity length scale ) . as stressed earlier in this review the idea of spacetime foam appears to appeal to everyone involved in quantum - spacetime research , but this is in part due to the fact that this idea is not really well defined , not by the qualitative intuitive picture proposed by wheeler . in order to set up a phenomenology for effects induced by this spacetime foam
i already discussed one possible such characterization , given in terms of distance fuzziness and associated strain noise for interferometry .
[ 220 , 221 ] ( other valuable perspectives on this subject can be found in refs .
[ 108 , 251 ] ) , focusing on the possibility that the rich dynamical properties of spacetime foam might act as a decoherence - inducing environment . the main focus of refs .
[ 220 , 221 ] has been the neutral - kaon system , whose remarkably delicate balance of scales provides opportunities not only for very sensitive tests of cpt symmetry , but also for very sensitive tests of decoherence .
[ 220 , 221 ] essentially propose a test theory , based on the mentioned liouville - strings idea , for spacetime - foam - induced decoherence in the neutral - kaon system .
this test theory adopts the formalism of density matrices and is centered on the following evolution equation for the neutral - kaon reduced density matrix : 67\documentclass[12pt]{minimal }
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\begin{document}$${\partial _
t}\rho = i[\rho , h ] + \delta h\;\rho,$$\end{document } where h is an ordinary - quantum - mechanics hamiltonian and hmn ( with indices m , n running from 1 to 4 : { m , n } { 1,2,3,4 } ) is the spacetime - foam - induced decoherence matrix , taken to be such that h1n = h2n = hn1 = hn2 = 0 , while h34 = h43 = 2 , h33 = 2 , and h44 = 2. therefore , the test theory is fully specified upon fixing h and giving some definite values to the parameters , , . it should be stressed that this test theory necessarily violates cpt symmetry whenever h 0 .
additional cpt violating features may be introduced in the ordinary - quantum - mechanics hamiltonian h , by allowing for differences in masses and/or differences in widths between particles and antiparticles .
therefore , this test theory is an example of a framework that could be used in a phenomenology looking simultaneously for departures from cpt symmetry of types admissible within ordinary quantum mechanics and for departures from cpt symmetry that require going beyond quantum mechanics ( by allowing for decoherence ) .
it is noteworthy that the two types of cpt violation ( within and beyond quantum mechanics ) can be distinguished experimentally . concerning more directly decoherence , various characterizations of the effects of this test theory
have been provided , and in particular a valuable description of how significant the decoherence effects are ( depending on the values given to , , ) is found looking at how the rate of kaon decay into a pair of pions , r2 , evolves as a function of time .
this time evolution will in general take the form 68\documentclass[12pt]{minimal }
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\begin{document}$${r_{2\pi}}(t ) = { c_s}{e^{- { \gamma _ s}t } } + { c_l}{e^{- { \gamma _ l}t } } + 2{c_i}{e^{- ( { \gamma _
l } + { \gamma _ s})t/2}}\cos [ ( { m_l } - { m_s})t - \phi ] , $ $ \end{document } where the indices s , l , i stand respectively for short - lived , long - lived , interference , and the combination \documentclass[12pt]{minimal }
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\begin{document}$\varsigma \equiv 1 - { c_i}/{\sqrt { { c_s}c_l}}$\end{document } provides a good phenomenological characterization of the amount of decoherence induced in the system . using data gathered by the cplear experiment , one can set bounds on , , at the levels 10 gev , 10 gev , and 10 gev .
a comparable limit on has been placed by dane s kloe experiment , and in that case the analysis was based [ 398 , 534 , 205 ] on entangled kaon states .
i should stress that this is clearly a quantum - spacetime picture ( at least in as much as it models spacetime foam ) and the objective of the associated research program is to introduce quantum / foamy properties of spacetime at the planck scale , but it is at present still unclear which levels of sensitivity to , , would correspond to foaminess of spacetime at the planck scale .
we are still unable to perform a derivation starting from foaminess at the planck scale and deriving corresponding values for , , . it is nonetheless encouraging that the present experimental limits on these ( dimensionful ) parameters are in a neighborhood of the planck - scale - inspired quantification mk / ep 10 ( but it should be noticed that as much planck - scale inspiration should be attributed , for example , to the scale \documentclass[12pt]{minimal }
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\begin{document}$m_k^2/e_p^2 \sim { 10^{- 38}}$\end{document } ) . another attempt to characterize spacetime foam as a decoherence - inducing medium was developed by percival and collaborators ( see , e.g. , refs .
this approach assumes that ordinary quantum systems should all be treated as open systems due to neglecting the degrees of freedom of the spacetime foam , but , rather than a formalization using density matrices , refs .
[ 452 , 453 , 454 ] adopt a formalism in which an open quantum system is represented by a pure state diffusing in hilbert space .
the dynamics of such states is formulated in terms of primary state diffusion , an alternative to quantum theory with only one free parameter , a time scale 0 , which one can set to be the planck time lp / c .
one way to charaterize 0 is through a formula for the proper time interval for a timelike segment , which is given by 69\documentclass[12pt]{minimal }
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\begin{document}$$\delta s \simeq
\vert \delta \xi ( x){\vert ^2 } + \delta \xi ( x)\sqrt { { \tau _ 0}},$$\end{document } where (x ) are point - dependent fluctuations induced by the foaminess / quantization of spacetime , which are modelled within the proposed theory . a key characteristic of this picture would be a suppression of the interference pattern for interferometers using beams of massive particles ( such that the original beam is first split and then reunited to seek an interference pattern ) . the suppression increases with the mass of the particles , so it could more easily be tested with atom interferometers ( rather than neutron interferometers ) .
unfortunately , a realistic analysis of an interferometer in the relevant primary - state - diffusion formalism is much beyond the level of answers one is ( at least presently ) able to extract from the primary - state - diffusion setup . ref . considered resorting to some simple - minded simplifications , including the assumption that the hamiltonian be given by the mass together with projectors onto the wave packets in the arms of the interferometer , neglecting the kinetic - energy terms . within such simplifications
one does find that values of 0 at or even a few orders of magnitude below the planck time would leave an observably large trace in modern atom interferometers .
however , these simplifications amount to a model of the interferometer that is much too crude ( as acknowledged by the authors themselves ) and this does not allow us to meaningfully explore the possibility of genuine planck - scale sensitivities being achieved by this strategy .
note that by taking 0 as the planck time it is not obvious that the effects are being introduced genuinely at the planck scale , since the nature of the effects is characterized not only by 0 but also by other aspects of the framework , such as the description of the fluctuations .
moreover , even if all other aspects of the picture were understood , the crudity of the model used for matter interferometers would still not allow us to investigate the planck - scale - sensitivity issue .
several aspects of the percival setup are maintained but different interpretations are applied in some aspects of the analysis .
removes some of the assumptions adopted by percival and collaborators , particularly in relation to the description of the quantum fluctuations of the metric , and proposes an estimate of the amount of suppression of the interference pattern,32 that is perhaps more intriguing from a phenomenology perspective , since it would suggest that the effect is just beyond present sensitivities ( but within the reach of sensitivities achievable by atom interferometers in the not - so - distant future ) .
for these recent proposals one is still ( for reasons analogous to these just discussed for the percival approach ) unable to meaningfully explore the issue of genuine planck - scale sensitivity , but they may represent a step in the direction of a more detailed description of spacetime foam , if intended as fluctuations of the metric .
the observations briefly discussed in the previous section 4.5 that are relevant for the study of manifestations of foam - induced decoherence in some laboratory experiments ( neutral - meson studies , atom interferometers ) can very naturally be applied to neutrino astrophysics as well , as discussed in ref . and references therein ( see also refs . [ 109 , 23 , 422 , 241 ] ) .
also in the neutrino context it is natural to attempt to develop test theories codifying the intuition that spacetime foam may act as an environment , so that neutrino observations would have to be analyzed considering the relevant neutrino system as an open system . and
the evolution of the neutrino density matrix could be described ( in the same sense as the description in eq .
( 67 ) for neutral - meson systems ) by an evolution equation of the type 70\documentclass[12pt]{minimal }
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\begin{document}$${\partial _
t}\rho = i[\rho , h ] + \delta h\;\rho.$$\end{document } it is argued in ref . that such a formalization of the effects of spacetime foam should generate a contribution to the mass difference between different netrinos , and could give rise to neutrino oscillations constituting a gravitational msw effect . as an alternative to the setup of eq .
( 70 ) one could consider [ 400 , 401 ] the possibility of random ( gaussian ) fluctuations of the background spacetime metric over which the neutrinos propagates .
for the random metric one can take [ 400 , 401 ] a formalization of the type 71\documentclass[12pt]{minimal }
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\begin{document}$${g^{\mu \nu } } = \left({\begin{array}{*{20}c } { - { { ( { a_1 } + 1)}^2 } + a_2 ^ 2 - { a_3}({a_1 } + 1 ) + { a_2}({a_4 } + 1 ) } \\ { - { a_3}({a_1 } + 1 ) + { a_2}({a_4 } + 1 ) - a_3 ^ 2 + { { ( { a_4 } + 1)}^2 } } \\ \end{array } } \right)$$\end{document } and enforce [ 400 , 401 ] for the random gaussian variables ai a parametrization based on parameters i ( one per ai ) such that i = 0 and aiaj = jiji . these fluctuations of the metric are found [ 400 , 401 ] to induce decoherence even when the neutrinos are assumed to evolve according to a standard hamiltonian setup , 72\documentclass[12pt]{minimal }
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\begin{document}$${\partial _
t}\rho = i[\rho , h].$$\end{document } but the decoherence effects generated in this framework with standard hamiltonian evolution in a nonstandard ( randomly - fluctuating ) metric , are significantly different from the ones generated with the nonstandard evolution equation ( 70 ) in a standard classical metric . in particular , in both cases one obtains neutrino - transition probabilities with decoherence - induced exponential damping factors in front of the oscillatory terms , but in the framework with evolution equation ( 70 ) the scaling with the oscillation length ( time ) is naturally linear [ 400 , 401 ] , whereas when adopting standard hamiltonian evolution in a fluctuating metric it is natural [ 400 , 401 ] to have quadratic scaling with the oscillation length ( time ) . the growing evidence for ordinary - physics neutrino oscillations , which one expects to be much more significant than the foam - induced ones , provides a formidable challenge for the phenomenology based on these test theories for foam - induced decoherence in the neutrino sector .
from the strict quantum - spacetime - phenomenology perspective of requiring one to establish that the relevant measurements could be sensitive to effects introduced genuinely at the planck scale , these neutrino - decoherence test theories must face challenges already discussed for a few other test theories : there is at present no rigorous / constructive derivation of the values of the parameters of these test theories from a description ( be it a full quantum - spacetime theory or simply a toy model ) of effects introduced genuinely at the planck scale , so one can only express these parameters in terms of the planck scale using some dimensional - analysis arguments .
a case for planck - scale sensitivity was recently made [ 97 , 99 ] for the hypothesis of possible violations of the pauli exclusion principle .
this has still not been metabolized by an appreciably - wide quantum - gravity community , but it certainly deserves to be highlighted briefly in this review , since the chances for gradually gaining a strong impact on quantum - spacetime phenomenology are rather high .
as observed already a few times in this review , the spin - statistics theorem assumes a classical spacetime with ordinary locality .
therefore , it is legitimate to speculate that small departures from the implications of the spin - statistics theorem may arise in a quantum spacetime . some earlier suggestions that this might be the case can be found , e.g. , in refs . [ 98 , 163 , 86 ] , but the setup then was not such that one could see an emerging case for planck - scale sensitivity .
the recent studies reported in refs . [ 97 , 99 ] investigated this issues assuming the specific form of spacetime noncommutativity given by 73\documentclass[12pt]{minimal }
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\begin{document}$$[{x_i},{x_j } ] = 0,\quad \quad [ { x_0},{x_i } ] = i\chi { \epsilon _ { kij}}{n_k}{x_j},$$\end{document } where nk are the components of a fixed spatial unit vector and the deformation length scale can be taken to be on the order of the planck length .
it is rather easy to show that this form of noncommutativity imposes a corresponding modification of the flip operator ,
i.e. , the operator that is used for symmetrization ( anti - symmetrization ) purposes in the commutative - spacetime case . in turn
and the end result is that certain transitions that would be pauli - forbidden in a commutative spacetime are actually allowed , although at a small rate ( suppressed by the smallness of ) .
( 73 ) is at present only possible by relying on an uncomfortable number of simplifying assumptions [ 97 , 99 ] , but the outcome is nonetheless intriguing since it suggests that sensitivity to values of on the order of the planck length is within reach .
this exploits the high sensitivity toward possible violations of the pauli exclusion principle at ongoing experiments , such as borexino and vip .
most of the quantum - spacetime phenomenology of this past decade has been inspired by results on spacetime noncommutativity and/or lqg .
i share the view of many quantum - spacetime phenomenologists who are looking at the approach based on causal dynamical triangulations [ 45 , 371 , 46 , 47 , 372 , 49 ] as a maturing opportunity for inspiring the phenomenology work . and
first indications are coming from the asymptotic safety approach [ 544 , 466 , 212 , 469 , 468 ] , on which i shall comment in relation to a tangible proposal for phenomenology later in this review . certainly in recent years
i place here an aside on this recent phenomenology inspired by the causal - set program , which also allows me to return , from a different perspective , on the important subject of non - systematic effects , already briefly discussed in section 4.3.2 . indeed ,
because of the perspective that guides that research program , most ( if not all ) new effects predicted within the causal set program will be of non - systematic type .
causal sets are a discretization of spacetime that allows the symmetries of gr to be preserved in the continuum approximation [ 131 , 470 , 284 ] . and
causal sets can be used to construct simple models suitable for exploring possible manifestations of fuzziness of quantum spacetime . moreover ,
the causal set proposal has recently been combined with the loop representation to formulate causal spin foams
, thereby establishing a link to an already mature source of inspiration for quantum - spacetime phenomenology . clearly ,
some of the manifestations one must expect from a causal - set setup fall within the class of phenomena already briefly described in section 4.3.1 : at a coarse - grained level of analysis a causal - set background should introduce an intrinsic limitation to the accuracy of lengths and durations .
several recent works were aimed at formalizing and modeling these aspects of fuzziness for propagation [ 311 , 312 , 215 ] .
the preliminary indications that are emerging appear to suggest that , if discreteness is indeed introduced at the planck scale , the effects are very soft ( hard to detect ) .
nonetheless we do already have a few examples of studies aiming for tangible predictions to be compared to actual data : for example , ref .
reports a causal - set - inspired analysis of possible fuzziness of arrival times ( the sort of effects already discussed in section 4.3.1 ) , relevant for studies conducted by gamma - ray telescopes .
an intriguing effect of random fluctuations in photon polarization can also be motivated by the causal - set framework .
the presently - available models of this causal - set - induced effect are to be viewed as very crude / preliminary , particularly since the present understanding of the framework is still not at the point of providing a definite model of photons propagating on a causal set background ( from which one could derive the polarization - fluctuation feature ) .
still , this appears a very promising direction , especially since experimental information on cmb polarization is improving quickly and will keep improving in the coming years .
presently the most tangible phenomenological plans inspired by the causal - set framework revolve around an effect [ 214 , 458 ] of lorentz invariant diffusion in the 4-momentum of massive particles .
this is an ornstein - uhlenbeck process , a diffusion process on the mass - shell that results in a stochastic evolution in spacetime .
, by considering a classical particle , of mass m propagating on a random spacetime lattice . the particle would then be constrained to move from point to point , but the discretization is such that in order to reach the next point
( remaining on the lattice ) the particle must swerve slightly , also adapting its velocity to the swerving ( also see ref . for a comparison of possible variants of the description of particle propagation in causal - set theory ) . the change in velocity amounts to the particle jumping to a different point on its mass shell .
the net result of this swerving is that [ 214 , 316 , 396 ] a collection of particles initially with an energy - momentum distribution (p ) will diffuse in momentum space along their mass shell according to the equation 74\documentclass[12pt]{minimal }
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\begin{document}$${{\partial \rho } \over { \partial \tau } } = \mathcal{k}\nabla _ \mathcal{p}^2\rho - { 1 \over m}{p^\mu}{\partial _ \mu}\rho,$$\end{document } where [ 214 , 316 , 396 ] is the diffusion constant , \documentclass[12pt]{minimal }
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\begin{document}$\nabla _ { \mathcal p}^2$\end{document } , is the laplacian in momentum space on the mass shell of the particle , is the proper time , and is an ordinary spacetime derivative . the tightest limit on is < 10 gev , and was obtained from limits on the amount of relic - neutrino contribution to hot dark matter .
this follows from the observation that energy on the mass shell is bound from below by the mass , so that particles close to rest , when swerving , can essentially only increase their energy .
interestingly these -governed effects can also be relevant for some of the phenomenology already discussed in this review , concerning the threshold requirements for certain particle - physics processes .
essentially the expected implications of swerving for these threshold analyses is similar to that already discussed in section 4.3.2 : in any given opportunity of interaction between a hard photon and a soft photon the swerving can effectively raise or lower ( from the perspective of the asymptotically - incoming states ) the threshold requirements for pion production .
however , it appears that the bound < 10 gev , if applicable also to protons,33 brings the magnitude of such effects safely beyond the reach of ongoing cosmic - ray studies .
i am focusing in this review on tests motivated by ( and on effects modeled within ) proposals of spacetime quantization at the planck scale , but concerning tests of the equivalence principle inspired by quantum - spacetime models there is some merit in making a small digression on tests of the equivalence principle in the semiclassical limit of quantum gravity ( where , by construction , no quantum - spacetime effects could be seen ) .
this will allow me to compellingly set - up the issue of testing the equivalence principle from a general quantum - gravity perspective and specifically from the perspective of spacetime quantization at the planck scale . as already discussed briefly in section 1
, there is a long tradition of phenomenological studies , concerning the semiclassical - gravity limit , based on a gravity version of the schrdinger equation of the form 75\documentclass[12pt]{minimal }
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\begin{document}$$\left [ { - \left({{1 \over { 2{m_i } } } } \right){{\vec \nabla}^2 } + { m_g}\phi ( \vec r ) } \right]\psi ( t,\vec r ) = i{{\partial \psi ( t,\vec r ) } \over { \partial t}},$$\end{document } describing the dynamics of matter ( with wave function \documentclass[12pt]{minimal }
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\begin{document}$\psi ( t,\vec r)$\end{document } , inertial mass mi and gravitational mass mg ) in an external gravitational potential \documentclass[12pt]{minimal }
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\begin{document}$\phi ( \vec r)$\end{document}. some of the most noteworthy results obtained within this framework are the interferometric studies of the type first set up by colella , overhauser and werner , which establish that the earth s gravitational field is strong enough to affect the evolution of the wave function in an observably - large manner , and the more recent evidence that ultracold neutrons falling towards a horizontal mirror do form gravitational quantum bound states .
of relevance here is the fact that some of the issues that have been most extensively considered by researchers involved in these studies concern the equivalence principle .
this is signaled by the adoption of separate notation for inertial and gravitational mass in eq .
the gravitational mass mg governs the accrual of gravity - induced phases , while the inertial mass mi intervenes in determining the ratio between wave vector and velocity vector in the galilean limit ( p m ) . and even for mg = mi the mass does not factor out of the free - fall evolution of the quantum state ( but for mg = mi one at least recovers a complete identification between the effects of gravitation and the effects of acceleration ) . besides neutrons
interestingly , one can also perform rather similar analyses in studying neutrino oscillations , finding ( see , e.g. , refs .
[ 252 , 272 , 148 ] and references therein ) , that gravity may induce neutrino oscillations if different neutrino flavors are coupled differently to the gravitational field , thereby violating the equivalence principle .
evidently , searches of possible violations of the equivalence principle in the semiclassical - gravity limit of quantum gravity have significant intrinsic interest . and some of these tests of the equivalence principle in semiclassical - gravity limit also find explicit motivation in approaches to the study of the full quantum - gravity problem : most notably the string - theory - inspired studies reported in refs .
[ 521 , 195 , 196 , 194 , 193 , 192 ] , and references therein , predict violations of the equivalence principle in the semiclassical - gravity limit . returning to the main subject of this review , i should stress that the idea of spacetime quantization at the planck scale provides a particularly crisp motivation for testing the equivalence principle . the simplest way to see this comes from observing the role of absolute and ideally sharp locality in the role that the equivalence principle plays in classical gravity , in contrast to the large class of qualitatively very severe ( though tiny ) anomalies for locality that the various known scenarios for spacetime quantization ( starting with spacetime noncommutativity for example ) provide .
unfortunately , our present level of mastery of the relevant formalisms often falls short of allowing us to investigate the fate of the equivalence principle .
therefore , i will briefly describe one illustrative example of promising attempt to model how spacetime foam could affect the equivalence principle .
this is the objective of recent studies , reported in ref . and references therein , in which spacetime foam is modeled in terms of small fluctuations of the metric on a given background metric.34 the analysis of ref . ,
which also involves an averaging procedure over a finite spacetime scale , ends up motivating the study of a modified schrdinger equation of the form 76\documentclass[12pt]{minimal }
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\begin{document}$$\left [ { - \left({{1 \over { 2 m } } } \right)({\delta ^{kl } } + { { \tilde \alpha}^{kl}}){\partial _ k}{\partial _ l } - m\phi ( \vec r ) } \right]\psi ( t,\vec r ) = i{\partial _ t}\psi ( t,\vec r),$$\end{document } where the tensor is a characterization of the spacetime foaminess , and it is natural to consider the tensor \documentclass[12pt]{minimal }
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\begin{document}${\tilde m^{kl}}$\end{document } , 77\documentclass[12pt]{minimal }
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\begin{document}$${({\tilde m^{kl}})^{- 1 } } \equiv { 1 \over m}({\delta ^{kl } } + { \tilde \alpha ^{kl}}),$$\end{document } as an anomalous inertial mass tensor that depends on the type of particle and on the fluctuation scenario .
the particle - dependent rescaling of the inertial mass provides a candidate key manifestation of foam - induced violations of the equivalence principle to be sought experimentally , in ways that are once again exemplified by the cow experiments .
this very recent proposal illustrates a type of path that could be followed to introduce violations of the equivalence principle originating genuinely from spacetime quantization at the planck scale : one might find a way to describe spacetime foaminess n terms of effects of genuinely planckian size , and then elaborate the implications of this spacetime foaminess for the equivalence principle . the formalization adopted in ref .
is still too crude to allow such an explicit link between the planck - scale picture of spacetime foam and the nature and magnitude of the effects , but provides a significant step in that direction .
work on planck - scale quantum - spacetime phenomenology is a rather recent development , with a significant effort taking place over only little more than a decade .
but one can already make a distinction between traditional and novel quantum - spacetime phenomenology approaches .
the proposals i have reviewed in the previous two sections 3 and 4 cover the scope of the traditional approach , considering uv effects that could be relevant for observations in astrophysics and/or in controlled - laboratory experiments .
i devote this and the next section 6 to the novel idea that planck - scale quantization of spacetime could have valuable phenomenological implications in some ir regimes and/or that the tests could rely on cosmology . considering that these novel areas of quantum - spacetime phenomenology are in a preliminarily exploratory phase i will adopt a lower standard in the selection of topics , meaning that i will even mention some proposals that have not fully established a link to a definite scheme of spacetime quantization and/or have not fully established the availability of sensitivities that could be compellingly linked with the introduction of spacetime quantization at the planck scale .
i will rather rely on an ( inevitably subjective ) assessment of whether the relevant proposals provide valuable first steps in the direction of establishing in the not - so - distant future robust planck - scale quantum - spacetime phenomenology . in the long [ 508 , 475 ] , and so far inconclusive ,
search for quantum gravity and quantum spacetime the main strategy was inspired by the discovery paradigm of the 20th century , the microscope paradigm with discovery potential measured in terms of the shortness of the distance scales probed .
but recent research has raised the possibility that by quantizing spacetime at the planck scale one might have not only some new phenomena in a far - uv regime , but also some new phenomena in a dual ir regime . actually , as compellingly stressed in ref .
, our present understanding of black - hole thermodynamics , and particularly the scaling s r of the entropy of a black hole of radius r , suggests that such effects of uv / ir mixing may be inevitable .
it is on the basis of apparently robust hypotheses concerning the behavior of quantum gravity in the uv ( planckian ) regime that we arrive at this quadratic dependence , which is surprising with respect to what one might expect in particular in quantum field theory , where cubic scaling ( s r ) naturally arises . but
this feature originating from the uv sector clearly should have its most profound implications in the large - distance / ir regime since the difference between quadratic dependence on the radius and cubic dependence on the radius becomes more and more significant as the radius is increased.35 another argument in favor of uv / ir mixing is found considering a popular intuition for quantum spacetime , which relies on the introduction of an uncertainty principle for spacetime itself ( in addition to the heisenberg one , which acts in phase space ) .
the link with uv / ir mixing can be already seen simply by considering a principle of the form \documentclass[12pt]{minimal }
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\begin{document}$\delta x\delta y \geq \lambda _ \ast^2$\end{document } for spatial coordinates , with * plausibly on the order of the planck length .
this type of uncertainty relation would evidently imply that small uncertainty in x should require large uncertainty in y , and this suggests a link between probing short distance scales ( small x ) and probing large distance scales ( large y ) .
for this last point we have more than general arguments : computations in a noncommutative spacetime compatible with this sort of uncertainty relations , the canonical spacetime , with non - commutativity of coordinates governed by [ x , x ]
this is particularly evident when analyzing mass renormalization within the most popular formalization of quantum field theories in such canonical noncommutative spacetimes .
at one loop one finds terms in mass renormalization of the form [ 213 , 516 , 397 ] ( for a
scalar field theory ) 78\documentclass[12pt]{minimal }
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\begin{document}$$\delta _ { { m^2}}^{{\rm{renorm } } } = { 1 \over { 32}}{{{g^2}\lambda _ { { \rm{eff}}}^2 } \over { { \pi ^2 } } } - { 1 \over { 32}}{{{g^2}{m^2 } } \over { { \pi ^2}}}\log { { \lambda _ { { \rm{eff}}}^2 } \over { { m^2 } } } + \mathcal{o}({g^4}),$$\end{document } where eff is a peculiar cutoff that can be expressed in terms of a standard uv cutoff , the noncommutativity matrix and the momentum q of the particle as follows 79\documentclass[12pt]{minimal }
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\begin{document}$$\lambda _ { { \rm{eff}}}^2 = { 1 \over { { \lambda ^{- 2 } } + { q_\rho}{{({\theta ^2})}^{\rho \sigma}}{q_\sigma}}}.$$\end{document } removing the cutoff ( ) one is left with \documentclass[12pt]{minimal }
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\begin{document}$\lambda _ { { \rm{eff}}}^2 = 1/[{q_\rho}{({\theta ^2})^{\rho \sigma}}{q_\sigma}]$\end{document } , so that 80\documentclass[12pt]{minimal }
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\begin{document}$$\delta _ { { m^2}}^{{\rm{renorm}}}\sim { { { g^2 } } \over { { q_\rho}{{({\theta ^2})}^{\rho \sigma}}{q_\sigma } } } + { g^2}{m^2}\log [ { m^2}{q_\rho}{({\theta ^2})^{\rho \sigma}}{q_\sigma}].$$\end{document } these power - law and logarithmic ir features are the result of the uv implications of noncommutativity , which manifest themselves in a rearrangement of the renormalization procedure [ 213 , 516 , 397 ] .
in general , the presence of such sharp features in the ir may be of some concern , since they have not ( yet ) been observed . and
however , it should be noticed that different choices of the matrix produce very different types of ir behavior , and it is well established that in the presence ( at least in the uv sector ) of supersymmetry only the logarithmic ir features survive ( the power - law corrections are removed by one of the standard supersymmetry - induced cancellation mechanisms ) .
the least virulent ir scenario is obtained by assuming the presence of uv supersymmetry and choosing a light - like noncommutativity matrix [ 19 , 90 ] ( = v = 0 ) , so that the main ir feature is a modification of the on - shell relation of the form 81\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { p^2 } + { \chi _
\theta}{m^2}\log \left({{{e + \vec p \cdot { { \hat u}_\theta } } \over m } } \right).$$\end{document } the unit vector describes a preferential direction determined by the matrix , while the dimensionless parameter also allows for an expected dependence of the magnitude of the effect on the specific particle under study : since the ir feature is found in the renormalization procedure , and this in turn has an obvious dependence on the interactions of a given field with other fields in the theory , the coefficient of the logarithmic ir correction has different value for different fields . note that in the ir regime ( small p ) one can rewrite ( 81 ) as follows 82\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { p^2 } + \chi m\vec p \cdot { \hat u_\theta},$$\end{document } so that the effect ultimately amounts to a correction that is linear in momentum . clearly , this is a scenario in which the ir implications of uv / ir mixing are particularly soft .
interestingly , canonical noncommutativity is not the only quantum - spacetime proposal that can motivate the study of uv / ir mixing .
this is suggested by the perspective on the semi - classical limit of lqg that provided motivation for the quantum - spacetime model of refs . [ 33 , 34 ] , that also inspired the models considered in refs .
[ 154 , 155 , 69 ] . in this lqg - inspired scenario one finds [ 33 , 34 ] modifications of the dispersion relation that are linear in momentum in the ir regime , and this has motivated a phenomenology based36 on the ir dispersion relation [ 154 , 155 , 69 ] 83\documentclass[12pt]{minimal }
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\begin{document}$${m^2 }
\simeq { e^2 } - { p^2 } + { \chi _ { \hat p}}\;m\;p,$$\end{document } where \documentclass[12pt]{minimal }
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\begin{document}${x_{\hat p}}$\end{document } is a phenomenological parameter37 analogous to . the long - wavelength behavior of the two scenarios for soft uv / ir mixing summarized here in eq .
( 82 ) and eq . ( 83 ) evidently differ only because of the fact that invariance under spatial rotations ( lost in eq .
therefore , one could simultaneously consider the two scenarios , by observing that the characterization of eq .
( 82 ) in terms of and is applicable to the scenario of eq .
( 83 ) by replacing with \documentclass[12pt]{minimal }
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\begin{document}${x_{\hat p}}$\end{document}. however , in light of the limited scope of my review of results on soft uv / ir mixing , i shall be satisfied with a simplified description , assuming space - rotation invariance and limiting my focus to the effects of dispersion relations of the form 84\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { p^2 } + \xi { { { m^2 } } \over { { e_p}}}p,$$\end{document } where i also introduced a change of definition of the dimensionless coefficient , rescaling it in a way that might be relevant for connecting the ir effects with the planck scale ( m / ep , which provides no loss of generality if is allowed to be particle dependent ) .
the phenomenology of models such as this requires a complete change of strategy with respect to the phenomenology of quantum - spacetime uv effects that i discussed in previous sections 3 and 4 of this review . whereas the typical search for those uv effects relied on low - precision high - energy data , for the type of ir effects that i am now considering the best options come from high - precision low - energy data .
, most notably with a ( however brief ) discussion of how a dispersion relation of type ( 84 ) could be relevant for lamb - shift measurements . indeed , assuming eq .
( 84 ) holds for the electron , then one should have a modification of the energy levels of the hydrogen atom . and in light of the high precision of certain lamb shift measurements ( which ref .
assesses as being better than one part in 10 , see also , e.g. , refs .
[ 328 , 546 ] ) one can use this observation to place valuable limits on parameters such as ( and ) for the electron .
evidently the ansatz ( 84 ) is such that if particles of different mass had the same value38 of then the effect would be seen more easily for heavier ( more massive ) types of particles .
i find particularly striking the case of measurements of the recoil of cesium ( and rubidium ) atoms . for cesium one would assume , following eq .
( 84 ) , that 85\documentclass[12pt]{minimal }
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\begin{document}$${m^2 } \simeq { e^2 } - { p^2 } + { \xi _ { cs}}{m^2}{p \over { { m_p}}},$$\end{document } where cs is the parameter for the case of cesium atoms . the measurement strategy we proposed in ref . for testing eq .
( 85 ) with atoms is applicable to measurements of the recoil frequency of atoms with experimental setups involving one or more two - photon raman transitions .
the strategy of the analysis is best described by setting aside initially the possibility of planck - scale effects , and looking at the recoil of an atom in a two - photon raman transition from the perspective adopted in ref . , which provides a convenient starting point for the planck - scale generalization that is of interest here .
one can impart momentum to an atom through a process involving absorption of a photon of frequency and ( stimulated ) emission , in the opposite direction , of a photon of frequency . the frequency is computed taking into account a resonance frequency * of the atom and the momentum the atom acquires , recoiling upon absorption of the photon : * + ( h * + p)/(2 m ) p/(2 m ) , where m is the mass of the atom ( e.g. , mcs 124 gev for cesium ) , and p is its initial momentum .
the emission of the photon of frequency must be such as to de - excite the atom and impart to it additional momentum : + ( 2h * + p)/(2 m ) * + ( h * + p)/(2 m ) . through this analysis one
establishes that by measuring , in cases in which * and p can be accurately determined , one actually measures h / m for the atoms : 86\documentclass[12pt]{minimal }
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\begin{document}$${{\delta \nu } \over { 2{\nu _ \ast}({\nu _ \ast } + p / h ) } } = { h \over m}.$$\end{document } this result has been confirmed experimentally with remarkable accuracy . a powerful way to illustrate this success
is provided by comparing the results of atom - recoil measurements of /[*( * + p / h ) ] and of measurements of , the square of the fine structure constant .
can be expressed in terms of the mass m of any given particle through the rydberg constant , r , and the mass of the electron , me , in the following way : \documentclass[12pt]{minimal }
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\begin{document}${\alpha ^2 } = 2{r_\infty}{m \over { { m_e}}}{h \over m}$\end{document}. therefore , according to eq .
( 86 ) one should have 87\documentclass[12pt]{minimal }
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\begin{document}$${{\delta \nu } \over { 2{\nu _ \ast}({\nu _ \ast } + p / h ) } } = { { { \alpha ^2 } } \over { 2{r_\infty}}}{{{m_e } } \over { { m_u}}}{{{m_u } } \over m},$$\end{document } where mu is the atomic mass unit and m is the mass of the atoms used in measuring /[*(
the outcomes of atom - recoil measurements , such as the ones with cesium reported in ref . , are consistent with eq .
( 86 ) has been verified to such a high degree of accuracy proves to be very valuable , since it turns out that modifications of the dispersion relation of type ( 85 ) require a modification of eq .
one easily finds 88\documentclass[12pt]{minimal }
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\begin{document}$$\delta \nu \simeq { { 2{\nu _ \ast}(h{\nu _ \ast } + p ) } \over m } + { \xi _ { cs}}{m \over { { m_p}}}{\nu _ \ast},$$\end{document } and in turn in place of eq .
( 87 ) one has 89\documentclass[12pt]{minimal }
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\begin{document}$${{\delta \nu } \over { 2{\nu _ \ast}({\nu _ \ast } + p / h)}}\left [ { 1 - { \xi _ { { \rm{cs}}}}\left({{m \over { 2{m_p } } } } \right)\left({{m \over { h{\nu _ \ast } + p } } } \right ) } \right ] = { { { \alpha ^2 } } \over { 2{r_\infty}}}{{{m_e } } \over { { m_u}}}{{{m_u } } \over m}.$$\end{document } this equation has been arranged so that on the left - hand side it is easy to recognize that the small quantum - spacetime effect in this specific context receives a sizable amplification by the large hierarchy of energy scales m/(hv * + p ) , which in typical experiments of the type here of interest can be of order 10 .
this turns out to be just enough to provide the desired planck - scale sensitivity : one easily finds that combining the measurements on cesium reported in ref .
it is interesting that , besides tests of ir modifications of the dispersion relation , these atom - recoil studies can also be used to investigate possible ir modifications of the law of conservation of momentum .
the use of atoms in quantum - spacetime phenomenology immediately confronts us with issues that are presently beyond the reach of available theoretical results .
a legitimate expectation is that quantum - spacetime effects for atoms could be weaker than for the particles that compose atoms , as a result of the sort of average - out effects that one is often expected in the quantum - spacetime literature .
this would have to be modeled by introducing an extra suppression factor ( a sort of compositeness factor ) in addition to the planck - scale suppression that is standard in quantum - spacetime phenomenology .
analyses not making room for such an additional suppression might overestimate the planck - scale - sensitivity reach of the relevant experiments . on the other hand we are at present not sure whether such compositeness - suppression factors are truly needed , or at least if they are needed in all contexts and in all quantum - spacetime models .
for example , it is not unreasonable to imagine that in appropriate quantum - spacetime models , when we achieve the ability to analyze them in detail , we might find that as long as a particle is to be handled as a quantum state ( far from its classical limit ) then it might be irrelevant for the magnitude of quantum - spacetime effects whether the particle is composite or fundamental .
this issue of compositeness will surely gradually take an important role in quantum - spacetime research , but at present it is at a very preliminary stage of investigation , and i shall therefore set it aside . however , do note that if particles composed of a very large number of constituent particles experience planck - scale effects unsuppressed by their compositeness , then not only atoms but also ( and perhaps more powerfully ) bose - einstein condensates could prove to be a very valuable opportunity for quantum - spacetime phenomenology . and
it is noteworthy that in the recent quantum - spacetime - phenomenology literature there has already been a surge of interest in the possibilities offered by bose - einstein condensates , as seen in refs .
[ 139 , 138 ] study bose - einstein condensates adopting a perspective on soft uv / ir mixing that is closely related to the one discussed for atoms in the previous section 5.3 .
perhaps the most tempting opportunity for the phenomenology of uv / ir mixing comes from studies of the low - energy beta decay spectrum of tritium , \documentclass[12pt]{minimal }
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\begin{document}$^3{\rm{h}}{\rightarrow ^3}{\rm{he + } } { { \rm{e}}^ - } + { \bar v_e}$\end{document } , which have produced so far some rather puzzling results [ 545 , 370 ] .
it is well understood ( see , e.g. , refs . [ 121 , 174 ] ) that these puzzles could be addressed by introducing deformed rules of kinematics . and
it is intriguing that studies conducted near the endpoint of tritium beta decay are the only known way to accurately investigate the properties of neutrinos in a non - relativistic ( non - ultrarelativistic ) regime , where their momenta could be comparable to their ( tiny ) masses .
so , it would seem to be a very natural opportunity for advocating uv / ir mixing as a possible explanation .
however , the evidence available so far is not very encouraging for the hope of attributing the magnitude of the reported anomalies to ir effects induced by the planck scale .
still , it is noteworthy that specifically the simple model for soft uv / ir mixing that i described in the previous sections 5.2 and 5.3 has just the right structure for producing the sort of anomalies that are being reported , as was first stressed in ref . .
the main point of ref . is centered on the properties of the function k(e ) conventionally used to characterize the kurie plot of tritium beta decay : 90\documentclass[12pt]{minimal }
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\begin{document}$$k(e ) = { \left [ { \int { d{p_\nu}p_\nu ^2\delta ( q - e - { e_\nu } ) } } \right]^{1/2}},$$\end{document } where q is the difference between initial and final masses of the process , \documentclass[12pt]{minimal }
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\begin{document}$q \simeq { m_{{3_{\rm{h } } } } } - { m_{{3_{\rm{h}}}_{\rm{e } } } } - { m_e}$\end{document } ( and , therefore , q is the sum of the neutrino energy , e , and the kinetic energy of the electron , e ) . using standard dispersion relations
one finds 91\documentclass[12pt]{minimal }
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\begin{document}$$k(e ) = { \left [ { ( q - e)\sqrt { { { ( q - e)}^2 } - m_\nu ^2 ) } } \right]^{1/2}},$$\end{document } which does not fit well with the available data near the endpoint [ 545 , 370 ] .
it was observed in ref . that instead using a modified dispersion relation of type ( 84 ) , for negative , one obtains better agreement , but this requires that \documentclass[12pt]{minimal }
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\begin{document}$\xi m_v^2/{e_p}$\end{document } have a value of a few ev . in turn
this implies a value39 of that is extremely large with respect to the natural quantum - spacetime estimate 1 , and as a result the case for a quantum - spacetime interpretation is rather weak at present .
still , this exciting experimental situation deserves to be further pursued : perhaps we are modeling soft uv / ir mixing correctly but we have developed the wrong intuition about the role the planck scale should play , or perhaps one should look at alternative ways to model uv / ir mixing .
in addition to precision measurements on particles of peculiarly low momentum , another very clear opportunity for uv - ir mixing is provided by data on the behavior of gravity on very large distance scales . and in that context speculating about new - physics phenomena is fully justified by the observed non - keplerian features of the rotation curves of galaxies or clusters .
these non - keplerian features are usually interpreted as motivation for introducing dark matter ( or other non - quantum - gravity new physics , such as mond ) , but , in light of the recent awareness of the possibility of uv / ir mixing , it is legitimate to speculate that they may be at least in part due to quantum - spacetime effects .
the perspective one might adopt in trying to profit from this opportunity is similar to when one works within standard quantum field theories and derives an effective potential ( usually obtained through the calculation of loop contributions ) that corrects the tree - level classical potential . interestingly , the type of modifications of dispersion relations that have been motivated by quantum - spacetime research do automatically suggest that the newtonian potential should receive some corresponding corrections .
in fact , the newtonian potential is produced by a static point source when the field that mediates the force described by the potential has energy - momentum space ( inverse ) propagator g(e , p ) = e p. in general , if the field that mediates the force has a different propagator , \documentclass[12pt]{minimal }
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\begin{document}$g_{def}^{- 1}(e , p)$\end{document } , the newtonian potential produced at the spatial point \documentclass[12pt]{minimal }
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\begin{document}$\vec r$\end{document } by a point - like mass m , located at the origin , is replaced by the potential obtained by computing 92\documentclass[12pt]{minimal }
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\begin{document}$$v(\vec r ) = l_p^2m\int { { { { d^3}p } \over { 2{\pi ^2}}}{g_{def}}(0,\vec p)\;{e^{i\vec
p \cdot \vec r}},}$$\end{document } i.e. , the potential is the spatial fourier transform of the propagator evaluated at e = 0 .
a more articulated argument for modifications of the newton potential at large distances from a quantum - spacetime perspective has been put forward as part of the mentioned research program on
this is done in ref . , which indeed adopts as a working assumption the availability of a quantum field theory of gravity whose underlying degrees of freedom are those of the spacetime metric , defined nonperturbatively as a fundamental , asymptotically - safe theory .
obtaining definite predictions for the rotation curves of galaxies or clusters within this formalism is presently well beyond our technical capabilities .
however , preliminary studies of the renormalization - group behavior provide encouragement for a certain level of analogy between this theory and non - abelian yang - mills theories , and , relying in part on this analogy , ref .
another opportunity for studies of uv / ir mixing is provided by measurements performed on neutron quantum states in the gravity field of the earth , such as the striking ones reported in refs .
i have nothing to report on this that would fit the main focus of this review , concerning planck - scale quantum pictures of spacetime , but it seemed worth mentioning this nonetheless , especially in light of the fact that this class of low - energy studies ( candidates for the investigation of uv / ir mixing ) have already been analyzed from the perspective of some quantum spacetimes , even though so far all such studies have introduced spacetime quantization at scales that are very far from the planck scale ( much lower energy scales , much greater distance scales ) .
since i am already here diverting from the main theme of the review , i shall be satisfied confining the discussion of quantum - spacetime studies of the gravitational quantum well to the particularly interesting points made in refs .
[ 118 , 102 , 483 ] all assumed canonical noncommutativity of spacetime coordinates : 93\documentclass[12pt]{minimal }
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\begin{document}$$[{x_j},{x_k } ] = i{\theta _ { jk}},\quad [ { x_j},{x_0 } ] = i{\theta _ { j0}},$$\end{document } where i separated the space / space noncommutativity ( jk = 0 ) from the space / time noncommutativity ( j0 0 ) . and refs .
[ 118 , 102 , 483 ] agree on the fact that pure space / space noncommutativity ( j0 = 0 ) has no significant implications for the gravitational quantum well . however , ref .
notices that with space / time noncommutativity ( j0 0 ) there are tangible consequences for the gravitational quantum well so that in turn one can use the measurement results of refs .
[ 428 , 429 ] to put bounds on space / time noncommutativity,40 although only at the level j0
< 10 m ( whereas interest from the planck - scale - quantum - spacetime side would focus in the neighborhood of j0 10 m ) . refs .
[ 118 , 102 ] make the choice of combining space / space noncommutativity with a noncommutativity of momentum space : 94\documentclass[12pt]{minimal }
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\begin{document}$$[{p_j},{p_k } ] = i{\psi _ { jk}}.$$\end{document } it then turns out that this noncommutativity of momentum space does tangibly affect the analysis of the gravitational quantum well .
[ 428 , 429 ] to place bounds at the level jk < 10 ev .
is an example of analysis of the gravitational quantum well not from the viewpoint of spacetime noncommutativity , but rather from the viewpoint of the scheme of spacetime quantization introduced in refs .
[ 323 , 322 ] , which is centered on a modification of the heisenberg principle 95\documentclass[12pt]{minimal }
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\begin{document}$$[{x_j},{p_k } ] = i{\delta _ { jk}}(1 + \beta { p^2}).$$\end{document } the parameter does turn out to affect the analysis of the gravitational quantum well , and using the measurement results of refs . [
428 , 429 ] one can place bounds at the level < 10 m .
in the previous sections 3 , 4 , 5 , i discussed several opportunities for investigating candidate quantum - spacetime effects through observations in astrophysics and , occasionally , some controlled laboratory setups .
however , it is likely that gradually cosmology will acquire more and more weight in the search for manifestations of quantum - spacetime effects . in the earliest stages of evolution of the universe ,
the typical energies of particles were much higher than the ones we can presently achieve , and high - energy particles are the ideal probes for the short - distance structure of spacetime . over these past few years , several studies that could be viewed as preparing the ground for this use of cosmology have been presented in the literature .
most of these proposals do not have the structure and robustness necessary for actual phenomenological analyses , such as the ones setting bounds on the parameters of a given quantum - spacetime picture .
but the overall picture emerging from these studies confirms the expectation that cosmology has the potential to be a key player in quantum - spacetime phenomenology . for a combination of reasons
, i shall review this recent literature in an even more sketchy way than other parts of this review .
this reflects the fact that i view this area as still at a very early stage of development : we are probably just starting to learn what could be the observable manifestations of the quantization of spacetime in cosmology . even in cases
when the quantum - spacetime side is reasonably well understood , the study of the implications for cosmology , when focused on possible observably - large manifestations , is still in its infancy .
moreover , most of the work done so far in this area does not even invoke a definite role for spacetime quantization , which is the main focus of this review , but rather finds inspiration in generic features of the quantum - gravity problem , or relies on string theory ( which , as stressed , is a fully legitimate quantum - gravity candidate , but is one such candidate that , as presently understood , would rather lead us to assume that quantum properties of spacetime are absent / negligible ) . in light of these considerations , the list of proposals and ideas that composes this section is not representative of the list of scenarios being considered in quantum - spacetime cosmology .
it mainly serves the purpose of offering some illustrative examples of how one might go about proposing a quantum - spacetime - cosmology scenario and giving some strength to my opening remarks foreseeing a great future for quantum - spacetime cosmology . in the last section 6.5 ,
i briefly mention some examples of quantum - gravity - cosmology proposals , which in their present formulation do not invoke a role for the quantization of spacetime ( but could inspire future reformulations centered on a quantum - spacetime perspective ) . in the long run , one of the most significant opportunities for quantum - spacetime phenomenology could be an aspect of quantum - spacetime cosmology : the trans - planckian problem .
inflation works in such a way that some of the scales that are presently of cosmological interest should have been trans - planckian scales at the beginning of inflation , and , therefore , can not be handled satisfactorily without ( the correct ) quantum gravity [ 134 , 393 , 197 ] . in extrapolating the evolution of cosmological perturbations according to linear theory to very early times ,
we are implicitly making the assumption that the theory remains perturbative to arbitrarily - high energies . and
it is easy to see that the expected new physics at the planck scale could affect our predictions .
for example , if there was a sharp planck - scale cutoff in the theory , then , if inflation lasts many e - folding , the modes which represent fluctuations on galactic scales today would not be present in the theory since their wavelength would have been smaller than the cutoff length at the beginning of inflation . while in the long run this might get very exciting , i feel we are at present only at a very early stage of exploration of the potentialities of this opportunity for quantum - spacetime phenomenology .
but there is growing awareness of this opportunity and the related literature starts to grow large ( see , e.g. , refs .
[ 393 , 197 , 435 , 136 , 320 , 172 , 410 , 217 , 266 , 198 , 321 , 145 , 274 , 510 , 383 , 135 ] , and references therein ) .
many of these studies [ 136 , 172 , 410 , 217 , 266 , 198 , 321 , 274 ] have probed the possibility that a short - distance cutoff might leave a trace in cosmology measurements such as the ones conducted on the cosmic microwave background . among the scenarios that have so far been considered in relation to the trans - planckian problem ,
the ones that are more directly linked with the study of spacetime quantization are those involving planck - scale modifications of the dispersion relation ( see , e.g. , refs .
for example , one may consider the possibility of dispersion relations with a trans - planckian branch , where energy increases with decreasing momenta , such as 96\documentclass[12pt]{minimal }
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\begin{document}$${\omega ^2 } \simeq { k^2 } - { \alpha _ 4}{k^4 } + { \alpha _ 6}{k^6}$$\end{document } for appropriate choices of the parameters 4 and 6 .
a radiation - dominated universe with particles governed by such modified dispersion relations ends up being characterized by negative radiation pressure and remarkably may be governed by an inflationary equation of state , even without introducing an inflaton field . these results ( and those of refs .
[ 248 , 318 , 437 , 273 ] ) establish a connection with previous attempts at replacing inflation by scenarios accommodating departures from lorentz symmetry , such as the scenario with a time - varying speed of light , introduced in works by moffat and in works by albrecht and magueijo ( see also ref . ) . by postulating an appropriate time variation of the speed of light
one can affect causality in a way that is somewhat analogous to inflation : very distant regions of the universe , which could have never been in causal contact with a time - independent speed of light , could have been in causal contact at very early times if at those early times the speed of light was much higher than at the present time . as argued in the recent review given in ref .
, this alternative to inflation is rather severely constrained but still to be considered a viable alternative to inflation . also relevant for cosmology
are the mentioned studies suggesting that spacetime quantization could effectively produce spacetime fuzziness / foam amenable to description in terms of a fluctuating spacetime metric [ 206 , 557 , 460 ] ( see also refs . [ 133 , 525 ] ) .
such fluctuations of the light - cone have implications for the arrival times of signals from distant sources that would result in a broadening of the spectra .
it was observed in ref . that starting with a thermal spectrum one would end up with a slightly different spectrum .
this can be summarized in a simple phenomenological formula for spectrum distorsion 97\documentclass[12pt]{minimal }
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\begin{document}$$f(\omega ) = { f_0}(\omega)[1 + f(\omega)],$$\end{document } where f0( ) is the spectrum expected without the light - cone fluctuation effects and f( ) encodes the corrections due to the lightcone fluctuations .
provides arguments in support of the possibility that the corrections due to lightcone fluctuations could get very large at large frequencies , with f( ) growing like . as we achieve better and better accuracy in the measurement of possible high - frequency departures from a thermal spectrum for the cosmic microwave background , we should then find evidence of such effects .
ref . also explored the possibility that lightcone fluctuations might have observable implications for the gravitational - wave background .
the gravitational - wave background is always emitted much before the cosmic microwave background , but it was found that the flat nature of the gravitational - wave - background spectrum is such that the effects of lightcone fluctuations are negligible .
an area of quantum - spacetime cosmology that is not directly linked to the tools and scenarios considered in other areas of quantum - spacetime phenomenology is the loop quantum cosmology ( lqc ) [ 125 , 94 , 126 , 92 ] .
this is a framework for implementing several effects seen to arise for the quantum spacetime of lqg in a cosmological setting
and one finds that the characteristic discreteness of the lqg spacetime quantization change the dynamics of expanding universe models .
these changes are particularly significant at high densities , giving rise to mechanisms avoiding classical singularities .
and one can also consider the novel quantum - spacetime effects at later stages of the universe expansion , when densities are lower and the corrections can be treated perturbatively in a gauge - invariant way .
this can be done in particular for linear perturbations around spatially flat friedmann - robertson - walker models , and the results are found to be primarily characterized in terms of inverse - volume corrections , due to the fact that the quantized densitized triad has a discrete spectrum , with the value zero contained in the spectrum .
essentially one finds that the lqg quantum - spacetime effects can be effectively described in terms of a novel repulsive force .
this repulsion can compete with the standard gravitational attraction , and can even become the dominant contribution , thereby evading the singularity , when the curvature is strong . in refs .
[ 126 , 92 , 127 ] , and references therein , readers can find a list of possible signatures of lqc . in my opinion , at present , such tests of predictions of lqc may tell us more about the choice of setup for incorporating the quantum - spacetime effects , rather than providing actual information on the quantum structure of spacetime .
but as this novel approach keeps maturing , it may well turn into a key resource for experimentally probing the quantum structure of spacetime . as mentioned earlier in this review ,
several formalisms relevant to the study of the quantum - gravity problem have recently been shown to host the mechanism of running spectral dimensions .
the spectral dimension of a spacetime is essentially defined by considering a fictitious diffusion process , with the spectral dimension given in terms of the average return probability for given ( fictitious ) diffusion time .
when the number of spectral dimensions matches the number of hausdorff dimensions of a spacetime , the return probability depends on diffusion time in a characteristic way that is indeed found in all models for large diffusion times .
but at short diffusion times one finds in several studies of interest for quantum - gravity and quantum - spacetime research that the average return probability has properties signaling a number of spectral dimensions smaller than the number of hausdorff dimensions .
first results of this type were found in studies done within the framework of causal dynamical triangulations , naturally working with four hausdorff dimensions and finding that the behavior at small diffusion times signaled two spectral dimensions .
two spectral dimensions for small diffusion times was then also found in studies inspired by asymptotic safety [ 360 , 467 ] and studies based on hoava - liftschitz gravity . a somewhat different situation is found in studies inspired by spacetime noncommutativity [ 110 , 87 , 31 ] and by spin foams , but still giving fewer than four spectral dimensions for small diffusion times ( also see refs . [ 506 , 153 ] ) .
these running spectral dimensions could have very significant implications for cosmology , as suggested at least intuitively by the definition of spectral dimensions based on the dependence of the return probability on the diffusion time .
at present these potentialities are still largely unexplored , but one possibility has been debated in refs .
, citing as motivation some of the quantum - gravity studies exhibiting running spectral dimensions , proposed that such studies should motivate the search for indirect evidence of the absence of gravitational degrees of freedom in the early universe .
more prudently observed that gravitational degrees of freedom are indeed absent in spacetimes with three or fewer hausdorff dimensions , but may well be present in spacetimes with four hausdorff dimensions but with three or fewer spectral dimensions .
so far in this section , consistent with the main goals of this review , i have focused on cosmology proposals that are based on ( or at least directly linkable to ) some theories of quantum spacetime . in this last part of the section ,
i mention just a few illustrative examples of ideas and proposals that are still based on the quantum - gravity problem , but without invoking any definite quantum properties of spacetime .
it is not unlikely that future exploitations of the associated phenomenological opportunities would involve spacetime quantization .
an active area of quantum - gravity cosmology focuses on lorentz - violating vector fields .
these studies do not have as reference scenarios for spacetime quantization , but they are being linked generically to the opportunities that the quantum - gravity problem provides for the emergence of lorentz - violating vector fields .
several possible implications are being considered , including the possibility that in the presence of such lorentz - violating vector fields the universe might experience a slower rate of expansion for a given matter content ( see , e.g. , ref . ) .
it is also emerging that the implications of such lorentz - violating vector fields would be rather significant for the cosmic microwave background .
in particular , as stressed in other parts of this review , the presence of lorentz - violating vector fields is often associated with energy - dependent birefringence . and the cosmic microwave background ,
since its radiation originates from the surface of last scattering , which is the most distant source of light , can be a very powerful opportunity to test anomalous features for the propagation of photons .
several techniques of data analysis have been developed that are capable of constraining the birefringence of photon propagation using cosmic - microwave - background data ( see , e.g. , refs.[317 , 315 , 344 , 270 ] and references therein ) .
cosmology is also an arena where some interest is attracted by studies of the semiclassical limit of quantum gravity .
these do not invoke a quantum - spacetime picture and may not even rely on any given quantum - gravity proposal .
they are rather viewed in analogy with the semiclassical limits of other quantum theories : one can consider , for example , the corrections to the classical maxwell action described by heisenberg and euler ( in a pre - qed era ) in terms of quantum fluctuations of electrons and positrons , which can now be rederived from qed by integrating out the fermions and expanding in powers of . these studies of the semiclassical limit of quantum gravity are often centered around the wheeler - dewitt equation . while for the development of a full quantum - gravity theory the wheeler - dewitt equation has proven to be extremely cumbersome , the fact that it is rather intuitively formulated is convenient for setting up a semiclassical approximation ( see , e.g. , refs .
a result that can be rather readily analyzed from a phenomenology perspective is the one providing correction terms for the schrdinger equation , obtained through a formal expansion of the wheeler - dewitt equation with respect to powers of the planck mass .
however , it is plausible that such a procedure could give rise to observably large effects in the description of the early stages of the evolution of the universe . in particular , the semiclassical approximation set up in ref .
could be used rather straightforwardly to describe corrections to the schrdinger equation for higher multipoles on a friedman background .
an interesting string - theory - inspired area of cosmology research revolves around a scenario for singularity avoidance linked to the availability of duality tranformations , which allow one to set up a suitable pre - big - bang scenario [ 253 , 143 , 142 ] . in this scenario
the universe starts inflating from an initial state characterized by very small curvature and weak interactions .
the small - curvature initial state is gravitationally unstable and would naturally evolve [ 253 , 143 , 142 ] into states with higher curvature , until string - size ( roughly planck - scale - size ) effects are strong enough to induce a bounce into a decreasing - curvature regime . instead of a conventional hot big bang
one would have [ 253 , 143 , 142 ] a hot big bounce in which in particular the heating mechanism is provided by the quantum production of particles in the pre - bounce phase characterized by high curvature and strong interactions . for this string - inspired pre - big - bang scenario several possible observational consequences have been discussed [ 253 , 143 , 142 ] , including the one of a stochastic background of gravity waves due to a background of gravitons from the pre - big - bang phase .
it appears to be plausible [ 253 , 143 , 142 ] that the magnitude of the associated effects might be within the range of sensitivities of modern gravity - wave interferometers .
most of the ideas for phenomenology i here reviewed are set up following a common strategy .
they reflect the expectation that the characteristic scale of quantum - spacetime effects should be within a few orders of magnitude of the planck scale , and that it should be possible ( for studies conducted at scales much below the planck scale ) to analyze quantum - spacetime effects using an expansion in powers of the planck length .
all this is inspired by analogous strategies that have been very fruitful in other areas of physics : many arguments indicate that the planck scale is the scale where the current theories break down , and usually the breakdown scale is also the scale that governs the magnitude of the effects of the new needed theory . in the case of quantum - spacetime research
the expectation of perturbative effects suppressed by a large scale finds further motivation in at least two observations :
the effects we expect from spacetime quantization are rather striking , qualitatively virulent departures from the structure of our current theories .
the fact that no trace of such easily noticeable effects has ever been seen surely provides further encouragement for the expectation of perturbative effects suppressed by an ultralarge scale.i would list the evidence in favor of grand unification as an even more significant source of additional encouragement for the expectation of perturbative effects suppressed by an ultralarge scale .
if that evidence is taken at face value ( as , i would argue , we should , at least as a natural working assumption ) it suggests that particle physics works well on its own up to a scale of about 10 the planck scale .
if quantum - spacetime effects were non - perturbative in ways affecting grand unification or if the scale of spacetime quantization was much lower than the planck scale , it would then be hard to explain the ( preliminary ) success of the grand - unification idea .
the effects we expect from spacetime quantization are rather striking , qualitatively virulent departures from the structure of our current theories .
the fact that no trace of such easily noticeable effects has ever been seen surely provides further encouragement for the expectation of perturbative effects suppressed by an ultralarge scale .
i would list the evidence in favor of grand unification as an even more significant source of additional encouragement for the expectation of perturbative effects suppressed by an ultralarge scale . if that evidence is taken at face value ( as , i would argue , we should , at least as a natural working assumption ) it suggests that particle physics works well on its own up to a scale of about 10 the planck scale .
if quantum - spacetime effects were non - perturbative in ways affecting grand unification or if the scale of spacetime quantization was much lower than the planck scale , it would then be hard to explain the ( preliminary ) success of the grand - unification idea . in light of this ,
surely quantum - spacetime phenomenologists should continue to focus most of their efforts on applications of the standard strategy , assuming perturbative effects suppressed by a scale in some neighborhood of the planck scale .
we are clearly presently unable to exclude that the correct quantum picture of spacetime might turn out to be unsuitable to the standard strategy of quantum - spacetime phenomenology . as a way to give some substance to this assessment
, i briefly discuss examples of mechanisms that could render ineffective the standard strategy of quantum - spacetime phenomenology .
can the scale characteristic of quantum - spacetime effects be much lower than the planck scale ?
we surely know at least one mechanism by which the quantum - gravity scale can be much lower than the planck scale , and therefore quantum - gravity models with spacetime quantization affected by this mechanism would describe quantum - spacetime effects at a relatively low scale .
one can achieve a sizeable reduction in the quantum - gravity scale with the introduction of d extra space dimensions [ 80 , 375 , 552 , 84 ] of finite size r*. then the fundamental length scale ld characteristic of quantum gravity in the 3+d+1-dimensional spacetime can be much bigger than the planck length .
the smallness of the planck length can emerge as the result of the fact that , as deduced from applying gauss s law in the 3+d+1-dimensional context , the strength of gravitation at distance scales larger than the size r * of the extra dimensions in the ordinary ( infinite - size ) 3 + 1-dimensional spacetime would be proportional to the square - root of the inverse of the volume of the external compactified space multiplied by an appropriate power of ld .
these scenarios need to be tuned rather carefully in order to get a phenomenologically - viable picture .
essentially the only truly appealing possibility is the one of 2 extra dimensions of relatively large size , somewhere below millimeter size ( perhaps 10 or 10 meters ) .
there might be other extra dimensions of smaller ( possibly planckian ) size , but for the desired phenomenology one needs two and only two extra dimensions of relatively large size ; otherwise one finds effects that either violate known experimental facts or are too small to ever be tested . but with these ( however contrived ) choices one does end up with a phenomenologically - exciting scenario in which the fundamental length scale of quantum gravity ld is somewhere in the neighborhood of the ( tev ) length scale , and therefore within the reach of particle - physics experiments ( see , e.g. , refs .
[ 260 , 258 , 207 , 82 ] ) . moreover , there are phenomenologically - relevant implications for the behavior of ( classical ) gravity at submillimeter distances [ 84 , 295 ] .
the large - extra - dimension scenario is an example of inapplicability of the standard setup of quantum - spacetime phenomenology due to the fact that , within that scenario , the characteristic scale of quantum gravity is not the planck scale .
there are also scenarios in which one may still assume that quantum - spacetime effects are fundamentally introduced at the planck scale , but the standard setup of quantum - spacetime phenomenology is inapplicable because the most characteristic effects are not describable in terms of an expansion in powers of the planck length .
i have already discussed this possibility in section 5 , devoted to soft uv / ir mixing . however , in that context one could fall back on roughly the standard strategy of quantum - spacetime phenomenology , by looking for an ir scale playing the role of characteristic scale of the ir manifestations of the quantum properties of spacetime .
let me just stress that an even more pervasive revision of the standard strategy of quantum - spacetime phenomenology would be required in the case of hard uv / ir mixing , which might take the form of correction terms with the behavior of inverse powers of momentum . with hard uv
/ ir mixing one should expect that in certain contexts the departures from known physical laws should be dramatic .
the most efficacious tests of this hypothesis might not take the shape of searches of small corrections to standard predictions in ordinary contexts , but rather be based on the identification of those peculiar contexts where the implications of uv / ir mixing are large .
some challenges for the standard setup of quantum - spacetime phenomenology may also be present when the effects are genuinely introduced at the planck scale and there is nothing peculiar about the ir sector .
in particular , just because this standard setup is based on a ( truncated ) expansion in powers of the planck length , it can happen that the formally sub - leading terms ( higher powers of the planck length ) , which are usually neglected in leading - order analyses , are actually not really negligible .
the fact that experiments suitable for quantum - spacetime phenomenology must host , as i stressed in several points of this review , some ultralarge ordinary - physics dimensionless amplifiers could play a role in these concerns : if some mechanism is allowing the tiny leading - order planck - length correction to be observably large it would not be so surprising to find that the same ( or some other ) amplifier is also such that some formally subleading planck - length corrections , neglected in the analysis , are significant . and another possible source of concern can originate from the fact that some of the contexts of interest for quantum - spacetime phenomenology are characterized by several length scales : expansions in powers of the planck length actually are expansions in powers of some dimensionless quantity obtained dividing the planck length by a characteristic length scale of the physical context of interest , and some pathologies may be encountered if there are several candidate length scales for the expansion . while i feel that these issues for the power expansion should not be ignored , it is partly reassuring that the only explicit examples we seem to be able to come up with are rather contrived .
for example , in order to illustrate the issues connected with the many length scales available in certain contexts of interest for quantum - spacetime phenomenology , i can not mention anything more appealing than the following ad hoc formulation of a deformation of the speed - energy relation applicable in the
relativistic regime ( e m ) : 98\documentclass[12pt]{minimal }
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\begin{document}$$v \simeq 1 - { { { m^2 } } \over { 2{e^2 } } } + \eta { l_p}e\left({\tanh \left({{{l_p^2{e^6 } } \over { { m^4 } } } } \right ) - 1 } \right).$$\end{document } at low ( but still relativistic ) energies this would fit within a picture that has been much studied from the quantum - spacetime - phenomenology perspective , that of the speed - energy relation v 1 m/(2e ) lpe . but whereas in the relevant literature it is assumed that the term lpe , if present , should always be the leading correction , up to particle energies on the order of the planck scale , e 1/lp , from eq .
( 98 ) one would find that the correction term is already no longer leading at particle energies on the order of e ( m / lp)/ , i.e. , well below the planck scale . here
( 98 ) is characterized by two distinct small quantities suitable for the expansion in powers of lp : the quantity lpe and the quantity lpe / m .
clearly , the most significant development of these first few years of quantum - spacetime phenomenology has been our ability to uncover some experimental / observational contexts in which , through appropriate data analyses , we could gain access to effects introduced genuinely at the planck scale . the compellingness of such instances of genuine planck - scale sensitivity , which are most simply and clearly illustrated in section 1.5 , should be contrasted to the more frequent case of dimensional - analysis planck - scale sensitivities , which typically involve a description of a plausible quantum - spacetime effect in terms of a dimensionless parameter , estimated arbitrarily as a ratio of the planck length and some characteristic length scale of the problem .
looking at the results summarized in this review , different readers , depending on how stringent their criteria for genuine planck - scale sensitivity , will only recognize one or two examples .
, as stressed , we do have , at this point , a rather encouraging list of contexts in which , while the availability of genuine planck - scale sensitivity has still not been fully established , it appears that sensitivity to effects introduced genuinely at the planck scale could be achieved in a not - so - distant future .
the fact that the development of this phenomenology is proving beneficial for the study of the idea of spacetime quantization is perhaps best testified by the fact that it is already managing to truly affect the directions taken by more formal work on spacetime quantization , especially in the areas of lqg and spacetime noncommutativity .
theorists in these areas follow the developments on the phenomenology side and do their best ( the technical challenges they are facing are very severe ) to derive results that can be exploited for the opportunities in phenomenology that are being established . in turn
the phenomenology takes notice of the developments on the theory side , finding in them new input for enlarging the list of candidate quantum - spacetime effects that one could attempt to investigate experimentally .
the goal of testing / falsifying rigorous theories of spacetime quantization appears to still be beyond our present reach .
but while most of the work in quantum - spacetime phenomenology so far has relied on simple - minded test theories describing candidate quantum - spacetime effects , i see first indications of a phase of further maturation of this phenomenology , in which we will actually test / falsify at least the most virulent rigorous formalizations of quantum spacetime .
planck - scale theories formulated in noncommutative versions of minkowski spacetime are the example where we are presently closer to this goal .
the ( however limited ) information presently available to us appears to provide a clear invitation to continue to focus most of our efforts in the search for effects describable in terms of a ( low - energy ) expansion in powers of the planck length , though other opportunities clearly should not be overlooked . concerning the type of data on which quantum - spacetime phenomenology can rely , i have attempted to maintain throughout this review some visible separations between different proposals on the basis of whether they concern astrophysics , cosmology or controlled laboratory experiments .
it is very clear that astrophysics has so far provided the most fruitful arena , but cosmology has the greatest potential reach ( although for the most part this potential has not yet materialized ) .
the role played so far in quantum - spacetime phenomenology by controlled laboratory experiments is rather marginal , but it would be important for the future development of quantum - spacetime phenomenology to find more opportunities for controlled laboratory experiments . | i review the current status of phenomenological programs inspired by quantum - spacetime research .
i stress in particular the significance of results establishing that certain data analyses provide sensitivity to effects introduced genuinely at the planck scale .
my main focus is on phenomenological programs that affect the directions taken by studies of quantum - spacetime theories . | Introduction and Preliminaries
Quantum-Gravity Theories, Quantum Spacetime, and Candidate Effects
Quantum-Spacetime Phenomenology of UV Corrections to Lorentz Symmetry
Other Areas of UV Quantum-Spacetime Phenomenology
Infrared Quantum-Spacetime Phenomenology
Quantum-Spacetime Cosmology
Quantum-Spacetime Phenomenology Beyond the Standard Setup
Closing Remarks | in particular , only over this recent period do we have the first cases of phenomenological programs that truly affect the directions taken by more formal work in quantum gravity . together with some ( however slow ) progress toward establishing the ability to falsify models and discriminate between models , the phenomenology work of this past decade
has also shown that the handful of examples of planck - scale sensitivities that generated excitement between 1997 and 2000 were not a one - time lucky streak :
the list of examples of experimental / observational contexts in which sensitivity to some effects introduced genuinely at the planck scale is established ( or found to be realistically within reach ) has continued to grow at a steady pace , as the content of this review will indicate , and the number of research groups joining the quantum - spacetime - phenomenology effort is also growing rapidly . it may be useful to now provide a simple example of analysis that illustrates some of the concepts i have discussed and renders more explicit the fact that some of the sensitivity levels now available experimentally do correspond to effects introduced genuinely at the planck scale . the analysis of sensitivities was the traditional exercise a decade ago , in the early days of modern quantum - spacetime phenomenology , since the key objective then was to establish that sensitivity to effects introduced genuinely at the planck scale is achievable . the largest area of quantum - spacetime - phenomenology research concerns the fate of lorentz ( /poincar ) symmetry at the planck scale , focusing on the idea that the conjectured new effects might become manifest at low energies ( the particle energies accessible to us , which are much below the planck scale ) in the form of uv corrections
, correction terms with powers of energy in the numerator and powers of the planck scale in the denominator . this is a very powerful tool for quantum - spacetime phenomenology [ 327 , 38 , 73 , 463 , 512 , 364 , 307 , 494 ] , and , in fact , at the beginning of this review , i chose the evaluation of the threshold energy for photopion production , p + cmber p + , as the basis for illustrating how the sensitivity levels that are within our reach can be placed in rather natural connection with effects introduced at the planck scale . over this past decade
there has been growing awareness of the fact that data analyses with good sensitivity to effects introduced genuinely at the planck scale are not impossible , as once thought . in approaches based on local field theory ,
it is natural to perform combined studies of cpt and lorentz symmetry.31 however , the notion of spacetime quantization at the planck scale involves some aspects of nonlocality ( at least the notion of points that coincide with accuracy better than the planck length is typically abandoned ) and in most quantum - spacetime studies of the fate of cpt symmetry the expectation is that these aspects of non - locality may be primarily responsible for the conjectured violations of cpt symmetry . i should stress that this is clearly a quantum - spacetime picture ( at least in as much as it models spacetime foam ) and the objective of the associated research program is to introduce quantum / foamy properties of spacetime at the planck scale , but it is at present still unclear which levels of sensitivity to , , would correspond to foaminess of spacetime at the planck scale . from the strict quantum - spacetime - phenomenology perspective of requiring one to establish that the relevant measurements could be sensitive to effects introduced genuinely at the planck scale , these neutrino - decoherence test theories must face challenges already discussed for a few other test theories : there is at present no rigorous / constructive derivation of the values of the parameters of these test theories from a description ( be it a full quantum - spacetime theory or simply a toy model ) of effects introduced genuinely at the planck scale , so one can only express these parameters in terms of the planck scale using some dimensional - analysis arguments . i am focusing in this review on tests motivated by ( and on effects modeled within ) proposals of spacetime quantization at the planck scale , but concerning tests of the equivalence principle inspired by quantum - spacetime models there is some merit in making a small digression on tests of the equivalence principle in the semiclassical limit of quantum gravity ( where , by construction , no quantum - spacetime effects could be seen ) . some challenges for the standard setup of quantum - spacetime phenomenology may also be present when the effects are genuinely introduced at the planck scale and there is nothing peculiar about the ir sector . clearly , the most significant development of these first few years of quantum - spacetime phenomenology has been our ability to uncover some experimental / observational contexts in which , through appropriate data analyses , we could gain access to effects introduced genuinely at the planck scale . , as stressed , we do have , at this point , a rather encouraging list of contexts in which , while the availability of genuine planck - scale sensitivity has still not been fully established , it appears that sensitivity to effects introduced genuinely at the planck scale could be achieved in a not - so - distant future . | [
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] |
the primary function of the immune system is effective protection against various types of infectious agents from the external environment .
the first line includes anatomical and physiological barriers that protect the inner layers of the body .
this natural microbial population competes with pathogens for nutrients and produces antibacterial substances to inhibit pathogen growth .
the protective role for the respiratory system is played by cough and sneeze reflexes , movement of cilia and pulmonary surfactant . in turn ,
the digestive system is protected by intestinal motility , presence of glycocalyx on the surface of epithelial cells , including enterocytes , and the low ph of the stomach .
the gastric juice , vaginal secretions and urine , which are acidic fluids , have an additional protective function . equally important
this group includes proteolytic enzymes , acute phase proteins , interferons , immunoglobulins and also antimicrobial peptides .
innate immunity is mediated by several humoral components ( complement proteins , some cytokines ) and cellular mechanisms involving neutrophils , monocytes , macrophages , mast cells , eosinophils , dendritic cells , and nk cells . finally , the third line of defense against pathogenic invasion is the adaptive immune response which involves expansion of the lymphocyte subpopulations and production of antibodies . in
the first steps of host defense against pathogens , antimicrobial peptides ( amps ) play an important role .
these peptides , also called natural antibiotics or cationic host defense peptides ( chdps ) , constitute a group of relatively small ( 12 - 50 amino acid residues ) molecules .
amps are found in both prokaryotes and eukaryotes and are produced by bacteria , plants , invertebrates , and vertebrates . to date , 2544 amps have been reported ( based on the antimicrobial peptide database ) .
these small molecules have a positive charge due to the presence of arginine and lysine residues and amphipathic structure .
chdps are able to kill microbes such as bacteria , viruses , fungi or protozoa in a very short time [ 13 ] .
various groups of organisms produce amps with different profiles . in mammals , two major groups of amps are defensins and cathelicidins .
defensins are classified into three subgroups : , and . the -defensin family has been identified in humans , monkeys , pigs , and several rodent species . so far , six distinct -defensins have been found in humans .
four of these were initially isolated from neutrophils , and therefore they are called human neutrophil peptides ( hnp-1 , hnp-2 , hnp-3 , and hnp-4 )
the human -defensins hd-5 and hd-6 are expressed mainly in paneth cells of the small intestine .
moreover , close -defensin homologs were found in snakes , platypus and sea anemones . in humans , -defensins
( hbd-1 , hbd-2 , hbd-3 ) are produced mainly by epithelial cells and keratinocytes .
-defensin family is found in the leukocytes of rhesus macaques [ 3 , 4 ] .
cathelicidins have been isolated from many mammalian species including rabbits , horses , pigs , rats , monkeys , cattle and humans .
the genes responsible for synthesis of cathelicidins are approximately 2 kb and show four - exon / three - intron organization .
the structure of precursor includes a highly conserved n - terminal segment , cathelin domain and a c - terminal domain with a varied sequence and length .
the n - terminal domain is composed of 29 - 30 amino acid residues and supports the release of biologically active peptides .
the cathelin domain is composed of 98 - 114 amino acid residues but its function has not been thoroughly examined . the c - terminal peptide ( 12 - 100 amino acid residues ) , as a mature peptide , exhibits broad antimicrobial activity against both gram - positive and gram - negative bacteria , viruses and fungi .
these peptides are produced by neutrophils , epithelial cells , keratinocytes , macrophages , mast cells , nk cells , dendritic cells , and lymphocytes [ 5 , 6 ] .
the only known member of the cathelicidin family expressed in humans is ll-37 ( leucine - leucine-37 ) .
this cationic molecule is a linear 37-amino acid - long peptide generated by cleavage of the c - terminus end of the hcap18 ( human cationic antimicrobial peptide 18 ) precursor protein .
the name hcap18 denotes the length of polypeptide ( 18 kda ) and the cationic character of the structure . in turn
, ll-37 refers to the number of residues with leu - leu at the n - terminus . ll-37 and
the human cathelicidin is produced mainly by neutrophils , monocytes , nk cells , mast cells , b cells and also colon enterocytes , epithelial cells and keratinocytes .
the molecule was detected in human wound fluid , seminal plasma , vernix and tracheal aspirates of newborns .
cathelicidins may be produced constitutively or synthesized in response to the presence of bacteria or their products .
the expression of ll-37 can be also regulated by various endogenous factors such as proinflammatory cytokines , growth factors and the active form of vitamin d .
cathelicidins exhibit a broad spectrum of antimicrobial activity against gram - positive and gram - negative bacteria [ 1 , 7 ] , enveloped viruses , and fungi .
it is commonly known that cathelicidins interact with the negatively charged bacterial and fungal membranes and induce membrane disruption .
the most accepted mechanisms of cathelicidin action are the carpet model , the barrel - stave model , and the toroidal pore model [ 9 , 10 ] . according to the carpet model ,
the pressure from the molecules is so strong that it causes a disruption of the cell membrane . in
mechanism , peptides are incorporated perpendicularly at the membrane creating the barrel with the peptides being the staves .
non - polar components of proteins interact with membrane lipids and polar parts form the pores in the cell membrane .
theory describes the formation of pores in the cell membrane where the hydrophilic components interact with the residues of phospholipids , while the hydrophobic parts with the lipids .
the effect of all mechanisms is the disintegration of the cell membrane and as a result , death of the pathogen .
additionally , cathelicidins may penetrate into the cell to bind or to degrade some molecules which are crucial to cell living .
furthermore , these peptides participate in neutralization of bacterial endotoxin , i.e. lipopolysaccharide ( lps ) , by blocking the interaction of the lipoglycan with lps binding protein ( lbp ) .
nowadays , however , more and more data indicate that apart from killing pathogens directly or indirectly , cathelicidins have a number of immunomodulatory functions that might be involved in the clearance of infection .
thus , considering the role of inflammation in host defense against pathogens it is extremely interesting to know and understand the influence of cathelicidins on inflammatory cell activities .
neutrophils are the most abundant type of white blood cells and it is absolutely proven that they are the first leukocytes rapidly and efficiently recruited to the site of injury and inflammation .
these cells are capable of expressing and/or producing various potent mediators , including numerous cytokines , chemokines , colony - stimulating factors , angiogenic factors and fibrogenic factors .
what is more , neutrophils express a lot of enzymes stored in different types of cytoplasmic granules .
it should be stressed that neutrophils also produce four types of -defensins , i.e. hnp-1 , hnp-2 , hnp-3 , and hnp-4 , accumulated in a subset of azurophilic granules .
moreover , neutrophils are the source of arachidonic acid metabolites , i.e. prostaglandins ( pgs ) and leukotrienes ( lts ) . finally , it is well known that neutrophil granulocytes are able to generate reactive oxygen species ( ros ) .
all these neutrophil - derived mediators influence extracellular matrix ( ecm ) components and strongly affect the surrounding cell activity , thereby neutrophils play a crucial role in acute and chronic inflammation .
in addition , neutrophil - derived mediators have a huge impact on immune cells functioning and consequently these cells participate in the course of innate and adaptive immunity .
it should be emphasized that neutrophils play a key role in host defense against viral , bacterial and fungal infections [ 1214 ] .
the average circulatory lifespan of human neutrophils is relatively short ( 5.4 days ) and ends by spontaneous apoptosis .
apoptotic neutrophils are phagocytosed without release of mediators , leading to the restriction of inflammation and tissue damage .
it should be noted that some host- and pathogen - derived substances may inhibit spontaneous neutrophil apoptosis .
the suppressed neutrophil apoptosis results in uncontrolled release of a variety of cytotoxic metabolites and pro - inflammatory mediators , which in turn leads to the enhancing of inflammatory response and tissue injury .
considering these data , observations that cathelicidins may influence neutrophil lifetime are of great importance .
[ 16 , 17 ] clearly demonstrated that ll-37 prolongs the lifespan of neutrophils by inhibition of spontaneous apoptosis .
inhibition of programmed cell death occurs through the increased expression of anti - apoptotic protein bcl - xl and by blocking the activation of caspase-3 , a key executor for apoptosis . of importance ,
human cathelicidin ll-37 elongates neutrophil lifespan in vitro over the concentration range from 10 ng / ml to 5 g / ml . interestingly , ll-37 induces suppression of neutrophil apoptosis acting via the activation of formyl peptide receptor ( fpr)1 and purinergic receptor p2x7 [ 17 , 18 ] .
cathelicidin ll-37 can also up - regulate the expression of p2x7 receptors on these cells .
it was also documented that in vitro ll-37 induces neutrophil migration and chemotaxis and this effect is mediated via fpr1 molecules [ 1922 ] . what is more , dose - dependent neutrophil migration in response to murine cathelicidin - related antimicrobial peptide ( cramp)-stimulation was observed .
porcine proline - arginine ( pr)-rich antibacterial peptide pr-39 induces chemotaxis of neutrophils , with a maximal response at 0.5 - 2 m , and stimulates extracellular ca influx in these cells , as well .
interestingly , ll-37 causes almost complete inhibition of serum amyloid a ( saa)-induced neutrophil chemotaxis .
it seems to be very important to note down that cathelicidin ll-37 reduces neutrophil surface expression of cxcr2 , but not cxcr1 [ 19 , 26 ] , i.e. the expression of receptor that mediates neutrophil migration to the sites of inflammation .
the mechanism of cxcr2 down - regulation by ll-37 is due to the receptor internalization .
human cathelicidin , at a concentration of 20 g / ml , stimulates neutrophils to the synthesis of pro - inflammatory cxcl8 under the control of mitogen - activated protein kinase ( mapk ) p38 and extracellular signal - regulated kinase ( erk ) [ 26 , 27 ] .
it is well known that this chemokine , acting via cxcr2 , induces chemotaxis of not only neutrophils but also other granulocytes , and stimulates neutrophils to phagocytosis . on the other hand ,
interesting data suggest that ll-37 inhibits saa - induced cxcl8 production and causes dramatic inhibition of erk and p38 mapk activities . in turn ,
ll-37 activates neutrophils to the release of interleukin-1 receptor antagonist ( il-1ra ) the natural antagonist of il-1 and at the same time induces a dose - dependent increase in hnp-1 , hnp-2 , and hnp-3 -defensin gene expression .
what is more , extracellular release of these -defensins in response to ll-37 stimulation was observed .
finally , ll-37 stimulates the generation of ros in neutrophils [ 2729 ] and this process is mediated by a flavoenzyme ( most probably nadph oxidase ) and via increase in intracellular ca concentrations .
without any doubt , in the course of acute and chronic inflammation an important role is played by monocytes and macrophages . given that these cell populations can produce both pro - inflammatory and anti - inflammatory mediators , it is obvious that they strongly direct the inflammatory processes and significantly influence an intensity of inflammation .
monocytes and macrophages have the ability to secrete a broad range of pro - inflammatory cytokines , such as il-1 , il-6 , il-12 , il-18 , il-23 , il-27 , and tumor necrosis factor ( tnf )
. these cells also produce various chemokines ( ccl2 , ccl5 , ccl7 , cxcl1 , cxcl2 , cxcl8 , cxcl9 , cxcl10 , cxcl11 ) as well as pgs , lts , and platelet activating factor ( paf ) .
moreover , activated macrophages synthesize ros and nitrogen intermediates that are highly toxic for microorganisms , but also induce tissue injury and lead to enhance inflammation . finally , monocytes / macrophages are the source of various proteolytic enzymes that can act directly on cells and ecm proteins leading to tissue damage . on the other hand , there are a lot of monocyte / macrophage - derived mediators involved in suppressing inflammation , including some anti - inflammatory cytokines , i.e. il-4 , il-10 , il-13 , il-19 , and growth factors , i.e. transforming growth factor ( tgf)- and vascular endothelial growth factor ( vegf ) .
in fact , macrophages can exhibit pro- or anti - inflammatory properties and this is determined by stimuli from their local microenvironment . thus , due to their cytokine / mediator profile two evidently distinct types of macrophages are designated .
m1 macrophages , differentiated under the influence of granulocyte - macrophage colony - stimulating factor ( gm - csf ) , with a pro - inflammatory signature , and m2 macrophages , differentiated under the influence of macrophage colony - stimulating factor ( m - csf ) , with an anti - inflammatory signature [ 30 , 31 ] .
it is well established that human cathelicidin ll-37 activates fpr2 molecules on monocytes , triggering monocytic 1- and 2-integrin conformational change toward active conformation that allows for monocyte adhesion . as in the case of neutrophils , ll-37 acts as potent chemoattractant for monocytes and
moreover , murine cramp also causes monocyte migration in a dose - dependent manner and the peak response was observed at 400 nm .
this cramp - induced monocyte chemotaxis seems to be mediated by a g protein coupled receptor ( gpcr ) , probably fpr2 molecule .
it is interesting that murine cramp acts as a chemoattractant for monocyte - derived macrophages as well .
this cathelicidin induces erk1/2 and p38 kinase phosphorylation and activation in monocytes , acting at a concentration as high as 50 g / ml .
however , in the presence of gm - csf , a cytokine found locally at sites of infection , ll-37 induces erk1/2 phosphorylation at lower concentrations , such as 5 - 10 g / ml . more and more data indicate that cathelicidins by themselves directly stimulate monocytes to produce and release some cytokines and chemokines that may strongly modulate the course and intensity of inflammation . it was demonstrated that genes encoding chemokines essential for cell recruitment to the site of inflammation , such as cxcl1 , cxcl8 , ccl2 , and ccl7 are significantly up - regulated in monocytes activated with ll-37 [ 34 , 35 ] . on the contrary , ll-37 down - regulates cxcr2 expression on monocytes .
it is interesting that ll-37 does not enhance mrna expression of pro - inflammatory cytokines tnf , il-1 , and il-6 and causes an increase in the transcription of the genes encoding anti - inflammatory cytokines such as il-10 and il-19 .
human cathelicidin elicits modest up - regulation of ccl7 and il-6 gene expression in peripheral blood mononuclear cells ( pbmcs ) as well .
moreover , ll-37 alone activates monocytes to the production of cxcl8 and ccl2 [ 34 , 35 ] and pbmcs to synthesis of cxcl8 , ccl2 , ccl5 , and ccl7 [ 35 , 36 ] .
it was noticed , however , that this peptide stimulates the production of anti - inflammatory cytokine il-1ra but does not induce the synthesis of il-10 in pbmcs .
furthermore , there is information that cathelicidins influence monocyte responsiveness to some endogenous pro - inflammatory factors .
co - stimulation of pbmcs with ll-37 and il-1 results in substantially augmented ccl7 and il-6 mrna accumulation and leads to an increase in the production of ccl2 , ccl7 , il-6 , and anti - inflammatory cytokine il-10 .
ll-37 substantially enhances the production of cxcl8 and il-6 , but not tnf , in synergy with il-1 . in the synergistic production of ccl7 induced by ll-37 and il-1 , gpcr and phosphatidylinositol 3-kinase ( pi3k ) , but not protein kinase ( pk)c and pka , a signaling pathway is involved .
ll-37 abrogates il-32-induced tnf and il-1 production by 97% in pbmcs and il-32-induced il-6 synthesis is suppressed by 50% in the presence of ll-37 .
in contrast , il-32-mediated chemokine cxcl1 and cxcl8 synthesis is not altered in the presence of this peptide .
moreover , ll-37 does not suppress il-32-mediated production of anti - inflammatory il-1ra but inhibits il-32-mediated il-10 synthesis in pbmcs .
it is also documented that ccl2 and il-6 production by pbmcs in response to gm - scf stimulation is significantly enhanced in the presence of ll-37 .
conversely , ll-37 has no effect on tnf - induced il-1 , il-6 , and cxcl8 production .
interestingly , cathelicidin ll-37 strongly inhibits the production of tnf and il-12 by monocytes stimulated with interferon ( ifn)- and suppresses ifn--induced ccl2 synthesis in pbmcs .
ccl2 production in response to ll-37 stimulation is inhibited in the presence of il-4 or il-12 .
extremely intriguing are observations that ll-37 has a general anti - inflammatory effect on toll - like receptor ( tlr ) stimulation , inhibiting pro - inflammatory cytokine release from monocytic cells stimulated with tlr2 , tlr4 , and tlr9 agonist .
it was demonstrated that ll-37 peptide significantly inhibits the expression of specific pro - inflammatory genes up - regulated by nuclear factor ( nf)-b in the presence of tlr4 ligand lps .
in contrast , ll-37 does not inhibit lps - induced genes which antagonize inflammation and certain chemokine genes classically considered pro - inflammatory .
moreover , ll-37 , at very low concentrations ( 1 g / ml ) , inhibits tnf synthesis and release from lps - induced monocytes .
this inhibitory effect increases to 95% with a dose of 20 g / ml of ll-37 .
similarly , ll-37 significantly inhibits tnf , il-1 , il-6 , and cxcl8 production in pbmcs stimulated with lps .
it should pointed out , however , that ll-37 stimulation of lps - primed monocytes leads to release of il-1 acting via p2x7 receptor [ 34 , 40 ] , and caspase-1 activation .
furthermore , acting on pbmcs , ll-37 significantly decreases the production of il-1 , il-6 , tnf , and cxcl8 resulting from activation of tlr2 molecules by lipoteichoic acid ( lta ) or synthetic tri - acylated lipopeptide pam3csk4 .
in addition , ll-37 reduces to 50% , cxcl8 secretion from pbmcs in response to stimulation with tlr9 agonist synthetic cpg oligodeoxynucleotide .
it is interesting that ll-37 very efficiently transports self - dna into monocytes , leading to the production of type i ifns in a tlr - independent manner .
peptide ll-37 ( re)directs m - csf - driven differentiation of monocytes toward macrophages with a pro - inflammatory instead of an anti - inflammatory signature .
these macrophages have a low expression of cd163 , little of il-10 and profound il-12 production on lps stimulation .
additionally , it is proved that under the influence of ll-37 fully differentiated m2 macrophages produce less il-10 and more il-12 . in like manner
ll-37 directly up - regulates 29 genes in macrophages , including those encoding chemokine ccl7 , anti - inflammatory cytokine il-10 , m - csf , many chemokine receptors , and several metalloproteinases ( mmps ) . on the contrary
, this peptide can down - regulate another 20 genes , including those encoding ccl3 and il-12 .
there are intriguing data that peptide ll-37 can be an endogenous regulator of eicosanoid - dependent inflammatory responses since it promotes ltb4 and thromboxane ( tx ) a2 generation by macrophages .
this cathelicidin , acting via p2x7 receptor , evokes ca mobilization , activation of erk1/2 and p38 mapks , as well as cytosolic phospholipase c(pl)a2 , and 5-lipoxygenase , leading to an early ( 1 hour ) release of ltb4 .
later on , 6 - 8 hours after ll-37-stimulation , internalized cathelicidin up - regulates cox-2 expression , promoting txa2 production . ll-37
significantly decreases neisseria meningitidis endotoxin lipooligosaccharide ( los)-induced both tnf and nitric oxide ( no ) release from macrophages .
this synergistic effect of ll-37 and tlr4 ligand los is a result of a direct effect exerted on the ros generating enzymes mainly the nadph oxidase complex .
what is more , the presence of ll-37 during the polarization of m1 macrophages by lps and ifn- results in strong inhibition of tnf and no production and in a slight decrease in ros synthesis .
similarly , tnf synthesis by polarized m2 macrophages in response to lps stimulation is effectively inhibited by ll-37 .
this peptide has no effect on tnf production by macrophages activated with tlr2 synthetic ligand pam3csk4 but dramatically inhibits lta - induced tnf synthesis .
scott et al . demonstrated that ll-37 causes a substantial inhibition of not only lps , but also lta- , and arabinosylated lipoarabinomannan ( aralam)-induced tnf synthesis by macrophages .
it was also documented that murine cathelicidin cramp exerts a strong inhibition of lps- , lta- , and flagellin-(tlr5 agonist ) induced p38 and erk phosphorylation in macrophages and strongly reduced tnf production by macrophages stimulated with lps or lta .
however , murine cramp does not affect the production of il-10 and chemokines cxcl1 and ccl3 by lps - activated macrophages .
observed that ll-37 activates caspase-1 , the central enzyme of the inflammasome , in both human and murine macrophages , resulting in release of active il-1 and il-18 .
ll-37 activation the nlrp3 inflammasome utilizes p2x7 receptor - mediated potassium efflux . on the other hand ,
it is worth noting that ll-37 inhibits lps / atp - induced il-1 expression , caspase-1 activation , inflammasome formation , as well as macrophage death by pyroptosis .
these cells are particularly numerous beneath the subepithelial layers of the skin , in the airways , gastrointestinal and genitourinary tracts , and adjacent to blood vessels and nerves .
they have the potential to secrete a wide spectrum of biologically active mediators , cytokines and chemokines . upon activation ,
mast cells rapidly release preformed , cytoplasmic granule - associated mediators , such as histamine , neutral proteases ( tryptase , chymase ) , proteoglycans , mmps , as well as various preformed cytokines including il-3 , il-4 , il-6 , il-10 , tnf , and cxcl8 .
moreover , in response to stimulation mast cells release various newly generated lipid mediators , including lts , pgs , txs , and paf , and many cytokines and chemokines .
these cells express numerous different receptors and thereby various endogenous and exogenous factors can activate mast cells to mediator/ cytokine generation .
. therefore , mast cells are important players in homeostasis maintenance via involvement in angiogenesis , tissue remodeling and repair , and regulation of vascular permeability [ 50 , 51 ] .
moreover , they strongly influence both innate and acquired immune responses and are critical components of host defense against bacteria and viruses [ 5254 ] .
mast cells take part in different pathological processes , including allergic disease as well [ 50 , 51 , 55 ] .
finally , inasmuch as mast cell - derived mediators elicit pro - inflammatory , anti - inflammatory and immunoregulatory effects , these cells are essential in both promoting and limiting the inflammatory processes [ 52 , 56 , 57 ] .
it has been proven that this cathelicidin induces , in a dose - dependent fashion , degranulation of rat mature mast cells , as assessed by histamine or -hexosaminidase release [ 5860 ] .
cathelicidin ll-37 promotes degranulation and preformed mediator release from human lung mast cells and stimulates degranulation and ca mobilization in immature lad2 human mast cells [ 62 , 63 ] as well .
moreover , this peptide activates lad2 mast cells to synthesis and releases the strong pro - inflammatory mediators such as ltc4 and pgd2 and rat cramp stimulates mature mast cells to production and release cysteinyl ( cys)lts .
importantly , anti - inflammatory cytokine il-10 , but not tnf , il-6 and ccl5 , strongly inhibits cramp - induced cyslt synthesis .
moreover , ll-37 activates mast cells to produce newly generated cytokines , including il-2 , il-4 , il-6 , il-31 , gm - csf , and tnf , and chemokines ccl4 and cxcl8 [ 60 , 63 , 64 ] .
this peptide , at a concentration of 20 g / ml , induces il-1 , ccl2 , and ccl3 mrna increase in rat mast cells .
the cathelicidin cramp stimulates mast cells to tnf and cxcl8 release and induces gmcsf , il-1 , ccl2 , and ccl3 , but not il-33 , mrna expression .
yoshioka et al . observed that ll-37 , at a concentration of 1 g / ml , stimulates lad2 mast cells to generate th2 cytokines il-4 and il-5 , but not il-10 , th1 cytokine il-2 , but not ifn- and tnf- , pro - inflammatory cytokines tnf and il-1 , but not il-6 and cxcl8 .
moreover , lps significantly reduced ll-37-induced il-4 and il-5 , but not il-1 , generation .
these intriguing findings suggest that ll-37 co - existing with the bacterial components may change the mast cell function toward innate immunity .
there are also very interesting data that cathelicidins ll-37 and cramp act as potent mast cell chemoattractants [ 60 , 65 , 66 ] , and thereby cathelicidins may additionally enhance inflammatory response via attracting mast cells to pathogen entry site .
besides , it was observed that ll-37 , at a concentration of 1 g / ml , up - regulates both tlr4 mrna and tlr4 protein expression on lad2 mast cells , and in that way , cathelicidin enhances mast cell capability to detect invading pathogen . up to date , the mechanisms by which cathelicidins activate mast cells are not fully clarified .
some data indicate that gpcrs mediate ll-37-induced signal transduction pathway in mast cells [ 58 , 66 ] .
there are some observations that ll-37 acts via mas - related gene ( mrg ) x2 receptor or through fpr2 and receptor p2x7 .
moreover , it was suggested that ll-37 activates mast cells in plc - dependent manner [ 58 , 66 ] .
in addition , some data indicate that in ll-37-dependent mast cell activation pi3k and mapk signaling pathways are involved [ 59 , 60 , 62 , 64 , 65 ] .
neutrophils , monocytes , macrophages , and mast cells undoubtedly play a key role in inflammation . however , it is not to overestimate the role of other populations of host cells in inflammatory processes .
limited data show that human cathelicidin ll-37 and mouse cramp can modulate activity of eosinophils , dendritic cells , epithelial cells , and keratinocytes . it is documented that ll-37 , at a concentration range from 15 to 30
g / ml , directly stimulates eosinophils to cys - lts and ltb4 release and priming with eosinophilopoietic cytokine gm - csf or il-5 significantly enhances ll-37-induced cyslt secretion .
moreover , in response to ll-37 activation , eosinophils release eosinophil cationic protein ( ecp ) .
it is also indicated that ll-37 enhances cytosolic pla2 activity , triggers intracellular translocation of 5-lipoxygenase and ltc4 synthase to perinuclear localization of lipid bodies .
human cathelicidin ll-37 , at a high concentration of 40 g / ml , induces eosinophil chemotaxis and this effect is mediated via fpr2 molecules . in the course of inflammatory processes dendritic cells take part to some extent , however the influence of ll-37 on dendritic cell functioning is currently almost unknown .
it was indicated that human cathelicidin ll-37 strongly inhibits lps - mediated pro - inflammatory cytokine , i.e. il-6 and tnf , generation [ 35 , 6971 ] .
similarly , murine cramp causes a decrease in il-6 , but not il-1 , synthesis by dendritic cells in response to lps stimulation .
ll-37 also blocks lps - induced chemokine cxcl8 synthesis [ 35 , 71 ] and declines lps - induced up - regulation of cxcr7 .
human cathelicidin ll-37 completely inhibits il-6 and tnf generation in response to lta and flagellin activation as well .
worthy of note is that ll-37 significantly abrogates lps- , lta- , and flagellin - mediated production of il-12 [ 69 , 70 ] , a pro - inflammatory cytokine with immunoregulatory functions which promotes differentiation of t cells into th1 cells .
di nardo et al . observed that pretreatment of monocyte - derived dendritic cells with ll-37 significantly reduces lps induction of il-10 . on the contrary , mookherjee et al .
it was established that this peptide stimulates airway epithelial cell migration and proliferation and this effect is mediated through gpcr and epidermal growth factor receptor ( egfr ) .
established that this peptide by itself induces il-6 , cxcl1 , and cxcl8 release from bronchial epithelial cells .
likewise , tjabringa et al . indicated that ll-37 , at concentrations as high as 50 - 100 g / ml , stimulates airway epithelial cells by activation of erk1/2 and increased release of cxcl8 .
it is interesting that ll-37 activates epithelial cells by transactivation of the egfr via metalloproteinase - dependent processing of egfr ligands .
furthermore , this host defense peptide augments tlr5-mediated ( flagellin ) production of il-6 by epithelial cells .
significantly higher levels of cxcl8 and il-6 are secreted by lung epithelial cells after stimulation with tlr4 agonist lps in the presence of ll-37 as well .
. showed that ll-37 , at concentrations of 2 - 3 g / ml , alters bronchial epithelial cell responses to pro - inflammatory stimuli . in combination with il-1 , tlr5 agonist flagellin , or tlr3
agonist polyinosinic : polycytidylic acid ( poly - i : c ) ll-37 causes synergistically an increase in cxcl8 production and in combination with tlr2 agonist pam3csk4 this cathelicidin synergistically induces transcription and release of both il-6 and cxcl8 from these cells .
the effect of ll-37 occurs via heparin - binding epidermal growth factor ( hb - egf)-mediated egfr transactivation and signal transducer and activator of transcription ( stat)1 , and stat3 intracellular molecules are involved [ 77 , 78 ] .
what is more , ll-37 by itself stimulates keratinocytes to synthesis and release of different pro - inflammatory and/or immunoregulatory cytokines , including il-6 , il-18 , il-20 , and gm - csf [ 77 , 79 , 80 ] . in response to ll-37 activation
, keratinocytes produce some chemokines , i.e. ccl2 , ccl5 , ccl20 , cxcl8 , and cxcl10 [ 77 , 79 , 80 ] .
it is important to note that human cathelicidin stimulates the synthesis of anti - inflammatory cytokine il-10 in keratinocytes and significantly up - regulates gene encoding cox2 as well .
cathelicidin peptide - induced cytokine / chemokine production in keratinocytes involves egfr , g protein , and plc signaling pathways [ 77 , 80 ] .
ll-37-induced il-18 secretion is probably via caspase-1-independent pathway and through p39 and erk1/2 mapk pathways .
very intriguing are observations that -defensins hbd-1 , -2 , -3 , and -4 have a synergistic effect on il-18 secretion by keratinocytes in response to ll-37 stimulation .
it should be also noted that ll-37 in combination with il-1 or tlr5 agonist synergistically increases cxcl8 synthesis by both proliferating and differentiated keratinocytes .
cathelicidins , like other amps , exhibit antimicrobial activities against a broad spectrum of microbes , including both gram - positive and gram - negative bacteria , enveloped viruses , and fungi .
these peptides directly kill the invaded microorganisms by perturbing their cell membranes and can neutralize biological activities of endotoxin . nowadays , more and more data indicate that cathelicidins , in addition to their antimicrobial properties , have the potential to influence and modulate , both directly and indirectly , the activity of various cell populations involved in inflammatory processes ( figure 1 ) .
first of all , it should be underlined that cathelicidins may intensify the course of inflammation via attracting neutrophils , monocytes , macrophages , eosinophils , and mast cells to pathogen entry site .
importantly , recruiting cells , and especially neutrophils , are a rich source of cathelicidins .
thus , high concentrations of these peptides can be most easily achieved at the sites of immune cell accumulation .
it is worthy of note , however , that cathelicidin ll-37 reduces cxcr2 expression on neutrophils and monocytes , i.e. the receptor that mediates chemokine - induced cell migration .
moreover , these peptides may exacerbate inflammatory reactions by suppressing apoptosis and prolonging the lifespan of neutrophils .
immunomodulatory activities of cathelicidins cathelicidins directly activate different cell populations to production and release of different pro - inflammatory and immunoregulatory cytokines , inter alia il-1 , il-6 , il-18 , il-20 , tnf , and gm - csf .
likewise , these peptides induce the production of various chemokines , including ccl2 , ccl5 , ccl7 , ccl20 , cxcl1 , cxcl4 , cxcl8 , and cxcl10 .
furthermore , in response to cathelicidin activation mast cells , macrophages , and eosinophils generate potent pro - inflammatory mediators , such as cyslts , ltb4 , txa2 , pgd2 and histamine .
it is crucial that cathelicidins stimulate production and release of ros , no , ecp , and some proteinases , which take part in tissue damage . on the other hand
, cathelicidins can induce the synthesis of some anti - inflammatory mediators ; macrophages and keratinocytes produce il-10 , monocytes synthesize il-10 and il-19 , and monocytes and neutrophils generate il-1ra . without any doubt
peptide ll-37 acting in synergy with il-1 causes a significant increase in cxcl8 production by epithelial cells and keratinocytes and in not only ccl2 , ccl7 , il-6 , but also il-10 , by monocytes .
this cathelicidin exerts synergistic effect on gm - csf - induced ccl2 and il-6 synthesis by monocytes and significantly enhances il-18 secretion from keratinocytes stimulated with -defensins . otherwise
, co - stimulation of monocytes and dendritic cells with ll-37 and ifn- causes an influential decrease in tnf synthesis , and monocyte co - stimulation with ll-37 and il-32 results in a decrease in tnf , il-6 , il-1 , and also il-10 generation .
in addition , cathelicidins also appear to have functions in modulating tlr - mediated cell responses .
these peptides strongly inhibit pro - inflammatory cytokine release , i.e. tnf , il-1 , il-6 and chemokine cxcl8 , from monocytes stimulated with tlr2 or tlr4 agonists .
it is important to stress that ll-37 significantly decreases cxcl8 production by monocytes activated with tlr9 ligand but enhances tnf and il-6 synthesis .
similarly , cathelicidins strongly decrease tlr2- and tlr4-mediated tnf and no synthesis in macrophages , but enhance ros release in response to tlr4 stimulation .
furthermore , cathelicidins strongly inhibit il-6 , il-12 , tnf , and cxcl8 generation by dendritic cells stimulated by tlr2 , tlr4 , or tlr5 ligands . on the contrary
, it is very interesting that synergistic action of ll-37 and tlr2 , tlr3 , tlr4 , or tlr5 ligands induces an increase in il-6 and cxcl8 generation by epithelial cells and keratinocytes . to date , it has not been entirely clear what the exact cathelicidin concentrations in physiological or pathophysiological conditions and in inflammatory milieu are . nevertheless , there are data that in healthy individuals the ll-37 levels were as low as 39.9 pg / ml in sputum , 1 - 4 ng / ml in tracheal aspirate samples obtained from new - born children , and 1.1 and 1.7 ng / ml in plasma of neonatal and maternal blood , respectively [ 83 , 84 ] .
some information indicates that in healthy individuals the concentrations of ll-37 are about 27.2 ng / ml in plasma and 30.5 ng / ml in saliva .
what is more , concentrations of ll-37 in sweat and bronchoalveolar lavage fluid ( bal ) of healthy infants are significantly higher and amount up to 4.47 g/ ml and 4.8 g / ml , respectively .
interestingly , the levels of ll-37 in sperm are as high as 86.5 g / ml . in pathological conditions , the concentrations of this cathelicidin are highly differentiated . in sputum obtained from patients suffering from cystic fibrosis ( cf ) , chronic obstructive pulmonary disease ( copd ) , and asthma , ll-37 concentrations were as low as 79.6 pg / ml , 75.3 pg / ml , and 13.6 pg / ml , respectively .
moreover , the concentration of ll-37 in plasma of patients with sepsis was 13.7 ng / ml , and 22.5 ng / ml in saliva of individuals suffering from chronic periodontitis .
chen et al . determined that concentrations of ll-37 in bal of patients with cf were from an undetectable level up to 15 g / ml . in turn , the level of this peptide was up to even 1.4 mg / ml in skin - biopsy samples from patients with psoriasis .
the cathelicidin effective concentrations used in the in vitro studies were in the range between 0.01 g / ml and 50 g / ml .
thus , it can be assumed that the cathelicidin concentrations used in experiments in vitro correspond to those occurring in vivo in different pathophysiological conditions , and especially during infection and inflammation in the local milieu . in conclusion , it is now obvious that cathelicidins have various immunomodulatory activities .
therefore , these peptides should be considered as the multifunctional molecules that strongly affect the course of inflammation .
it is important to state , however , that the exact role of these peptides in inflammation is ambiguous , as cathelicidins might promote or reduce the inflammatory intensity .
moreover , without any doubt these host derived peptides affect host defense against microorganisms by modulating epithelial responses to infecting pathogens , by balancing the tlr - mediated cell response to bacteria and viruses , and by influencing the inflammatory processes at sites of pathogen entry . without any doubt , further studies are necessary to fully clarify the exact role of cathelicidins in inflammation and innate immunity developed during infection . | cathelicidins , like other antimicrobial peptides , exhibit direct antimicrobial activities against a broad spectrum of microbes , including both gram - positive and gram - negative bacteria , enveloped viruses , and fungi .
these host - derived peptides kill the invaded pathogens by perturbing their cell membranes and can neutralize biological activities of endotoxin . nowadays , more and more data indicate that these peptides , in addition to their antimicrobial properties , possess various immunomodulatory activities .
cathelicidins have the potential to influence and modulate , both directly and indirectly , the activity of various cell populations involved in inflammatory processes and in host defense against invading pathogens .
they induce migration of neutrophils , monocytes / macrophages , eosinophils , and mast cells and prolong the lifespan of neutrophils .
these peptides directly activate inflammatory cells to production and release of different pro - inflammatory and immunoregulatory mediators , cytokines , and chemokines , however cathelicidins might mediate the generation of anti - inflammatory cytokines as well .
cathelicidins also modulate epithelial cell / keratinocyte responses to infecting pathogens . what is more , they affect activity of monocytes , dendritic cells , keratinocytes , or epithelial cells acting in synergy with cytokines or -defensins .
in addition , these peptides indirectly balance tlr - mediated responses of monocytes , macrophages , dendritic cells , epithelial cells , and keratinocytes .
this review discusses the role and significance of cathelicidins in inflammation and innate immunity against pathogens . | Introduction
Cathelicidins affect neutrophil activity
Cathelicidins affect monocyte/macrophage activity
Cathelicidins affect mast cell activity
Cathelicidins affect other inflammatory cell activity
Concluding remarks
None | innate immunity is mediated by several humoral components ( complement proteins , some cytokines ) and cellular mechanisms involving neutrophils , monocytes , macrophages , mast cells , eosinophils , dendritic cells , and nk cells . the c - terminal peptide ( 12 - 100 amino acid residues ) , as a mature peptide , exhibits broad antimicrobial activity against both gram - positive and gram - negative bacteria , viruses and fungi . these peptides are produced by neutrophils , epithelial cells , keratinocytes , macrophages , mast cells , nk cells , dendritic cells , and lymphocytes [ 5 , 6 ] . ll-37 and
the human cathelicidin is produced mainly by neutrophils , monocytes , nk cells , mast cells , b cells and also colon enterocytes , epithelial cells and keratinocytes . cathelicidins exhibit a broad spectrum of antimicrobial activity against gram - positive and gram - negative bacteria [ 1 , 7 ] , enveloped viruses , and fungi . nowadays , however , more and more data indicate that apart from killing pathogens directly or indirectly , cathelicidins have a number of immunomodulatory functions that might be involved in the clearance of infection . given that these cell populations can produce both pro - inflammatory and anti - inflammatory mediators , it is obvious that they strongly direct the inflammatory processes and significantly influence an intensity of inflammation . monocytes and macrophages have the ability to secrete a broad range of pro - inflammatory cytokines , such as il-1 , il-6 , il-12 , il-18 , il-23 , il-27 , and tumor necrosis factor ( tnf )
. on the other hand , there are a lot of monocyte / macrophage - derived mediators involved in suppressing inflammation , including some anti - inflammatory cytokines , i.e. more and more data indicate that cathelicidins by themselves directly stimulate monocytes to produce and release some cytokines and chemokines that may strongly modulate the course and intensity of inflammation . they have the potential to secrete a wide spectrum of biologically active mediators , cytokines and chemokines . finally , inasmuch as mast cell - derived mediators elicit pro - inflammatory , anti - inflammatory and immunoregulatory effects , these cells are essential in both promoting and limiting the inflammatory processes [ 52 , 56 , 57 ] . neutrophils , monocytes , macrophages , and mast cells undoubtedly play a key role in inflammation . limited data show that human cathelicidin ll-37 and mouse cramp can modulate activity of eosinophils , dendritic cells , epithelial cells , and keratinocytes . what is more , ll-37 by itself stimulates keratinocytes to synthesis and release of different pro - inflammatory and/or immunoregulatory cytokines , including il-6 , il-18 , il-20 , and gm - csf [ 77 , 79 , 80 ] . cathelicidins , like other amps , exhibit antimicrobial activities against a broad spectrum of microbes , including both gram - positive and gram - negative bacteria , enveloped viruses , and fungi . these peptides directly kill the invaded microorganisms by perturbing their cell membranes and can neutralize biological activities of endotoxin . nowadays , more and more data indicate that cathelicidins , in addition to their antimicrobial properties , have the potential to influence and modulate , both directly and indirectly , the activity of various cell populations involved in inflammatory processes ( figure 1 ) . first of all , it should be underlined that cathelicidins may intensify the course of inflammation via attracting neutrophils , monocytes , macrophages , eosinophils , and mast cells to pathogen entry site . immunomodulatory activities of cathelicidins cathelicidins directly activate different cell populations to production and release of different pro - inflammatory and immunoregulatory cytokines , inter alia il-1 , il-6 , il-18 , il-20 , tnf , and gm - csf . furthermore , in response to cathelicidin activation mast cells , macrophages , and eosinophils generate potent pro - inflammatory mediators , such as cyslts , ltb4 , txa2 , pgd2 and histamine . without any doubt
peptide ll-37 acting in synergy with il-1 causes a significant increase in cxcl8 production by epithelial cells and keratinocytes and in not only ccl2 , ccl7 , il-6 , but also il-10 , by monocytes . moreover , without any doubt these host derived peptides affect host defense against microorganisms by modulating epithelial responses to infecting pathogens , by balancing the tlr - mediated cell response to bacteria and viruses , and by influencing the inflammatory processes at sites of pathogen entry . | [
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chronic myeloid leukemia ( cml ) is a myeloproliferative disorder of blood stem cells.1 the causative molecular defect is the bcr - abl protein , which is encoded by the philadelphia chromosome ( ph).2 this genetic anomaly arises from an exchange of genetic material between chromosomes 9 and 22 , which results in the fusion of the breakpoint cluster region ( bcr ) and the abelson leukemia virus ( abl ) proto - oncogene.3,4 the resulting gene encodes a constitutively active protein kinase that activates a number of proteins involved in cell - cycle regulation that hasten cell division and affect dna repair.58 imatinib , also known as gleevec ( novartis , basel , switzerland ) , is a selective inhibitor of not only abl but also kit and pdgfr kinases , and exerts significant antileukemic activity in the majority of cml patients . according to the current national comprehensive cancer network ( nccn ) guidelines9 and the european leukemianet ( eln ) recommendations,10 a response outcome to imatinib therapy is classified as optimal , suboptimal , and failure based on the level of response achieved at various time points throughout the course of treatment using specific hematologic , cytogenetic and molecular criteria .
optimal responses proposed by the nccn closely corresponding to the eln recommendations and defined as the achievement of a ccyr after 12 months of imatinib at 400 mg daily . in patients with suboptimal responses to first - line imatinib , the use of high - dose ( 600 or 800 mg / day ) is recommended as an alternative therapeutic option by both the eln and the nccn .
failure to achieve any cytogenetic response from standard - dose imatinib after 6 months of therapy or major cytogenetic response and a ccyr after 12 and 18 months of therapy , respectively , are considered treatment failure by the nccn . in such cases ,
it is recommended that treatment should be changed for a second - generation tyrosine kinase inhibitor ( tki ) .
these criteria became particularly important as several kinase targeted therapies with long half - lives were developed for the treatment of patients who fail or are intolerant to imatinib therapy . according to the most recent results from the international randomized study of interferon versus sti571 ( iris ) trial ,
the estimated overall survival for patients still on imatinib was 85% at 8 years , or 93% when only cml - related deaths or deaths prior to stem cell transplant were included .
a total of 92% of the patients were free of disease progression . among those who achieved ccyr , only 3% progressed during the 8-year follow - up.11 nevertheless , of the 259 patients receiving imatinib as front - line therapy who had treatment discontinuation , 30 ( 5.4% ) and 77 ( 13.9% ) patients stopped their therapy because of intolerance or unsatisfactory therapeutic effect , respectively .
the inability of patients to tolerate treatment and the emergence of bcr - abl mutations that reduced the binding affinity of imatinib prompted pharmaceutical research that led to the discovery of several similarly effective , targeted , second generation tkis such as nilotinib ( tasigna , amn107 , novartis , basel , switzerland ) and dasatinib ( sprycel ; bristol - myers squibb , new york , ny).1214 evidence from an in vitro study has indicated that nilotinib is 20 times more potent than imatinib against cells expressing the wild - type bcrabl .
15 dasatinib is a multi - targeted kinase inhibitor that is structurally unrelated to imatinib and is able to bind and inhibit both the active and inactive conformations of abl , resulting in 100- to 300-fold higher activity than imatinib for unmutated bcr - abl , and greater inhibition against mutants with high levels of imatinib resistance ( except for t315i and relative insensitivity to f317l).1517 dasatinib is generally well tolerated . however ,
adverse events ( aes ) associated with its use , such as pleural effusions , hemorrhage , and febrile neutropenia have been frequently reported in the advanced stage of the disease ( reviewed by wong18 ) .
the nccn guidelines9 recommend that at the onset of grade 3/4 thrombocytopenia ( platelet count < 50,000/mm ) , therapy with dasatinib should be held .
once the toxicity has resolved ( platelet count 50,000/mm ) , dasatinib should be resumed at original starting dose if recovery occurs within 7 days or reduced one dose level if platelet count decreased to < 25,000/mm for more than 7 days .
attempts to identify the optimum and effective dose of dasatinib continue and include modification of scheduling , management of toxicity and dose optimization . to determine the optimum administration schedule
, the efficacy , response and tolerability of dasatinib has been investigated in several clinical trials .
it is now apparent that a scheduled once daily starting dose of 100 mg offers an improved safety profile and shows similar efficacy for patients with chronic phase cml ( cp cml ) compared to a 70 mg twice daily dose.1921 the recommended dosage schedule for patients in the advanced phase of the disease is 140 mg once daily.22,23 however , some intolerant patients may require a dose adjustment from 140 to 100 mg / day or from 100 to 80 mg / day to continue treatment and thus maintain control of their disease . even at a dose of 80 mg / day
, some patients require further dose reduction due to substantial toxicities . because of the clinical difficulty posed by this subgroup , we report the effects of an unusually low dosage of dasatinib during the treatment of four patients ( 2 in chronic phase and 2 in accelerated phase ) with proven efficacy almost identical to that seen with conventional dosage .
a 31-year - old man was admitted to our department in march 2008 with leukocytosis ( wbc 88.900/mm ) and splenomegaly of cml .
the patient was treated with imatinib mesylate at 400 mg / day in july 2008 .
one month after starting the therapy , the patient developed a grade ( g ) 4 thrombocytopenia ( 8.000/mm platelets ) with epistaxis and needed a platelet transfusion .
the patient had a therapy break of 4 months . in october 2008 , after resolution of thrombocytopenia to the g1 level , he was restarted on a reduced imatinib dose of 300 mg per day , but therapy was again discontinued for thrombocytopenia g3 ( 26.000/mm platelets ) . in february 2009 , because of severe intolerance to imatinib , dasatinib was started at a dose of 100 mg / day .
after 2 weeks of treatment under this therapy , the patient developed a g4 thrombocytopenia .
as a result , treatment was suspended until ( march 2009 ) recovery to g1 ( 78.000/mm ) and then resumed at a dose of 80 mg / day . however , the patient experienced the same side effects one week after restarting dasatinib .
the dose was then decreased to 50 mg / day with gradual increase of platelets . in june 2009 , after 4 months of treatment , the patient achieved a ccyr .
the patient continued the same dasatinib dose and in july 2009 , the platelet count reached 130.000/mm and molecular analysis showed a bcr - abl / bcr ratio of 0.01% indicating that the patient had achieved a major molecular response ( mmr ) .
a 46-year - old male was admitted to our department in july 2008 with cp cml .
this patient had two breaks in his imatinib therapy due to the recurring of g 3 thrombocytopenia .
after the second break , the patient progressed to ap cml without evidence of mutations in the abl kinase domain . in march 2009
, dasatinib was prescribed to this patient at 140 mg / day and after four weeks of therapy , the patient developed g3 thrombocytopenia , after which we decided to maintain the therapy at a lower dose of 70 mg / day . in june 2009 , after three months of treatment , the patient reached a ccyr and his thrombocytopenia improved to g1 . to achieve mmr
, we decided to increase the therapeutic dose to 80 mg / day in may 2010 . by august 2010 , on his last evaluation , this patient s bcr - abl /
a 69-year - old male was admitted to our department in march 2009 with a history of cml , first diagnosed in august 2007 in japan .
six months before the referral , he complained of fatigue , weight loss and splenomegaly .
a diagnosis of ap cml was confirmed with the appearance of additional chromosomal aberrations besides the ph chromosome ( 47xy , + 8 , t(9,22 ) , add(13)(q33 ) in 20/20 metaphase analyzed ) and , in march 2009 , imatinib was initiated at a daily dose of 600 mg .
after 1 month , the patient experienced g3 neutropenia ( 700/mm ) and g2 thrombocytopenia ( 62.000/mm ) .
the patient continued imatinib at the same dose with 300 g granulocyte - colony - stimulating factor ( g - csf ) added simultaneously every week to stimulate myelopoiesis . in october 2009 ,
the patient experienced g3 thrombocytopenia ( 41.000/mm ) and his bcr - abl / bcr ratio was 7.68% without any cytogenetic response after 6 months of therapy . at this time , sequencing of the bcr - abl kinase domain revealed no evidence of mutations in the abl kinase domain . based on these results ,
the patient was , therefore , considered resistant to imatinib and , in november 2009 , was placed on dasatinib at a daily dose of 140 mg . after the first 3 weeks of treatment , dasatinib was reduced to 100 mg / day because of the occurrence of g2 thrombocytopenia ( 61.000/mm ) .
two weeks later , his platelet count dropped to 50.000/mm . at this time , dasatinib was lowered to a dose of 70 mg in an attempt to maintain a stable platelet count .
however , after another 2 weeks , in january 2010 , the patient developed g3 neutropenia ( 900/mm ) and his platelet count dropped to 26.000/mm . at this time , the patient s bcr - abl / bcr ratio was 9.87% .
as dasatinib was the only option for the treatment of this patient s disease , it was again reduced to a dose of 50 mg / day with g - csf . in february 2010 ,
the patient s bcr - abl / bcr ratio was decreased to 0.81% and his platelet numbers started to increase gradually . as a result , g - csf was stopped and the patient continued dasatinib at the same dose .
five months after initiation of dasatinib , in april 2010 , cytogenetic analysis revealed a ccyr and complete disappearance of the initially detected additional chromosomal aberrations . in may 2010 , dasatinib was increased to 60 mg once daily to achieve mmr and no toxicity was noted on follow - up examination .
one month later , in june 2010 , the patient achieved mmr , with a transcript ratio of 0.01% .
the patient , a 58-year - old man , was presented to our hospital in march 2009 with a clinical history of rheumatoid arthritis , hypertension , gastritis and chronic renal failure and undergoing conservative therapy . at presentation , a routine blood count showed leukocytosis with a left shift and physical examination revealed no splenomegaly .
a diagnosis of cp cml was made after further laboratory studies and , in april 2009 , imatinib was initiated at a daily dose of 400 mg .
after a one month , despite a complete hematological response , the patient s pancreatic enzymes elevated to g1 and then gradually increased in severity to g4 in may 2009 .
after a one month period of interruption , imatinib was resumed at the same dose . however , the patient experienced the same side effects again and treatment was permanently discontinued .
one month later , the patient s pancreatic enzyme values returned to normal but he lost his hematologic response . at that time
, dasatinib was initiated at a dose of 100 mg / day . during follow - up ,
the patient had two breaks in the dasatinib therapy at a daily dose of 100 mg and 40 mg because of recurring g2 and g4 pancreatitis , respectively .
after the second break , a 10-week interruption , the serum lipase level was returned to normal and dasatinib was reintroduced at a further reduced dose of 20 mg / day taken in combination with pancrelipase ( creon ) capsules . at a subsequent clinic visit ,
the patient presented with g2 pancreatitis and the treatment schedule and doses were maintained . in february 2010 , the patient underwent a switch to alternate - day dasatinib at dosages of 20 and 40 mg every other day . in march 2010 , after 3 months of taking a lower dosage ( 20 mg / day ) of dasatinib , the patient achieved a ccyr and his bcr - abl / bcr showed a ratio of 0.1% .
the measurement of his transcript was repeated in august 2010 and confirmed the mmr ( bcr - abl / bcr ratio = 0.018% ) . at that time , the patient had mild pancreatitis with serum lipase level and was scored as grade 1 .
a 31-year - old man was admitted to our department in march 2008 with leukocytosis ( wbc 88.900/mm ) and splenomegaly of cml .
the patient was treated with imatinib mesylate at 400 mg / day in july 2008 .
one month after starting the therapy , the patient developed a grade ( g ) 4 thrombocytopenia ( 8.000/mm platelets ) with epistaxis and needed a platelet transfusion .
the patient had a therapy break of 4 months . in october 2008 , after resolution of thrombocytopenia to the g1 level , he was restarted on a reduced imatinib dose of 300 mg per day , but therapy was again discontinued for thrombocytopenia g3 ( 26.000/mm platelets ) . in february 2009 , because of severe intolerance to imatinib , dasatinib was started at a dose of 100 mg / day .
after 2 weeks of treatment under this therapy , the patient developed a g4 thrombocytopenia .
as a result , treatment was suspended until ( march 2009 ) recovery to g1 ( 78.000/mm ) and then resumed at a dose of 80 mg / day . however , the patient experienced the same side effects one week after restarting dasatinib .
the dose was then decreased to 50 mg / day with gradual increase of platelets . in june 2009 , after 4 months of treatment , the patient achieved a ccyr .
the patient continued the same dasatinib dose and in july 2009 , the platelet count reached 130.000/mm and molecular analysis showed a bcr - abl / bcr ratio of 0.01% indicating that the patient had achieved a major molecular response ( mmr ) .
a 46-year - old male was admitted to our department in july 2008 with cp cml .
this patient had two breaks in his imatinib therapy due to the recurring of g 3 thrombocytopenia .
after the second break , the patient progressed to ap cml without evidence of mutations in the abl kinase domain . in march 2009
, dasatinib was prescribed to this patient at 140 mg / day and after four weeks of therapy , the patient developed g3 thrombocytopenia , after which we decided to maintain the therapy at a lower dose of 70 mg / day . in june 2009 , after three months of treatment , the patient reached a ccyr and his thrombocytopenia improved to g1 . to achieve mmr
, we decided to increase the therapeutic dose to 80 mg / day in may 2010 . by august 2010 , on his last evaluation , this patient s bcr - abl / bcr ratio showed 0.8% .
a 69-year - old male was admitted to our department in march 2009 with a history of cml , first diagnosed in august 2007 in japan .
six months before the referral , he complained of fatigue , weight loss and splenomegaly .
a diagnosis of ap cml was confirmed with the appearance of additional chromosomal aberrations besides the ph chromosome ( 47xy , + 8 , t(9,22 ) , add(13)(q33 ) in 20/20 metaphase analyzed ) and , in march 2009 , imatinib was initiated at a daily dose of 600 mg . after 1 month
, the patient experienced g3 neutropenia ( 700/mm ) and g2 thrombocytopenia ( 62.000/mm ) .
the patient continued imatinib at the same dose with 300 g granulocyte - colony - stimulating factor ( g - csf ) added simultaneously every week to stimulate myelopoiesis . in october 2009 ,
the patient experienced g3 thrombocytopenia ( 41.000/mm ) and his bcr - abl / bcr ratio was 7.68% without any cytogenetic response after 6 months of therapy . at this time , sequencing of the bcr - abl kinase domain revealed no evidence of mutations in the abl kinase domain . based on these results ,
the patient was , therefore , considered resistant to imatinib and , in november 2009 , was placed on dasatinib at a daily dose of 140 mg . after the first 3 weeks of treatment , dasatinib was reduced to 100 mg / day because of the occurrence of g2 thrombocytopenia ( 61.000/mm ) .
two weeks later , his platelet count dropped to 50.000/mm . at this time , dasatinib was lowered to a dose of 70 mg in an attempt to maintain a stable platelet count .
however , after another 2 weeks , in january 2010 , the patient developed g3 neutropenia ( 900/mm ) and his platelet count dropped to 26.000/mm . at this time , the patient s bcr - abl / bcr ratio was 9.87% .
as dasatinib was the only option for the treatment of this patient s disease , it was again reduced to a dose of 50 mg / day with g - csf . in february 2010 , the patient s bcr - abl / bcr ratio was decreased to 0.81% and his platelet numbers started to increase gradually . as a result ,
five months after initiation of dasatinib , in april 2010 , cytogenetic analysis revealed a ccyr and complete disappearance of the initially detected additional chromosomal aberrations . in may 2010
, dasatinib was increased to 60 mg once daily to achieve mmr and no toxicity was noted on follow - up examination .
one month later , in june 2010 , the patient achieved mmr , with a transcript ratio of 0.01% .
the patient , a 58-year - old man , was presented to our hospital in march 2009 with a clinical history of rheumatoid arthritis , hypertension , gastritis and chronic renal failure and undergoing conservative therapy . at presentation , a routine blood count showed leukocytosis with a left shift and physical examination revealed no splenomegaly .
a diagnosis of cp cml was made after further laboratory studies and , in april 2009 , imatinib was initiated at a daily dose of 400 mg . after a one month , despite a complete hematological response , the patient s pancreatic enzymes elevated to g1 and then gradually increased in severity to g4 in may 2009 .
after a one month period of interruption , imatinib was resumed at the same dose . however , the patient experienced the same side effects again and treatment was permanently discontinued .
one month later , the patient s pancreatic enzyme values returned to normal but he lost his hematologic response . at that time
, dasatinib was initiated at a dose of 100 mg / day . during follow - up ,
the patient had two breaks in the dasatinib therapy at a daily dose of 100 mg and 40 mg because of recurring g2 and g4 pancreatitis , respectively .
after the second break , a 10-week interruption , the serum lipase level was returned to normal and dasatinib was reintroduced at a further reduced dose of 20 mg / day taken in combination with pancrelipase ( creon ) capsules . at a subsequent clinic visit ,
the patient presented with g2 pancreatitis and the treatment schedule and doses were maintained . in february 2010 , the patient underwent a switch to alternate - day dasatinib at dosages of 20 and 40 mg every other day . in march 2010 , after 3 months of taking a lower dosage ( 20 mg / day ) of dasatinib , the patient achieved a ccyr and his bcr - abl / bcr showed a ratio of 0.1% .
the measurement of his transcript was repeated in august 2010 and confirmed the mmr ( bcr - abl / bcr ratio = 0.018% ) . at that time , the patient had mild pancreatitis with serum lipase level and was scored as grade 1 .
dasatinib is a highly effective targeted therapy in patients with cml who are unresponsive to imatinib , or as an alternative where imatinib is poorly tolerated . as such
, it provides a valuable therapeutic option for those patients who are at high risk for disease progression .
although dasatinib is generally well tolerated , serious toxicities can occur in some patients , even at a lower recommended dose of 80 mg / day , and can pose particular challenges to clinicians caring for this subgroup of patients .
patients intolerant of dasatinib and other tkis are unlike those resistant to therapy ; they often suffer increased treatment interruptions and compromised therapeutic benefits . therefore , to fit an individual s unique profile , adjustment of dose on the basis of the patient s response and tolerability is required .
our experience in this short case series demonstrate that dasatinib at a dose well below the minimum recommended dosage has clinical activity associated with only minor toxicity in 4 dasatinib - intolerant patients who had previously experienced imatinib - related hematological ( 3 cases ) and non - hematological complications ( 1 case ) . at the time of initiation of dasatinib therapy ,
all 4 patients had been initially treated with imatinib as a first - line therapy at either 400 mg / day or 600 mg / day and all but one developed hematological toxicities ( table 1 ) .
the same pre - existing complications that occurred during imatinib therapy were observed after crossover to dasatinib .
it is possible that the complications manifested in our patients during imatinib therapy may have increased the likelihood of these abnormalities shortly after initiation of dasatinib .
alternatively , it is also possible that tkis predisposed our patients to these aes . in all patients , dasatinib , was the only second generation tkis available at that time , had to be reduced to a lower dose because of the dose - limiting toxicity .
the mean time to a ccyr in the first 3 patients was 3 and 5.5 months ; similar to durations of < 6 months that have been reported previously for standard doses.21,24 administration of dasatinib at a reduced daily dose thus induces remissions in a similar time frame compared with administration of a conventional dosage amount .
moreover , under continuous treatment with dasatinib under low dosage , there was progressive improvement of the platelet count in all of our first three patients , with progression to g1 thrombocytopenia ( case 1 and 2 ) and complete normalization ( case 3 ) .
although the third patient developed g3 neutropenia with dasatinib , it was easily managed by the weekly administration of g - csf .
the myelosupression can occur as a result of a sudden hematopoiesis suppression that was maintained by ph+ cells without equilibrium of the reappearance of the ph - cells .
the fourth patient developed g4 pancreatitis after being initiated on imatinib mesylate and recurred with the same symptoms after switching to dasatinib even at the lower dose of 40 mg / day .
the patient had no risk factors for pancreatitis and was taking no medications known to cause pancreatitis other than imatinib .
the reason why it was unlikely for this patient to develop pancreatitis due to cml is because the onset of this complication appeared during the imatinib treatment and then gradually improved upon suspension of therapy .
pancreatitis is an extremely rare adverse event which occurs after the use of tkis.23,2529 the pathophysiology of tkis - induced pancreatitis remains speculative , but has been proposed by plandari and colleagues26 to be due either to the inhibition of c - abl , which might interfere with the molecular mechanisms that regulate pancreatic cell death and thus induce pancreatic damage , or to the indirect effect of the drug on the release of calcium from the intracellular acinar stores , which regulate exocrine pancreatic secretion , and may enhance the accumulation of fatty acid inside the pancreatic acinar cell , which disturbs exocytosis . at the time of this report ,
it is possible that the greater potency of bcr - abl inhibition of dasatinib compared to imatinib contributed to the induction of remissions in our patients . because of its greater potency ( 325-fold )
, it has previously been reported that exposure to low or subnanomolar concentrations of dasatinib is sufficient to commit cell lines expressing bcr - abl to apoptotic cell death.15,16 these results may help to explain the improved tolerability and efficacy observed in our patients .
an increasing number of investigators are exploring the use of a lower dose intensity of dasatinib for the treatment of therapy intolerant patients .
in one recent study , the frequency and significance of dose reductions and treatment holidays among 280 patients treated with 2nd generation tkis were retrospectively analyzed.30 the results revealed that 176 ( 63% ) of these patients required treatment interruptions and/or dose reduction at least once during therapy .
the authors conclude that lower doses of dasatinib and nilotinib may potentially have similar efficacy in the therapy of cml as standard doses . in another small study ,
bergeron et al31 investigated pleural and pulmonary complications in 40 patients who received dasatinib ( 70 mg twice daily ) for imatinib resistance or intolerance .
dasatinib treatment was interrupted in eight patients , either immediately after diagnosis of lung involvement ( five patients ) , or after a course of diuretics ( three patients ) . after treatment holidays ,
lung abnormalities were resolved in all cases and did not recur in three out of four patients when dasatinib was reintroduced at a dose of 40 mg twice daily .
in addition , breccia et al32 treated a 34-year - old ph+ cml female patient who had a molecular relapse after haploidentical bmt with chronic liver gvhd and was severely intolerant to both imatinib and dasatinib at a standard dose ; she had a cmr to dasatinib at a reduced dose of 20 mg / day without serious adverse events . a recent case report published by yamaguchi et al.33 described a successful treatment of a 70-year - old man with cml who developed a megakaryoblastic crisis concomitant with myelofibrosis despite imatinib therapy with dasatinib at a starting dose of 80 mg / day . despite the successful clinical response in these patients , conclusions drawn from this series
should be interpreted with caution and considered tentative until experimental studies to define the minimum effective dose and optimal dosing schedule for dasatinib - intolerant patients are carried out .
however , our series lend support to previous reports that showed a comparable efficacy of standard and low - dose dasatinib with very minor toxicities ; thus , for patients intolerant to dasatinib at a standard dose , a gradual dose reduction may be tried before drug discontinuation . | we report our experience in 4 patients with chronic myeloid leukemia ( cml ) who had discontinued imatinib as a result of adverse events and had switched to dasatinib . the chronic phase ( n 2 ) and accelerated phase ( n 2 ) cml patients received dasatinib at starting dose of 100 and 140 mg once daily , respectively .
reappearance of hematological toxicity was observed in 3 patients and pancreatitis in one patient .
treatment was given at a lower dose and patients were followed .
the median follow - up was 13 months and the median dose of dasatinib until achievement of complete cytogenetic remission ( ccyr ) was 60 mg daily ( range = 20 to 120 mg ) .
all four patients had achieved ccyr at a median of 4 months ( range = 3 to 5 months ) and among them , three had also achieved major molecular remission .
we conclude that low - dose dasatinib therapy in intolerant patients appears safe and efficacious and may be tried before drug discontinuation . | Introduction
Case one
Case two
Case three
Case four
Discussion | chronic myeloid leukemia ( cml ) is a myeloproliferative disorder of blood stem cells.1 the causative molecular defect is the bcr - abl protein , which is encoded by the philadelphia chromosome ( ph).2 this genetic anomaly arises from an exchange of genetic material between chromosomes 9 and 22 , which results in the fusion of the breakpoint cluster region ( bcr ) and the abelson leukemia virus ( abl ) proto - oncogene.3,4 the resulting gene encodes a constitutively active protein kinase that activates a number of proteins involved in cell - cycle regulation that hasten cell division and affect dna repair.58 imatinib , also known as gleevec ( novartis , basel , switzerland ) , is a selective inhibitor of not only abl but also kit and pdgfr kinases , and exerts significant antileukemic activity in the majority of cml patients . among those who achieved ccyr , only 3% progressed during the 8-year follow - up.11 nevertheless , of the 259 patients receiving imatinib as front - line therapy who had treatment discontinuation , 30 ( 5.4% ) and 77 ( 13.9% ) patients stopped their therapy because of intolerance or unsatisfactory therapeutic effect , respectively . it is now apparent that a scheduled once daily starting dose of 100 mg offers an improved safety profile and shows similar efficacy for patients with chronic phase cml ( cp cml ) compared to a 70 mg twice daily dose.1921 the recommended dosage schedule for patients in the advanced phase of the disease is 140 mg once daily.22,23 however , some intolerant patients may require a dose adjustment from 140 to 100 mg / day or from 100 to 80 mg / day to continue treatment and thus maintain control of their disease . because of the clinical difficulty posed by this subgroup , we report the effects of an unusually low dosage of dasatinib during the treatment of four patients ( 2 in chronic phase and 2 in accelerated phase ) with proven efficacy almost identical to that seen with conventional dosage . as a result , treatment was suspended until ( march 2009 ) recovery to g1 ( 78.000/mm ) and then resumed at a dose of 80 mg / day . in may 2010 , dasatinib was increased to 60 mg once daily to achieve mmr and no toxicity was noted on follow - up examination . during follow - up ,
the patient had two breaks in the dasatinib therapy at a daily dose of 100 mg and 40 mg because of recurring g2 and g4 pancreatitis , respectively . as a result , treatment was suspended until ( march 2009 ) recovery to g1 ( 78.000/mm ) and then resumed at a dose of 80 mg / day . in march 2009
, dasatinib was prescribed to this patient at 140 mg / day and after four weeks of therapy , the patient developed g3 thrombocytopenia , after which we decided to maintain the therapy at a lower dose of 70 mg / day . in may 2010
, dasatinib was increased to 60 mg once daily to achieve mmr and no toxicity was noted on follow - up examination . during follow - up ,
the patient had two breaks in the dasatinib therapy at a daily dose of 100 mg and 40 mg because of recurring g2 and g4 pancreatitis , respectively . our experience in this short case series demonstrate that dasatinib at a dose well below the minimum recommended dosage has clinical activity associated with only minor toxicity in 4 dasatinib - intolerant patients who had previously experienced imatinib - related hematological ( 3 cases ) and non - hematological complications ( 1 case ) . at the time of initiation of dasatinib therapy ,
all 4 patients had been initially treated with imatinib as a first - line therapy at either 400 mg / day or 600 mg / day and all but one developed hematological toxicities ( table 1 ) . the mean time to a ccyr in the first 3 patients was 3 and 5.5 months ; similar to durations of < 6 months that have been reported previously for standard doses.21,24 administration of dasatinib at a reduced daily dose thus induces remissions in a similar time frame compared with administration of a conventional dosage amount . in addition , breccia et al32 treated a 34-year - old ph+ cml female patient who had a molecular relapse after haploidentical bmt with chronic liver gvhd and was severely intolerant to both imatinib and dasatinib at a standard dose ; she had a cmr to dasatinib at a reduced dose of 20 mg / day without serious adverse events . however , our series lend support to previous reports that showed a comparable efficacy of standard and low - dose dasatinib with very minor toxicities ; thus , for patients intolerant to dasatinib at a standard dose , a gradual dose reduction may be tried before drug discontinuation . | [
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] |
due to population ageing , primary care systems throughout the world are encountering great challenges urging innovation in the organization of elderly care .
elderly individuals will gradually experience complex age - related problems in the physical , psychological , cognitive and social domains of daily functioning .
this condition is known as frailty and is found to increase the risk of negative health and social outcomes .
frailty is related to poor quality of life and becoming more care dependent , with an increased likelihood of hospitalization and institutionalization ( 1 ) .
while budget cuts reduce health and social care expenditures , there is , thus , a strong need for providing high - quality care in order to maintain elderly s quality of life .
it is frequently questioned whether the current approach to care delivery provides good value for money , given its fragmentation and its lack of responsiveness to the needs of frail elderly ( 2 ) .
integrated care is defined as a well - planned and well - organised set of services and care processes , targeted at multi - dimensional needs / problems of an individual client , or a category of persons with similar needs / problems ( 3 ) .
first , integrated care is demand - oriented , addressing client s needs by professionals from different disciplines and sectors ( 2 ) . second , integrated care aims to promote continuity : the set of services is delivered coherently , seamlessly and in accordance with clients changing needs over time ( 3 ) .
common elements of integrated care models proven to be effective for community - dwelling frail elderly are a single entry point , geriatric assessments , case management , multidisciplinary teams ( 4 ) , multidisciplinary protocols and discussions , web - based patient files and a network structure ( 5 ) .
even though integrated care largely aims at cost - effectiveness , research comparing the associated costs and effects of interventions is scarce , limiting conclusions on the cost - effectiveness of integrated care interventions ( 6 ) .
some interventions for community - dwelling frail elderly have shown to be cost - effective compared with care as usual ( 69 ) , whereas other studies have shown that integrated care is not cost - effective ( 10,11 ) .
the wide variation in the interventions , costs and effects considered in these studies , limits the possibility to draw conclusions regarding what promotes cost - effectiveness in integrated care for community - dwelling frail elderly .
this study adds knowledge by exploring the cost - effectiveness of a specific integrated care intervention : the walcheren integrated care model ( wicm ) .
in contrast to earlier studies that used a narrow health care perspective ( 6,7,9 ) , we adopted a societal perspective , which is strongly recommended given its policy relevance at the macro level ( 12 ) .
second , our intervention comprises all integrated care elements that have been identified as effective in prior research rather than a selection of elements .
therefore , we provide valuable insights regarding the cost - effectiveness of a comprehensive integrated care model for community - dwelling frail elderly . this study aimed to answer the following research question : is the wicm cost - effective from a societal perspective after 12 months ?
the design of this study was quasi - experimental and included before and after measurements with a control group providing care as usual [ for a more detailed description of the methods , see ref .
the cost - effectiveness analysis was conducted from a societal perspective and thus considered all costs related to the intervention , irrespective of who pays for these expenses ( 12 ) . in the wicm , the gp functions as care coordinator and as a partner in prevention .
the gp practice is a single entry point for the elderly , their informal caregivers and health professionals .
gps detect frailty in their patient population using the groningen frailty indicator , a validated 15-item instrument that measures decreases in physical , cognitive , social and psychological functioning .
elderly patients with a score of 4 or higher are visited by a nurse practitioner who assesses their functional , cognitive , mental and psychological functioning using easycare , an evidence - based instrument used to assess care needs . a multidisciplinary treatment plan
multidisciplinary meetings are attended by the gp , the nurse practitioner and other professionals , depending on the care required by the frail elderly .
the wicm requires task reassignment and delegation between nurses and doctors , and among gps , nursing home doctors and geriatricians .
, a steering group serves as an umbrella organization under which the wicm is developed and disseminated .
the steering group , which consists of representatives from all involved organizations , forms a joint governing board that provides the necessary provider network .
all patient representatives support the project , and the health insurer cz provides financial support for the project . compared with the wicm ,
care as usual in the netherlands is fragmented and reactive . in the dutch health care systems ,
patients need a referral from their gp to obtain care from the primary , secondary and tertiary echelons .
care as usual is fragmented , as professionals merely communicate bilaterally through referral letters and sporadic telephone calls .
moreover , care as usual is reactive ; patients solely receive care for specific ( health ) problems on their own initiative .
the gps in the control group were unable to implement elements of the integrated model during the study period because they did not receive financial support from the health insurer to implement the integrated care activities of the wicm .
accordingly , participants in the control group were not systematically screened for frailty , their care needs were not assessed , multidisciplinary treatment plan were not formulated and case management was not provided .
the gps in the control group had a monodisciplinary focus ; they did not organize multidisciplinary meetings or implement multidisciplinary protocols and web - based files .
furthermore , the gps in the control group could not treat the frail elderly patients differently , as these gps were not given information on who participated in the study .
the study population consisted of the entire elderly patient population of the gps in both the experimental and control groups ( see fig .
were included in the experimental group , and 249 frail elderly from six gp practices in the control group .
the frail elderly were asked whether they received informal care , including care from non - professionals and unpaid care provided by partners , family , close friends or neighbours . at baseline ,
144 frail elderly in the experimental group reported receiving informal care compared with 118 frail elderly in the control group .
after 12 months , the final study population included 184 frail elderly and 83 informal caregivers in the experimental group and 193 frail elderly and 76 informal caregivers in the control group .
flow chart of selection and loss to follow - up of study participants in experimental and control groups the primary outcome of the intervention was quality of life , which was operationalized with health - related quality of life measured with the eq-5d instrument .
the eq-5d has five dimensions : mobility , self - care , usual activities , pain / discomfort and anxiety / depression .
each dimension has three answering categories : ( i ) no problems ; ( ii ) some problems and ( iii ) extreme problems .
the answer to each of these 5 dimensions leads to a combination of 5 numbers and 243 possible health states ( e.g. health state 21232 means : having some problems in walking about , having no problems with self - care ; having some problems with performing usual activities ; having extreme pain or discomfort ; being moderately anxious or depressed ) .
the health states unconscious and dead were added , which makes a total of 245 health states that were valued by the dutch audience on their desirability . in previous research a general sample of the dutch audience
was asked to indicate what period of time in perfect health ( 11111 ) was equal to 10 years in a specific health state ( e.g. 21232 ) ( 14 ) .
the weights obtained in this research were used to calculate the utility scores of the frail elderly of our study population .
measurements of these utility scores were obtained at baseline , 3 and 12 months and were used to calculate quality - adjusted life years ( qalys ) for each respondent .
qalys combine both quantity and quality of life in one single measure ; 1 qaly means 1 year in perfect health ( 14 ) .
health care costs , intervention costs and informal care costs were calculated by multiplying the volume of care by its corresponding cost price .
health care volumes were collected through questionnaires and gp file research ( see table 1 ) . in the questionnaires ,
the frail elderly were asked to indicate the volume of care in assisted living facilities and nursing homes , in day care centres and in home care .
information on the volume of care in assisted living facilities and nursing homes was sought retrospectively after 3 and 12 months .
the volumes of day care and home care were measured in the questionnaire at baseline , 3 and 12 months .
these volumes were extrapolated with a calculation rule to obtain the volume of care over 12 months .
the volume at baseline was considered to be the volume for the first month , the volume at 3 months was considered the volume for the second and third months and the volume at 12 months was considered to be the volume for the last 9 months .
the gp file research led to data regarding the volume of care within gp practices , hospitals and paramedical and psychological care .
data were not extrapolated , as the files provided the exact date of care consumption .
costs of care and data collection
the cost price differs per group health care professionals and is calculated for each group separately .
information on intervention costs was obtained from time registrations of the case managers and notes from the multidisciplinary meetings .
the exact intervention time and therefore intervention costs could be calculated for each individual frail elderly person .
informal care volumes were assessed by questionnaires completed by informal caregivers of the frail elderly at baseline , 3 and 12 months .
the volume of informal care was measured using the objective burden of informal care instrument ( 15 ) that distinguishes time spent on household , personal care and instrumental tasks .
the same calculation rule was applied as for the health care costs assessed in the questionnaire of the frail elderly .
cost prices were determined in euros for the year 2011 and were corrected for inflation .
first , the background characteristics of the experimental and control participants at baseline were compared by chi - square tests for the categorical variables and t - tests for the continuous variables .
second , the average volume of care and corresponding costs during the 12-month period were compared between the experimental and control groups with t - tests ( 17 ) .
the cost - effectiveness of the wicm was determined by calculating the incremental cost - effectiveness ratio ( icer ) .
the icer is calculated by dividing the difference between costs of the experimental group and control group ( incremental costs ) by the difference in effects between the experimental and control groups ( incremental effects ) .
we determined the reliability of the icer with the bootstrap method , which is a statistical method with repetitive computation to determine the confidence interval ( ci ) of the icer . by sampling from both the distribution of costs and effects concurrently ,
the design of this study was quasi - experimental and included before and after measurements with a control group providing care as usual [ for a more detailed description of the methods , see ref .
the cost - effectiveness analysis was conducted from a societal perspective and thus considered all costs related to the intervention , irrespective of who pays for these expenses ( 12 ) .
in the wicm , the gp functions as care coordinator and as a partner in prevention . the gp practice is a single entry point for the elderly , their informal caregivers and health professionals .
gps detect frailty in their patient population using the groningen frailty indicator , a validated 15-item instrument that measures decreases in physical , cognitive , social and psychological functioning .
elderly patients with a score of 4 or higher are visited by a nurse practitioner who assesses their functional , cognitive , mental and psychological functioning using easycare , an evidence - based instrument used to assess care needs . a multidisciplinary treatment plan
multidisciplinary meetings are attended by the gp , the nurse practitioner and other professionals , depending on the care required by the frail elderly .
the wicm requires task reassignment and delegation between nurses and doctors , and among gps , nursing home doctors and geriatricians .
, a steering group serves as an umbrella organization under which the wicm is developed and disseminated .
the steering group , which consists of representatives from all involved organizations , forms a joint governing board that provides the necessary provider network .
all patient representatives support the project , and the health insurer cz provides financial support for the project . compared with the wicm , care as usual in the netherlands is fragmented and reactive . in the dutch health care systems ,
patients need a referral from their gp to obtain care from the primary , secondary and tertiary echelons .
care as usual is fragmented , as professionals merely communicate bilaterally through referral letters and sporadic telephone calls .
moreover , care as usual is reactive ; patients solely receive care for specific ( health ) problems on their own initiative .
the gps in the control group were unable to implement elements of the integrated model during the study period because they did not receive financial support from the health insurer to implement the integrated care activities of the wicm .
accordingly , participants in the control group were not systematically screened for frailty , their care needs were not assessed , multidisciplinary treatment plan were not formulated and case management was not provided .
the gps in the control group had a monodisciplinary focus ; they did not organize multidisciplinary meetings or implement multidisciplinary protocols and web - based files .
furthermore , the gps in the control group could not treat the frail elderly patients differently , as these gps were not given information on who participated in the study .
the study population consisted of the entire elderly patient population of the gps in both the experimental and control groups ( see fig .
were included in the experimental group , and 249 frail elderly from six gp practices in the control group .
the frail elderly were asked whether they received informal care , including care from non - professionals and unpaid care provided by partners , family , close friends or neighbours . at baseline ,
144 frail elderly in the experimental group reported receiving informal care compared with 118 frail elderly in the control group .
after 12 months , the final study population included 184 frail elderly and 83 informal caregivers in the experimental group and 193 frail elderly and 76 informal caregivers in the control group .
flow chart of selection and loss to follow - up of study participants in experimental and control groups
the primary outcome of the intervention was quality of life , which was operationalized with health - related quality of life measured with the eq-5d instrument .
the eq-5d has five dimensions : mobility , self - care , usual activities , pain / discomfort and anxiety / depression .
each dimension has three answering categories : ( i ) no problems ; ( ii ) some problems and ( iii ) extreme problems .
the answer to each of these 5 dimensions leads to a combination of 5 numbers and 243 possible health states ( e.g. health state 21232 means : having some problems in walking about , having no problems with self - care ; having some problems with performing usual activities ; having extreme pain or discomfort ; being moderately anxious or depressed ) .
the health states unconscious and dead were added , which makes a total of 245 health states that were valued by the dutch audience on their desirability . in previous research a general sample of the dutch audience
was asked to indicate what period of time in perfect health ( 11111 ) was equal to 10 years in a specific health state ( e.g. 21232 ) ( 14 ) .
the weights obtained in this research were used to calculate the utility scores of the frail elderly of our study population .
measurements of these utility scores were obtained at baseline , 3 and 12 months and were used to calculate quality - adjusted life years ( qalys ) for each respondent .
qalys combine both quantity and quality of life in one single measure ; 1 qaly means 1 year in perfect health ( 14 ) .
health care costs , intervention costs and informal care costs were calculated by multiplying the volume of care by its corresponding cost price .
health care volumes were collected through questionnaires and gp file research ( see table 1 ) . in the questionnaires ,
the frail elderly were asked to indicate the volume of care in assisted living facilities and nursing homes , in day care centres and in home care .
information on the volume of care in assisted living facilities and nursing homes was sought retrospectively after 3 and 12 months .
the volumes of day care and home care were measured in the questionnaire at baseline , 3 and 12 months .
these volumes were extrapolated with a calculation rule to obtain the volume of care over 12 months .
the volume at baseline was considered to be the volume for the first month , the volume at 3 months was considered the volume for the second and third months and the volume at 12 months was considered to be the volume for the last 9 months .
the gp file research led to data regarding the volume of care within gp practices , hospitals and paramedical and psychological care .
data were not extrapolated , as the files provided the exact date of care consumption .
costs of care and data collection
the cost price differs per group health care professionals and is calculated for each group separately .
information on intervention costs was obtained from time registrations of the case managers and notes from the multidisciplinary meetings . the exact intervention time and
informal care volumes were assessed by questionnaires completed by informal caregivers of the frail elderly at baseline , 3 and 12 months .
the volume of informal care was measured using the objective burden of informal care instrument ( 15 ) that distinguishes time spent on household , personal care and instrumental tasks .
the same calculation rule was applied as for the health care costs assessed in the questionnaire of the frail elderly .
cost prices were determined in euros for the year 2011 and were corrected for inflation .
the primary outcome of the intervention was quality of life , which was operationalized with health - related quality of life measured with the eq-5d instrument .
the eq-5d has five dimensions : mobility , self - care , usual activities , pain / discomfort and anxiety / depression .
each dimension has three answering categories : ( i ) no problems ; ( ii ) some problems and ( iii ) extreme problems .
the answer to each of these 5 dimensions leads to a combination of 5 numbers and 243 possible health states ( e.g. health state 21232 means : having some problems in walking about , having no problems with self - care ; having some problems with performing usual activities ; having extreme pain or discomfort ; being moderately anxious or depressed ) .
the health states unconscious and dead were added , which makes a total of 245 health states that were valued by the dutch audience on their desirability . in previous research a general sample of the dutch audience
was asked to indicate what period of time in perfect health ( 11111 ) was equal to 10 years in a specific health state ( e.g. 21232 ) ( 14 ) .
the weights obtained in this research were used to calculate the utility scores of the frail elderly of our study population .
measurements of these utility scores were obtained at baseline , 3 and 12 months and were used to calculate quality - adjusted life years ( qalys ) for each respondent .
qalys combine both quantity and quality of life in one single measure ; 1 qaly means 1 year in perfect health ( 14 ) .
health care costs , intervention costs and informal care costs were calculated by multiplying the volume of care by its corresponding cost price .
health care volumes were collected through questionnaires and gp file research ( see table 1 ) . in the questionnaires , the frail elderly were asked to indicate the volume of care in assisted living facilities and nursing homes , in day care centres and in home care .
information on the volume of care in assisted living facilities and nursing homes was sought retrospectively after 3 and 12 months .
the volumes of day care and home care were measured in the questionnaire at baseline , 3 and 12 months .
these volumes were extrapolated with a calculation rule to obtain the volume of care over 12 months .
the volume at baseline was considered to be the volume for the first month , the volume at 3 months was considered the volume for the second and third months and the volume at 12 months was considered to be the volume for the last 9 months .
the gp file research led to data regarding the volume of care within gp practices , hospitals and paramedical and psychological care .
data were not extrapolated , as the files provided the exact date of care consumption .
costs of care and data collection
the cost price differs per group health care professionals and is calculated for each group separately .
information on intervention costs was obtained from time registrations of the case managers and notes from the multidisciplinary meetings . the exact intervention time and
informal care volumes were assessed by questionnaires completed by informal caregivers of the frail elderly at baseline , 3 and 12 months .
the volume of informal care was measured using the objective burden of informal care instrument ( 15 ) that distinguishes time spent on household , personal care and instrumental tasks .
the same calculation rule was applied as for the health care costs assessed in the questionnaire of the frail elderly .
cost prices were determined in euros for the year 2011 and were corrected for inflation .
first , the background characteristics of the experimental and control participants at baseline were compared by chi - square tests for the categorical variables and t - tests for the continuous variables .
second , the average volume of care and corresponding costs during the 12-month period were compared between the experimental and control groups with t - tests ( 17 ) . the cost - effectiveness of the wicm was determined by calculating the incremental cost - effectiveness ratio ( icer ) .
the icer is calculated by dividing the difference between costs of the experimental group and control group ( incremental costs ) by the difference in effects between the experimental and control groups ( incremental effects ) .
we determined the reliability of the icer with the bootstrap method , which is a statistical method with repetitive computation to determine the confidence interval ( ci ) of the icer . by sampling from both the distribution of costs and effects concurrently ,
the study population consisted of frail elderly patients with an average age of 82 years and an average score of 6 on the groningen frailty indicator ( table 2 ) .
women were over - represented in both groups and the majority of the frail elderly lived alone and independently .
the health - related quality of life was equal in both groups . compared with the control group ,
the experimental group consisted of significantly more women and frail elderly who lived in assisted living facilities .
characteristics of the study participants in experimental and control groups at baseline frail elderly patients most commonly used care from the gp , hospital and home care ( table 3 ) .
all experimental participants used gp care , as it was the single entry point of care for the intervention . in the control group , 4% of the frail elderly did not use any gp care over the 1-year period .
only limited differences were observed in the health care utilization of the experimental and control group . for two types of care ,
the first type was gp care : the costs were significantly higher in the experimental group than in the control group .
furthermore , because the intervention costs were 0 in the control group , these costs were significantly higher in the experimental group .
the average total costs in the experimental group were 17,089 euros for each frail elderly person over a 1-year period ( table 4 ) .
the costs were lower in the control group , with an average of 15,189 euros for each frail elderly person .
the average effect in the experimental group was 0.00 compared with 0.01 in the control group ; this difference was not significant .
effects and total costs of care in experimental and control groups 012 months the wicm was not found to be cost - effective after 12 months .
the intervention does not achieve incremental effects , meaning that no additional effects were gained .
the incremental costs of the intervention are 1970 euros so the wicm is more expensive than care as usual .
the results indicate an icer of 412450 euros , implying that on average 412450 should be spent to gain 1 additional qaly ( 1 year in perfect health ) .
the results of the bootstrap analysis are presented in the cost - effectiveness plane ( fig .
2 ) . very few of the bootstrap results , 0.21% , appear in the southeast quadrant , meaning that the intervention is more effective and generates lower costs than care as usual .
cost - effectiveness plane costs ( euros ) versus effects ( qaly ) of wicm versus care as usual
in this study , we performed an economic evaluation of the wicm , a comprehensive integrated care intervention for community - dwelling frail elderly including several effective integrated care elements and differing considerably from standard care ( in the netherlands ) .
the main conclusion is that the wicm is not cost - effective from a societal perspective over a 12-month period , as the costs do not outweigh the effects and the costs per qaly are high . because studies of the cost - effectiveness of integrated care show mixed results , our study both confirms and contradicts current evidence . with regard to the effects ,
these limited effects do not depend on the effect measures , as studies have adopted different effect measures , e.g. functional performance , mental health ( 6 ) , frailty state ( 9 ) and health - related quality of life ( 811 ) . in our cost - effectiveness analysis , we also chose to explore effects on quality of life because this refers to the subjective appraisal of the frail elderly themselves ( 1 ) .
moreover , we focused on health - related quality of life because this measure is primarily used for interventions that expect effects on patient health ( 12 ) .
however , comparability between the studies is limited ; it is uncertain what results would have been observed if all studies had chosen the same effect measures .
the main difference between our study and earlier research concerns the costs included ( i.e. health care costs , intervention costs and informal care costs ) . with regard to the health care costs ,
our study included a wide range of costs because the intervention focused on physical , psychological and social functioning of the elderly .
accordingly , we included costs of both paramedical and psychological care , which were not or partially considered in other studies from a societal perspective ( 8,11 ) .
furthermore , intervention costs were calculated differently in our study than in other studies . in these studies ,
the total intervention costs were calculated and divided by the number of intervention participants ( 811 ) .
the wicm involved specific investments , such as case management and time spent on multidisciplinary meetings by all professionals .
finally , informal care costs were considered only in studies adopting a societal perspective ( 8,11 ) .
three of the interventions that were considered to be cost - effective ( 6,7,9 ) adopted a health care perspective that did not include the assessment of informal care costs .
our quasi - experimental design was chosen to ensure that the frail elderly patients could stay with their own gp .
as randomization of the frail elderly made this impossible , a quasi - experimental design was the second best choice . however , quasi - experimental designs may risk baseline differences between the experimental and control groups . in our study ,
the experimental group consisted of more women and more elderly living in assisted living facilities compared with the control group .
however , these differences did not influence our results , as previous research has shown no clear association between sex and quality of life ( 18 ) or between living in an assisted living facility and quality of life ( 19 ) .
this also applies to the costs of care , which were not found to be higher for women ( 20 ) or for elderly in assisted living ( 21 ) .
additionally , with the quasi - experimental design , we might have selectively included gps in the experimental group who initially already had a more proactive attitude toward the delivery of care to frail elderly patients . because a proactive attitude has an effect on elderly s quality of life ( 1 ) , the choice not to randomize the gps might have led to a smaller effect on the change in quality of life for the experimental group .
although the quality of life at baseline did not significantly differ in the two groups , we have no information regarding changes in the quality of life prior to the beginning of the intervention .
the selection of intervention gps could also mean that these gps are more likely to participate in care activities for the frail elderly , leading to higher care costs irrespective of the costs associated with the wicm .
, precise data on the volume of some types of formal and informal care were lacking because the elderly patients did not keep records of the care they received , a method which is a commonly used in cost - effectiveness analyses .
instead , we extrapolated the volume based on their health care use at three explicit moments in time ( at baseline , after 3 and after 12 months ) .
this method could have led to an underestimation or overestimation of health care use and informal care and , consequently , of the costs of care .
additional analyses also showed that the volume of care used at the three moments in time rarely differed .
third , we did not account for all costs in the cost - effectiveness analysis , e.g. costs regarding medication and assistive devices .
we selected the seemingly most important types of care because it remains unknown what specific types of health and social care should be considered in cost - effectiveness analyses of integrated care interventions for the frail elderly .
furthermore , the costs of schooling and training were not accounted for because consideration of such costs would lead to unrealistically high costs for the experimental group , as the return on investment for these costs requires > 12 months .
it remains unclear whether integrated care for the frail elderly can achieve one of its major aims of being cost - effective and thereby providing value for money . in current health care systems ,
this implies that further research of evaluation studies on integrated care should include a cost - effectiveness analysis from a societal perspective with similar types of care considered . adopting a societal perspective , i.e. considering the costs of informal care ,
this is necessary because informal caregivers have become increasingly important in the care of frail elderly patients .
it is crucial to consider similar costs and effects in cost - effectiveness analyses to ensure comparability among studies .
more comparable cost - effectiveness analyses may help researchers to draw conclusions regarding what combinations of integrated care elements are cost - effective .
however , performing such research requires determination of the types of care and health issues can be influenced by integrated care interventions for the frail elderly and should thus be considered relevant costs and effects in future cost - effectiveness analyses .
second , future research may explore whether other goals of the wicm are achieved , such as improvements in the quality of care and consumer satisfaction .
because of a possible trade - off between the various goals of integrated care , focusing solely on cost - effectiveness might impede the implementation of a potentially successful integrated care arrangement for frail elderly patients .
funding : the netherlands organisation for health research and development ( zonmw ; grant number 313030201 ) as part of the national care for the elderly program in the netherlands .
ethics approval : the study ( design ) was reviewed by the medical ethics committee of the erasmus medical center , rotterdam , the netherlands , under protocol number mec-2013 - 058 .
this committee waived further examination because the rules established in the medical research involving human subjects act did not apply . | background.an important aim of integrated care for frail elderly is to generate more cost - effective health care . however , empirical research on the cost - effectiveness of integrated care for community - dwelling frail elderly is limited.objective.this study reports on the cost - effectiveness of the walcheren integrated care model ( wicm ) after 12 months from a societal perspective.methods.the design of this study was quasi - experimental . in total ,
184 frail elderly patients from 3 gp practices that implemented the wicm were compared with 193 frail elderly patients of 5 gp practices that provided care as usual .
effects were determined by health - related quality of life ( eq-5d questionnaire ) .
costs were assessed based on questionnaires , gp files , time registrations and reports from multidisciplinary meetings .
average costs and effects were compared using t - tests . the incremental cost - effectiveness ratio ( icer ) was calculated , and bootstrap methods were used to determine its reliability.results.neither the wicm nor care as usual resulted in a change in health - related quality of life .
the average total costs of the wicm were higher than care as usual ( 17089 euros versus 15189 euros ) .
the incremental effects were 0.00 , whereas the incremental costs were 1970 euros , indicating an icer of 412450 euros.conclusions.the wicm is not cost - effective , and the costs per quality - adjusted life year are high .
the costs of the integrated care intervention do not outweigh the limited effects on health - related quality of life after 12 months .
more analyses of the cost - effectiveness of integrated care for community - dwelling frail elderly are recommended as well as consideration of the specific costs and effects . | Introduction
Methods
Design
Intervention
Participants
Measures
Effects
Costs
Statistical analysis
Results
Conclusions
Declaration | common elements of integrated care models proven to be effective for community - dwelling frail elderly are a single entry point , geriatric assessments , case management , multidisciplinary teams ( 4 ) , multidisciplinary protocols and discussions , web - based patient files and a network structure ( 5 ) . even though integrated care largely aims at cost - effectiveness , research comparing the associated costs and effects of interventions is scarce , limiting conclusions on the cost - effectiveness of integrated care interventions ( 6 ) . some interventions for community - dwelling frail elderly have shown to be cost - effective compared with care as usual ( 69 ) , whereas other studies have shown that integrated care is not cost - effective ( 10,11 ) . the wide variation in the interventions , costs and effects considered in these studies , limits the possibility to draw conclusions regarding what promotes cost - effectiveness in integrated care for community - dwelling frail elderly . this study adds knowledge by exploring the cost - effectiveness of a specific integrated care intervention : the walcheren integrated care model ( wicm ) . therefore , we provide valuable insights regarding the cost - effectiveness of a comprehensive integrated care model for community - dwelling frail elderly . this study aimed to answer the following research question : is the wicm cost - effective from a societal perspective after 12 months ? the design of this study was quasi - experimental and included before and after measurements with a control group providing care as usual [ for a more detailed description of the methods , see ref . the cost - effectiveness analysis was conducted from a societal perspective and thus considered all costs related to the intervention , irrespective of who pays for these expenses ( 12 ) . flow chart of selection and loss to follow - up of study participants in experimental and control groups the primary outcome of the intervention was quality of life , which was operationalized with health - related quality of life measured with the eq-5d instrument . measurements of these utility scores were obtained at baseline , 3 and 12 months and were used to calculate quality - adjusted life years ( qalys ) for each respondent . the cost - effectiveness of the wicm was determined by calculating the incremental cost - effectiveness ratio ( icer ) . by sampling from both the distribution of costs and effects concurrently ,
the design of this study was quasi - experimental and included before and after measurements with a control group providing care as usual [ for a more detailed description of the methods , see ref . flow chart of selection and loss to follow - up of study participants in experimental and control groups
the primary outcome of the intervention was quality of life , which was operationalized with health - related quality of life measured with the eq-5d instrument . measurements of these utility scores were obtained at baseline , 3 and 12 months and were used to calculate quality - adjusted life years ( qalys ) for each respondent . the primary outcome of the intervention was quality of life , which was operationalized with health - related quality of life measured with the eq-5d instrument . measurements of these utility scores were obtained at baseline , 3 and 12 months and were used to calculate quality - adjusted life years ( qalys ) for each respondent . the cost - effectiveness of the wicm was determined by calculating the incremental cost - effectiveness ratio ( icer ) . effects and total costs of care in experimental and control groups 012 months the wicm was not found to be cost - effective after 12 months . the incremental costs of the intervention are 1970 euros so the wicm is more expensive than care as usual . cost - effectiveness plane costs ( euros ) versus effects ( qaly ) of wicm versus care as usual
in this study , we performed an economic evaluation of the wicm , a comprehensive integrated care intervention for community - dwelling frail elderly including several effective integrated care elements and differing considerably from standard care ( in the netherlands ) . the main conclusion is that the wicm is not cost - effective from a societal perspective over a 12-month period , as the costs do not outweigh the effects and the costs per qaly are high . because studies of the cost - effectiveness of integrated care show mixed results , our study both confirms and contradicts current evidence . functional performance , mental health ( 6 ) , frailty state ( 9 ) and health - related quality of life ( 811 ) . in our cost - effectiveness analysis , we also chose to explore effects on quality of life because this refers to the subjective appraisal of the frail elderly themselves ( 1 ) . moreover , we focused on health - related quality of life because this measure is primarily used for interventions that expect effects on patient health ( 12 ) . in current health care systems ,
this implies that further research of evaluation studies on integrated care should include a cost - effectiveness analysis from a societal perspective with similar types of care considered . however , performing such research requires determination of the types of care and health issues can be influenced by integrated care interventions for the frail elderly and should thus be considered relevant costs and effects in future cost - effectiveness analyses . because of a possible trade - off between the various goals of integrated care , focusing solely on cost - effectiveness might impede the implementation of a potentially successful integrated care arrangement for frail elderly patients . | [
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] |
brachytherapy procedures have been used for treatment of cancer patients by radiation emitted from small encapsulated sources . in this treatment modality ,
the common radionuclides of brachytherapy sources are co , cs , ir , au , i , and pd [ 14 ] .
ir can be accounted as the most commonly radionuclide used for both low - dose - rate ( ldr ) and high - dose - rate ( hdr ) brachytherapy sources .
recently , new radionuclides have been considered as alternatives to the above noted sources in brachytherapy treatments .
these sources include cs , yb , co , gd , and tm [ 512 ] .
radial dose function of cs is similar to those of i sources but it has radiobiological advantages in permanent brachytherapy implants over other sources due to its shorter half - life [ 5 , 6 ] .
yb and tm with average energies of 93 kev and 66.39 kev and half - lives of 32 days and 128.6 days , respectively , are interesting for use as hdr brachytherapy sources [ 710 ] .
co emits photons with energies of 122 and 136 kev and half - life of 272 days .
two dimensional ( 2d ) anisotropy function of co is comparable to that of ir and its radial dose function has an increasing trend in some distances , which could lead to larger dose to deeper tissue .
gd radionuclide with average energy of 60.9 kev was proposed as a low dose rate or pulsed dose rate brachytherapy source .
have shown that this source can be used for interstitial brachytherapy with rotating shield in needle .
these new radionuclides , despite having average energies lower than ir , have high enough energy to minimize photoelectric interactions in soft tissue . with these sources , a smaller shielding
the drawbacks of some of the new hypothetical isotopes can be related to their short half - life , beta contamination , and bremsstrahlung contributions .
for example , the yield of the photons emission of tm is lower than the yield its electron emission ( 6 photons per 100 electrons emitted ) .
the energy of the photon emission and half - life of rhodium-101 ( rh ) make this radionuclide a possible candidate as a brachytherapy source .
rh can be produced by nuclear reactions of ru(d , 2n)rh or ru(d , 3n)rh in a cyclotron .
prototype radionuclides have been produced at the isochronous cyclotron at the hiskp ( helmholtz - instituts fr strahlen - und kernphysik ) of the university of bonn ( germany ) .
a natural ruthenium foil ( with 20 mm cross - section and 0.3 mm thickness ) was applied as target , which included ru and ru isotopes .
a number of typical factors used in this nuclear reaction are : 1 ) deuteron energy : 27 mev ; cross section : 1000 mbarn ; deuteron beam intensity : 1 a ; 2 ) irradiation time : 2 days ; rh activity after cool - down period of 43 days : 1 mbq .
advantages of rh over the existing ir may be due to its relatively longer half - life of 3.3 years , higher specific activity of 397 tbq / g , and lower mean photon energy of 121.5 kev as shown in tables 1 and 2 .
these adequate physical characteristics made this radioisotope interesting for this project for evaluation as a possible alternative for the hdr brachytherapy source .
the purpose of this work is to evaluate the dosimetric parameters of a hypothetical rh source using task group no .
43 ( tg-43 ) recommendations and verify if it would be a potential hdr brachytherapy source .
the photon energy spectrum emitted by rh radionuclide
characteristics of the co , rh and ir sources
in this project , the geometric structure of the hypothetical rh source was designed to be similar to the flexisource ir hdr source , just for the ease of the comparison of the dosimetric parameters between the two sources .
the active core of the source is a pure rh cylinder ( density of 12.41 g / cm ) with an active length of 3.5 mm and an active diameter of 0.6 mm .
the active core is covered by a 304 stainless steel capsule ( composition by weight fe : 67.92% , cr : 19% , ni : 10% , mn : 2% ,
si : 1% and c : 0.08% ; density : 8.0 g / cm ) .
the outer dimensions of the source are 0.85 mm in diameter and 4.6 mm in total length .
the 304 stainless steel cable ( density of 8.0 g / cm ) has been considered as a cylinder with a length of 5 mm and a diameter of 0.5 mm .
the energy spectrum of the rh photons considered in this study is listed in table 1 , .
this figure is not to scale based on updated tg-43 formalism , dose rate at a point is calculated from the following formula:1d.(r,)=skg(r,)g(r0,0)g(r)f(r, )
in the above formula , s
k , , g(r, ) , g(r ) and f(r, ) present air kerma strength , dose rate constant , geometry function , radial dose function , and 2d anisotropy function , respectively . the mcnpx monte carlo ( mc ) code ( version 2.4.0 ) was used for obtaining dosimetric parameters of the new source design . in the calculations , only photons emitted by the rh were defined in the source definition card .
it was assumed that the beta particles emitted by the source ( ranging 118.78 - 541 kev with the most probable energy of 215.77 ) are absorbed by the encapsulation of source , therefore they were ignored . for estimation of air kerma strength ,
a torus with a minor diameter of 0.5 mm and major radii of 20 cm was defined on the transverse plane of the hypothetical source .
f6 tally , which is a commonly used tally in mcnp simulations , was used for these calculations and the results were obtained in terms of kerma of the source in mev/(g.photon ) .
this torus is composed of air and the surrounding phantom material is composed of vacuum .
air kerma strength then was calculated from the air kerma strength formula presented in the updated tg-43 formalism using the kerma value and the corresponding units and other conversion factors ( such as ev to joule , grams to kilograms , photon yields , etc . ) . for determination of dose rate constant
, the hypothetical source was positioned at the center of a spherical water phantom with radius of 50 cm in the simulations .
a torus with 0.5 mm minor diameter and major radii of 1.0 cm was defined on the transverse plane and * f8 tally was calculated .
this torus was also composed of water . using * f8 tally ( in terms of mev ) in mcnp it is feasible to score the absorbed energy inside a tally cell .
therefore , dose rate is output of * f8 tally divided by the mass of the torus considering the units for conversion factors ( for example , ev to joule , grams to kilograms , yields , etc . ) .
dose rate constant then was obtained as the dose rate divided by the air kerma strength . as an example for conversion of the mc output ( for * f8 tally divided per mass in terms of : mev / g per photon ) to absorbed dose rate in units of cgy/(h.mci ) the following formula can be used:2dose rate(cgy / h)=mc output(mev / g per photon)106(ev / mev)1.60210 - 19(j / ev)103(g / kg)1.0mci10 - 3(ci / mci)3.71010(bq / ci)1(dis / s per bq)photon yield of101rh(photon
/ dis)100(cgy / gy)3600(s / h )
with this regard , the value of photon yield of the rh radionuclide is equal to 2.37 photons / dis .
this value can be obtained from summation and normalization of the prevalences listed in table 1 .
the activity of rh source , used in this conversion was equal to 1.0 mci . in order to calculate radial dose function of the new source design , a spherical water phantom with 50 cm radius
torus voxels were considered at distances of 0.1 - 15.0 cm on the transverse plane of the source in the phantom .
the thicknesses of the tori were 0.4 mm at distances up to 1.0 cm , and 1.0 mm for other distances up to 15.0 cm .
the outputs of * f4 tally was used for calculation of dose rate at various radial distances . to have an acceptable level of uncertainty
, this type of tally was used to speed up the calculations in the mcnp simulations .
finally , radial dose function was calculated from dose rate and geometry function values at various distances . for calculation of 2d anisotropy function ,
the same phantom ( i.e. spherical water phantom with 50 cm radius ) was defined as that described in calculation of radial dose function .
for these simulations , the calculation points were located on a circular path around the source with the same radial distances , and different polar angles , ranging from 0 to 180 angles at 5 degrees intervals .
these points were divided into two groups , those that were along the longitudinal axis of the source , and those that were off the longitudinal axis .
spherical voxels with 0.1 cm diameter were utilized to score the dose on the longitudinal axis of the source . for the other points , tori with minor diameter of 0.4 mm
the minor diamaters of these tori were 1 mm for larger distances ( larger than 1.0 cm , up to 15.0 cm ) .
the reason for different tally voxel sizes at various distances is due to the presence of steep dose gradient as a function of distance from the source , which is an inherent effect in brachytherapy .
therefore , using larger voxel size at shorter distances may introduce artifacts in the calculations by volume - averaging .
the outputs of * f4 was used for calculation of 2d anisotropy functions . from the selected tallies at various distances ,
the 2d anisotropy function values were calculated for distances ranging between 0.5 - 15.0 cm and for angles between 0 to 180 with 5.0 degrees intervals . in calculation of tg-43 parameters ,
an attempt was also made to use toroid cells not having large dimensions at various reference directions to avoid volume - averaging artifacts .
as an example , for calculation of anisotropy function at 2.0 cm distance and 30 angle , a torus with minor diameter of 1 mm at horizontal and vertical directions was utilized . it should be noticed that in these calculations , the source delivery cable is coincident on the angle of 180 degrees . in all calculations , the energy cut off of 1 kev was defined for photons and electrons .
for calculation of tg-43 dosimetric parameters , except for 2d anisotropy function , the input files were run for 2.5 10 particles . for calculation of 2d anisotropy function , in order to reduce the calculation uncertainties at larger distances from the source , the input file was run for 10 photons . the maximum type a errors or statistical uncertainties in monte carlo calculations for air kerma strength , dose rate constant and radial dose function over all the evaluated distances were 0.3% , 3.32% and 0.44% , respectively .
the reason for having larger uncertainty in dose rate constant than the other two parameters was due to the use of * f8 tally in this case compared to f6 or * f4 tallies for the others . for similar particle scoring numbers , using * f8 tally in mcnp normally incorporates to larger uncertainty compared to the other tallies . the maximum type a uncertainty in calculation of 2d anisotropy function was 4.42% .
it should be noted that these values are type a uncertainties , by having ignored type b uncertainties and with a coverage factor ( k ) of 2.0 , the expanded uncertainties ( u ; u = ku
c , in which u
c is the combined uncertainty ) will be twice these values .
a coverage factor of 2.0 corresponds to 95% confidence level . with 95% confidence level , the true value is in the calculated value
2u calculated value + 2u interval with the probability of 0.95 .
the input programs were run using a personal computer having 64-bit windows 7.0 operating system , 3.20 ghz intel ( r ) core i7 cpu , and 2.00 gb ram . with this computer ,
the running time for the air kerma strength , dose rate constant , radial dose function , anisotropy function , with the aforementioned numbers of particles histories , was 5.25 h , 24.75 h , 25.50 h , and 134.25 h , respectively . finally , the tg-43 dosimetric parameters of the hypothetical rh source were compared with the data for another hypothetical co source and flexisource ir source .
in this project , the geometric structure of the hypothetical rh source was designed to be similar to the flexisource ir hdr source , just for the ease of the comparison of the dosimetric parameters between the two sources .
the active core of the source is a pure rh cylinder ( density of 12.41 g / cm ) with an active length of 3.5 mm and an active diameter of 0.6 mm .
the active core is covered by a 304 stainless steel capsule ( composition by weight fe : 67.92% , cr : 19% , ni : 10% , mn : 2% ,
si : 1% and c : 0.08% ; density : 8.0 g / cm ) .
the outer dimensions of the source are 0.85 mm in diameter and 4.6 mm in total length .
the 304 stainless steel cable ( density of 8.0 g / cm ) has been considered as a cylinder with a length of 5 mm and a diameter of 0.5 mm .
the energy spectrum of the rh photons considered in this study is listed in table 1 , .
based on updated tg-43 formalism , dose rate at a point is calculated from the following formula:1d.(r,)=skg(r,)g(r0,0)g(r)f(r, )
in the above formula , s
k , , g(r, ) , g(r ) and f(r, ) present air kerma strength , dose rate constant , geometry function , radial dose function , and 2d anisotropy function , respectively . the mcnpx monte carlo ( mc ) code ( version 2.4.0 ) was used for obtaining dosimetric parameters of the new source design . in the calculations , only photons emitted by the rh were defined in the source definition card .
it was assumed that the beta particles emitted by the source ( ranging 118.78 - 541 kev with the most probable energy of 215.77 ) are absorbed by the encapsulation of source , therefore they were ignored . for estimation of air kerma strength ,
a torus with a minor diameter of 0.5 mm and major radii of 20 cm was defined on the transverse plane of the hypothetical source .
f6 tally , which is a commonly used tally in mcnp simulations , was used for these calculations and the results were obtained in terms of kerma of the source in mev/(g.photon ) .
this torus is composed of air and the surrounding phantom material is composed of vacuum .
air kerma strength then was calculated from the air kerma strength formula presented in the updated tg-43 formalism using the kerma value and the corresponding units and other conversion factors ( such as ev to joule , grams to kilograms , photon yields , etc . ) . for determination of dose rate constant
, the hypothetical source was positioned at the center of a spherical water phantom with radius of 50 cm in the simulations .
a torus with 0.5 mm minor diameter and major radii of 1.0 cm was defined on the transverse plane and * f8 tally was calculated .
this torus was also composed of water . using * f8 tally ( in terms of mev ) in mcnp
therefore , dose rate is output of * f8 tally divided by the mass of the torus considering the units for conversion factors ( for example , ev to joule , grams to kilograms , yields , etc . ) .
dose rate constant then was obtained as the dose rate divided by the air kerma strength . as an example for conversion of the mc output ( for * f8 tally divided per mass in terms of : mev / g per photon ) to absorbed dose rate in units of cgy/(h.mci )
the following formula can be used:2dose rate(cgy / h)=mc output(mev / g per photon)106(ev / mev)1.60210 - 19(j / ev)103(g / kg)1.0mci10 - 3(ci / mci)3.71010(bq / ci)1(dis / s per bq)photon yield of101rh(photon
/ dis)100(cgy / gy)3600(s / h )
with this regard , the value of photon yield of the rh radionuclide is equal to 2.37 photons / dis .
this value can be obtained from summation and normalization of the prevalences listed in table 1 .
the activity of rh source , used in this conversion was equal to 1.0 mci . in order to calculate radial dose function of the new source design , a spherical water phantom with 50 cm radius
torus voxels were considered at distances of 0.1 - 15.0 cm on the transverse plane of the source in the phantom .
the thicknesses of the tori were 0.4 mm at distances up to 1.0 cm , and 1.0 mm for other distances up to 15.0 cm .
the outputs of * f4 tally was used for calculation of dose rate at various radial distances . to have an acceptable level of uncertainty ,
this type of tally was used to speed up the calculations in the mcnp simulations .
finally , radial dose function was calculated from dose rate and geometry function values at various distances . for calculation of 2d anisotropy function ,
the same phantom ( i.e. spherical water phantom with 50 cm radius ) was defined as that described in calculation of radial dose function . for these simulations ,
the calculation points were located on a circular path around the source with the same radial distances , and different polar angles , ranging from 0 to 180 angles at 5 degrees intervals .
these points were divided into two groups , those that were along the longitudinal axis of the source , and those that were off the longitudinal axis .
spherical voxels with 0.1 cm diameter were utilized to score the dose on the longitudinal axis of the source . for the other points , tori with minor diameter of 0.4 mm
the minor diamaters of these tori were 1 mm for larger distances ( larger than 1.0 cm , up to 15.0 cm ) .
the reason for different tally voxel sizes at various distances is due to the presence of steep dose gradient as a function of distance from the source , which is an inherent effect in brachytherapy .
therefore , using larger voxel size at shorter distances may introduce artifacts in the calculations by volume - averaging .
the outputs of * f4 was used for calculation of 2d anisotropy functions . from the selected tallies at various distances ,
the 2d anisotropy function values were calculated for distances ranging between 0.5 - 15.0 cm and for angles between 0 to 180 with 5.0 degrees intervals . in calculation of tg-43 parameters ,
an attempt was also made to use toroid cells not having large dimensions at various reference directions to avoid volume - averaging artifacts . as an example , for calculation of anisotropy function at 2.0 cm distance and 30 angle , a torus with minor diameter of 1 mm at horizontal and vertical directions was utilized .
it should be noticed that in these calculations , the source delivery cable is coincident on the angle of 180 degrees . in all calculations ,
for calculation of tg-43 dosimetric parameters , except for 2d anisotropy function , the input files were run for 2.5 10 particles .
for calculation of 2d anisotropy function , in order to reduce the calculation uncertainties at larger distances from the source , the input file was run for 10 photons .
the maximum type a errors or statistical uncertainties in monte carlo calculations for air kerma strength , dose rate constant and radial dose function over all the evaluated distances were 0.3% , 3.32% and 0.44% , respectively .
the reason for having larger uncertainty in dose rate constant than the other two parameters was due to the use of * f8 tally in this case compared to f6 or * f4 tallies for the others . for similar particle scoring numbers , using * f8 tally in mcnp normally incorporates to larger uncertainty compared to the other tallies . the maximum type a uncertainty in calculation of 2d anisotropy function was 4.42% .
it should be noted that these values are type a uncertainties , by having ignored type b uncertainties and with a coverage factor ( k ) of 2.0 , the expanded uncertainties ( u ; u = ku
c , in which u
c is the combined uncertainty ) will be twice these values .
, the true value is in the calculated value 2u calculated value + 2u interval with the probability of 0.95 .
the input programs were run using a personal computer having 64-bit windows 7.0 operating system , 3.20 ghz intel ( r ) core i7 cpu , and 2.00 gb ram . with this computer ,
the running time for the air kerma strength , dose rate constant , radial dose function , anisotropy function , with the aforementioned numbers of particles histories , was 5.25 h , 24.75 h , 25.50 h , and 134.25 h , respectively .
finally , the tg-43 dosimetric parameters of the hypothetical rh source were compared with the data for another hypothetical co source and flexisource ir source .
air kerma strength per activity and dose rate constant values for the hypothetical rh source were obtained , and found to be 1.09 0.01 u / mci and 1.18 0.08 cgy/(hu ) , respectively .
the errors in these values are reported with a coverage factor of 2. a comparison of other characteristics of these three sources is shown in table 2 .
these characteristics include specific activity , half - life , average photons energy , etc . it can be noticed that the specific activity was calculated theoretically from : s = ( n
a)/m .
radial dose function of the hypothetical rh source as a function of radial distance is shown in table 3 .
2d anisotropy function values of the hypothetical rh as a function of radial distance and polar angles were listed in table 4 .
radial dose function for rh hypothetical source 2d anisotropy function for rh hypothetical source a comparison of the radial dose function of the hypothetical rh source with the published data for the hypothetical co source , and flexisource ir source is shown in figure 2 .
moreover , figure 3 shows the comparison between the 2d anisotropy function of the hypothetical rh source and the values from hypothetical co source and flexisource ir source , at the distances of 0.5 , 1.0 , 5.0 , and 10.0 cm from the source .
the data presented in figures 2 and 3 for the hypothetical co source was obtained via personal communication .
the data for the flexisource ir source in these figures were extracted from the data reported by granero et al . .
a comparison of the radial dose function for the hypothetical rh source , hypothetical co source and flexisource ir source a comparison of the 2d anisotropy function for the hypothetical rh source , hypothetical co source and flexisource ir source at the distances of ( a ) : 0.5 cm ; ( b ) : 1.0 cm ; ( c ) : 5.0 cm ; ( d ) : 10.0 cm from the source
in the present study , tg-43 dosimetric parameters of a hypothetical rh source were calculated and compared with those of a hypothetical co and a commercially available flexisource ir sources .
based on the data in table 2 , air kerma strength per mci activity for the hypothetical rh source is higher ( factor of 2.37 ) than that of the hypothetical co source and less ( factor of 3.32 ) than that of ir source .
since all the three sources have the same geometrical structure , this effect is due to the differences in yield and specific activities , energy spectra of photons emitted from the radionuclides and self - absorptions inside the active cores .
higher air kerma strength per mci is an advantage for ir brachytherapy source over rh .
however , this source ( rh ) has more than twice air kerma strength per mci higher than that for co. air kerma strength per mci activity indicates the level of self - absorption inside a source , and depends on the energy spectrum of a radionuclide and source design . on the other hand ,
dose rate constant of the hypothetical rh source is approximately the same as than those for co and ir sources .
therefore , it can be concluded that per u of air kerma strength , these sources can release the same dose rates at the reference distance ( 1.0 cm ) from the source in water .
based on the data presented in figure 2 , radial dose function for the hypothetical rh source is larger than the values for ir source for distances greater than 1.0 cm .
this comparison indicates that there would be a larger dose delivered to the tissues located at larger distances from the hypothetical rh source than the ir source .
for example , at a distance of 8.0 cm , the radial dose functions of the rh and ir sources are 1.16 and 0.96 , respectively .
therefore , at this distance , rh delivers approximately 21% larger dose than ir source .
this is an excellent advantage for brachytherapy treatment of many different cancer patients , such as cervical or deep seated vaginal cancer .
moreover , the skin sparing characteristics of the rh source may become superior to the ir sources for some treatments such as the breast cancer with accuboost system .
interestingly , this graph indicates that the co source delivers larger dose than both rh and ir sources , which can be accounted as an advantage for this source .
figure 3 shows that , except at very small angles ( < 20 degrees ) , the 2d anisotropy function data of rh very similar to the ir and co data for all distances .
2d anisotropy function explains the non - isotropic feature of dose distribution around the source due to self - absorption within the source and distribution of the activity with a linear pattern .
it should be clear that the present results of tg-43 parameters for the rh source are only valid for the geometry design that is defined in this study and they can not be used for the clinical purposes . therefore , the dose distribution and the tg-43 parameters will change by variation in the source design for the hypothetical rh sources .
in other words , the results of this work , especially anisotropy , depend on the structural details of the source design .
moreover , anisotropy can not be used as a criterion for advocating the use of a new radionuclide for brachytherapy .
there are cases , in which highly anisotropic sources can be used effectively , provided their anisotropy function is accurately known .
this leaves air kerma strength , dose rate constant , and radial dose function as tg-43 quantities meaningful for the evaluation of a candidate radionuclide for brachytherapy .
their appropriateness , however , should be assessed in the context of a specific application . as a sample ,
the study by lymperopoulou et al . compares yb with ir for use as sources in prostate brachytherapy .
the same evaluations can be performed for the rh radionuclide for application in prostate brachytherapy . in the following text ,
some aspects of use of this source in interstitial rotating shield brachytherapy ( i - rsbt ) are described .
based on the data presented in table 2 , the energy of photons emitted by rh is on average lower than ir .
therefore , for medium energy photons emitted by rh , the required thickness of the shielding ( with half - value layer of 0.0331 mm lead ) is much lower than that needed for protection against ir brachytherapy source ( 2.97 mm lead ) .
it should be noticed that having compared only the average photon energies of these sources ( table 2 ) , the hvl differences are not justified .
the lower thickness for shielding requirement will reduce the costs of treatment room shielding for the rh source .
other advantages of rh are relatively long half - life of 3.3 years and high specific activity of 397 tbq / g .
these suitable characteristics made rh radionuclide interesting for application as a brachytherapy source . on the other hand ,
air kerma strength per mci activity of the rh source with the proposed geometry is lower than that of ir source . while this effect will depend on the geometries of these two sources with the assumed geometries
this is because that with the same and specific activities for these two sources , with ir the source strength will be higher and this is corresponded to a lower treatment time duration in a single treatment session . as listed in table 2 ,
dose rate constant of the rh is 1.18 cgy/(h.u ) , which is approximately 6% larger than the ir value of 1.114 .
therefore , the rh with the specified source design is able to deliver approximately 6% larger dose per air kerma strength than the ir source .
the significance of this difference should be evaluated by considering the radiobiology of the treatment site .
enger et al . has reported a dose rate at 1.0 cm in water of 4.18 10 cgy / h for a varisource ir source with activity of 370 gbq ( i.e. equivalent to 10 ci ) .
based on our monte carlo calculations , this quantity for the rh hypothetical source with a flexisource design is equal to 35.29 cgy/(h.gbq ) .
while this variable for rh source is lower than that of ir source , the source designs should be taken into account , since varisource ir source has an active length of 10.0 mm but flexisource design have a 3.5 mm one .
this comparison was performed roughly for these two sources and a precise comparison should be performed with sources with the same geometries because self - absorption inside a source and encapsulation geometry and composition will affect the dose rate at 1.0 cm distance inside a water phantom .
generally , a lower dose rate per activity at 1.0 cm will need to higher activity or higher treatment time for a standard prescribed dose regime , and , therefore , for having a reasonable treatment time a multiple - source choice may be relevant . for having a higher activity , the production costs will be higher due to longer irradiation time needed for the target in a cyclotron or nuclear reactor . with this regard ,
a comparison more consistent with tg-43 formalism can be performed with comparison of dose rate constants of the sources . since in dose rate constant ,
dose rate is normalized to air kerma strength ( instead of activity in mci ) and having mci to u conversion factors for the ir and rh sources is necessary to have a conversion from dose rate at 1.0 cm per activity to dose rate constant . in a study by lymperopoulou et al .
, monte carlo simulation was utilized and yb was compared to ir for breast hdr brachytherapy with multiple catheter implants .
the results using dose volume histogram indicated that yb could be at least as effective as ir in delivering the same dose to the lung and with slightly less dose to the skin on breast .
the finding implied that for the sources with intermediate photon energy such as yb , there is a need for the modification of calculation algorithms used in clinical treatment planning applied in particular brachytherapy practices .
. assumed a hypothetical yb source in evaluation of the use of this radionuclide for prostate hdr brachytherapy .
yb proved to be at least equivalent to ir , independent to the prostate volume in the situation that the radiation scattering be overcompensated for absorption in intermediate energies and distances in prostate hdr brachytherapy .
having reviewed the methods used in the aforementioned studies , and the point that the rh source was evaluated only from tg-43 and general dosimetric specifications , it is recommended that this radionuclide be evaluated further in comparison with ir in brachytherapy applications for various cancers .
one may need to consider two other aspects of the rh source in application of this source in brachytherapy .
it is mentioned in the introduction section that rh is produced by deuteron irradiation of isotopes of ru . theoretically , there are also other possible reactions for rh production .
some examples are : rh(n , 3n)rh ; pd(n , np)rh and pd(n , d)rh .
these reactions can be achieved by neutron irradiation of the target in a nuclear reactor .
it should be noticed that these reactions require fast neutrons and are not accounted as fission reactions .
the cross sections of these deuteron or neutron reactions are of importance when having enough activity of the rh product is aimed .
recently , a study was performed and it was disclosed that with 100 h cyclotron run time having 1 a beam current , about 900 mbq of rh can be produced . in other words ,
a maximum beam current of 1 a could be achieved in the experiments with bonn cyclotron .
rh decays to rh with a half - life of 4.34 days , and due to the short half - life of rh and long half - life of rh , production of rh with a cyclotron can be an alternative for achievement of rh . as another pathway for production of rh , it was also reported that irradiation of y by c can be proposed as a pathway to produce rh .
an issue , which could be considered with this process is the contamination of rh by other radionuclides and its influence on the tg-43 parameters .
the cross section of rh as compared to those of ir is relevant in these considerations .
ir is produced as a fission product in a nuclear reactor by irradiation of the target by thermal neutrons .
production of a radioisotope in a nuclear reactor result to lower production costs compared to a cyclotron .
although , due to lower energy photons emitted by rh relative to ir , the construction costs of shielding for treatment room may be lower , the production costs for rh source may be more than that of ir source .
i - rsbt method was evaluated for gd radioisotope in a study by adams et al . .
in that study a novel needle , catheter and source system was presented for ( i - rsbt ) application in brachytherapy of prostate .
their justification for application of a shielded source and catheter system was their aim to reduce the dose received by urethra , rectum , and bladder .
the reason for use of gd source instead of i and ir sources was that i sources has normally rapid dose fall - off in soft tissue and the thickness for shielding of ir sources would be large and can not be fitted in catheters , which are normally used in prostate brachytherapy .
however , rh emits a number of relatively high energy photons albeit with lower prevalence ( table 1 ) .
the average photon energy and half - value layer ( hvl ) in lead for rh is much lower than that of ir .
a comparison between the hvl for gd and rh may be interesting for use in i - rsbt .
the hvl ( in mm pb ) for gd and rh are 0.0783 and 0.0331 , respectively .
it is evident that with rh a lower thickness of shielding is required in this method .
however , a quantitative evaluation of shielding requirements for application in i - rsbt as a subject of further research in this field and will be interesting . the evaluations on the rh radionuclide could be accomplished simply using point source approximation that is time efficient but still meaningful with the purpose of only indication of an effect .
a number of previous studies have used this approximation in their evaluations . in a report on dose calculation in brachytherapy , it was proposed as a consideration for high energy photon emitting brachytherapy sources that modeling of sources using point source approximation is facilitated by averaging dose anisotropy over all angles .
this method of calculation can be used in permanent prostate brachytherapy dose calculation , in which seed orientation is not distinguishable for clinical non - stranded application due to the large number of seed orientations .
utilizing a point source could be easier and faster to perform but we preferred to execute a precise evaluation on this source similar to other articles on hypothetical sources by simulation of the source in its complete geometry [ 1012 ] .
based on the aapm and estro report on high - energy sources , spherical phantom radius of 40 cm was recommended .
the size of the spherical phantom used in this study is 50 cm and therefore the full scatter condition is obtained .
this report also announced recommendations on maximum voxel sizes , which are being used for scoring the dosimetric variables to minimize the volume - averaging artifacts : ( 0.1 mm ) voxels for r 1 cm ; 0.5 0.5 0.5 mm voxels for 1 cm < r 5 cm ; 1 1 1 mm voxels for 5 cm < r 10 cm and 2 2 2 mm voxels for 10 cm < r 20 cm . in the present study , voxels are not cubic and are in the form of toroidal cells with 0.4 mm in thickness for r < 1 cm and with 1 mm thickness for the other distances .
there are minor differences between our methodology for the thickness of the tally cells and those recommended by the aapm and estro report at close distances from the source , and this point should be noticed in the further methodologies .
the spectral data of rh radionuclide used in the simulations in this study ( table 1 ) were extracted from a database presented by lund university . on the other hand , a joint report by american association of physicists in medicine ( aapm ) , and european society for therapeutic radiology and oncology ( estro )
recommends that national nuclear data center ( nndc ) data be used in application of brachytherapy sources , which are clinically related .
nndc includes three datasets for energy spectra of the rh radionuclide . in a study by rivard et al .
, the influence of the choice of energy spectrum on kerma and dose distributions of three brachytherapy sources was evaluated .
it was observed that there were water - kerma differences of about 2% , 2% , and 0.7% with various spectrum choices for ir , i , and pd sources , respectively .
furthermore , the influence of photon spectrum on the dose rate constant and the radial dose function ranged from 0.1 - 2% . as a rough evaluation , the photon yields from the spectrum used in this study ( table 1 ) and the dataset no .
3 of nndc for rh are 2.3705 photons / dis . and 2.3670 photons / dis .
the details are not presented herein but the spectrum used in the present study for the rh has more detailed energies than that announced by nndc .
while it is predicted that relatively the same results will be obtained with various choices of reference spectra for the hypothetical rh source , it is recommended that the spectrum from the nndc website be used in the future studies on this source .
treatment planning in brachytherapy has advancements starting from simple look - up tables up to computerized dose calculation algorithms .
the current algorithms are based on the tg-43 formalism with recent advances in calculation of dose distributions for single sources . however , this formalism has limitations for calculation of patient dose .
various dose calculation algorithms are being developed based on : monte carlo methods , collapsed cone , etc .
the scopes of current advancements in brachytherapy include : improved dose calculation tools , planning systems to account for heterogeneities , scattering conditions , radiobiology , and image guidance brachytherapy .
following literature reports announcing the deficiencies involved in the approximations of conventional brachytherapy dosimetry , model - based dosimetry algorithms were incorporated in commercial brachytherapy treatment planning systems .
the primary calculations of these algorithms are defined , having criteria established by the developers with the purpose of optimization of computation speed and accuracy . on the other hand ,
a basic realization of the limitations of these algorithms in commissioning step and their further evaluations compared to the conventional ones is necessary .
186 provided guidance for early adopters of model - based dose calculation algorithms for brachytherapy users .
dose calculation accuracy in brachytherapy highly depends on the scatter conditions and photoelectric cross - sections relative to water . in some situations
, differences between the tg-43 and model - based algorithms can lead to dose differences exceeding a factor of 10 .
model - based dose calculation algorithms raise the major aspects , which are not addressed by current guidelines : dose sensitivity to the dose specification medium , dose calculation for the local medium in heterogeneous medium , and the dose in a small volume of water in heterogeneous medium .
was evaluated from only general and tg-43 dosimetric parameters , further evaluation of this source from the view of model - based dose calculation algorithms can be a subject of more complementary studies .
in the present study , tg-43 dosimetric parameters of a hypothetical rh source were calculated and compared with those of a hypothetical co and a commercially available flexisource ir sources .
based on the data in table 2 , air kerma strength per mci activity for the hypothetical rh source is higher ( factor of 2.37 ) than that of the hypothetical co source and less ( factor of 3.32 ) than that of ir source .
since all the three sources have the same geometrical structure , this effect is due to the differences in yield and specific activities , energy spectra of photons emitted from the radionuclides and self - absorptions inside the active cores .
higher air kerma strength per mci is an advantage for ir brachytherapy source over rh .
however , this source ( rh ) has more than twice air kerma strength per mci higher than that for co. air kerma strength per mci activity indicates the level of self - absorption inside a source , and depends on the energy spectrum of a radionuclide and source design . on the other hand ,
dose rate constant of the hypothetical rh source is approximately the same as than those for co and ir sources .
therefore , it can be concluded that per u of air kerma strength , these sources can release the same dose rates at the reference distance ( 1.0 cm ) from the source in water .
based on the data presented in figure 2 , radial dose function for the hypothetical rh source is larger than the values for ir source for distances greater than 1.0 cm .
this comparison indicates that there would be a larger dose delivered to the tissues located at larger distances from the hypothetical rh source than the ir source .
for example , at a distance of 8.0 cm , the radial dose functions of the rh and ir sources are 1.16 and 0.96 , respectively .
therefore , at this distance , rh delivers approximately 21% larger dose than ir source .
this is an excellent advantage for brachytherapy treatment of many different cancer patients , such as cervical or deep seated vaginal cancer .
moreover , the skin sparing characteristics of the rh source may become superior to the ir sources for some treatments such as the breast cancer with accuboost system .
interestingly , this graph indicates that the co source delivers larger dose than both rh and ir sources , which can be accounted as an advantage for this source .
figure 3 shows that , except at very small angles ( < 20 degrees ) , the 2d anisotropy function data of rh very similar to the ir and co data for all distances .
2d anisotropy function explains the non - isotropic feature of dose distribution around the source due to self - absorption within the source and distribution of the activity with a linear pattern .
it should be clear that the present results of tg-43 parameters for the rh source are only valid for the geometry design that is defined in this study and they can not be used for the clinical purposes . therefore , the dose distribution and the tg-43 parameters will change by variation in the source design for the hypothetical rh sources .
in other words , the results of this work , especially anisotropy , depend on the structural details of the source design .
moreover , anisotropy can not be used as a criterion for advocating the use of a new radionuclide for brachytherapy .
there are cases , in which highly anisotropic sources can be used effectively , provided their anisotropy function is accurately known .
this leaves air kerma strength , dose rate constant , and radial dose function as tg-43 quantities meaningful for the evaluation of a candidate radionuclide for brachytherapy .
their appropriateness , however , should be assessed in the context of a specific application . as a sample ,
the study by lymperopoulou et al . compares yb with ir for use as sources in prostate brachytherapy .
the same evaluations can be performed for the rh radionuclide for application in prostate brachytherapy . in the following text ,
some aspects of use of this source in interstitial rotating shield brachytherapy ( i - rsbt ) are described .
based on the data presented in table 2 , the energy of photons emitted by rh is on average lower than ir .
therefore , for medium energy photons emitted by rh , the required thickness of the shielding ( with half - value layer of 0.0331 mm lead ) is much lower than that needed for protection against ir brachytherapy source ( 2.97 mm lead ) .
it should be noticed that having compared only the average photon energies of these sources ( table 2 ) , the hvl differences are not justified .
the lower thickness for shielding requirement will reduce the costs of treatment room shielding for the rh source .
other advantages of rh are relatively long half - life of 3.3 years and high specific activity of 397 tbq / g .
these suitable characteristics made rh radionuclide interesting for application as a brachytherapy source . on the other hand ,
air kerma strength per mci activity of the rh source with the proposed geometry is lower than that of ir source . while this effect will depend on the geometries of these two sources with the assumed geometries
this is because that with the same and specific activities for these two sources , with ir the source strength will be higher and this is corresponded to a lower treatment time duration in a single treatment session . as listed in table 2 ,
dose rate constant of the rh is 1.18 cgy/(h.u ) , which is approximately 6% larger than the ir value of 1.114 .
therefore , the rh with the specified source design is able to deliver approximately 6% larger dose per air kerma strength than the ir source .
the significance of this difference should be evaluated by considering the radiobiology of the treatment site .
enger et al . has reported a dose rate at 1.0 cm in water of 4.18 10 cgy / h for a varisource ir source with activity of 370 gbq ( i.e. equivalent to 10 ci ) .
based on our monte carlo calculations , this quantity for the rh hypothetical source with a flexisource design is equal to 35.29 cgy/(h.gbq ) .
while this variable for rh source is lower than that of ir source , the source designs should be taken into account , since varisource ir source has an active length of 10.0 mm but flexisource design have a 3.5 mm one .
this comparison was performed roughly for these two sources and a precise comparison should be performed with sources with the same geometries because self - absorption inside a source and encapsulation geometry and composition will affect the dose rate at 1.0 cm distance inside a water phantom .
generally , a lower dose rate per activity at 1.0 cm will need to higher activity or higher treatment time for a standard prescribed dose regime , and , therefore , for having a reasonable treatment time a multiple - source choice may be relevant . for having a higher activity , the production costs will be higher due to longer irradiation time needed for the target in a cyclotron or nuclear reactor . with this regard ,
a comparison more consistent with tg-43 formalism can be performed with comparison of dose rate constants of the sources . since in dose rate constant ,
dose rate is normalized to air kerma strength ( instead of activity in mci ) and having mci to u conversion factors for the ir and rh sources is necessary to have a conversion from dose rate at 1.0 cm per activity to dose rate constant . in a study by lymperopoulou et al .
, monte carlo simulation was utilized and yb was compared to ir for breast hdr brachytherapy with multiple catheter implants .
the results using dose volume histogram indicated that yb could be at least as effective as ir in delivering the same dose to the lung and with slightly less dose to the skin on breast .
the finding implied that for the sources with intermediate photon energy such as yb , there is a need for the modification of calculation algorithms used in clinical treatment planning applied in particular brachytherapy practices .
. assumed a hypothetical yb source in evaluation of the use of this radionuclide for prostate hdr brachytherapy .
yb proved to be at least equivalent to ir , independent to the prostate volume in the situation that the radiation scattering be overcompensated for absorption in intermediate energies and distances in prostate hdr brachytherapy .
having reviewed the methods used in the aforementioned studies , and the point that the rh source was evaluated only from tg-43 and general dosimetric specifications , it is recommended that this radionuclide be evaluated further in comparison with ir in brachytherapy applications for various cancers .
one may need to consider two other aspects of the rh source in application of this source in brachytherapy .
it is mentioned in the introduction section that rh is produced by deuteron irradiation of isotopes of ru . theoretically , there are also other possible reactions for rh production .
some examples are : rh(n , 3n)rh ; pd(n , np)rh and pd(n , d)rh .
these reactions can be achieved by neutron irradiation of the target in a nuclear reactor .
it should be noticed that these reactions require fast neutrons and are not accounted as fission reactions .
the cross sections of these deuteron or neutron reactions are of importance when having enough activity of the rh product is aimed .
recently , a study was performed and it was disclosed that with 100 h cyclotron run time having 1 a beam current , about 900 mbq of rh can be produced . in other words ,
a maximum beam current of 1 a could be achieved in the experiments with bonn cyclotron .
rh decays to rh with a half - life of 4.34 days , and due to the short half - life of rh and long half - life of rh , production of rh with a cyclotron can be an alternative for achievement of rh . as another pathway for production of rh , it was also reported that irradiation of y by c can be proposed as a pathway to produce rh .
an issue , which could be considered with this process is the contamination of rh by other radionuclides and its influence on the tg-43 parameters .
the cross section of rh as compared to those of ir is relevant in these considerations .
ir is produced as a fission product in a nuclear reactor by irradiation of the target by thermal neutrons .
production of a radioisotope in a nuclear reactor result to lower production costs compared to a cyclotron .
although , due to lower energy photons emitted by rh relative to ir , the construction costs of shielding for treatment room may be lower , the production costs for rh source may be more than that of ir source .
i - rsbt method was evaluated for gd radioisotope in a study by adams et al . .
in that study a novel needle , catheter and source system was presented for ( i - rsbt ) application in brachytherapy of prostate .
their justification for application of a shielded source and catheter system was their aim to reduce the dose received by urethra , rectum , and bladder .
the reason for use of gd source instead of i and ir sources was that i sources has normally rapid dose fall - off in soft tissue and the thickness for shielding of ir sources would be large and can not be fitted in catheters , which are normally used in prostate brachytherapy .
however , rh emits a number of relatively high energy photons albeit with lower prevalence ( table 1 ) .
the average photon energy and half - value layer ( hvl ) in lead for rh is much lower than that of ir .
a comparison between the hvl for gd and rh may be interesting for use in i - rsbt .
the hvl ( in mm pb ) for gd and rh are 0.0783 and 0.0331 , respectively .
it is evident that with rh a lower thickness of shielding is required in this method .
however , a quantitative evaluation of shielding requirements for application in i - rsbt as a subject of further research in this field and will be interesting .
the evaluations on the rh radionuclide could be accomplished simply using point source approximation that is time efficient but still meaningful with the purpose of only indication of an effect .
a number of previous studies have used this approximation in their evaluations . in a report on dose calculation in brachytherapy , it was proposed as a consideration for high energy photon emitting brachytherapy sources that modeling of sources using point source approximation is facilitated by averaging dose anisotropy over all angles .
this method of calculation can be used in permanent prostate brachytherapy dose calculation , in which seed orientation is not distinguishable for clinical non - stranded application due to the large number of seed orientations .
utilizing a point source could be easier and faster to perform but we preferred to execute a precise evaluation on this source similar to other articles on hypothetical sources by simulation of the source in its complete geometry [ 1012 ] .
based on the aapm and estro report on high - energy sources , spherical phantom radius of 40 cm was recommended .
the size of the spherical phantom used in this study is 50 cm and therefore the full scatter condition is obtained .
this report also announced recommendations on maximum voxel sizes , which are being used for scoring the dosimetric variables to minimize the volume - averaging artifacts : ( 0.1 mm ) voxels for r 1 cm ; 0.5 0.5 0.5 mm voxels for 1 cm < r 5 cm ; 1 1 1 mm voxels for 5 cm < r 10 cm and 2 2 2 mm voxels for 10 cm < r 20 cm . in the present study ,
voxels are not cubic and are in the form of toroidal cells with 0.4 mm in thickness for r < 1 cm and with 1 mm thickness for the other distances .
there are minor differences between our methodology for the thickness of the tally cells and those recommended by the aapm and estro report at close distances from the source , and this point should be noticed in the further methodologies .
the spectral data of rh radionuclide used in the simulations in this study ( table 1 ) were extracted from a database presented by lund university . on the other hand , a joint report by american association of physicists in medicine ( aapm ) , and european society for therapeutic radiology and oncology ( estro )
recommends that national nuclear data center ( nndc ) data be used in application of brachytherapy sources , which are clinically related .
nndc includes three datasets for energy spectra of the rh radionuclide . in a study by rivard et al .
, the influence of the choice of energy spectrum on kerma and dose distributions of three brachytherapy sources was evaluated .
it was observed that there were water - kerma differences of about 2% , 2% , and 0.7% with various spectrum choices for ir , i , and pd sources , respectively .
furthermore , the influence of photon spectrum on the dose rate constant and the radial dose function ranged from 0.1 - 2% . as a rough evaluation , the photon yields from the spectrum used in this study ( table 1 ) and the dataset no .
3 of nndc for rh are 2.3705 photons / dis . and 2.3670 photons / dis . , respectively .
the details are not presented herein but the spectrum used in the present study for the rh has more detailed energies than that announced by nndc .
while it is predicted that relatively the same results will be obtained with various choices of reference spectra for the hypothetical rh source , it is recommended that the spectrum from the nndc website be used in the future studies on this source .
treatment planning in brachytherapy has advancements starting from simple look - up tables up to computerized dose calculation algorithms .
the current algorithms are based on the tg-43 formalism with recent advances in calculation of dose distributions for single sources . however , this formalism has limitations for calculation of patient dose .
various dose calculation algorithms are being developed based on : monte carlo methods , collapsed cone , etc .
the scopes of current advancements in brachytherapy include : improved dose calculation tools , planning systems to account for heterogeneities , scattering conditions , radiobiology , and image guidance brachytherapy .
following literature reports announcing the deficiencies involved in the approximations of conventional brachytherapy dosimetry , model - based dosimetry algorithms were incorporated in commercial brachytherapy treatment planning systems .
the primary calculations of these algorithms are defined , having criteria established by the developers with the purpose of optimization of computation speed and accuracy . on the other hand ,
a basic realization of the limitations of these algorithms in commissioning step and their further evaluations compared to the conventional ones is necessary .
186 provided guidance for early adopters of model - based dose calculation algorithms for brachytherapy users .
dose calculation accuracy in brachytherapy highly depends on the scatter conditions and photoelectric cross - sections relative to water . in some situations
, differences between the tg-43 and model - based algorithms can lead to dose differences exceeding a factor of 10 .
model - based dose calculation algorithms raise the major aspects , which are not addressed by current guidelines : dose sensitivity to the dose specification medium , dose calculation for the local medium in heterogeneous medium , and the dose in a small volume of water in heterogeneous medium .
was evaluated from only general and tg-43 dosimetric parameters , further evaluation of this source from the view of model - based dose calculation algorithms can be a subject of more complementary studies .
advantages of rh to ir are having relatively longer half - life ( 3.3 years versus 74 days ) , higher specific activity ( 397 tbq / g versus 341 tbq / g)k , and very low half - value layer ( 0.0331 mm in lead versus 2.97 mm of lead ) .
air kerma strength per activity for hypothetical rh source is about twice than that of hypothetical co source and it has a dose rate constant comparable to hypothetical co and ir sources .
radial dose function for the rh hypothetical source is greater than that of ir source for distances greater than 1.0 cm .
the 2d anisotropy function of rh is very similar to that of ir , which can be taken into account as another advantage of this new proposed source . with these suitable physical properties , | purposerecently a number of hypothetical sources have been proposed and evaluated for use in brachytherapy . in the present study , a hypothetical 101rh source with mean
photon energy of 121.5 kev and half - life of 3.3 years , has been evaluated as an alternative to the existing high - dose - rate ( hdr ) sources .
dosimetric characteristics of this source model have been determined following the recommendation of the task group 43 ( tg-43 ) of the american association of the physicist in medicine ( aapm ) , and the results are compared with the published data for 57co source and flexisource 192ir sources with similar geometries.material and methodsmcnpx monte carlo code was used for simulation of the 101rh hypothetical hdr source design .
geometric design of this hypothetical source was considered to be similar to that of flexisource 192ir source .
task group no .
43 dosimetric parameters , including air kerma strength per mci , dose rate constant , radial dose function , and two dimensional ( 2d ) anisotropy functions were calculated for the 101rh source through simulations.resultsair kerma strength per activity and dose rate constant for the hypothetical 101rh source were 1.09 0.01 u / mci and 1.18 0.08 cgy/(h.u ) , respectively . at distances beyond 1.0 cm in phantom , radial dose function for the hypothetical 101rh source is higher than that of 192ir .
it has also similar 2d anisotropy functions to the flexisource 192ir source.conclusions101rh is proposed as an alternative to the existing hdr sources for use in brachytherapy .
this source provides medium energy photons , relatively long half - life , higher dose rate constant and radial dose function , and similar 2d anisotropy function to the flexisource 192ir hdr source design .
the longer half - life of the source reduces the frequency of the source exchange for the clinical environment . | Purpose
Material and methods
Design for the hypothetical
Calculation of TG-43 dosimetric parameters
Results
Discussion
Dosimetric comparison with other radionuclides
Production of
Use of
Limitations and suggestions for further research
Conclusions
Disclosure | advantages of rh over the existing ir may be due to its relatively longer half - life of 3.3 years , higher specific activity of 397 tbq / g , and lower mean photon energy of 121.5 kev as shown in tables 1 and 2 . the photon energy spectrum emitted by rh radionuclide
characteristics of the co , rh and ir sources
in this project , the geometric structure of the hypothetical rh source was designed to be similar to the flexisource ir hdr source , just for the ease of the comparison of the dosimetric parameters between the two sources . (r,)=skg(r,)g(r0,0)g(r)f(r, )
in the above formula , s
k , , g(r, ) , g(r ) and f(r, ) present air kerma strength , dose rate constant , geometry function , radial dose function , and 2d anisotropy function , respectively . the maximum type a errors or statistical uncertainties in monte carlo calculations for air kerma strength , dose rate constant and radial dose function over all the evaluated distances were 0.3% , 3.32% and 0.44% , respectively . with this computer ,
the running time for the air kerma strength , dose rate constant , radial dose function , anisotropy function , with the aforementioned numbers of particles histories , was 5.25 h , 24.75 h , 25.50 h , and 134.25 h , respectively . in this project , the geometric structure of the hypothetical rh source was designed to be similar to the flexisource ir hdr source , just for the ease of the comparison of the dosimetric parameters between the two sources . (r,)=skg(r,)g(r0,0)g(r)f(r, )
in the above formula , s
k , , g(r, ) , g(r ) and f(r, ) present air kerma strength , dose rate constant , geometry function , radial dose function , and 2d anisotropy function , respectively . the maximum type a errors or statistical uncertainties in monte carlo calculations for air kerma strength , dose rate constant and radial dose function over all the evaluated distances were 0.3% , 3.32% and 0.44% , respectively . with this computer ,
the running time for the air kerma strength , dose rate constant , radial dose function , anisotropy function , with the aforementioned numbers of particles histories , was 5.25 h , 24.75 h , 25.50 h , and 134.25 h , respectively . air kerma strength per activity and dose rate constant values for the hypothetical rh source were obtained , and found to be 1.09 0.01 u / mci and 1.18 0.08 cgy/(hu ) , respectively . radial dose function for rh hypothetical source 2d anisotropy function for rh hypothetical source a comparison of the radial dose function of the hypothetical rh source with the published data for the hypothetical co source , and flexisource ir source is shown in figure 2 . moreover , figure 3 shows the comparison between the 2d anisotropy function of the hypothetical rh source and the values from hypothetical co source and flexisource ir source , at the distances of 0.5 , 1.0 , 5.0 , and 10.0 cm from the source . a comparison of the radial dose function for the hypothetical rh source , hypothetical co source and flexisource ir source a comparison of the 2d anisotropy function for the hypothetical rh source , hypothetical co source and flexisource ir source at the distances of ( a ) : 0.5 cm ; ( b ) : 1.0 cm ; ( c ) : 5.0 cm ; ( d ) : 10.0 cm from the source
in the present study , tg-43 dosimetric parameters of a hypothetical rh source were calculated and compared with those of a hypothetical co and a commercially available flexisource ir sources . based on the data in table 2 , air kerma strength per mci activity for the hypothetical rh source is higher ( factor of 2.37 ) than that of the hypothetical co source and less ( factor of 3.32 ) than that of ir source . based on the data in table 2 , air kerma strength per mci activity for the hypothetical rh source is higher ( factor of 2.37 ) than that of the hypothetical co source and less ( factor of 3.32 ) than that of ir source . this leaves air kerma strength , dose rate constant , and radial dose function as tg-43 quantities meaningful for the evaluation of a candidate radionuclide for brachytherapy . air kerma strength per activity for hypothetical rh source is about twice than that of hypothetical co source and it has a dose rate constant comparable to hypothetical co and ir sources . | [
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] |
tubo - ovarian abscess ( toa ) is a serious complication of pelvic inflammatory disease ( pid ) .
it occurs usually in women aged 2049 years and carries a significant risk of morbidity , occasional mortality , and long - term sequelae , which may include chronic pelvic pain and infertility .
treatment of toa historically was surgical , with most women having radical surgery with total abdominal hysterectomy ( tah ) and bilateral salpingo - oophorectomy ( bso).1 management of toa has changed drastically in the past 4 decades with the advent of broad - spectrum antibiotics and continues to evolve with improved imaging and drainage techniques .
studies have demonstrated an overall 16%95% success rate with conservative medical management , with the majority of studies demonstrating a success rate of 70%.1 current practice is to start with broad - spectrum intravenous ( iv ) antibiotics for 4872 hours and then resort to surgical or interventional radiology drainage , should no significant improvement be noted.13 surgical and/or radiological drainage has been shown in recent studies to decrease the hospitalization period and rehospitalization with the same complaint within 3 months.4 early resort to drainage has also been shown to hasten resolution of toa and decrease scarring of pelvic organs and the long - term sequelae , especially for women with a desire for fertility preservation.5 the size of toa should be taken into consideration when managing patients with large toa ( > 10 cm ) and early resort to drainage is advisable .
an audit at middlemore hospital conducted prior to this study had found a high incidence of recurrent admissions for patients with toa .
we therefore hypothesized that our department may have not been resorting to surgical drainage of toas as often as was ideally required ; hence , this study aims to further investigate this issue .
this was a retrospective cohort study ; all patients admitted with the diagnosis of toa to middlemore hospital between january 01 , 2008 , and december 31 , 2010 , were included and were followed up until june 30 , 2011 .
toas were coded as follows :
n700 : acute salpingitis and oophoritis , n701 : chronic salpingitis and oophoritis , n709 : salingitis and oophoritis unspecified .
n700 : acute salpingitis and oophoritis , n701 : chronic salpingitis and oophoritis , n709 : salingitis and oophoritis unspecified .
an application was lodged with the study protocol to the northern regional health and disability ethics committee with approval number ntx/11/exp/078 , dated april 26 , 2011 .
the ethics committee also deemed that written patient consent was not needed as this was a retrospective study .
data were collected from individual patient s notes , patient s information systems including concerto , and in certain instances general practitioners and/or patients were directly contacted to complete missing information .
the national health index was used to identify individual patients and to follow them up , but another identifying number was assigned to each patient for confidentiality purposes .
covariate data recorded included demographic factors , risk factors , and clinical factors as described below :
demographic factors : age , marital status , gravidity , and parity.risk factors ( at time of index admission ) : self - reported sexually active status , history of current or previous pid / sexually transmitted disease ( std ) , recent insertion or removal of intrauterine device ( iud ) ( within 3 months ) , prolonged use of iud ( > 5 years ) , and recent pelvic surgery ( within 6 weeks).clinical factors ( at time of index admission ) : abdominal pain , vaginal discharge , fever , cervical motion tenderness , adnexal tenderness , white blood cell ( wbc ) and neutrophil counts , c - reactive protein ( crp ) , chlamydia status on cervicovaginal swabs / urine , gonorrhea status on cervicovaginal swabs , bacterial vaginosis ( bv ) status on cervicovaginal swabs , bacterial culture when applicable , radiological diagnosis ultrasound scan ( uss ) or computer tomography ( ct ) scan or both , magnetic resonance imaging ( mri ) , unilaterality or bilaterality of toa , and size of toa.management factors : duration of iv / oral antibiotics , duration of hospitalization , and management plan ( medical , surgical , or interventional radiology ) .
the following supplementary data were collected conditionally on the management strategy level :
medical management : surgical management : team performing surgery ( general surgery / obstetrics and gynecology ; obstetrics and gynecology ) , type of surgery ( laparoscopy or laparotomy ) , actual procedure ( drainage only versus drainage and excision ) , surgical complications ( primary surgery versus surgery after failure of medical or interventional radiology).interventional radiology : type of drainage a ( aspiration versus aspiration and drain placement ) , type of drainage b ( ct versus uss drainage ) , site of drainage ( transvaginal , transgluteal , or transabdominal ) , number of interventions , and complications .
demographic factors : age , marital status , gravidity , and parity . risk factors ( at time of index admission ) : self - reported sexually active status , history of current or previous pid / sexually transmitted disease ( std ) , recent insertion or removal of intrauterine device ( iud ) ( within 3 months ) , prolonged use of iud ( > 5 years ) , and recent pelvic surgery ( within 6 weeks ) . clinical factors ( at time of index admission ) : abdominal pain , vaginal discharge , fever , cervical motion tenderness , adnexal tenderness , white blood cell ( wbc ) and neutrophil counts , c - reactive protein ( crp ) , chlamydia status on cervicovaginal swabs / urine , gonorrhea status on cervicovaginal swabs , bacterial vaginosis ( bv ) status on cervicovaginal swabs , bacterial culture when applicable , radiological diagnosis ultrasound scan ( uss ) or computer tomography ( ct ) scan or both , magnetic resonance imaging ( mri ) , unilaterality or bilaterality of toa , and size of toa .
management factors : duration of iv / oral antibiotics , duration of hospitalization , and management plan ( medical , surgical , or interventional radiology ) .
the following supplementary data were collected conditionally on the management strategy level :
medical management : surgical management : team performing surgery ( general surgery / obstetrics and gynecology ; obstetrics and gynecology ) , type of surgery ( laparoscopy or laparotomy ) , actual procedure ( drainage only versus drainage and excision ) , surgical complications ( primary surgery versus surgery after failure of medical or interventional radiology).interventional radiology : type of drainage a ( aspiration versus aspiration and drain placement ) , type of drainage b ( ct versus uss drainage ) , site of drainage ( transvaginal , transgluteal , or transabdominal ) , number of interventions , and complications .
surgical management : team performing surgery ( general surgery / obstetrics and gynecology ; obstetrics and gynecology ) , type of surgery ( laparoscopy or laparotomy ) , actual procedure ( drainage only versus drainage and excision ) , surgical complications ( primary surgery versus surgery after failure of medical or interventional radiology ) .
interventional radiology : type of drainage a ( aspiration versus aspiration and drain placement ) , type of drainage b ( ct versus uss drainage ) , site of drainage ( transvaginal , transgluteal , or transabdominal ) , number of interventions , and complications .
primary endpoint includes the clinical and radiological resolutions of toa within 6 months of index admission with toa .
secondary endpoints include duration of hospitalization , duration of iv and oral antibiotics , and readmission with the same complaint , repeat interventional radiology drainage , and repeat surgical intervention within 6 months of index admission with toa .
baseline characteristics were summarized according to mean and standard deviation , as well as median and interquartile range , for continuous covariates .
logistic regression was used to obtain odds ratios ( ors ) for the outcomes considered associated with each management strategy .
propensity functions ( imai and van dyk , 2004 ) based on the other observed covariates were used to appropriately reweight the outcomes according to the management strategy .
outcomes within a given management strategy were analyzed using the strategy - specific data as covariates .
times to event ( secondary outcomes ) were analyzed similarly using survival analysis tools , including cox regression and nonparametric inferential methods .
all data were entered on an excel spreadsheet , and analysis was performed using sas version 9.2 and r version 2.12 .
primary endpoint includes the clinical and radiological resolutions of toa within 6 months of index admission with toa .
secondary endpoints include duration of hospitalization , duration of iv and oral antibiotics , and readmission with the same complaint , repeat interventional radiology drainage , and repeat surgical intervention within 6 months of index admission with toa .
baseline characteristics were summarized according to mean and standard deviation , as well as median and interquartile range , for continuous covariates .
logistic regression was used to obtain odds ratios ( ors ) for the outcomes considered associated with each management strategy .
propensity functions ( imai and van dyk , 2004 ) based on the other observed covariates were used to appropriately reweight the outcomes according to the management strategy .
outcomes within a given management strategy were analyzed using the strategy - specific data as covariates .
times to event ( secondary outcomes ) were analyzed similarly using survival analysis tools , including cox regression and nonparametric inferential methods .
all data were entered on an excel spreadsheet , and analysis was performed using sas version 9.2 and r version 2.12 .
of the 277 files reviewed , 158 patients fulfilled the inclusion criteria of the study , that is , those patients with either radiologically proven toa or surgically proven toa . of those 158 patients , 15 patients represented after the follow - up period of 6 months with either a new toa ( three ) or persistent and/or recurrent ipsilateral toa ( eleven , including one patient who had three index admissions over 18 months ) .
the total number of patient / index admission with toa was , therefore , 174 .
two patients left the country after their index admission , could not be followed up , and were therefore removed from the assessment of primary and secondary outcomes but were included in the descriptive analysis . early in the data analysis
, we realized that there were only two cases that were managed with interventional radiology drainage and could not be set as a group apart ; therefore , we decided that those two cases be included in the medical management group for statistical purposes .
the mean age of patients in this study was 37.8 years , and 43% of patients were in the age group of 4049 years , 20% were older than 30 years , and 6.9% of patients were older than 50 years .
one hundred sixteen ( 66% ) patients were registered as married , 82% reported that they were sexually active , 37% had a prior history of pid / std , two ( 1.1% ) patients had a recent iud inserted , 26 ( 15.1% ) patients had prolonged use of iud
( > 5 years ) , and 19 ( 10.9% ) patients had recent pelvic surgery / intervention ( table 1 ) . regarding clinical signs , 97.7% ( 170/174 ) of patients presented with abdominal pain , 33% ( 58/174 ) presented with abnormal vaginal discharge , and 48.3% ( 84/174 ) of patients presented with fever ( table 1 ) .
physical signs included abdominal tenderness 95.4% ( 166/174 ) , cervical motion tenderness 32.2% ( 56/174 ) , and adnexal tenderness 48.3% ( 84/174 ) ( table 1 ) .
the mean maximum size of the toa was 7.2 cm with a right of 6.9 cm and a left of 6.8 cm ( table 2 ) .
diagnosis was confirmed at the time of surgery for 33 ( 18.9% ) patients , by uss of pelvis for 147 ( 84.4% ) patients , or by other radiological modalities including ct scan of abdomen and pelvis for 57 patients and mri of pelvis for seven patients .
ninety patients had negative cultures , and 80 patients had positive cultures with a mixture or a dominant pathogen ( table 3 ) .
there were 130 patients who had medical treatment only ( mp1 ) with hospitalization and iv antibiotics followed by oral antibiotics as per middlemore hospital guidelines for the management of pid / toa and 44 patients who were managed with antibiotics and surgical drainage ( mp2 ) .
this protocol for nonpregnant patients included cefoxitin 2 g iv q8h , doxycycline 100 mg bd po 14 days , metronidazole 400 mg bd po 14 days , and gentamycin if not settling after 2448 hours for patients not allergic to penicillin and clindamycin 450600 mg iv q8h , gentamycin 5 mg / kg ( ideal body weight ) iv daily followed by doxycycline 100 mg bd po 14 days , and metronidazole 400 mg bd po 14 days for patients with severe allergy to penicillin .
forty - five ( 25.8% ) patients did not have antibiotics according to protocol ; of these patients , most of them had cefuroxime or augmentin ( amoxicillin and clavulinic acid ) instead of cefoxitine or had another combination of antibiotics as advised by infectious disease team .
surgical procedures were done through laparoscopy for 23 of 44 patients and laparotomy for 21 of 44 patients with one case having a hymenotomy and drainage of an infected hematocolpos with toa .
procedures performed were lavage and drainage alone for 20 patients , excision for twelve patients , and appendicectomy for five patients .
no significant complications were noted for laparoscopies except one case of pulmonary embolus and one case of prolonged ileus .
laparotomies were associated with more complications including wound infection and dehiscence in six cases , two bowel injuries and two ureteral injuries , and one case of pneumonia likely due to sepsis ( table 4 ) .
of the 128 patients who had medical management ( mp1 ) , patients excluding the two patients lost to follow - up , 99 ( 77.3% ) patients had complete resolution of toa and 29 ( 22.6% ) patients had no resolution of toa within 6 months of index admission .
of the 44 patients who had surgical management ( mp2 ) , 41 ( 93.2% ) patients had complete resolution of toa and only three ( 6.8% ) patients had no resolution by the end of the 6 months following the index admission .
unfortunately , only 22.2% ( 38/172 ) of our study group had an imaging modality to confirm radiological resolution .
nonetheless , those patients and/or their general practitioners were contacted confirming the complete clinical resolution of their symptoms .
some of them even opted not to have the follow - up imaging and/or the clinical follow - up . instead of excluding those patients from the study
, we decided to keep them and consider that since those patients had no further complaints relating to their toas , we could also presume that those patients had complete radiological resolution .
the mean overall duration of iv antibiotics was 4.5 days , and the mean overall duration of oral antibiotics was 20.1 days .
the overall duration of iv antibiotics was 4.3 days for medical management and 4.9 days for surgical management .
the overall duration of oral antibiotics was 21.7 days for medical management and 15.4 days for surgical management .
overall , 38 ( 21.8% ) patients were readmitted with toa within 6 months of index admission , of whom 32 of 130 ( 24% ) patients were from the medical management group ( mp1 ) and six of 44 ( 13.6% ) patients were from the surgical treatment group ( mp2 ) .
overall , three of the 174 ( 1.7% ) patients needed subsequent radiological drainage ; all patients were from the medical management group .
overall , three of the 174 patients needed subsequent / repeat surgical intervention , of whom one patient was from the medical management group and two patients were from the surgical management group . when the surgical management group ( mp2 ) was compared with the medical management group ( mp1 ) , it appeared that patients who were managed surgically were more likely to have complete resolution of toa within 6 months of index admission with an or of 4 and a p - value of 0.029 , which is statistically significant .
there was no statistically significant difference in the secondary outcomes namely of readmission with toa ( or : 0.47 ) and the need for repeat surgical or radiological drainage ( or : 1.48 ) .
nonetheless , the relative duration of hospitalization was longer for the surgical group ( mp2 ) with a p - value of < 0.0001 .
the relative duration on antibiotics was shorter for the surgical group ( mp2 ) , with a statistically significant additive duration and p - value of 0.0002 ( table 5 ) .
several models were fitted on each group of the clinical factors , and further analyses were carried out on the resolution of toa outcome only .
crp and the size of the toa were found to be significant factors in the resolution of the toa within 6 months of index admission .
every unit increase in the crp increased the chance of resolution of toa b~10% ; this was not very well understood but may likely be due to the fact that patients who had raised crp were more likely to have surgery , although this was barely statistically significant with a p - value of 0.037 .
the size of the toa was also a significant covariate ; with every increase of 1 cm in the overall size of the toa , there was a 23% reduction in the resolution of toa ( table 6 ) .
patients with larger toa size were more likely to be readmitted to hospital within 6 months of index admission ( or : 1.2 [ 1.041.39 ] and p - value 0.02 ) ( table 7 ) .
every unit increase in the crp count was noted to be associated with ~2% reduction in the repeat surgical or radiological drainage of toa ( table 8) .
gamma regression analysis was carried out on the duration of hospital stay by age group .
comparison was made in the younger age group patients aged < 30 years , and this was found to be statistically significant at the 10% level .
this indicated that the older the patient was , the more likely she would have to stay longer when compared with the younger group of patients aged < 30 years ( table 9 , model 10 ) .
it appeared that patients who reported to be sexually active were more likely to have shorter hospital stay after adjusting for all age groups ( table 9 , model 1 ) .
patients who had fever were more likely to have a longer hospital stay ( table 9 , model 5 ) .
every unit rise in the crp count increased hospital stay by ~0.2% ( table 9 , model 6 ) .
patients with bilateral toa compared to those with unilateral toa were more likely to have a longer hospital stay ( table 9 , model 8) .
the size of the toa was also an important covariate influencing hospital stay ; in fact , with every 1 cm increase in the size of toa , there was ~6% increase in the duration of hospitalization .
in other words , with every 2 cm increase in the size of the toa , the duration of hospitalization was increased by 1 day ( table 9 , model 9 ) .
normal regression was carried out on the duration of antibiotics according to age group at index admission .
the age category was found to be not significant at the 10% level , and it was not included in all the analyses of the outcome .
patients who were sexually active were noted to be more likely to have a shorter duration of antibiotics ( p - value = 0.0002 ) when compared with those who were not sexually active .
in addition , patients who presented with abdominal pain were more likely to have a shorter duration of antibiotic treatment ( p - value = 0.015 ) when compared with those who had no pain on admission .
patients who presented with fever were more likely to have a longer duration of antibiotics ( p - value = 0.0033 ) when compared with those who had no fever .
again those patients with higher crp count and those patients with larger size toa were more likely to have a longer antibiotic treatment , with the p - values of 0.0096 and 0.009 , respectively .
of the 277 files reviewed , 158 patients fulfilled the inclusion criteria of the study , that is , those patients with either radiologically proven toa or surgically proven toa . of those 158 patients , 15 patients represented after the follow - up period of 6 months with either a new toa ( three ) or persistent and/or recurrent ipsilateral toa ( eleven , including one patient who had three index admissions over 18 months ) .
the total number of patient / index admission with toa was , therefore , 174 .
two patients left the country after their index admission , could not be followed up , and were therefore removed from the assessment of primary and secondary outcomes but were included in the descriptive analysis . early in the data analysis
, we realized that there were only two cases that were managed with interventional radiology drainage and could not be set as a group apart ; therefore , we decided that those two cases be included in the medical management group for statistical purposes .
the mean age of patients in this study was 37.8 years , and 43% of patients were in the age group of 4049 years , 20% were older than 30 years , and 6.9% of patients were older than 50 years .
one hundred sixteen ( 66% ) patients were registered as married , 82% reported that they were sexually active , 37% had a prior history of pid / std , two ( 1.1% ) patients had a recent iud inserted , 26 ( 15.1% ) patients had prolonged use of iud
( > 5 years ) , and 19 ( 10.9% ) patients had recent pelvic surgery / intervention ( table 1 ) . regarding clinical signs , 97.7% ( 170/174 ) of patients presented with abdominal pain , 33% ( 58/174 ) presented with abnormal vaginal discharge , and 48.3% ( 84/174 ) of patients presented with fever ( table 1 ) .
physical signs included abdominal tenderness 95.4% ( 166/174 ) , cervical motion tenderness 32.2% ( 56/174 ) , and adnexal tenderness 48.3% ( 84/174 ) ( table 1 ) .
the mean maximum size of the toa was 7.2 cm with a right of 6.9 cm and a left of 6.8 cm ( table 2 ) .
diagnosis was confirmed at the time of surgery for 33 ( 18.9% ) patients , by uss of pelvis for 147 ( 84.4% ) patients , or by other radiological modalities including ct scan of abdomen and pelvis for 57 patients and mri of pelvis for seven patients .
ninety patients had negative cultures , and 80 patients had positive cultures with a mixture or a dominant pathogen ( table 3 ) .
there were 130 patients who had medical treatment only ( mp1 ) with hospitalization and iv antibiotics followed by oral antibiotics as per middlemore hospital guidelines for the management of pid / toa and 44 patients who were managed with antibiotics and surgical drainage ( mp2 ) .
this protocol for nonpregnant patients included cefoxitin 2 g iv q8h , doxycycline 100 mg bd po 14 days , metronidazole 400 mg bd po 14 days , and gentamycin if not settling after 2448 hours for patients not allergic to penicillin and clindamycin 450600 mg iv q8h , gentamycin 5 mg / kg ( ideal body weight ) iv daily followed by doxycycline 100 mg bd po 14 days , and metronidazole 400 mg bd po 14 days for patients with severe allergy to penicillin .
forty - five ( 25.8% ) patients did not have antibiotics according to protocol ; of these patients , most of them had cefuroxime or augmentin ( amoxicillin and clavulinic acid ) instead of cefoxitine or had another combination of antibiotics as advised by infectious disease team .
surgical procedures were done through laparoscopy for 23 of 44 patients and laparotomy for 21 of 44 patients with one case having a hymenotomy and drainage of an infected hematocolpos with toa .
procedures performed were lavage and drainage alone for 20 patients , excision for twelve patients , and appendicectomy for five patients .
no significant complications were noted for laparoscopies except one case of pulmonary embolus and one case of prolonged ileus .
laparotomies were associated with more complications including wound infection and dehiscence in six cases , two bowel injuries and two ureteral injuries , and one case of pneumonia likely due to sepsis ( table 4 ) .
of the 128 patients who had medical management ( mp1 ) , patients excluding the two patients lost to follow - up , 99 ( 77.3% ) patients had complete resolution of toa and 29 ( 22.6% ) patients had no resolution of toa within 6 months of index admission .
of the 44 patients who had surgical management ( mp2 ) , 41 ( 93.2% ) patients had complete resolution of toa and only three ( 6.8% ) patients had no resolution by the end of the 6 months following the index admission .
unfortunately , only 22.2% ( 38/172 ) of our study group had an imaging modality to confirm radiological resolution .
nonetheless , those patients and/or their general practitioners were contacted confirming the complete clinical resolution of their symptoms .
some of them even opted not to have the follow - up imaging and/or the clinical follow - up . instead of excluding those patients from the study
, we decided to keep them and consider that since those patients had no further complaints relating to their toas , we could also presume that those patients had complete radiological resolution .
the mean overall duration of iv antibiotics was 4.5 days , and the mean overall duration of oral antibiotics was 20.1 days .
the overall duration of iv antibiotics was 4.3 days for medical management and 4.9 days for surgical management .
the overall duration of oral antibiotics was 21.7 days for medical management and 15.4 days for surgical management .
overall , 38 ( 21.8% ) patients were readmitted with toa within 6 months of index admission , of whom 32 of 130 ( 24% ) patients were from the medical management group ( mp1 ) and six of 44 ( 13.6% ) patients were from the surgical treatment group ( mp2 ) .
overall , three of the 174 ( 1.7% ) patients needed subsequent radiological drainage ; all patients were from the medical management group .
overall , three of the 174 patients needed subsequent / repeat surgical intervention , of whom one patient was from the medical management group and two patients were from the surgical management group .
when the surgical management group ( mp2 ) was compared with the medical management group ( mp1 ) , it appeared that patients who were managed surgically were more likely to have complete resolution of toa within 6 months of index admission with an or of 4 and a p - value of 0.029 , which is statistically significant .
there was no statistically significant difference in the secondary outcomes namely of readmission with toa ( or : 0.47 ) and the need for repeat surgical or radiological drainage ( or : 1.48 ) . nonetheless , the relative duration of hospitalization was longer for the surgical group ( mp2 ) with a p - value of < 0.0001 .
the relative duration on antibiotics was shorter for the surgical group ( mp2 ) , with a statistically significant additive duration and p - value of 0.0002 ( table 5 ) .
several models were fitted on each group of the clinical factors , and further analyses were carried out on the resolution of toa outcome only .
crp and the size of the toa were found to be significant factors in the resolution of the toa within 6 months of index admission .
every unit increase in the crp increased the chance of resolution of toa b~10% ; this was not very well understood but may likely be due to the fact that patients who had raised crp were more likely to have surgery , although this was barely statistically significant with a p - value of 0.037 .
the size of the toa was also a significant covariate ; with every increase of 1 cm in the overall size of the toa , there was a 23% reduction in the resolution of toa ( table 6 ) .
patients with larger toa size were more likely to be readmitted to hospital within 6 months of index admission ( or : 1.2 [ 1.041.39 ] and p - value 0.02 ) ( table 7 ) .
every unit increase in the crp count was noted to be associated with ~2% reduction in the repeat surgical or radiological drainage of toa ( table 8) .
gamma regression analysis was carried out on the duration of hospital stay by age group .
comparison was made in the younger age group patients aged < 30 years , and this was found to be statistically significant at the 10% level .
this indicated that the older the patient was , the more likely she would have to stay longer when compared with the younger group of patients aged < 30 years ( table 9 , model 10 ) .
it appeared that patients who reported to be sexually active were more likely to have shorter hospital stay after adjusting for all age groups ( table 9 , model 1 ) .
patients who had fever were more likely to have a longer hospital stay ( table 9 , model 5 ) .
every unit rise in the crp count increased hospital stay by ~0.2% ( table 9 , model 6 ) .
patients with bilateral toa compared to those with unilateral toa were more likely to have a longer hospital stay ( table 9 , model 8) .
the size of the toa was also an important covariate influencing hospital stay ; in fact , with every 1 cm increase in the size of toa , there was ~6% increase in the duration of hospitalization .
in other words , with every 2 cm increase in the size of the toa , the duration of hospitalization was increased by 1 day ( table 9 , model 9 ) .
normal regression was carried out on the duration of antibiotics according to age group at index admission .
the age category was found to be not significant at the 10% level , and it was not included in all the analyses of the outcome .
patients who were sexually active were noted to be more likely to have a shorter duration of antibiotics ( p - value = 0.0002 ) when compared with those who were not sexually active . in addition , patients who presented with abdominal pain were more likely to have a shorter duration of antibiotic treatment ( p - value = 0.015 ) when compared with those who had no pain on admission .
patients who presented with fever were more likely to have a longer duration of antibiotics ( p - value = 0.0033 ) when compared with those who had no fever .
again those patients with higher crp count and those patients with larger size toa were more likely to have a longer antibiotic treatment , with the p - values of 0.0096 and 0.009 , respectively .
pid usually occurs because of an ascending infection affecting the uterus ( endometritis and myometritis ) , fallopian tubes ( salpingitis ) and surrounding structures , ovary ( oophoritis ) , broad ligaments ( parametritis ) , and abdomen ( peritonitis ) .
the anatomy of the abdomen is significant in the development of pid and sepsis . in men ,
in contrast , in women , the peritoneal cavity is perforated by the free ends of the fallopian tubes.6 peritoneal reflections and mesenteric attachments compartmentalize the intraperitoneal space and facilitate the spread of exudates to sites that are often distant leading to more risk of disseminated peritoneal infection compared to men.6 one in ten women suffers from pid during her reproductive years.7 approximately 60% of pid cases may be triggered by a sexually transmitted infection ( sti ) , such as chlamydia , gonorrhea , or mycoplasma genitalium , and ~30% are not caused by an sti.1,8 there is also a known association between bv and pid.9 because of varying degrees of clinical appearance and the lack of specific laboratory tests , medical therapy is often delayed and almost one in four women with pid experiences long - term sequelae , such as chronic abdominal pain , ectopic pregnancy , and infertility.7 it has been demonstrated that no single subjective complaint , physical examination finding , or laboratory test is highly sensitive or specific for the diagnosis of pid .
thus , the diagnosis of pid requires careful consideration of the combination of patient s risk factors , physical examination findings , laboratory findings , and overall clinical presentation . because of this , the diagnosis of pid is imprecise with the clinical diagnosis of pid having a positive predictive value of 65%90% in even the most experienced practitioners hands.1 that is why the us center for disease control and prevention recommends a low threshold for treating patients if they are at risk of pid and if on physical examination they exhibit any signs of uterine , adnexal , or cervical tenderness without other apparent causes.10 if pid is inadequately treated a pelvic abscess , typically a toa develops .
toas are generally reported as complicating 10%15% of hospitalized cases of pid.7,11 toa is the most common cause of an intraabdominal abscess in premenopausal women.12 risk factors for toa are similar to those of pid and include a previous history of pid , multiple sexual partners , iud , and immunosuppression.7 in our study , two patients with toa had a recent iud insertion and 15.1% ( 26/174 ) of patients had prolonged use of iucd for > 5 years .
the percentage of iud users diagnosed with toa ranges from 20% to 54%.7 it is well known in the literature that recent insertion of iud is associated with a short - term increased risk of pid.6 a recent swedish study has demonstrated that the use of a copper iud for a period of > 5 years may be a risk factor for toa , therefore challenging the current swedish medical products agency recommendation that a woman nearing her reproductive phase could safely use the same iud for a period of > 5 years.13,14 the mechanisms by which toas are formed have been difficult to establish due to various presentations and degrees of tubo - ovarian damage present when the infection is diagnosed .
however , in many women with toa , no symptoms or signs of sti / std can be traced.7 in our study , only 12 patients had chlamydia and only two patients had gonorrhea present in their genital tract .
it has been suggested that the initial step in the formation of toa is damage and necrosis of fallopian tube epithelium by a pathogen usually an anaerobic bacterium , thereby establishing a favorable environment for anaerobic invasion and growth .
the destruction of the fallopian tube results in the production of purulent exudates.7,11 after a while , the ovary becomes involved in the inflammation .
the abscess may also engage neighboring structures , such as bowel , bladder , and the contralateral fallopian tube and ovary .
if the inflammation is not stopped , tissue planes are lost and the identification of pelvic structures and organs becomes difficult . at this stage ,
rupture of the abscess may occur , causing life - threatening peritonitis.7 other complications may include small bowel obstruction15,16 and bowel perforation especially involving the sigmoid colon.17 in our study , seven patients had recent removal of iud , four patients had a recent hysteroscopy dilatation and curettage , two patients had a recent vaginal delivery , one patient had a recent pipelle biopsy , and one patient had a recent oocyte retrieval for ivf .
surgical procedures of the female genital tract , abdominal , or vaginal , place the patient at increased risk for pid , with ~15% of pelvic infections occurring after procedures that break the cervical mucus barrier.18,19 such infections have been reported after dilatation and curettage,18,19 oocytes vaginal retrieval for ivf,19,20 essure insertions even few years later with essure acting either as a nidus or as a conduit for infection,21 and pipelle sampling.19 toa can also be secondary to an infection by contiguity originating in the small bowel secondary to crohn s disease,22 or in the appendix with acute appendicitis , subsequent perforation , and periappendiceal abscess formation that would later involve the adjacent right ovary and tube.23 the infection could also originate in the large bowel with diverticulitis and abscess formation involving especially the left ovary being very close to the sigmoid colon where most diverticulae occur.5,24 occasionally , the infection could involve the upper gastrointestinal tract with one case of toa in the literature due to a perforated peptic ulcer.25 the infection could also be due to hematogenous spread such as mycobacterium tuberculosis ; we had two cases in our study .
there are reported cases in the literature of toa due to pasteurella mutocida secondary to bacteremia from cat scratch disease.26 we also had one case with the same bacterium .
complications of abdominal and pelvic malignancy can also lead to a toa.26 in our study , one patient had a toa with lymphoma and another patient had a toa with a serous ovarian carcinoma .
the mean age of patients presenting with toa was 37.8 years with ~43% in the age group of 4049 years and 6.9% in the age group of > 50 years , which is similar to that found in the study of halperin et al conducted in israel .
it seems that our study likewise indicates a change in the epidemiology of toa.27 in halperin et als27 study , the older group of patients had a more aggressive disease likely explained by more aggressive microorganisms and/or by the delay in diagnosis and treatment of pid . in our study , patients older than 40 years had longer hospital stay compared to patients younger than 30 years . and
patients who were sexually active , usually younger , had shorter hospital stay , which again may be similar to the findings by halperin et al , although our studies aims and analyses were different .
demitras et al28 also found a mean age for patients with toa of 41.4 years . in our study , 90 patients had negative cultures and 80 patients had positive cultures with a mixture or a dominant pathogens ( table 3 ) .
two patients had streptococcus pneumoniae toa , which is unusual but has also been reported by seshadri et al.29
actinomyces species that are usually associated with long - term iud use7 were present in six of our patients , of whom four of them were associated with prolonged iud use .
two principles are essential for the understanding of the pathogenesis of female genital tract infections .
the first one is that except for a few microorganisms , such as group a-hemolytic streptoccocus , chlamydia trachomatis , and neisseria gonorrhea , the pathogens causing genital tract infections arise from the microflora of the vagina and cervix .
the second principle is that pelvic infections are usually of polymicrobic etiology.7 the microbial content of toa is usually a mixture of anaerobic , aerobic , and facultative microorganisms .
some microorganisms frequently cultured include escherichia coli ( 37% ) , bacteroides fragilis ( 22% ) , various bacteroides species ( 26% ) , peptostreptococci ( 18% ) , and peptococci ( 11%).7 patients typically present with lower abdominal pain 98% ( 97.7% in our study ) , with or without fever and chills 50% ( 48% had fever in our study ) , with a history of previous pid / std ~50% ( 37% in our study ) , and with abnormal vaginal discharge 28% ( 33% in our study).7 abdominal examination commonly elicits lower abdominal tenderness with or without guarding ( 96% in our study ) .
vaginal examination often demonstrates mucopurulent cervicovaginal discharge , cervical motion tenderness ( 32% in our study ) , and adnexal tenderness ( 48% in our study ) with sometimes the suspicion of an adnexal mass , which is difficult to ascertain when usually the pain precludes an adequate bimanual examination.7 in our study , the mean wbc count was 16.1 and the mean neutrophil count was 13.4 .
laboratory tests in patients with toa usually show an elevated wbc count and neutrophil count with also elevated inflammatory markers , including erythrocyte sedimentation rate , crp , and pentraxin 3 ( ptx3 ) , which is a novel acute inflammatory protein secreted by macrophages , dendritic cells , monocytes , smooth muscle cells , fibroblasts , and epithelial cells.8 demirtas et al28 have indicated in their study that crp values were observed to have a statistical significance in the diagnosis of an abscess , whereas the leukocyte count did not .
crp had a diagnostic value of 73% with a specificity of 83% and a sensitivity of 79% based on a cutoff value of 11.5 mg / l on the other hand , and because of the low diagnostic value of the leukocyte count , a cutoff value could not be specified.28 gngrdk et al3 found that at a cutoff of 21 mg / l crp had a diagnostic value of 80.4% with a specificity of 82.3% and a sensitivity of 65.8% .
erythrocyte sedimentation rate was requested only for a few patients in our study , and most of our patients had crp instead .
ptx3 is a test that is not yet available in new zealand , but it appears from chang et al study that this test has higher sensitivity ( 84.38% ) and lower false negative rates ( 15.63% ) when compared to crp for the diagnosis of severe pid and toa.8 patients with toa usually have higher plasma levels of ptx3 when compared to patients with pid without toa .
there are other markers of inflammation mentioned in the literature , such as osteopontin , neutrophil gelatinase - associated lipocalin , and ykl-40 , whose levels also correlate with the severity of pid and toa , and these are beyond the scope of this study.28,30 the high level of crp was associated with longer duration of hospitalization and disease severity.28 this was also echoed in our study ; patients with a higher crp were more likely to have longer hospital stay .
every unit rise in the crp count increased hospital stay by ~0.2% ( table 9 , model 6 ) .
nonetheless , it was also demonstrated in our study that every unit increase in the crp increases the chance of resolution of toa by ~10% , this was not very well understood but may likely to be due to the fact that patients who had raised crp were more likely to have surgery , although this was barely statistically significant with a p - value of 0.037 .
again those patients with higher crp count and those with larger size toa were more likely to require a longer antibiotics treatment with the p - values of 0.0096 and 0.009 , respectively ( table 9 ) . every unit increase in the crp count was noted to be associated with ~2% reduction in the repeat surgical or radiological drainage of toa .
vaginal wet smears have been evaluated in the literature to have a high sensitivity for pid / toa ( 87%91% ) if three or more wbc were seen per high - power field with a negative predictive value of 94.5% if none is seen;1 this was not evaluated in our study .
most of our patients ( 84% ) had an uss pelvis , which in most cases was a combination of transabdominal and transvaginal scanning with the use of doppler studies : 57 patients had a ct scan and seven patients had an mri scan .
pelvic uss is the initial modality of choice for assessing patients with suspected pid / toa with a diagnostic sensitivity of 56%93% and a specificity of 86%98%.12,3136 mri has a diagnostic sensitivity of 100% and a specificity of 90% , which is superior to that of uss.31,34 ct scan is generally not indicated for differential diagnosis of pelvic masses because of poor soft tissue discrimination except for fatty tissue and calcification and the disadvantages of irradiation . in our study , most ct scan would have been requested by the general surgical team or by the gynecology team when complications of toa , for example , toa rupture was suspected .
ultrasonography is most useful for differentiating toa from other stages of pid . in pid ,
the most common sonographic findings include thickened , heterogeneous endometrium , an enlarged uterus with fluid in the cavity and fluid - filled fallopian tubes.32 the next step is a tubo - ovarian complex , which is a pelvic inflammatory mass without any collection of pus within a cavity , in which edematous , adherent , infected ovaries , and tubes can still be visualized but can not be separated by an endovaginal probe . in toa , there is a loss of normal boundaries between the fallopian tube and ovary due to pus - filled , edematous - inflamed tissue .
a variety of sonographic appearances result , as described by timor - tritsch et al , as presented in the paper by adhikari et al.32 the typical sonographic appearance of a toa is a complex adnexal mass of varying echogenicity with debris , septations , and irregular margins .
the other sonographic markers of toa are pyosalpinx and loculated or speckled , echogenic fluid in the cul - de - sac.32,33,36 thickening of uterosacral ligament is visible when inflammation extends posteriorly , internal gas bubbles , strong indicators of toa , are rarely seen , and the rectosigmoid and the ureter are the most common organs that can be involved with toa.36 the mri appearance of toa is described as a thick - walled adnexal mass with low signal intensity contents on t2-weighted images .
however , this appearance is variable ; there are cases of toa demonstrating intense or increased signal intensity on t1-weighted images , and some have heterogeneous signal intensity on t2-weighted images.35 in our study , we aimed to include patients with a radiologically / ultrasonographically proven toa , but in fact there is no such entity of a radio - logically proven toa ; the diagnosis of toa is more complex than that and should be based on history and physical examination and aided by laboratory findings and medical imaging.33,35 the ultrasonographic findings of toa are not specific and could resemble and mimic or be mistaken for other cystic ovarian masses , including an endometrioma , hemorrhagic cyst , sebaceous dermoid , or some malignant ovarian neoplasms.35,37 even with mri and positron emission tomography scans , a toa can be misdiagnosed as a pelvic malignancy .
rakheja et al35 reported on a case of 20 years old woman with an intensely f - fdg avid bilateral toa mistakenly diagnosed as ovarian malignancy .
the diagnosis of toa was confirmed by surgery , mainly laparoscopy , for ~18% of patients , laparoscopy being the gold standard for the diagnosis of toa.32,33 in our study , conservative medical treatment including the two cases that had radiological drainage was successful in ~77% , which is in line with the majority of studies demonstrating a success rate of 70%.1 moreover , it appears that in our institution , there was no differentiation in management for patients with unruptured toa versus ruptured toa .
if a patient was well enough , medical treatment would be instituted first and surgery would be decided depending on worsening clinical signs and symptoms .
most toas respond well to broad spectrum iv antibiotics followed by oral antibiotics over a prolonged period of time .
triple antibiotic therapy , like the regimen used by us , seems to be the treatment of choice with a success rate varying between 16% and 95%.1,7 historically , surgical management ranging from posterior colpotomy , transabdominal surgical drainage , and unilateral salpingo - oophorectomy to tah and bso in combination with antibiotics was performed in women with toa .
although this approach offered high cure rates , it resulted in hormone deficiency and left women of child - bearing age without reproductive potential .
in addition , due to the presence of friable inflammatory tissues and adhesions , surgery in this group of patients is often technically difficult and is associated with complications .
thus , kaplan et al , as presented in the paper by granberg et al reported that bowel injury occurred in 8.4% of the patients who underwent laparotomy.7 in our study , we had two bowel injuries with our 20 laparotomies , which is ~10% of our patients .
midline laparotomy is preferred over pfannesteil incisions as the former allow surgeons to extend the incision cephalad to get better exposure , and retroperitoneal dissection may also be used as demonstrated by sharma et al to avoid bowel injury.38 posterior colpotomy is limited in that it is restricted to patients who meet the criterion of a fluctuant mass in the midline that dissects the rectovaginal septum .
additionally , it must be adherent to the parietal peritoneum . and given the complications rates and the limited patient selection , this approach should not be used in the management of toa.5 none of our patients underwent this procedure .
today the aim of surgical management is to be as minimally invasive and as conservative as possible .
this means that when surgery is undertaken , lysis of adhesions , drainage of the abscess , excision of infected and necrotic tissues , and irrigation of the peritoneal cavity are usually conducted.7 in our study , this principle was generally followed , with procedures performed being drainage and lavage alone for 20 patients , excision for 12 patients , appendicectomy for five patients with tah , and bso performed on five patients ( table 4 ) . in our study , among the 23 patients managed by operative laparoscopy , there was only one complication , which was a pulmonary embolus , that again confirms , in line with the other studies , the safety of this type of procedure .
in many centers , laparoscopy has been the gold standard for the diagnosis and treatment of toa for many years.5,7,4042 yang et al42 recommend the use of hasson open entry technique to minimize inadvertent injury to visceral organs .
henry - suchet39 carried out laparoscopic adhesiolysis and drainage of abscess in combination with antibiotics in 50 women . in 45 patients ( 90% ) , the approach was successful , while 5% required further surgery .
similar to the study of henry - suchet,7,39 reich et al , as presented in the paper by buchweitz et al reported no complications following laparoscopic and organ - preserving management of toa in 25 patients.41 in some studies , no drain is necessary after laparoscopic drainage.38 buchweitz et al41 compared the intraoperative and postoperative safety and prospects of fertility in women who had laparoscopic incision of the abscess cavity and lavage only to women who had laparoscopic salpingectomy or salpingo - oophorectomy .
they found that a significantly higher incidence of complications occurred when ablative treatment was performed when compared with organ - preserving surgery.41 some of the complications noted with the ablative group were intestinal perforation , bowel serosal injury , greater omentum injury , lacerations of blood vessels collaterals of the internal iliac artery , bowel obstruction , deep venous thrombosis , and readmission with lower abdominal pain .
there were more live term pregnancies in the drainage group compared to the ablation group with no ectopic pregnancies reported.7 in 1995 , raiga et al , as presented in the paper by yang et al evaluated the effects on fertility with the use of laparoscopy in the treatment of toa.42 all patients were treated with lysis of adhesions , drainage of purulent material , and irrigation with saline , and some patients needed further laparoscopic drainage with linear salpingotomies and/or fimbrioplasty to facilitate drainage of abscess .
all patients had complete response , and 12% of patients needed further surgery at a later stage with up to 9 years of follow - up .
the spontaneous pregnancy rate was 63% , and there was a tendency toward reduced adhesions on the second - look laparoscopies , which is likely attributed to the nearby healthy tissue having decreased exposure to necrotic and inflamed material .
hence , rosen et al in 2009 recommended considering early / immediate laparoscopy to allow for accurate diagnosis and effective treatment under magnification , with minimal complications , with possibly faster response rates , with shorter hospitalization times , and with decreased infertility.5 mitchell added that if active surgical management is not performed , there is a risk of rupture of toa and life - threatening peritonitis , with medical management alone , which is ~3%4%.5 our study was not designed to look in particular at patients who had laparoscopy but combined also those who had laparotomy .
overall , 93.2% of our patients who had surgical management reported complete resolution , which is again in line with 90%100% of initial response rate with the use of immediate laparoscopy reported by mitchell et al .
patients who had surgical management in our study had longer hospital stay with shorter duration of antibiotics compared to the medical management group .
however , we do share the opinion of mitchell et al that we should resort more frequently to laparoscopy to manage patients with suspected toa , and this study has demonstrated clearly that there are benefits to patients from this attitude .
it is of note that most of our laparoscopies in this study were performed by the surgical team in their quest to rule out a perforated / inflamed appendix , and it appears that those patients may be considered lucky to have their toa dealt with actively , with benefits in complete resolution , less days on antibiotics , possibly less scarring and adhesions , and fertility preservation for those who were still young and contemplating pregnancies .
image - guided drainage in combination with antibiotics is the third modality of management of toa . in our study
, only two patients had primary transabdominal drainage of toa : one patient was ct guided and one patient was uss guided and one patient had complete resolution and one patient did not .
three patients had secondary drainage , with all having no resolution after 6 months of index admission . during the past 23 decades ,
several studies have described image - guided drainage of pelvic abscesses with concomitant iv antibiotics to be an efficacious mode of treatment .
various approaches for abscess drainage have been reported , including transabdominal , transgluteal , transvaginal , and transrectal with the use of either computer tomographic guidance or ultrasonographic guidance . in a norwegian study,43 published in 2005 , by gjelland
et al involving 302 women with toa , it was demonstrated that transvaginal needle aspiration of abscess content together with iv antibiotics was successful in 282 ( 93.4% ) of the patients . in 20 women ( 6.6% ) , a laparoscopy or laparotomy was required .
one hundred ninety - seven ( 65.2% ) women had only one needle aspiration , 80 ( 26.5% ) women needed two aspirations , and 15 ( 5% ) women needed three aspirations , while at least ten ( 3.2% ) women needed four aspirations . the success of the procedure
no procedure - related complications such as bowel injury were recorded.43 in another study by goharkhay et al,4 the outcome of the treatment of toa by image - guided drainage and iv antibiotics versus antibiotics alone , of the total of 58 patients , 50 patients were initially treated with iv antibiotics and eight patients had initial ultrasound - guided drainage and irrigation of abscess cavity with clindamycin .
complete response was noted in 29 ( 58% ) patients treated with iv antibiotics alone .
of the 21 women who failed medical treatment , two women underwent surgery and 19 women underwent salvage drainage with ultrasound guidance for eleven patients and ct guidance for eight patients .
patients with primary drainage group had shorter hospital stay and showed more rapid resolution of fever than women who had antibiotics only.4,13 harisinghani et al44 in their study published in 2003 showed that the computer tomographic percutaneous transgluteal approach for drainage of deep pelvic abscesses with catheter placement was a safe and effective alternative to surgery for deep pelvic abscesses .
one hundred thirty - four of 140 ( 96% ) patients in their series had complete resolution of the abscess without subsequent surgery.44 it seems that this modality of management of toa is underused in our institution , and we recommend that image - guided drainage be used more frequently to improve the outcome of patients as clearly demonstrated in the aforementioned studies . is toa size associated with the duration of hospitalization and complications ? in our study , the size of the toa was a significant factor , and with every increase of 1 cm in the overall size of the toa , there was a 23% reduction in the resolution of toa .
patients with larger toa size were more likely to be readmitted to hospital within 6 months of index admission ( or : 1.2 [ 1.041.39 ] and p - value 0.02 ) .
the size of the toa was also an important factor influencing hospital stay ; in fact , with every 1 cm increase in the size of toa , there was ~6% increase in the duration of hospitalization .
in other words , with every 2 cm increase in the size of the toa , the duration of hospitalization was increased by 1 day ( table 9 , model 9 ) , which is similar to that of dewitt et al45 who demonstrated that with every 1 cm increase in the size of the abscess , there was an increase of hospitalization by 0.4 days .
again patient with larger toa was more likely to require longer antibiotics treatment with the p - value of 0.009 .
several authors have investigated the impact of toa size on the outcome of antimicrobial therapy , the need for surgery , and the rate of surgery - associated complications .
reed et al , as presented in the paper by goharkhay et al found surgery to be necessary in 60% of patients with a toa size of > 10 cm compared to 20% of those with a toa size of < 5 cm.3 doganay et al46 reported that laparotomy was performed in 72% of patients with toa diameter of > 10 cm and in 26% of patients with an abscess diameter of < 5 cm .
dewitt et al45 showed that abscesses of > 8 cm required drainage or surgery more often than abscesses of smaller sizes and toas of > 8 cm in size were associated with longer duration of hospitalization .
gngrdk et al3 found that a toa size of > 7 cm was associated with adverse outcomes , including failed response to antibiotic therapy , longer duration of hospitalization , and higher risk of surgical complications .
a toa size of 6.5 cm predicted the need for surgery with a sensitivity of 77.6% and a specificity of 65.0%.3 in our study , 15 patients had endometriosis complicated by toa who required surgery , ten patients had complete resolution within 6 months , and five patients needed surgery at a later stage , one patient needing bowel resection and permanent colostomy , and one ureter reimplantation , and another one bowel resection with reanastomosis .
of the 16 index readmissions after 6 months , nine index readmissions were for patients with endometriosis .
those patients with this combined pathology posed also a diagnostic dilemma with the endometrioma looking like toa on uss and with pathological findings of endometrioma without toa in certain cases which may indicate either resolution of the infection prior to surgery or absence of toa in the first place .
the development of toa in the context of pelvic endometriosis has been described by several authors,9,42,4750 and may be due to increased susceptibility to infection particularly in the altered immune environment seen with endometrial glands and stroma although there are no epidemiologic data to support this theory.49 an endometrioma may become a nidus for infection especially with assisted reproductive technology ; mainly oocyte retrieval with inadvertent puncture of an endometrioma .
elizur et al49 found that women with endometriosis had more severe pelvic infection pid / toa which was more refractory to conservative antibiotic treatment , often requiring surgical intervention .
elizur et al49 have also added that endometriosis is a risk factor for the development of severe pid / toa , particularly after ivf treatment .
romero et al recommended vaginal douching with povidone iodine followed by saline irrigation and adherence to strict aseptic technique to decrease the rate of infection for patients with endometriosis / endometrioma undergoing ivf treatment.50 our study was not organized to look at this group of patients in particular .
nonetheless ; with our limited analysis of those above cases , we do share the opinion of elizur et al,49 about the severity and complexity of patients with toa and endometriosis .
our study showed that toa is a complication of pid that affects the older gynecological patient and may have a pathophysiology that is different from that of pid with less involvement of the known stds caused typically by chlamydia and gonorrhea .
the diagnosis of toa is complex and relies on history , physical examination , and laboratory tests including full blood count and crp , which is aided by ultrasonography , ct scan and mri and may also be ascertained by laparoscopy or laparotomy .
crp and the size of the toa were the important prognostic factors to consider for the duration of hospitalization and the need for surgical and/or radiological drainage .
medical management alone was successful in 77% of patients , while medical and surgical drainage was superior with 93% success rate although the duration of hospitalization was longer for those who had surgery and the duration of antibiotic treatment was longer for the medical treatment group .
radiological drainage was underused and could be resorted to more frequently to improve clinical outcome .
toa with endometriosis poses a diagnostic and therapeutic dilemma with a protracted and complicated course usually requiring definitive extirpative surgery .
the major limitation of our study was the fact that it was a retrospective study and that at times proper follow - up was not arranged for all patients .
future prospective randomized studies may be able to better elucidate some of the issues raised in this study regarding , for example , the duration of hospitalization and the cost effectiveness of medical versus surgical management . | aimthe aim of this paper was to study the characteristics of patients presenting to middlemore hospital with tubo - ovarian abscess ( toa ) and to compare the outcomes of conservative medical management versus medical management with surgical drainage and medical management with radiological drainage.methodsall patients admitted with a radiologically or surgically proven toa between january 01 , 2008 and december 31 , 2010 , were included and followed up until june 30 , 2011 .
the total number of patient / index admission was 174.resultsthe mean age of patients was 37.8 years .
one hundred thirty patients had medical treatment only with hospitalization and antibiotics , and 44 patients were managed with antibiotics and surgical drainage .
complete resolution of toa was 77.3% ( 99/128 ) for patients managed medically and 93.2% ( 41/44 ) for patients managed surgically .
when the two groups were compared , patients who were managed surgically were more likely to have complete resolution of toa within 6 months of index admission with an odds ratio ( or ) of 4 and a p - value of 0.029 .
there was no statistically significant difference in the secondary outcomes namely of readmission with toa ( or : 0.47 ) and the need for repeat surgical or radiological drainage ( or : 1.48 ) . nonetheless , the relative duration of hospitalization was longer for the surgical group with a p - value of < 0.0001 . the c - reactive protein and the size of toa were the significant factors involved in the resolution of toa.conclusionthe results of this study confirmed our initial hypothesis that we should consider surgical drainage more often , probably earlier , especially for the younger patients still desiring fertility preservation and for larger abscesses .
laparoscopic surgical drainage is safe and could be used as the procedure of choice .
conservative medical management is still acceptable with good cure rates of 77% .
c - reactive protein and the size of the abscess were the important factors to consider when managing patients with toa . | Introduction
Methods
Primary endpoint
Secondary endpoints
Statistical analysis plan
Results
Descriptive statistical analysis
Inferential statistical analysis
Discussion and review of literature
Conclusion | studies have demonstrated an overall 16%95% success rate with conservative medical management , with the majority of studies demonstrating a success rate of 70%.1 current practice is to start with broad - spectrum intravenous ( iv ) antibiotics for 4872 hours and then resort to surgical or interventional radiology drainage , should no significant improvement be noted.13 surgical and/or radiological drainage has been shown in recent studies to decrease the hospitalization period and rehospitalization with the same complaint within 3 months.4 early resort to drainage has also been shown to hasten resolution of toa and decrease scarring of pelvic organs and the long - term sequelae , especially for women with a desire for fertility preservation.5 the size of toa should be taken into consideration when managing patients with large toa ( > 10 cm ) and early resort to drainage is advisable . this was a retrospective cohort study ; all patients admitted with the diagnosis of toa to middlemore hospital between january 01 , 2008 , and december 31 , 2010 , were included and were followed up until june 30 , 2011 . there were 130 patients who had medical treatment only ( mp1 ) with hospitalization and iv antibiotics followed by oral antibiotics as per middlemore hospital guidelines for the management of pid / toa and 44 patients who were managed with antibiotics and surgical drainage ( mp2 ) . of the 128 patients who had medical management ( mp1 ) , patients excluding the two patients lost to follow - up , 99 ( 77.3% ) patients had complete resolution of toa and 29 ( 22.6% ) patients had no resolution of toa within 6 months of index admission . when the surgical management group ( mp2 ) was compared with the medical management group ( mp1 ) , it appeared that patients who were managed surgically were more likely to have complete resolution of toa within 6 months of index admission with an or of 4 and a p - value of 0.029 , which is statistically significant . there was no statistically significant difference in the secondary outcomes namely of readmission with toa ( or : 0.47 ) and the need for repeat surgical or radiological drainage ( or : 1.48 ) . nonetheless , the relative duration of hospitalization was longer for the surgical group ( mp2 ) with a p - value of < 0.0001 . crp and the size of the toa were found to be significant factors in the resolution of the toa within 6 months of index admission . every unit increase in the crp increased the chance of resolution of toa b~10% ; this was not very well understood but may likely be due to the fact that patients who had raised crp were more likely to have surgery , although this was barely statistically significant with a p - value of 0.037 . patients with larger toa size were more likely to be readmitted to hospital within 6 months of index admission ( or : 1.2 [ 1.041.39 ] and p - value 0.02 ) ( table 7 ) . there were 130 patients who had medical treatment only ( mp1 ) with hospitalization and iv antibiotics followed by oral antibiotics as per middlemore hospital guidelines for the management of pid / toa and 44 patients who were managed with antibiotics and surgical drainage ( mp2 ) . of the 128 patients who had medical management ( mp1 ) , patients excluding the two patients lost to follow - up , 99 ( 77.3% ) patients had complete resolution of toa and 29 ( 22.6% ) patients had no resolution of toa within 6 months of index admission . when the surgical management group ( mp2 ) was compared with the medical management group ( mp1 ) , it appeared that patients who were managed surgically were more likely to have complete resolution of toa within 6 months of index admission with an or of 4 and a p - value of 0.029 , which is statistically significant . there was no statistically significant difference in the secondary outcomes namely of readmission with toa ( or : 0.47 ) and the need for repeat surgical or radiological drainage ( or : 1.48 ) . nonetheless , the relative duration of hospitalization was longer for the surgical group ( mp2 ) with a p - value of < 0.0001 . crp and the size of the toa were found to be significant factors in the resolution of the toa within 6 months of index admission . every unit increase in the crp increased the chance of resolution of toa b~10% ; this was not very well understood but may likely be due to the fact that patients who had raised crp were more likely to have surgery , although this was barely statistically significant with a p - value of 0.037 . nonetheless , it was also demonstrated in our study that every unit increase in the crp increases the chance of resolution of toa by ~10% , this was not very well understood but may likely to be due to the fact that patients who had raised crp were more likely to have surgery , although this was barely statistically significant with a p - value of 0.037 . crp and the size of the toa were the important prognostic factors to consider for the duration of hospitalization and the need for surgical and/or radiological drainage . | [
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] |
multiple sclerosis ( ms ) is a progressive autoimmune disease that invokes an inflammatory attack on the central nervous system ( cns ) resulting in an accumulating disability [ 1 , 2 ] .
experimental allergic encephalomyelitis ( eae ) is a primary animal model of ms which is widely used for the evaluation of different drugs in ms treatment .
eae includes the breakdown of the blood - brain barrier , infiltration of the cns by cd t cells and macrophages , and activation of microglia and astrocytes .
activated microglia and astrocytes have been implicated in the secretion of a number of proinflammatory mediators , such as tnf- , ifn- , and metalloproteinases , which act as inflammatory mediators and tissue damaging agents in the onset of eae [ 1 , 6 , 7 ] .
the remission phase of eae is accompanied by increased production of immunoregulatory cytokine transforming growth factor- ( tgf- ) [ 8 , 9 ] .
as the disease progresses , defective remyelination due to the loss of oligodendrocytes and axonal degeneration can lead to the increase of clinical handicaps .
in addition to that , secondary neuronal loss and astrogliosis underlie the deterioration of eae [ 11 , 12 ] .
the dynamic process of transendothelial migration includes the initial tethering of leukocytes to the vessel wall , followed by the rolling of these cells along the endothelium , forming tight adhesion to the endothelial surface , and ultimately moving through the intercellular junctions into the underlying tissue [ 13 , 14 ] .
integrins are the molecular limbs of a cell , enabling the trafficking and entry of pathogenic leukocytes into inflamed tissues .
3 is the most promiscuous member of the integrin family , which allows endothelial cells to interact with a wide variety of extracellular matrix components [ 1316 ] .
it is preferentially expressed on angiogenic blood vessels and supposed to have the function of improving angiogenesis [ 1316 ] .
previous studies have also revealed that occupancy of the 3 integrin could decrease monocyte binding to intercellular adhesion molecule-1 ( icam-1 ) and block the process of monocytes migration across the endothelium [ 14 , 15 ] .
the synthetic c16 peptide ( kafdityvrlkf ) , representing a functional laminin domain , may selectively bind to 3 integrin , interfering with a leukocyte ligand required for transmigration and attenuating monocytes transmigration across endothelial cell layer in vitro [ 15 , 16 ] .
it also alleviates monocytes extravasation and macrophages activation in spinal cord contusion models in vivo [ 16 , 17 ] .
most importantly , a previous study has shown that the c16 peptide does not affect systemic leukocyte counts and is not an immune - suppressant . in ms ,
large numbers of leukocytes infiltrate through the blood - brain barrier into the cns resulting in widespread tissue damage without any apparent infection [ 1821 ] .
previous researches have suggested that infiltration of such a large number of leukocytes plays a central role in the development and progression of ms and eae [ 18 , 2225 ] .
therefore , targeting neuroinflammatory reaction has been an important remedial point to alleviate the pathological features and clinical motor symptoms . since
c16 has been shown to exert anti - inflammatory activity in traumatic models of cns , we hypothesize that intravenous administration of c16 would also limit inflammatory cells infiltration in eae models . in order to optimize the application dose ,
a c16 dose response study was carried out and the most appropriate therapeutic time window was discussed .
a total of 100 adult male lewis rats were obtained from zhejiang university laboratory animal services centre . of these ,
4 were taken as normal control and the remaining 96 were randomly assigned into two vehicle control groups ( n = 32 , 16/group ) and four c16 treatment groups ( n = 64 , 16/group ) .
experiments were carried out in accordance with nih guidelines for the care and use of laboratory animals , with approval from the animal ethics committee at zhejiang university .
eae was induced in the rats for c16 treatment and the vehicle control groups by subcutaneously injection of 0.2 ml 1 : 1 mixture of guinea pig spinal cord homogenate ( gpsch ) and complete freud adjuvant ( cfa ) , containing 0.5 mg of heat killed mycobacterium tuberculosis ( difco laboratories , detroit , mi ) .
the rats in the normal control group were injected with cfa emulsified 1 : 1 with 0.9% saline [ 5 , 20 ] . immediately after cfa injection , and again after 48 hours , the rats received intraperitoneal injection of 300 ng pertussis toxin ( sigma ) twice . beginning on day 7 ,
the animals were weighed and assessed for clinical signs of disease on a daily basis .
disease severity was assessed using a scale ranging from 0 to 5 : grade 0 = no signs , grade 1 = partial loss of tail tonicity , grade 2 = total loss of tail tonicity , grade 3 = unsteady gait and mild paralysis , grade 4 = hind limb paralysis and incontinence , and grade 5 = moribund or death .
the eae model was generally considered a success if its score exceeded 2 , and scoring was continuallyd performed by people blinded to the treatments of the animals until the time of sacrifice .
the peptide solution was sterilized through a 0.22 m disc filter and neutralized to ph 7.4 with naoh .
this solution was buffered by adding an equal volume of sterile phosphate - buffered saline and the final concentration was 2 mg / ml .
the vehicle solution was prepared in the same manner without adding the peptide . for the dose
dependent test , the c16 application rats were divided into low ( 0.5 mg / per day , n = 16 ) , medium ( 1 mg / day , n = 16 ) , and high ( 2 mg / day , n = 16 ) treatment groups ; each received 1 ml of c16 solution of different concentrations . the control ( vehicle ) group ( n = 16 ) was treated with 1 ml of vehicle solution , via intravenous injection of the tail vein .
the first dose was given immediately after receiving eae induction ( following the gpsch injection and pertussis toxin injection ) ; thereafter , the solutions were injected intravenously each day for a period of two weeks . in order to establish the appropriate therapeutic time window ,
another 32 rats were assigned into a vehicle and a c16 late treated group ( 16/group ) . in the latter group ,
the first dose of c16 ( 1 mg / day ) was given 10 days after immunization , when the clinical handicaps of motor function began to emerge .
animals of vehicle control and c16 treatment groups were sacrificed at 2 weeks and 8 weeks ( 5/time point in each group ) after immunization .
rats were anesthetized with sodium pentobarbital , and then after receiving an in vivo mri scan , they were perfused intracardially with cold saline followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) .
the spinal cord and brain tissues were carefully harvested and dissected . 1 cm of the lumbar spinal cord and half of each animal 's brain were fixed in the same fixative for 4 h and then transferred into 30% sucrose in pbs until the tissue sank to the bottom of the container .
twenty m thick sections were cut on freezing microtome through the coronal plane of the brain and the transverse plane of spinal cord using a leica cryostat and then mounted onto 0.02% poly - l - lysine - coated slides .
the remains of the central nervous tissue were fixed in 2.5% glutaraldehyde solution and then examined by transmission electron microscope [ 15 , 23 ] .
mr scanning was performed using a clinical 3.0 t mri machine ( signa hdxt 3 t ) equipped with a dedicated solenoid rat coil .
t2-weighted sequences ( 15 contiguous coronal slices of 1.5 mm ) were collected with the following characteristics : te = 120 ms , tr = 3200 ms , slice thickness = 1.5 mm , slice = 15 , 320 192 matrix , field of view = 60 60 mm , flip angle = 90 .
hematoxylin and eosin ( h&e ) staining and cresyl violet ( nissl ) staining were employed to assess inflammation and neuron survival , respectively .
digital images were collected using a nikon te-300 microscope in 3 vision fields / section with 200 magnification under a bright field .
neuron counts from both spinal cord anterior horns were performed and restricted to the neurons with well - defined nuclei as well as a cell body with adequate amount of endoplasmic reticulum .
an assessment of the severity of inflammatory cell infiltration was done by a conventional h&e staining and scaled as follows : 0 , no inflammation ; 1 , cellular infiltrates only around blood vessel and meninges ; 2 , mild cellular infiltrates in parenchyma ( 110/section ) ; 3 , moderate cellular infiltrates in parenchyma ( 11100/section ) ; and 4 , serious cellular infiltrates in parenchyma ( 100/section ) .
luxol fast blue ( lfb ) staining was used to evaluate the degree of axon demyelination , as previously described .
demyelination was scored as follows using the following scale : 0 , normal white matter ; 1 , rare foci ; 2 , a few areas of demyelination ; 3 , confluent perivascular or subpial demyelination ; 4 , massive perivascular and subpial demyelination involving one half of the spinal cord with presence of cellular infiltrates in the cns parenchyma ; and 5 , extensive perivascular and subpial demyelination involving the whole cord section with presence of cellular infiltrates in the cns parenchyma .
axonal loss was assessed using the following scale : 0 , no axonal loss ; 1 , a few foci of superficial axonal loss involving less than 25% of the tissues ; 2 , foci of deep axonal loss that encompassed over 25% of the tissue ; and 3 , diffused and widespread axonal loss .
a ring of wax was applied around the sections with a pap pen ( invitrogen , carlsbad , ca , usa ) . after rinsing in 0.01 m tris - buffered saline ( tbs ) for 10 min ,
the sections were permeabilized and blocked with 0.3% triton x-100/10% normal goat serum in 0.01 m pbs for 30 min and then incubated with polyclonal rabbit antibodies : anti - cd4 ( 1 : 500 , abcam , cambridge , ma ) , anti - tumor - necrosis - factor alpha ( tnf- 1 : 1000 , prosci incorporated , ca , usa ) , anti - caspase-3 ( 1 : 500 ; cayman chemical , ann arbor , mi ) , and mouse antimyelin basic protein ( mbp , 1 : 500 , abcam , cambridge , ma ) overnight at 4c .
the process of immunohistochemical staining was performed as described previously ; primary antibody omission controls were used in order to confirm further the specificity of the immunohistochemical labeling .
five sections from the motor cortex and anterior horns of spinal cord of each animal were randomly selected and images were photographed under 200 magnification in 3 vision fields / section .
the caspase-3 and tnf- immunoreactive cells were counted , and the immunoreactive areas for cd4 and mbp were analyzed with nih image software .
the sections were pretreated with the same method described above and incubated with primary monoclonal mouse antibodies cd45 and cd68/ed1 ( 1 : 100 ; santa cruz biotechnology , santa cruz , ca ) and polyclonal rabbit anti - glial fibrillary acidic protein ( gfap 1 : 200 , thermo fisher scientific , waltham , ma ) .
the process of immunofluorescence staining was performed as described previously ; primary antibody omission controls were used in order to confirm further the specificity of the immunohistochemical labeling .
immunoreactive areas of gfap , cd45 , and cd68 were analyzed with nih image software .
postfixed tissues were placed in 1% osmium tetroxide at 4c overnight and then washed 3 times with 0.1 m pb . the processing for electron microscopy was performed as described previously .
images were captured first at low resolution and then at higher magnification in different regions of the white matter .
peripheral blood samples were collected from rats that had been sacrificed by decapitation at 2 weeks and 8 weeks after immunization ( n = 3 per time point / each group ) .
plasma samples were collected on ice ( centrifuged for 20 minutes at 1000 g within 30 minutes of collection , and then at 10,000 g for 10 minutes at 4c for complete platelet removal ) using heparin as an anticoagulant .
to assess cytokine expression , plasma samples were incubated in 96-well plates precoated with antibodies to ifn- ( biolegend inc . , san diego , ca ) and tgf- ( r&d systems , minneapolis , mn ) at 37c for 60 min .
hrp - conjugated goat anti - rabbit igg ( bio - rad ) diluted at 1 : 2,000 was used as the secondary antibody .
optical density was measured at 450 nm on model 680 microplate reader ( bio - rad laboratories , corston , uk ) .
the optical density was quantified by graphpad prism 4 ( graphpad software , inc ) .
the rats were sacrificed by decapitation at 2 weeks and 8 weeks after immunization ( n = 3 per time point / each group ) , and from each animal , the whole brain cortex and a 10 mm lumbar spinal cord segment were prepared for western blotting , which was performed as described previously .
total proteins were extracted with 2 mm pmsf in 1 ml ice - cold ripa buffer added protease inhibitor cocktail .
sds - page was performed on 12% polyacrylamide slab gel , and separated proteins were electrophoretically transferred to pvdf membrane in a bio - rad transblot apparatus . after blocking nonspecific binding sites with bovine serum albumin ,
each membrane was incubated for 12 h at room temperature with primary rabbit polyclonal anti - gfap ( 1 : 500 ) , anti - tnf- ( 1 : 2000 ) , and anti - caspase-3 ( 1 : 500 ) . to normalize protein bands to a gel loading control
, membranes were washed in tbst and reprobed with rabbit anti--actin ( 1 : 5,000 , abcam , ma ) followed by an incubation with peroxidase - conjugated goat anti - rabbit ( 1 : 5,000 , santa cruz , ca ) and ecl detection . for the negative control
kruskal - wallis nonparametric analysis was conducted and followed by a nonparametric mann - whitney test on each pair of data .
data were analyzed by spss 13.0 software , and p values less than 0.05 were considered statistically significant .
at the peak time of the disease ( 2 weeks after immunization ) , h&e staining of the cerebral cortex and spinal cord ( figure 1 ) revealed a significant increase of the cellular density in eae rats treated with vehicle .
diffuse infiltration of inflammatory cells appeared in surrounding blood vessels and under the meninges ; there was also wide infiltration of brain tissue parenchyma ( figure 1(d ) ) .
clear lesion focus with hyperintensity was detectable in the white matter by mri t2w scanning ( figure 1(c ) ) . in the spinal cord ,
the gray matter showed a marked cellular infiltration which was not confined to the anterior or dorsal horns but extended throughout the gray matter and white matter ( figure 1(n ) ) .
after c16 treatment for 2 weeks at different doses , less severe perivascular and parenchymal infiltration of inflammatory cells ( mixed with macrophages , microglia , and lymphocytes ) within the spinal cord and brain tissue was observed ( figure 1 ) . in general , when treated with a low dose of c16 ( 0.5 mg / day ) , the extent of inflammatory cells infiltration , the number of perivascular infiltration focus points , and the size of hyperintensity lesion sites in t2w images were significantly more abundant and larger in size than in the medium ( 1 mg / day ) and in high ( 2 mg / day ) dose c16 treated groups ( figures 1(e ) and 1(f ) ) .
there were no significant differences in inflammatory cell infiltration between the medium and high dose groups ( p > 0.05 , figure 1(q ) ) .
the c16 late treated group , which received c16 application 10 days after immunization , also presented a decrease of inflammation score compared with the vehicle control ( p < 0.01 , figure 1(r ) ) .
eight weeks after immunization , visible lesion sites could still be detected by t2w scan ( figure 1(i ) ) , but the extent of infiltration and the number of perivascular infiltration focus sites decreased compared with 2 weeks after immunization in the vehicle control ( figures 1(j)1(l ) ) .
the low dose c16 treatment did not show evident amelioration of inflammation , but the medium and high dose treatments all exhibited remarkable improvement in suppressing the inflammatory cells infiltration ( p < 0.001 , figure 1(q ) ) . in the c16
late treated case , the escaping inflammatory cells also noticeably decreased compared with the vehicle control ( p < 0.001 , figure 1(r ) ) . for determination of the types of inflammatory cells ,
the immunostaining of cd4 ( a marker for extravasated t lymphocytes ) , cd45 ( a pan - leukocyte marker for leukocytes ) , and cd68 ( for activated microglia and extravasated macrophages ) was done .
the results showed that cells positive for cd4 , cd68 , and cd45 all increased remarkably in both grey and white matter of cns in the vehicle control rats ( figure 2 ) .
an evident decline of t lymphocyte infiltration , leukocytes extravasation , and macrophages activation was detected in c16 treated group ( figures 2(c ) , 2(d ) , 2(f ) , 2(g ) , 2(i ) , 2(j ) , 2(l ) , 2(m ) , 2(o ) , and 2(p ) ) , especially in the medium and high dose treated groups ( p < 0.001 , figures 3(a ) , 3(c ) , and 3(e ) ) .
the c16 late treatment also reduced the cd4 , cd68 and cd45 labeled inflammatory cells in both week 2 and 8 after immunization ( p < 0.01 , figures 3(b ) , 3(d ) , and 3(f ) ) . by luxol
fast blue ( lfb ) staining and mbp ( one of the major central myelin proteins ) immunohistochemical staining , we checked the total demyelination condition of each group .
massive perivascular and confluent demyelinated areas were present in the parenchyma of the cns of vehicle control rats at the peak time of disease ( week 2 after immunization ) ( figures 4(b ) , 4(k ) , and 4(l ) ) .
at the same time point , when treated with different doses of c16 peptide , the visible areas of demyelination gradually declined ( figures 4(c ) , 4(d ) , 4(m ) , and 4(n ) ) . in the high dose c16 treated group , only rare foci of demyelination could be found ( figures 4(c ) and 4(g ) ) . at week 8 after immunization , the demyelination condition in vehicle control was still obvious , while the myelin loss was significantly reduced by medium and high dose c16 treatment ( figures 4(e)4(g ) ) .
similarly , the late c16 treatments also remarkably reduced the demyelination score and improved the mbp labeled myelin area compared with vehicle control at the same time point ( figures 4(d ) , 4(h ) , 4(o ) , and 4(p ) ; tables 1 and 2 ) .
the vehicle control rats displayed severe axonal loss both in white and gray matters of cns at week 2 after immunization ; a similar condition existed in week 8 after immunization .
the bielschowsky staining impregnation revealed that the injured axons have swelling , with a deformed and ovoid formation ( figures 5(c ) and 5(d ) ) .
compared with vehicle control , there was no notable difference in axonal number in the low dose c16 treated group , but more axons with relatively normal formation were kept in medium and high dose treated eae rats ( figures 5(e ) , 5(i ) , 5(j ) , and 5(m ) ) .
even when the treatment was delayed to day 10 post immunization , medium dose c16 application still effectively suppressed the axonal loss score compared with the vehicle control of the same time point ( figures 5(f ) , 5(k ) , 5(l ) , and 5(n ) ) .
transmission electron microscopy examination further revealed that a considerable amount of the myelin sheath displayed splitting and vacuoles changes in the vehicle control group ( figures 6(b ) and 6(c ) ) .
meanwhile , the axons were covered by disrupted myelin sheaths , and in some places , the axons even disappeared ( figure 6(c ) ) .
the neurons showed apoptotic signs of shrunken nuclei with condensed , fragmented , and marginated nuclear chromatin ( figure 6(d ) ) .
the low dose c16 treated eae rats revealed similar disrupted myelin and shrunken axons ( figure 6(e ) ) , but more lightly vacuolated myelin sheaths were found in medium and high dose c16 treated eae rats and the corresponding axons and neighboring nuclei were close to the normal ultrastructure ( figures 6(f)6(h ) ) .
however , more seriously loosened myelin appeared in the cns of late c16 treated eae rats compared with the early treated rats of the same dose ( figure 6(i ) ) . at week 8 after immunization in the vehicle control group , some myelin lamellae were still undergoing vesicular disintegration ( figure 6(j ) ) .
the disrupted myelin sheaths had been invaded by macrophages containing myelin debris ( figure 6(k ) ) , and the nucleus of neurons exhibited apoptotic signs ( figure 6(l ) ) , and some were undergoing remyelination . in the meantime , more remyelinated fibers appeared both in early and late c16 treated eae rats ( figures 6(m)6(p ) ) . in the vehicletreated rats ,
the acute phase of the disease began with a sharp increase of motor symptoms ( average clinical score : 3.54.0 ) which peaked at week 2 after immunization ( figure 7 ) .
thereafter , the clinical score gradually declined . at week 8 after immunization , the clinical score of surviving vehicletreated animals returned to a level of 1.5 - 2 ( figures 7(a)7(d ) ) .
although animals treated with c16 showed a similar disease course to the vehicle control group , the low , medium , and high dose c16 treatments all could clearly suppress the clinical score in the peak stage ( weeks 24 after immunization ) and the medium and high dose treatments also accelerated the progress of functional recovery . with the increasing dosage , the amelioration of clinical signs was more conspicuous ( p < 0.01 in high dose treatment , figures 7(a)7(c ) ) .
as for the late treated groups , there was no visible difference in clinical signs at the first two weeks after immunization , whereas the c16 treated group exhibited a significant decline in disease severity at weeks 3 - 4 after immunization ( figure 7(d ) ) . to assess whether c16 treatments could inhibit eae - induced reactive gliosis at the chronic stage , we examined expression of gfap , a marker for astrocytes , with western blot analysis and immunofluorescence label .
westerns blot revealed that the expression of gfap increased both in spinal cord and cerebral cortex of vehicle control rats ( figure 8) .
immunolabeling showed that the astrocytes proliferated and formed visible glial scar ( figures 9(b ) and 9(h ) ) .
the gfap expression level and glial scar formations were significantly decreased in both the early and late c16 treated groups ( p < 0.01 , figure 8(c ) ; p < 0.05 , figures 9(j ) and 9(k ) ) .
however , the medium and high dose c16 treatments , but not the low dose treatment , showed a remarkable inhibition to astrocytes proliferation when compared with the vehicle control ( p < 0.01 , figures 8 and 9 ) . between week 2 and week 8 aftert immunization , accompanied with severe inflammation , there was a remarkable increase in the number of caspase-3 ( an enzyme involved in the execution of the mammalian apoptotic cell death program ) immunoreactive neuronal cells in the spinal cords and motor cortexes of vehicle control rats ( figure 10 ) .
western blot analysis also revealed a significant increase of active caspase-3 expression level in vehicle control , which was significantly reversed by c16 treatment , especially in the high dose c16 treated rats ( p < 0.01 , figure 11 ) .
moreover , the caspase-3 expression level and caspase-3 ir neural cell numbers were also declined evidently in late c16 treated groups from 2 to 8 weeks after immunization ( figure 10 ) .
meanwhile , remarkable neuron loss appeared in the anterior horn of spinal cords and motor cortexes of the vehicle control rats ( p < 0.01 versus normal control , figure 12 ) , especially at the late stage of the clinical course ( p < 0.005 versus normal control , week 8 after immunization ) . compared with vehicle control , medium and
high dose c16 treatment significantly ameliorated the neuronal loss , both at weeks 2 and 8 after immunization ( p < 0.01 , figure 12(q ) ) . in the late treated group
, significant differences were found between the vehicle and c16 treated rats in the anterior horn of the spinal cord ( p < 0.05 ) but not in the motor cortex ( p > 0.05 , figure 12(r ) ) . at the early ( week 2 ) and late ( week 8) stage of the clinical course , extensive expression of tnf-was found in neurons and other neuronal cells in the motor cortex and the spinal cord of vehicle control treated eae rats ( figures 13(a ) , 13(b ) , 13(g ) , and 13(h ) ) .
however , the tnf- ir neuronal cells were much less in c16 treated eae rats ( figures 13(c)13(f ) , and figures 13(i)13(l ) ) .
the western blot also demonstrated a remarkable decrease of tnf- expression in each c16 treated group ( p < 0.01 , figure 14 ) .
such a declining trend could remain from week 2 to 8 after immunization , and was also detected in c16 late treated group ( figures 13(m ) and 13(n ) , figure 14 .
results showed that both medium and high doses of c16 application could noticeably reduce the expression of ifn- and the late treated c16 also possessed similar effects ( p < 0.05 , figures 15(a ) and 15(b ) ) .
nevertheless , as expected , the high dose c16 application induced higher tgf- expression ( p < 0.05 versus normal rats group ) while there was no significantly difference in tgf- expression between low dose c16 treatment and vehicle control rats ( p > 0.05 , figures 15(c ) and 15(d ) ) .
in our study , a peptide that can specifically recognize and bind to 3 , the integrin that plays an important role in the leukocyte accumulation and adhesion process [ 1417 ] , was employed to competitively block transmigration of leukocytes when they were crossing the endothelium . as the antibody to integrin
4 has been already reported to prevent leukocyte infiltration in guinea pig eae models , 3 integrin inhibition was also discovered to reduce leukocyte - endothelium interaction in a pressure - induced reperfusion model . in agreement with former research
, our data demonstrated that the widespread perivascular and parenchymal infiltrations of leukocytes , lymphocytes ( labeled by pan - leukocyte marker cd45 and lymphocytes marker cd4 ) , and activated microglia and extravasated macrophages ( labeled by cd68 ) in the cns of rodent eae model were all significantly suppressed by consecutive intravenous injections of c16 .
although we gave daily injection of c16 for 2 weeks , half of the animals survived for 8 weeks .
thus , these sustained neuroprotective effects observed at the later stage of the eae model ( week 8 after immunization ) suggested that neurotrophic treatments may have lasting effects , even when treatment has been halted .
in addition to taking part in the infiltration process of inflammatory cells , 3 integrin is thought to be an important receptor that regulates macrophage differentiation and macrophage responses to external signaling .
previous studies hypothesize that 3 activation can maintain chronic inflammatory processes in pathological conditions ; thus , besides interfering with the leukocyte transmigration , blockage of 3 may also directly inhibit macrophage - related inflammation .
in addition to inflammation , the demyelination , axonal injury , and neuronal loss all underlie the accumulation of disability and the disease progression .
extensive demyelination has been confirmed by reduced lfb staining and the loss of mbp immunoreactivity in our vehicle control rats , in which subsequent axonal loss was also prominent in the spinal cords and brain cortexes .
the inflammatory scores were also clearly decreased compared with that of the vehicle control . since extravasated inflammatory cells could activate a series of noxious factors that contribute to secondary injury , the improvement of the microenvironment in c16 treated eae rats should alleviate secondary injury that would otherwise lead to subsequent demyelination , axon loss , and further tissue damage . therefore , a significant decrease in demyelination areas and relief of axonal damage were detected in c16 treated groups both at week 2 and week 8 after immunization .
the less abnormal ultrastructure and the more remyelinated axons under electron microscopy further confirmed the positive effects of c16 on myelin and axons .
the notable increase of the active caspase-3 expression was detected in vehicle treated eae rats .
the higher number of intact neurons in the brain cortex and spinal cord anterior horn ( showed by nissel staining and cell counting ) of c16 treated eae rats confers that the improved micro - environment could suppress apoptosis of neural cells .
other than neurons , the oligodendrocytes were also highly vulnerable to an aggravated micro - environment .
at chronic stages of neuroinflammation , a large number of oligodendrocytes underwent apoptosis at sites distant from the vicinity of primary injury [ 3133 ] , which , led to denudement of axons and deterioration of their conductive abilities , thereby exacerbating the impediment of function .
the reduction in neuron and oligodendrocyte apoptosis may contribute to ameliorating disease progression and alleviating disease severity at peak time of eae , leading to more rapid recovery of locomotor function in c16 treated groups .
cytokines produced by the myelin specific cd4 t helper 1 ( th1 ) cells subset , such as il-6 , il-12 , tnf- , and ifn- , tend to act in a pro - inflammatory manner , thereby worsening eae disease process .
the proinflammatory cytokines tnf- and ifn- could further induce a local influx of inflammatory cells into the cns .
inhibition of these pro - inflammatory cytokines has been shown to be effective in downregulating the eae disease process [ 2 , 36 ] .
on the other hand , tgf- , the cytokine produced by cd4 t helper 2 ( th2 ) subset , is anti - inflammatory ; it could ameliorate the eae disease pathogenesis .
our results showed that c16 application could noticeably reduce the expression of tnf- and ifn- but had no considerable effects on tgf- levels when compared with the vehicle treated eae rats .
since c16 treatment has been shown to block the accumulation and infiltration of cd4 labeled t cells , pro - inflammatory factors produced by these cells would decrease accordingly , which further inferred the downstream signal transduction pathway and reduced the secondary transmigration of inflammatory cells . on the other hand , the lack of a positive effect on anti - inflammatory cytokine tgf- may be due to similar suppressing effects on th2 cells subset .
furthermore , the slight increase in tgf- levels over the normal control may , in fact , be a compensation reaction in the inflammatory microenvironments .
reactive astroglia accumulated within and at the margins of demyelination lesions in ms and eae , and contributed to the inflammatory response by synthesizing proinflammatory cytokines and presenting peptide antigens to t lymphocytes .
a long term result of the astrocytic reaction could be the formation of a glial scar at the lesion site , which may inhibit axonal regeneration or remyelination . with improved regional microenvironment , decreased demyelination , and less pro - inflammatory cytokines , c16 treatment evidently ameliorated reactive astrocytes proliferation , which would be propitious in relieving disability in locomotor function . among a variety of eae models in rodents ,
the acute eae model induced in lewis rats is a well - established model of ms , characterized by a single peak of paralysis after which animals recover spontaneously .
the utilization of this model gives us an opportunity to elucidate the induction , peak , and resolution of the inflammation - based immune response of ms .
although animals recover spontaneously in this eae model , c16 treated groups had less severe clinical score at the same time point in peak and resolution stages when compared with the vehicle control .
daily intravenous injections of c16 at a higher dose provided significantly better protection to white matter than the low dose , which suggested a dose - dependent effect of c16 application . in another group ,
the daily injection of c16 started 10 days after immunization , when the symptoms of motor disability would have clinical symptoms , so the case could be diagnosed and intravenous treatments could start
. the clinical score was not improved at week 2 after immunization in the c16 late treated group when the c16 injection was only performed for 4 days . however , even in this case , the infiltration of inflammatory cells has been partly reduced and the proinflammatory cytokines / apoptotic signals have also been suppressed . moreover , a clearly alleviated disease severity and more rapid recovery in locomotor function were detected at week 8 after immunization , after receiving c16 treatment for a 2-week duration .
the results implied that delayed c16 treatment could also , at least to a certain extent , offer neuroprotective effects for eae .
the dose and duration of application were important factors in evaluating the effects of c16 .
our data suggests that c16 peptide might act as a protective agent by attenuating inflammatory progression , improving micro - environment , and affecting the expression of some proinflammatory cytokines in neuroinflammatory disease .
although we have explored a part of the underlying mechanism of c16 effects , further molecular mechanisms still need extensive investigation . combination with different innovative factors that target different pathways of neuroprotection should be one more effective route to enhanced therapies .
for instance , neurotrophic factors may promote remyelination and prevent neuronal damage ; it is possible that they have the potential to be used in a combination of therapeutic drugs that targets neuroprotection pathways other than c16 one . | previous studies have shown that prevention of leukocyte infiltration by targeting integrins involved in transendothelial migration may suppress the clinical and pathological features of neuroinflammatory disease .
this study was designed to investigate the effects of c16 , an 3 integrin - binding peptide , in an acute experimental allergic encephalomyelitis ( eae ) rat model .
multiple histological and immunohistochemical staining , electron microscopy observation , elisa assay , western blot , and magnetic resonance imaging ( mri ) were employed to assess the degree of inflammation , axonal loss , neuronal apoptosis , white matter demyelination , and extent of gliosis in the brain and spinal cord of differently treated eae models .
the results showed that c16 treatment could inhibit extensive leukocyte and macrophage accumulation and infiltration and reduce cytokine tumor necrosis factor- ( tnf- ) and interferon- ( ifn- ) expression levels .
a significantly lower clinical score at the peak time of disease was also demonstrated in the c16 treated group .
moreover , astrogliosis , demyelination , neuronal death , and axonal loss were all alleviated in c16 treated eae animals , which may be attributed to the improvement of microenvironment .
the data suggests that c16 peptide may act as a protective agent by attenuating inflammatory progression and thus affecting the expression of some proinflammatory cytokines during neuroinflammatory disease . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion | experimental allergic encephalomyelitis ( eae ) is a primary animal model of ms which is widely used for the evaluation of different drugs in ms treatment . at the peak time of the disease ( 2 weeks after immunization ) , h&e staining of the cerebral cortex and spinal cord ( figure 1 ) revealed a significant increase of the cellular density in eae rats treated with vehicle . in general , when treated with a low dose of c16 ( 0.5 mg / day ) , the extent of inflammatory cells infiltration , the number of perivascular infiltration focus points , and the size of hyperintensity lesion sites in t2w images were significantly more abundant and larger in size than in the medium ( 1 mg / day ) and in high ( 2 mg / day ) dose c16 treated groups ( figures 1(e ) and 1(f ) ) . the results showed that cells positive for cd4 , cd68 , and cd45 all increased remarkably in both grey and white matter of cns in the vehicle control rats ( figure 2 ) . an evident decline of t lymphocyte infiltration , leukocytes extravasation , and macrophages activation was detected in c16 treated group ( figures 2(c ) , 2(d ) , 2(f ) , 2(g ) , 2(i ) , 2(j ) , 2(l ) , 2(m ) , 2(o ) , and 2(p ) ) , especially in the medium and high dose treated groups ( p < 0.001 , figures 3(a ) , 3(c ) , and 3(e ) ) . massive perivascular and confluent demyelinated areas were present in the parenchyma of the cns of vehicle control rats at the peak time of disease ( week 2 after immunization ) ( figures 4(b ) , 4(k ) , and 4(l ) ) . at the same time point , when treated with different doses of c16 peptide , the visible areas of demyelination gradually declined ( figures 4(c ) , 4(d ) , 4(m ) , and 4(n ) ) . compared with vehicle control , there was no notable difference in axonal number in the low dose c16 treated group , but more axons with relatively normal formation were kept in medium and high dose treated eae rats ( figures 5(e ) , 5(i ) , 5(j ) , and 5(m ) ) . although animals treated with c16 showed a similar disease course to the vehicle control group , the low , medium , and high dose c16 treatments all could clearly suppress the clinical score in the peak stage ( weeks 24 after immunization ) and the medium and high dose treatments also accelerated the progress of functional recovery . to assess whether c16 treatments could inhibit eae - induced reactive gliosis at the chronic stage , we examined expression of gfap , a marker for astrocytes , with western blot analysis and immunofluorescence label . western blot analysis also revealed a significant increase of active caspase-3 expression level in vehicle control , which was significantly reversed by c16 treatment , especially in the high dose c16 treated rats ( p < 0.01 , figure 11 ) . at the early ( week 2 ) and late ( week 8) stage of the clinical course , extensive expression of tnf-was found in neurons and other neuronal cells in the motor cortex and the spinal cord of vehicle control treated eae rats ( figures 13(a ) , 13(b ) , 13(g ) , and 13(h ) ) . such a declining trend could remain from week 2 to 8 after immunization , and was also detected in c16 late treated group ( figures 13(m ) and 13(n ) , figure 14 . results showed that both medium and high doses of c16 application could noticeably reduce the expression of ifn- and the late treated c16 also possessed similar effects ( p < 0.05 , figures 15(a ) and 15(b ) ) . since extravasated inflammatory cells could activate a series of noxious factors that contribute to secondary injury , the improvement of the microenvironment in c16 treated eae rats should alleviate secondary injury that would otherwise lead to subsequent demyelination , axon loss , and further tissue damage . the higher number of intact neurons in the brain cortex and spinal cord anterior horn ( showed by nissel staining and cell counting ) of c16 treated eae rats confers that the improved micro - environment could suppress apoptosis of neural cells . the reduction in neuron and oligodendrocyte apoptosis may contribute to ameliorating disease progression and alleviating disease severity at peak time of eae , leading to more rapid recovery of locomotor function in c16 treated groups . our results showed that c16 application could noticeably reduce the expression of tnf- and ifn- but had no considerable effects on tgf- levels when compared with the vehicle treated eae rats . the clinical score was not improved at week 2 after immunization in the c16 late treated group when the c16 injection was only performed for 4 days . our data suggests that c16 peptide might act as a protective agent by attenuating inflammatory progression , improving micro - environment , and affecting the expression of some proinflammatory cytokines in neuroinflammatory disease . | [
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the natural course of aortic stenosis ( as ) is characterised by among others cardiac arrhythmia and sudden death in up to 34% of the patients. surgical aortic valve replacement ( avr ) has shown to reduce the risk of sudden death .
whether this also holds for patients who undergo transcatheter aortic valve implantation ( tavi ) , which are characterised by more advanced age and co - morbid conditions than patients who undergo avr , remains to be elucidated .
consistent with the findings of thomas , et al . , we recently found that sudden death accounts for approximately 10% of all - cause 1-year mortality after tavi .
although not per se cardiac in origin , it is conceivable that late sudden death may be explained or caused by abnormalities in ventricular repolarisation .
corrected qt dispersion ( cqtd ) can be used to assess disturbances in the homogeneity of ventricular repolarisation and may , therefore , help to identify patients who are at increased risk of unexplained late death .
an increased cqtd reflects inhomogeneity of ventricular repolarisation and is associated with an increased risk of cardiac death in patients with a variety of heart diseases. a reduction in cqtd has been reported early after avr most likely due to a reduction in afterload , improved coronary flow and early left ventricular mass regression. similar effects may be expected after tavi , yet , as mentioned these patients are older with more comorbidities and may also have been exposed to a longer period of increased left ventricular afterload .
we , therefore , sought to explore the changes in cqtd and the frequency , predictors and prognostic effects of defective cqtd recovery at six months after tavi .
between november 2005 and january 2012 , a total of 222 consecutive patients with as underwent trans - arterial tavi with the medtronic corevalve system ( mcs ) . in all patients , a pre- , post- and follow - up 12-lead ecg was available .
patients with atrial fibrillation and those taking class i or iii antiarrhytmics or on chronic haemodialysis were excluded . for the purpose of an accurate assessment of cqtd changes over time , patients with a new conduction abnormality
[ left bundle branch block ( lbbb ) , right bundle branch block ( rbbb ) , or paced rhythm ] immediately or six months after tavi and those with a pre - existent conduction abnormality that progressed to a higher degree av block were excluded as well .
the final study population , therefore , consisted of 45 patients ( figure 1 ) .
details of the patient selection , prosthesis and subsequent phases of the transfemoral tavi procedure have been previously published. briefly , all patients referred for tavi were first seen on the out - patient clinic and then discussed in the heart team .
treatment decision was based upon consensus between an interventional cardiologist and a cardiac surgeon and since september 2010 by a team consisting of an interventional cardiologist , cardiac surgeon and a clinical cardiologist and/or an echocardiographist .
treatment decision was based upon a weighted assessment of risk and benefit of the various treatment modalities on the basis of all clinical and technical information of the patient .
the procedure was performed with the patient under general anaesthesia with a temporary pacemaker wire positioned in the right ventricle and with default femoral arterial access through an 18f sheath .
this wire was exchanged for a stiff support wire followed by balloon valvuloplasty ( 22 or 23 mm 4 cm ) under rapid right ventricular pacing .
the valve ( available in 26 , 29 and 31 mm ) was then advanced over the aortic arch into the aortic annulus with a target deployment of 46 mm below the annulus .
patients were extubated before leaving the catheterization laboratory or within 2 h after arrival in the cardiac care unit .
all pre- , post- and follow - up [ median : 181 , interquartile range ( iqr ) : 110 - 279 days ] 12-lead ecgs were recorded at a paper speed of 25 mm / s and reprinted at a speed of 50 mm / s to improve sensitivity and accuracy of the manual measurements .
, inter - observer variability of cqtd measurements were evaluated by comparing the results of cqtd analysis between two observers who independently from one another analysed the ecgs .
inter - observer variability , expressed as the mean absolute difference between observers , was 8 16 ms as determined in a consecutive series of 10 patients ( 30 ecgs ) .
the following time intervals were measured : pr , qrs , rr , qt and jt . the qt interval was considered to reflect the ventricular depolarization and repolarisation whereas the jt interval only reflects ventricular repolarisation because it does not encompass the depolarization phase .
the qt interval was calculated by measuring the beginning of the qrs complex until the visual return of the t wave to the tp baseline .
when a t wave was complicated by a u wave , the end of the t wave was defined as the nadir between the t and the u waves .
wherever possible , three consecutive cycles were measured and corrected for the heart rate using the bazett 's formula . , the average of the three consecutive cycles was used . the cjt interval for each interval
cqtd was calculated by measuring the cqt interval of all 12 ecg leads and defined by the substraction of the shortest from the longest interval ( maximum minus minimum cqt interval ) . in case of uncertainty of the definition of the end of a t wave ,
defective cqtd recovery was defined as any progression beyond the baseline cqtd as assessed using follow - up ecgs . the cqtd variation ( cqtd )
was calculated as the difference between cqtd before vs. after and before vs. 6 months after tavi . left and right bundle branch block was defined according to the wold health organization and international society and federation for cardiology task force .
follow - up information was collected during structured out - patient clinic visits after discharge .
survival and cause of death was obtained every 6 months by contacting the dutch civil register and/or referring cardiologists and/or the general practitioners .
sudden and unexpected death was defined as a natural , unexpected death due to cardiac causes , heralded by an abrupt loss of consciousness within 1 h of the onset of acute symptoms in patients who were previously stable .
categorical variables are presented as frequencies and percentages and were compared with the chi square test or fisher 's exact test .
normal and skewed continuous variables are presented as means sd and medians [ interquartile range ( iqr ) ] , respectively . the wilcoxon signed - rank test ( for continuous variables ) and
the mcnemar test conducted by exact methods ( for categorical variables ) were used to perform paired comparisons for pre - treatment vs. post - treatment and pre - treatment vs. follow - up ecg findings .
multivariable logistic regression including all variables with p < 0.05 in the univariable analysis was performed to determine the predictive factors of defective cqtd recovery .
given the small number of deaths during follow - up ( n = 7 ) , a univariable cox regression analysis was performed to explore the potential association between defective cqtd recovery and late mortality .
a two - sided p < 0.05 was considered to indicate significance and all statistical analyses were performed with spss software ( version 17 ) . in accordance with the institutions policies , every patient gave written informed consent for tavi and the use of anonymous clinical , procedural and follow - up data for research in accordance with institutional review board approval .
between november 2005 and january 2012 , a total of 222 consecutive patients with as underwent trans - arterial tavi with the medtronic corevalve system ( mcs ) . in all patients , a pre- , post- and follow - up 12-lead ecg was available .
patients with atrial fibrillation and those taking class i or iii antiarrhytmics or on chronic haemodialysis were excluded . for the purpose of an accurate assessment of cqtd changes over time , patients with a new conduction abnormality
[ left bundle branch block ( lbbb ) , right bundle branch block ( rbbb ) , or paced rhythm ] immediately or six months after tavi and those with a pre - existent conduction abnormality that progressed to a higher degree av block were excluded as well .
the final study population , therefore , consisted of 45 patients ( figure 1 ) .
details of the patient selection , prosthesis and subsequent phases of the transfemoral tavi procedure have been previously published. briefly , all patients referred for tavi were first seen on the out - patient clinic and then discussed in the heart team .
treatment decision was based upon consensus between an interventional cardiologist and a cardiac surgeon and since september 2010 by a team consisting of an interventional cardiologist , cardiac surgeon and a clinical cardiologist and/or an echocardiographist .
treatment decision was based upon a weighted assessment of risk and benefit of the various treatment modalities on the basis of all clinical and technical information of the patient .
the procedure was performed with the patient under general anaesthesia with a temporary pacemaker wire positioned in the right ventricle and with default femoral arterial access through an 18f sheath .
this wire was exchanged for a stiff support wire followed by balloon valvuloplasty ( 22 or 23 mm 4 cm ) under rapid right ventricular pacing .
the valve ( available in 26 , 29 and 31 mm ) was then advanced over the aortic arch into the aortic annulus with a target deployment of 46 mm below the annulus .
patients were extubated before leaving the catheterization laboratory or within 2 h after arrival in the cardiac care unit .
all pre- , post- and follow - up [ median : 181 , interquartile range ( iqr ) : 110 - 279 days ] 12-lead ecgs were recorded at a paper speed of 25 mm / s and reprinted at a speed of 50 mm / s to improve sensitivity and accuracy of the manual measurements .
, inter - observer variability of cqtd measurements were evaluated by comparing the results of cqtd analysis between two observers who independently from one another analysed the ecgs .
inter - observer variability , expressed as the mean absolute difference between observers , was 8 16 ms as determined in a consecutive series of 10 patients ( 30 ecgs ) .
the following time intervals were measured : pr , qrs , rr , qt and jt . the qt interval was considered to reflect the ventricular depolarization and repolarisation whereas the jt interval only reflects ventricular repolarisation because it does not encompass the depolarization phase .
the qt interval was calculated by measuring the beginning of the qrs complex until the visual return of the t wave to the tp baseline .
when a t wave was complicated by a u wave , the end of the t wave was defined as the nadir between the t and the u waves .
intervals preceded by premature beats were not measured . for each lead , wherever possible , three consecutive cycles were measured and corrected for the heart rate using the bazett 's formula . , the average of the three consecutive cycles was used . the cjt interval for each interval
cqtd was calculated by measuring the cqt interval of all 12 ecg leads and defined by the substraction of the shortest from the longest interval ( maximum minus minimum cqt interval ) . in case of uncertainty of the definition of the end of a t wave , the lead disclosing this uncertainty
defective cqtd recovery was defined as any progression beyond the baseline cqtd as assessed using follow - up ecgs . the cqtd variation ( cqtd )
was calculated as the difference between cqtd before vs. after and before vs. 6 months after tavi . left and right bundle branch block was defined according to the wold health organization and international society and federation for cardiology task force
follow - up information was collected during structured out - patient clinic visits after discharge .
survival and cause of death was obtained every 6 months by contacting the dutch civil register and/or referring cardiologists and/or the general practitioners .
sudden and unexpected death was defined as a natural , unexpected death due to cardiac causes , heralded by an abrupt loss of consciousness within 1 h of the onset of acute symptoms in patients who were previously stable .
categorical variables are presented as frequencies and percentages and were compared with the chi square test or fisher 's exact test .
normal and skewed continuous variables are presented as means sd and medians [ interquartile range ( iqr ) ] , respectively . the wilcoxon signed - rank test ( for continuous variables ) and
the mcnemar test conducted by exact methods ( for categorical variables ) were used to perform paired comparisons for pre - treatment vs. post - treatment and pre - treatment vs. follow - up ecg findings .
multivariable logistic regression including all variables with p < 0.05 in the univariable analysis was performed to determine the predictive factors of defective cqtd recovery .
given the small number of deaths during follow - up ( n = 7 ) , a univariable cox regression analysis was performed to explore the potential association between defective cqtd recovery and late mortality .
a two - sided p < 0.05 was considered to indicate significance and all statistical analyses were performed with spss software ( version 17 ) . in accordance with the institutions policies , every patient gave written informed consent for tavi and the use of anonymous clinical , procedural and follow - up data for research in accordance with institutional review board approval .
the baseline characteristics and the serial ecg changes are summarized in table 1 and 2 , respectively .
overall , the average heart rate , qrs duration and minimum cqt interval increased immediately after tavi , yet all changes returned to baseline values at 6 months .
the cqtd decreased immediately after tavi , with a further decline at 6 months ( 47 vs. 45 vs. 40 ms , p = 0.049 ) . in a subgroup of 27 patients without conduction abnormality before , after and 6 months after tavi , the cqtd decreased from 50 ms at baseline to 38 ms at 6 months ( reduction of 24% , p = 0.006 ) .
data are expressed as means sd , medians ( iqr ) or numbers ( % ) .
data are expressed as means sd , medians ( iqr ) or n ( % ) .
tavi : transcatheter aortic valve implantation ; iqr : interquartile range . in 27 out of the 45 patients ( 60% ) , a reduction in cqtd between baseline and follow - up was observed , whereas in 18 patients ( 40% ) an increase was observed ( defective cqtd recovery ) .
details of the baseline characteristics and perioperative results grouped according to the occurrence of defective cqtd recovery are shown in table 3 and 4 , respectively .
patients with defective cqtd recovery had a worse ejection fraction ( 45% vs. 54% , p = 0.026 ) , longer minimum cqt interval before tavi ( 432 ms vs. 407 ms , p = 0.001 ) and an increase in cqtd immediately after tavi ( cqtd : + 13 ms vs. 13 ms , p = 0.002 ) in comparison to patients without defective cqtd recovery .
the median follow - up was 14 months ( iqr : 723 ) ; seven patients ( 16% ) died at a median of 11 months ( iqr : 615 ) after tavi .
four deaths occurred in patients with defective cqtd recovery ( cardiac death : 50% ) and three deaths in patients without defective cqtd recovery ( cardiac death : 0% ) . by univariable analysis , a higher baseline serum creatinine ( per 10 mmol / l increase , hr : 1.09 ; 95% ci : 1.031.16 ) and defective cqtd recovery ( cqtd at 6 months , per 10 ms decrease , hr : 1.52 ; 95% ci : 1.052.17 ) were significantly associated with late mortality .
data are expressed as means sd , medians ( iqr ) or n ( % ) .
cqtd : corrected qt dispersion ; iqr : interquartile range ; tavi : transcatheter aortic valve implantation .
in 27 out of the 45 patients ( 60% ) , a reduction in cqtd between baseline and follow - up was observed , whereas in 18 patients ( 40% ) an increase was observed ( defective cqtd recovery ) .
details of the baseline characteristics and perioperative results grouped according to the occurrence of defective cqtd recovery are shown in table 3 and 4 , respectively .
patients with defective cqtd recovery had a worse ejection fraction ( 45% vs. 54% , p = 0.026 ) , longer minimum cqt interval before tavi ( 432 ms vs. 407 ms , p = 0.001 ) and an increase in cqtd immediately after tavi ( cqtd : + 13 ms vs. 13 ms , p = 0.002 ) in comparison to patients without defective cqtd recovery .
the median follow - up was 14 months ( iqr : 723 ) ; seven patients ( 16% ) died at a median of 11 months ( iqr : 615 ) after tavi .
four deaths occurred in patients with defective cqtd recovery ( cardiac death : 50% ) and three deaths in patients without defective cqtd recovery ( cardiac death : 0% ) . by univariable analysis , a higher baseline serum creatinine ( per 10 mmol / l increase , hr : 1.09 ; 95% ci : 1.031.16 ) and defective cqtd recovery ( cqtd at 6 months , per 10 ms decrease , hr : 1.52 ; 95% ci : 1.052.17 ) were significantly associated with late mortality .
data are expressed as means sd , medians ( iqr ) or n ( % ) .
cqtd : corrected qt dispersion ; iqr : interquartile range ; tavi : transcatheter aortic valve implantation .
in this study of 45 patients with aortic stenosis , we found that the average cqtd - time was significantly reduced at 6 months after tavi . yet
, defective cqtd recovery was present in 40% of the patients . worsening of cqtd immediately after tavi was an independent predictor of defective cqtd recovery at 6 months , which in turn showed a directional signal of a higher risk of late mortality .
cqtd can be used to determine non - uniform ventricular repolarization and is increased in a variety of cardiac conditions such as long qt syndrome , chronic heart failure , hypertrophic cardiomyopathy , myocardial infarction and in patients with left ventricular hypertrophy associated with aortic stenosis. in these patients , increased cqtd has been associated with susceptibility to ventricular arrhythmias , cardiac mortality and unexplained sudden death . yet , in patients with aortic stenosis , cqtd has been shown to reduce early after surgical aortic valve replacement , which is attributed to a reduced afterload , improved coronary flow and early left ventricular mass regression. this has also been demonstrated in patients with severe congenital as undergoing percutaneous balloon valvuloplasty .
ckmb : creatine kinase mb ; cqtd : corrected qt dispersion ; tavi : transcatheter aortic valve implantation .
the findings of the present study indicate that cqtd recovery after tavi does not occur immediately after the procedure but is a time related phenomenon that is not complete at 6 months ( 40% of the patients still had a defective cqtd recovery ) .
this is in contrast with the findings of sarubbi , et al . who showed a 23% cqtd reduction immediately after balloon valvuloplasty and those of tsai , et al . who demonstrated a 48% reduction by 6 days after avr
the evident and immediate cqtd reduction following avr contrasts with our findings of a delayed and incomplete cqtd reduction after tavi .
this can be explained by patient - related factors since tavi patients are in general older than patients who undergo surgical valve replacement and , therefore , have more pronounced age - related degenerative changes of the myocardium . in addition , tavi patients may suffer from longer periods of elevated afterload in comparison to surgical patients due to a longer time interval to clinical detection and treatment delay . , they may therefore , have more extensive cqtd and less response to early changes following afterload correction . the observed increase in cqtd immediately after tavi in patients with a defective cqtd recovery during follow - up could be due to an early impairment of the conductive system not appearing at the 12-lead ecg evaluation . in these particular patients ,
the reduction in afterload , improved coronary flow and left ventricular mass regression may not be sufficient to improve the cqtd at follow - up .
this phenomenon could be particularly common after corevalve implantation considering the frequent occurrence of new conduction defects associated with this device .
the latter may also explain why a significant number of patients had discordant cqtd changes ( i.e. , an initial decrease in cqtd from baseline to post - tavi , followed by an increase at follow - up ) , which occurred in 46% of patients .
yet , some studies demonstrated that cqtd is strongly associated with malignant arrhythmias and cardiac mortality .
, as mentioned , cqtd recovery was incomplete at six months in the present population .
although we lack the power to demonstrate an association with malignant arrhythmia and sudden cardiac death , it is conceivable that these patients are at increased risk of late sudden death . of note
, the causes of death in patients with defective cqtd recovery were sudden death ( n = 1 ) , myocardial infarction ( n = 1 ) and terminal kidney failure ( n = 2 ) , whereas patients without defective cqtd recovery died because of a pneumonia ( n = 1 ) , sepsis ( n = 1 ) and a perforated diverticulitis ( n = 1 ) .
the clinical relevance of defective cqtd recovery was previously demonstrated by chalil , et al . who demonstrated that increased cqtd duration independently predicts sudden death / resuscitation in patients undergoing cardiac resynchronization therapy .
cqtd is known to be an approximate of repolarisation abnormalities . yet , given the absence of an underlying pathophysiologic explanation , the observation of ( defective ) cqtd recovery is of observational nature .
we also acknowledge the fact that drugs other than class i or iii antiarrhytmic drugs ( i.e. , antidepressants ) may have affected the cqt recovery assessment and the fact that only quarter of the patients who underwent tavi were eligible mainly because of the frequent occurrence of new conduction abnormalities associated with tavi .
another limitation of the present study is that it can not indicate which measures have to be taken in case of defective cqtd recovery .
the question is whether one should first stop all drugs that may have such an effect or whether one even should consider the implantation of an automated implantable cardioverter - defibrillator .
data in larger series of patients stemming from multicenter observations are needed to confirm or to rule out the current observations , the precise timing and/or delay of recovery and to elucidate the role of potential measures to be taken . at last
, the inter- and intra - observer variability of approximately 20% should be taken into account when interpreting the study results , and warrants future research to confirm our findings .
tavi was associated with a gradual reduction of cqtd during the follow - up period .
this was predominantly seen in patients with an increase in cqtd immediately after tavi and was associated with a higher risk of late mortality .
in addition to the regular follow - up examinations after tavi , it is conceivable that more detailed analysis of the ecg may help to identify patients at risk of late unexpected death .
cqtd is known to be an approximate of repolarisation abnormalities . yet , given the absence of an underlying pathophysiologic explanation , the observation of ( defective ) cqtd recovery is of observational nature .
we also acknowledge the fact that drugs other than class i or iii antiarrhytmic drugs ( i.e. , antidepressants ) may have affected the cqt recovery assessment and the fact that only quarter of the patients who underwent tavi were eligible mainly because of the frequent occurrence of new conduction abnormalities associated with tavi .
another limitation of the present study is that it can not indicate which measures have to be taken in case of defective cqtd recovery .
the question is whether one should first stop all drugs that may have such an effect or whether one even should consider the implantation of an automated implantable cardioverter - defibrillator .
data in larger series of patients stemming from multicenter observations are needed to confirm or to rule out the current observations , the precise timing and/or delay of recovery and to elucidate the role of potential measures to be taken .
at last , the inter- and intra - observer variability of approximately 20% should be taken into account when interpreting the study results , and warrants future research to confirm our findings .
tavi was associated with a gradual reduction of cqtd during the follow - up period .
this was predominantly seen in patients with an increase in cqtd immediately after tavi and was associated with a higher risk of late mortality .
in addition to the regular follow - up examinations after tavi , it is conceivable that more detailed analysis of the ecg may help to identify patients at risk of late unexpected death . | backgroundcorrected qt dispersion ( cqtd ) has been correlated with non - uniform ventricular repolarisation and increased mortality . in patients with aortic stenosis , cqtd has been shown improved after surgical valve replacement , but the effects of transcatheter aortic valve implantation ( tavi ) are unknown . therefore , we sought to explore the frequency , predictors and prognostic effects of defective cqtd recovery at 6 months after tavi.methodsa total of 222 patients underwent tavi with the medtronic - corevalve system between november 2005 and january 2012 .
patients who were on class i or iii antiarrhythmics or on chronic haemodialysis or who developed atrial fibrillation , a new bundle branch block or became pacemaker dependent after tavi were excluded . as a result , pre- , post- and follow - up ecg ( median : 6 months ) analysis was available in 45 eligible patients .
defective cqtd recovery was defined as any progression beyond the baseline cqtd at 6 months.resultsin the 45 patients , the mean cqtd was 47 23 ms at baseline , 45 17 ms immediately after tavi and 40 16 ms at 6 months ( 15% reduction , p = 0.049 ) . compared to baseline , cqtd at 6 months
was improved in 60% of the patients whereas defective cqtd recovery was present in 40% .
cqtd increase immediately after tavi was an independent predictor of defective cqtd recovery at 6 months ( per 10 ms increase ; or : 1.89 , 95% ci : 1.153.12 ) . by univariable analysis ,
defective cqtd recovery was associated with late mortality ( hr : 1.52 , 95% ci : 1.052.17).conclusionsdespite a gradual reduction of cqtd after tavi , 40% of the patients had defective recovery at 6 months which was associated with late mortality .
more detailed ecg analysis after tavi may help to avoid late death . | Introduction
Methods
Patients and procedure
Electrocardiographic recordings and measurements
Follow-up
Statistical analysis
Results
Frequency and predictors of defective cQTD recovery
Prognostic effects of defective cQTD recovery
Discussion
Limitations
Conclusions | corrected qt dispersion ( cqtd ) can be used to assess disturbances in the homogeneity of ventricular repolarisation and may , therefore , help to identify patients who are at increased risk of unexplained late death . we , therefore , sought to explore the changes in cqtd and the frequency , predictors and prognostic effects of defective cqtd recovery at six months after tavi . between november 2005 and january 2012 , a total of 222 consecutive patients with as underwent trans - arterial tavi with the medtronic corevalve system ( mcs ) . patients with atrial fibrillation and those taking class i or iii antiarrhytmics or on chronic haemodialysis were excluded . for the purpose of an accurate assessment of cqtd changes over time , patients with a new conduction abnormality
[ left bundle branch block ( lbbb ) , right bundle branch block ( rbbb ) , or paced rhythm ] immediately or six months after tavi and those with a pre - existent conduction abnormality that progressed to a higher degree av block were excluded as well . in case of uncertainty of the definition of the end of a t wave ,
defective cqtd recovery was defined as any progression beyond the baseline cqtd as assessed using follow - up ecgs . between november 2005 and january 2012 , a total of 222 consecutive patients with as underwent trans - arterial tavi with the medtronic corevalve system ( mcs ) . in all patients , a pre- , post- and follow - up 12-lead ecg was available . patients with atrial fibrillation and those taking class i or iii antiarrhytmics or on chronic haemodialysis were excluded . for the purpose of an accurate assessment of cqtd changes over time , patients with a new conduction abnormality
[ left bundle branch block ( lbbb ) , right bundle branch block ( rbbb ) , or paced rhythm ] immediately or six months after tavi and those with a pre - existent conduction abnormality that progressed to a higher degree av block were excluded as well . in case of uncertainty of the definition of the end of a t wave , the lead disclosing this uncertainty
defective cqtd recovery was defined as any progression beyond the baseline cqtd as assessed using follow - up ecgs . the cqtd decreased immediately after tavi , with a further decline at 6 months ( 47 vs. 45 vs. 40 ms , p = 0.049 ) . in a subgroup of 27 patients without conduction abnormality before , after and 6 months after tavi , the cqtd decreased from 50 ms at baseline to 38 ms at 6 months ( reduction of 24% , p = 0.006 ) . patients with defective cqtd recovery had a worse ejection fraction ( 45% vs. 54% , p = 0.026 ) , longer minimum cqt interval before tavi ( 432 ms vs. 407 ms , p = 0.001 ) and an increase in cqtd immediately after tavi ( cqtd : + 13 ms vs. 13 ms , p = 0.002 ) in comparison to patients without defective cqtd recovery . by univariable analysis , a higher baseline serum creatinine ( per 10 mmol / l increase , hr : 1.09 ; 95% ci : 1.031.16 ) and defective cqtd recovery ( cqtd at 6 months , per 10 ms decrease , hr : 1.52 ; 95% ci : 1.052.17 ) were significantly associated with late mortality . by univariable analysis , a higher baseline serum creatinine ( per 10 mmol / l increase , hr : 1.09 ; 95% ci : 1.031.16 ) and defective cqtd recovery ( cqtd at 6 months , per 10 ms decrease , hr : 1.52 ; 95% ci : 1.052.17 ) were significantly associated with late mortality . in this study of 45 patients with aortic stenosis , we found that the average cqtd - time was significantly reduced at 6 months after tavi . yet
, defective cqtd recovery was present in 40% of the patients . worsening of cqtd immediately after tavi was an independent predictor of defective cqtd recovery at 6 months , which in turn showed a directional signal of a higher risk of late mortality . yet , in patients with aortic stenosis , cqtd has been shown to reduce early after surgical aortic valve replacement , which is attributed to a reduced afterload , improved coronary flow and early left ventricular mass regression. the findings of the present study indicate that cqtd recovery after tavi does not occur immediately after the procedure but is a time related phenomenon that is not complete at 6 months ( 40% of the patients still had a defective cqtd recovery ) . | [
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the family serrasalmidae comprises approximately 80 species distributed among 15 genera , with a number of commercially important species of the genera colossoma , mylossoma , and piaractus , which are valued for fishing or aquaculture in the amazon region ( arajo - lima and goulding , 1998 ; oliveira and arajo - lima , 1998 ; jgu , 2003 ; nelson , 2006 ) .
the species of this family are popularly known as pacus , piranhas and tambaqui ( c. macropomum ) , which have high , laterally compressed bodies with abdominal spines and long dorsal fins .
their distribution is exclusively neotropical and they inhabit environments such as floodplains , seasonally flooded forests , white - waters , and rivers main channels throughout south america , mainly in the amazon , paraguay , and orinoco river basins ( goulding , 1980 ; jgu , 2003 ) . according to calcagnotto et al .
( 2005 ) , serrasalmidae is strongly supported as a monophyletic family and , according to the phylogeny based on mitochondrial dna proposed by ort et al .
( 2008 ) , the species of this family can be divided into herbivorous clades represented by pacus and myleus and a carnivorous clade composed of piranhas .
pacus are considered basal and piranhas are thought to be the most derived clade . supporting this classification ,
cytogenetic studies have demonstrated variations in the diploid numbers in the family , with 2n = 54 for the pacus clade ( nirchio et al . , 2003 ; nakayama et al . ,
2012 ) , 2n = 58 for the myleus clade ( porto jir , 1999 , phd thesis .
inpa / ufam , manaus , am ) ( garca - parra wj , 2000 , phd thesis .
inpa / ufam , manaus , am ) , and 2n = 58 to 64 for the most derived piranhas clade ( muramoto et al . , 1968 ; nakayama et al . ,
although the karyotypes of many species of this family have been described ( almeida - toledo et al . , 1987 ; cestari and galetti jr , 1992a , b ; nakayama et al . , 2000 , 2001 , 2002 , 2008 , 2012
inpa / ufam , manaus , am ) , analyses using molecular cytogenetic techniques , mainly in the most basal genera ( colossoma , mylossoma and piaractus ) , are still scarce .
fluorescence in situ hybridization ( fish ) allows mapping specific dna sequences on the chromosomes , with repetitive sequences , such as telomeric , 18s and 5s rdna , being the most commonly studied ( martins , 2007 ) .
mapping of these sequences has been useful in studying questions related to karyotypic evolution , sexual and super - numerary chromosomes origin , and genomic organization in many fish species ( voltolin et al . , 2010 ; gross et al .
, 2010 ; schneider et al . , 2012 ; terencio et al . , 2012 ) . within this context , the present work aimed to investigate the genomic organization of 18s rdna , 5s rdna and telomeric sequences in species of the basal clade of serrasalmidae in order to understand the family chromosomal evolution .
four species belonging to the serrasalmidae family were analyzed : colossoma macropomum , mylossoma aureum , m. duriventre , and piaractus mesopotamicus .
thirty - eight specimens collected in the central amazon region or from pisciculture farms ( table 1 ) were anesthetized with eugenol ( a 5 ml stock solution diluted into 12 l of water ) and sacrificed to obtain chromosome preparations .
mitotic chromosomes were obtained from kidney cells ( bertollo et al . , 1978 ) .
constitutive heterochromatin detection was achieved using the c - banding technique ( sumner , 1972 ) , and the nucleolus organizing regions ( nors ) were evidenced by silver nitrate impregnation ( howell and black , 1980 ) .
total dna extraction was performed using muscle tissue samples of c. macropomum , m. aureum and m. duriventre following sambrook et al .
( 1989 ) , and quantification was performed in agarose gels by comparison with a standard lambda marker .
the 18s and 5s rdna genes were amplified by polymerase chain reaction ( pcr ) , employing the oligonucleotide primers 18sf ( 5-ccg ctt tgg tga ctc ttg at-3 ) and 18sr ( 5-ccg aggacc tca cta aac ca-3 ) ( gross et al . , 2010 ) , 5sa ( 5-tac gcc cga tct cgt ccg atc-3 ) , and 5sb ( 5- caggct ggt atg gcc gta agc-3 ) ( martins and galetti jr . , 1999 ) .
pcr reactions were performed in a final volume of 25 l containing genomic dna ( 200 ng ) , 10 buffer with 1.5 mm mgcl2 , taq dna polymerase ( 5 u/l ) , dntps ( 1 mm ) , the primer pairs ( 5 mm ) , and deionized water .
conditions for the 18s rdna amplification reaction were : 1 min at 95 c , 35 cycles of 1 min at 94 c , 1 min at 56 c , and 90 s at 72 c ; with a final extension of 5 min at 72 c .
conditions for the 5s rdna amplification reaction were : 1 min at 95 c , followed by 30 cycles of 1 min at 94 c , 1 min at 59 c and 90 s at 72 c ; with a final extension of 5 min at 72 c .
pcr reactions for the telomeric sequences ( ttaggg)n were performed in a final volume of 25 l containing 10 buffer with 1.5 mm of mgcl2 , dntps ( 1 mm ) , 0.2 l ( ttaggg)5 primer , 0.2 l ( ccctaa)5 primer , and 2 u of taq dna polymerase ( ijdo et al . , 1991 ) .
the first part of the amplification process was conducted under low stringency ( 4 min at 94 c ; followed by 12 cycles of 1 min at 94 c , 45 s at 52 c and 90 s at 72 c ) , followed by 35 cycles at high stringency conditions ( 1 min at 94 c , 90 s at 60 c and 90 s at 72 c ) .
telomeric sequences and 18s rdna products were labeled with digoxigenin-11-dutp ( dig nick translation mix ; roche ) , whereas the 5s rdna products were labeled with biotin-14-datp ( biotin nick translation mix ; roche ) , according to the manufacturer s instructions .
avidin - fitc ( sigma - aldrich ) , biotinylated anti - avidin ( sigma - aldrich ) , and anti - digoxigenin - rhodamine ( roche ) were used to immunodetect the probes .
( 1986 ) for a 77% stringency ( 2.5 ng/l of 18s rdna , 5s rdna , or telomeric probe , 50% formamide , 10% dextran sulfate , and 2 ssc at 37c for 18 h ) .
chromosomes were counterstained with dapi ( 2 mg / ml ) in vectashield ( vector ) mounting medium . after fish
images were captured using a coupled olympus dp71 digital camera and the image - pro mc 6.3 software .
metaphases were processed with adobe photoshop cs3 and the chromosomes were measured with image j and classified according to the nomenclature proposed by levan et al .
the four species analyzed had diploid numbers of 2n = 54 chromosomes and fundamental numbers fn = 108 - although their karyotypic formulas differed .
the karyotypic formulas were : 26m+28sm in colossoma macropomum ( figure 1a ) ; 24m+30sm in piaractus mesopotamicus ( figure 1b ) ; 40m+14sm in mylossoma aureum ( figure 1c ) , and 28m+12sm+14st in m. duriventre ( figure 1d ) .
the constitutive heterochromatin of c. macropomum , m. duriventre , and p. mesopotamicus was concentrated in the centromeric regions of several chromosomes ( figure 1e , f , h ) . in m.
aureum , besides the heterochromatic blocks in the centromeric regions , pairs 2 , 6 , 12 , 19 and 23 had heterochromatin at interstitial positions of the long arms and pair 21 on its short arm ( figure 1 g ) .
nucleolus organizing regions ( nor ) were located on the terminal portions of the long arms of metacentric pairs 6 and 12 of c. macropomum ( figure 1a ) , in terminal regions of chromosome 4 in p. mesopotamicus ( figure 1b ) and in the short arms of one homologue of pair 12 in species of mylossoma ( figure 1c , d ) . in situ hybridization experiments with 18s sequences as probes were performed numerous times in mylossoma species but no labeling of ribosomal sites could be detected .
this result may reflect methodological problems or may be due to the small size of these sites which would prevent their visualization .
the 18s rdna sequences were visualized in the terminal regions of the long arms of metacentric pairs 6 , 10 and 12 of c. macropomum ( figure 2a ) , with pairs 6 and 12 coinciding with the ag - nor . in p. mesopotamicus
, signals were observed in the terminal regions of the long arms of metacentric pairs 4 and 6 ( figure 2b ) .
5s rdna sequences mapped to euchromatic and heterochromatic regions of two chromosome pairs in each of the four species ( figure 3e , f , g , h ) .
labeling was observed in an interstitial position of pair 11 and in the terminal region of the short arm of pair 13 in c. macropomum and p. mesopotamicus ( figure 3a , b ) . in both species , the 5s rdna sites of pair 11 co - localized with heterochromatic blocks and the 5s rdna sites of pair 13 were adjacent to heterochromatic regions ( figure 3e , f ) .
the 5s rdna sites were present in pairs 4 and 12 of mylossoma aureum ( figure 3c , g ) and in pairs 4 and 8 of m. duriventre ( figure 3d ) .
the interstitial signals in the long arms coincided with the heterochromatic blocks in both species .
fish with telomeric probes revealed signals at the terminal portions of both arms of all chromosomes in the four species ( figure 4a , b , c , d ) .
an interstitial telomeric site ( its ) was observed in one metacentric pair of c. macropomum ( figure 4a ) and coincided with a positive c - banding region .
the four species analyzed had diploid numbers of 2n = 54 chromosomes and fundamental numbers fn = 108 - although their karyotypic formulas differed .
the karyotypic formulas were : 26m+28sm in colossoma macropomum ( figure 1a ) ; 24m+30sm in piaractus mesopotamicus ( figure 1b ) ; 40m+14sm in mylossoma aureum ( figure 1c ) , and 28m+12sm+14st in m. duriventre ( figure 1d ) .
the constitutive heterochromatin of c. macropomum , m. duriventre , and p. mesopotamicus was concentrated in the centromeric regions of several chromosomes ( figure 1e , f , h ) . in m.
aureum , besides the heterochromatic blocks in the centromeric regions , pairs 2 , 6 , 12 , 19 and 23 had heterochromatin at interstitial positions of the long arms and pair 21 on its short arm ( figure 1 g ) .
nucleolus organizing regions ( nor ) were located on the terminal portions of the long arms of metacentric pairs 6 and 12 of c. macropomum ( figure 1a ) , in terminal regions of chromosome 4 in p. mesopotamicus ( figure 1b ) and in the short arms of one homologue of pair 12 in species of mylossoma ( figure 1c , d ) .
in situ hybridization experiments with 18s sequences as probes were performed numerous times in mylossoma species but no labeling of ribosomal sites could be detected . this result may reflect methodological problems or may be due to the small size of these sites which would prevent their visualization .
the 18s rdna sequences were visualized in the terminal regions of the long arms of metacentric pairs 6 , 10 and 12 of c. macropomum ( figure 2a ) , with pairs 6 and 12 coinciding with the ag - nor . in p. mesopotamicus
, signals were observed in the terminal regions of the long arms of metacentric pairs 4 and 6 ( figure 2b ) .
5s rdna sequences mapped to euchromatic and heterochromatic regions of two chromosome pairs in each of the four species ( figure 3e , f , g , h ) .
labeling was observed in an interstitial position of pair 11 and in the terminal region of the short arm of pair 13 in c. macropomum and p. mesopotamicus ( figure 3a , b ) . in both species , the 5s rdna sites of pair 11 co - localized with heterochromatic blocks and the 5s rdna sites of pair 13 were adjacent to heterochromatic regions ( figure 3e , f ) .
the 5s rdna sites were present in pairs 4 and 12 of mylossoma aureum ( figure 3c , g ) and in pairs 4 and 8 of m. duriventre ( figure 3d ) .
the interstitial signals in the long arms coincided with the heterochromatic blocks in both species .
fish with telomeric probes revealed signals at the terminal portions of both arms of all chromosomes in the four species ( figure 4a , b , c , d ) .
an interstitial telomeric site ( its ) was observed in one metacentric pair of c. macropomum ( figure 4a ) and coincided with a positive c - banding region .
( 2008 ) , the serrasalmidae family may be divided into three main clades : pacus , myleus , and piranhas .
all of the species analyzed herein belong to the pacus clade , which comprises the piaractus , colossoma , and mylossoma genera , considered basal among serrasalmidae ( ort et al . , 1996 ) .
the evolutionary relationships among serrasalmidae are not well defined and have been the focus of several studies .
molecular data have been used to supplement morphological studies that confirm the monophyly of this fish group from species to family and/or subfamily levels ( calcagnotto et al . , 2005 ;
( 1996 ) phylogenetic proposal for serrasalmidae - with the pacus group , considered the most basal , having 2n = 54 biarmed chromosomes , the myleus
inpa / ufam , manaus , am ) ( garca - parra wj , 2000 , phd thesis .
inpa / ufam , manaus , am ) , and the piranhas clade , considered the most derived , presenting diploid numbers that vary from 58 to 64 and the presence of acrocentric pairs ( muramoto et al . , 1968 ;
this scenario suggests an evolutionary trend towards increasing chromosome numbers mainly due to chromosomal fissions .
all the species analyzed here shared the same diploid number , although their karyotypic formulas differed , indicating the occurrence of non - robertsonian rearrangements . this kind of rearrangements have been reported for a number of other fish species ( garcia and moreira - filho , 2005 ; mazzuchelli et al .
, 2007 ; nakayama et al . , 2008 , 2012 ; schneider et al . ,
clade is the only one whose species consistently present only biarmed chromosomes ( metacentric , submetacentric , and subtelocentric ) .
the karyotypic formula of our specimens of c. macropomum differed from that described by nirchio et al .
( 2003 ) ; p. mesopotamicus presented the same karyotypic formula described when this species was still classified as colossoma mitrei ( almeida - toledo et al . , 1987 ) , but differences were detected in the distribution of heterochromatic blocks , with small quantities of constitutive heterochromatin observed in the present work .
the great diversity of habitats may expose organisms to different selection pressures that may activate or inactivate certain genes through epigenetic mechanisms .
the appearance of distinct heterochromatic regions in different populations could well reflect adaptive processes that are part of the necessary metabolic fine - tuning for species survival . according to richards et al .
( 2010 ) , epigenetic changes induced by hybridization or environmental stress may be passed on for many generations and contribute for the adaptation of these fish to new environments .
fluorescence in situ hybridization with 18s rdna sequences showed multiple signals in two species , as previously reported for other serrasalmidae ( nakayama et al . , 2008 , 2012 ) .
the 18s rdna distribution in three chromosome pairs of c. macropomum corroborated earlier findings for that same species ( nirchio et al . , 2003 ;
2012 ) , in contrast to the ag - nor staining that evidenced only two pairs .
the pattern observed in p. mesopotamicus ( two labeled pairs ) differed , however , from that described in other species of the same genera ( p. brachypomus ) which presented a single 18s rdna - bearing chromosome pair ( nirchio et al . , 2003 )
. the smaller number of nor evidenced by silver nitrate could be explained by the fact that this agent only interacts with proteins in recently active regions , whereas fish detects all rdna sites despite their recent activity . if the presence of single nor are considered a plesiomorphic characteristic , as in other fish groups ( e.g. , feldberg et al .
, 2003 - family cichlidae ) , a similar situation may well exist in the serrasalmidae family , with the basal species p. brachypomus ( orti et al . , 2008 ) bearing 18s rdna sequences in a single chromosome pair ( nirchio et al . , 2003 ) , followed by other species of the same genus with two 18s rdna - bearing pairs and by c. macropomum with these sequences present in three pairs .
clade has five to eight chromosome pairs bearing 18s rdna sites ( nakayama et al . , 2012 ) .
fish with 5s rdna sequences resulted in two chromosome pairs labeled in the four species .
the pairs bearing the 5s rdna sites ( pairs 11 and 13 ) in c. macropomum and p. mesopotamicus appeared to be homeologs , as their sizes and morphologies were very similar ( figure 3a , b ) .
although the mylossoma species also showed 5s rdna sites in two chromosome pairs , these did not seem to be intra- or intergeneric homeologs , as the morphology and interstitial labeling location in one of the pairs differed from those observed in the other species ( figure 3c , d ) .
additionally , the 5s rdna localization in c. macropomum differed from that previously reported for this same species , in which the single 5s rdna site coincided with a heterochromatic region ( nakayama et al . , 2012 ) .
this variation in the number of chromosome pairs with multiple 5s rdna sites in c. macropomum contradicts nakayama et al .
( 2012 ) observation that the pattern of a single 5s rdna - bearing site found in serrasalmus was a characteristic shared by the entire family .
additionally , the synteny of the 5s and 18s rdna sites previously described in c. macropomum was not observed in the present work , perhaps due to the fact that their chromosomes morphological similarities led to errors in their identification and the5s and 18s rdna probes were not hybridized simultaneously .
these observations also do not corroborate the hypothesis that the localization of interstitial sites on a single chromosome pair is characteristic of basal species ( martins and galetti jr . , 1999 ) . in the serrasalmidae family , for example , the opposite pattern was observed , with the species of the basal genera ( colossoma , mylossoma , and piaractus ) having two 5s rdna - bearing chromosome pairs and the most derived species ( of the genus serrasalmus ) showing a single chromosome with 5s rdna sequences ( nakayama et al . , 2012 ) .
the location of the 5s rdna sites in heterochromatic regions was the only similarity observed in relation to other serrasalmids .
telomeric sequences were present in the terminal regions of all the chromosome arms in all the analyzed species , confirming the hypothesis that the observed variations between the karyotype were due to inversions . interstitial telomeric sequences ( itss ) in one chromosome pair were only observed in c. macropomum and did not seem to result from fusion , as the diploid number was maintained , neither to be due to inversions .
other repetitive dna sequences present in constitutive heterochromatin regions have been associated with telomeric sequences ( schneider et al . , 2012 ) .
additionally , its observed in centromeric positions ( as in c. macropomum ) may be related to satellite dnas transferred from other regions by processes such as unequal crossing - over , transpositions , or duplications in the heterochromatin ( schneider et al . , 2012 ) .
based on the data presented herein , we conclude that the basal clade of serrasalmidae , composed of c. macropomum , m. aureum , m. duriventre , and p. mesopotamicus has karyotypic features consistent with the previously published data for these species , including their 2n = 54 .
the data presented herein demonstrate the need of additional molecular studies within this clade to aid the identification of the distribution patterns of the repetitive sequences that are effectively determining the evolutionary trend within this group .
( 2008 ) , the serrasalmidae family may be divided into three main clades : pacus , myleus , and piranhas .
all of the species analyzed herein belong to the pacus clade , which comprises the piaractus , colossoma , and mylossoma genera , considered basal among serrasalmidae ( ort et al . , 1996 ) .
the evolutionary relationships among serrasalmidae are not well defined and have been the focus of several studies .
molecular data have been used to supplement morphological studies that confirm the monophyly of this fish group from species to family and/or subfamily levels ( calcagnotto et al . , 2005 ;
( 1996 ) phylogenetic proposal for serrasalmidae - with the pacus group , considered the most basal , having 2n = 54 biarmed chromosomes , the myleus
inpa / ufam , manaus , am ) ( garca - parra wj , 2000 , phd thesis .
inpa / ufam , manaus , am ) , and the piranhas clade , considered the most derived , presenting diploid numbers that vary from 58 to 64 and the presence of acrocentric pairs ( muramoto et al . , 1968 ;
this scenario suggests an evolutionary trend towards increasing chromosome numbers mainly due to chromosomal fissions .
all the species analyzed here shared the same diploid number , although their karyotypic formulas differed , indicating the occurrence of non - robertsonian rearrangements . this kind of rearrangements have been reported for a number of other fish species ( garcia and moreira - filho , 2005 ; mazzuchelli et al .
, 2007 ; nakayama et al . , 2008 , 2012 ; schneider et al . ,
clade is the only one whose species consistently present only biarmed chromosomes ( metacentric , submetacentric , and subtelocentric ) .
the karyotypic formula of our specimens of c. macropomum differed from that described by nirchio et al .
( 2003 ) ; p. mesopotamicus presented the same karyotypic formula described when this species was still classified as colossoma mitrei ( almeida - toledo et al . , 1987 ) , but differences were detected in the distribution of heterochromatic blocks , with small quantities of constitutive heterochromatin observed in the present work .
the great diversity of habitats may expose organisms to different selection pressures that may activate or inactivate certain genes through epigenetic mechanisms .
the appearance of distinct heterochromatic regions in different populations could well reflect adaptive processes that are part of the necessary metabolic fine - tuning for species survival . according to richards et al .
( 2010 ) , epigenetic changes induced by hybridization or environmental stress may be passed on for many generations and contribute for the adaptation of these fish to new environments .
fluorescence in situ hybridization with 18s rdna sequences showed multiple signals in two species , as previously reported for other serrasalmidae ( nakayama et al . , 2008 , 2012 ) .
the 18s rdna distribution in three chromosome pairs of c. macropomum corroborated earlier findings for that same species ( nirchio et al . , 2003 ; nakayama et al . , 2012 ) , in contrast to the ag - nor staining that evidenced only two pairs .
the pattern observed in p. mesopotamicus ( two labeled pairs ) differed , however , from that described in other species of the same genera ( p. brachypomus ) which presented a single 18s rdna - bearing chromosome pair ( nirchio et al . , 2003 )
. the smaller number of nor evidenced by silver nitrate could be explained by the fact that this agent only interacts with proteins in recently active regions , whereas fish detects all rdna sites despite their recent activity . if the presence of single nor are considered a plesiomorphic characteristic , as in other fish groups ( e.g. , feldberg et al .
, 2003 - family cichlidae ) , a similar situation may well exist in the serrasalmidae family , with the basal species p. brachypomus ( orti et al . ,
2008 ) bearing 18s rdna sequences in a single chromosome pair ( nirchio et al . , 2003 ) , followed by other species of the same genus with two 18s rdna - bearing pairs and by c. macropomum with these sequences present in three pairs .
clade has five to eight chromosome pairs bearing 18s rdna sites ( nakayama et al . , 2012 ) .
fish with 5s rdna sequences resulted in two chromosome pairs labeled in the four species .
the pairs bearing the 5s rdna sites ( pairs 11 and 13 ) in c. macropomum and p. mesopotamicus appeared to be homeologs , as their sizes and morphologies were very similar ( figure 3a , b ) .
although the mylossoma species also showed 5s rdna sites in two chromosome pairs , these did not seem to be intra- or intergeneric homeologs , as the morphology and interstitial labeling location in one of the pairs differed from those observed in the other species ( figure 3c , d ) . additionally , the 5s rdna localization in c. macropomum differed from that previously reported for this same species , in which the single 5s rdna site coincided with a heterochromatic region ( nakayama et al . , 2012 ) .
this variation in the number of chromosome pairs with multiple 5s rdna sites in c. macropomum contradicts nakayama et al .
( 2012 ) observation that the pattern of a single 5s rdna - bearing site found in serrasalmus was a characteristic shared by the entire family .
additionally , the synteny of the 5s and 18s rdna sites previously described in c. macropomum was not observed in the present work , perhaps due to the fact that their chromosomes morphological similarities led to errors in their identification and the5s and 18s rdna probes were not hybridized simultaneously .
these observations also do not corroborate the hypothesis that the localization of interstitial sites on a single chromosome pair is characteristic of basal species ( martins and galetti jr . , 1999 ) . in the serrasalmidae family , for example
, the opposite pattern was observed , with the species of the basal genera ( colossoma , mylossoma , and piaractus ) having two 5s rdna - bearing chromosome pairs and the most derived species ( of the genus serrasalmus ) showing a single chromosome with 5s rdna sequences ( nakayama et al . , 2012 ) .
the location of the 5s rdna sites in heterochromatic regions was the only similarity observed in relation to other serrasalmids .
telomeric sequences were present in the terminal regions of all the chromosome arms in all the analyzed species , confirming the hypothesis that the observed variations between the karyotype were due to inversions .
interstitial telomeric sequences ( itss ) in one chromosome pair were only observed in c. macropomum and did not seem to result from fusion , as the diploid number was maintained , neither to be due to inversions .
other repetitive dna sequences present in constitutive heterochromatin regions have been associated with telomeric sequences ( schneider et al . , 2012 ) .
additionally , its observed in centromeric positions ( as in c. macropomum ) may be related to satellite dnas transferred from other regions by processes such as unequal crossing - over , transpositions , or duplications in the heterochromatin ( schneider et al . , 2012 ) .
based on the data presented herein , we conclude that the basal clade of serrasalmidae , composed of c. macropomum , m. aureum , m. duriventre , and p. mesopotamicus has karyotypic features consistent with the previously published data for these species , including their 2n = 54 .
the data presented herein demonstrate the need of additional molecular studies within this clade to aid the identification of the distribution patterns of the repetitive sequences that are effectively determining the evolutionary trend within this group . | the serrasalmidae family is composed of a number of commercially interesting species , mainly in the amazon region where most of these fishes occur . in the present study , we investigated the genomic organization of the 18s and 5s rdna and telomeric sequences in mitotic chromosomes of four species from the basal clade of the serrasalmidae family : colossoma macropomum , mylossoma aureum , m. duriventre , and piaractus mesopotamicus , in order to understand the chromosomal evolution in the family .
all the species studied had diploid numbers
2n = 54 and exclusively biarmed chromosomes , but variations of the karyotypic formulas were observed .
c - banding resulted in similar patterns among the analyzed species , with heterochromatic blocks mainly present in centromeric regions .
the 18s rdna mapping of c. macropomum and p. mesopotamicus revealed multiple sites of this gene ; 5s rdna sites were detected in two chromosome pairs in all species , although not all of them were homeologs .
hybridization with a telomeric probe revealed signals in the terminal portions of chromosomes in all the species and an interstitial signal was observed in one pair of c. macropomum . | Introduction
Material and Methods
Results
Karyotypic descriptions
Repetitive sequences mapping
Discussion
Cytogenetic descriptions
Repetitive sequences mapping | the family serrasalmidae comprises approximately 80 species distributed among 15 genera , with a number of commercially important species of the genera colossoma , mylossoma , and piaractus , which are valued for fishing or aquaculture in the amazon region ( arajo - lima and goulding , 1998 ; oliveira and arajo - lima , 1998 ; jgu , 2003 ; nelson , 2006 ) . within this context , the present work aimed to investigate the genomic organization of 18s rdna , 5s rdna and telomeric sequences in species of the basal clade of serrasalmidae in order to understand the family chromosomal evolution . four species belonging to the serrasalmidae family were analyzed : colossoma macropomum , mylossoma aureum , m. duriventre , and piaractus mesopotamicus . the constitutive heterochromatin of c. macropomum , m. duriventre , and p. mesopotamicus was concentrated in the centromeric regions of several chromosomes ( figure 1e , f , h ) . nucleolus organizing regions ( nor ) were located on the terminal portions of the long arms of metacentric pairs 6 and 12 of c. macropomum ( figure 1a ) , in terminal regions of chromosome 4 in p. mesopotamicus ( figure 1b ) and in the short arms of one homologue of pair 12 in species of mylossoma ( figure 1c , d ) . labeling was observed in an interstitial position of pair 11 and in the terminal region of the short arm of pair 13 in c. macropomum and p. mesopotamicus ( figure 3a , b ) . an interstitial telomeric site ( its ) was observed in one metacentric pair of c. macropomum ( figure 4a ) and coincided with a positive c - banding region . the constitutive heterochromatin of c. macropomum , m. duriventre , and p. mesopotamicus was concentrated in the centromeric regions of several chromosomes ( figure 1e , f , h ) . nucleolus organizing regions ( nor ) were located on the terminal portions of the long arms of metacentric pairs 6 and 12 of c. macropomum ( figure 1a ) , in terminal regions of chromosome 4 in p. mesopotamicus ( figure 1b ) and in the short arms of one homologue of pair 12 in species of mylossoma ( figure 1c , d ) . labeling was observed in an interstitial position of pair 11 and in the terminal region of the short arm of pair 13 in c. macropomum and p. mesopotamicus ( figure 3a , b ) . an interstitial telomeric site ( its ) was observed in one metacentric pair of c. macropomum ( figure 4a ) and coincided with a positive c - banding region . although the mylossoma species also showed 5s rdna sites in two chromosome pairs , these did not seem to be intra- or intergeneric homeologs , as the morphology and interstitial labeling location in one of the pairs differed from those observed in the other species ( figure 3c , d ) . in the serrasalmidae family , for example , the opposite pattern was observed , with the species of the basal genera ( colossoma , mylossoma , and piaractus ) having two 5s rdna - bearing chromosome pairs and the most derived species ( of the genus serrasalmus ) showing a single chromosome with 5s rdna sequences ( nakayama et al . telomeric sequences were present in the terminal regions of all the chromosome arms in all the analyzed species , confirming the hypothesis that the observed variations between the karyotype were due to inversions . based on the data presented herein , we conclude that the basal clade of serrasalmidae , composed of c. macropomum , m. aureum , m. duriventre , and p. mesopotamicus has karyotypic features consistent with the previously published data for these species , including their 2n = 54 . in the serrasalmidae family , for example
, the opposite pattern was observed , with the species of the basal genera ( colossoma , mylossoma , and piaractus ) having two 5s rdna - bearing chromosome pairs and the most derived species ( of the genus serrasalmus ) showing a single chromosome with 5s rdna sequences ( nakayama et al . telomeric sequences were present in the terminal regions of all the chromosome arms in all the analyzed species , confirming the hypothesis that the observed variations between the karyotype were due to inversions . based on the data presented herein , we conclude that the basal clade of serrasalmidae , composed of c. macropomum , m. aureum , m. duriventre , and p. mesopotamicus has karyotypic features consistent with the previously published data for these species , including their 2n = 54 . | [
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despite declines in cardiovascular ( cv ) disease ( cvd ) mortality , cvd remains the leading cause of death in the united states .
clinical practice focuses on primary prevention , specifically the prevention of incident cvd among individuals classified at high risk based on clinical risk factors , such as hypertension ( htn ) and high cholesterol .
numerous risk prediction tools can estimate an individual 's shortterm ( ie , 10year ) risk of cvd and the american heart association ( aha ) endorses the use of these tools in the clinical setting however , because the shortterm risk of developing cvd for the majority of middleaged adults is low , yet the longterm risk is high , these prediction models do not capture the true cumulative burden of cvd in this population .
furthermore , more than half of all cvd events occur in individuals who are not classified as high risk on the basis of clinical risk factors .
conversely , adults with optimal levels of risk factors at midlife have a 70% to 80% lower risk of coronary heart disease ( chd ) and cvd mortality .
the absence of established risk factors at the age of 55 is associated with a lifetime risk of cvd of 5% to 8% .
thus , the prevention of cvd risk factor development , or primordial prevention , is of paramount importance to minimize an individual 's longterm cvd risk . in clinical trials ,
intensive lifestyle modification significantly improves risk factors , such as blood pressure and lipid profiles .
furthermore , in longterm observational epidemiology studies , an overall healthy lifestyle , including prudent diet , not smoking , healthy weight , and physical activity , in midlife may prevent the development of cvd risk factors and ultimately cvd events .
we sought to develop and validate a risk prediction model based on modifiable lifestyle factors , which may be used as a tool for risk assessment in the primordial prevention setting .
we developed this risk model among healthy middleaged men and women , who are the ideal population to focus longterm prevention efforts clinically .
we derived the healthy heart score , a lifestylebased risk score , among women enrolled in the nurses health study ( nhs ) , a prospective cohort of 121 700 female nurses ages 30 to 55 years at baseline in 1976 and men in the health professionals followup study ( hpfs ) , a cohort of 51 529 u.s .
male health professionals , ages 40 to 75 years , in 1986 . participants in both cohorts provided information on medical history , lifestyle factors , and newly diagnosed diseases on selfreported questionnaires throughout followup .
the institutional review boards at the harvard school of public health ( boston , ma ) and brigham and women 's hospital ( boston , ma ) approved the study protocols and return of the questionnaire implied consent .
this analysis included 61 025 women and 34 478 men who provided a complete ascertainment of pertinent lifestyle factors on a questionnaire administered in 1986 and who were free of previously diagnosed cvd ( myocardial infarction [ mi ] , angina , stroke , transient ischemic attack , and coronary revascularization ) and cancer at baseline . because diabetes is considered a chd risk equivalent , we also excluded individuals with a history of diabetes at baseline .
the outcome for this study was ischemic cvd , which included nonfatal mi , fatal chd , and ischemic stroke , in line with current aha recommendation for risk assessment algorithms .
mi was defined according to world health organization criteria and strokes were confirmed using the national survey of stroke criteria and both were adjudicated by study investigators through medical record review .
strokes were classified as ischemic stroke ( thrombotic or embolic occlusion of a cerebral artery ) , hemorrhagic stroke ( subarachnoid and intraparenchymal hemorrhage ) , or stroke of probable or unknown subtype , when the subtype could not be ascertained because of unobtainable medical records .
we excluded confirmed hemorrhagic strokes in our endpoint , but included confirmed ischemic and unknown subtypes , because 80% of all unknown strokes are ischemic in origin .
fatal cvd events were identified by next of kin , postal authorities , or the national death index and confirmed by medical records , autopsy reports , and death certificates with chd or ischemic stroke listed as the underlying cause .
criteria for consideration included the strength and consistency of association with risk of cvd in the literature and availability of data among study participants .
a full list of variables considered in the model can be found in table 1 .
dietary and lifestyle factors considered for inclusion in the healthy heart score for physical activity , men and women were asked how many hours per week , on average , they engaged in specific activities ( walking , jogging , running , bicycling , swimming , tennis , squash / racquetball , rowing , and calisthenics ) using a previously validated physical activity questionnaire .
we calculated the average hours per week spent in moderate ( 3 to 6 metabolic equivalent tasks [ mets ] ) and vigorous ( 6 mets ) activity , which included walking at a pace of 3 mph or greater and other activities .
we calculated body mass index ( bmi ; kg / m ) from selfreported height and weight ; selfreported weight was highly correlated with directly measured weight ( r=0.96 ) .
smoking status was defined as never , past , or current , with various definitions of past smokers and current smokers as shown in table 1 .
information on diet and alcohol was assessed through a validated 131item food frequency questionnaire ( ffq ) .
total nutrient intake was calculated by multiplying the frequency of consumption of each food item by its nutrient content ( from the harvard university food composition database ) of the specified portion and then summing the nutrient values across all contributing foods .
we calculated average alcohol intake in g / day , assuming 12.8 g of alcohol in 12 ounces ( oz ) of beer , 11.0 g of alcohol in 4 oz of wine , and 14.0 g of alcohol in 1.5 oz of liquor .
all analyses were performed separately in the nhs and hpfs cohorts using the structure outlined below .
each participant contributed followup time from the return of the 1986 questionnaire until the date of diagnosis of an ischemic cvd event , date of death , or end of followup ( june 2010 in women and february 2010 in men ) . for each cohort , we randomly assigned two thirds of the study participants to a derivation cohort ( n=40 680 women ; n=23 026 men ) , and the remaining one third of the participants were reserved as a validation cohort ( n=20 345 women ; n=11 452 men ) . a priori
, we decided to create a lifestyle score that included the same risk indicators for both men and women to provide a consistent clinical and public health message about cvd prevention ; thus , we included components that met the inclusion criteria in at least 1 , but not necessarily both , cohorts .
the models were computed with risk at 20 years , because lifestyle factors likely play a greater role in the longterm , rather than shortterm , prevention of cvd events . over 99% of men and 89% of women
first , we derived a composite diet score within the derivation cohort . to be included in the composite diet score ,
first , the dietary component had to be a significant and independent predictor of ischemic cvd risk in multivariable cox proportional hazards models that included other dietary factors .
second , the addition of the dietary variable had to minimize the model bayes information criterion ( bic ) .
therefore , the model with the lowest bic is the bestfitting , most parsimonious model . for all dietary factors , we explored the relation with cvd risk modeled in multiple forms , including as continuous and categorical , examining various cutpoints . to minimize the effect of influential outliers for continuous dietary factors
the summary diet score was calculated by multiplying each component by its cohortspecific beta coefficient from the cox model and summing across all components .
we derived the best overall prediction algorithm based on hazard ratios estimated in cox proportional hazards models within the derivation data set .
as with the dietary factors , we explored the relation of each lifestyle factor with cvd risk modeled in multiple forms , including continuous , with higherorder functions when necessary , and categorical variables .
we truncated the distribution of the continuous lifestyle factors at the 1st and 99th percentiles .
we included all variables that were independent predictors of cvd risk for inclusion into the prediction model in multivariable models .
we assessed model goodness of fit by the gronnesbyborgan test statistic , a more robust statistic for survival data compared to hosmerlemeshow 's statistic for calibration .
as suggested by d'agostino and nam , chisquare ( ) values > 20 ( p<0.01 ) suggest a lack of adequate fit .
we used harrell 's cindex for survival data to assess model discrimination , which measures the ability to distinguish between individuals who experience a cvd event from those who do not .
we compared model discrimination of the lifestylebased cvd risk score to models with age only .
we also assessed discrimination of a prediction model that added selfreported physiciandiagnosed risk factors at baseline ( htn and hypercholesterolemia or high cholesterol ) to this lifestylerisk risk score . to assess model calibration , we used calibration plots , plotting the average predicted risk within deciles of predicted risk against the observed risk in that decile . in sensitivity analyses , we examined model performance stratified by presence and absence of baseline clinical risk factors , hypercholesterolemia or high cholesterol and/or htn , which are key risk factors for cvd and included in most clinically based risk prediction models .
we derived the healthy heart score , a lifestylebased risk score , among women enrolled in the nurses health study ( nhs ) , a prospective cohort of 121 700 female nurses ages 30 to 55 years at baseline in 1976 and men in the health professionals followup study ( hpfs ) , a cohort of 51 529 u.s .
male health professionals , ages 40 to 75 years , in 1986 . participants in both cohorts provided information on medical history , lifestyle factors , and newly diagnosed diseases on selfreported questionnaires throughout followup .
the institutional review boards at the harvard school of public health ( boston , ma ) and brigham and women 's hospital ( boston , ma ) approved the study protocols and return of the questionnaire implied consent .
this analysis included 61 025 women and 34 478 men who provided a complete ascertainment of pertinent lifestyle factors on a questionnaire administered in 1986 and who were free of previously diagnosed cvd ( myocardial infarction [ mi ] , angina , stroke , transient ischemic attack , and coronary revascularization ) and cancer at baseline .
because diabetes is considered a chd risk equivalent , we also excluded individuals with a history of diabetes at baseline .
the outcome for this study was ischemic cvd , which included nonfatal mi , fatal chd , and ischemic stroke , in line with current aha recommendation for risk assessment algorithms .
mi was defined according to world health organization criteria and strokes were confirmed using the national survey of stroke criteria and both were adjudicated by study investigators through medical record review .
strokes were classified as ischemic stroke ( thrombotic or embolic occlusion of a cerebral artery ) , hemorrhagic stroke ( subarachnoid and intraparenchymal hemorrhage ) , or stroke of probable or unknown subtype , when the subtype could not be ascertained because of unobtainable medical records .
we excluded confirmed hemorrhagic strokes in our endpoint , but included confirmed ischemic and unknown subtypes , because 80% of all unknown strokes are ischemic in origin .
fatal cvd events were identified by next of kin , postal authorities , or the national death index and confirmed by medical records , autopsy reports , and death certificates with chd or ischemic stroke listed as the underlying cause .
criteria for consideration included the strength and consistency of association with risk of cvd in the literature and availability of data among study participants .
a full list of variables considered in the model can be found in table 1 .
dietary and lifestyle factors considered for inclusion in the healthy heart score for physical activity , men and women were asked how many hours per week , on average , they engaged in specific activities ( walking , jogging , running , bicycling , swimming , tennis , squash / racquetball , rowing , and calisthenics ) using a previously validated physical activity questionnaire .
we calculated the average hours per week spent in moderate ( 3 to 6 metabolic equivalent tasks [ mets ] ) and vigorous ( 6 mets ) activity , which included walking at a pace of 3 mph or greater and other activities .
we calculated body mass index ( bmi ; kg / m ) from selfreported height and weight ; selfreported weight was highly correlated with directly measured weight ( r=0.96 ) .
smoking status was defined as never , past , or current , with various definitions of past smokers and current smokers as shown in table 1 .
information on diet and alcohol was assessed through a validated 131item food frequency questionnaire ( ffq ) .
total nutrient intake was calculated by multiplying the frequency of consumption of each food item by its nutrient content ( from the harvard university food composition database ) of the specified portion and then summing the nutrient values across all contributing foods .
we calculated average alcohol intake in g / day , assuming 12.8 g of alcohol in 12 ounces ( oz ) of beer , 11.0 g of alcohol in 4 oz of wine , and 14.0 g of alcohol in 1.5 oz of liquor .
all analyses were performed separately in the nhs and hpfs cohorts using the structure outlined below .
each participant contributed followup time from the return of the 1986 questionnaire until the date of diagnosis of an ischemic cvd event , date of death , or end of followup ( june 2010 in women and february 2010 in men ) . for each cohort , we randomly assigned two thirds of the study participants to a derivation cohort ( n=40 680 women ; n=23 026 men ) , and the remaining one third of the participants were reserved as a validation cohort ( n=20 345 women ; n=11 452 men ) . a priori
, we decided to create a lifestyle score that included the same risk indicators for both men and women to provide a consistent clinical and public health message about cvd prevention ; thus , we included components that met the inclusion criteria in at least 1 , but not necessarily both , cohorts .
the models were computed with risk at 20 years , because lifestyle factors likely play a greater role in the longterm , rather than shortterm , prevention of cvd events . over 99% of men and 89% of women were followed for a minimum of 20 years .
first , we derived a composite diet score within the derivation cohort . to be included in the composite diet score ,
first , the dietary component had to be a significant and independent predictor of ischemic cvd risk in multivariable cox proportional hazards models that included other dietary factors .
second , the addition of the dietary variable had to minimize the model bayes information criterion ( bic ) .
therefore , the model with the lowest bic is the bestfitting , most parsimonious model . for all dietary factors , we explored the relation with cvd risk modeled in multiple forms , including as continuous and categorical , examining various cutpoints . to minimize the effect of influential outliers for continuous dietary factors
the summary diet score was calculated by multiplying each component by its cohortspecific beta coefficient from the cox model and summing across all components .
we derived the best overall prediction algorithm based on hazard ratios estimated in cox proportional hazards models within the derivation data set .
as with the dietary factors , we explored the relation of each lifestyle factor with cvd risk modeled in multiple forms , including continuous , with higherorder functions when necessary , and categorical variables .
we truncated the distribution of the continuous lifestyle factors at the 1st and 99th percentiles .
we included all variables that were independent predictors of cvd risk for inclusion into the prediction model in multivariable models .
we assessed model goodness of fit by the gronnesbyborgan test statistic , a more robust statistic for survival data compared to hosmerlemeshow 's statistic for calibration .
as suggested by d'agostino and nam , chisquare ( ) values > 20 ( p<0.01 ) suggest a lack of adequate fit .
we used harrell 's cindex for survival data to assess model discrimination , which measures the ability to distinguish between individuals who experience a cvd event from those who do not .
we compared model discrimination of the lifestylebased cvd risk score to models with age only .
we also assessed discrimination of a prediction model that added selfreported physiciandiagnosed risk factors at baseline ( htn and hypercholesterolemia or high cholesterol ) to this lifestylerisk risk score . to assess model calibration
, we used calibration plots , plotting the average predicted risk within deciles of predicted risk against the observed risk in that decile . in sensitivity analyses , we examined model performance stratified by presence and absence of baseline clinical risk factors , hypercholesterolemia or high cholesterol and/or htn , which are key risk factors for cvd and included in most clinically based risk prediction models .
among 61 025 women who were free of known cvd , diabetes , and cancer at baseline and had a median age of 52 years ( interquartile range [ iqr ] , 46 to 58 ) , 3775 cvd cases ( 57% chd and 43% ischemic stroke ) occurred ; among 34 478 apparently healthy men , median age 51 years ( iqr , 44 to 59 ) , a total of 3506 cvd cases ( 79% chd and 21% ischemic stroke ) occurred .
the prediction model was derived among 2525 events in women and 2375 in men and validated among 1250 events in women and 1129 in men .
lifestyle characteristics were similar between the derivation and validation cohorts for both men and women ( table 2 ) .
the median period of followup was 23.9 ( iqr , 23.6 , 23.9 ) years among women and 23.7 ( iqr , 23.3 , 23.8 ) among men .
demographic and lifestyle characteristics in the derivation and validation data sets among men and women at baseline means ( sd ) for continuous variables .
five dietary components , fruits+vegetables , nuts , cereal fiber , red+processed meat , and sugarsweetened beverages , which have been associated with cvd previously , met the inclusion criteria for the composite diet score in at least 1 cohort and were included in the final score ( table 3 ) .
five lifestyle factors , smoking , bmi , moderatetovigorous exercise , alcohol intake and the diet score , in addition to age , were included in the overall lifestylebased risk score ( table 4 ) .
each lifestyle factor was associated with risk of cvd linearly , except alcohol , which had a ushaped association .
multivariable hazard ratios ( hr ) of cardiovascular disease for dietary components in the derivation data sets ( n=40 680 women and 23 026 men ) serving sizes : 1 medium piece of fruit , cup of berries , cup of vegetables , 1 cup of green leafy vegetables , 1 can / bottle / glass of sugarsweetened beverages , 4 ounces ( oz ) of unprocessed meat and 1.5 oz of processed meat , and 1 oz of nuts or 1 tablespoon ( tbsp ) of nut butter .
multivariable hr of cvd for lifestyle factors in the derivation data sets ( n=40 680 women and 23 026 men ) bmi indicates body mass index ; cvd , cardiovascular disease ; hr , hazards ratio .
beer=12.8 g of alcohol , 4oz wine=11.0 g of alcohol , and 1.5oz liquor=14.0 g of alcohol
. hours per week spent walking ( 3 mph ) , jogging , running , bicycling , swimming , tennis , squash / racquetball , rowing , and calisthenics . in the validation cohorts , gronnesbyborgan 's test statistic indicated adequate model fit and harrell 's cstatistic suggested good discrimination between cases and controls ( table 5 ) .
the lifestyle factors significantly increased the cstatistic , compared to models with just age alone .
further addition of selfreported htn and hypercholesterolemia or high cholesterol to the risk model significantly increased the cstatistic from 0.72 to 0.73 ( 95% confidence interval [ ci ] , 0.72 , 0.74 ; pdiff<0.001 ) in women and from 0.77 to 0.78 ( 95% ci , 0.76 , 0.79 ; pdiff=0.001 ) in men .
finally , the model was well calibrated for 20year cvd risk prediction based on the calibration plots in women ( figure 1a ) .
in men , the prediction model slightly underestimated cvd risk at high predicted risk levels ( figure 1b ) .
summary statistics to assess model performance of the lifestylebased risk score within the validation data sets ( n=20 345 women and 11 452 men ) ci indicates confidence interval .
p value , test for difference in cstatistic comparing model adjusted for age and models adjusted for age+lifestyle factors .
calibration plots of predicted 20year cardiovascular disease ( cvd ) risk within deciles against the observed 20year cvd risk in the validation data set ( n=20 345 women and 11 452 men ) .
data are plotted among all women ( a : black diamonds ) , women without cvd risk factors of hypertension and hypercholesterolemia at baseline ( a : white circles ) , and women with risk factors of hypertension and hypercholesterolemia at baseline ( a : black circles ) and all men ( b : black diamonds ) , men without cvd risk factors of hypertension and hypercholesterolemia at baseline ( b : white circles ) , and men with risk factors of hypertension and hypercholesterolemia at baseline ( b : black circles ) . whereas model fit was good among individuals with and without clinical risk factors at baseline ( table 5 ) , model discrimination ( table 5 ) and calibration ( figure 1a and 1b )
were better in the absence of baseline clinical risk factors ( htn and/or hypercholesterolemia or high cholesterol ) , compared to the presence of either risk factors . given that the model performed well in the independent validation data set , we combined the derivation and validation data sets in each cohort and refit the final model in the full data set to obtain moreprecise estimates of the coefficients for our lifestylebased cvd risk score .
the equations for the diet score calculation and the healthy heart score , based on the full data set , are presented in figure 2 .
formula to estimate the 20year risk of cvd based on healthy heart score derived in the full data set ( n=61 025 women and 34 478 men ) . serving sizes :
1 medium piece of fruit ; cup of berries ; cup of vegetables ; 1 cup of green leafy vegetables ; 1 can / bottle / glass of sugarsweetened beverages ; 4 ounces ( oz ) of unprocessed meat and 1.5 oz of processed meat ; and 1 oz nuts or 1 tablespoon ( tbsp ) of nut butter . as a practical example
, we estimated the 20year risk of cvd , relative to a healthy lifestyle , for individuals with various combinations of ages and lifestyle habits ( figure 3 ) .
compared to a 45yearold individual with very healthy lifestyle habits ( nonsmoker , bmi of 23 kg / m , 3.5 hours per week of moderatetovigorous physical activity , diet score of 5 points , and moderate alcohol intake [ 15 g / day in women and 30 g / day in men ] ) , the 20year risk of cvd for 45yearold individuals with unhealthy lifestyle habits ( current smoker , bmi of 35 kg / m , no physical activity , diet score of 0 points , and 0 g / day of alcohol ) was over 7fold higher in women and 6fold higher in men . estimated 20year risk of cvd for women ( n=61 025 ) and men ( n=34 478 ) across varying lifestyle habits , relative to the healthiest lifestyle , according to the healthy heart score
healthiest lifestyle : never smoker , bmi : 23 kg / m , moderate exercise : 3.5 hours per week , moderate alcohol : 15 g / day in women and 30 g / day in men , diet score : 5 points ; average lifestyle : never smoker , bmi : 28 kg / m , moderate exercise : 1.5 hours per week , moderate alcohol : 5 g / day , diet score : 2.5 points ; least healthy lifestyle : never smoker , bmi : 35 kg / m , moderate exercise : 0 hours per week , moderate alcohol : 0 g / day , diet score : 0 points .
five dietary components , fruits+vegetables , nuts , cereal fiber , red+processed meat , and sugarsweetened beverages , which have been associated with cvd previously , met the inclusion criteria for the composite diet score in at least 1 cohort and were included in the final score ( table 3 ) .
five lifestyle factors , smoking , bmi , moderatetovigorous exercise , alcohol intake and the diet score , in addition to age , were included in the overall lifestylebased risk score ( table 4 ) .
each lifestyle factor was associated with risk of cvd linearly , except alcohol , which had a ushaped association .
multivariable hazard ratios ( hr ) of cardiovascular disease for dietary components in the derivation data sets ( n=40 680 women and 23 026 men ) serving sizes : 1 medium piece of fruit , cup of berries , cup of vegetables , 1 cup of green leafy vegetables , 1 can / bottle / glass of sugarsweetened beverages , 4 ounces ( oz ) of unprocessed meat and 1.5 oz of processed meat , and 1 oz of nuts or 1 tablespoon ( tbsp ) of nut butter .
multivariable hr of cvd for lifestyle factors in the derivation data sets ( n=40 680 women and 23 026 men ) bmi indicates body mass index ; cvd , cardiovascular disease ; hr , hazards ratio .
12oz ( ounce ) beer=12.8 g of alcohol , 4oz wine=11.0 g of alcohol , and 1.5oz liquor=14.0 g of alcohol .
hours per week spent walking ( 3 mph ) , jogging , running , bicycling , swimming , tennis , squash / racquetball , rowing , and calisthenics .
in the validation cohorts , gronnesbyborgan 's test statistic indicated adequate model fit and harrell 's cstatistic suggested good discrimination between cases and controls ( table 5 ) .
the lifestyle factors significantly increased the cstatistic , compared to models with just age alone .
further addition of selfreported htn and hypercholesterolemia or high cholesterol to the risk model significantly increased the cstatistic from 0.72 to 0.73 ( 95% confidence interval [ ci ] , 0.72 , 0.74 ; pdiff<0.001 ) in women and from 0.77 to 0.78 ( 95% ci , 0.76 , 0.79 ; pdiff=0.001 ) in men .
finally , the model was well calibrated for 20year cvd risk prediction based on the calibration plots in women ( figure 1a ) . in men ,
the prediction model slightly underestimated cvd risk at high predicted risk levels ( figure 1b ) .
summary statistics to assess model performance of the lifestylebased risk score within the validation data sets ( n=20 345 women and 11 452 men ) ci indicates confidence interval .
p value , test for difference in cstatistic comparing model adjusted for age and models adjusted for age+lifestyle factors .
calibration plots of predicted 20year cardiovascular disease ( cvd ) risk within deciles against the observed 20year cvd risk in the validation data set ( n=20 345 women and 11 452 men ) .
data are plotted among all women ( a : black diamonds ) , women without cvd risk factors of hypertension and hypercholesterolemia at baseline ( a : white circles ) , and women with risk factors of hypertension and hypercholesterolemia at baseline ( a : black circles ) and all men ( b : black diamonds ) , men without cvd risk factors of hypertension and hypercholesterolemia at baseline ( b : white circles ) , and men with risk factors of hypertension and hypercholesterolemia at baseline ( b : black circles ) . whereas model fit was good among individuals with and without clinical risk factors at baseline ( table 5 ) , model discrimination ( table 5 ) and calibration ( figure 1a and 1b ) were better in the absence of baseline clinical risk factors ( htn and/or hypercholesterolemia or high cholesterol ) , compared to the presence of either risk factors .
given that the model performed well in the independent validation data set , we combined the derivation and validation data sets in each cohort and refit the final model in the full data set to obtain moreprecise estimates of the coefficients for our lifestylebased cvd risk score .
the equations for the diet score calculation and the healthy heart score , based on the full data set , are presented in figure 2 .
formula to estimate the 20year risk of cvd based on healthy heart score derived in the full data set ( n=61 025 women and 34 478 men ) .
serving sizes : 1 medium piece of fruit ; cup of berries ; cup of vegetables ; 1 cup of green leafy vegetables ; 1 can / bottle / glass of sugarsweetened beverages ; 4 ounces ( oz ) of unprocessed meat and 1.5 oz of processed meat ; and 1 oz nuts or 1 tablespoon ( tbsp ) of nut butter . as a practical example , we estimated the 20year risk of cvd , relative to a healthy lifestyle , for individuals with various combinations of ages and lifestyle habits ( figure 3 ) . compared to a 45yearold individual with very healthy lifestyle habits ( nonsmoker , bmi of 23 kg / m , 3.5 hours per week of moderatetovigorous physical activity , diet score of 5 points , and moderate alcohol intake [ 15 g / day in women and 30 g / day in men ] ) , the 20year risk of cvd for 45yearold individuals with unhealthy lifestyle habits ( current smoker , bmi of 35 kg / m , no physical activity , diet score of 0 points , and 0 g / day of alcohol ) was over 7fold higher in women and 6fold higher in men . estimated 20year risk of cvd for women ( n=61 025 ) and men ( n=34 478 ) across varying lifestyle habits , relative to the healthiest lifestyle , according to the healthy heart score .
healthiest lifestyle : never smoker , bmi : 23 kg / m , moderate exercise : 3.5 hours per week , moderate alcohol : 15 g / day in women and 30 g / day in men , diet score : 5 points ; average lifestyle : never smoker , bmi : 28 kg / m , moderate exercise : 1.5 hours per week , moderate alcohol : 5 g / day , diet score : 2.5 points ; least healthy lifestyle : never smoker , bmi : 35 kg / m , moderate exercise : 0 hours per week , moderate alcohol : 0 g / day , diet score : 0 points .
in this study , we derived the healthy heart score , an algorithm based on modifiable lifestyle factors that effectively predicts risk of cvd among men and women 40 years of age , particularly among individuals without htn or hypercholesterolemia or high cholesterol .
the healthy heart score included smoking status , bmi , physical activity levels , alcohol consumption , and dietary intake and performed well in a validation data set , as demonstrated by goodness of model fit , calibration , and discrimination .
to our knowledge , the healthy heart score is the first prediction model to assess cvd risk formally based on healthy lifestyle factors .
clinical practice focuses on the prevention of cvd through modification and pharmacological treatment of elevated clinical risk factors in an effort to prevent a cardiovascular event .
physicians spend little time assessing or advising patients on healthy lifestyle behaviors , such as physical activity and diet , particularly among patients classified as lowrisk by the framingham risk score ( frs ) , and who are the ideal population to target primordial prevention efforts .
the prevalence of healthy behaviors in the united states is low , and there has been little improvement in prevalence over the past decade .
thus , the use of a tool such as the healthy heart score may facilitate clinicians in the assessment of a limited number of critical lifestyle factors in an effort to identify individuals at high risk for cvd .
furthermore , this lifestylebased tool may heighten awareness to explore true primordial prevention through interventions on underlying unhealthy behaviors to prevent the development of risk factors initially , rather than treating risk factors only when they become elevated .
the healthy heart score , which identifies individuals at high risk based on lifestyle behaviors , could be used in combination with clinically based primary prevention models . for adults with a high shortterm risk of cvd , guidelines are in place for the optimal course of treatment .
however , among individuals with low shortterm risk , the healthy heart score may provide additional important information about longterm cvd risk and overall burden of cvd .
the addition of a lifestylebased evaluation in the primary care setting may improve risk assessment and may be particularly applicable among middleaged adults for whom current prediction models underestimate cvd risk burden .
ultimately , research is needed to assess the feasibility and effectiveness of this lifestyleonly risk assessment tool on health behavior modification , cvd risk factor improvement , and overall cvd risk assessment when incorporated into the clinical care setting , particularly in combination with shortterm , clinically based risk models .
other risk calculators , which incorporate both clinical and lifestyle factors for cvd risk prediction are available , including mylifecheck ( based on life 's simple 7 from the aha ) and your disease risk ( from washington university in st louis , mo ) .
many traditional risk factors are downstream of lifestyle factors and may mediate the effect of lifestyle on cvd risk , therefore diminishing the predictive value of lifestyle factors after the development of clinical risk factors .
therefore , it may be more appropriate to develop risk models separately for the primordial and primary prevention of cvd , rather than developing a risk prediction model comprehensive of lifestyle and clinical risk factors . for example
, the healthy heart score performed better in the absence of clinical risk factors , which suggests that lifestyle factors provide the most information about cvd risk preceding the development of clinical risk factors .
furthermore , a lack of clinical measurements may increase the applicability of a lifestyleonly score beyond the clinical setting . as a broader public health screening tool
, the risk score could be used to assess and motivate a much larger audience who may not have laboratorybased measures available because of irregular checkups or lack of health care resources .
our diet score and final prediction model are not fully inclusive of all lifestyle factors that are predictive of cvd .
adults who have healthy levels of the behaviors included in the lifestyle score likely adhere to other behaviors associated with lower risk of cvd , such as good sleep habits , less time spent in sedentary behavior , low intake of sodium and trans fat , and high intake of marine n3 fatty acids .
we created the most parsimonious , rather than comprehensive , model for cvd risk prediction to identify the most critical factors necessary to assess cvd risk .
thus , the physicians need to inquire about only 3 lifestyle factors and 6 dietary components , avoiding the use of a long ffq and minimizing the burden of data collection on both the patient and clinician .
the healthy heart score predicts total ischemic cvd risk , although the underlying etiology of chd and stroke differ .
thus , some of the disparities in the model performance between the cohorts may be explained by the different distribution of the cvd endpoints .
however , from a public health perspective , the similarities for chd and stroke prevention provide strong rationale for the prediction of total ischemic cvd , rather than developing individual prediction models .
the model performance among men was modest , and better in women , likely because some risk factors were more strongly associated with risk among women .
additional studies are warranted to determine whether these differences are an artifact of the population characteristics or true biological difference between genders .
the nhs and hpfs are the ideal populations to develop a lifestylebased longterm cvd prediction algorithm given the long and rigorous followup over 20 years , which is particularly important in the setting of primordial prevention .
furthermore , we have detailed assessment of numerous diet and lifestyle factors across a broad range of values .
given the large sample size , we were able to validate the risk scores in an independent subset of the populations .
additionally , we estimated cvd risk empirically as a direct function of individual lifestyle risk factors , accounting for the impact of all lifestyle factors simultaneously and the gradient in risk across the full distribution of lifestyle factor levels , in contrast to other calculators that were based on risk estimates from the published literature , have not been independently validated in terms of cvd risk prediction and use simple categorization of lifestyle factors .
first , we derived these risk equations using conditional models , and though the relative risks for the lifestyle factors are valid , we recognize that absolute risk in these cohorts may be overestimated .
however , in contrast , the absolute risk of cvd among these primarily caucasian health professionals , within a narrow socioeconomic status ( ses ) range , is likely lower than the risk of cvd in the general population . to address these concerns , we present the 20year risk of cvd , relative to a healthy lifestyle , in figure 3 , as well as in the accompanying online calculator .
before a risk tool can be used in the clinical setting , it is imperative to evaluate its performance in external populations , particularly in populations with diversity in race , ses , and education .
although we would expect the underlying biology of these lifestyle factors to be similar across these demographics , simple calibration or potential reestimation may be required to estimate risk in morediverse populations accurately .
importantly , other clinical risk scores , such as the frs and reynolds risk score , were developed in nonrepresentative populations , yet are commonly used .
additionally , we lack direct measures of clinical risk factors and biomarkers used in other clinical prediction algorithms , and thus we can not compare directly the predictive ability of the lifestyle model to existing risk models in these populations . finally , we were unable to develop this primordial prevention risk score among individuals with no clinical risk factors ( htn and hypercholesterolemia or high cholesterol ) as a result of the decreased precision with fewer cvd cases .
despite being the leading cause of mortality and morbidity , cvd is largely preventable through primordial and primary prevention .
current clinical practice focuses on the primary prevention of cvd events among asymptomatic , but highrisk , individuals .
the healthy heart score , based on smoking status , bmi , physical activity , dietary intake , and alcohol consumption , could serve as an important tool for the longterm prevention of cvd , which is needed to achieve optimal populationwide cv health .
dr chiuve had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis . a userfriendly calculator to assess the 20year risk of cvd based on the healthy heart score is freely accessible at http://www.healthyheartscore.org .
the goal of the web application is to engage website visitors to learn their personal risk of cvd , understand the lifestyle habits that affect their risk of heart attack and stroke , and serve as a cue to action for behavioral change . upon completion ,
the patients will receive a summary that is color coded ( red , yellow , and green to represent high , medium , or low risk , respectively ) and purposely very visual to provide the patient with a quick snapshot of areas that need focused prevention .
the user also receives practical tips for maintaining or improving each lifestyle factor and simple explanations for why each factor is important for cvd health .
all data are saved as a pdf that can be printed and brought to their clinician 's office ( if completed at home ) or for the patient to take home ( if completed in the clinic ) . | backgroundclinical practice focuses on the primary prevention of cardiovascular ( cv ) disease ( cvd ) through the modification and pharmacological treatment of elevated risk factors .
prediction models based on established risk factors are available for use in the primary prevention setting . however , the prevention of risk factor development through healthy lifestyle behaviors , or primordial prevention , is of paramount importance to achieve optimal populationwide cv health and minimize longterm cvd risk.methods and resultswe developed a lifestylebased cvd prediction model among 61 025 women in the nurses health study and 34 478 men in the health professionals followup study , who were free of chronic disease in 1986 and followed for 24 years .
lifestyle factors were assessed by questionnaires in 1986 . in the derivation step
, we used the bayes information criterion to create parsimonious 20year risk prediction models among a random two thirds of participants in each cohort separately .
the scores were validated in the remaining one third of participants in each cohort . over 24 years , there were 3775 cases of cvd in women and 3506 cases in men .
the healthy heart score included age , smoking , body mass index , exercise , alcohol , and a composite diet score . in the validation cohort , the risk score demonstrated good discrimination ( harrell 's cindex , 0.72 ; 95% confidence interval [ ci ] , 0.71 , 0.74 [ women ] ; 0.77 ; 95% ci , 0.76 , 0.79 [ men ] ) , fit , and calibration , particularly among individuals without baseline hypertension or hypercholesterolemia.conclusionsthe healthy heart score accurately identifies individuals at elevated risk for cvd and may serve as an important clinical and public health screening tool for the primordial prevention of cvd . | Introduction
Methods
Study Participants and Endpoint Ascertainment
Ascertainment of CVD
Ascertainment of Lifestyle Factors
Statistical Analysis
Derivation of a Composite Diet Score
Derivation of the LifestyleBased CVD Risk Score
Validation of the Healthy Heart Score
Results
Derivation of the Composite Diet Score and the LifestyleBased CVD Risk Score
Assessment of Model Performance in the Validation Data Set
Practical Example of Risk Estimation
Discussion
Conclusion
Acknowledgments | thus , the prevention of cvd risk factor development , or primordial prevention , is of paramount importance to minimize an individual 's longterm cvd risk . we derived the healthy heart score , a lifestylebased risk score , among women enrolled in the nurses health study ( nhs ) , a prospective cohort of 121 700 female nurses ages 30 to 55 years at baseline in 1976 and men in the health professionals followup study ( hpfs ) , a cohort of 51 529 u.s . this analysis included 61 025 women and 34 478 men who provided a complete ascertainment of pertinent lifestyle factors on a questionnaire administered in 1986 and who were free of previously diagnosed cvd ( myocardial infarction [ mi ] , angina , stroke , transient ischemic attack , and coronary revascularization ) and cancer at baseline . for each cohort , we randomly assigned two thirds of the study participants to a derivation cohort ( n=40 680 women ; n=23 026 men ) , and the remaining one third of the participants were reserved as a validation cohort ( n=20 345 women ; n=11 452 men ) . we derived the healthy heart score , a lifestylebased risk score , among women enrolled in the nurses health study ( nhs ) , a prospective cohort of 121 700 female nurses ages 30 to 55 years at baseline in 1976 and men in the health professionals followup study ( hpfs ) , a cohort of 51 529 u.s . this analysis included 61 025 women and 34 478 men who provided a complete ascertainment of pertinent lifestyle factors on a questionnaire administered in 1986 and who were free of previously diagnosed cvd ( myocardial infarction [ mi ] , angina , stroke , transient ischemic attack , and coronary revascularization ) and cancer at baseline . for each cohort , we randomly assigned two thirds of the study participants to a derivation cohort ( n=40 680 women ; n=23 026 men ) , and the remaining one third of the participants were reserved as a validation cohort ( n=20 345 women ; n=11 452 men ) . further addition of selfreported htn and hypercholesterolemia or high cholesterol to the risk model significantly increased the cstatistic from 0.72 to 0.73 ( 95% confidence interval [ ci ] , 0.72 , 0.74 ; pdiff<0.001 ) in women and from 0.77 to 0.78 ( 95% ci , 0.76 , 0.79 ; pdiff=0.001 ) in men . formula to estimate the 20year risk of cvd based on healthy heart score derived in the full data set ( n=61 025 women and 34 478 men ) . compared to a 45yearold individual with very healthy lifestyle habits ( nonsmoker , bmi of 23 kg / m , 3.5 hours per week of moderatetovigorous physical activity , diet score of 5 points , and moderate alcohol intake [ 15 g / day in women and 30 g / day in men ] ) , the 20year risk of cvd for 45yearold individuals with unhealthy lifestyle habits ( current smoker , bmi of 35 kg / m , no physical activity , diet score of 0 points , and 0 g / day of alcohol ) was over 7fold higher in women and 6fold higher in men . further addition of selfreported htn and hypercholesterolemia or high cholesterol to the risk model significantly increased the cstatistic from 0.72 to 0.73 ( 95% confidence interval [ ci ] , 0.72 , 0.74 ; pdiff<0.001 ) in women and from 0.77 to 0.78 ( 95% ci , 0.76 , 0.79 ; pdiff=0.001 ) in men . compared to a 45yearold individual with very healthy lifestyle habits ( nonsmoker , bmi of 23 kg / m , 3.5 hours per week of moderatetovigorous physical activity , diet score of 5 points , and moderate alcohol intake [ 15 g / day in women and 30 g / day in men ] ) , the 20year risk of cvd for 45yearold individuals with unhealthy lifestyle habits ( current smoker , bmi of 35 kg / m , no physical activity , diet score of 0 points , and 0 g / day of alcohol ) was over 7fold higher in women and 6fold higher in men . in this study , we derived the healthy heart score , an algorithm based on modifiable lifestyle factors that effectively predicts risk of cvd among men and women 40 years of age , particularly among individuals without htn or hypercholesterolemia or high cholesterol . clinical practice focuses on the prevention of cvd through modification and pharmacological treatment of elevated clinical risk factors in an effort to prevent a cardiovascular event . the healthy heart score , based on smoking status , bmi , physical activity , dietary intake , and alcohol consumption , could serve as an important tool for the longterm prevention of cvd , which is needed to achieve optimal populationwide cv health . | [
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dismembered pyeloplasty for pelviureteric junction obstruction ( pujo ) has a high success rate . considering
that majority of patients will experience a favorable outcome following pyeloplasty , a large number of patients routinely undergo post - operative evaluation by which problems occurring in some patients can be detected .
usg and radionuclide renograms are commonly used to document success following pyeloplasty . while differential functions have been sufficiently evaluated
, a number of studies have shown that the function and curve pattern do not always improve on renograms after pyeloplasty .
ultrasound is a useful , non - invasive tool to assess hydronephrosis , but only few studies have evaluated its role after pyeloplasty . besides the inability to quantify the functional status of the kidney , the use of ultrasound as a marker of success of pyeloplasty is limited due to the persistence and , at times , worsening of dilatation of pelvicalyceal system after pyeloplasty . to overcome these drawbacks ,
previous investigators have proposed using ultrasound to calculate pelvis / cortex ratio and percent improvement in anteroposterior diameter ( apd ) to predict the success of pyeloplasty .
but these have not gained widespread acceptance and the technique concerned has not been standardized . in this study
, we used a standardized ultrasound technique to evaluate the apd of the renal pelvis in patients underwent pyeloplasty and compared the measurements between supine and prone positions with the aim to determine whether the difference between the two helps to exclude obstruction in dilated renal system post pyeloplasty and predict the success of pyeloplasty .
the study was carried out in two different sites with a standardized technique that has been described subsequently .
pediatric patients ( age 0 - 15 years ) who had undergone pyeloplasty ( either in the same institute or other institutes ) were consecutively recruited to the study from january 2012 to december 2013 .
these subjects attended the institute either for follow - up following pyeloplasty or for other non - urological problems .
as there was no institutional review board , consents were obtained from the parents and management of the hospital .
those patients who did not have a pre - pyeloplasty renogram were excluded from the study .
the second institution is a tertiary referral center for pediatric urology in a developed country .
all subjects ( age 0 - 15 years ) underwent pre - operative assessment and pyeloplasty in the same institute , and were being regularly followed up after the surgery .
as there is no institutional review board in the first institute , consent of the management of the hospital was taken while in the second institute approval from the head of department of ultrasonography was taken .
ultrasound examination was performed by a single operator ( vyfl , sonographer > 20 years of experience and as , radiologist > 7 years of experience ) in each tertiary care center , respectively using the prosound alpha 6 machine from hitachi - aloka medical america , inc .
the imaging procedure was standardized as follows : all children were instructed to have adequate fluid intake prior to the study as the measurement of apd could be variably affected by hydration status of the subject , particularly in children . just before the ultrasound scanning
, all subjects were instructed to void ( in infants , we ensured an almost empty bladder was visualized before scanning started ) as a full bladder could impair drainage of urine from the kidneys into the bladder and affect the apd measurement .
apd was first measured in the transverse plane at the level of renal hilum with the patient in supine position and with the ultrasound probe placed laterally in mid - axillary line .
appropriate magnification was done for correct measurements . during this examination , the presence or absence of calyceal dilatation
the percentage change of apd was calculated with the following formula : % change in apd = ( average apd in supine position - average apd in prone position)/average apd in supine position 100 .
a % change of greater than + 10% was taken to be an indicator of non - obstructed collecting system .
the reproducibility and degree of agreement of supine and prone apd measurements of 10 subjects were compared between the two operators [ vyfl , wcwc ( radiolog ist with > 20 years experience ) ] in the second institute . to determine the intra - observer error ,
three measurements were obtained for apd in both supine and prone positions by each operator . to determine inter - observer error ,
all patients included in this study had also undergone a pre - operative and at least one post operative diuretic radionuclide renogram ( symbia e gamma camera , siemens ; usa ) using either ec[ethylene dicysteine ] or mag3 [ mercapto acetyl triglycine ] . the difference in differential renal functions before and after pyeloplasty was evaluated . in the second institute
, reference was also made for patients who had more than one post - op pyeloplasty renograms performed .
if there was no interval deterioration ( in which deterioration was defined as an interval drop of > 5% differential function ) or there was improvement in differential renal function post pyeloplasty , and the patient was asymptomatic , the pyeloplasty was regarded as successful .
the primary hypothesis of the study was to see if a decrease in apd in prone position was indicative of successful pyeloplasty .
the secondary objective was to see if those who did not show a decrease in apd in prone position had deterioration of renal function by > 5% suggesting persistent obstruction requiring a secondary pyeloplasty .
the results were tested using wilcoxon signed rank sum test for difference in apd between supine and prone positions . the relationship between change in apd and success of pyeloplasty as indicated by the results of diuretic radionuclide renogram
both statistical tests were performed at 5% level of significance using the statistical analysis software spss version 19 [ ibm corp . released 2010 .
the study was carried out in two different sites with a standardized technique that has been described subsequently .
pediatric patients ( age 0 - 15 years ) who had undergone pyeloplasty ( either in the same institute or other institutes ) were consecutively recruited to the study from january 2012 to december 2013 .
these subjects attended the institute either for follow - up following pyeloplasty or for other non - urological problems .
as there was no institutional review board , consents were obtained from the parents and management of the hospital .
those patients who did not have a pre - pyeloplasty renogram were excluded from the study .
the second institution is a tertiary referral center for pediatric urology in a developed country .
all subjects ( age 0 - 15 years ) underwent pre - operative assessment and pyeloplasty in the same institute , and were being regularly followed up after the surgery .
as there is no institutional review board in the first institute , consent of the management of the hospital was taken while in the second institute approval from the head of department of ultrasonography was taken .
ultrasound examination was performed by a single operator ( vyfl , sonographer > 20 years of experience and as , radiologist > 7 years of experience ) in each tertiary care center , respectively using the prosound alpha 6 machine from hitachi - aloka medical america , inc .
the imaging procedure was standardized as follows : all children were instructed to have adequate fluid intake prior to the study as the measurement of apd could be variably affected by hydration status of the subject , particularly in children . just before the ultrasound scanning
, all subjects were instructed to void ( in infants , we ensured an almost empty bladder was visualized before scanning started ) as a full bladder could impair drainage of urine from the kidneys into the bladder and affect the apd measurement .
apd was first measured in the transverse plane at the level of renal hilum with the patient in supine position and with the ultrasound probe placed laterally in mid - axillary line .
appropriate magnification was done for correct measurements . during this examination , the presence or absence of calyceal dilatation
at the end of each examination , the percentage change of apd was calculated with the following formula : % change in apd = ( average apd in supine position - average apd in prone position)/average apd in supine position 100 .
a % change of greater than + 10% was taken to be an indicator of non - obstructed collecting system .
the reproducibility and degree of agreement of supine and prone apd measurements of 10 subjects were compared between the two operators [ vyfl , wcwc ( radiolog ist with > 20 years experience ) ] in the second institute . to determine the intra - observer error ,
three measurements were obtained for apd in both supine and prone positions by each operator .
to determine inter - observer error , the mean values from all apd measurements were compared between the two operators .
all patients included in this study had also undergone a pre - operative and at least one post operative diuretic radionuclide renogram ( symbia e gamma camera , siemens ; usa ) using either ec[ethylene dicysteine ] or mag3 [ mercapto acetyl triglycine ] .
the difference in differential renal functions before and after pyeloplasty was evaluated . in the second institute ,
reference was also made for patients who had more than one post - op pyeloplasty renograms performed .
if there was no interval deterioration ( in which deterioration was defined as an interval drop of > 5% differential function ) or there was improvement in differential renal function post pyeloplasty , and the patient was asymptomatic , the pyeloplasty was regarded as successful .
the primary hypothesis of the study was to see if a decrease in apd in prone position was indicative of successful pyeloplasty .
the secondary objective was to see if those who did not show a decrease in apd in prone position had deterioration of renal function by > 5% suggesting persistent obstruction requiring a secondary pyeloplasty .
the results were tested using wilcoxon signed rank sum test for difference in apd between supine and prone positions . the relationship between change in apd and success of pyeloplasty as indicated by the results of diuretic radionuclide renogram
were performed at 5% level of significance using the statistical analysis software spss version 19 [ ibm corp .
in the first institution , out of 48 patients , 12 did not have a pre - pyeloplasty renogram and hence were excluded from the study . in the second institution , seven patients with complete record were recruited . in the total cohort of 43 patients
included ( 31 males and 12 females ; age from 5 months to 18 years ) , 33 had undergone left - sided and 9 had undergone right - sided dismembered pyeloplasty .
the intraclass correlation [ icc ] for the single operator was 0.986 ( 95% ci 0.978 - 0.991 ) .
there was also very good reproducibility between the two operators ( icc = 0.85 , 95% ci = 0.70 - 0.94 ) .
there was no hydronephrosis seen in one patient and this patient did not show any deterioration of renal function in the post - pyeloplasty renogram [ no . 18 in table 1 ] .
there were 36 patients who had a decrease of apd by > 10% in prone position [ figures 13 ] .
they had either improvement or no deterioration of renal function in their post - pyeloplasty renograms as compared to their pre - pyeloplasty renograms [ table 1 , figure 1 ] .
this association was statistically significant ( p < 0.005 by chi - square test ) .
this confirmed the primary hypothesis of the study that a decrease in apd by > 10% in prone position suggests a successful pyeloplasty .
demographic data , ultrasound findings , and differential renal function in children who has reduced apd from supine to prone position ( negative value at % change in apd ) ( a ) post - pyeloplasty ultrasound showing apd of left kidney in supine 23 mm and in prone position 12 mm ( b ) pre - pyeloplasty renogram showing left kidney having 23% differential function ( c ) post - pyeloplasty renogram showing 33.3% of left kidney improved renal function suggesting a successful pyeloplasty post pyeloplasty ultrasound showing apd of 1.42 cm in supine and 1.18 cm in prone position post pyeloplasty ultrasound showing apd of 14 mm in supine and 10 mm in prone position there were five patients who had an increase in apd in prone position [ figure 4 ] , and in one patient , there was no change in apd in prone position [ table 2 ] .
three of these patients had deterioration of function of the operated kidney , as compared to the pre - pyeloplasty renograms [ figure 4 ] .
this was taken as indicative of secondary pujo and these subjects were advised a second pyeloplasty .
( a ) post - pyeloplasty ultrasound showing apd of left kidney in supine 44 mm and in prone position 46 mm ( b ) pre - pyeloplasty renogram showing 46% differential function of left kidney ( c ) post - pyeloplasty renogram showing 24.9% differential function of left kidney , indicating obstruction demographic data , ultrasound findings , and differential renal function in children who have either no change or increased apd from supine to prone position ( zero or positive value at % change in apd ) in three subjects who showed increase in apd ( range from 8.8 to 27.4% ) in prone position , the differential renal function on mag3 renogram was either static or improved .
review of dynamic images of the radionuclide renogram showed sluggish drainage from the dilated pelvis into the ureter but no definite accumulation of tracer with time .
all three subjects underwent another follow - up renogram within the next 6 months which confirmed static differential renal function .
thus , the secondary objective , that is , an absence of decrease in apd in prone position / increase in apd in prone position indicates a failed pyeloplasty , could not be ascertained as statistically significant .
the success of pyeloplasty is conventionally evaluated with ultrasound by demonstrating a decrease in hydronephrosis and with radionuclide renograms by showing improvement in function and/or drainage pattern .
diuretic renography has been regarded as the gold standard to document surgical success of pyeloplasty , but is invasive , expensive , and is associated with the risk of radiation . at times , despite pyeloplasty , the renal function and drainage pattern show no improvement on radionuclide renogram , leading to a diagnostic dilemma for the clinician and anxiety among patients and parents .
furthermore , residual hydronephrosis ( even though to a lesser degree compared with the pre - op status ) is common after pyeloplasty . patients who undergo pyeloplasty later in life also have persistence of hydronephrosis and poor radiological improvement due to the decreased resilience of the renal pelvis .
patients are , therefore , advised to have repeated assessment by ultrasound and/or diuretic renogram .
studies have shown that repeated nuclear studies are not necessary as it is uncommon for patients to have significant fluctuations in split function . also , given the > 95% success rate of pyeloplasty , the large number of renograms currently performed in daily practice are not cost - effective for identifying just a small portion of patients who might be benefitted ; therefore , a more selective approach is advocated .
ultrasound alone has been proposed for the initial assessment after pyeloplasty , with renogram reserved for those patients who do not show improvement on ultrasonography . though ultrasound is easily accessible and non - invasive , it is , by nature , operator dependent .
the status of pre - ultrasound hydration and the amount of urine in the bladder can affect the degree of hydronephrosis .
amling et al . found that hydronephrosis could remain the same or worsen in the first several months following pyeloplasty even in those kidneys which show good response to surgery ultimately .
kis et al . found slow improvement in the pelvic dilatation in the first postoperative year .
park et al . found faster improvement in grade iii hydronephrosis as compared to grade iv hydronephrosis , but even when all grades of hydronephrosis were considered , only 56% of patients achieved normalization .
found that majority of cases took more than 6 months to improve on ultrasound . to the best of our knowledge , currently there is no international recommended standardized protocol to guide ultrasound measurement for determining the severity of hydronephrosis .
investigators have tried to determine the success of pyeloplasty on ultrasound by using various parameters such as calyx / parenchyma ratio , parenchyma / pelvicalyceal area ratio , pelvis / cortex ratio , and percent improvement in apd .
very few studies have evaluated the usefulness of resistive index and resistive index ratio , as determined by doppler evaluation of the kidneys , in patients who have undergone pyeloplasty .
though these parameters correlate well with the results of diuretic renography , they can not differentiate a non obstructed collecting system from pujo , especially if there has been severe damage initially .
there is usually a dilemma of differentiating a dilated but non - obstructive system from a dilated and obstructed system in post - pyeloplasty patients .
the above scenario is particularly complicated in situations where patients usually come to seek medical advice with no previous records .
some patients do have regular follow - up , but radionuclide renograms might not be available .
ultrasound is commonly used as an investigatory tool for any abdominal problem . if hydronephrosis is found , it is not easy to distinguish whether it is due to residual hydronephrosis or secondary pujo .
we sought to look for a solution to this dilemma by comparing the renal pelvis apd in supine and prone positions .
the rationale of the design of the study is based on the fact that the renal pelvis and collecting system drains better in prone position . in the prone position ,
the proximal end of ureter is placed more anteriorly than the upper pole of the kidney .
the non - obstructed dilated pelvis would drain better in prone position and would have an apd less than that in the supine position . on the other hand , the obstructed systems would not drain better in prone position , and hence , the apd of these systems , in prone position , would be more or equal to the apd in supine position . the same rationale has been employed and proved to be valid by the authors in evaluation of prenatally detected puj obstruction . in the previous study , children who had a decrease in apd in prone position by > 10% as compared with supine position showed resolution or no deterioration of hydronephrosis and did not require pyeloplasty , while those who showed no decrease in apd in prone position had worsening of hydronephrosis and needed pyeloplasty .
based on this , we took the 10% cut - off value as indicative of successful pyeloplasty in the current study .
we found that all patients who had a decrease in the apd in prone position by > 10% as compared to supine position had better renal function after pyeloplasty .
therefore , we propose that a decrease of apd > 10% in prone position is indicative of a successful pyeloplasty and no further radionuclide renogram is required .
this finding of absence of calyceal dilatation in cases that have undergone a successful pyeloplasty is in accordance with the findings of other investigators who reported that calyceal dilatation resolves early and pelvic dilatation persists longer .
recently , lantz et al . have shown that prone position for renograms facilitates drainage and is a more accurate representation of postoperative outcome after pyeloplasty .
of the six patients who did not meet the above criteria , three ( 50% ) were confirmed to have secondary pujo with deteriorating renal function which required a second surgery . in the other three subjects ( 50% ) ,
hence , we propose that those who fail to achieve a decrease of apd > 10% in prone position should be under close follow - up with ultrasonography . a further diuretic renogram is recommended to differentiate secondary obstruction from sluggish drainage pattern .
interestingly , all cases with deteriorating renal function [ cases 1 - 3 in table 2 ] were found to have co - existing calyceal dilatation , while those without deteriorating renal function [ cases 4 - 6 in table 2 ] did not show calyceal dilatation .
thus , in addition to increased apd in prone position , calyceal dilatation might be a useful sign with higher predictive value to identify dilated system with obstruction .
the above statement / suggestion , however , needs to be validated with a larger sample size .
the timing of ultrasound postoperatively was variable , ranging from 3 months to 15 years after pyeloplasty .
the operative details of many patients , with regard to whether they had undergone a reduction of pelvis or only a simple excision of the pelvi - ureteric junction , were not available .
however , these limitations are a regular scenario in many developing countries where radionuclide studies are not easily available , patients are lost to follow - up , many get diagnosed late in life , and a single - center follow - up is uncommon due to variable health care practices .
a patient who has undergone pyeloplasty in the past comes to seek medical advice for a non - renal pathology .
an ultrasound examination is performed which detects hydronephrosis and the sonologist faces the dilemma of differentiating a dilated but non - obstructed system from a dilated and obstructed system .
this dilemma is further compounded when the clinician gets a renogram done , but the differential renal function and the drainage curves can not answer the question of differentiating a dilated non - obstructed system from an obstructed system , especially in the absence of pre pyeloplasty renograms .
it is in these scenarios that a simple bedside ultrasound evaluation and measurement of apd of renal pelvis in supine and prone positions can be useful .
the aforementioned limitations were partially overcome by a collaborative study carried out in the second institution , which is a tertiary referral center for pediatric urology . in this latter institution ,
all subjects had pre - op ultrasound and diuretic renogram , as well as regular imaging follow - up post pyeloplasty , if there was persistent hydronephrosis .
the surgical notes were also available and all subjects underwent standardized procedure of pyeloplasty by the same team of surgeons .
firstly , the ultrasound examination was validated by clearly laid out protocol including patient preparation and scanning technique .
intra - observer and inter - observer variability was measured and proved that ultrasound technique was reliable and reproducible . secondly , serial follow - up was available for patients with discrepancies in the first supine - prone ultrasound and diuretic renogram , which enhances a better understanding about the interpretation of the ultrasound findings .
a proposed algorithm for investigating children with previous pujo and treated with pyeloplasty is given in flow chart 1 .
we propose to use a simple ultrasound technique to measure the apd of renal pelvis in both supine and prone positions to differentiate a dilated non - obstructed system from a possibly obstructed system in children after pyeloplasty .
a decrease in apd in prone position by > 10% as compared to supine position indicates a successful pyeloplasty , thus obviating the need for any further investigation with diuretic renogram . in those patients who do not show a decrease in apd in prone position or even an increase in apd in prone position , diuretic radionuclide study is recommended .
if our proposal is validated by larger - scale study , ultrasound may become a reliable diagnostic tool and lead to a new guideline for imaging children after pyeloplasty .
this new protocol , hopefully , will help to alleviate parents anxiety , saving time and money for further investigation , and reduce the radiation exposure in this group of children by limiting redundant radionuclide studies . | objective : to evaluate patients who had undergone pyeloplasty for pelviureteric junction obstruction , by measuring the anteroposterior diameter ( apd ) of the renal pelvis in supine and prone positions , and determine whether a decrease in apd in prone position can exclude obstruction in dilated renal system.materials and methods : from january 2012 to december 2013 , patients who had undergone pyeloplasty were evaluated by ultrasound in two centers .
the difference of apd of the renal pelvis in supine and prone positions was obtained .
correlation was made with the pre- and post - pyeloplasty renal function by radionuclide renogram.results:there were 42 patients ( 31 males , 11 females ; age range 5 months to 18 years ) .
residual hydronephrosis was detected in 41 patients of whom 35 patients ( 85% ) showed decrease in apd by > 10% in prone position .
these patients and the one without hydronephrosis showed either no deterioration or improvement in renal function .
six patients ( 15% ) showed either no change or increase in apd in prone position .
three patients ( 7.5% ) were confirmed to have decrease in renal function indicating obstruction .
three patients ( 7.5% ) showed no deterioration of renal function , but sluggish drainage on radionuclide renogram.conclusion:demonstration of decreased apd of renal pelvis in prone position by ultrasound is useful to differentiate obstructed from non - obstructed dilated renal system , and it correctly identified 85% candidates with successful pyeloplasty . in patients with no decrease or increase in apd at prone position , further follow - up
is recommended to rule out obstruction . | Introduction
Materials and Methods
Patient selection
Technique of ultrasonography
Analysis of change in APD
Nuclear studies
Outcomes
Statistical analysis
Results
Discussion
Conclusion | in this study
, we used a standardized ultrasound technique to evaluate the apd of the renal pelvis in patients underwent pyeloplasty and compared the measurements between supine and prone positions with the aim to determine whether the difference between the two helps to exclude obstruction in dilated renal system post pyeloplasty and predict the success of pyeloplasty . pediatric patients ( age 0 - 15 years ) who had undergone pyeloplasty ( either in the same institute or other institutes ) were consecutively recruited to the study from january 2012 to december 2013 . the secondary objective was to see if those who did not show a decrease in apd in prone position had deterioration of renal function by > 5% suggesting persistent obstruction requiring a secondary pyeloplasty . pediatric patients ( age 0 - 15 years ) who had undergone pyeloplasty ( either in the same institute or other institutes ) were consecutively recruited to the study from january 2012 to december 2013 . the primary hypothesis of the study was to see if a decrease in apd in prone position was indicative of successful pyeloplasty . the secondary objective was to see if those who did not show a decrease in apd in prone position had deterioration of renal function by > 5% suggesting persistent obstruction requiring a secondary pyeloplasty . in the total cohort of 43 patients
included ( 31 males and 12 females ; age from 5 months to 18 years ) , 33 had undergone left - sided and 9 had undergone right - sided dismembered pyeloplasty . there were 36 patients who had a decrease of apd by > 10% in prone position [ figures 13 ] . this confirmed the primary hypothesis of the study that a decrease in apd by > 10% in prone position suggests a successful pyeloplasty . demographic data , ultrasound findings , and differential renal function in children who has reduced apd from supine to prone position ( negative value at % change in apd ) ( a ) post - pyeloplasty ultrasound showing apd of left kidney in supine 23 mm and in prone position 12 mm ( b ) pre - pyeloplasty renogram showing left kidney having 23% differential function ( c ) post - pyeloplasty renogram showing 33.3% of left kidney improved renal function suggesting a successful pyeloplasty post pyeloplasty ultrasound showing apd of 1.42 cm in supine and 1.18 cm in prone position post pyeloplasty ultrasound showing apd of 14 mm in supine and 10 mm in prone position there were five patients who had an increase in apd in prone position [ figure 4 ] , and in one patient , there was no change in apd in prone position [ table 2 ] . ( a ) post - pyeloplasty ultrasound showing apd of left kidney in supine 44 mm and in prone position 46 mm ( b ) pre - pyeloplasty renogram showing 46% differential function of left kidney ( c ) post - pyeloplasty renogram showing 24.9% differential function of left kidney , indicating obstruction demographic data , ultrasound findings , and differential renal function in children who have either no change or increased apd from supine to prone position ( zero or positive value at % change in apd ) in three subjects who showed increase in apd ( range from 8.8 to 27.4% ) in prone position , the differential renal function on mag3 renogram was either static or improved . in the previous study , children who had a decrease in apd in prone position by > 10% as compared with supine position showed resolution or no deterioration of hydronephrosis and did not require pyeloplasty , while those who showed no decrease in apd in prone position had worsening of hydronephrosis and needed pyeloplasty . we found that all patients who had a decrease in the apd in prone position by > 10% as compared to supine position had better renal function after pyeloplasty . of the six patients who did not meet the above criteria , three ( 50% ) were confirmed to have secondary pujo with deteriorating renal function which required a second surgery . in the other three subjects ( 50% ) ,
hence , we propose that those who fail to achieve a decrease of apd > 10% in prone position should be under close follow - up with ultrasonography . it is in these scenarios that a simple bedside ultrasound evaluation and measurement of apd of renal pelvis in supine and prone positions can be useful . we propose to use a simple ultrasound technique to measure the apd of renal pelvis in both supine and prone positions to differentiate a dilated non - obstructed system from a possibly obstructed system in children after pyeloplasty . a decrease in apd in prone position by > 10% as compared to supine position indicates a successful pyeloplasty , thus obviating the need for any further investigation with diuretic renogram . in those patients who do not show a decrease in apd in prone position or even an increase in apd in prone position , diuretic radionuclide study is recommended . | [
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patients undergoing cardiopulmonary bypass ( cpb ) surgery frequently develop systematic inflammatory response syndrome , ranging from mild to severe complications such as pericardial , pleural and/or abdominal effusion , liver enlargement and edema .
these complications are characterized by increased capillary permeability , a shift of fluid and protein from the intravascular to the interstitial space and may further progress into hypovolemia , massive generalized edema , acute respiratory distress syndrome , or even capillary leak syndrome ( cls ) or multiple organ dysfunction ( mod ) or failure , with a substantial morbidity and mortality . although the incidence of postoperative effusion in children is substantial ( > 25% )
nearly 97,000 ( germany 1998 ) and 800,000 ( usa 1996 , american heart association , ) patients undergo cpb surgery annually ( ~10% for congenital heart disease ) , hence postoperative complications constitute a significant clinical problem . the extensive contact between heparin anticoagulated blood and foreign surfaces of the extracorporal circuit during cpb , in combination with anesthetics and other medication used during and after surgery stimulates the immune system .
tumor necrosis factor- ( tnf- ) , interleukin ( il)-6 and il-8 ( proinflammatory cytokines ) may contribute to myocardial dysfunction and increased apoptosis and increased neutrophil activation , and il-10 may contribute to immune depression and increased susceptibility to infection .
there is some evidence that patients who later develop postoperative complications may be identified in the early perioperative or even in the preoperative period .
several scoring systems use clinical and/or laboratory data acquired during or after therapy to predict cardiac patients outcome with informative serum parameters like soluble endothelial ( se)-selectin for restenosis or perioperative c - reactive protein ( crp ) , lactate , il-6 or altered blood coagulation after open heart surgery .
the prediction of patients at risk for postoperative complications is important for the individual preoperative prophylactic treatment .
preoperative prediction is based on the hypothesis that the primed immune system amplifies the immune response to cardiosurgical trauma ; for example , tnf- or fibronectin primed neutrophils respond more strongly to stimulation in vitro . priming in the patients may be caused by an allergic / atopic predisposition but can also be a result of fresh or reactivated viral infection .
a recent study in this journal indicates gender as a predisposing factor for mod in children . in a recent study we showed that children who suffered from postoperative effusions and edema ( poee ) are , 24 hours before surgery , already exhibiting altered antigen expression on leukocytes , by which risk assessment would be possible using discriminant analysis .
based on these results we hypothesized that children at risk of poee have an altered preoperative level of markers of immunoactivation , allergic / atopic predisposition or t - helper type 2 ( th2 ) phenotype , which may be used as predictors for risk assessment .
in addition , we also included readily available standard laboratory parameters in order to test predictive strength .
the advantage of a serological classifier over that based on antigen expression data by flow cytometry is that these data and methods are accessible for virtually all clinical facilities and are easily standardized . in the present study
we show that children at risk of poee are already predisposed to the condition and can be predicted from these data .
this prospective non - randomized study was conducted between november 1995 and may 2001 following approval by the ethical committee of the medical faculty at the university of leipzig , germany .
a total of 75 patients who underwent cardiac surgery with cpb were analyzed [ inclusion criteria : aged 318 years , body weight > 12 kg ; exclusion criteria : missing informed consent of parents , palliative cardiac surgery ( e.g. if single ventricle circulation was the aim of surgery , ( glenn , fontan or total cavopulmonary connection [ tcpc ] ) .
the surgical procedures included were : closure of atrial septal defect ( n = 39 ) or ventricular septal defect ( n = 11 ) ; replacement of pulmonary valve by an allogeneic heart valve ( n = 18 ) ; resection of an aortic subvalvular stenosis resulting from a subaortic membrane or fibrous cap ( n = 6 ) ; correction of tetralogy of fallot ( n = 1 ) .
all children received similar anesthesia , medication and intraoperative and postoperative care and cpb as detailed elsewhere .
after delivery to the intensive care unit postoperatively , the incidence of pericardial- , pleural- and/or abdominal - effusion was monitored by echocardiography , chest x - ray or sonogra - phy . if patients developed detectable effusions after removal of the thoracic drainage ( which was usually one day after surgery ) until discharge they were allocated into the poee group ( n = 29 ) , or into the non - poee group ( no effusion , n = 46 ) .
as evaluated visually , all poee patients had edema of the face and/or hands and/or feet .
incidence of edema was not used for poee discrimination because quantitative measures of extravascular body fluid volume ( such as scintigraphy following labelling of the extravascular fluid by radiolabelled sulphide or bromide ) were ethically not feasible in children .
massive generalized edema , cls or mod as defined by seghaye et al . was not observed in any of the patients .
however , 65% of the poee patients fulfilled at least one mod criterion as defined by trotter et al . .
postpericar - diotomy syndrome with effusions and fever of non - infectious origin within a week , or later , of surgery was not present in any of the patients .
blood was obtained one day ( median : 20 hours ) before surgery in untreated tubes as well as in ethylenediamine - tetracetic acid ( edta ) and heparin tubes , centrifuged at 2800 g for 10 min at 4c and the supernatant was collected .
urine was sampled in untreated tubes . within 1 hour after collection , serum , edta - plasma and urine samples were stored in aliquots at -80c .
the concentration of the complement components ( c3 , c4 , c5 , c1-inhibitor , c3d ) and immunoglobulin ( ig)g2 was determined by radial immune diffusion ( the binding site , heidelberg , germany ) with serum or edta - plasma ( c3d ) and total hemolytic complement ch100 by lysis of antibody - coated sheep erythrocytes ( the binding site ) .
all other parameters were quantified using enzyme - linked immunosorbent assay [ ige , interleukin ( il)-1 , tnf- , interferon- , rantes , histamine : beckman - coulter , krefeld , germany ; il-4 , il-10 , il-13 , soluble intracellular adhesion molecule-1 ( sicam-1 ) , platelet endothelial cell adhesion molecule-1 ( pecam ) : bender medsystems , vienna , austria ; il-5 , il-6 high sensitivity , il-10 high sensitivity , il-12 p40/p70 , soluble leukocytic ( sl)-selectin , se - selectin : r&d systems gmbh , wiesbaden , germany ; il-2 , il-2-receptor , serum and urine neopterin : dpc biermann gmbh , bad nauheim , germany ; il-4 high sensitivity , il-11 : natutec , frankfurt , germany ; il-12 p70 , il-13 : biozol diagnostica ver - trieb gmbh , eching , germany ; c5a : behringwerke ag , marburg , germany ] . the complement fragment ratios c3d
/ c3 , c5a / c5 and immunoglobulin ratio ige / igg2 , were calculated as measures for complement activation and th2/th1 imbalance , respectively .
additionally , routine laboratory and clinical chemistry parameters were determined ( cell count , differential blood count , crp , creatinine , electrolytes , protein , hematocrit , blood coagulation parameters ) .
in total 56 parameters were analyzed per patient including age , gender and body weight .
data are displayed as mean standard deviation ( sd ) . between - group comparison
was undertaken by unpaired student 's t - test or mann - whitney u - test as appropriate [ statistical program for social sciences version 8.0 ( spss ) , knowledge dynamics , canyon lake , tx ] .
discrimination of patients into the poee and control group was tested by data pattern analysis using two different methods as detailed .
this classifier was optimized by increasing the f - probability followed by determination of the unstandardized canonical discriminant function .
, the triple matrix data pattern analyzer classif1 was used as an algorithmic data mining approach . with classif1 no replacement of missing data values and no mathematical assumptions on parameter distributions are required .
this prospective non - randomized study was conducted between november 1995 and may 2001 following approval by the ethical committee of the medical faculty at the university of leipzig , germany .
a total of 75 patients who underwent cardiac surgery with cpb were analyzed [ inclusion criteria : aged 318 years , body weight > 12 kg ; exclusion criteria : missing informed consent of parents , palliative cardiac surgery ( e.g. if single ventricle circulation was the aim of surgery , ( glenn , fontan or total cavopulmonary connection [ tcpc ] ) .
the surgical procedures included were : closure of atrial septal defect ( n = 39 ) or ventricular septal defect ( n = 11 ) ; replacement of pulmonary valve by an allogeneic heart valve ( n = 18 ) ; resection of an aortic subvalvular stenosis resulting from a subaortic membrane or fibrous cap ( n = 6 ) ; correction of tetralogy of fallot ( n = 1 ) .
all children received similar anesthesia , medication and intraoperative and postoperative care and cpb as detailed elsewhere .
after delivery to the intensive care unit postoperatively , the incidence of pericardial- , pleural- and/or abdominal - effusion was monitored by echocardiography , chest x - ray or sonogra - phy . if patients developed detectable effusions after removal of the thoracic drainage ( which was usually one day after surgery ) until discharge they were allocated into the poee group ( n = 29 ) , or into the non - poee group ( no effusion , n = 46 ) .
as evaluated visually , all poee patients had edema of the face and/or hands and/or feet .
incidence of edema was not used for poee discrimination because quantitative measures of extravascular body fluid volume ( such as scintigraphy following labelling of the extravascular fluid by radiolabelled sulphide or bromide ) were ethically not feasible in children .
massive generalized edema , cls or mod as defined by seghaye et al . was not observed in any of the patients .
however , 65% of the poee patients fulfilled at least one mod criterion as defined by trotter et al . .
postpericar - diotomy syndrome with effusions and fever of non - infectious origin within a week , or later , of surgery was not present in any of the patients .
blood was obtained one day ( median : 20 hours ) before surgery in untreated tubes as well as in ethylenediamine - tetracetic acid ( edta ) and heparin tubes , centrifuged at 2800 g for 10 min at 4c and the supernatant was collected .
urine was sampled in untreated tubes . within 1 hour after collection , serum , edta - plasma and urine samples were stored in aliquots at -80c .
the concentration of the complement components ( c3 , c4 , c5 , c1-inhibitor , c3d ) and immunoglobulin ( ig)g2 was determined by radial immune diffusion ( the binding site , heidelberg , germany ) with serum or edta - plasma ( c3d ) and total hemolytic complement ch100 by lysis of antibody - coated sheep erythrocytes ( the binding site ) .
all other parameters were quantified using enzyme - linked immunosorbent assay [ ige , interleukin ( il)-1 , tnf- , interferon- , rantes , histamine : beckman - coulter , krefeld , germany ; il-4 , il-10 , il-13 , soluble intracellular adhesion molecule-1 ( sicam-1 ) , platelet endothelial cell adhesion molecule-1 ( pecam ) : bender medsystems , vienna , austria ; il-5 , il-6 high sensitivity , il-10 high sensitivity , il-12 p40/p70 , soluble leukocytic ( sl)-selectin , se - selectin : r&d systems gmbh , wiesbaden , germany ; il-2 , il-2-receptor , serum and urine neopterin : dpc biermann gmbh , bad nauheim , germany ; il-4 high sensitivity , il-11 : natutec , frankfurt , germany ; il-12 p70 , il-13 : biozol diagnostica ver - trieb gmbh , eching , germany ; c5a : behringwerke ag , marburg , germany ] . the complement fragment ratios c3d
/ c3 , c5a / c5 and immunoglobulin ratio ige / igg2 , were calculated as measures for complement activation and th2/th1 imbalance , respectively .
additionally , routine laboratory and clinical chemistry parameters were determined ( cell count , differential blood count , crp , creatinine , electrolytes , protein , hematocrit , blood coagulation parameters ) .
in total 56 parameters were analyzed per patient including age , gender and body weight .
data are displayed as mean standard deviation ( sd ) . between - group comparison
was undertaken by unpaired student 's t - test or mann - whitney u - test as appropriate [ statistical program for social sciences version 8.0 ( spss ) , knowledge dynamics , canyon lake , tx ] .
discrimination of patients into the poee and control group was tested by data pattern analysis using two different methods as detailed .
this classifier was optimized by increasing the f - probability followed by determination of the unstandardized canonical discriminant function .
the triple matrix data pattern analyzer classif1 was used as an algorithmic data mining approach . with classif1 no replacement of missing data values and no mathematical assumptions on parameter distributions
clinical data are comparable in the control group and among those patients at risk for poee . data on patients and surgical parameters were grouped according to the clinical outcome in non - poee and poee groups ( table 1 ) .
patients with poee were of similar age and gender , while duration of surgery + anesthesia and extracorporal circulation were longer .
other parameters , including priming and infusion volume , duration of hypothermia and hemofiltration volume ( i.e. volume of fluid that has been removed from the blood to accomplish normal hematocrit values at the end of surgery ) were not significantly different ( not shown ) .
poee patients had a higher body weight ( table 2 ) and stayed in hospital one day longer after surgery .
clinical and surgical data of poee and non - poee patients ( means sd ) chi - squared test , ns = not significant , two - tailed student 's t - test , mann - whitney u - test .
cpb , cardiopulmonary bypass ; icu , intensive care unit ; poee , postoperative effusions and edema .
patients at risk of poee exhibited signs of inflammation . children with poee had preoperatively significantly higher levels of several complement components , tnf- , neutrophilic granulocyte count and percentage ( table 2 ) .
these data indicate increased immune activation / alteration of at risk patients . at risk patients
the use of single parameters for individual risk assessment is insufficient , as most data for the poee patients ( > 75% ) showed significant overlap with non - poee patients .
the highest discrimination by a single parameter was obtained withc3 ( specificity : 55% ; sensitivity : 67% ) .
on multivariate analysis , however , the majority of patients from both groups were correctly classified irrespective of the classification program applied ( spss / classif1 ; specificity : 80.4%/97.8% ; sensitivity : 86.2%/72.4% ; and negative : 90.2%/84.9% ; and positive : 73.5%/91.3% predictive values ) ( table 3 ) . only nine of the 56 parameters were required for these classifications ( table 4 ) .
five parameters were unique to each classifier , while increased c5 and sl - selectin serum concentration , increased neutrophil percentage or count and elevated hematocrit were selected by both classification methods as discriminant factors .
misclassifications were not assigned to a certain type of cardiac defect ( chi - squared test ; see also table 3 , classification of subgroups ) , indicating that poee prediction is independent of the surgery performed .
this interpretation is also supported by the result that atrial septal defect patients and the patients who underwent other types of surgeries were both classified with nearly identical sensitivity , specificity and negative and positive predictive values ( table 3 ) .
first , that cardiac surgery patients with problematic postoperative disease already exhibit elevated serum concentration of complement components c3 and c5 , tnf- and neutrophils ( count and percentage ) one day preoperatively .
second , that preopera - tive risk assessment based on serological and clinical chemistry data is possible , with high levels of accuracy .
the preoperative predictive risk assessment represents a clear advantage over assays relying on data acquired during or after cardiac intervention .
preoperative differences , as selected by our explorative data analysis , indicate a preopera - tive activation of the immune system , for example , by a subclinical inflammatory response , an atopic / allergic predisposition or a condition resulting from the congenital heart disease .
in contrast to the recent report that mod in children is gender related , gender was not a predisposing factor in our study .
twenty - four hour preoperative serum parameters in post operative non - poee and poee patients ( means sd ) . from the 56 determined parameters , those selected by one of the classification programs or exhibiting significant differences
are shown two - tailed student 's t - test , mann - whitney u - test .
parameter used by s = spss classifier , c = classif1 classifier or s , c= both classifiers .
il , interleukin ; poee , postoperative effusions and edema ; sl - selectin , soluble leukocytic - selectin ; tnf- , tumor necrosis factor - alpha
. classification of poee and non - poee patients ( confusion matrices ) of 24 h preoperative serological parameters by the spss and the classif1 classifiers ( see table 4 ) classification result shown separately for asd patients or the residual patients applying the identical classification algorithms as for the total group of patients .
simultaneous classification non - poee / poee for one patient increases line sum above 100% .
preoperative parameters and coefficients for prediction of postoperative cardiac surgery outcome by the spss and classif1 classifiers formula of the discriminant function : constant + , resulting value < 0 , non - poee risk , if > 0 , poee risk .
parameter on average above ( + ) or below ( - ) the 2575% percentile thresholds for c1-inhibitor : 300/377 mg / l ( 25%/75% ) ; c3 : 1181/1456 mg / l ; c5 : 100/131 mg / l ; sl - selection : 1102/1501
g / l , neutrophil count : 3900/5520cells/l ; eosinophil count : 85/269cells/l ; hematocrit : 34.0/39.8% ; partial thrombin time : 33.3/37.9s ; k :
non - poee patients have , on average , all parameters unchanged ( 0 ) between the 2575% percentile thresholds .
unknown patients are classified according to the highest number of positional coincidences , with the poee or the non - poee patients classification mask .
the parameters selected by the two classifiers in this study indicate increased poee risk for patients with elevated inflammatory response by increased complement and neutrophil activation and coagulation ( see table 4 ) . in different cardiac situations , crp , se - selectin , sicam-1 and neutrophil adhesion molecule expression
have been discussed as risk factors . as already suggested by others , preoperatively altered blood coagulation values such as partial thrombin time were found to be prognostic for postoperative blood loss .
fibrinogen and fibrin are ligands for mac-1 , inducing neutrophil , monocyte or resting platelet activation .
our study indicates this activation by elevated sl - selectin level as an important discriminant parameter .
cpb is associated with major qualitative and quantitative alterations of humoral pathways and changes in leukocyte subsets , generating a systemic inflammatory response with interactions between vascular endothelium , platelets and leukocytes including signal exchanges , adhesion molecule expression and secretion of cytokines or chemokines in a multi - step process .
patients with an altered immune profile before surgery might show a more pronounced or sustained immune response after surgery . in an unstimulated immune system
, cpb exposure constitutes the initial stimulus that might prime the system for postoperative complications . in patients with a primed or predisposed immune profile , cpb as the second stimulus may facilitate an enhanced immune response , which , in turn , may lead to poee , cls or multiple organ failure .
the main discriminators of at risk patients ( elevated levels of complement and activated complement components , tnf- and il-10 ) indicate the significance of complement system and monocyte activation .
il-10 release is specific to cpb surgery and patients with poee or mod release higher quantities of il-10 .
increased il-10 release as an indicator of mod or effusions is also supported by the finding that perioperative methyl - prednisolone administration , that enhances il-10 release during cpb surgery in adults , aggravates postoperative effusions and bleeding in children with postcardiotomy syndrome .
an observation that contrasts with the finding that children with mod had reduced il-10 serum levels prior to cpb .
patients from our study had no gender - related differences in any of the analyzed laboratory parameters .
we have no explanation for this discrepancy , but differences in the age distribution and the congenital heart diseases of patients included in our study , as compared to trotter et al .
severe allergic reactions with cardiac surgery and allergic predisposition in adults at risk for cardiovascular death have been reported .
the interpretation of allergic / atopic predisposition in poee risk was indicated by our recent observation of elevated leukocyte function asscoiated molecule-1 ( lfa-1 ) expression on leukocytes of at risk patients , as lfa-1 expression is increased on leukocytes of atopic children .
we reported earlier that patients at risk for poee also had increased preoperative histamine and eosinophil counts , among others .
the results from the present study do not clearly support the hypothesis of risk prevalence for atopic / allergic patients because only few of the selected markers could indicate an atopic / allergic predisposition ( e.g. tnf- and il-10 ) .
we conclude from these differences that both increased inflammatory status and allergic / atopic predisposition are predictors of increased poee in children . taken together ,
risk patients might have : ( i ) latent infection ; ( ii ) atopic / allergic predisposition ; or ( iii ) immune alterations as a result of the congenital heart disease .
these hypotheses have to be further scrutinized by future studies . because children with postoperative complications usually have a longer stay on the icu , a longer period of mechanical ventilation and stay longer in hospital ,
preoperative risk assessment is of clear therapeutic advantage and can be cost - effective by reducing any stay in intensive care . by prospective classification , up to 86% of the patients at risk were correctly identified preoperatively . in view of the fact that such predictions were not possible at all until now , these predictive values are promising .
however , the classifier will be optimized by increasing the number of patients enrolled in studies and by combining this serological classifier with additional parameters such as flow - cytometric data .
individual risk assessment before cardiac surgery of this type might open new ways to develop individual treatment strategies with two possible clinical consequences : first , postponement of surgery until the normalization of clinical parameters ( e.g. elimination of stress or a latent infection ) ; and , second , application of individual prophylaxis in the case of endogenous reasons for immune system alterations .
the hypothesis that postponement or individual prophylaxis will reduce poee has to be scrutinized in additional studies .
the proposed serological classifier should permit individual risk assessment in hospitals with lower patient numbers .
it is planned to set up and optimize an on - line classifier for poee risk assessment on the internet .
one of the practical consequences of this would be that diseases could be categorized at institutions where no sufficient database can be generated in a reasonable time period .
risk assessments for patients at other institutions can be calculated for test purposes using the indicated spss classifier formula ( table 4 ) .
the local data correction factor is obtained as a ratio between the parameter mean from non - poee patients from table 2 of this study and the mean of the respective parameter from the local non - poee group of 20 to 40 complication - free patients . the local data correction factor for the establishment of the individual patient 's triple matrix for the classif1 classification
the parameters selected by the two classifiers in this study indicate increased poee risk for patients with elevated inflammatory response by increased complement and neutrophil activation and coagulation ( see table 4 ) . in different cardiac situations , crp , se - selectin , sicam-1 and neutrophil adhesion molecule expression
preoperatively altered blood coagulation values such as partial thrombin time were found to be prognostic for postoperative blood loss .
fibrinogen and fibrin are ligands for mac-1 , inducing neutrophil , monocyte or resting platelet activation .
our study indicates this activation by elevated sl - selectin level as an important discriminant parameter .
cpb is associated with major qualitative and quantitative alterations of humoral pathways and changes in leukocyte subsets , generating a systemic inflammatory response with interactions between vascular endothelium , platelets and leukocytes including signal exchanges , adhesion molecule expression and secretion of cytokines or chemokines in a multi - step process .
patients with an altered immune profile before surgery might show a more pronounced or sustained immune response after surgery . in an unstimulated immune system
, cpb exposure constitutes the initial stimulus that might prime the system for postoperative complications . in patients with a primed or predisposed immune profile , cpb as the second stimulus may facilitate an enhanced immune response , which , in turn , may lead to poee , cls or multiple organ failure .
the main discriminators of at risk patients ( elevated levels of complement and activated complement components , tnf- and il-10 ) indicate the significance of complement system and monocyte activation .
il-10 release is specific to cpb surgery and patients with poee or mod release higher quantities of il-10 .
increased il-10 release as an indicator of mod or effusions is also supported by the finding that perioperative methyl - prednisolone administration , that enhances il-10 release during cpb surgery in adults , aggravates postoperative effusions and bleeding in children with postcardiotomy syndrome .
an observation that contrasts with the finding that children with mod had reduced il-10 serum levels prior to cpb .
patients from our study had no gender - related differences in any of the analyzed laboratory parameters .
we have no explanation for this discrepancy , but differences in the age distribution and the congenital heart diseases of patients included in our study , as compared to trotter et al .
severe allergic reactions with cardiac surgery and allergic predisposition in adults at risk for cardiovascular death have been reported .
the interpretation of allergic / atopic predisposition in poee risk was indicated by our recent observation of elevated leukocyte function asscoiated molecule-1 ( lfa-1 ) expression on leukocytes of at risk patients , as lfa-1 expression is increased on leukocytes of atopic children .
we reported earlier that patients at risk for poee also had increased preoperative histamine and eosinophil counts , among others .
the results from the present study do not clearly support the hypothesis of risk prevalence for atopic / allergic patients because only few of the selected markers could indicate an atopic / allergic predisposition ( e.g. tnf- and il-10 ) .
we conclude from these differences that both increased inflammatory status and allergic / atopic predisposition are predictors of increased poee in children .
risk patients might have : ( i ) latent infection ; ( ii ) atopic / allergic predisposition ; or ( iii ) immune alterations as a result of the congenital heart disease .
because children with postoperative complications usually have a longer stay on the icu , a longer period of mechanical ventilation and stay longer in hospital , preoperative risk assessment is of clear therapeutic advantage and can be cost - effective by reducing any stay in intensive care . by prospective classification ,
up to 86% of the patients at risk were correctly identified preoperatively . in view of the fact that such predictions were not possible at all until now
however , the classifier will be optimized by increasing the number of patients enrolled in studies and by combining this serological classifier with additional parameters such as flow - cytometric data .
individual risk assessment before cardiac surgery of this type might open new ways to develop individual treatment strategies with two possible clinical consequences : first , postponement of surgery until the normalization of clinical parameters ( e.g. elimination of stress or a latent infection ) ; and , second , application of individual prophylaxis in the case of endogenous reasons for immune system alterations .
the hypothesis that postponement or individual prophylaxis will reduce poee has to be scrutinized in additional studies .
the proposed serological classifier should permit individual risk assessment in hospitals with lower patient numbers .
it is planned to set up and optimize an on - line classifier for poee risk assessment on the internet .
one of the practical consequences of this would be that diseases could be categorized at institutions where no sufficient database can be generated in a reasonable time period .
risk assessments for patients at other institutions can be calculated for test purposes using the indicated spss classifier formula ( table 4 ) .
the local data correction factor is obtained as a ratio between the parameter mean from non - poee patients from table 2 of this study and the mean of the respective parameter from the local non - poee group of 20 to 40 complication - free patients . the local data correction factor for the establishment of the individual patient 's triple matrix for the classif1 classification
the development of postoperative edema and effusion ( poee ) in children after cardiopulmonary bypass surgery can be predicted preoperatively .
the immune activation has cellular ( neutrophil , eosinophil , monocyte counts , hematocrit ) and humoral ( c1-inhibitor , c3 , c5a / c5 , il-10 , sl - selectin , partial thrombin time , serum potassium ) components .
preoperative normalization of the immune activation status has the potential of decreasing the intensive care treatment and the overall level of postoperative complications .
cls , capillary leak syndrome ; cpb , cardiopulmonary bypass ; crp , c - reactive protein ; edta , ethylenediaminetetracetic acid ; ig , immunoglobulin ; il , interleukin ; lfa-1 , leukocyte function associated molecule-1 ; mod , multiple organ dysfunction ; poee , postoperative effusions and edema ; se - selectin , soluble endothelial - selectin ; sl - selectin , soluble leukocytic - selectin ; th1/2 , t - helper type 1/2 ; tnf , tumor necrosis factor .
the authors thank mrs jacqueline richter for excellent technical help . a grant to undertake this study
was provided by the schsisches minis - terium fr wissenschaft und kunst ( smwk , research grant p.o . ) , dresden , and deutsche stiftung fr herzforschung , frankfurt , germany . | aimpostoperative effusions and edema and capillary leak syndrome in children after cardiac surgery with cardiopulmonary bypass constitute considerable clinical problems .
overshooting immune response is held to be the cause . in a prospective study we investigated whether preoperative immune status differences exist in patients at risk for postsurgical effusions and edema , and to what extent these differences permit prediction of the postoperative outcome.methodone-day preoperative serum levels of immunoglobulins , complement , cytokines and chemokines , soluble adhesion molecules and receptors as well as clinical chemistry parameters such as differential counts , creatinine , blood coagulation status ( altogether 56 parameters )
were analyzed in peripheral blood samples of 75 children ( aged 318 years ) undergoing cardiopulmonary bypass surgery ( 29 with postoperative effusions and edema within the first postoperative week).resultspreoperative elevation of the serum level of c3 and c5 complement components , tumor necrosis factor- , percentage of leukocytes that are neutrophils , body weight and decreased percentage of lymphocytes ( all p < 0.03 ) occurred in children developing postoperative effusions and edema . while single parameters did not predict individual outcome , > 86% of the patients with postoperative effusions and oedema were correctly predicted using two different classification algorithms .
data mining by both methods selected nine partially overlapping parameters .
the prediction quality was independent of the congenital heart defect.conclusionindicators of inflammation were selected as risk indicators by explorative data analysis .
this suggests that preoperative differences in the immune system and capillary permeability status exist in patients at risk for postoperative effusions .
these differences are suitable for preoperative risk assessment and may be used for the benefit of the patient and to improve cost effectiveness . | Introduction
Methods
Study groups
Complement, cytokines, soluble adhesion molecules
Statistical analysis
Results
Conclusion
Inflammatory response
Clinical implications
Key messages
Competing interests
Abbreviations
Acknowledgment | these complications are characterized by increased capillary permeability , a shift of fluid and protein from the intravascular to the interstitial space and may further progress into hypovolemia , massive generalized edema , acute respiratory distress syndrome , or even capillary leak syndrome ( cls ) or multiple organ dysfunction ( mod ) or failure , with a substantial morbidity and mortality . the prediction of patients at risk for postoperative complications is important for the individual preoperative prophylactic treatment . in a recent study we showed that children who suffered from postoperative effusions and edema ( poee ) are , 24 hours before surgery , already exhibiting altered antigen expression on leukocytes , by which risk assessment would be possible using discriminant analysis . based on these results we hypothesized that children at risk of poee have an altered preoperative level of markers of immunoactivation , allergic / atopic predisposition or t - helper type 2 ( th2 ) phenotype , which may be used as predictors for risk assessment . a total of 75 patients who underwent cardiac surgery with cpb were analyzed [ inclusion criteria : aged 318 years , body weight > 12 kg ; exclusion criteria : missing informed consent of parents , palliative cardiac surgery ( e.g. incidence of edema was not used for poee discrimination because quantitative measures of extravascular body fluid volume ( such as scintigraphy following labelling of the extravascular fluid by radiolabelled sulphide or bromide ) were ethically not feasible in children . additionally , routine laboratory and clinical chemistry parameters were determined ( cell count , differential blood count , crp , creatinine , electrolytes , protein , hematocrit , blood coagulation parameters ) . a total of 75 patients who underwent cardiac surgery with cpb were analyzed [ inclusion criteria : aged 318 years , body weight > 12 kg ; exclusion criteria : missing informed consent of parents , palliative cardiac surgery ( e.g. incidence of edema was not used for poee discrimination because quantitative measures of extravascular body fluid volume ( such as scintigraphy following labelling of the extravascular fluid by radiolabelled sulphide or bromide ) were ethically not feasible in children . additionally , routine laboratory and clinical chemistry parameters were determined ( cell count , differential blood count , crp , creatinine , electrolytes , protein , hematocrit , blood coagulation parameters ) . first , that cardiac surgery patients with problematic postoperative disease already exhibit elevated serum concentration of complement components c3 and c5 , tnf- and neutrophils ( count and percentage ) one day preoperatively . preoperative differences , as selected by our explorative data analysis , indicate a preopera - tive activation of the immune system , for example , by a subclinical inflammatory response , an atopic / allergic predisposition or a condition resulting from the congenital heart disease . il , interleukin ; poee , postoperative effusions and edema ; sl - selectin , soluble leukocytic - selectin ; tnf- , tumor necrosis factor - alpha
. the main discriminators of at risk patients ( elevated levels of complement and activated complement components , tnf- and il-10 ) indicate the significance of complement system and monocyte activation . by prospective classification , up to 86% of the patients at risk were correctly identified preoperatively . in different cardiac situations , crp , se - selectin , sicam-1 and neutrophil adhesion molecule expression
preoperatively altered blood coagulation values such as partial thrombin time were found to be prognostic for postoperative blood loss . the main discriminators of at risk patients ( elevated levels of complement and activated complement components , tnf- and il-10 ) indicate the significance of complement system and monocyte activation . we have no explanation for this discrepancy , but differences in the age distribution and the congenital heart diseases of patients included in our study , as compared to trotter et al . by prospective classification ,
up to 86% of the patients at risk were correctly identified preoperatively . the local data correction factor for the establishment of the individual patient 's triple matrix for the classif1 classification
the development of postoperative edema and effusion ( poee ) in children after cardiopulmonary bypass surgery can be predicted preoperatively . cls , capillary leak syndrome ; cpb , cardiopulmonary bypass ; crp , c - reactive protein ; edta , ethylenediaminetetracetic acid ; ig , immunoglobulin ; il , interleukin ; lfa-1 , leukocyte function associated molecule-1 ; mod , multiple organ dysfunction ; poee , postoperative effusions and edema ; se - selectin , soluble endothelial - selectin ; sl - selectin , soluble leukocytic - selectin ; th1/2 , t - helper type 1/2 ; tnf , tumor necrosis factor . | [
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] |
short - term intensive insulin therapy prescribed early on in the course of type 2 diabetes can improve -cell function and insulin resistance by eliminating glucotoxicity and lead to drug - free glycemic remission for up to 2 years [ 1 , 2 ] .
however , intensive insulin therapy is not generally suitable for outpatients who are often unable to undergo multiple insulin injections ; as a result many patients are prescribed oral hypoglycemic agents ( ohas ) when they are no longer in glycemic remission .
basal - supported oral therapy ( bot ) , comprising a basal insulin and ohas , is an alternative option in this clinical setting [ 35 ] .
however , conventional bot using sulfonylureas ( sus ) mainly controls fasting plasma glucose ( fpg ) and does not necessarily correct post - prandial glycemia ; as a consequence , adequate glycemic control is not always achieved and frequent hypoglycemic episodes may occur .
dipeptidyl peptidase-4 ( dpp-4 ) inhibitors are used in clinical practice for the treatment of type 2 diabetes [ 7 , 8 ] .
these agents have an advantage over sus as they regulate insulin secretion in a glucose - dependent manner [ 9 , 10 ] , thereby minimizing the risk of hypoglycemia and reducing glycemic fluctuations [ 12 , 13 ] .
a few studies evaluating bot using dpp-4 inhibitors have demonstrated a glucose - lowering effect when used in conjunction with ongoing insulin therapy in patients with type 2 diabetes [ 1416 ] .
the aim of the present study was to evaluate whether starting bot with sitagliptin , a dpp-4 inhibitor , achieves adequate glycemic control and allows subsequent switching from bot to sitagliptin - based oral therapy .
patients aged between 30 and 70 years were enrolled in the study if they had a new or recent diagnosis of type 2 diabetes , had not been treated with an antidiabetic agent in the previous 2 years , and had poor glycemic control ( hemoglobin a1c [ hba1c ] 9.0% ) .
patients were excluded if they had type 1 diabetes , chronic liver disease , advanced kidney disease , were taking corticosteroids , or were diagnosed with diabetic ketoacidosis .
patients were educated in diet therapy [ 25 kcal ideal body weight ( kg ) per day ] by a dietician and instructed to take 12,000 steps per day as exercise , according to the japanese treatment guide for diabetes .
adherence to diet and exercise therapy was evaluated by the proportion of patients attaining at least 80% of the instructed levels using self - dietary records and pedometer , respectively .
patients were treated with a combination of sitagliptin and insulin glargine for 24 weeks .
sitagliptin was initiated and maintained at a dose of 50 mg / day , and insulin glargine was initiated at a dose of 4 u at bedtime and adjusted if needed at monthly clinic visit ( increased by 1 unit when fpg > 180 mg / dl , and decreased by 1 unit when fpg < 90 mg / dl ) .
patients who achieved an hba1c of < 7% during the 24-week treatment period were referred to as achievers and discontinued their basal insulin therapy .
they were then maintained on sitagliptin ( 50 mg / day ) monotherapy or on a combination of sitagliptin with metformin ( 5001,500 mg / day ) .
patients who did not achieve an hba1c of < 7% ( non - achievers ) continued on bot throughout the 24-week treatment period .
the primary end point was the proportion of patients who achieved an hba1c < 7.0% during bot and discontinued basal insulin during the 24-week treatment period .
secondary end points were changes of fpg and hba1c from baseline in achievers and non - achievers during the 24-week study period .
c - peptide index ( cpi ) , a measure of insulin secretion capacity , was calculated at week 12 using the following formula : [ 100 fasting c - peptide immunoreactivity ( cpr ) ( ng / ml)]/[fpg ( mg / dl ) ] .
in addition , 20/[fasting cpr ( mmol / l ) fpg ( mmol / l ) ] , a potential index of insulin resistance , was also calculated at week 12 . lower value of this index indicates being more insulin resistant .
hba1c levels were determined using high - performance liquid chromatography with an automated ah-8280 analyzer ( arkray , kyoto , japan ) at every clinical visit .
hypoglycemic episodes were counted by documentation of any hypoglycemic episodes and any symptoms derived from hypoglycemia based on patients reports .
all statistical analyses were performed using the statview software package version 5.0 ( abacus concept , berkeley , ca ) .
changes in fpg and hba1c at 4 , 8 , 12 , and 24 weeks versus baseline were evaluated using anova followed by bonferroni s test .
all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 .
patients aged between 30 and 70 years were enrolled in the study if they had a new or recent diagnosis of type 2 diabetes , had not been treated with an antidiabetic agent in the previous 2 years , and had poor glycemic control ( hemoglobin a1c [ hba1c ] 9.0% ) .
patients were excluded if they had type 1 diabetes , chronic liver disease , advanced kidney disease , were taking corticosteroids , or were diagnosed with diabetic ketoacidosis .
patients were educated in diet therapy [ 25 kcal ideal body weight ( kg ) per day ] by a dietician and instructed to take 12,000 steps per day as exercise , according to the japanese treatment guide for diabetes .
adherence to diet and exercise therapy was evaluated by the proportion of patients attaining at least 80% of the instructed levels using self - dietary records and pedometer , respectively .
patients were treated with a combination of sitagliptin and insulin glargine for 24 weeks .
sitagliptin was initiated and maintained at a dose of 50 mg / day , and insulin glargine was initiated at a dose of 4 u at bedtime and adjusted if needed at monthly clinic visit ( increased by 1 unit when fpg > 180 mg / dl , and decreased by 1 unit when fpg < 90 mg / dl ) .
patients who achieved an hba1c of < 7% during the 24-week treatment period were referred to as achievers and discontinued their basal insulin therapy .
they were then maintained on sitagliptin ( 50 mg / day ) monotherapy or on a combination of sitagliptin with metformin ( 5001,500 mg / day ) .
patients who did not achieve an hba1c of < 7% ( non - achievers ) continued on bot throughout the 24-week treatment period .
the primary end point was the proportion of patients who achieved an hba1c < 7.0% during bot and discontinued basal insulin during the 24-week treatment period .
secondary end points were changes of fpg and hba1c from baseline in achievers and non - achievers during the 24-week study period .
c - peptide index ( cpi ) , a measure of insulin secretion capacity , was calculated at week 12 using the following formula : [ 100 fasting c - peptide immunoreactivity ( cpr ) ( ng / ml)]/[fpg ( mg / dl ) ] .
in addition , 20/[fasting cpr ( mmol / l ) fpg ( mmol / l ) ] , a potential index of insulin resistance , was also calculated at week 12 .
hba1c levels were determined using high - performance liquid chromatography with an automated ah-8280 analyzer ( arkray , kyoto , japan ) at every clinical visit .
hypoglycemic episodes were counted by documentation of any hypoglycemic episodes and any symptoms derived from hypoglycemia based on patients reports .
all statistical analyses were performed using the statview software package version 5.0 ( abacus concept , berkeley , ca ) .
changes in fpg and hba1c at 4 , 8 , 12 , and 24 weeks versus baseline were evaluated using anova followed by bonferroni s test .
all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 .
a total of 22 patients were enrolled in the study and 19 of the participants completed the study protocol .
patient baseline characteristics are summarized in table 1.table 1baseline clinical characteristics of the study populationage ( years )
48.7 8.3sex : male / female ( n)16/3duration of diabetes ( years )
3.1 2.3body weight ( kg)78.2 16.0bmi ( kg / m)26.9 5.1fpg ( mg / dl)215 48hba1c ( % )
11.0 1.5triglyceride ( mg / dl)160
77hdl - c ( mg / dl )
50 11ldl - c ( mg / dl )
111 29hypertension , n ( % ) 6 ( 31.5%)dyslipidemia , n ( % ) 9 ( 47.4% )
bmi body mass index , fpg fasting plasma glucose , ldl - c low - density lipoprotein cholesterol , hdl - c high - density lipoprotein cholesterol , hba1c hemoglobin a1c
continuous data are means ( sd ) baseline clinical characteristics of the study population
bmi body mass index , fpg fasting plasma glucose , ldl - c low - density lipoprotein cholesterol , hdl - c high - density lipoprotein cholesterol , hba1c hemoglobin a1c
continuous data are means ( sd ) during the 24-week treatment period , 12 patients ( 63.2% ) achieved an hba1c < 7% using initial bot ( achievers).the mean time to achieve hba1c < 7.0% among the achievers was 13.7 5.6 weeks . all achievers switched from bot to sitagliptin monotherapy or in combination with metformin during the study period .
the remaining seven patients ( 36.8% ) failed to achieve hba1c levels < 7% during 24 weeks of treatment ( non - achievers ) .
rates of adherence to diet and exercise were higher in achievers ( diet 75% ; exercise 67% ) than in non - achievers ( diet 43% ; exercise 29% ) .
both fpg and hba1c in achievers significantly dropped at 4 , 8 , 12 and 24 weeks from baseline , while those in non - achievers significantly decreased at 12 and 24 weeks from baseline , but failed to reach at target glycemic control ( fig . 1 ) .
there were statistically significant differences in fpg at 4 , 8 , 12 and 24 weeks and in hba1c at 8 , 12 and 24 weeks between achievers and non-achievers.fig . 1
a change in fasting plasma glucose ( fpg ) and b hemoglobin a1c ( hba1c ) with time among achievers and non - achievers during the 24-week study period .
achievers : patients who achieved hba1c of < 7.0% and switched from bot to sitagliptin - based oral therapy during the 24-week study period .
non - achievers : patients who failed to achieve hba1c of < 7.0% and continued on bot during the 24-week study period . * p < 0.05 , * * p < 0.01 ( vs. week 0 )
p < 0.05 ,
p < 0.01 ( achievers vs. non - achievers )
a change in fasting plasma glucose ( fpg ) and b hemoglobin a1c ( hba1c ) with time among achievers and non - achievers during the 24-week study period .
achievers : patients who achieved hba1c of < 7.0% and switched from bot to sitagliptin - based oral therapy during the 24-week study period .
non - achievers : patients who failed to achieve hba1c of < 7.0% and continued on bot during the 24-week study period . * p < 0.05 , * * p < 0.01 ( vs. week 0 )
p < 0.05 ,
p < 0.01 ( achievers vs. non - achievers ) the achievers experienced a significant reduction in body weight and bmi at 12 and 24 weeks versus baseline , while the non - achievers did not ( table 2 ) .
the insulin requirement for achievers ( 3.8 0.8 u / day ) was significantly lower than that for non - achievers ( 7.3 3.3 u / day ) during bot .
concomitant metformin dosage was significantly lower in achievers ( 500 0 mg / day ; n = 2 ) than in non - achievers ( 1,050 450 mg / day ; n = 5 ) .
there was no significant difference in cpi between achievers and non - achievers ; however , non - achievers showed a significant insulin resistance index ( value of 20/[fasting cpr
fpg ] ) compared to achievers.table 2comparisons between achievers and non - achieversachieversnon - achievers
n
127sex : male / female ( n)11/15/2age ( years )
49.6 9.647.1 5.8duration of diabetes ( years )
2.9 2.03.6 2.7body weight ( kg)78.1 13.978.3 20.4 body weight ( kg )
12 weeks4.2 3.7*0.4 1.4 24 weeks6.6 5.2**0.7 2.2bmi ( kg / m)26.3 3.828.0 7.0 bmi ( kg / m ) 12 weeks1.8 1.3**0.1 0.9 24 weeks1.8 1.7**0.3 0.9insulin dose during bot ( u / day)3.8 0.8**7.3 3.3cpi at 12 weeks1.7 0.51.4 0.620/(fpg cpr ) at 12 weeks6.2 2.1 *
3.8 1.3
bot basal - supported oral therapy , cpr c - peptide immunoreactivity , cpi c - peptide index , fpg fasting plasma glucose * p < 0.05 , * * p < 0.01 by unpaired t test ( achievers vs. non - achievers )
continuous data are means ( sd ) . body weight : change from baseline , bmi : change from baseline comparisons between achievers and non - achievers
bot basal - supported oral therapy , cpr c - peptide immunoreactivity , cpi c - peptide index , fpg fasting plasma glucose * p < 0.05 , * * p < 0.01 by unpaired t test ( achievers vs. non - achievers )
continuous data are means ( sd ) .
body weight : change from baseline , bmi : change from baseline two patients in the achiever group and one patient in the non - achiever group experienced hypoglycemia ; there were no cases of severe hypoglycemia .
during the 24-week treatment period , 12 patients ( 63.2% ) achieved an hba1c < 7% using initial bot ( achievers).the mean time to achieve hba1c < 7.0% among the achievers was 13.7 5.6 weeks .
all achievers switched from bot to sitagliptin monotherapy or in combination with metformin during the study period .
the remaining seven patients ( 36.8% ) failed to achieve hba1c levels < 7% during 24 weeks of treatment ( non - achievers ) .
rates of adherence to diet and exercise were higher in achievers ( diet 75% ; exercise 67% ) than in non - achievers ( diet 43% ; exercise 29% ) .
both fpg and hba1c in achievers significantly dropped at 4 , 8 , 12 and 24 weeks from baseline , while those in non - achievers significantly decreased at 12 and 24 weeks from baseline , but failed to reach at target glycemic control ( fig . 1 ) .
there were statistically significant differences in fpg at 4 , 8 , 12 and 24 weeks and in hba1c at 8 , 12 and 24 weeks between achievers and non-achievers.fig . 1
a change in fasting plasma glucose ( fpg ) and b hemoglobin a1c ( hba1c ) with time among achievers and non - achievers during the 24-week study period .
achievers : patients who achieved hba1c of < 7.0% and switched from bot to sitagliptin - based oral therapy during the 24-week study period .
non - achievers : patients who failed to achieve hba1c of < 7.0% and continued on bot during the 24-week study period . * p < 0.05 , * * p < 0.01 ( vs. week 0 )
p < 0.05 ,
p < 0.01 ( achievers vs. non - achievers )
a change in fasting plasma glucose ( fpg ) and b hemoglobin a1c ( hba1c ) with time among achievers and non - achievers during the 24-week study period .
achievers : patients who achieved hba1c of < 7.0% and switched from bot to sitagliptin - based oral therapy during the 24-week study period .
non - achievers : patients who failed to achieve hba1c of < 7.0% and continued on bot during the 24-week study period . * p < 0.05 , * * p < 0.01 ( vs. week 0 )
p < 0.05 ,
p < 0.01 ( achievers vs. non - achievers ) the achievers experienced a significant reduction in body weight and bmi at 12 and 24 weeks versus baseline , while the non - achievers did not ( table 2 ) .
the insulin requirement for achievers ( 3.8 0.8 u / day ) was significantly lower than that for non - achievers ( 7.3 3.3 u / day ) during bot .
concomitant metformin dosage was significantly lower in achievers ( 500 0 mg / day ; n = 2 ) than in non - achievers ( 1,050 450 mg / day ; n = 5 ) .
there was no significant difference in cpi between achievers and non - achievers ; however , non - achievers showed a significant insulin resistance index ( value of 20/[fasting cpr fpg ] ) compared to achievers.table 2comparisons between achievers and non - achieversachieversnon - achievers
n
127sex : male / female ( n)11/15/2age ( years )
49.6 9.647.1 5.8duration of diabetes ( years )
2.9 2.03.6 2.7body weight ( kg)78.1 13.978.3 20.4 body weight ( kg )
12 weeks4.2 3.7*0.4 1.4 24 weeks6.6 5.2**0.7 2.2bmi ( kg / m)26.3 3.828.0 7.0 bmi ( kg / m ) 12 weeks1.8 1.3**0.1 0.9 24 weeks1.8 1.7**0.3 0.9insulin dose during bot ( u / day)3.8 0.8**7.3 3.3cpi at 12 weeks1.7 0.51.4 0.620/(fpg cpr ) at 12 weeks6.2 2.1 * 3.8 1.3
bot basal - supported oral therapy , cpr c - peptide immunoreactivity , cpi c - peptide index , fpg fasting plasma glucose * p < 0.05 , * * p < 0.01 by unpaired t test ( achievers vs. non - achievers )
continuous data are means ( sd ) . body weight : change from baseline , bmi : change from baseline comparisons between achievers and non - achievers
bot basal - supported oral therapy , cpr c - peptide immunoreactivity , cpi c - peptide index , fpg fasting plasma glucose * p < 0.05 , * * p < 0.01 by unpaired t test ( achievers vs. non - achievers )
continuous data are means ( sd ) .
two patients in the achiever group and one patient in the non - achiever group experienced hypoglycemia ; there were no cases of severe hypoglycemia .
the current study demonstrated that initiating bot with sitagliptin successfully improved glycemic control and allowed patients to switch from bot to sitagliptin monotherapy or combination therapy , maintaining adequate glycemic control during a 24-week period in untreated patients with type 2 diabetes .
bot is often prescribed for outpatients because once daily injection is more acceptable than multiple insulin injections .
however , one of the biggest problems associated with conventional bot is postprandial hyperglycemia while fpg is within normal range .
dpp-4 inhibitors , as monotherapy or combination therapy , have several advantages over sus , since these agents enhance insulin secretion in a glucose - dependent manner and suppress glucagon secretion [ 9 , 10 ] . in the current study , 12 patients ( 63.2% ) achieved hba1c < 7% using initial bot , and subsequently discontinued basal insulin .
these achievers showed a significant decrease in hba1c at 4 , 8 , 12 and 24 weeks versus baseline , and versus non - achievers at 8 , 12 and 24 weeks .
the basal insulin dose was relatively low and the duration of bot was short to achieve target glycemic control .
in contrast , the remaining seven patients ( 36.8% ) failed to achieve a target hba1c < 7% during the 24-week treatment period , although in common with the achievers they adhered to their instructed insulin injection .
it is reported that insulin therapy is usually required in japanese type 2 diabetic patients with a cpi ( insulin secretion capacity ) lower than 0.8 .
cpi was measured at 12 weeks because glucotoxicity might have influenced this index at baseline . in comparison to the achievers , the non - achievers had a similar cpi , but a significantly lower 20/[fasting cpr fpg ] level , a possible marker of insulin resistance at 12 weeks .
lower value of this index indicates being more insulin resistant . body weight and bmi in non - achievers
were not significantly reduced from baseline , probably because these patients did not strictly adhere to diet and exercise therapy .
these results suggest that insulin resistance was not sufficiently improved to allow non - achievers to discontinue basal insulin . in the add - on lantus to oral hypoglycemic agents ( aloha ) study in insulin - nave
3,515 japanese patients starting insulin glargine ( mean dose 8.5 u / day ) plus ohas , mainly sus , 15.5% of participants achieved hba1c < 7.0% at 24 weeks , whereas 84.5% of participants did not .
in such a study , a shorter duration of diabetes ( < 1 year ) and lower hba1c ( < 8.5% ) at baseline were significantly associated with a higher rate of achieving target hba1c ( < 7.0% ) .
previous bot trials [ 1416 ] with dpp-4 inhibitors added to ongoing insulin therapy in patients with type 2 diabetes have demonstrated a significant glucose - lowering effect ; however , the background of previous studies [ 1416 ] where duration of diabetes was over 10 years , daily insulin dose exceeded 15 u / day and concomitant ohas were frequently used is quite different from the current study .
recently , harashima et al . have demonstrated in a 52-week study of ongoing bot with sus that replacement of basal insulin with sitagliptin was associated with a decrease in hba1c level ( < 7.0% ) in 67.4% of patients with a mean diabetes duration of 12.1 years , while 32.6% of subjects with a longer duration of diabetes ( ~18.9 years ) and receiving higher doses of concomitant sus could not replace basal insulin by sitagliptin .
taken together , the efficacy of bot in type 2 diabetes patients may be related to several factors of duration of diabetes , pre - treatment or insulin resistance .
first , this was a single arm , single center study involving a small sample of patients .
it was unable to compare the efficacy and convenience of initial treatments between the current study of bot with sitagliptin and other therapies by bot with su or multiple insulin injection regimen .
second , the study findings can not be generalized to patients with type 2 diabetes who would not meet the study inclusion criteria , i.e. , drug - nave patients with recently diagnosed , poorly controlled type 2 diabetes .
third , the titration of basal insulin and adherence to diet and exercise may have been insufficient for the non - achievers . on this basis
, a large prospective study is needed to validate the findings of the current study .
the current study showed that initial introduction of bot with sitagliptin was effective for achievement of glycemic control and subsequent switching from bot to dpp - iv inhibitor - based therapy in recent onset and untreated patients with type 2 diabetes .
all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation ( institutional and national ) and with the helsinki declaration of 1975 , as revised in 2000 and 2008 .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . | introductionthe present study assessed the efficacy of initial basal - supported oral therapy ( bot ) with sitagliptin for achievement of glycemic control and subsequent switching from bot to sitagliptin - based oral therapy.methodsnineteen recently diagnosed type 2 diabetic patients who had received no antidiabetic medication in the previous 2 years were sequentially examined for the 24-week study .
patients were initially treated with a combination of insulin glargine and sitagliptin .
sitagliptin was initiated and maintained at a dose of 50 mg / day , and insulin glargine was started at a dose of 4 u at bedtime and adjusted if needed.resultsduring the 24-week treatment period , 12 patients ( 63% ) achieved hba1c levels < 7% ( mean bot duration 13.7 5.6 weeks ) and switched from bot to sitagliptin monotherapy or in combination with metformin ( achievers ) .
the remaining seven patients ( 37% ) failed to achieve hba1c levels < 7% ( non - achievers ) and continued on bot .
both fpg and hba1c in achievers significantly dropped at 4 , 8 , 12 and 24 weeks from baseline , while those in non - achievers significantly decreased at 12 and 24 weeks from baseline , but failed to reach target glycemic control .
there were statistically significant differences in fpg at 4 , 8 , 12 and 24 weeks and in hba1c at 8 , 12 and 24 weeks between achievers and non - achievers .
body weight and bmi in achievers were significantly reduced at 12 and 24 weeks , but those in non - achievers did not change significantly .
dosage of concomitant insulin during bot was significantly lower in achievers compared to non - achievers .
non - achievers had a similar cpi , a measure of insulin secretion capacity , to achievers , but significantly showed an insulin resistance index ( value of 20/[fasting cpr fpg ] ) , in comparison to achievers.conclusioninitiating bot with sitagliptin followed by sitagliptin - based oral therapy is a useful option in untreated and poorly controlled patients with type 2 diabetes . | Introduction
Materials and Methods
Patients
Treatment
Study End Points
Safety Assessment
Statistical Analysis
Ethics Statement
Results
Efficacy
Safety
Discussion
Conclusion
Conflict of interest
Compliance with ethics guidelines
Open Access | sitagliptin was initiated and maintained at a dose of 50 mg / day , and insulin glargine was initiated at a dose of 4 u at bedtime and adjusted if needed at monthly clinic visit ( increased by 1 unit when fpg > 180 mg / dl , and decreased by 1 unit when fpg < 90 mg / dl ) . both fpg and hba1c in achievers significantly dropped at 4 , 8 , 12 and 24 weeks from baseline , while those in non - achievers significantly decreased at 12 and 24 weeks from baseline , but failed to reach at target glycemic control ( fig . there was no significant difference in cpi between achievers and non - achievers ; however , non - achievers showed a significant insulin resistance index ( value of 20/[fasting cpr
fpg ] ) compared to achievers.table 2comparisons between achievers and non - achieversachieversnon - achievers
n
127sex : male / female ( n)11/15/2age ( years )
49.6 9.647.1 5.8duration of diabetes ( years )
2.9 2.03.6 2.7body weight ( kg)78.1 13.978.3 20.4 body weight ( kg )
12 weeks4.2 3.7*0.4 1.4 24 weeks6.6 5.2**0.7 2.2bmi ( kg / m)26.3 3.828.0 7.0 bmi ( kg / m ) 12 weeks1.8 1.3**0.1 0.9 24 weeks1.8 1.7**0.3 0.9insulin dose during bot ( u / day)3.8 0.8**7.3 3.3cpi at 12 weeks1.7 0.51.4 0.620/(fpg cpr ) at 12 weeks6.2 2.1 *
3.8 1.3
bot basal - supported oral therapy , cpr c - peptide immunoreactivity , cpi c - peptide index , fpg fasting plasma glucose * p < 0.05 , * * p < 0.01 by unpaired t test ( achievers vs. non - achievers )
continuous data are means ( sd ) . both fpg and hba1c in achievers significantly dropped at 4 , 8 , 12 and 24 weeks from baseline , while those in non - achievers significantly decreased at 12 and 24 weeks from baseline , but failed to reach at target glycemic control ( fig . there was no significant difference in cpi between achievers and non - achievers ; however , non - achievers showed a significant insulin resistance index ( value of 20/[fasting cpr fpg ] ) compared to achievers.table 2comparisons between achievers and non - achieversachieversnon - achievers
n
127sex : male / female ( n)11/15/2age ( years )
49.6 9.647.1 5.8duration of diabetes ( years )
2.9 2.03.6 2.7body weight ( kg)78.1 13.978.3 20.4 body weight ( kg )
12 weeks4.2 3.7*0.4 1.4 24 weeks6.6 5.2**0.7 2.2bmi ( kg / m)26.3 3.828.0 7.0 bmi ( kg / m ) 12 weeks1.8 1.3**0.1 0.9 24 weeks1.8 1.7**0.3 0.9insulin dose during bot ( u / day)3.8 0.8**7.3 3.3cpi at 12 weeks1.7 0.51.4 0.620/(fpg cpr ) at 12 weeks6.2 2.1 * 3.8 1.3
bot basal - supported oral therapy , cpr c - peptide immunoreactivity , cpi c - peptide index , fpg fasting plasma glucose * p < 0.05 , * * p < 0.01 by unpaired t test ( achievers vs. non - achievers )
continuous data are means ( sd ) . | [
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the liver has an important role in lipid metabolism , which includes the mobilization and synthesis of free fatty acids as well as the storage and export of lipids and lipoproteins .
various drugs , nutritional factors , and genetic defects in energy metabolism can result in excess hepatic triacylglycerol accumulation ( hepatic steatosis ) .
hepatic steatosis can be a benign condition , or it may evolve with inflammation ( steatohepatitis ) , fibrosis , and cirrhosis , an altered spectrum termed nonalcoholic fatty liver disease ( nafld ) .
insulin resistance is a condition wherein higher than normal insulin levels are needed to provoke normal metabolic responses or where normal metabolic responses are not achieved with normal insulin concentrations .
depending on the primary site of involvement , the insulin resistance can be central ( liver ) or peripheral ( muscle or fat tissue ) . in nafld
, the initial site appears to be in the periphery , followed by or resulting in hepatic steatosis , which exacerbates hepatic insulin resistance and thus the degree of overall insulin resistance .
peripheral insulin resistance increases the serum levels of free fatty acids derived from the lipolysis of triacylglycerol from white adipose tissue ; these fatty acids are taken up by the liver and used in the production of triacylglycerol .
in addition , chronic hyperinsulinemia resulting from overall insulin resistance promotes de novo hepatic lipogenesis through the upregulation of lipogenic transcription factors .
a transcription factor that participates in the development of fatty liver in rodents is srebp-1c ( sterol regulatory element - binding protein-1c ) . in the nucleus ,
peroxisome proliferator - activated receptor gamma ( ppar ) is another transcription factor involved in the development of hepatic steatosis in rodents . in the liver ,
ppar is usually expressed at very low levels , but its expression is markedly increased in animal models exhibiting insulin resistance and fatty livers .
increases in oxidative stress and in factors that promote proinflammatory cytokine expression , such as interleukin ( il)-6 and tumor necrosis factor- ( tnf ) , are implicated in the development of nonalcoholic steatohepatitis ( nash ) , with the nuclear factor kappa b ( nf-b ) playing a critical role in the modulation of proinflammatory transcription . moreover ,
several studies have documented the association of insulin resistance ( liver and adipose tissue ) with inflammation .
protein restriction is associated with hepatic steatosis . in our laboratory , we have studied an experimental model of protein restriction in early life that demonstrates deficits in insulin secretion and liver insulin resistance in adulthood .
we use soybean flour diets as an alternative low - cost and high - quality protein source to feed these animals in an attempt to prevent and/or to treat the long - term consequences of malnutrition .
this choice was motivated by scientific evidence showing that the consumption of soy protein and isoflavones may exert beneficial effects on glucoregulation , lipotoxicity in the liver , and lipidemia by acting on a wide spectrum of biochemical and molecular activities [ 21 , 22 ] .
interestingly , we observed that serum insulin concentrations were increased in animals reared on a soybean diet , but alterations occurred in the early steps of the hepatic insulin signal transduction pathway , indicating hepatic insulin resistance [ 23 , 24 ] .
moreover , the weight and lipid content of the white adipose tissue as well as the lipolysis rate by isoproterenol in white adipocytes were decreased in rats fed the soybean diet .
thus , the aim of this study was to investigate the effects of nutritional recovery after weaning with a soybean flour diet on de novo hepatic lipogenesis and inflammation in adult rats exposed to protein restriction during intrauterine life and lactation .
the experimental procedures involving rats were performed in accordance with the guidelines of the brazilian college for animal experimentation ( cobea ) and were approved by the ethics committee at the federal university of mato grosso .
male and virgin female wistar rats ( 8590 days old ) were obtained from the university 's own breeding colony .
mating was performed by housing males with females overnight ( 1 male and 4 females ) , and pregnancy was confirmed by the presence of sperm in vaginal smears .
pregnant females were separated at random and maintained from the first day of pregnancy until the end of lactation on isocaloric diets containing 6% ( low protein ( lp ) diet , n = 6 ) or 17% ( control ( c ) diet , n = 8) protein .
spontaneous delivery occurred at day 22 of pregnancy , after which , at 3 days of age , large litters were reduced to eight pups to ensure a standard litter size per mother . after weaning in the 4th week , the males were divided into five groups : cc , consisting of offspring born to and suckled by mothers fed a c diet and subsequently fed the same diet after weaning until 90 days of age ; cs , consisting of offspring born to and suckled by mothers fed a c diet and subsequently fed a soybean flour diet with 17% protein after weaning until 90 days of age ; ll , consisting of offspring born to mothers fed an lp diet and subsequently fed the same diet after weaning until 90 days of age ; lc , consisting of offspring born to mothers fed an lp diet and subsequently fed a c diet after weaning until 90 days of age ; and ls , consisting of offspring born to mothers fed an lp diet and subsequently fed a soybean flour diet containing 17% protein after weaning until 90 days of age .
the diets have been previously described [ 23 , 26 ] . throughout the experimental period ,
the homeostasis model assessment of insulin resistance ( homa - ir ) index was assessed from the basal glucose and insulin concentrations using the following formula : basal glucose ( mmol / l ) basal insulin ( u / ml)/22.5
. fed rats were euthanized , and liver tissue samples were quickly removed , frozen immediately in liquid nitrogen , and stored at 80c to determine malic enzyme ( me ) and citrate lyase ( cly ) activities ; hepatic fat content ; and nsrebp-1c ( nuclear sterol regulatory element - binding protein-1c ) , ppar , me , cly , tnf , nf-b , and il-10 expression levels by immunoblotting .
the mrna expression levels of tnf , il-6 , il-10 , and nf-b were determined by real - time pcr .
liver samples were obtained for in vivo lipogenesis measurements from another group of rats in the fed state .
blood glucose concentrations were determined using a portable glucose meter ( accu - chek , roche diagnostics , mannheim , germany ) .
after euthanasia , aliquots of serum obtained by centrifugation were used to measure total serum protein , serum triacylglycerol , and hepatic aminotransferase concentrations ( bt-3000 plus , wiener lab , rosario , argentina ) .
me activity was assayed by the method of ochoa in accordance with the modifications proposed by hsu and lardy .
h2o ( 3 mci ) dissolved in 0.3 ml saline was administered to anesthetized rats by intraperitoneal injection 1 h before the experiment .
h2o was removed from the inferior phase ( predominantly chloroform ) by washing three times with a superior - phase mixture . after each shaking ,
the tubes were briefly centrifuged to sharpen the phase boundary , and the superior phase was aspirated and discarded .
isolation and counting of h2o - labeled fatty acids from the inferior phase was performed as previously described . for the calculations of lipid synthesis in experiments with h2o
, it was assumed that the specific activity of intracellular water was identical to that of plasma water , which was determined directly in aliquots of diluted ( 20 times ) plasma .
the rates of tissue lipid synthesis were calculated assuming that each fatty acid incorporated into triacylglycerol contained 13.3 atoms of tritium .
all sections were coded and analyzed blindly by the pathologist without knowledge of related characteristics or diet .
the degree of fat accumulation was graded on a scale of 0 to 4 as follows : 0 = no evidence of or barely visible microvesicular fat ; 1 + = < 25% ; 2 + = 25% to 49% ; 3 + = 50% to 75% ; 4 + = fat involving > 75% of the lobule .
one frozen liver fragment was homogenized in freshly prepared buffer ( 1% triton x-100 , 100
mmol / l tris - hcl ( ph 7.4 ) , 100 mmol / l sodium pyrophosphate , 100 mmol / l sodium fluoride , 10 mmol / l edta , 10 mmol / l sodium orthovanadate , 2.0 mmol / l pmsf , and 0.1 mg aprotinin / ml ) .
insoluble material was removed by centrifugation for 20 min at 12000 g at 4c .
samples containing 40 g of protein from each experimental group were incubated with 4x concentrated laemmli sample buffer and 15 mg of dtt and assayed on polyacrylamide gels at 120 v for 90 min ( 10% gels for me and cly ) .
the electrotransfer of proteins to nitrocellulose membranes ( bio - rad ) was per formed for 90 min at 120 v in buffer containing methanol and sds . after ascertaining transfer efficiency by ponceau staining ,
the membranes were blocked with 5% albumin in tween - tris buffered saline ( ttbs ) ( 10 mmol / l tris , 150 mmol / l nacl , 0.5% tween 20 ) overnight at 4c . me , cly , tnf , nf-b , and
il-10 were detected in the membranes after a 2 h incubation at room temperature with anti - me1 rabbit monoclonal igg ( sigma - aldrich , st .
louis , mo , usa ) , anti - cly polyclonal igg ( cell signaling technology inc . ,
usa ) , anti - tnf rabbit polyclonal igg , anti - nf-b mouse monoclonal igg , or anti - il-10 goat polyclonal igg ( santa cruz biotechnology , usa ) diluted 1 : 500 in ttbs containing 3% dry albumin .
enhanced chemiluminescence was performed ( supersignal west pico , pierce ) after incubation with the appropriate horseradish peroxidase - conjugated secondary antibody .
nuclear extracts ( 20 g ) were separated by sds - page in 10% gels according to milanski et al . .
the membranes were blocked with 5% skim milk in tween - tris - buffered saline ( ttbs ) ( 10 mmol / l tris , 150 mmol / l nacl , 0.5% tween 20 ) overnight at 4c .
nsrebp-1c and ppar were detected in the membranes after a 2 h incubation at room temperature with anti - srebp-1c rabbit polyclonal igg and anti - ppar mouse polyclonal igg ( santa cruz biotechnology ; diluted 1 : 500 in ttbs containing 3% dry skimmed milk ) .
histone was used as a marker of the subcellular fraction and a protein loading marker .
enhanced chemiluminescence was performed ( super signal west pico , pierce ) after incubation with the appropriate horseradish peroxidase - conjugated secondary antibody .
total rna was isolated from frozen liver samples by trizol reagent ( invitrogen , usa ) , according to the supplier 's instructions .
three micrograms of total rna were transcribed into cdna with high capacity reverse transcriptase ( applied biosystems ) .
primers specific for rat tnf ( rn00563005_m1 ) , il-6 ( rn99999011_m1 ) , il-10 ( rn00563409_m1 ) , nf-b ( rn01399583_m1 ) , and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ( rn01775763_g1 ) were obtained from applied biosystems .
pcr was carried out in duplicate on a step one system using taqman gene expression master mix ( applied biosystems ) .
the cdna was amplified under the following conditions : 95c for 10 min for denaturation and then 40 cycles of 95c for 15 s , 60c for 20 s , and 72c for 15 s , followed by extension at 72c for 10 min .
real - time data were analyzed using the step one system ( applied biosystems ) .
the results are expressed as the means with their respective standard deviations for the number of rats indicated in parentheses .
bartlett 's test for the homogeneity of variances was initially used to determine whether the data complied with the assumptions necessary for a parametric anova .
when necessary , the data were log transformed to correct for variance in heterogeneity or nonnormality . a two - way anova ( i.e. , effects of nutritional status in early life and diet ) was used to compare the data from the cc , cs , lc , and ls groups . a one - way anova was used to assess whether the diets were effective at improving the nutritional status of the lc , ls , and ll groups .
when necessary , these analyses were complemented by the least significant difference test to determine the significance of the individual differences .
all statistical comparisons were conducted using the statistica software package ( stat - soft ) .
at the beginning of the recovery phase , lc , ls , and ll rats had similar body weights , and in all cases , these were significantly lower than those of the cc and cs rats .
body weight at the end of the experimental period was significantly lower in the lc and ls groups than in the cc and cs groups ( p < 0.001 ) .
rats maintained on a soybean flour diet after weaning ( ls and cs groups ) had a lower final body weight than those fed a casein diet ( lc and cc groups ) ( p < 0.001 ) . although ls rats reached a higher final body weight than ll rats ( p < 0.001 ) , their weights were still significantly lower than those of lc rats ( p < 0.001 ) .
total serum protein concentrations did not differ among the cc , cs , lc , and ls groups .
the ls and lc rats had higher total serum protein levels than rats from the ll group ( p < 0.05 ) .
serum triacylglycerol levels were higher in the lc group than in the ls , cs , and cc groups . in the ls group ,
serum triacylglycerol concentrations were lower relative to those of lc rats but did not differ from those exhibited by the ll rats .
serum insulin concentrations were higher in rats fed a soybean diet ( cs and ls groups ) than in rats fed a casein diet ( cc and lc groups ) ( p < 0.0001 ) .
the homa - ir index was higher in rats fed a soybean diet than in those reared on casein ( p < 0.0001 ) .
similar glycemia levels , but higher insulinemia and homa - ir indices , were recorded in ls rats relative to the ll and lc groups .
there was no difference in homa - ir index or serum insulin concentrations between the ll and lc groups .
serum levels of alanine aminotransferase ( alt ) were higher in the ls and lc groups than in the cs and cc groups ( p < 0.01 ) and in rats maintained on a soybean diet ( ls and cs rats ) relative to those fed a casein diet ( lc and cc rats ) ( p < 0.05 ) .
there was no difference in alt levels between the ll , ls , and lc groups .
levels of serum gamma - glutamyl - transpeptidase ( gt ) in the ls group were significantly higher than those of the cs group but similar to those of the cc and lc groups .
the ls rats had significantly higher gt levels than the ll rats , but they were similar to those of the lc group .
alkaline phosphatase ( alp ) levels were significantly higher in the ls and lc groups than in the cs and cc groups ( p < 0.01 ) .
the ls and lc groups showed alp concentrations that were similar to each other and significantly lower than those observed in the ll group ( table 1 ) .
hepatocellular lipid accumulation ( steatosis ) was assessed in liver sections using the conventional hematoxylin - eosin staining technique ( figure 1(a ) ) .
liver histology revealed that rats fed a soybean diet did not show an abnormal accumulation of fat in their livers because 100% of liver specimens from the ls and cs groups were graded as 0 . in the lc group , 60% of liver specimens
were graded as 0 and 40% as 1 or 2 , whereas in the cc group , 80% were graded 1 or 2 and 20% as 3 or 4 . in the ll group ,
60% were graded 1 or 2 and 40% as 3 or 4 ( figure 1(b ) ) .
the hepatic fat content ( figure 1(c ) ) was reduced in rats fed a soybean diet ( ls and cs groups ) compared with rats fed a casein diet ( lc and cc groups ) ( p < 0.001 ) .
moreover , rats fed the soybean flour diet ( cs and ls groups ) exhibited lower fatty acid synthesis rates compared with those fed the casein diet ( cc and lc rats ) ( p < 0.001 ) .
the fatty acid synthesis rate in the ls group was lower than that of the ll group and similar to that observed in the lc group ( figure 1(d ) ) .
nsrebp-1c protein levels were similar across the cs , cc , ls , and lc groups .
however , the ls and lc groups showed significantly higher nsrebp-1c than the ll group ( figure 2(a ) ) .
rats fed the soybean diet ( cs and ls groups ) had lower nppar levels than those fed the casein diet ( cc and lc groups ) ( p < 0.05 ) .
there was no difference in nppar levels among the ll , ls , and lc groups ( figure 2(b ) ) .
liver me and cly contents were significantly lower in rats fed the soybean diet ( cs and ls groups ) relative to those fed the casein diet ( cc and cs groups ) ( p < 0.05 and p < 0.01 , resp . ) .
livers from the ls group also exhibited lower me and cly contents than livers from the ll and lc groups ( figures 3(a ) and 3(b ) ) .
the cs and ls groups showed lower me and cly activities compared with the cc and lc groups ( p < 0.05 and p < 0.01 , resp . ) .
moreover , me and cly activities were lower in the ls rats than in the lc rats , and these activities were significantly lower in both groups relative to the ll rats ( figures 3(c ) and 3(d ) ) .
mrna levels of nf-b ( figure 4(a ) ) and il-6 ( figure 4(b ) ) were significantly reduced in the lc and ls groups relative to the cc and cs groups ( p < 0.0001 ; p < 0.005 ) .
no difference was observed in the nf-b and il-6 mrna expression among the lc , ls , and ll groups .
tnf mrna levels ( figure 4(c ) ) were significantly higher in rats fed the soybean diet ( ls and cs groups ) than in those fed casein ( lc and cc groups ) ( p < 0.01 ) .
the tnf mrna levels of the ls group were similar to those of the lc group and higher relative to the ll group ( p < 0.05 ) .
il-10 mrna expression ( figure 4(d ) ) was not significantly different among the cc , cs , lc , and ls groups .
however , the ls group exhibited significantly higher il-10 mrna expression than that observed in the ll and lc groups ( p < 0.01 ) .
nf-b ( figure 5(a ) ) and il-10 ( figure 5(b ) ) protein levels did not differ among the cc , cs , lc , and ls groups . however , ls and lc rats exhibited higher nf-b protein levels than ll rats .
tnf protein levels were higher in the cs and ls groups than in the cc and lc groups ( p < 0.05 ) , but no difference in tnf levels was observed among the ll , ls , and lc groups ( figure 5(c ) ) .
in this study , rats recovered with the soybean diet showed low body weight and normalization of serum triacylglycerol concentrations , corroborating the well - known favorable effect of soy protein containing various levels of isoflavones on somatic parameters [ 39 , 40 ] and serum triacylglycerol levels [ 41 , 42 ] .
however , contrary to the observations that soy protein reduces serum insulin levels [ 42 , 43 ] , we verify here , as in previous studies [ 23 , 24 , 26 ] , that the consumption of a soybean flour diet increased serum insulin concentrations .
this effect may be attributable to genistein , which increases insulin secretion due to its ability to activate the camp - pka pathway .
interestingly , our animals fed with soybean protein showed low liver fat content ( assessed by gravimetric and histologic assays ) and liver insulin resistance as determined by the homa - ir index and confirmed by high serum alt concentrations ( a hepatic enzyme whose appearance reflects hepatic insulin resistance and nafld ) .
it is also noteworthy that abundant lipid droplets ( observed histologically ) as well as elevated liver fat contents were observed in the rats from the cc group .
hepatic steatosis has also been reported by others , who attributed this effect to the elevated amounts of carbohydrates and calories provided by the ain-93 diet . however , the cc rats exhibited homa - ir index values and serum alt concentrations not compatible with metabolic alterations or hepatocellular injury .
moreover , although liver fat contents appeared to be comparable between the cc and ll groups , twice as many liver specimens from the ll group were graded as 3 or 4 .
liver insulin resistance results in the overexpression of srebp-1c , whereas soy protein and genistein suppress the srebp-1c levels .
thus , in this and in a previous study , the interplay between liver insulin resistance , which induces srebp-1c overexpression , and other factors ( e.g. , genistein and soy protein ) that suppress its expression resulted in no change in nsrebp-1c contents or srebp-1c mrna expression .
an adequate explanation for the paradoxical association of liver insulin resistance with unmodified nsrebp-1c concentrations is the mild hyperinsulinemia and the euglycemia observed in our animals fed with soybean , as noted here and previously .
srebp-1c expression has been shown to be stimulated by glucose and insulin and repressed by glucagon [ 9 , 10 , 48 ] .
interestingly , the repression of srebp-1c expression by soy protein has been attributed to decreased insulin : glucagon ratios . in our animals , we previously observed reduced insulin : glucagon ratios in lc and ll rats compared with ls rats . in that prior study
, we observed that both the lc and ll groups exhibited lower serum insulin concentrations , lower liver insulin resistance , and basal glucose concentrations that were similar to those of ls rats .
surprisingly , only ll rats exhibited low nsrebp-1c levels , but ll and lc rats had similar liver fat storage levels , and both had higher liver fat content than ls rats .
in contrast , we observed that the soybean diet resulted in reduced expression of ppar , which targets the me and cly genes [ 49 , 50 ] . in agreement with this finding and with the observation that soy protein
reduces the expression of me , we verified low contents of both me and cly in liver samples from rats fed the soybean diet .
interestingly , studies have shown that the genetic deletion of ppar in the livers of lipodystrophic transgenic mice markedly attenuates the development of nafld , independent of the presence of hyperinsulinemia or hyperglycemia [ 50 , 51 ] .
liver fat storage is also regulated by the integrated activities of cellular enzymes that catalyze lipid synthesis .
insulin acts as a key regulator of triglyceride biosynthesis in the liver by modulating enzymatic activities involved in the synthesis of fatty acids . in this study , both cly and me activities were reduced , and consequently , a decrease in de novo lipogenesis was observed in rats fed with soybean .
it is well known that soy protein reduces me activity in the liver ; however , in contrast to our observations , this suppressive effect was accompanied by low serum insulin concentrations and a reduced insulin : glucagon ratio .
in contrast to the animals fed soybean , in rats maintained on a low - protein diet , cly and me activity levels were elevated , resulting in an increase in the rate of de novo fatty acid synthesis , despite low levels of serum insulin .
two factors may have contributed to the increased activities of these enzymes : an increased sensitivity to insulin and the high carbohydrate levels of the low - protein diet .
some lines of evidence suggest that cly and me activity is stimulated by insulin and high carbohydrate intake [ 52 , 53 ] .
thus , the high cly and me activity levels appeared to determine the elevated rate of de novo fatty acid synthesis in the livers of rats from the ll group .
curiously , the animals that were fed a soybean diet did not exhibit lipid accumulation in hepatocytes but did have high serum alt concentrations , a marker of liver injury .
a potential induction factor for the hepatocellular injury and fibrosis observed in nafld is oxidative stress .
it has been shown that chronic genistein supplementation at more than 500 mg / kg / day adversely affects liver structure and function .
moreover , genistein increases the level and activity of ppar [ 22 , 56 ] , which , in the liver , plays a pivotal role in fatty acid catabolism by upregulating the expression of numerous genes involved in mitochondrial and peroxisomal fatty acid oxidation .
consequently , activation of ppar can prevent and decrease hepatic fat storage , but the oxidation of fatty acids remains an important source of reactive oxygen species ( ros ) in fatty livers .
this hypothesis is weakened by the fact that the amount of genistein contained in our diet was determined to be lower than 500 mg / kg body weight / day , yet our soybean - fed animals exhibited reduced ppar mrna expression and unchanged ppar protein contents in the liver . however , these animals also exhibited reduced expression of liver acetyl - coenzyme a carboxylase beta ( acc ) , an enzyme localized in the mitochondrial membrane , where it is believed to regulate local malonyl - coenzyme a levels , carnitine palmitoyltransferase i ( cpt-1 ) activity , and fat oxidation .
the lack of acc is associated with continuous fatty acid oxidation and reduced fat storage .
we also previously observed a reduction in nonprotein thiol levels in the livers of soybean - treated animals , indicating the consumption of nonprotein thiols by enhanced free radical generation ( unpublished data ) .
hence , elevated alt may reflect inflammation , which impairs insulin signaling in the liver .
however , alt is also a gluconeogenic enzyme , and increased alt levels could therefore indicate impaired insulin signaling instead of liver injury . in our rats that were fed the soybean diet ,
the increased alt , combined with high tnf mrna and protein levels , is consistent with inflammation .
however , the unchanged nf-b expression in livers from these soybean - fed rats is indicative of the absence of fibrosis because nf-b is considered a profibrotic marker .
the rats that were rescued from malnutrition in early life showed reduced transcription of nf-b and il-6 and increased transcription of il-10 ( an anti - inflammatory cytokine ) . however , neither nf-b nor il-10 protein levels were altered under these conditions . given the elevated tnf content , it is reasonable to suggest that the soybean diet increased nf-b activity .
interestingly , in the livers of rats maintained on the soybean diet , the expression of tnf was correlated with the homa - ir index , confirming the role of this cytokine in the development of hepatic insulin resistance . in conclusion , the present study showed that a soybean diet prevented steatosis at least in part through reduced lipogenesis but resulted in tnf-mediated inflammation . | we evaluated the effects of postweaning nutritional recovery with a soybean flour diet on de novo hepatic lipogenesis and inflammation in adult rats exposed to protein restriction during intrauterine life and lactation .
rats from mothers fed with protein ( casein ) in a percentage of 17% ( control , c ) or 6% ( low , l ) during pregnancy and lactation were fed with diet that contained 17% casein ( cc and lc groups , resp . ) or soybean ( cs and ls groups , resp . ) after weaning until 90 days of age .
ls and cs rats had low body weight , normal basal serum triglyceride levels , increased alt concentrations , and high homa - ir indices compared with lc and cc rats .
the soybean diet reduced ppar as well as malic enzyme and citrate lyase contents and activities .
the lipogenesis rate and liver fat content were lower in ls and cs rats relative to lc and cc rats .
tnf mrna and protein levels were higher in ls and cs rats than in lc and cc rats .
nf-b mrna levels were lower in the lc and ls groups compared with the cc and lc groups .
thus , the soybean diet prevented hepatic steatosis at least in part through reduced lipogenesis but resulted in tnf-mediated inflammation . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | thus , the aim of this study was to investigate the effects of nutritional recovery after weaning with a soybean flour diet on de novo hepatic lipogenesis and inflammation in adult rats exposed to protein restriction during intrauterine life and lactation . after weaning in the 4th week , the males were divided into five groups : cc , consisting of offspring born to and suckled by mothers fed a c diet and subsequently fed the same diet after weaning until 90 days of age ; cs , consisting of offspring born to and suckled by mothers fed a c diet and subsequently fed a soybean flour diet with 17% protein after weaning until 90 days of age ; ll , consisting of offspring born to mothers fed an lp diet and subsequently fed the same diet after weaning until 90 days of age ; lc , consisting of offspring born to mothers fed an lp diet and subsequently fed a c diet after weaning until 90 days of age ; and ls , consisting of offspring born to mothers fed an lp diet and subsequently fed a soybean flour diet containing 17% protein after weaning until 90 days of age . body weight at the end of the experimental period was significantly lower in the lc and ls groups than in the cc and cs groups ( p < 0.001 ) . rats maintained on a soybean flour diet after weaning ( ls and cs groups ) had a lower final body weight than those fed a casein diet ( lc and cc groups ) ( p < 0.001 ) . serum triacylglycerol levels were higher in the lc group than in the ls , cs , and cc groups . serum insulin concentrations were higher in rats fed a soybean diet ( cs and ls groups ) than in rats fed a casein diet ( cc and lc groups ) ( p < 0.0001 ) . similar glycemia levels , but higher insulinemia and homa - ir indices , were recorded in ls rats relative to the ll and lc groups . serum levels of alanine aminotransferase ( alt ) were higher in the ls and lc groups than in the cs and cc groups ( p < 0.01 ) and in rats maintained on a soybean diet ( ls and cs rats ) relative to those fed a casein diet ( lc and cc rats ) ( p < 0.05 ) . alkaline phosphatase ( alp ) levels were significantly higher in the ls and lc groups than in the cs and cc groups ( p < 0.01 ) . the hepatic fat content ( figure 1(c ) ) was reduced in rats fed a soybean diet ( ls and cs groups ) compared with rats fed a casein diet ( lc and cc groups ) ( p < 0.001 ) . moreover , rats fed the soybean flour diet ( cs and ls groups ) exhibited lower fatty acid synthesis rates compared with those fed the casein diet ( cc and lc rats ) ( p < 0.001 ) . rats fed the soybean diet ( cs and ls groups ) had lower nppar levels than those fed the casein diet ( cc and lc groups ) ( p < 0.05 ) . liver me and cly contents were significantly lower in rats fed the soybean diet ( cs and ls groups ) relative to those fed the casein diet ( cc and cs groups ) ( p < 0.05 and p < 0.01 , resp . ) the cs and ls groups showed lower me and cly activities compared with the cc and lc groups ( p < 0.05 and p < 0.01 , resp . ) moreover , me and cly activities were lower in the ls rats than in the lc rats , and these activities were significantly lower in both groups relative to the ll rats ( figures 3(c ) and 3(d ) ) . mrna levels of nf-b ( figure 4(a ) ) and il-6 ( figure 4(b ) ) were significantly reduced in the lc and ls groups relative to the cc and cs groups ( p < 0.0001 ; p < 0.005 ) . tnf mrna levels ( figure 4(c ) ) were significantly higher in rats fed the soybean diet ( ls and cs groups ) than in those fed casein ( lc and cc groups ) ( p < 0.01 ) . tnf protein levels were higher in the cs and ls groups than in the cc and lc groups ( p < 0.05 ) , but no difference in tnf levels was observed among the ll , ls , and lc groups ( figure 5(c ) ) . in our rats that were fed the soybean diet ,
the increased alt , combined with high tnf mrna and protein levels , is consistent with inflammation . in conclusion , the present study showed that a soybean diet prevented steatosis at least in part through reduced lipogenesis but resulted in tnf-mediated inflammation . | [
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] |
changes in prostate cancer ( pc ) incidence of migrant populations and geographical differences in pc incidence rates [ 2 , 3 ] have motivated the study of possible lifestyle and environmental factors involved in the development of pc , including diet .
pc is the second most commonly diagnosed cancer among men globally . among argentinean men , pc is the most frequently diagnosed cancer and it is the third most common cause of cancer death .
however , the etiology of prostate carcinoma is mostly unknown and the role of dietary habits is rather controversial .
high intakes of some foods , such as dairy products , red meats , and processed meats , have been suggested as possible risk factors .
additionally , nutrients including -linolenic acid and calcium seem to play a role in prostate carcinogenesis . despite the increasing number of published papers addressing the relationship between dietary habits and pc from different approaches ,
the issue is still open to discussion . due to the complexity of dietary intake and
the potential for effect modifications among dietary components , a dietary eating patterns approach could be more suitable than the traditional analysis of isolated foods and nutrients . factor analysis has been broadly used in research into diet and pc associations in the last two decades to describe diet and disease associations .
dietary patterns approach deals with the issue of collinearity of nutrients and possible interdependencies between foods and nutrients .
in addition , it simplifies the interpretation of a complex and multidimensional phenomenon such as dietary intake .
several studies have examined population dietary patterns related to pc in the last decade [ 913 ] . however , this strategy has not yet been addressed in argentina for the study of pc .
traditionally , in populations of the region known as the southern cone ( that includes argentina , uruguay , and chile ) , the contribution of meat ( especially red meat ) to energy intake has been of considerable magnitude , providing in some cases about 50 percent of total daily energy [ 14 , 15 ] . according to the fao food balance sheets , in argentina
the per capita food supply of meat was 193 g / day in 2011 , ranking first of all countries of america , while the united states comes on second place with 183 g / day .
several observational studies found that higher intakes of total meat as well as red and processed meat were associated with the occurrence of pc when they were analyzed as an individual food group or as a characteristic of a dietary pattern [ 12 , 13 ] .
furthermore , and according to the global nutritional transition process , in the southern cone population there were changes in food consumption related to the inclusion of high - energy refined foods .
consequently , additional attention must be paid to the study of cancer risk in this region due to its population 's eating habits .
thus , it is necessary to consider the complex process of food consumption , intercrossed by many other cultural habit characteristics of subjects and populations .
the objective of this study was to estimate the effect of characteristic dietary patterns on the occurrence of pc in argentinean men .
the study was conducted within the framework of the environmental epidemiology of cancer in crdoba ( eecc ) project . in addition to case - control studies about dietary and other environmental exposures related to the cancers of highest incidence , the project includes the study of incidence analysis and spatial distribution and mortality trends and patterns .
this case - control study was conducted from january 2008 to december 2013 in crdoba , the second most populated argentinean province ( 3,067,000 inhabitants , according to the 2010 census ) , located in the center of the country .
cases were men with incident , histologically confirmed pc ( icd-10th edition , icie10:c61 ) with no previous diagnosis of cancer in other sites . they were identified in public and private health institutions registered at the crdoba tumor registry ( ctr ) .
two controls per case , frequency matched by age ( 5 years ) and area of residence , were randomly chosen from the census list and included only after verifying the absence of any neoplastic or related condition as well as diseases or other conditions that generate long - term modifications to dietary habits .
a total of 147 men with pc aged 4889 ( median age 72 ) and 300 controls aged 4689 years ( median age 71 ) were included . on average ,
10% of cases and 10% of controls invited to take part in the interview refused to participate .
subjects interviewed were from rural ( 54% ) and urban ( 46% ) areas ( including the most populated area , crdoba city , with 1,300,000 inhabitants ) , in representative proportions of the total population of crdoba province .
a structured questionnaire was completed including information about sociodemographic characteristics , occupational history , smoking habits , alcohol consumption , self - reported anthropometric characteristics , physical activity , medical insurance , personal medical history , and family history of cancer . to assess dietary exposure , a validated food frequency questionnaire ( ffq ) of 127 items
subjects were asked about their dietary intake over the 5 years prior to diagnosis ( cases ) or interview ( controls ) .
the ffq was coupled with an also validated photographical atlas based on standard portion sizes in argentina . the seasonal pattern of consumption of each vegetable or fruit was also taken into account .
frequency and duration of physical activity were then expressed as metabolic equivalent of tasks ( mets ) . in the present work ,
a principal component factor analysis ( pcfa ) and a varimax rotation method were applied on 300 male controls to characterization of dietary patterns .
the food items contained in the dietary ffq were classified into 24 predefined food groups based on similarities in the nutrient profile and culinary usage in the argentinean diet : milk / yogurt , cheese , lean red meat , fatty red meat , processed meat , offal , chicken , fish , eggs , fruits , nonstarchy vegetables , starchy vegetables , nuts , refined cereals , whole grains , bakery products , pulses , added sugar and sweets ( sugar , jam , honey , and caramels ) , candies ( dulce de leche ( milk jam ) , ice cream , chocolates , and peanut butter ) , vegetable oils , fats , infusions , sugary drinks , and alcoholic drinks .
factor analysis was then applied to reduce the food groups to a small number of factors that explained the maximum fraction of the variance .
the factorability of the correlation matrix was evaluated by applying the same criteria used previously . to determine the number of components to be retained , the eigenvalues ( greater than 1 ) and the scree test were considered .
furthermore , the percentage of variance explained by each factor and the interpretability of the factors were taken into account .
each factor was named according to its dominant food groups and those with an absolute rotated factor loading 0.40 were considered .
each pattern was then correlated with life style and sociodemographic characteristics , using direct and partial correlation coefficients . as a second step ,
after that , all participants were categorized into quartiles of adherence to each factor score .
a multilevel logistic regression ( mlr ) model for the binary response ( 1 if a case , 0 otherwise ) was fitted . a hierarchical structure in the data was assumed : subjects ( level 1 ) , in order to assess individual - level variable effects such as dietary patterns to the outcome , clustered into a second level of aggregation , the family history of cancer , defined according three categories , first- or second - degree relatives with pc , first- or second - degree relatives with other cancer , or no family history of cancer .
identified dietary patterns , energy intake , body mass index ( bmi ) , and occupational exposure ( industrial exposure to chemical contaminants recognized by iarc as carcinogens , i.e. , industries such as dyes , paints , textiles , plastics , rubber , leather , herbicides , automotive , chemical , and coal industry , for at least two years ) were included as first - level covariates .
a period of two years or more was considered because in the exploratory analysis higher risk was identified from this time onwards .
mor can be conceptualized as the increased risk that ( in median ) a subject would have if moving from one context of family history of cancer to another . in this study
, mor shows the extent to which the individual probability of having pc is determined by belonging to the family history of pc group .
a multiple probabilistic sensitivity analysis was performed by assigning conventional probability density distributions to the values of the bias parameters .
differential misclassification of exposure was assumed by drawing the sensitivities and specificities from different trapezoidal distributions for cases and controls .
minimum values equal to 0.70 and 0.75 and maximum ones equal to 0.90 and 1 were assigned in cases and controls specificity , respectively , while both sensitivities ranged from 0.75 to 1 .
lower specificity in the cases group was assigned taking into account the possibility of recall bias .
moreover , a higher probability to select unexposed cases and controls was assumed as respondents could have an increased interest in health - related issues and have healthier habits than nonrespondents . however , a small association between respondents - nonrespondents and pc is to be expected .
thus , we assigned a prior log - normal distribution to the selection - bias factor with mean 0 and standard deviation 0.21 .
this value of standard deviation is such that it permits the bias factor to fall 95% of times between 0.7 and 1.5 ( exp(1.960.21 ) and exp(1.960.21 ) ) , which yields 95% prior probability of the bias factor falling between exp(1.960.21 ) = 0.7 and exp(1.960.21 ) = 1.5 .
finally , the potential confounding effect introduced by the effect of central obesity was considered as this condition could be associated with pc and with a risky dietary pattern ( such as the traditional pattern identified in this study ) .
thus , a prevalence of the confounder of 0.2 to 0.3 and 0.1 to 0.2 among those exposed and unexposed to traditional pattern was assigned , respectively . a log - normal prior probability distribution for the confounder - pc or , with 95% confidence limits of ln(0.4 ) and ln(0.9 ) , was specified .
thus , the mean of this prior distribution is { ln(0.4 ) + ln(0.9)}/2 = 1.1268 with standard deviation { ln(0.4 ) + ln(0.9)}/(21.96 ) = 0.0575 .
the multiple probabilistic sensitivity analysis was applied to the effect of traditional pattern on the risk of pc as it is the most characterizing pattern of the argentinean diet [ 16 , 20 ] .
both groups had a similar distribution of age , socioeconomic status , smoking habits , physical activity , energy intake , and bmi .
on the other hand , occupational exposure and low educational level displayed higher percentages among cases compared with controls .
factor loadings for food groups and the variance explained by each factor are shown in table 2 .
four distinct dietary patterns were identified from the factor analysis explaining 31.5% of total variance .
the first factor , labeled traditional pattern , was positively loaded for fatty red meats , offal , processed meat , starchy vegetables , added sugars and sweets , candies , fats , and vegetable oils .
the second factor , consisted of high loadings for nonstarchy vegetables , whole grains , and low loadings for alcoholic drinks , was named prudent pattern .
the third factor , characterized by high loadings for sodas / juices and bakery products , was named carbohydrate pattern .
the last factor was labeled cheese pattern because it was positively loaded for this food group and negatively loaded for fish . in this last pattern ,
even if both foods groups had similar loadings values , the name was chosen based on the food group more frequently consumed .
the traditional pattern correlates strongly with total energy intake , intake of carbohydrates , lipids , cholesterol , calcium , and vitamin e ( table 3 ) .
a higher score for the traditional pattern was associated with a lower proportion of men undertaking physical activity , while the inverse was found for the carbohydrate ( table 4 ) .
also , in higher quartiles for the prudent pattern as well as the cheese pattern a lower proportion of smokers was found .
distributions for the rest of the characteristics studied were similar across quartiles . the traditional pattern and carbohydrate pattern were significantly associated with pc risk ( or 2.54 ; 95% ci 1.4914.342 and or 2.10 ; 95% ci 1.4003.164 , resp . , quartile 1 as baseline ) ,
while the prudent and cheese patterns were not significantly associated ( or 1.31 ; 95% ci 0.4933.508 and or 1.02 ; 95% ci 0.5381.932 , resp . )
it was observed that over 30% of the variance of the outcome is attributable to this clustering ( icc = 0.33 ) .
a mor of 3.38 indicated that moving from no familiar history of cancer to a family history of pc increased by three times the individual odds of pc occurrence when randomly picking out two persons in different groups .
besides , probabilistic sensitivity analysis showed that systematic and random error - adjusted median ors ( 1.34 ) had slight differences with conventional ors ( 1.33 ) , and the ratio of 95% simulation limits including systematic and random error is nearly two times higher than the conventional one ( table 5 ) .
in total , four distinct dietary patterns in men participating in this study were identified ; these were labeled traditional pattern , prudent pattern , carbohydrate pattern , and cheese pattern . the higher adherence to traditional and carbohydrate patterns conferred an increased risk for pc .
the variance portions explained by each dietary pattern are similar to those reported in other studies of dietary patterns and pc which range from 1.72% to 11% [ 10 , 11 , 13 ] .
remarkably , two of the most characteristic patterns that emerged in this population had a promoting effect for pc occurrence .
the pattern labeled traditional was the most representative pattern and , with high loadings found for fatty red meats , offal , and processed meats , coincides with the main characteristics of argentinean food habits described in previous studies [ 14 , 15 ] .
frequently , patterns with red meat and/or processed meat and eggs in other studies were named
( which include also sugar and candies ) , processed , or carnic
high adherence to these patterns increased the risk of pc [ 10 , 11 , 13 , 27 , 28 ] , in agreement with our results .
nevertheless , in other studies similar patterns do not show association with this disease [ 29 , 30 ] , including one of the largest studies published to date on dietary patterns and pc .
however , among men aged > 65 years , greater adherence to the western pattern suggested an increased risk of pc in the aforementioned study .
the consumption of red meat in some countries of south america is among the highest in the world .
uruguay and argentina rank first and second , respectively , with about 60 kg per year per capita . specifically in this study , subjects with higher adherence to the traditional pattern consumed a mean of 313 grams of fatty red meat daily , including the intake of offal , frequently added to the traditional argentinean barbecue or parrillada . in fact , charcoal grilling is one of the most common methods for cooking meat in argentina which results in a high formation of heterocyclic amines and polycyclic aromatic hydrocarbons , such as benzo(a)pyrene , both of which are considered carcinogens in animals .
several case - control studies [ 3337 ] have reported that meat overall and salted and red meat intake have a positive relation with pc , all of them showing ors of 1.5 or higher .
the basis of the association between pc and high consumption of red meat is not known .
this mineral is essential for testosterone synthesis and may have other effects on the prostate .
additionally , meat is the major contributor to fat intake in the southern cone diet .
thus , risk increase shown with high adherence to traditional pattern may reflect the high exposure to saturated fat from fatty meats and from fats , also present in this pattern .
high fat intake ( mainly saturated fatty acids and linoleic acid ) appears to be associated with an increased risk of pc [ 6 , 38 ] .
the main dietary oil consumed in this population is sunflower oil , which is predominantly a mixture of oleic ( omega-9 ) and linoleic fatty acids ( omega-6 ) .
however , ma and chapman , after reviewing numerous studies , concluded there was not enough evidence to draw conclusions about polyunsaturated fatty acid intake .
also high intakes of eggs , starchy vegetables , and added sugar and sweets , the other food groups characterizing the traditional pattern , when analyzed separately were associated with a high risk of pc [ 4044 ] . nevertheless , the promoting effect for pc possibly results from the combination of food groups characterizing the traditional pattern . at the same time
some authors include the deficiency of vegetable foods intake as another hypothesis of high meat intake and pc association [ 37 , 41 ]
. the increasing risk of pc with high adherence to the carbohydrate pattern could be linked to the high carbohydrate content and glycemic index of food groups that characterize this pattern .
hyperglycemia induced by the intake of these beverages and foods stimulates high insulin secretions , which act per se as a growth factor and induce an increase of igf-1 ( insulin - like growth factor ) .
igf-1 stimulates anabolic metabolism , cell proliferation , and cell differentiation and can also inhibit apoptosis .
high intake of sodas , juices , sweets , added sugar , and other high glycemic index foods was associated with an increase in pc in other epidemiological studies [ 44 , 45 ] whereas others found no associations [ 46 , 47 ] . in most other studies , patterns comparable to the prudent pattern identified in the present study report similar results on pc risk .
the prudent pattern in a prospective study using data from several cohort studies in the united states showed no association with pc risk .
similar results were found in other studies regarding a prudent pattern ( that in some cases also included dairy foods or fish ) and its association with pc occurrence [ 10 , 11 , 13 , 27 , 29 , 31 ] .
nonstarchy vegetables and fruits intake have a protective role for diverse tumors , possibly associated with a high content of antioxidant compounds , specially carotenoids and vitamins c and e . however , the evidence in epidemiologic studies regarding vegetable intake and pc risk association is considered insufficient .
diverse studies showed suggestive , but not definitive , evidence that dairy products , as well as the nutrients they provide , may increase the risk of pc [ 5 , 49 ] .
cheeses are sometimes high in fat , animal protein , and calcium but also contain vitamin d and conjugated linoleic acid that may be protective [ 5052 ] .
negative loading for fish of this factor also reflects the low intake of fish of cordobesian population .
about 30% of subject in this study did not consume fish , and the mean intake among those who do consume is 21 g / day ( data not shown ) . in the present study heterogeneity of responses coming from a second level of aggregation such as the family history of cancer was considered in the risk estimation process .
family history of cancer was selected based on the known heritability of this disease derived from either genetic susceptibility or exposure to common environmental factors . in accordance
thus , the risk of pc in a subject without a family history of any cancer would increase if , given the same individual - level covariates , he had family history of pc .
systematic errors are frequent in observational epidemiologic studies ; however they seldom are measured quantitatively . in the present study
the possibility of selection bias as well as recall bias , a classification bias caused by
additionally , to avoid potentially important bias due to confounders , similar distribution of age and place of residence in cases and controls was sought , and both groups were interviewed in the same period . however , residual unmeasured confounders may be present , such as the presence of abdominal obesity , given its association with pc and with a high adherence to a risky dietary pattern .
ors adjusted through quantitative bias analysis were slightly different compared with conventional ones . therefore , the sensitivity analysis performed based on the possibility of systematic errors mentioned showed no major evidence of influence of bias .
ffqs may be prone to error ; however the reproducibility of our 5-year ffq has been accurately tested for epidemiological cancer studies .
this case - control study also benefits from the use of population rather than hospital based data , thus avoiding berkson 's bias ( where hospital controls might not represent the prevalence of exposure in the community from which cases arise ) .
the use of the principal component analysis for identifying dietary patterns has potential strengths and limitations . in the first place ,
the labeling of the patterns is mainly subjective and derived from the authors ' criteria . besides , resulting patterns in a posteriori methodologies are specific to the population from which they emerge , which results in difficulty comparing with other studies .
even so , pcfa is the main statistical method proposed today to derive dietary patterns in cancer epidemiology research .
the study of information of multiple food intakes summarized in a unique exposition measurement constitutes a methodological advantage since it addresses the problem of multicollinearity and simplifies the interpretation of results .
epidemiological and statistical literature provide asymptotic formulas for the computation of case - control sample sizes required for odds ratios , unadjusted or adjusted for a confounder .
however , all these recommendations only take into account fixed effects of covariates , including the intercept . the limited number of parameters imposed in the model ( one for each pattern ) and the constraint on the sources of variability ( a variance component to quantify the intraclass correlation ) constitute a suitable effort to compensate for the small size of our study .
the multilevel modeling approach constitutes a statistic and interpretative advantage as it proposes a theoretical construct for addressing diet - cancer relationship , based on an idea of reality organized hierarchically on dimensions ( familial or contextual ) [ 56 , 57 ] .
this is considered especially important in the study of health determinants , given that it provides relevant information that allows for assessing the importance of the context in different individual results in health .
the present work adds evidence about the effect of particular dietary patterns on pc occurrence , coupled with the association with the family history dimension .
we concluded that the traditional and the carbohydrate patterns could be associated with pc , possibly due to the presence of high loadings of fatty meats , eggs , starchy vegetables , and foods groups rich in sugar and fat , coupled with an absence of fresh vegetables , especially focusing on populations with a family history of pc . | there is increasing evidence that dietary habits play a role in prostate cancer ( pc ) occurrence .
argentinean cancer risk studies require additional attention because of the singular dietary pattern of this population . a case - control study ( 147 pc cases , 300 controls )
was conducted in crdoba ( argentina ) throughout 20082013 .
a principal component factor analysis was performed to identify dietary patterns .
a mixed logistic regression model was applied , taking into account family history of cancer .
possible bias was evaluated by probabilistic bias analysis .
four dietary patterns were identified : traditional ( fatty red meats , offal , processed meat , starchy vegetables , added sugars and sweets , candies , fats , and vegetable oils ) , prudent ( nonstarchy vegetables , whole grains ) , carbohydrate ( sodas / juices and bakery products ) , and cheese ( cheeses ) .
high adherence to the traditional ( or 2.82 , 95%ci : 1.5695.099 ) and carbohydrate patterns ( or 2.14 , 95%ci : 1.4703.128 ) showed a promoting effect for pc , whereas the prudent and cheese patterns were independent factors . pc occurrence was also associated with family history of pc .
bias adjusted ors indicate that the validity of the present study is acceptable .
high adherence to characteristic argentinean dietary patterns was associated with increased pc risk .
our results incorporate original contributions to knowledge about scenarios in south american dietary patterns and pc occurrence . | 1. Background
2. Methods
3. Results
4. Discussion
5. Conclusion | changes in prostate cancer ( pc ) incidence of migrant populations and geographical differences in pc incidence rates [ 2 , 3 ] have motivated the study of possible lifestyle and environmental factors involved in the development of pc , including diet . the study was conducted within the framework of the environmental epidemiology of cancer in crdoba ( eecc ) project . this case - control study was conducted from january 2008 to december 2013 in crdoba , the second most populated argentinean province ( 3,067,000 inhabitants , according to the 2010 census ) , located in the center of the country . in the present work ,
a principal component factor analysis ( pcfa ) and a varimax rotation method were applied on 300 male controls to characterization of dietary patterns . the food items contained in the dietary ffq were classified into 24 predefined food groups based on similarities in the nutrient profile and culinary usage in the argentinean diet : milk / yogurt , cheese , lean red meat , fatty red meat , processed meat , offal , chicken , fish , eggs , fruits , nonstarchy vegetables , starchy vegetables , nuts , refined cereals , whole grains , bakery products , pulses , added sugar and sweets ( sugar , jam , honey , and caramels ) , candies ( dulce de leche ( milk jam ) , ice cream , chocolates , and peanut butter ) , vegetable oils , fats , infusions , sugary drinks , and alcoholic drinks . a hierarchical structure in the data was assumed : subjects ( level 1 ) , in order to assess individual - level variable effects such as dietary patterns to the outcome , clustered into a second level of aggregation , the family history of cancer , defined according three categories , first- or second - degree relatives with pc , first- or second - degree relatives with other cancer , or no family history of cancer . the multiple probabilistic sensitivity analysis was applied to the effect of traditional pattern on the risk of pc as it is the most characterizing pattern of the argentinean diet [ 16 , 20 ] . the first factor , labeled traditional pattern , was positively loaded for fatty red meats , offal , processed meat , starchy vegetables , added sugars and sweets , candies , fats , and vegetable oils . the second factor , consisted of high loadings for nonstarchy vegetables , whole grains , and low loadings for alcoholic drinks , was named prudent pattern . the third factor , characterized by high loadings for sodas / juices and bakery products , was named carbohydrate pattern . the traditional pattern and carbohydrate pattern were significantly associated with pc risk ( or 2.54 ; 95% ci 1.4914.342 and or 2.10 ; 95% ci 1.4003.164 , resp . , quartile 1 as baseline ) ,
while the prudent and cheese patterns were not significantly associated ( or 1.31 ; 95% ci 0.4933.508 and or 1.02 ; 95% ci 0.5381.932 , resp . ) a mor of 3.38 indicated that moving from no familiar history of cancer to a family history of pc increased by three times the individual odds of pc occurrence when randomly picking out two persons in different groups . in total , four distinct dietary patterns in men participating in this study were identified ; these were labeled traditional pattern , prudent pattern , carbohydrate pattern , and cheese pattern . remarkably , two of the most characteristic patterns that emerged in this population had a promoting effect for pc occurrence . the pattern labeled traditional was the most representative pattern and , with high loadings found for fatty red meats , offal , and processed meats , coincides with the main characteristics of argentinean food habits described in previous studies [ 14 , 15 ] . frequently , patterns with red meat and/or processed meat and eggs in other studies were named
( which include also sugar and candies ) , processed , or carnic
high adherence to these patterns increased the risk of pc [ 10 , 11 , 13 , 27 , 28 ] , in agreement with our results . also high intakes of eggs , starchy vegetables , and added sugar and sweets , the other food groups characterizing the traditional pattern , when analyzed separately were associated with a high risk of pc [ 4044 ] . high intake of sodas , juices , sweets , added sugar , and other high glycemic index foods was associated with an increase in pc in other epidemiological studies [ 44 , 45 ] whereas others found no associations [ 46 , 47 ] . in most other studies , patterns comparable to the prudent pattern identified in the present study report similar results on pc risk . in the present study heterogeneity of responses coming from a second level of aggregation such as the family history of cancer was considered in the risk estimation process . we concluded that the traditional and the carbohydrate patterns could be associated with pc , possibly due to the presence of high loadings of fatty meats , eggs , starchy vegetables , and foods groups rich in sugar and fat , coupled with an absence of fresh vegetables , especially focusing on populations with a family history of pc . | [
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] |
spontaneous intracerebral hemorrhage ( ich ) is the second most common cause of stroke , occurring globally at a rate of 24.6 per 100000 person - years and causing severe disability and high mortality . in spite of recent clinical trials aimed at improving the outcome of ich patients ,
the median case fatality has not decreased over time according to a recent meta - analysis1,15,27,28,38 ) .
ct angiography ( cta ) spot sign refers to one or more foci with contrast enhancement within an ich as a radiologic surrogate marker for hematoma expansion and clinical outcome in acute ich11 ) .
hematoma growth is also one of the major determinants of mortality and poor outcome in acute ich6,8 ) .
several factors associated with the cta spot sign have been reported in the western population , such as large hematoma volume , anticoagulation , and the apoe 2 allele3,4,12 ) .
oral anticoagulant medication , hemostatic proteins , a cta spot sign , and high systolic blood pressure have also been reported as contributing factors to hematoma growth in acute ich1,11,14,26,34 ) . while the incidence of ich varies among different countries and
is relatively high in the asian population16,31 ) , there are few studies investigating risk factors and the relationship of the cta spot sign to hematoma growth in asian countries25,32,40 ) .
the current study was conducted to investigate the predictive factors associated with the cta spot sign and hematoma growth of acute spontaneous ich patients in korea .
from 2005 - 2012 , we collected retrospective data for consecutive patients with spontaneous supratentorial ich that were admitted to the stroke and cerebrovascular center at our hospital , which serves a population of approximately 560000 .
the inclusion criteria were the following : 1 ) age 18 years old and 2 ) cta - proven spontaneous supratentorial hemorrhage .
patients with a cerebral aneurysm rupture , traumatic ich , tumor bleeding , arteriovenous malformation - related ich , dural arteriovenous fistula associated ich , cavernous hemangioma , moyamoya disease , moyamoya syndrome , probable moyamoya disease , venous thrombosis , hemorrhagic transformation after cerebral infarction , ich after thrombolysis , or posterior reversible leukoencephalopathy were excluded . out of 713 eligible patients with intracerebral hemorrhage from 2005 - 2012
, 287 patients were excluded due to secondary causes of ich . of the remaining 426 ich patients
, we excluded those who arrived at our hospital more than 12 hours from ictus , as well as those who did not undergo cta at admission or at follow - up to determine the factors associated with a spot sign in ich at the acute stage ( fig .
we scrutinized baseline characteristics of the patients including onset age , hypertension , stroke history , antiplatelet drug medication , warfarin medication , reasons for antithrombotics medication , and aggravation of neurologic status indicated by a decrease in the glasgow coma scale ( gcs ) score 2 or a decrease in the national institutes of health stroke scale 4 within 24 hours of ictus .
the hematocrit , international normalized ratio ( inr ) , platelet count , and alanine transaminase ( gpt ) levels were recorded .
radiologic profiles were also assessed to determine the location of the hemorrhage , volume of the ich ( using the abc/2 method ) , location of the spot sign on an enhanced computed tomography ( ct ) image , intraventricular hemorrhage ( ivh ) degree ( indicating the number of hemorrhage - involved ventricles ) and the time from ictus until the ct scan was performed .
hemorrhages originating from the basal ganglia , thalamus , or corpus callosum were categorized as deep - seated hemorrhages .
the spot sign was defined as a contrast enhancement focus that was greater than 1 mm in size within the ich on the axial image of the cta regardless of the shape of the enhancement ( fig .
hematoma growth was defined as an absolute increase > 6 ml or a relative increase > 33% compared with the initial ct image , or an increased ivh determined on a follow - up ct scan within 14 days of ictus .
all patients presenting with intracranial hemorrhage arrived at our emergency department and underwent brain ct angiography with a multi - detector 64 slice ct scanner ( aquilion , toshiba medical system corporation , tokyo , japan ) .
secondary causes of ich were thoroughly ruled out through further studies , such as digital subtraction angiography , brain mr imaging , or cerebral venography .
a non - contrast ct examination was performed using an axial technique ( 120 kv , 70 ma , and 5 mm slice thickness reconstruction ) ; a multidetector ct scan was subsequently conducted by scanning from the base of the c7 vertebral body to the vertex using an axial technique ( 0.641 pitch , 0.5-mm collimation , 350 maximal ma , 120 kvp , 2 mm slice thickness ) with 80 - 100 ml of iodinated contrast material administered using a power injector at 3 - 4 ml per second into the antecubital vein with an approximately 16- to 18-second delay between the onset of contrast injection and the start of scanning .
protamine sulfate and vitamin k were administered as antidotes to patients with heparin - related and warfarin - related hemorrhage to induce hemostasis .
aminomethylbenzoic acid ( 150 mg ) was routinely injected with normal saline fluid for 3 days .
blood pressure was reduced to < 140 mm hg for systolic blood pressure ( sbp ) and < 90 mm hg for diastolic blood pressure ( dbp ) through nicardipine or labetalol iv injections for patients with an initial sbp < 180 mm hg or dbp < 110 mm hg . in the case of an initial sbp
> 180 mm hg or dbp > 110 mm hg , the initial sbp or dbp was reduced by approximately 15% .
brain ct or mr scanning was followed - up routinely as follows : 1 ) between 24 and 72 hours from admission , 2 ) 4 hours later if a spot sign was found on the initial enhanced ct image , 3 ) immediately if neurological deterioration occurred , or 4 ) 7 to 14 days from admission .
the hematoma volume was calculated using the formula abc/2 , where a is the greatest diameter on the largest hematoma image , b is perpendicular to line a and c is the vertical depth of the hematoma . the time to reach the target blood pressure according to our center 's guideline
screening was conducted by a neurological surgeon , and baseline characteristics were investigated by two training residents .
clinical outcomes were assessed by two trained stroke nurses who were not involved in the current study and unaware of the baseline treatments . for the descriptive analysis
, all baseline values are displayed as percentage , and the mean valuesstandard deviation or the median with the interquartile range are shown .
normality tests were performed on all potential continuous variables , and t - tests were conducted to confirm intergroup differences in cases with normal distributions . for categorical variables , a chi - square test was performed between independent two groups .
a binary logistic regression method was used to calculate association predictors for the cta spot sign and hematoma growth .
if the p value of a potential factor was less than 0.05 in the univariable analysis , it was entered into the multivariable analysis model , but backward - eliminated to p<0.1 .
the hosmer - lemeshow test was used as a goodness - of - fit test .
the mann - whitney u test was used to compare variables between two groups with non - normal distributions .
from 2005 - 2012 , we collected retrospective data for consecutive patients with spontaneous supratentorial ich that were admitted to the stroke and cerebrovascular center at our hospital , which serves a population of approximately 560000 .
the inclusion criteria were the following : 1 ) age 18 years old and 2 ) cta - proven spontaneous supratentorial hemorrhage .
patients with a cerebral aneurysm rupture , traumatic ich , tumor bleeding , arteriovenous malformation - related ich , dural arteriovenous fistula associated ich , cavernous hemangioma , moyamoya disease , moyamoya syndrome , probable moyamoya disease , venous thrombosis , hemorrhagic transformation after cerebral infarction , ich after thrombolysis , or posterior reversible leukoencephalopathy were excluded . out of 713 eligible patients with intracerebral hemorrhage from 2005 - 2012
, 287 patients were excluded due to secondary causes of ich . of the remaining 426 ich patients
, we excluded those who arrived at our hospital more than 12 hours from ictus , as well as those who did not undergo cta at admission or at follow - up to determine the factors associated with a spot sign in ich at the acute stage ( fig .
we scrutinized baseline characteristics of the patients including onset age , hypertension , stroke history , antiplatelet drug medication , warfarin medication , reasons for antithrombotics medication , and aggravation of neurologic status indicated by a decrease in the glasgow coma scale ( gcs ) score 2 or a decrease in the national institutes of health stroke scale 4 within 24 hours of ictus .
the hematocrit , international normalized ratio ( inr ) , platelet count , and alanine transaminase ( gpt ) levels were recorded .
radiologic profiles were also assessed to determine the location of the hemorrhage , volume of the ich ( using the abc/2 method ) , location of the spot sign on an enhanced computed tomography ( ct ) image , intraventricular hemorrhage ( ivh ) degree ( indicating the number of hemorrhage - involved ventricles ) and the time from ictus until the ct scan was performed .
hemorrhages originating from the basal ganglia , thalamus , or corpus callosum were categorized as deep - seated hemorrhages .
the spot sign was defined as a contrast enhancement focus that was greater than 1 mm in size within the ich on the axial image of the cta regardless of the shape of the enhancement ( fig .
hematoma growth was defined as an absolute increase > 6 ml or a relative increase > 33% compared with the initial ct image , or an increased ivh determined on a follow - up ct scan within 14 days of ictus .
all patients presenting with intracranial hemorrhage arrived at our emergency department and underwent brain ct angiography with a multi - detector 64 slice ct scanner ( aquilion , toshiba medical system corporation , tokyo , japan ) .
secondary causes of ich were thoroughly ruled out through further studies , such as digital subtraction angiography , brain mr imaging , or cerebral venography .
a non - contrast ct examination was performed using an axial technique ( 120 kv , 70 ma , and 5 mm slice thickness reconstruction ) ; a multidetector ct scan was subsequently conducted by scanning from the base of the c7 vertebral body to the vertex using an axial technique ( 0.641 pitch , 0.5-mm collimation , 350 maximal ma , 120 kvp , 2 mm slice thickness ) with 80 - 100 ml of iodinated contrast material administered using a power injector at 3 - 4 ml per second into the antecubital vein with an approximately 16- to 18-second delay between the onset of contrast injection and the start of scanning .
protamine sulfate and vitamin k were administered as antidotes to patients with heparin - related and warfarin - related hemorrhage to induce hemostasis .
aminomethylbenzoic acid ( 150 mg ) was routinely injected with normal saline fluid for 3 days .
blood pressure was reduced to < 140 mm hg for systolic blood pressure ( sbp ) and < 90 mm hg for diastolic blood pressure ( dbp ) through nicardipine or labetalol iv injections for patients with an initial sbp < 180 mm hg or dbp < 110 mm hg . in the case of an initial sbp
> 180 mm hg or dbp > 110 mm hg , the initial sbp or dbp was reduced by approximately 15% .
brain ct or mr scanning was followed - up routinely as follows : 1 ) between 24 and 72 hours from admission , 2 ) 4 hours later if a spot sign was found on the initial enhanced ct image , 3 ) immediately if neurological deterioration occurred , or 4 ) 7 to 14 days from admission .
the hematoma volume was calculated using the formula abc/2 , where a is the greatest diameter on the largest hematoma image , b is perpendicular to line a and c is the vertical depth of the hematoma . the time to reach the target blood pressure according to our center 's guideline
screening was conducted by a neurological surgeon , and baseline characteristics were investigated by two training residents .
clinical outcomes were assessed by two trained stroke nurses who were not involved in the current study and unaware of the baseline treatments .
for the descriptive analysis , all baseline values are displayed as percentage , and the mean valuesstandard deviation or the median with the interquartile range are shown .
normality tests were performed on all potential continuous variables , and t - tests were conducted to confirm intergroup differences in cases with normal distributions . for categorical variables , a chi - square test was performed between independent two groups .
a binary logistic regression method was used to calculate association predictors for the cta spot sign and hematoma growth .
if the p value of a potential factor was less than 0.05 in the univariable analysis , it was entered into the multivariable analysis model , but backward - eliminated to p<0.1 .
the hosmer - lemeshow test was used as a goodness - of - fit test .
the mann - whitney u test was used to compare variables between two groups with non - normal distributions .
all statistical calculations were performed using sas version 9.13 ( sas institute inc . , cary , nc ) .
the mean age of the 287 patients was 61.7 ( standard deviation 12.7 ) years .
of these patients , 161 ( 56.1% ) were male and 126 ( 43.9% ) were female .
hypertension was identified in 151 ( 52.6% ) , diabetes in 32 ( 11.5% ) , hyperlipidemia or statin medication in 62 ( 21.6% ) , current smoking in 103 ( 35.9% ) , daily alcohol consumption at an amount > 60 g in 41 ( 14.3% ) , antiplatelet agent medication in 51 ( 17.8% ) , warfarin medication in 7 ( 2.4% ) , ischemic stroke history in 11 ( 3.8% ) , and hemorrhagic stroke in 6 ( 2.1% ) patients .
forty ( 13.9% ) patients had lobar hemorrhage and 247 ( 86.1% ) had deep - seated hemorrhage .
the median hematoma volume was 16 ml ( range : 1 - 160 ml ) .
the cta spot sign was identified in 40 ( 13.9% ) patients . among the 40 patients with the spot sign , 27 ( 67.5% )
had 1 spot , 10 ( 25% ) had 2 spots , 1 ( 2.5% ) had 3 spots , and 2 ( 5% ) had 4 spots .
the median hounsfield units of the spot sign was 152 ( range : 74 - 297 ) .
thirty six ( 90% ) of 40 spot signs were identified within 3 hours from ictus .
baseline demography is described in detail in table 1 , 2 . in the univariable analysis ,
reasons for antithrombotics medication , time from ictus to ct scan <3 hours , hemorrhagic volume 30 ml , and intraventricular extension were significantly associated with a cta spot sign ( table 1 ) .
these variables , except for antithrombotics medication due to heterogeneous disease entity , were entered into the final multivariable binary logistic regression analysis model ( table 3 ) .
an inr 1.8 , alanine aminotransferase 40 iu , hemorrhage volume 30 ml , and a spot sign were significantly associated with hematoma growth ( table 2 ) in univariable analysis and then entered into multivariable logistic regression analysis ( table 4 ) .
warfarin medication and hematocrit were also entered into a multivariable model due to a borderline probability for hematoma growth ( p<0.1 ) ( table 2 , 4 ) .
after adjustment of potential factors , time from ictus to ct scan <3 hours was the only factor identified as a significant predictor for the cta spot sign ( or , 5.14 ; 95% ci , 1.76 - 15.02 ; p=0.068 ) .
intraventricular extension was revealed as a borderline association factor ( or , 1.96 ; 95% ci , 0.95 - 4.05 ; p=0.002 ) for the cta spot sign ( table 3 ) . on the other hand , a cta spot sign ( or , 5.70 ; 95% ci , 2.70 - 12.01 ; p<0.001 ) and elevated gpt level > 40 iu ( or , 2.01 ; 95% ci , 1.01 - 4.01 ; p=0.047 ) were independent predicting factors of hematoma growth . though an inr of 1.8 or greater was marginally related to hematoma growth in the multivariable binary logistic regression model 1 , an inr 1.8 or warfarin medication
was significantly associated with hematoma growth in model 2 ( table 4 ) . in the subgroup analysis of 78 hematoma
growth patients , the time from ictus to hematoma growth was shorter in the group with spot signs than in the group without any spot sign ( fig .
in addition to a spot sign ( or , 3.47 ; 95% ci , 1.15 - 10.48 ; p=0.027 ) and large hemorrhage volume of 30 ml or more ( or , 3.63 ; 95% ci , 1.25 - 10.57 ; p=0.018 ) , antiplatelet medication ( or , 4.92 ; 95% ci , 1.31 - 18.50 ; p=0.019 ) showed a significant association with hematoma growth within 6 hours from ictus in multivariable regression analysis ( table 5 ) .
in the univariable analysis , reasons for antithrombotics medication , time from ictus to ct scan <3 hours , hemorrhagic volume 30 ml , and intraventricular extension were significantly associated with a cta spot sign ( table 1 ) .
these variables , except for antithrombotics medication due to heterogeneous disease entity , were entered into the final multivariable binary logistic regression analysis model ( table 3 ) .
an inr 1.8 , alanine aminotransferase 40 iu , hemorrhage volume 30 ml , and a spot sign were significantly associated with hematoma growth ( table 2 ) in univariable analysis and then entered into multivariable logistic regression analysis ( table 4 ) .
warfarin medication and hematocrit were also entered into a multivariable model due to a borderline probability for hematoma growth ( p<0.1 ) ( table 2 , 4 ) .
after adjustment of potential factors , time from ictus to ct scan <3 hours was the only factor identified as a significant predictor for the cta spot sign ( or , 5.14 ; 95% ci , 1.76 - 15.02 ; p=0.068 ) .
intraventricular extension was revealed as a borderline association factor ( or , 1.96 ; 95% ci , 0.95 - 4.05 ; p=0.002 ) for the cta spot sign ( table 3 ) . on the other hand , a cta spot sign ( or , 5.70 ; 95% ci , 2.70 - 12.01 ; p<0.001 ) and elevated gpt level
> 40 iu ( or , 2.01 ; 95% ci , 1.01 - 4.01 ; p=0.047 ) were independent predicting factors of hematoma growth . though an inr of 1.8 or greater was marginally related to hematoma growth in the multivariable binary logistic regression model 1 , an inr 1.8 or warfarin medication
was significantly associated with hematoma growth in model 2 ( table 4 ) . in the subgroup analysis of 78 hematoma
growth patients , the time from ictus to hematoma growth was shorter in the group with spot signs than in the group without any spot sign ( fig .
in addition to a spot sign ( or , 3.47 ; 95% ci , 1.15 - 10.48 ; p=0.027 ) and large hemorrhage volume of 30 ml or more ( or , 3.63 ; 95% ci , 1.25 - 10.57 ; p=0.018 ) , antiplatelet medication ( or , 4.92 ; 95% ci , 1.31 - 18.50 ; p=0.019 ) showed a significant association with hematoma growth within 6 hours from ictus in multivariable regression analysis ( table 5 ) .
our study showed that an elapsed time from ictus to ct scan of 3 hours or less was an independent predictor for a cta spot sign . additionally , a cta spot sign and elevated gpt level were independent predictors for hematoma growth of acute ich patients in korea .
elevated inr was marginally associated with hematoma growth , and antiplatelet medication was particularly related to hematoma growth within 6 hours of ictus . only an elapsed time to ct scan of 3 hours or less was strongly associated with a cta spot sign in the current study ( table 3 ) . similar to our results , an elapsed time to cta scan of 3 hours or less as well as an intraventricular extension were shown to be predictors of a cta spot sign by delgado almandoz et al.9,10 ) . however , radmanesh et al.33 ) reported that a hemostatic factor , such as warfarin medication , was a shared predictor for a cta spot sign for both deep and lobar ich , while the time to cta scan was identified as a risk factor only for deep ich . on the contrary ,
in the present study , there was no difference in the occurrence of a cta spot sign between deep and lobar hemorrhages ( table 1 ) , and intraventricular extension showed a borderline association with the cta spot sign ( table 3 ) .
3 demonstrates that the time from ictus to hematoma growth of ich with a cta spot sign was shorter than that without a cta spot sign .
furthermore , in hyper - acute ich within 3 hours of ictus , there may be a high probability of the presence of a cta spot sign , which leads to early hematoma growth .
many recent studies have reported that a cta spot sign may be a good radiologic indicator for hematoma growth as well as poor outcome for acute ich patients in western countries9,11,39 ) .
hemostatic factors , such as inr elevation , have been shown in many studies to be risk factors for hematoma growth in acute ich but not factors in initial ich volume5,17,20 ) .
the cta spot sign has also been an independent and strong predictor for hematoma growth and poor outcome2,9,11,19,25,39 ) . in our results , the presence of a cta spot sign was also found to be markedly related to hematoma growth and poor outcome ( table 2 , 4 ) .
gpt elevation was newly identified as an association factor for hematoma growth . in a large epidemiological study in asian countries ,
gpt elevation was revealed to be associated with ich development in east asia , and a korean medical insurance corporation study also showed the association between gpt elevation and ich occurrence21,22 ) .
therefore , further research examining gpt elevation and hematoma growth in acute ich may be necessary .
several previous studies have demonstrated an association between warfarin use and hematoma growth5,17,20 ) , but the present study did not show a significant association due to testing different populations and a different sampling design .
in contrast to the larger percentage of patients who had used warfarin in the prior study ( 23.0%)17 ) , our sample population contained a small proportion of only 7 ( 2.4% ) patients who had used warfarin before ich development ; therefore , the effect of warfarin on the size of hematoma growth might be underestimated in our results ( table 2 ) .
one japanese cohort study was consistent with our finding that anticoagulant medication was not significantly different between patients with and without hematoma growth36 ) .
moreover , for hemostatic status , an inr elevation of 1.8 or more was a marginally significant predictor of hematoma growth ( table 4 , model 1 ) , and an inr 1.8 combined with warfarin medication showed a significant association with hematoma growth ( table 4 , model 2 ) .
therefore , a vitamin k antagonist may be a risk factor for hematoma growth in acute ich .
with regard to antiplatelet medication , some investigators have reported increased early hematoma growth , but others could not show that the previous antiplatelet medication was associated with hematoma growth in acute ich29,30,35,37 ) . in our results ,
antiplatelet medication was a significant risk factor for hyper - acute hematoma growth within 6 hours of ictus ( table 5 ) .
an ongoing randomized clinical trial to confirm the efficacy and safety of platelet transfusion in ich may address this issue7 ) .
to date , the cta spot sign and contrast extravasation have not been definitively differentiated as early radiologic markers for hematoma growth in acute ich2,9,18,39 ) . moreover ,
the cta spot sign has been variously defined as a foci of enhancement within the hematoma on cta source images ( proposed by wada et al.)39 ) , and the criteria have included such characteristics as one or more foci of contrast pooling within the ich , an attenuation of 120 hounsfield units or more , a discontinuity from normal or abnormal vasculature adjacent to the ich , as well as having any size or morphology ( suggested by delgado almandoz et al.)9 ) . compared with the rates of cta spot sign in studies performed in western countries ( 22 - 41%)11,13,19,39 ) , the spot sign rate in our study ( 13.9% ) was relatively low and in accordance with that of another study in korea ( 15%)32 ) .
first , this finding may be attributable to the relatively longer time window of enrollment for patients in our study ( as was used in the study by park et al.32 ) and to the inhomogeneous definition of cta spot sign compared with the results in the literature9,10,11,19,25,39,40 ) .
second , the first pass cta may have a lower spot sign detection rate than delayed enhanced ct imaging23 ) .
prior studies in western countries have reported that hematoma growth is a strong predictor of early mortality and poor functional outcome5,6,8,29 ) . in the current study ,
hematoma growth increased the probability of early death in acute ich patients ( p=0.018 ) ( table 2 ) ; this finding is consistent with that of another study in korea32 ) .
therefore , hematoma growth may be accepted as one of the important predictors of mortality and poor outcome in korean patients with acute ich .
the 1-month mortality in this study ( table 1 ) was also comparable to those of other studies for korean patients with acute ich24,32 ) .
this study revealed the association and risk factors between the cta spot sign and hematoma growth , as well as the clinical impact for early outcome in korean patients with acute ich .
however , there were some limitations , including the retrospective study design , multiple testing problems , and a generalizability issue due to the single - center sample .
our results were consistent with those of previous other populations in the way that the cta spot sign showed a strong probability of being identified in the hyper - acute stage and was an independent strong predictor for hematoma growth in korean patients with spontaneous ich .
oral antithrombotics including vitamin k antagonists or antiplatelet drugs might also be associated with hyper - acute hematoma growth in our population . unlike prior studies ,
elevated gpt was newly identified as a predictor for hematoma growth and a further prospective study to confirm this association between elevated gpt and hematoma growth in korean patients with acute ich . | objectivethis study was conducted to clarify the association factors and clinical significance of the ct angiography ( cta ) spot sign and hematoma growth in korean patients with acute intracerebral hemorrhage ( ich).methodswe retrospectively collected the data of 287 consecutive patients presenting with acute ich who arrived within 12 hours of ictus .
baseline clinical and radiological characteristics as well as the mortality rate within one month were assessed .
a binary logistic regression was conducted to obtain association factors for the cta spot sign and hematoma growth.resultswe identified a cta spot sign in 40 patients ( 13.9% ) and hematoma growth in 78 patients ( 27.2% ) . an elapsed time to ct scan of less than 3 hours ( or , 5.14 ; 95% ci , 1.76 - 15.02 ; p=0.003 )
was associated with the spot sign . a cta spot sign ( or , 5.70 ; 95% ci , 2.70 - 12.01 ; p<0.001 ) , elevated alanine transaminase ( gpt ) level > 40 iu ( or , 2.01 ; 95% ci , 1.01 - 4.01 ; p=0.047 ) , and an international normalized ratio 1.8 or warfarin medication ( or , 5.64 ; 95% ci , 1.29 - 24.57 ; p=0.021 ) were independent predictors for hematoma growth .
antiplatelet agent medication ( or , 4.92 ; 95% ci , 1.31 - 18.50 ; p=0.019 ) was significantly associated with hematoma growth within 6 hours of ictus.conclusionas previous other populations , cta spot sign was a strong predictor for hematoma growth especially in hyper - acute stage of ich in korea .
antithrombotics medication might also be associated with hyper - acute hematoma growth . in our population ,
elevated gpt was newly identified as a predictor for hematoma growth and its effect for hematoma growth is necessary to be confirmed through a further research . | INTRODUCTION
MATERIALS AND METHODS
Populations and study design
Baseline characteristics, laboratory and radiologic profiles
CT protocol, ICH management, and outcome measurements
Statistics
RESULTS
Predictors for CTA spot sign and hematoma growth
DISCUSSION
CONCLUSION | after adjustment of potential factors , time from ictus to ct scan <3 hours was the only factor identified as a significant predictor for the cta spot sign ( or , 5.14 ; 95% ci , 1.76 - 15.02 ; p=0.068 ) . intraventricular extension was revealed as a borderline association factor ( or , 1.96 ; 95% ci , 0.95 - 4.05 ; p=0.002 ) for the cta spot sign ( table 3 ) . on the other hand , a cta spot sign ( or , 5.70 ; 95% ci , 2.70 - 12.01 ; p<0.001 ) and elevated gpt level > 40 iu ( or , 2.01 ; 95% ci , 1.01 - 4.01 ; p=0.047 ) were independent predicting factors of hematoma growth . though an inr of 1.8 or greater was marginally related to hematoma growth in the multivariable binary logistic regression model 1 , an inr 1.8 or warfarin medication
was significantly associated with hematoma growth in model 2 ( table 4 ) . in addition to a spot sign ( or , 3.47 ; 95% ci , 1.15 - 10.48 ; p=0.027 ) and large hemorrhage volume of 30 ml or more ( or , 3.63 ; 95% ci , 1.25 - 10.57 ; p=0.018 ) , antiplatelet medication ( or , 4.92 ; 95% ci , 1.31 - 18.50 ; p=0.019 ) showed a significant association with hematoma growth within 6 hours from ictus in multivariable regression analysis ( table 5 ) . after adjustment of potential factors , time from ictus to ct scan <3 hours was the only factor identified as a significant predictor for the cta spot sign ( or , 5.14 ; 95% ci , 1.76 - 15.02 ; p=0.068 ) . intraventricular extension was revealed as a borderline association factor ( or , 1.96 ; 95% ci , 0.95 - 4.05 ; p=0.002 ) for the cta spot sign ( table 3 ) . on the other hand , a cta spot sign ( or , 5.70 ; 95% ci , 2.70 - 12.01 ; p<0.001 ) and elevated gpt level
> 40 iu ( or , 2.01 ; 95% ci , 1.01 - 4.01 ; p=0.047 ) were independent predicting factors of hematoma growth . though an inr of 1.8 or greater was marginally related to hematoma growth in the multivariable binary logistic regression model 1 , an inr 1.8 or warfarin medication
was significantly associated with hematoma growth in model 2 ( table 4 ) . in addition to a spot sign ( or , 3.47 ; 95% ci , 1.15 - 10.48 ; p=0.027 ) and large hemorrhage volume of 30 ml or more ( or , 3.63 ; 95% ci , 1.25 - 10.57 ; p=0.018 ) , antiplatelet medication ( or , 4.92 ; 95% ci , 1.31 - 18.50 ; p=0.019 ) showed a significant association with hematoma growth within 6 hours from ictus in multivariable regression analysis ( table 5 ) . additionally , a cta spot sign and elevated gpt level were independent predictors for hematoma growth of acute ich patients in korea . furthermore , in hyper - acute ich within 3 hours of ictus , there may be a high probability of the presence of a cta spot sign , which leads to early hematoma growth . compared with the rates of cta spot sign in studies performed in western countries ( 22 - 41%)11,13,19,39 ) , the spot sign rate in our study ( 13.9% ) was relatively low and in accordance with that of another study in korea ( 15%)32 ) . this study revealed the association and risk factors between the cta spot sign and hematoma growth , as well as the clinical impact for early outcome in korean patients with acute ich . our results were consistent with those of previous other populations in the way that the cta spot sign showed a strong probability of being identified in the hyper - acute stage and was an independent strong predictor for hematoma growth in korean patients with spontaneous ich . unlike prior studies ,
elevated gpt was newly identified as a predictor for hematoma growth and a further prospective study to confirm this association between elevated gpt and hematoma growth in korean patients with acute ich . | [
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the basic method of treatment for chronic low back pain is conservative treatment , consisting of pharmacotherapy , kinesiotherapy , and physical therapy treatments .
dynamic development of biomedical engineering results in new technical solutions applied in creating new medical devices .
the devices that are currently used in physical therapy treatments support , and at times even replace , pharmacological treatment methods .
in addition , due to their rare adverse effects , physical methods shorten treatment period , improve quality of life , and reduce therapy costs .
one of the physical treatments most frequently used in back pain syndromes is electrical therapy .
the main goal of using electrical stimulation in treating low back pain syndromes is an attempt to alleviate both pain and inflammation , as well as to reduce muscle tension in the affected regions [ 13 ] .
however , on the basis of analysis of the available literature on electrotherapeutical methods of low back pain treatment , most studies fail to meet the basic criteria of evidence - based physiotherapy , which makes it extremely difficult to carry out a clear and objective analysis of clinical efficacy of treatments that are widely used in everyday practice . in the currently available research publications on electrical therapy of low back pain , generally no control groups or detailed randomization
were used , and such studies were often conducted with relatively small groups of patients , based solely on subjective questionnaires and pain assessment scales ( lacking measurement methods to objectify the therapeutic progress ) .
the available literature also lacks any comprehensive and large - scale clinical study analyzing clinical efficacy of the numerous electrotherapeutic methods popular in physiotherapy in a single study , with consistent statistical calculations , which would allow for a reliable assessment of the studied treatments and their comparison to one another , and in relation to a representative control group .
therefore , the aim of this study was to assess the effects of treating low back pain using selected electrical therapies .
the study assesses the influence of individual electrotherapeutic treatments on reduction of pain , improvement of the range of movement in lower section of the spine , and improvement of motor functions and mobility .
the research project was approved by the bioethics commission of the academy of physical education in katowice ( no .
the study included patients with low back pain , referred for physical therapy to the clinical research lab of the public higher medical professional school in opole , poland .
qualification of patients was made by a team composed of an orthopedist , a neurologist , a neurosurgeon , an internist , a radiologist , and a physiotherapist .
patients qualified for the study suffered from the l5-s1 discopathy , chronic radiating pain , and pseudoradicular syndrome , and had no previous surgical procedures within the spinal area .
the patients were at least 18 years old and had valid certificates of the mri examination , confirming the diagnosis of low back pain syndrome ( at least type iii changes in accordance with the modic classification in the l5s1 spine section ) .
on the other hand , the allocation of patients who positively underwent the qualification procedure to specific groups was random ( based on a computer number generator ) .
exclusion criteria referred to patients who were diagnosed with : acute pain ( less than 6 months ) , the radicular syndrome , discopathy of other spine regions ( patients with early type i and ii changes were not excluded from the study , only the type iii degeneration , as per the modic classification , constituted grounds for exclusion ) , no pain or reduction of mobility in the lumbosacral section , other disorders of the spine ( spondylolisthesis , fractures , tumors , rheumatic diseases , the cauda equina syndrome ) , pregnancy , defect symptoms , cardiovascular diseases , pacemakers , metal implants within the application area , sensory disorders , mental disorders , cancer , skin lesions within the area of application of the electrodes , psoriasis , scleroderma , and viral or bacterial infections .
patients who had undergone spinal surgery , as well as those taking painkillers or anti - inflammatory drugs , were also excluded from the study .
exclusion criteria referred also to patients with damage to the vestibule and/or a part of the vestibulocochlear nerve , with mnire syndrome , vestibular neuritis , sudden loss of function of the inner ear , as well as damage to the cerebellum , spinal cord and brainstem , resulting in balance disorders .
a total of 127 patients were qualified for the therapy ( ultimately , 124 patients completed the study ) and assigned to 6 comparison groups ( a flow diagram is presented in figure 1 ) .
group a consisted of 20 patients ( all participants in this group completed the entire study program ) .
treatment parameters were : alternating current , rectangular impulse , impulse duration 100 s , frequency of 100 hz , subjective dosage ( until a distinctive sensation of the current flow was experienced , during the patient s habituation to the electrical stimulus , the therapist gradually increased the intensity during treatment to maintain the desired sensations ) , and 60-min duration of a single treatment .
group b consisted of 20 patients ( as in the previous group , all the patients completed the therapy ) .
patients were treated with the transcutaneous electrical nerve stimulation ( acupuncture - like tens ) .
treatment parameters were : alternating current , rectangular impulse , impulse duration 200 s , frequency of 10 hz , subjective dosage ( until distinctive muscle contraction , during habituation and decrease of the motion effect , the therapist gradually increased the intensity during treatment to maintain the desired muscle stimulation threshold ) , and 60-min duration of a single treatment .
group c initially consisted of 22 patients , but 2 participants withdrew from further therapy due to viral infection and did not complete the treatment series ( 1 participant resigned after 4 sessions and the other after 6 sessions ) .
one person had to discontinue the therapy after 3 sessions due to the occurrence of skin lesions within the area of application of the electrodes . in total ,
treatment parameters were : output voltage 100 v , alternating current , spike impulse , impulse duration 100 s , frequency of 100 hz , subjective dosage ( until a distinctive sensation of the current flow was experienced , during the patient s habituation to the electrical stimulus , the therapist gradually increased intensity during treatment to maintain the desired sensations ) , and 50-min duration of a single treatment .
group d initially consisted of 22 patients , but 1 person had to resign from further therapy due to aggravation of symptoms and started taking analgesics .
subjects in group d were treated with electrotherapy using medium - frequency currents ( interferential current stimulation ) .
treatment parameters were : alternating current , sinusoidal impulse , impulse duration 100 s , basic frequency 4000 hz , alternating frequency 50100 hz , subjective dosage ( until a distinctive sensation of the flowing current was experienced , during the patient s habituation to the electrical stimulus , the therapist gradually increased the intensity during treatment to maintain the desired sensations ) , and 20 min duration of a single treatment .
treatment parameters were : pulsed current , sinusoidal impulse , impulse duration and frequency ( sequentially df 10 ms , 100 hz , cp 10 ms , 50100 hz , lp 10 ms , 50100 hz , but with an alternating amplitude ) , subjective dosage ( until a distinctive sensation of the flowing current is experienced , during the patient s habituation to the electrical stimulus the therapist gradually increased the intensity during treatment to maintain the desired sensations ) , and 9-min duration of a single treatment ( df , lp , and cp were 3 min each ) .
in all patients treated with physical therapy , the electrodes were placed in the lumbar region in the posterior axillary line ( figures 2 , 3 ) .
however , patients in group f ( 21 patients , control group ) were treated only by means of motor improvement exercises .
the stabilization training [ 57 ] included : myofascial release techniques for the erector spinae muscle , activation techniques for the neutral position of the lumbo - pelvic - hip complex and deep muscles , training the activation of proper breathing and the transversus abdominis muscle , exercising the coordination of superficial and deep trunk muscles , and postural and dynamic training .
duration of a training session was 45 min ( 5 times a week , from monday to friday ) . in the comparison groups a
, b , c , d , and e , patients treated with electrical therapy performed basic therapy exercises in accordance with the same methodology as patients in group f. patients in all the comparison groups ( with the exception of group f , in which only the daily motor improvement exercises were used for 3 weeks ) were subjected to a series of 15 treatments , 5 times a week ( monday to friday ) for a period of 3 weeks .
the treatments were performed with the ionoson expert current generators ( physio med electromedizin , germany ) , which were calibrated before treatment of each patient using the measurement system and the serial connection of the ionoson device to a cathodal oscilloscope and a decade resistor ( electrical circuit loaded with the resistance of 10 k , as the average resistance of the human body ) in order to verify the repeatability , durability , and stability of the treatment parameters generated by the electrostimulator .
patients in all the comparison groups were homogeneous in terms of basic characteristics specific for the studied populations ( table 1 ) .
the groups were also homogeneous as regards the initial measurements concerning pain assessment , functional state , mobility range , and body posture . in order to analyze the therapeutic progress for the subjective assessment of pain and functional capacity as well as the assessment of the degree of disability ,
the following tests were performed : the vas ( visual analogue scale ) pain assessment scale was used for subjective assessment of the experienced pain , in which the patient assesses the experienced pain on a simple scale from 0 to 10 , where 0 denotes lack of pain and 10 denotes the strongest pain .
the modified laitinen pain scale was used to assess 4 indicators : pain intensity , frequency of pain occurrence , use of analgesics , and limitations of mobility . the oswestry questionnaire ( the oswestry low back pain disability questionnaire , oswestry disability index [ odi ] )
was used to evaluate the functional ability of patients ; it is a widely recognized and reliable scale for evaluation of patients with low back pain .
when answering the individual questions , the patient can choose 1 of the 6 options scored from 0 to 5 : a 0 points ; b 1 point ; c 2 points ; d 3 points ; e 4 points ; f 5 points .
after summing the scores for all questions , the oswestry disability index is as follows : no disability ( 04 points ) ; minimal disability ( 514 points ) ; moderate disability ( 1524 points ) ; severe disability ( 2534 points ) ; and full disability ( 3550 points ) .
the roland - morris disability questionnaire ( rm ) was used to assess the degree of disability in patients with low back pain and reflects the condition of the patient on the day of the examination .
each yes answer scores 1 point and each no answer scores 0 points . after summing the scores for all questions , the roland - morris disability index is as follows : no disability ( 03 points ) ; minimal disability ( 410 points ) ; moderate disability ( 1117 points ) ; severe disability ( 1824 points ) .
the lasgue test was used to measure the mobility range in the hip joint on the side of the herniated disc in the course of spinal discopathy .
the examiner then slowly lifts one of the patient s legs while the knee is straight at the joint , until pain occurs .
while the patient is in a standing position , the examiner marks 2 points on the patient s skin : at 10 cm above the line connecting the posterior superior iliac spines , and then at 5 cm below that line .
the patient then slowly bends down as far as possible , while keeping the knees straight .
the mobility range measurements were carried out by the same technician ( each measurement was an arithmetic mean of 5 trials ) .
for the purposes of this clinical study , a self - estimation of error of the person performing the measurements was calculated .
for each of 15 randomly selected participants , 20 more measurements were taken using the lasgue test and the schober s test ( 600 measurements in total ) . the absolute measurement error ( x )
was calculated using the formula : then the relative error ( x ) was estimated , using the formula : the mean percentage error ( relative error expressed in percentage points ) was then calculated for all the 20 measurements for the lasgue and schober tests .
the resulting measurement error , in accordance with the proprietary calculations , was as follows : the arithmetic mean of the measurement error was 5.88% , and the standard deviation was 3.73% for the lasgue test , and the mean was 3.45% , and the standard deviation was 1.04% for the schober test .
the evaluation was performed using a double - plate stabilographic platform equipped with a computer - aided posturographic system , manufactured by cq elektronik system ( poland ) , model cq stab2p .
the measurement error was 0.86% . for each patient , 2 trials were carried out : the first trial with eyes open in full visual control , and the second trial with eyes closed , without visual control .
the subjects were in a habitual , upright position , standing barefoot on the posturographic platform ( feet apart in line with their hips , arms down along their bodies , head facing forward , with eyes fixed on a designated point placed at eye level about 1.5 m away ) ( figure 4 ) .
statistical analyses of the basic posturographic parameters were performed to compare balance conditions in the tested group of patients .
the following parameters were analyzed : total path length [ mm ] , i.e. the total sway of the center of pressure of the subject s feet during the trial ( 30 s ) , in millimeters ; anterio / posterior path length [ mm ] ; medio / lateral path length [ mm ] ; mean amplitude ( radius ) [ mm ] ; and mean anterio / posterior amplitude [ mm ] .
the above postural stability tests were carried out both before the therapy process and after its completion .
the homogeneity of distribution of patients characteristics in all groups was analyzed with the chi - square test in the highest reliability version ( ) and the kruskal - wallis homogeneity test .
for dependent variables , the nonparametric wilcoxon s matched pairs test was used , and for independent variables we used the nonparametric kruskal - wallis variance analysis .
the tukey post hoc multiple comparisons test was used to identify the exact dependencies resulting from the variance analysis between individual groups .
the research project was approved by the bioethics commission of the academy of physical education in katowice ( no .
the study included patients with low back pain , referred for physical therapy to the clinical research lab of the public higher medical professional school in opole , poland .
qualification of patients was made by a team composed of an orthopedist , a neurologist , a neurosurgeon , an internist , a radiologist , and a physiotherapist .
patients qualified for the study suffered from the l5-s1 discopathy , chronic radiating pain , and pseudoradicular syndrome , and had no previous surgical procedures within the spinal area .
the patients were at least 18 years old and had valid certificates of the mri examination , confirming the diagnosis of low back pain syndrome ( at least type iii changes in accordance with the modic classification in the l5s1 spine section ) .
on the other hand , the allocation of patients who positively underwent the qualification procedure to specific groups was random ( based on a computer number generator ) .
exclusion criteria referred to patients who were diagnosed with : acute pain ( less than 6 months ) , the radicular syndrome , discopathy of other spine regions ( patients with early type i and ii changes were not excluded from the study , only the type iii degeneration , as per the modic classification , constituted grounds for exclusion ) , no pain or reduction of mobility in the lumbosacral section , other disorders of the spine ( spondylolisthesis , fractures , tumors , rheumatic diseases , the cauda equina syndrome ) , pregnancy , defect symptoms , cardiovascular diseases , pacemakers , metal implants within the application area , sensory disorders , mental disorders , cancer , skin lesions within the area of application of the electrodes , psoriasis , scleroderma , and viral or bacterial infections .
patients who had undergone spinal surgery , as well as those taking painkillers or anti - inflammatory drugs , were also excluded from the study .
exclusion criteria referred also to patients with damage to the vestibule and/or a part of the vestibulocochlear nerve , with mnire syndrome , vestibular neuritis , sudden loss of function of the inner ear , as well as damage to the cerebellum , spinal cord and brainstem , resulting in balance disorders .
a total of 127 patients were qualified for the therapy ( ultimately , 124 patients completed the study ) and assigned to 6 comparison groups ( a flow diagram is presented in figure 1 ) .
group a consisted of 20 patients ( all participants in this group completed the entire study program ) .
treatment parameters were : alternating current , rectangular impulse , impulse duration 100 s , frequency of 100 hz , subjective dosage ( until a distinctive sensation of the current flow was experienced , during the patient s habituation to the electrical stimulus , the therapist gradually increased the intensity during treatment to maintain the desired sensations ) , and 60-min duration of a single treatment .
group b consisted of 20 patients ( as in the previous group , all the patients completed the therapy ) .
patients were treated with the transcutaneous electrical nerve stimulation ( acupuncture - like tens ) .
treatment parameters were : alternating current , rectangular impulse , impulse duration 200 s , frequency of 10 hz , subjective dosage ( until distinctive muscle contraction , during habituation and decrease of the motion effect , the therapist gradually increased the intensity during treatment to maintain the desired muscle stimulation threshold ) , and 60-min duration of a single treatment
. group c initially consisted of 22 patients , but 2 participants withdrew from further therapy due to viral infection and did not complete the treatment series ( 1 participant resigned after 4 sessions and the other after 6 sessions ) .
one person had to discontinue the therapy after 3 sessions due to the occurrence of skin lesions within the area of application of the electrodes . in total ,
treatment parameters were : output voltage 100 v , alternating current , spike impulse , impulse duration 100 s , frequency of 100 hz , subjective dosage ( until a distinctive sensation of the current flow was experienced , during the patient s habituation to the electrical stimulus , the therapist gradually increased intensity during treatment to maintain the desired sensations ) , and 50-min duration of a single treatment .
group d initially consisted of 22 patients , but 1 person had to resign from further therapy due to aggravation of symptoms and started taking analgesics .
subjects in group d were treated with electrotherapy using medium - frequency currents ( interferential current stimulation ) .
treatment parameters were : alternating current , sinusoidal impulse , impulse duration 100 s , basic frequency 4000 hz , alternating frequency 50100 hz , subjective dosage ( until a distinctive sensation of the flowing current was experienced , during the patient s habituation to the electrical stimulus , the therapist gradually increased the intensity during treatment to maintain the desired sensations ) , and 20 min duration of a single treatment .
treatment parameters were : pulsed current , sinusoidal impulse , impulse duration and frequency ( sequentially df 10 ms , 100 hz , cp 10 ms , 50100 hz , lp 10 ms , 50100 hz , but with an alternating amplitude ) , subjective dosage ( until a distinctive sensation of the flowing current is experienced , during the patient s habituation to the electrical stimulus the therapist gradually increased the intensity during treatment to maintain the desired sensations ) , and 9-min duration of a single treatment ( df , lp , and cp were 3 min each ) .
in all patients treated with physical therapy , the electrodes were placed in the lumbar region in the posterior axillary line ( figures 2 , 3 ) .
however , patients in group f ( 21 patients , control group ) were treated only by means of motor improvement exercises .
the stabilization training [ 57 ] included : myofascial release techniques for the erector spinae muscle , activation techniques for the neutral position of the lumbo - pelvic - hip complex and deep muscles , training the activation of proper breathing and the transversus abdominis muscle , exercising the coordination of superficial and deep trunk muscles , and postural and dynamic training .
duration of a training session was 45 min ( 5 times a week , from monday to friday ) . in the comparison groups a
, b , c , d , and e , patients treated with electrical therapy performed basic therapy exercises in accordance with the same methodology as patients in group f. patients in all the comparison groups ( with the exception of group f , in which only the daily motor improvement exercises were used for 3 weeks ) were subjected to a series of 15 treatments , 5 times a week ( monday to friday ) for a period of 3 weeks .
the treatments were performed with the ionoson expert current generators ( physio med electromedizin , germany ) , which were calibrated before treatment of each patient using the measurement system and the serial connection of the ionoson device to a cathodal oscilloscope and a decade resistor ( electrical circuit loaded with the resistance of 10 k , as the average resistance of the human body ) in order to verify the repeatability , durability , and stability of the treatment parameters generated by the electrostimulator .
patients in all the comparison groups were homogeneous in terms of basic characteristics specific for the studied populations ( table 1 ) .
the groups were also homogeneous as regards the initial measurements concerning pain assessment , functional state , mobility range , and body posture .
in order to analyze the therapeutic progress for the subjective assessment of pain and functional capacity as well as the assessment of the degree of disability , the following tests were performed : the vas ( visual analogue scale ) pain assessment scale was used for subjective assessment of the experienced pain , in which the patient assesses the experienced pain on a simple scale from 0 to 10 , where 0 denotes lack of pain and 10 denotes the strongest pain .
the modified laitinen pain scale was used to assess 4 indicators : pain intensity , frequency of pain occurrence , use of analgesics , and limitations of mobility . the oswestry questionnaire ( the oswestry low back pain disability questionnaire , oswestry disability index [ odi ] ) was used to evaluate the functional ability of patients ; it is a widely recognized and reliable scale for evaluation of patients with low back pain .
when answering the individual questions , the patient can choose 1 of the 6 options scored from 0 to 5 : a 0 points ; b 1 point ; c 2 points ; d 3 points ; e 4 points ; f 5 points . after summing the scores for all questions , the oswestry disability index is as follows : no disability ( 04 points ) ; minimal disability ( 514 points ) ; moderate disability ( 1524 points ) ; severe disability ( 2534 points ) ; and full disability ( 3550 points ) . the roland - morris disability questionnaire ( rm ) was used to assess the degree of disability in patients with low back pain and reflects the condition of the patient on the day of the examination .
each yes answer scores 1 point and each no answer scores 0 points . after summing the scores for all questions , the roland - morris disability index is as follows : no disability ( 03 points ) ; minimal disability ( 410 points ) ; moderate disability ( 1117 points ) ; severe disability ( 1824 points ) .
the lasgue test was used to measure the mobility range in the hip joint on the side of the herniated disc in the course of spinal discopathy .
the examiner then slowly lifts one of the patient s legs while the knee is straight at the joint , until pain occurs .
while the patient is in a standing position , the examiner marks 2 points on the patient s skin : at 10 cm above the line connecting the posterior superior iliac spines , and then at 5 cm below that line .
the patient then slowly bends down as far as possible , while keeping the knees straight .
the mobility range measurements were carried out by the same technician ( each measurement was an arithmetic mean of 5 trials ) .
for the purposes of this clinical study , a self - estimation of error of the person performing the measurements was calculated . for each of 15 randomly selected participants ,
20 more measurements were taken using the lasgue test and the schober s test ( 600 measurements in total ) .
the absolute measurement error ( x ) was calculated using the formula : then the relative error ( x ) was estimated , using the formula : the mean percentage error ( relative error expressed in percentage points ) was then calculated for all the 20 measurements for the lasgue and schober tests . the resulting measurement error , in accordance with the proprietary calculations , was as follows : the arithmetic mean of the measurement error was 5.88% , and the standard deviation was 3.73% for the lasgue test , and the mean was 3.45% , and the standard deviation was 1.04% for the schober test .
the evaluation was performed using a double - plate stabilographic platform equipped with a computer - aided posturographic system , manufactured by cq elektronik system ( poland ) , model cq stab2p .
2 trials were carried out : the first trial with eyes open in full visual control , and the second trial with eyes closed , without visual control .
the subjects were in a habitual , upright position , standing barefoot on the posturographic platform ( feet apart in line with their hips , arms down along their bodies , head facing forward , with eyes fixed on a designated point placed at eye level about 1.5 m away ) ( figure 4 ) .
statistical analyses of the basic posturographic parameters were performed to compare balance conditions in the tested group of patients .
the following parameters were analyzed : total path length [ mm ] , i.e. the total sway of the center of pressure of the subject s feet during the trial ( 30 s ) , in millimeters ; anterio / posterior path length [ mm ] ; medio / lateral path length [ mm ] ; mean amplitude ( radius ) [ mm ] ; and mean anterio / posterior amplitude [ mm ] .
the above postural stability tests were carried out both before the therapy process and after its completion .
the studied parameters were analyzed using the statistica statistical software ver . 10.0 ( statsoft , dell inc .
the homogeneity of distribution of patients characteristics in all groups was analyzed with the chi - square test in the highest reliability version ( ) and the kruskal - wallis homogeneity test .
for dependent variables , the nonparametric wilcoxon s matched pairs test was used , and for independent variables we used the nonparametric kruskal - wallis variance analysis .
the tukey post hoc multiple comparisons test was used to identify the exact dependencies resulting from the variance analysis between individual groups .
after completion of the therapy , all the comparison groups demonstrated a statistically significant reduction of pain as compared to the initial values , measured using the vas scale ( table 2 ) .
similarly , a subjective reduction of pain was recorded using the laitinen questionnaire ( table 3 ) .
however , the intergroup analysis demonstrated that the highest analgesic effect was recorded in group d ( interferential current stimulation ) , which proved to be a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( high - voltage electrical stimulation [ hves ] ) .
no statistically significant differences were observed between groups a , b , and c. the lowest analgesic effect was observed in group e ( diadynamic currents [ dd ] ) and f ( control group ) , at a similar level in both groups .
the above was confirmed both by the results of measurements performed using the vas scale ( figure 5 ) and the laitinen questionnaire ( figure 6 ) .
after 3 weeks of treatment , all the comparison groups demonstrated a statistically significant improvement of functional ability , measured using the oswestry questionnaire ( table 4 ) .
similarly , a subjective improvement of patients ability was recorded using the roland - morris questionnaire ( table 5 ) .
however , on the basis of an intergroup analysis , we found that the highest percentual improvement of the patients functional ability measured with the oswestry questionnaire took place in group d ( interferential current stimulation ) , giving a significantly more beneficial effect than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) .
no statistically significant differences were observed between groups a , b , and c. the lowest effect was observed in group e ( dd ) and f ( control group ) , at a similar level in both groups ( figure 7 ) . in case of intergroup comparisons regarding the subjective experience of ability measured with the roland - morris questionnaire ,
the greatest progress was observed in group d ( interferential current stimulation ) . in the remaining groups ,
immediately after completion of therapy , the comparison groups demonstrated a statistically significant increase of mobility in the hip joint as compared to the pre - therapy state , measured using the lasgue test ( table 6 ) .
an improved mobility in the low back region was also recorded in the schober s test ( table 7 ) . on the basis of an intergroup analysis
, we found that the highest percentual improvement in the lasgue test took place in group d ( interferential current stimulation ) , giving a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) .
no statistically significant differences were observed between groups a , b , and c. the least progress was recorded in group e ( dd ) and f ( control group ) , at a similar level in both groups ( figure 9 ) .
the comparison of intergroup results of the schober s test indicated that groups a ( conventional tens ) , b ( pseudo - acupuncture tens ) , c ( hves ) , and d ( interference currents ) gained a significant advantage over groups e ( dd ) and f ( control group ) ( figure 10 ) .
after completion of therapy , tests on the stabilometric platform clearly demonstrated a statistically significant improvement in body posture as regards the total path length of sway of the center of pressure during a time trial ( table 8) and the path length in the anterio - posterior sway ( table 9 ) in comparison to the initial state .
these parameters were significantly reduced in all the comparison groups ( in particular with eyes closed ) , which confirms that patients with low back pain had a more stable body posture after the therapy . in case of measurement of the path in the lateral sway ,
a beneficial reduction was also recorded in the studied groups , although these changes were not statistically significant ( table 10 ) .
interesting changes occurred in measurements of the mean radius of the center of pressure sway , as the beneficial reduction of this parameter and improvement of postural stability took place in the closed - eyes trial ( table 11 ) . in trials under visual control ,
the radius was also reduced in all groups , but this change was significantly smaller .
the analysis of variance showed that the greatest percentual reduction in the length of the total path of the center of pressure sway in a time trial occurred in group d ( interference currents ) .
these parameters were also slightly reduced in the remaining groups , especially group a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) , but despite this trend , no statistically significant difference was obtained in relation to groups e ( dd ) and f ( control group ) .
a similar situation was observed in the case of closed - eyes trial , where group d had an even more significant advantage over the other groups .
the analysis of percentual shortening of the path length in the anterio - posterior sway of the patients center of pressure indicates that the outcome of this process was the most beneficial in group d ( interferential current stimulation ) . in the remaining groups , the phenomenon occurred with a slightly lower intensity .
on the other hand , in the percentage analysis of reduction of the mean radius of center of pressure sway in trials under visual control , there were no significant changes , and the recorded changes occurred at a similar level in all groups .
interestingly , in the eyes - closed trial , patients in group d ( interferential current stimulation ) had better scores than those obtained in other groups .
however , no statistically significant differences were detected , although an evident trend was observed regarding changes in benefit of group d in relation to the other groups .
observation of the percentual reduction of the mean lateral sway of the center of pressure in the eyes - open trial did not show a visible improvement of this parameter in the studied groups ( reduction of sway was small and occurred at a similar level in all patients irrespectively of the treatment method ) .
no statistically significant intergroup differences were noted . all the more interesting was the significant advantage of group d ( interferential current stimulation ) in relation to the other groups during the trial without visual control . in group d , a statistically significant difference was noted in relation to other groups .
after completion of the therapy , all the comparison groups demonstrated a statistically significant reduction of pain as compared to the initial values , measured using the vas scale ( table 2 ) .
similarly , a subjective reduction of pain was recorded using the laitinen questionnaire ( table 3 ) .
however , the intergroup analysis demonstrated that the highest analgesic effect was recorded in group d ( interferential current stimulation ) , which proved to be a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( high - voltage electrical stimulation [ hves ] ) .
no statistically significant differences were observed between groups a , b , and c. the lowest analgesic effect was observed in group e ( diadynamic currents [ dd ] ) and f ( control group ) , at a similar level in both groups .
the above was confirmed both by the results of measurements performed using the vas scale ( figure 5 ) and the laitinen questionnaire ( figure 6 ) .
after 3 weeks of treatment , all the comparison groups demonstrated a statistically significant improvement of functional ability , measured using the oswestry questionnaire ( table 4 ) .
similarly , a subjective improvement of patients ability was recorded using the roland - morris questionnaire ( table 5 ) . however
, on the basis of an intergroup analysis , we found that the highest percentual improvement of the patients functional ability measured with the oswestry questionnaire took place in group d ( interferential current stimulation ) , giving a significantly more beneficial effect than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) .
no statistically significant differences were observed between groups a , b , and c. the lowest effect was observed in group e ( dd ) and f ( control group ) , at a similar level in both groups ( figure 7 ) . in case of intergroup comparisons regarding the subjective experience of ability measured with the roland - morris questionnaire ,
the greatest progress was observed in group d ( interferential current stimulation ) . in the remaining groups ,
immediately after completion of therapy , the comparison groups demonstrated a statistically significant increase of mobility in the hip joint as compared to the pre - therapy state , measured using the lasgue test ( table 6 ) .
an improved mobility in the low back region was also recorded in the schober s test ( table 7 ) . on the basis of an intergroup analysis
, we found that the highest percentual improvement in the lasgue test took place in group d ( interferential current stimulation ) , giving a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) .
no statistically significant differences were observed between groups a , b , and c. the least progress was recorded in group e ( dd ) and f ( control group ) , at a similar level in both groups ( figure 9 ) .
the comparison of intergroup results of the schober s test indicated that groups a ( conventional tens ) , b ( pseudo - acupuncture tens ) , c ( hves ) , and d ( interference currents ) gained a significant advantage over groups e ( dd ) and f ( control group ) ( figure 10 ) .
after completion of therapy , tests on the stabilometric platform clearly demonstrated a statistically significant improvement in body posture as regards the total path length of sway of the center of pressure during a time trial ( table 8) and the path length in the anterio - posterior sway ( table 9 ) in comparison to the initial state .
these parameters were significantly reduced in all the comparison groups ( in particular with eyes closed ) , which confirms that patients with low back pain had a more stable body posture after the therapy . in case of measurement of the path in the lateral sway ,
a beneficial reduction was also recorded in the studied groups , although these changes were not statistically significant ( table 10 ) .
interesting changes occurred in measurements of the mean radius of the center of pressure sway , as the beneficial reduction of this parameter and improvement of postural stability took place in the closed - eyes trial ( table 11 ) . in trials under visual control ,
the radius was also reduced in all groups , but this change was significantly smaller .
the analysis of variance showed that the greatest percentual reduction in the length of the total path of the center of pressure sway in a time trial occurred in group d ( interference currents ) .
these parameters were also slightly reduced in the remaining groups , especially group a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) , but despite this trend , no statistically significant difference was obtained in relation to groups e ( dd ) and f ( control group ) .
a similar situation was observed in the case of closed - eyes trial , where group d had an even more significant advantage over the other groups .
the analysis of percentual shortening of the path length in the anterio - posterior sway of the patients center of pressure indicates that the outcome of this process was the most beneficial in group d ( interferential current stimulation ) . in the remaining groups , the phenomenon occurred with a slightly lower intensity .
on the other hand , in the percentage analysis of reduction of the mean radius of center of pressure sway in trials under visual control , there were no significant changes , and the recorded changes occurred at a similar level in all groups .
interestingly , in the eyes - closed trial , patients in group d ( interferential current stimulation ) had better scores than those obtained in other groups .
however , no statistically significant differences were detected , although an evident trend was observed regarding changes in benefit of group d in relation to the other groups .
observation of the percentual reduction of the mean lateral sway of the center of pressure in the eyes - open trial did not show a visible improvement of this parameter in the studied groups ( reduction of sway was small and occurred at a similar level in all patients irrespectively of the treatment method ) .
all the more interesting was the significant advantage of group d ( interferential current stimulation ) in relation to the other groups during the trial without visual control . in group d
this condition may also cause pain radiating along the lower limbs , muscle weakness due to compression and irritation of nerve roots , as well as limitation of spinal mobility .
these symptoms may appear at any stage of the disease , disturbing the function of the locomotor system .
they are also a cause of disability and related social factors , which entails increased expenditure on , among others , diagnostics and treatment .
therefore , it is necessary to search for effective treatment methods for back pain syndromes .
one of the treatment methods is physical therapy , which helps to reduce the disease symptoms .
although the available literature contains a large number of proposals for conservative treatment of low back pain using electrical therapy , such publications usually concern only 1 type ( or , in some cases , 2 types in a direct comparison under 1 clinical study ) of electric current .
such articles contain a number of limitations and weaknesses , which makes it difficult to carry out an unequivocal clinical assessment .
studies conducted by brazilian researchers confirm the high effectiveness of interference currents and transcutaneous electrical nerve stimulation ( tens ) in low back pain therapy .
compared the effects of the tens treatment and interferential current stimulation ( ifc ) treatment in patients with nonspecific chronic low back pain .
the study involved a group of 150 patients , randomly assigned to 3 comparison groups .
the first group consisted of patients who were treated with tens therapy , the second group was treated with the ifc currents , and the third group was not treated with any physical stimulus .
patients in the first and second group underwent treatment for 30 min during 10 consecutive days . in all patients ,
pain level was assessed using the visual analogue pain scale ( vas ) and the mcgill pain questionnaire .
results of the study demonstrate that the tens and ifc therapy brought about significant effects as in reduction of pain intensity , improvement of disability , and reduction in the amount of non - steroidal anti - inflammatory drugs used , in comparison to the control group .
however , no significant differences were noted between patients from the first and the second group .
other brazilian researchers evaluated the effectiveness of the interferential current stimulation ( ifc ) therapy .
correa et al . conducted a randomized clinical trial involving 150 patients with a chronic , nonspecific low back pain syndrome , which confirmed the beneficial therapeutic effect of the ifc .
. demonstrated in their publication that the effectiveness of interferential currents was higher than that of superficial massage applied to the lumbar region of the spine .
patients from the first group were subjected to a series of treatments using ifc , whereas patients in the second group underwent a series of massage treatments . in both groups ,
treatments were performed for 10 weeks ( a total of 20 treatments , 2 times a week ) .
subjective pain assessment was performed using the visual analogue pain scale ( vas ) , while the oswestry and the roland - morris questionnaires were used to evaluate the functional ability of patients .
after the completed therapy , it was found that in comparison to superficial massage , the interference current led to significantly more improvement of disability , reduction of pain , and increase of quality of life .
turkish researchers demonstrated that tens and exercise increased quality of life and reduced pain in patients with low back pain .
in the first group patients performed aerobic and general development exercises , the second group performed general development exercises and was treated with physical therapy ( conventional tens ) , and in the third group the patients performed only general development exercises . before the therapy and after its completion , spine mobility was assessed with schober s test , pain intensity was assessed with the visual analogue pain scale ( vas ) , and the disability assessment was performed using the roland - morris questionnaire .
an assessment was also made of the general mental condition of patients , using the beck depression inventory ( bdi ) .
on the basis of the obtained effects , only the second study group demonstrated a statistically significant improvement as regards the measured parameters and the experienced pain .
however , the literature also provides examples of cases where electrotherapy had low effectiveness or only a short - term analgesic effect .
mcloughlin et al . indicated a lack of analgesic effect after the application of high - voltage electrical stimulation .
ambiguous results of a study in which tens was used in therapy of low back pain was presented in a publication by buchmuller et al . .
the study involved 236 persons suffering from low back pain , from 21 treatment centers in france .
the first group was made up of 117 patients who were treated with active tens treatments , while the second group , consisting of 119 patients , was subjected to simulated tens treatments .
the functional state of patients was assessed using the roland - morris questionnaire , and pain intensity was measured in accordance with the visual analogue pain scale ( vas ) .
surveys were conducted before the commencement of the therapy , after 2 and 6 weeks , and at 3 months after completion of the study .
independently of the study report , patients kept a diary to record their assessment of the intensity of pain .
analysis of the obtained results demonstrated similar reactions in the group of patients who underwent the active tens treatments and in the placebo group as regards the assessment of functional state and in relation to the vas scale .
however , the assessments of pain intensity recorded in patients diaries demonstrated a highly significant difference in favor of the active tens treatments , which shows a strong placebo effect of this method . in the meta - analysis presented by khadilkar et al . , whose aim was to determine the effectiveness of tens in the therapy of chronic pain of the lumbar section of the spine , a series of equivocal data was recorded , confirming the use of the tens as an analgesic .
the authors , while performing an in - depth assessment , noted inter alia the non - uniform methodology , the discrepancies in inclusion and exclusion criteria , and the differing therapy durations .
it would be valuable to have results from a large , multi - center research project using control groups ( quasi - electrotherapy ) and thorough randomization , as well as to unify the methodology of the performed treatments .
particular attention should be given to the long - term benefits which may possibly be observed after the application of tens .
the novelty of our study consists in an unambiguous assessment of popular methods of electrical therapy used in low back pain treatment under a single , prospective , randomized clinical pilot trial , based on strict inclusion and exclusion criteria , using both modern objective measurements and subjective measurements , with a uniform statistical analysis and a comprehensive and multi - faceted observation of the achieved results . to the best of our knowledge ,
the present study is the first wide - ranging scientific study of low back pain electrotherapy in accordance with the guidelines of the evidence - based physiotherapy .
none of the current publications made such a detailed diagnostics or a classification based on the radiological modic assessment criteria .
this allowed for a very representative population , which was recruited for the purpose of this project .
in addition , other researchers did not use calibration of the electrostimulator before each treatment .
irrespective of the innovative character of our pilot research and the elements of novelty , this work also contains a number of limitations .
it would certainly be worthwhile to supplement the project in the future with other modern and objective measurement tools , such as muscle electromyography , the biodex system movement analysis , and tensiometry .
also , the study did not involve blind trials or placebo effect assessment . in 1 of the comparison groups ,
patients underwent only the standard functional training and were not treated with any electrotherapeutical treatment whatsoever , which constituted a point of reference in relation to the exposed groups and which is permitted in the methodology of medical research publications .
however , using the simulated treatments and the so - called single - blind trial
would certainly constitute an interesting addition and would also significantly raise the profile of this study .
a weakness of this research was also the relatively high measurement error ( although in most cases the researchers do not even perform this kind of analysis or self - reflection ) during the observation of mobility of the hip joint and the lumbar region of the spine .
we endeavored to mitigate it by making our own error estimations and by making sure that all measurements were made by the same person ( an average of the 5 performed trials ) .
, it will also be necessary to perform an assessment of remote results , which will allow us to estimate the sustainability of remission achieved due to electrical therapy .
the novelty of our study consists in an unambiguous assessment of popular methods of electrical therapy used in low back pain treatment under a single , prospective , randomized clinical pilot trial , based on strict inclusion and exclusion criteria , using both modern objective measurements and subjective measurements , with a uniform statistical analysis and a comprehensive and multi - faceted observation of the achieved results . to the best of our knowledge ,
the present study is the first wide - ranging scientific study of low back pain electrotherapy in accordance with the guidelines of the evidence - based physiotherapy .
none of the current publications made such a detailed diagnostics or a classification based on the radiological modic assessment criteria .
this allowed for a very representative population , which was recruited for the purpose of this project .
in addition , other researchers did not use calibration of the electrostimulator before each treatment .
irrespective of the innovative character of our pilot research and the elements of novelty , this work also contains a number of limitations .
it would certainly be worthwhile to supplement the project in the future with other modern and objective measurement tools , such as muscle electromyography , the biodex system movement analysis , and tensiometry .
also , the study did not involve blind trials or placebo effect assessment . in 1 of the comparison groups ,
patients underwent only the standard functional training and were not treated with any electrotherapeutical treatment whatsoever , which constituted a point of reference in relation to the exposed groups and which is permitted in the methodology of medical research publications .
however , using the simulated treatments and the so - called single - blind trial would certainly constitute an interesting addition and would also significantly raise the profile of this study .
a weakness of this research was also the relatively high measurement error ( although in most cases the researchers do not even perform this kind of analysis or self - reflection ) during the observation of mobility of the hip joint and the lumbar region of the spine .
we endeavored to mitigate it by making our own error estimations and by making sure that all measurements were made by the same person ( an average of the 5 performed trials ) .
, it will also be necessary to perform an assessment of remote results , which will allow us to estimate the sustainability of remission achieved due to electrical therapy .
our conducted research indicates that using electrostimulation with interferential current stimulation penetrating deeper into the tissues results in a significant and long - term elimination of pain , and an improvement of functional ability of patients suffering from low back pain on the basis of an analysis of both subjective and objective parameters .
although tens currents and hves are helpful in treatment of discopathy of the lower region of the spine , use of interference currents led to greater remission of symptoms . on the other hand
, the research indicates that the use of diadynamic currents appears to be useless in the course of degenerative proliferative disease of the spine ( within the scope studied in this paper ) . | backgroundin the currently available research publications on electrical therapy of low back pain , generally no control groups or detailed randomization were used , and such studies were often conducted with relatively small groups of patients , based solely on subjective questionnaires and pain assessment scales ( lacking measurement methods to objectify the therapeutic progress ) .
the available literature also lacks a comprehensive and large - scale clinical study .
the purpose of this study was to assess the effects of treating low back pain using selected electrotherapy methods .
the study assesses the influence of individual electrotherapeutic treatments on reduction of pain , improvement of the range of movement in lower section of the spine , and improvement of motor functions and mobility.material/methodsthe 127 patients qualified for the therapy ( ultimately , 123 patients completed the study ) and assigned to 6 comparison groups : a conventional tens , b acupuncture - like tens , c high - voltage electrical stimulation , d interferential current stimulation , e diadynamic current , and f control group.resultsthe research showed that using electrical stimulation with interferential current penetrating deeper into the tissues results in a significant and more efficient elimination of pain , and an improvement of functional ability of patients suffering from low back pain on the basis of an analysis of both subjective and objective parameters .
the tens currents and high voltage were helpful , but not as effective .
the use of diadynamic currents appears to be useless.conclusionsselected electrical therapies ( interferential current , tens , and high voltage ) appear to be effective in treating chronic low back pain . | Background
Material and Methods
Patients qualification
Inclusion and exclusion criteria
Interventions characteristics
Pain measurements and functional testing
Stabilometric platform measurements
Statistical analysis
Results
Analysis of results regarding the influence of electrical therapy on the subjective experience of spinal pain
Analysis of results regarding the influence of electrical therapy on the subjective experience of functional ability in patients with low back pain
Analysis of results regarding the influence of electrical therapy on hip mobility range on the affected side and in the low back region
Analysis of the results regarding the influence of electrical therapy on body posture parameters measured using the stabilometric platform
Discussion
Strength/weakness of the study
Conclusions | however , on the basis of analysis of the available literature on electrotherapeutical methods of low back pain treatment , most studies fail to meet the basic criteria of evidence - based physiotherapy , which makes it extremely difficult to carry out a clear and objective analysis of clinical efficacy of treatments that are widely used in everyday practice . in the currently available research publications on electrical therapy of low back pain , generally no control groups or detailed randomization
were used , and such studies were often conducted with relatively small groups of patients , based solely on subjective questionnaires and pain assessment scales ( lacking measurement methods to objectify the therapeutic progress ) . the available literature also lacks any comprehensive and large - scale clinical study analyzing clinical efficacy of the numerous electrotherapeutic methods popular in physiotherapy in a single study , with consistent statistical calculations , which would allow for a reliable assessment of the studied treatments and their comparison to one another , and in relation to a representative control group . therefore , the aim of this study was to assess the effects of treating low back pain using selected electrical therapies . the study assesses the influence of individual electrotherapeutic treatments on reduction of pain , improvement of the range of movement in lower section of the spine , and improvement of motor functions and mobility . a total of 127 patients were qualified for the therapy ( ultimately , 124 patients completed the study ) and assigned to 6 comparison groups ( a flow diagram is presented in figure 1 ) . a total of 127 patients were qualified for the therapy ( ultimately , 124 patients completed the study ) and assigned to 6 comparison groups ( a flow diagram is presented in figure 1 ) . however , the intergroup analysis demonstrated that the highest analgesic effect was recorded in group d ( interferential current stimulation ) , which proved to be a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( high - voltage electrical stimulation [ hves ] ) . however , on the basis of an intergroup analysis , we found that the highest percentual improvement of the patients functional ability measured with the oswestry questionnaire took place in group d ( interferential current stimulation ) , giving a significantly more beneficial effect than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) . on the basis of an intergroup analysis
, we found that the highest percentual improvement in the lasgue test took place in group d ( interferential current stimulation ) , giving a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) . these parameters were also slightly reduced in the remaining groups , especially group a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) , but despite this trend , no statistically significant difference was obtained in relation to groups e ( dd ) and f ( control group ) . however , the intergroup analysis demonstrated that the highest analgesic effect was recorded in group d ( interferential current stimulation ) , which proved to be a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( high - voltage electrical stimulation [ hves ] ) . however
, on the basis of an intergroup analysis , we found that the highest percentual improvement of the patients functional ability measured with the oswestry questionnaire took place in group d ( interferential current stimulation ) , giving a significantly more beneficial effect than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) . on the basis of an intergroup analysis
, we found that the highest percentual improvement in the lasgue test took place in group d ( interferential current stimulation ) , giving a significantly better result than in groups a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) . these parameters were also slightly reduced in the remaining groups , especially group a ( conventional tens ) , b ( acupuncture - like tens ) , and c ( hves ) , but despite this trend , no statistically significant difference was obtained in relation to groups e ( dd ) and f ( control group ) . although the available literature contains a large number of proposals for conservative treatment of low back pain using electrical therapy , such publications usually concern only 1 type ( or , in some cases , 2 types in a direct comparison under 1 clinical study ) of electric current . , whose aim was to determine the effectiveness of tens in the therapy of chronic pain of the lumbar section of the spine , a series of equivocal data was recorded , confirming the use of the tens as an analgesic . our conducted research indicates that using electrostimulation with interferential current stimulation penetrating deeper into the tissues results in a significant and long - term elimination of pain , and an improvement of functional ability of patients suffering from low back pain on the basis of an analysis of both subjective and objective parameters . on the other hand
, the research indicates that the use of diadynamic currents appears to be useless in the course of degenerative proliferative disease of the spine ( within the scope studied in this paper ) . | [
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] |
for thousands of years , several plants have been used in popular medicine . despite being
considered the main source of antimutagenics and antioxidants ( elik and aslantrk , 2010 ) some of their phytochemicals may
cause adverse reactions or have potential of interacting with other medications ,
generating toxic , cytotoxic , mutagenic and genotoxic effects ( pawlowski et al . , 2012 ; ping et al . , 2012 ; ray et al . ,
however , for many of them
there is little information available regarding the potential health risks at short ,
mid- or long term ( nunes et al . ,
2012 ) .
jatropha gossypiifolia l. ( euphorbiaceae ) , commonly known as bellyache
bush , black physicnut or cotton - leaf physicnut , is a shrub that contains a
characteristic latex largely used for medicinal purposes , though in an empirical way
( cordeiro and secco , 2014 ) .
the leaves are
used in natura or in compresses , and are considered to have
anti - malarian ( jansen et al . ,
2010 ) , insecticidal ( valencia et
al . , 2006 ) , anti - inflammatory ( oliveira et al . , 2010 ) and antimicrobial ( dhale and birari , 2010 ; gaikwad et al . , 2012 ) properties .
the root and stem have
cytotoxic ( nazeema and girija , 2013 ) ,
anti - malarian , leishmanicidal , antimicrobial , insecticidal , molluscicidal ( reviewed by
sabandar et al . , 2013 ) and
anti - inflammatory ( bhagat et al . ,
2013 ) properties . the seeds and fruits
are used against influenza , and also as
laxative ( sabandar et al . ,
2013 ) , sedative , analgesic or anti - diarrheal agents ( apu et al . , 2013 ) .
the latex , in turn , is
bactericidal ( gaikwad et al . ,
2012 ) and molluscicidal ( matos , 2004 ) .
in brazil ,
the topical application of latex in natura is employed
against wounds and bites of venomous animals ( stasi and
hiruma - lima , 2002 ) , and its ingestion in diluted form is used for treatment of
diarrhea by indigenous peoples ( curto , 1993 ) . in
india , the solution of latex with mustard oil ( brassica campestris ) and
clove oil ( syzygium aromaticum ) is applied onto painful gum or teeth
( punjaji , 2012 ) , whereas in the south of
nigeria it is used to reduce nosebleed and wounded skin ( oduola et al . , 2007 ) . despite its medicinal properties , the latex of j. gossypiifolia in
natura , in direct contact with the skin ,
moreover , the
aqueous extract of latex was shown to be toxic for fish as well as upon intraperitoneal
administration to house mice , presenting significant reduction of acetylcholinesterase
( ache ) inhibition in the first ( singh and singh ,
2012 ; pratap and singh , 2013 ) and
seizures in the latter ( singh and singh , 2012 ) .
the inhibition of ache prevents the degradation of choline neurotransmitters , such as
acetylcholine , and prolongs the signal transmission through synapses .
consequently , it
may lead to lethal paralysis by hyperstimulation of the nervous system ( stansley , 1993 ) .
the toxic effect may be mainly associated to
cyclic peptides of the latex ( horsten et
al . , 1996 ; auvin - guette et
al . , 1997 ;
pratap and singh ,
2013 ) , which show cytotoxic activity , as related for other
jatropha species ( reviewed by sabandar et al . , 2013 ) .
therefore , complementary studies are
still necessary for the latex of j. gossypiifolia to be safely employed
for medicinal purposes , starting with the evaluation of its toxic potential .
whereas the toxicity tests in animals lead to their death , alternative analysis should
be considered . in this sense ,
genotoxicity tests using the
allium cepa test system showed a good correlation with the test
system of mammals ( rank and nielsen , 1994 ) ,
indicating its use as an alternative for monitoring the genotoxic potential of chemical
compounds ( fachinetto et al . ,
2007 ) .
additionally , a. cepa stands out among other plants
due to presenting large chromosomes and in few number ( 2n = 16 ) in its
cells ( fiskesjo , 1985 ) .
moreover , the a.
cepa test system has high sensitivity in detecting chemical and
environmental agents ( leme and marin - morales ,
2009 ) .
this system is easy to use and presents itself as a suitable
bioindicator for the first screening of genotoxicity , thanks to its low cost ,
reliability and concordance with other genotoxicity tests .
this way it contributes in
the preliminary genotoxicity assessment of compounds for medicinal purposes ( bagatini et al . , 2007 ) .
thus , the
present work aimed at analyzing the toxic , cytotoxic and genotoxic effects of the latex
from j. gossypiifolia by means of the a.
cepa test system .
latex of j. gossypiifolia was collected from an adult plant in
teresina ( pi , brazil ) in january 2013 .
herbarium specimens containing leaves , flowers
and fruits were stored in the afrnio fernandes at the state university of piau
herbarium ( uespi teresina , brazil ) ; voucher specimen number : haf 03111 .
vale ouro ipa 11 ) used in the bioassays were
kindly provided by the agronomic institute of pernambuco ( ipa , recife , brazil ) .
the latex of j. gossypiifolia was extracted following removal of the
leaf petioles using pruning shears , at 8 to 9 am .
the latex was immediately stored in
falcon tubes wrapped in aluminum foil in order to reduce the oxidation process .
the
latex was then transported , in cooling box containing ice , to the laboratory of plant
genetics and bio - technology ( genetics department , ufpe ) where it was diluted in
distilled water to yield five different latex concentrations ( 1.25 ; 2.5 ; 5 ; 10 and 20
vale ouro ipa -11 ) were germinated
in petri dishes containing filter paper moistened with distilled water , at room
temperature .
when the rootlets reached about 1 cm in length , they were transferred to
the five cited latex concentrations ( one dish for each concentration ) for 24 h.
distilled water was used as negative control ( nc ) ; mms ( methyl methanesulfonate 4
10 m ) , a drug with clastogenic activity , and the herbicide
trifluralin ( 0.84 ppm of the active agent ) , a substance with aneugenic activity
( fernandes et al . , 2007 ) ,
when rootlets reached about 1.5 cm length , the
material was fixed in carnoy ( ethanol : acetic acid 3:1 , v / v ) for 68 h , at room
temperature , and stored at 20 c until slide preparation . for slide preparation , the root tips were washed three times in distilled water , for
5 min each time , and hydrolyzed at 60 c for 10 min in hcl 1 n. after hydrolysis , the
root tips were again washed in distilled water and transferred to amber glass bottles
containing schiffs reagent , in which they remained for 2 h in the dark .
after this
time , the root tips were washed until complete removal of the re - agent , transferred
onto slides , squashed with one drop of 2% acetic carmine , and mounted with
entellan .
latex toxicity was evaluated according to the mean root length variation ( in
centimeters ) of 30 roots per treatment .
the experimental unit consisted of one
individual root ( one root per specimen ) .
cytotoxicity and genotoxicity were evaluated
by scoring 5,000 meristematic cells ( experimental unit : slide with 500 cells , with a
total of 10 analyzed slides per treatment ) under light microscope ( 400 ) .
the
assessed aspects were : ( 1 ) mitotic index ( cytotoxicity ) and ( 2 ) chromosome alteration
index ( genotoxicity ) .
the last one includes alterations resulting from aneugenic
effects ( e.g. c - metaphases , metaphase with chromosome adherences ,
loss chromosomes , multipolar anaphases , binucleate cells , polyploid metaphases , and
other alterations ) or clastogenic effects ( e.g. chromosome fragments
in meta - phase or anaphase , chromosome bridges and other alterations ) .
photographs of the best cells were taken in a leica conventional microscope ( dm 500 )
( 1000 ) , using a digital camera ( nikon 14.0 megapixels ) .
images were adjusted for
brightness and contrast , in gray tones using adobe photoshop cs6 .
the toxicity values were expressed as averages , whereas the values of cytotoxicity
and genotoxicity were expressed as frequencies . statistical analysis to evaluate data
distribution with regard to normality was carried out by lilliefors test ( d ) in the
program assistat 7.7 ( silva and azevedo ,
2002 ) , while homogeneity of variance was evaluated by the cochran test in the
program bioestat 5.3 ( ayres et al . ,
2007 ) .
data which did neither present normal distribution nor homogeneity
were analyzed by the non - parametric test of kruskal - wallis , followed by the a
posteriori student - newman - keuls test ( p < 0.05 ) in the program
bioestat 5.3 ( ayres et al . ,
2007 ) .
the data on cytotoxicity were the only presenting normal
distribution and homogeneity of variance , and were analyzed by the parametric test of
scott - knott ( p < 0.05 ) in the program assistat 7.7 ( silva and azevedo , 2002 ) .
latex of j. gossypiifolia was collected from an adult plant in
teresina ( pi , brazil ) in january 2013 .
herbarium specimens containing leaves , flowers
and fruits were stored in the afrnio fernandes at the state university of piau
herbarium ( uespi teresina , brazil ) ; voucher specimen number : haf 03111 .
vale ouro ipa 11 ) used in the bioassays were
kindly provided by the agronomic institute of pernambuco ( ipa , recife , brazil ) .
the latex of j. gossypiifolia was extracted following removal of the
leaf petioles using pruning shears , at 8 to 9 am .
the latex was immediately stored in
falcon tubes wrapped in aluminum foil in order to reduce the oxidation process .
the
latex was then transported , in cooling box containing ice , to the laboratory of plant
genetics and bio - technology ( genetics department , ufpe ) where it was diluted in
distilled water to yield five different latex concentrations ( 1.25 ; 2.5 ; 5 ; 10 and 20
vale ouro ipa -11 ) were germinated
in petri dishes containing filter paper moistened with distilled water , at room
temperature .
when the rootlets reached about 1 cm in length , they were transferred to
the five cited latex concentrations ( one dish for each concentration ) for 24 h.
distilled water was used as negative control ( nc ) ; mms ( methyl methanesulfonate 4
10 m ) , a drug with clastogenic activity , and the herbicide
trifluralin ( 0.84 ppm of the active agent ) , a substance with aneugenic activity
( fernandes et al . , 2007 ) ,
were used as positive controls .
when rootlets reached about 1.5 cm length , the
material was fixed in carnoy ( ethanol : acetic acid 3:1 , v / v ) for 68 h , at room
temperature , and stored at 20 c until slide preparation . for slide preparation , the root tips were washed three times in distilled water , for
5 min each time , and hydrolyzed at 60 c for 10 min in hcl 1 n. after hydrolysis , the
root tips were again washed in distilled water and transferred to amber glass bottles
containing schiffs reagent , in which they remained for 2 h in the dark .
after this
time , the root tips were washed until complete removal of the re - agent , transferred
onto slides , squashed with one drop of 2% acetic carmine , and mounted with
entellan .
latex toxicity was evaluated according to the mean root length variation ( in
centimeters ) of 30 roots per treatment .
the experimental unit consisted of one
individual root ( one root per specimen ) .
cytotoxicity and genotoxicity were evaluated
by scoring 5,000 meristematic cells ( experimental unit : slide with 500 cells , with a
total of 10 analyzed slides per treatment ) under light microscope ( 400 ) .
the
assessed aspects were : ( 1 ) mitotic index ( cytotoxicity ) and ( 2 ) chromosome alteration
index ( genotoxicity ) .
the last one includes alterations resulting from aneugenic
effects ( e.g. c - metaphases , metaphase with chromosome adherences ,
loss chromosomes , multipolar anaphases , binucleate cells , polyploid metaphases , and
other alterations ) or clastogenic effects ( e.g. chromosome fragments
in meta - phase or anaphase , chromosome bridges and other alterations ) .
photographs of the best cells were taken in a leica conventional microscope ( dm 500 )
( 1000 ) , using a digital camera ( nikon 14.0 megapixels ) .
images were adjusted for
brightness and contrast , in gray tones using adobe photoshop cs6 .
the toxicity values were expressed as averages , whereas the values of cytotoxicity
and genotoxicity were expressed as frequencies . statistical analysis to evaluate data
distribution with regard to normality was carried out by lilliefors test ( d ) in the
program assistat 7.7 ( silva and azevedo ,
2002 ) , while homogeneity of variance was evaluated by the cochran test in the
program bioestat 5.3 ( ayres et al . ,
2007 ) .
data which did neither present normal distribution nor homogeneity
were analyzed by the non - parametric test of kruskal - wallis , followed by the a
posteriori student - newman - keuls test ( p < 0.05 ) in the program
bioestat 5.3 ( ayres et al . ,
2007 ) .
the data on cytotoxicity were the only presenting normal
distribution and homogeneity of variance , and were analyzed by the parametric test of
scott - knott ( p < 0.05 ) in the program assistat 7.7 ( silva and azevedo , 2002 ) .
the tests of toxicity and cytotoxicity carried out with seeds of a.
cepa exposed to the different concentrations of latex from j.
gossypiifolia showed a significant reduction in root mean growth and in
mitotic index ( mi ) values of all tested latex concentrations , when compared to nc ,
except for 1.25 ml / l . in these analyses ,
the reduction was partially or entirely
dose - dependent for root mean growth and in mitotic index , respectively ( table 1 ) .
significant in the kruskal - wallis test with a posteriori
student - newman - keuls test scott - knott test ( p < 0.05 ; averages followed by the same lowercase letter
are not significantly different ) .
the results refer to analysis of 5,000 cells
per treatment . with regards to chromosome alterations ( ca ) , the increase in the total indexes ( table 2 ; figure
1 ) was highly significant when compared with nc results , except for the 10 and
20 ml / l concentrations .
from these results , we may infer that j.
gossypiifolia latex has a genotoxic action . on the other hand , the nemployed
empirically in the popular medicine of maon - significant ca frequencies found for the
higher latex concentrations ( 10 and 20
significant in the kruskal - wallis test with a posteriori
student - newman - keuls test ( the results refer to analysis of 5,000 cells per treatment . when the chromosome alterations were analyzed separately , metaphases with chromosome
adherences ( figure 1a ) have been found in all the
treatments carried out with the latex ; nevertheless significant nc related results were
only recorded for the three lower concentrations ( 1.25 ; 2.5 and 5 ml / l ) .
the presence of
c - metaphases ( figure 1b ) was significant only for
2.5 and 5 ml / l concentrations ( table 2 ) .
other non - significant alterations in chromosome segregation during anaphase and
telophase were registered , such as chromosome losses ( figure 1c ) and multipolarities ( figure
1d e ; table 2 ) . additionally ,
polyploid ( figure 1f ) and binucleated cells ( figure 1 g ) , nuclear buds ( figure 1h ) and lobulated nuclei ( figure 1i ) have been found in all analyzed treatments , although no
significant alterations have been observed for any concentration ( table 2 ) .
chromosome fragments ( figure 1j ) and chromosome
bridges ( figure 1k ; table 2 ) , arising from clastogenic effects , were also
registered , but only the presence of chromosome bridges was significant for
concentrations 1.25 , 5 and 10 ml / l .
the latex of j. gossypiifolia is broadly used in popular medicine in
many countries . however , this plant has phyto - chemicals in its composition such as
alkaloids and cyclic peptides , which may be toxic to users ( singh and singh , 2012 ; pratap
and singh , 2013 ) . in the present study ,
the test system a.
cepa was used to evaluate the toxic , cytotoxic and genotoxic effects of
different concentrations ( 1.25 ; 2.5 ; 5 ; 10 and 20
the concentrations tested were selected based on a usage
concentration recommended in popular medicine ( approximately 10 ml / l , corresponding to
one tablespoon in one liter of water ) .
apart from the recommended concentration , others
were also tested based on results of latex toxicity in fish , which varied from 10 to
21.5 ml / l ( singh and singh , 2012 ; pratap and singh , 2013 ) , but mainly on antimicrobial activity
data , which varied from 1.9 to 4.4 ml / l ( patil
et al . , 2012 ) for the latex of j.
gossypiifolia and from 0.5 to 10
ml / l for the latex of j.
curcas ( arekemase et al . ,
2011 ) .
the bioassays carried out here revealed dose - dependent toxicity ( mean root growth ) and
cytotoxicity ( mitotic index ) for all analyzed treatments except for the lowest
concentration ( 1.25 ml / l ) .
these effects may be attributed to various chemical
substances present in the latex of j. gossypiifolia , mainly cyclic
peptides ( cpi ) , such as cyclogossines a ( horsten
et al . , 1996 ) and b ( auvin - guette et al . , 1997 ) , terpenes ( patil et al . , 2012 ) and alkaloids that have
cytotoxic activity ( sabandar et al . ,
2013 ) .
peptides present in other species of the genus
jatropha have also been reported as cytotoxic , such as
integerrimides a and b of j. integerrima ( mongkolvisut et al . , 2006 ) and the
curcacyclins a and b of j. curcas ( insanu et al . , 2012 ) .
cyclic peptides have a cyclical conformation ( sakai
et al . , 1996 ) , which facilitates their passing through
cell membranes , as well as absent exposure of the c- and n - terminal groups to
exopeptidases ( wu et al . , 2007 )
these characteristics may be related to the ease with which these peptides enter and
remain inside cells of a. cepa , causing toxic effects , as observed here
and in previous studies with latex of j. curcas in artemia
salina as well as in cell culture of human ovarian cancer ( insanu et al . , 2012 ) .
on the other hand ,
terpenes , such as diterpenes , are some of the most toxic compounds in
jatropha ( devappa et
al . , 2011 ) , whereas lipophilic nature ( wink , 2012 ) facilitates their entry and remaining inside cells
of a. cepa , causing toxic effects .
additionally , terpenes in latex may
be associated with a reduction in calcium concentration , thus inhibiting protein kinase
c ( pkc ) , as observed in leaf ethanol extract of j. gossypiifolia ( silva et al . , 1995 ; paes et al . ,
2012 ) , decreasing cell
proliferation ( alberts et al . ,
2010 ) .
similar results were observed with inhibitors of pkc in a.
cepa ( blume et al . ,
2008 ) , arabidopsis thaliana ( sheremet et al . , 2010 ) and nicotiana
tabacum ( sheremet et al . ,
2012 ) .
in the current study , most chromosome alterations ( ca ) , such as chromosome adherences
and c - metaphases , were considered a result of genotoxic effects , since they represent
damage to the genetic material which is not necessarily fixed in the organism ( leme and marin - morales , 2009 ; mazzeo et al . , 2011 ) . when these alterations
can be repaired , they are not transmitted to descendant cells ( ventura - camargo et al . ,
the presence
of chromo - some adherences and c - metaphases confirms the interference of phytochemicals
of j. gossypiifolia latex in the assembly , stabilization and/or
inactivation of spindle fibers , characterizing an aneugenic activity of the latex .
aneugenic activities in metaphase may generate other types of cell abnormalities such as
multipolar ana - phases , nuclear buds , lobulated nuclei , and polyploid cells ( fernandes et al . , 2009 ) . however ,
these alterations were not significant in the present study , what indicates that
chromosome adherences and c - metaphases did not contribute to further alterations , and
suggests a reversible mechanism for them ( odeigah
et al . , 1997 ) .
chromosome bridges , chromosome fragments and part of micronucleus formation are related
to clastogenic activity ( fenech et al . ,
2011 ) .
chromosome bridges and fragments , for instance , could be the result of
chromosome breakage - fusion - bridge cycles , elucidated for the first time by barbara
mcclintock in the 1930s in maize chromosomes ( reviewed by jones , 2005 ) .
however , chromosome fragments could also be
induced by several factors involved in dna breaks ( fenech , 2000 ) . on the other hand
, micronuclei formation could be induced by
both aneugenic and clastogenic activities , related to entire chromosomes or chromosome
fragments ( respectively ) not incorporated into the main nucleus during the cell cycle
( fenech et al . , 2011 ) .
in the
present results , from these three clastogenic alterations , only chromosome bridges had a
significant increase .
this type of alteration represents damage to the genetic material at
chromosome level that could not be repaired by the cell , being possibly transmitted to
descendant cells ( grant , 1978 ) .
furthermore , j.
gossypiifolia latex caused toxicity and cytotoxicity at the tested concentrations ,
except for 1.25 ml / l .
it also generated significant alterations in the assembly and
stabilization of the mitotic spindle fibers , resulting in disturbance of the cell cycle
and chromosome alterations for all concentrations .
based on these last results , it can
be inferred that the mechanism of action of the latex also has aneugenic nature . although j. gossypiifolia is considered an important potential plant
for the generation of pharmacological and/or biotechnological products ( flix - silva et al . ,
2014 ) , overall ,
the tests performed indicated toxicity , cytotoxicity and genotoxicity of j.
gossypiifolia latex , including the concentrations acknowledged as
antimicrobial ( 1.9 and 4.4 ml / l ) ( patil et
al . , 2012 ) .
considering that the latex of j.
gossypiifolia is employed empirically in the popular medicine of many
countries , and that the a. cepa test system presents good correlation
with the tests carried out in mammals ( rank and nielsen ,
1994 ) , it should be pointed out that it may potentially harm the human health
especially if ingested . in addition ,
other studies with this plant material are
necessary in order to elucidate the mechanisms of action of its bioactive compounds , and
to reduce its toxicity while keeping its therapeutic action , for further use of isolated
latex compounds in the pharmaceutical industry . |
jatropha gossypiifolia l. ( euphorbiaceae ) , popularly known as
cotton - leaf physicnut , is a milky shrub notable for its medicinal properties .
the
present study aimed to evaluate the toxic , cytotoxic and genotoxic effects of the
latex of j. gossypiifolia , using allium cepa l. as
test system .
seeds of a. cepa were exposed to five concentrations of
the latex ( 1.25 ; 2.5 ; 5 ; 10 and 20
ml / l ) in order to evaluate parameters of toxicity
( evaluation of root growth ) , cytotoxicity ( mitotic index frequency ) and genotoxicity
( frequency of chromosome alterations ) .
the latex showed a significant decrease in
root mean growth value as well as mitotic index for the tested concentrations , except
for 1.25
ml / l , when compared to results from the negative control .
the 1.25 , 2.5 and
5 ml / l concentrations induced significant chromo - some adherences , c - metaphases and/or
chromosome bridges , as genotoxic effects .
the significant frequency of chromosome
bridges also indicated mutagenic potential for chromosomes of j.
gossypiifolia as discussed in the paper . considering that the latex is
used in popular therapies , and that the test system a. cepa presents
good correlation with tests carried out in mammals
, it can be pointed out that its
use for medicinal purposes may be harmful to human health especially if ingested . | Introduction
Material and Methods
Biological material
Statistical Analysis
Results
Discussion | jatropha gossypiifolia l. ( euphorbiaceae ) , commonly known as bellyache
bush , black physicnut or cotton - leaf physicnut , is a shrub that contains a
characteristic latex largely used for medicinal purposes , though in an empirical way
( cordeiro and secco , 2014 ) . despite its medicinal properties , the latex of j. gossypiifolia in
natura , in direct contact with the skin ,
moreover , the
aqueous extract of latex was shown to be toxic for fish as well as upon intraperitoneal
administration to house mice , presenting significant reduction of acetylcholinesterase
( ache ) inhibition in the first ( singh and singh ,
2012 ; pratap and singh , 2013 ) and
seizures in the latter ( singh and singh , 2012 ) . thus , the
present work aimed at analyzing the toxic , cytotoxic and genotoxic effects of the latex
from j. gossypiifolia by means of the a.
cepa test system . the
latex was then transported , in cooling box containing ice , to the laboratory of plant
genetics and bio - technology ( genetics department , ufpe ) where it was diluted in
distilled water to yield five different latex concentrations ( 1.25 ; 2.5 ; 5 ; 10 and 20
vale ouro ipa -11 ) were germinated
in petri dishes containing filter paper moistened with distilled water , at room
temperature . the
latex was then transported , in cooling box containing ice , to the laboratory of plant
genetics and bio - technology ( genetics department , ufpe ) where it was diluted in
distilled water to yield five different latex concentrations ( 1.25 ; 2.5 ; 5 ; 10 and 20
vale ouro ipa -11 ) were germinated
in petri dishes containing filter paper moistened with distilled water , at room
temperature . the tests of toxicity and cytotoxicity carried out with seeds of a.
cepa exposed to the different concentrations of latex from j.
gossypiifolia showed a significant reduction in root mean growth and in
mitotic index ( mi ) values of all tested latex concentrations , when compared to nc ,
except for 1.25 ml / l . with regards to chromosome alterations ( ca ) , the increase in the total indexes ( table 2 ; figure
1 ) was highly significant when compared with nc results , except for the 10 and
20 ml / l concentrations . when the chromosome alterations were analyzed separately , metaphases with chromosome
adherences ( figure 1a ) have been found in all the
treatments carried out with the latex ; nevertheless significant nc related results were
only recorded for the three lower concentrations ( 1.25 ; 2.5 and 5 ml / l ) . the presence of
c - metaphases ( figure 1b ) was significant only for
2.5 and 5 ml / l concentrations ( table 2 ) . chromosome fragments ( figure 1j ) and chromosome
bridges ( figure 1k ; table 2 ) , arising from clastogenic effects , were also
registered , but only the presence of chromosome bridges was significant for
concentrations 1.25 , 5 and 10 ml / l . in the present study ,
the test system a.
cepa was used to evaluate the toxic , cytotoxic and genotoxic effects of
different concentrations ( 1.25 ; 2.5 ; 5 ; 10 and 20
the concentrations tested were selected based on a usage
concentration recommended in popular medicine ( approximately 10 ml / l , corresponding to
one tablespoon in one liter of water ) . the bioassays carried out here revealed dose - dependent toxicity ( mean root growth ) and
cytotoxicity ( mitotic index ) for all analyzed treatments except for the lowest
concentration ( 1.25 ml / l ) . ,
the presence
of chromo - some adherences and c - metaphases confirms the interference of phytochemicals
of j. gossypiifolia latex in the assembly , stabilization and/or
inactivation of spindle fibers , characterizing an aneugenic activity of the latex . furthermore , j.
gossypiifolia latex caused toxicity and cytotoxicity at the tested concentrations ,
except for 1.25 ml / l . considering that the latex of j.
gossypiifolia is employed empirically in the popular medicine of many
countries , and that the a. cepa test system presents good correlation
with the tests carried out in mammals ( rank and nielsen ,
1994 ) , it should be pointed out that it may potentially harm the human health
especially if ingested . | [
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the biomaterial mineral trioxide aggregate ( mta ) was approved for endodontic use by the
u.s .
mta has been shown to promote favorable tissue reactions
characterized by the absence of severe inflammation , the presence of a fibrous capsule ,
and the induction of mineralized repair tissue .
mta is highly
biocompatible when used for pulp capping , root perforations and retrograde fillings .
however , despite its positive characteristics , mta does not have the
appropriate physical properties for use as a sealer .
some manufacturers have recently added specific components to improve the ease of
handling and insertion material properties of mta in order to create mta - based sealers .
some examples of such materials currently on the market are proroot endo
sealer ( dentsply , tulsa , ok , usa ) , endo - cpm - sealer ( egeo srl ,
buenos aires , buenos aires , argentina ) and mta fillapex ( angelus , londrina ,
pr , brazil ) . according to the manufacturer
, endo - cpm - sealer has a chemical composition
similar to that of mta but with the addition of calcium carbonate to reduce the post - set
ph level from 12.5 to 10.0 .
this restricts the necrosis of the surface in contact with
the material , enabling the action of alkaline phosphatase .
endo - cpm - sealer has satisfactory plasticity ,
adhesion , flow , radiopacity , and antimicrobial activity .
the
biocompatibility of endo - cpm - sealer is similar to that of angelus
mta : both produced a moderate chronic inflammatory response ( apparent on
the 7 and 15 days post - procedure ) that decreased with
time .
this material has also
been shown to stimulate mineralization of the subcutaneous tissue of rats .
mta fillapex is composed of salicylate resin , resin diluent , natural resin ,
bismuth oxide , silica nanoparticles , and mineral trioxide aggregate . according to the
manufacturer
, it has the following physical properties : working time , 35 min , flow
capacity , 27.66 mm , setting time , 130 min , optical density , 77% , and solubility , 0.1% .
mta fillapex presented solubility values
higher than required by the ansi / ada .
according to a cytotoxicity assay , mta fillapex was more cytotoxic in
fibroblast cultures in the beginning but it developed the best behavior after 48
hours .
a subcutaneous study
showed that mta fillapex produced a moderate chronic inflammatory reaction
evident on the 7 day that resolved over a short period of time ( 15 days ) ,
similar to that induced by angelus mta and faster than that induced by
sealapex .
however , the
behaviour of mta fillapex has not yet been evaluated in an application model
study , such as in the treatment of chronic apical lesions in canine teeth .
therefore , this study aimed to evaluate the healing of periapical lesions in canine
teeth after a single endodontic treatment using sealapex ( sybronendo ,
glendora , ca , usa ) , endo - cpm - sealer or mta fillapex as sealers .
sixty - four roots of 2 male beagle dogs were used in this study : eight central incisors ,
eight intermediate incisors , eight lateral incisors , eight canines , four second superior
premolars ( 2 roots each ) , four third superior premolars ( 2 roots each ) , four third
inferior premolars ( 2 roots each ) and four fourth inferior premolars ( 2 roots each ) .
the
care of the animals was in accordance with the guidelines of the araatuba school of
dentistry - unesp ethical committee , which approved the project before the beginning of
the experiments .
the animals were anaesthetized by intramuscular injection of a combination of xylazine
( cristlia chemicals pharmaceuticals , ltd . , itapira , sp , brazil ; 0.05 ml / kg body weight )
and a mixture of tiletamine hydrochloride and zolazepam hydrochloride ( zoletil-50 ;
virbac , so paulo , sp , brazil ; 0.2 ml/ kg body weight ) . the teeth studied were subjected
to coronary opening and pulp extirpation up to the apical barrier .
the canals were left
open and exposed to the oral environment in order to induce periapical lesions .
after 6
months , control radiographs were taken and the teeth were fitted with rubber dams and
subjected to endodontic treatment .
the fifty - four experimental root canals were biomechanically prepared up to the apical
barrier with a # 40 k - file and abundantly irrigated with 2.5% sodium hypochlorite ( naocl )
throughout the instrumentation process .
over - instrumentation with # 20 k files was then
performed in such a way as to create an artificial main canal foramen .
the canals were
irrigated , dried with paper points , and filled with 17% edta for 3 min .
the edta was
removed by irrigation with 2.5% naocl followed by a final irrigation with saline
solution .
the root canals were
filled to 1 mm before the apex with gutta - percha points and sealed by the active lateral
condensation technique with sealapex , endo - cpm - sealer , or mta
fillapex .
the pulp chambers were cleaned and the access cavities sealed with
intermediate restorative material ( coltosol ; vigodent , rio de janeiro , rj , brazil ) and
composite resin ( tph spectrum ; dentsply , tulsa , ok , usa ) .
these procedures divided the
roots into 3 experimental treatment groups of 18 roots each as follows : group i :
sealapex , group ii : endo - cpm - sealer , and group iii : mta
fillapex .
as positive controls ( group iv ) , 4 roots were selected for pulp extirpation and their
canals left exposed to the oral environment with no additional treatment for 6 months
( after which the animals were sacrificed ) . as negative controls ( group v ) , 4 roots
remained completely healthy until the end of the experiment once they were not subjected
to any treatment .
six months after treatment , the animals were sacrificed with an overdose of anaesthetic .
the maxilla and mandibles containing the roots were removed , fixed in 10%
neutral - buffered formalin solution , and decalcified in 17% edta solution .
segments of
the jaws , each containing 1 root , were prepared for histological examination using
standard procedures .
briefly , the specimens were embedded in paraffin , serially
sectioned at 6-m thickness , and stained with hematoxylin and eosin or by the brown and
brenn technique .
some roots were discarded due to histological artefacts , and only 12 roots treated with
each material were tested and 4 roots from each control group were analyzed .
the
histological details of the newly formed cementum ( thickness , extension , and biological
closure of the main and accessory canals ) , cementum resorption , bone tissue resorption ,
inflammatory reaction ( chronic or acute , number of cells , and extension of the
reaction ) , periodontal ligament ( thickness and organisation ) , root canal filling limit ,
and presence of debris , giant cells , and microorganisms were noted .
all of the
above - listed histomorphological events were scored from 1 to 4 ( from the best to the
worst ) as described in figure 6 and according to a
previous study .
the statistical
significance of the results was analyzed by the kruskal wallis test ( p=0.05 ) .
( a ) radiographic examination
( * ) 180 days after pulpectomy [ original ; rx ] .
( b ) panoramic view [ 100 ;
hematoxylin & eosin ( h.e . ) ] .
( c ) severe inflammatory infiltrate and extensive
areas of resorption ( arrow ) [ 400 ; h.e . ] .
( d ) gram - negative microorganisms in the
dentinal tubules ( arrows ) [ 400 ; brown and brenn ( b.b . ) ] representative images of the negative control group . ( a ) radiographic examination
( * )
( b ) panoramic view [ 100 ; hematoxylin &
eosin ( h.e . ) ] .
( c , d ) mild inflammatory infiltrate and tissue organization ( arrows )
[ 400 ; h.e . ] .
( e ) absence of microorganisms in cementum lacunae ( arrows ) [ 400
brown and brenn ( b.b . ) ] representative images of the sealapex group .
( a ) radiographic examination ( * ) 180
days after filling [ original ; rx ] .
( b ) panoramic view showing lack of complete
closure of the main canal [ 100 ; hematoxylin & eosin ( h.e . ) ] . ( c )
( d ) pulp stump with mild
inflammatory infiltrate and mineralized tissue ( arrows ) [ 400 ; h.e . ] . ( e )
gram - negative microorganisms in cementum lacunae ( arrows ) [ 400 ; brown and brenn
( b.b . ) ] .
( g ) newly formed cementum in the
vicinity of the main canal ( arrow ) [ 400 ; h.e . ]
representative images of the endo - cpm - sealer group . ( a ) radiographic examination
( * ) 180 days after filling [ original ; rx ] .
( b ) panoramic view showing areas of
resorption and the absence of newly formed cementum ( arrows ) [ 100 ; hematoxylin
& eosin ( h.e . ) ] . ( c ) gram - negative microorganisms in cementum lacunae ( arrows )
( d ) severe inflammatory infiltrate ( arrow ) [ 400 -
h.e . ] .
( f ) pulp stump with moderate inflammatory
infiltrate and mineralized tissue ( arrow ) [ 400 ; h.e . ]
( a ) radiographic examination ( * )
180 days after filling [ original ; rx ] .
( b ) panoramic view showing areas of
resorption and the absence of newly formed cementum ( arrow ) [ 100 ; hematoxylin
& eosin ( h.e . ) ] .
( c ) severe inflammatory infiltrate ( arrow ) [ 400 ; h.e . ] .
( d )
pulp stump with mild inflammatory infiltrate and mineralized tissue ( arrows )
[ 400 ; h.e . ] .
( e ) gram - positive and gramnegative microorganisms in cementum
lacunae ( arrows ) [ 400 ; brown and brenn ( b.b . ) ] .
( g ) newly formed cementum in the vicinity of the main canal , unrepaired areas of
resorption , and severe inflammatory infiltrate ( arrows ) [ 400 ; he ] distribution of specimens of each group according to scores of histopathologic
parameters
the areas
of resorption were either active or inactive without signs of healing ( figure 1b ) .
the apical periodontal space was
intensely and extensively invaded by tissue containing acute and chronic inflammatory
infiltrates ; the latter was characterized by lymphocytes , plasma cells , and
macrophages ( figure 1c ) .
brown and brenn staining showed gram - negative microorganisms in the
main canal , apical accessory canals , and cementum lacunae of all specimens ( figure 1d ) .
no areas
of active resorption of cementum or bone were observed in any specimen ( figure 2b ) .
mild inflammatory infiltrates were
observed within the ramifications or adjacent to the foramina in all specimens ( figures 2c , d ) .
the thickness of the periodontal ligament was variable , ranging up to 200
m , and the ligament was inserted into the cementum and the bone across the apical
portion in all specimens .
eosinophilic newly formed cementum was observed in 9 specimens analyzed , but the new
cementum repaired only 1/3 or fewer of the areas of resorption in 8 specimens .
active
resorption in the lateral surfaces of the roots was observed in 6 specimens .
only 1 specimen showed complete closure of the delta by the newly formed cement ,
while 2 specimens showed complete closure of the main canal ( figures 3f , g ) .
ten
specimens showed severe chronic inflammatory infiltration of the periodontal ligament
( figure 3c ) .
however , the periapical tissue
that grew into the canal showed mild inflammatory infiltrates close to the material
and mineralized tissue in close contact with the material in all specimens ( figure 3d ) .
the root canal filling limit was beyond the intended limit in 9 specimens and short
of the intended limit in only 3 specimens .
inactive bone was observed in 4 specimens ,
resorption with a few active areas in 6 specimens , and resorption with many active
areas in 2 specimens .
no debris was observed in 8 specimens , while discrete pockets
of debris were present in 4 specimens .
many giant cells were observed in 8 specimens ,
moderate numbers in 3 specimens , and discrete giant cells in 1 specimen .
brown and brenn staining showed gram - positive and gram - negative microorganisms , with
gram - negative organisms predominating in the ramifications of the main canal and
especially in the cementum lacunae in all specimens ( figure 3e ) .
eosinophilic newly formed cementum was observed in 5 of the specimens analyzed but
repaired only 1/3 or fewer of the areas of resorption in 4 specimens
no complete closure of the apical delta and only 1 example of complete closure of the
main canal by the newly formed cement was observed ( figure 4e ) .
severe chronic inflammatory infiltrates were observed in 11
specimens ( figure 4d ) and a moderate
inflammatory infiltrate in 1 additional specimen .
moderate inflammatory infiltrates
were observed in the pulp stump in all specimens , and mineralized tissue was noted in
close contact with the material in most specimens ( figure 4f ) .
the limit of filling was beyond the intended apical limit in 9 specimens and short of
the intended limit in only 3 specimens .
resorption of bone tissue with few active
areas was observed in 10 specimens and resorption with many active areas in 2
specimens .
brown and brenn staining showed gram - negative microorganisms in the ramifications of
the main canal and especially in the cementum lacunae of all specimens ( figure 4c ) .
eosinophilic newly formed cementum was observed in 9 specimens analyzed and repaired
1/3 or fewer of the areas of resorption in all 9 cases .
complete closure of the apical delta in a few ramifications was observed in 9
specimens and complete closure of the main canal in 1 specimen ( figures 5f , g ) .
moderate
chronic inflammatory infiltrates were observed in 2 specimens and severe inflammatory
infiltrates in 10 specimens ( figure 5c ) .
mild
inflammatory infiltrates and the presence of mineralized tissue in close contact with
the material were seen in the pulp stumps of all specimens ( figure 5d ) .
the root canal filling limit was beyond the intended apical limit in 11 specimens and
short of the intended limit in only one specimen .
resorption of bone tissue with many
active areas was observed in only 1 specimen , resorption with few active areas in 9
specimens , and no active areas of resorption in 2 specimens .
no debris was observed
in 7 specimens and discrete pockets of debris in 5 specimens ( figures 5b , d ) .
brown and brenn staining showed gram - negative microorganisms in the ramifications of
the main canal and especially in the cementum lacunae of all specimens ( figure 5e ) .
the scores assigned to the various histomorphological events were subjected to the
kruskal - wallis test .
this test ranked the experimental groups from the best to the
worst as follows : ( a ) sealapex , ( b ) mta fillapex , and ( c )
endo - cpm - sealer .
the scores did not differ significantly among the
groups ( p>0.05 ) ( figures 6 and 7 ) , owing mainly to the absence of periapical
tissue healing .
however , the pulp stump exhibited mild inflammatory reaction only in
the sealapex and mta fillapex groups .
the areas
of resorption were either active or inactive without signs of healing ( figure 1b ) .
the apical periodontal space was
intensely and extensively invaded by tissue containing acute and chronic inflammatory
infiltrates ; the latter was characterized by lymphocytes , plasma cells , and
macrophages ( figure 1c ) .
brown and brenn staining showed gram - negative microorganisms in the
main canal , apical accessory canals , and cementum lacunae of all specimens ( figure 1d ) .
no areas
of active resorption of cementum or bone were observed in any specimen ( figure 2b ) .
mild inflammatory infiltrates were
observed within the ramifications or adjacent to the foramina in all specimens ( figures 2c , d ) .
the thickness of the periodontal ligament was variable , ranging up to 200
m , and the ligament was inserted into the cementum and the bone across the apical
portion in all specimens .
eosinophilic newly formed cementum was observed in 9 specimens analyzed , but the new
cementum repaired only 1/3 or fewer of the areas of resorption in 8 specimens .
active
resorption in the lateral surfaces of the roots was observed in 6 specimens .
only 1 specimen showed complete closure of the delta by the newly formed cement ,
while 2 specimens showed complete closure of the main canal ( figures 3f , g ) .
ten
specimens showed severe chronic inflammatory infiltration of the periodontal ligament
( figure 3c ) .
however , the periapical tissue
that grew into the canal showed mild inflammatory infiltrates close to the material
and mineralized tissue in close contact with the material in all specimens ( figure 3d ) .
the root canal filling limit was beyond the intended limit in 9 specimens and short
of the intended limit in only 3 specimens .
inactive bone was observed in 4 specimens ,
resorption with a few active areas in 6 specimens , and resorption with many active
areas in 2 specimens .
no debris was observed in 8 specimens , while discrete pockets
of debris were present in 4 specimens .
many giant cells were observed in 8 specimens ,
moderate numbers in 3 specimens , and discrete giant cells in 1 specimen .
brown and brenn staining showed gram - positive and gram - negative microorganisms , with
gram - negative organisms predominating in the ramifications of the main canal and
especially in the cementum lacunae in all specimens ( figure 3e ) .
eosinophilic newly formed cementum was observed in 5 of the specimens analyzed but
repaired only 1/3 or fewer of the areas of resorption in 4 specimens .
no complete closure of the apical delta and only 1 example of complete closure of the
main canal by the newly formed cement was observed ( figure 4e ) .
severe chronic inflammatory infiltrates were observed in 11
specimens ( figure 4d ) and a moderate
inflammatory infiltrate in 1 additional specimen .
moderate inflammatory infiltrates
were observed in the pulp stump in all specimens , and mineralized tissue was noted in
close contact with the material in most specimens ( figure 4f ) .
the limit of filling was beyond the intended apical limit in 9 specimens and short of
the intended limit in only 3 specimens .
resorption of bone tissue with few active
areas was observed in 10 specimens and resorption with many active areas in 2
specimens .
brown and brenn staining showed gram - negative microorganisms in the ramifications of
the main canal and especially in the cementum lacunae of all specimens ( figure 4c ) .
eosinophilic newly formed cementum was observed in 9 specimens analyzed and repaired
1/3 or fewer of the areas of resorption in all 9 cases .
complete closure of the apical delta in a few ramifications was observed in 9
specimens and complete closure of the main canal in 1 specimen ( figures 5f , g ) .
moderate
chronic inflammatory infiltrates were observed in 2 specimens and severe inflammatory
infiltrates in 10 specimens ( figure 5c ) .
mild
inflammatory infiltrates and the presence of mineralized tissue in close contact with
the material were seen in the pulp stumps of all specimens ( figure 5d ) .
the root canal filling limit was beyond the intended apical limit in 11 specimens and
short of the intended limit in only one specimen .
resorption of bone tissue with many
active areas was observed in only 1 specimen , resorption with few active areas in 9
specimens , and no active areas of resorption in 2 specimens .
no debris was observed
in 7 specimens and discrete pockets of debris in 5 specimens ( figures 5b , d ) .
brown and brenn staining showed gram - negative microorganisms in the ramifications of
the main canal and especially in the cementum lacunae of all specimens ( figure 5e ) .
the scores assigned to the various histomorphological events were subjected to the
kruskal - wallis test .
this test ranked the experimental groups from the best to the
worst as follows : ( a ) sealapex , ( b ) mta fillapex , and ( c )
endo - cpm - sealer .
the scores did not differ significantly among the
groups ( p>0.05 ) ( figures 6 and 7 ) , owing mainly to the absence of periapical
tissue healing .
however , the pulp stump exhibited mild inflammatory reaction only in
the sealapex and mta fillapex groups .
the experimental model of lesion induction used in this study was based on previously
established criteria .
moreover , in
the present study , the root canal filling materials were evaluated without the use of an
intracanal dressing ; this choice was made to reduce the number of variables , not
necessarily as an option for clinical use .
the results obtained in this study for the healing of periapical lesions in canine teeth
were unsatisfactory regardless of the sealer used .
mta fillapex gave results
similar to those obtained with sealapex and endo - cpm - sealer with
regard to the different histopathological criteria investigated .
the similarity of the
results may be because all contain calcium oxide ( cao ) and may therefore have similar
dominant mechanisms of biological action .
cao may react with water or tissue fluids to
form calcium hydroxide , which then dissociates into calcium and hydroxyl ions and
alkalizes the tissue to form calcite crystals .
several factors can influence the results of endodontic treatment of teeth with
periapical lesions , including the biomechanical preparation ( instrumentation and
irrigation ) , intracanal dressing , and filling .
the instruments penetrate almost the entire main root canal but
not the ramifications and cementum lacunae , in which microorganisms may lodge and cause
apical periodontitis . the use of irrigation solutions is intended to reduce the number of microorganisms ,
remove debris , and neutralize organic compounds ; however , due to the risk of leakage
through the apical foramen , the irrigation solutions must be biocompatible and
non - irritating to the periapical tissues . at high concentrations , naocl has potent antimicrobial have
activity due to the release of secondary chlorates , which lead to increased tissue
dissolution , and it is recommended for the treatment of teeth with periapical
lesions . the antimicrobial activity of 2.5% naocl against enterococcus faecalis ,
porphyromonas endodontalis , porphyromonas gingivalis , prevotella intermedia ,
prevotella nigrescens , streptococcus mutans , streptococcus sanguis , and
streptococcus sobrinus and its effectiveness at disinfecting the
root canal have been demonstrated in some in vitro studies .
furthermore , naocl solutions at concentrations of 1% , 2.5% , and 5%
have been shown to have similar antimicrobial activities .
although irrigation with 2.5% naocl was used in
association with instrumentation in this study , bacteria were still observed in most
specimens of all 3 experimental groups .
the results of this study showed that the filling materials used did not effectively
combat endodontic infection , since the periapical tissue was not completely repaired .
although sealers have antimicrobial activity , this was evidently
insufficient to control the infection as an intracanal treatment , especially after the
setting time .
furthermore , these
results were similar to those of previously reported studies that showed that teeth
treated in a single appointment did not heal adequately compared with those treated with
calcium hydroxide paste as an intracanal dressing
12 , 22 .
consistent with this
fact , the complexity of the root canal system formed by the main canals and
ramifications must be considered an important factor in the disinfection strategy that
justifies the use of an intracanal dressing
12 , 19 .
calcium hydroxide
dressing is widely used as an intracanal dressing because it inhibits bacterial enzymes
and activates tissue enzymes such as alkaline phosphatase resulting in a mineralizing
effect . radiographic study
evaluating the healing of periapical lesions treated in one or two visits using calcium
hydroxide as an intracanal dressing and found that the additional disinfecting with
calcium hydroxide resulted in a 10% increasing on the healing rates .
an interesting positive observation was that mta fillapex and
sealapex produced less inflammatory reaction in the region of the pulp
stump in close contact with the filling materials , demonstrating the superior
biocompatibility of these materials in an application model .
these findings are in
accordance with results obtained in a rat model that showed that mta
fillapex and sealapex were biocompatible and stimulated
mineralization 8- 10 ,
13 .
these calcifications are thought to originate from the cao present
in mta fillapex and in sealapex .
when in contact with water , cao
can be converted into ca(oh)2 and dissociated into ca and
oh .
the diffusion of hydroxyl ions from the root canal increases the ph
level at the surface of the root adjacent to the periodontal tissues , possibly
interfering with osteoclastic activity and promoting alkalinization in the adjacent
tissues , which favors healing .
this study demonstrated that even with the use of biocompatible materials that can
stimulate tissue mineralization , the complete repair did not occur without the root
canal system disinfection .
it can be concluded that the endodontic treatment performed in a single session using
these materials can not support complete healing of the periapical tissues of canine
teeth . | some manufacturers have recently added specific components to improve the ease of
handling and insertion material properties of mta in order to create mta - based
sealers.objective:the aim of this study was to evaluate the healing of periapical lesions in canine
teeth after a single session of endodontic treatment with mta fillapex
compared with sealapex or endo - cpm - sealer. material and methods : sixty - two root canals were performed on two 1-year - old male dogs .
after coronal
access and pulp extirpation , the canals were exposed to the oral cavity for 6
months in order to induce periapical lesions .
the root canals were prepared ,
irrigated with a solution of 2.5% sodium hypochlorite and filled with gutta - percha
and different sealers , according to the following groups : 1 ) sealapex ; 2 )
endo - cpm - sealer ; and 3 ) mta fillapex . some teeth with periapical
lesions
were left untreated for use as positive controls .
healthy teeth were used
as negative controls .
after 6 months , the animals were sacrificed and serial
sections from the roots were prepared for histomorphologic analysis and stained
with hematoxylin and eosin and the brown and brenn technique .
the lesions were
scored according to pre - established histomorphologic parameters and the scores
statistically analyzed using the kruskal - wallis test .
results : all 3 materials produced similar patterns of healing ( p>0.05 ) ; in particular ,
persistent inflammation and absence of complete periapical tissue healing were
consistently noted .
conclusions : preparation of the infected root canals followed by filling with the materials
studied was insufficient to provide complete healing of the periapical
tissues . | INTRODUCTION
MATERIAL AND METHODS
RESULTS
Positive control group
Negative control group
Sealapex
Endo-CPM-Sealer
MTA Fillapex
Comparison among the groups
DISCUSSION
CONCLUSIONS | some manufacturers have recently added specific components to improve the ease of
handling and insertion material properties of mta in order to create mta - based sealers . some examples of such materials currently on the market are proroot endo
sealer ( dentsply , tulsa , ok , usa ) , endo - cpm - sealer ( egeo srl ,
buenos aires , buenos aires , argentina ) and mta fillapex ( angelus , londrina ,
pr , brazil ) . according to the manufacturer
, endo - cpm - sealer has a chemical composition
similar to that of mta but with the addition of calcium carbonate to reduce the post - set
ph level from 12.5 to 10.0 . however , the
behaviour of mta fillapex has not yet been evaluated in an application model
study , such as in the treatment of chronic apical lesions in canine teeth . therefore , this study aimed to evaluate the healing of periapical lesions in canine
teeth after a single endodontic treatment using sealapex ( sybronendo ,
glendora , ca , usa ) , endo - cpm - sealer or mta fillapex as sealers . the canals were left
open and exposed to the oral environment in order to induce periapical lesions . after 6
months , control radiographs were taken and the teeth were fitted with rubber dams and
subjected to endodontic treatment . the fifty - four experimental root canals were biomechanically prepared up to the apical
barrier with a # 40 k - file and abundantly irrigated with 2.5% sodium hypochlorite ( naocl )
throughout the instrumentation process . the root canals were
filled to 1 mm before the apex with gutta - percha points and sealed by the active lateral
condensation technique with sealapex , endo - cpm - sealer , or mta
fillapex . these procedures divided the
roots into 3 experimental treatment groups of 18 roots each as follows : group i :
sealapex , group ii : endo - cpm - sealer , and group iii : mta
fillapex . as positive controls ( group iv ) , 4 roots were selected for pulp extirpation and their
canals left exposed to the oral environment with no additional treatment for 6 months
( after which the animals were sacrificed ) . briefly , the specimens were embedded in paraffin , serially
sectioned at 6-m thickness , and stained with hematoxylin and eosin or by the brown and
brenn technique . all of the
above - listed histomorphological events were scored from 1 to 4 ( from the best to the
worst ) as described in figure 6 and according to a
previous study . however , the periapical tissue
that grew into the canal showed mild inflammatory infiltrates close to the material
and mineralized tissue in close contact with the material in all specimens ( figure 3d ) . the scores assigned to the various histomorphological events were subjected to the
kruskal - wallis test . this test ranked the experimental groups from the best to the
worst as follows : ( a ) sealapex , ( b ) mta fillapex , and ( c )
endo - cpm - sealer . the scores did not differ significantly among the
groups ( p>0.05 ) ( figures 6 and 7 ) , owing mainly to the absence of periapical
tissue healing . however , the periapical tissue
that grew into the canal showed mild inflammatory infiltrates close to the material
and mineralized tissue in close contact with the material in all specimens ( figure 3d ) . the scores assigned to the various histomorphological events were subjected to the
kruskal - wallis test . this test ranked the experimental groups from the best to the
worst as follows : ( a ) sealapex , ( b ) mta fillapex , and ( c )
endo - cpm - sealer . the scores did not differ significantly among the
groups ( p>0.05 ) ( figures 6 and 7 ) , owing mainly to the absence of periapical
tissue healing . the results obtained in this study for the healing of periapical lesions in canine teeth
were unsatisfactory regardless of the sealer used . mta fillapex gave results
similar to those obtained with sealapex and endo - cpm - sealer with
regard to the different histopathological criteria investigated . several factors can influence the results of endodontic treatment of teeth with
periapical lesions , including the biomechanical preparation ( instrumentation and
irrigation ) , intracanal dressing , and filling . it can be concluded that the endodontic treatment performed in a single session using
these materials can not support complete healing of the periapical tissues of canine
teeth . | [
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] |
the ubiquitination process is carried out by an enzymatic cascade that includes an activating enzyme ( e1 ) , a conjugating enzyme ( e2 ) and a ligase ( e3 ; dye & schulman , 2007 ) .
the transfer of the ubiquitin moiety from the thioester - linked e3 ( in hect - type ligases ) to the acceptor lysine on the substrate is the last step of this process .
subsequent chain elongation requires the modification of specific lysine residues in consecutive ubiquitin moieties . with few exceptions ( petroski & deshaies , 2005
; jin et al , 2008 ) , little is known about the mechanisms of ubiquitin - chain assembly , although various models have been proposed ( hochstrasser , 2006 ) .
the nedd4 family of hect domain e3 ligases is a well - characterized class of enzymes that present a conserved modular organization with an amino - terminal c2 domain that is crucial for membrane localization , between two and four ww domains that recognize substrates and adaptor proteins and a carboxy - terminal catalytic hect domain . in humans ,
there are nine members of this family that are implicated in a range of biological processes such as endocytosis , protein transport , viral budding , signalling , cellular growth and proliferation ( rotin & kumar , 2009 ) .
this class of e3 enzymes seems to use a sequential addition mechanism , by which ubiquitin molecules are added one at a time from the catalytic cysteine to the distal lysine of the growing chain ( kim & huibregtse , 2009 ) .
a key question is how e3 enzymes deal with the shifting position of the acceptor site during chain elongation .
two groups have recently identified a surface implicated in non - covalent ubiquitin binding on the hect - type e3 ligases rsp5 and smurf2 ( french et al , 2009 ; ogunjimi et al , 2010 ) .
this surface was proposed to have a role in regulating polyubiquitination , although opposite mechanisms were suggested by the groups , with the surface being required to either restrict the length of polyubiquitin chains synthesized by the hect domain ( french et al , 2009 ) or to promote polyubiquitination ( ogunjimi et al , 2010 ) . in this study , we show the crystal structure of the hect domain of nedd4 alone and in complex with ubiquitin , and we present molecular insights into the mechanism by which nedd4 catalyses polyubiquitination .
we characterized the interaction of the isolated hect domain of nedd4 ( hect ) with ubiquitin in detail .
the ubiquitin - binding ability resides in the n - lobe , does not show preference for lys 63- or lys 48-polyubiquitin chains and requires the canonical hydrophobic patch on ubiquitin , centred on ile 44 ( supplementary fig s1 online ) .
we extended this analysis to the other mammalian nedd4 family members and found that only a subset of these hect domains binds to ubiquitin , namely nedd4 , nedd4-like and smurf2 ( fig 1a ) . to understand how ubiquitin binds to the hect
, we determined the crystal structure of the hect in isolation ( at 2.5 ) and in complex with ubiquitin ( at 2.7 ) by molecular replacement ( supplementary table s1 online and supplementary fig s2 online ) . in both structures , hect displays the typical hect fold ( huang et al , 1999 ; verdecia et al , 2003 ; ogunjimi et al , 2005 ) composed of two lobes connected by a flexible hinge ( fig 1b , c ) .
the n - lobe , an elongated array of helices and -hairpins , consists of two moieties , known as the large and small subdomains ( fig 1b ) .
the small subdomain , which hosts the e2-binding site , comprises helices 68 and -sheets 56 ( huang et al , 1999 ; kamadurai et al , 2009 ) .
the large subdomain of the n - lobe is present below the c - lobe , an / sandwich domain that carries the catalytic cysteine .
the orientation of the c - lobe differs in the two hect structures , and both orientations are distinct from those of previously reported hect domain structures ( supplementary fig s3 online ) .
this highlights the freedom of movement of the c - lobe , which is key for the catalytic function of hect domains ( verdecia et al , 2003 ; kamadurai et al , 2009 ) .
non - covalent ubiquitin binding to hect conceals a solvent - accessible interaction surface area on ubiquitin of approximately 900 , the largest surface identified so far for ubiquitin - binding domains ( supplementary table s2 online ) .
ubiquitin makes contact with glu 554 and neighbouring residues of helix 1 ; tyr604 , tyr 605 and tyr 610 from the region comprising helix 3 and strand 3 ; asn628 and glu 629 from helix 4 and the ensuing loop ; and phe707 and neighbouring residues of the 56 beta hairpin .
these residues are distributed in both the small and the large subdomains of the n - lobe ( figs 1e,2a ) . in the absence of ubiquitin ,
the relative orientation of the small and large subdomains is not fixed and varies for different structures ( huang et al , 1999 ; verdecia et al , 2003 ; ogunjimi et al , 2005 ) .
ubiquitin binding might therefore be expected to stabilize a specific reciprocal orientation of the n - lobe subdomains .
indeed , superposition of the large subdomains of hect and hect : ubiquitin ( root mean square deviation of 0.6 over 181 c ) clearly indicates a relative movement of the 56 hairpin of the small subdomain in the hect : ubiquitin structure by approximately 5 towards the large subdomain of the n - lobe ( fig 1d ) . as predicted by the defective behaviour of the i44a ubiquitin mutant ( supplementary fig s1c online ) ,
the interaction surface on ubiquitin involves the canonical ile 44 hydrophobic patch , which also includes gly 47 , leu 8 and val 70 ( fig 2a ) .
however , the surface of interaction is not limited to this patch , but extends to a second hydrophobic patch ' , including the residues ile 36/leu 71/leu 73 , the role of which has recently been discussed in the context of the e2-to - hect ubiquitin transfer ( kamadurai et al , 2009 ; fig 2a ; supplementary fig s4 online ) .
the asn 628 , tyr 634 and glu 554 side chains on nedd4 form hydrogen bonds with the main chain nitrogen atoms of ubiquitin - leu 73 , arg74 and gly 75 , whereas the ubiquitin - leu 73 side chain is stacked between tyr 634 and tyr 605 of nedd4 .
we generated nedd4 mutants that substantiate the functional importance of both interacting patches on ubiquitin .
mutation of tyr 605 to ala ( y605a ) or phe 707 to ala ( f707a ) almost abolished hect binding to lys 63 ubiquitin ( fig 2b ) .
phe 707 to tyr ( f707y ) , asn 628 to ala ( n628a ) or glu 629 to ala ( e629a ) mutations had milder effects , preserving the association with higher molecular weight lys 63 ubiquitin to varying degrees ( fig 2b ) .
we confirmed these results by measuring the interaction between the hect domains and monomeric and dimeric ubiquitin by fluorescence polarization and surface plasmon resonance ( spr ) assay ( fig 2c ; supplementary fig s1d f online ) .
both ubiquitin ligands interact with the hect with rapid kinetics ( fast kon and koff rate constants , data not shown ) .
wild - type hect displays a moderate affinity ( kd approximately 11 m ) in the range of those reported for ubiquitin - binding domains ( supplementary table s2 online ) , whereas y605a and f707a mutations show from 20- to 30-fold decreases in binding ( fig 2c ; supplementary fig s1f online ) .
, the ubiquitin - binding surface might have a role at three stages of the e3 catalysis : binding to the e2 , transthiolation process from e2 to e3 or substrate ubiquitination .
we tested all of these possibilities using the isolated hect , which retains the ability to ubiquitinate itself as well as substrates , albeit with reduced efficiency ( not shown ) .
pull - down and spr assays showed that mutants have no significant impairment in binding with either the apo or the ubiquitin thioester - linked form of e2 enzyme ube2d3 ( fig 3a and data not shown ) .
indeed , the e2 binding is built on an adjacent but non - overlapping surface on the large subdomain of the n - lobe ( huang et al , 1999 ; fig 4c ) .
we then tested the importance of the ubiquitin - binding surface in the e2-to - hect transthiolation process by using a pulse - chase protocol ( fig 3b ) .
again , no appreciable transthiolation defects were observed for the mutants , supporting the notion that the ubiquitin - binding surface is not involved in the upstream steps of the enzymatic cascade . of note ,
the thioester hectubiquitin bond is unstable and the ubiquitin moiety is immediately transferred to the lysine / s of the hect , as demonstrated by the appearance of higher molecular - weight proteins that are resistant to dithiothreitol treatment ( fig 3b , lower panels ) .
these results led us to propose that the ubiquitin - binding surface on the hect might act to bind a ubiquitin moiety that is already conjugated to a substrate , thus promoting polyubiquitination .
indeed , when we assayed f707a and y605a in an in vitro ubiquitination reaction , we found that mutations in the ubiquitin - binding surface strongly impaired free - chain formation and ubiquitination of all the substrates tested ( fig 3c ; supplementary fig s5 online ) .
the mutant enzymes were efficient in the first cycle of substrate ubiquitination and in ubiquitin dimer formation , using free ubiquitin as a pseudosubstrate ( fig 3c ; supplementary fig s5 online ) .
this was confirmed by using a ubiquitin lys0 mutant and quantification of the results repeated as fold differences between wild - type hect and mutants ( supplementary fig s5a online ) .
interestingly , f707a and y605a mutations did not affect self - ubiquitination of nedd4 ( fig 3b , lower panels and fig 3d ) , indicating that this in cis reaction is a catalytically distinct process that can not be used as a surrogate assay for ligase activity on substrates .
most hect e3s synthesize polyubiquitin chains with specific linkages ( wang et al , 2006 ; kim et al , 2007 ) . to gain insight into the type of chains synthesized by nedd4 , we tested substrate ubiquitination using ubiquitin - bearing individual lysine - to - arginine mutations ( kr mutants ) .
we found that nedd4 has a strong preference for building lys 63-chains on substrates , a feature retained by the f707a mutant ( fig 4a ) .
consistent with previous data , however , f707a has defective chain elongation on substrate and shorter free chains , regardless of the type of ubiquitin used ( fig 4a ) .
therefore , we conclude that the ubiquitin - binding surface on the hect acts to promote substrate polyubiquitination , but it does not dictate the preference for a specific lysine during elongation .
indeed , recent observations suggest that the c - lobe , rather than the ubiquitin - binding n - lobe , is the crucial determinant of lysine selection during elongation ( kim & huibregtse , 2009 ) .
collectively , our results support the hypothesis that the ubiquitin - binding surface is required for the processivity of the polyubiquitination reaction ( ogunjimi et al , 2010 ) , rather than for limiting chain elongation , as suggested previously ( french et al , 2009 ) .
it is tempting to envision a model in which the distal ubiquitin on the substrate occupies this surface , promoting retention of the ubiquitinated substrate to the e3 , and also keeping the small subdomain of the n - lobe in a conformation that favours processive ubiquitin addition .
this could be achieved by either reducing the gap between the catalytic cysteine of the hect and the c - terminus of ubiquitin linked to the e2 enzyme ( kamadurai et al , 2009 ) or by facilitating the transfer of a subsequent ubiquitin to the hect - bound substrate .
in support of this model , we found that the non - covalent ubiquitin - binding surface that we mapped remains accessible in the complex of the hect domain of nedd4-like with the ubiquitin - loaded e2 ( kamadurai et al , 2009 ; fig 4c ) .
our data support the notion that nedd4 adopts a simple sequential - addition model to build a chain on a substrate ; after the first ubiquitin is attached , the chain is elongated through lys 63 linkage , because of the ability of the n - lobe to maintain the growing polyubiquitin chain in close proximity .
a similar conclusion about the role of the hect ubiquitin - binding site in promoting chain elongation was reached in the accompanying study by kim et al ( 2011 ) on rsp5 .
although the position of lys 63 on bound ubiquitin does not seem to be able to orient the growing chain towards the e2 catalytic cysteine ( fig 4b ) , the average b factors for the ubiquitin molecules are considerably higher than those of their hect counterparts ( approximately 76 a for ubiquitin molecules , approximately 48 a for hect domains ; supplementary table s1 online ) , suggesting freedom of movement of ubiquitin on its docking site .
this could imply that the binding of ubiquitin to the e3 is strong enough to promote polyubiquitination , yet loose enough to allow chain growth , possibly through a slippage mechanism by which the ubiquitin - binding surface specifically binds to the distal ubiquitin at the end of the chain .
the moderate affinity and fast kinetic rates of the hect : ubiquitin interaction fit well with such a mechanism .
future structural studies with nedd4 in complex with a ubiquitinated substrate might be required to provide a definitive picture of this dynamic process .
the detailed molecular view provided by our structure allows the identification of the crucial residues required for binding ( fig 1e ) , which can be used to predict the hect e3 enzymes that are able to bind to ubiquitin .
it remains to be established whether the presence of this binding surface might determine the mechanism of chain synthesis adopted by the different hect ligases to become processive . at our level of understanding , generalizing the mechanisms that underlie polyubiquitination
would be premature , but it is interesting to note from the comparison of the small nedd4 family of e3 , that nature has used a variety of protein architectures to ensure specificity .
we characterized the interaction of the isolated hect domain of nedd4 ( hect ) with ubiquitin in detail .
the ubiquitin - binding ability resides in the n - lobe , does not show preference for lys 63- or lys 48-polyubiquitin chains and requires the canonical hydrophobic patch on ubiquitin , centred on ile 44 ( supplementary fig s1 online ) .
we extended this analysis to the other mammalian nedd4 family members and found that only a subset of these hect domains binds to ubiquitin , namely nedd4 , nedd4-like and smurf2 ( fig 1a ) . to understand how ubiquitin binds to the hect
, we determined the crystal structure of the hect in isolation ( at 2.5 ) and in complex with ubiquitin ( at 2.7 ) by molecular replacement ( supplementary table s1 online and supplementary fig s2 online ) . in both structures , hect displays the typical hect fold ( huang et al , 1999 ; verdecia et al , 2003 ; ogunjimi et al , 2005 ) composed of two lobes connected by a flexible hinge ( fig 1b , c ) .
the n - lobe , an elongated array of helices and -hairpins , consists of two moieties , known as the large and small subdomains ( fig 1b ) .
the small subdomain , which hosts the e2-binding site , comprises helices 68 and -sheets 56 ( huang et al , 1999 ; kamadurai et al , 2009 ) .
the large subdomain of the n - lobe is present below the c - lobe , an / sandwich domain that carries the catalytic cysteine .
the orientation of the c - lobe differs in the two hect structures , and both orientations are distinct from those of previously reported hect domain structures ( supplementary fig s3 online ) .
this highlights the freedom of movement of the c - lobe , which is key for the catalytic function of hect domains ( verdecia et al , 2003 ; kamadurai et al , 2009 ) .
non - covalent ubiquitin binding to hect conceals a solvent - accessible interaction surface area on ubiquitin of approximately 900 , the largest surface identified so far for ubiquitin - binding domains ( supplementary table s2 online ) .
ubiquitin makes contact with glu 554 and neighbouring residues of helix 1 ; tyr604 , tyr 605 and tyr 610 from the region comprising helix 3 and strand 3 ; asn628 and glu 629 from helix 4 and the ensuing loop ; and phe707 and neighbouring residues of the 56 beta hairpin .
these residues are distributed in both the small and the large subdomains of the n - lobe ( figs 1e,2a ) . in the absence of ubiquitin ,
the relative orientation of the small and large subdomains is not fixed and varies for different structures ( huang et al , 1999 ; verdecia et al , 2003 ; ogunjimi et al , 2005 ) .
ubiquitin binding might therefore be expected to stabilize a specific reciprocal orientation of the n - lobe subdomains .
indeed , superposition of the large subdomains of hect and hect : ubiquitin ( root mean square deviation of 0.6 over 181 c ) clearly indicates a relative movement of the 56 hairpin of the small subdomain in the hect : ubiquitin structure by approximately 5 towards the large subdomain of the n - lobe ( fig 1d ) . as predicted by the defective behaviour of the i44a ubiquitin mutant ( supplementary fig s1c online ) ,
the interaction surface on ubiquitin involves the canonical ile 44 hydrophobic patch , which also includes gly 47 , leu 8 and val 70 ( fig 2a ) .
however , the surface of interaction is not limited to this patch , but extends to a second hydrophobic patch ' , including the residues ile 36/leu 71/leu 73 , the role of which has recently been discussed in the context of the e2-to - hect ubiquitin transfer ( kamadurai et al , 2009 ; fig 2a ; supplementary fig s4 online ) .
the asn 628 , tyr 634 and glu 554 side chains on nedd4 form hydrogen bonds with the main chain nitrogen atoms of ubiquitin - leu 73 , arg74 and gly 75 , whereas the ubiquitin - leu 73 side chain is stacked between tyr 634 and tyr 605 of nedd4 .
we generated nedd4 mutants that substantiate the functional importance of both interacting patches on ubiquitin .
mutation of tyr 605 to ala ( y605a ) or phe 707 to ala ( f707a ) almost abolished hect binding to lys 63 ubiquitin ( fig 2b ) .
phe 707 to tyr ( f707y ) , asn 628 to ala ( n628a ) or glu 629 to ala ( e629a ) mutations had milder effects , preserving the association with higher molecular weight lys 63 ubiquitin to varying degrees ( fig 2b ) .
we confirmed these results by measuring the interaction between the hect domains and monomeric and dimeric ubiquitin by fluorescence polarization and surface plasmon resonance ( spr ) assay ( fig 2c ; supplementary fig s1d f online ) .
both ubiquitin ligands interact with the hect with rapid kinetics ( fast kon and koff rate constants , data not shown ) .
wild - type hect displays a moderate affinity ( kd approximately 11 m ) in the range of those reported for ubiquitin - binding domains ( supplementary table s2 online ) , whereas y605a and f707a mutations show from 20- to 30-fold decreases in binding ( fig 2c ; supplementary fig s1f online ) .
, the ubiquitin - binding surface might have a role at three stages of the e3 catalysis : binding to the e2 , transthiolation process from e2 to e3 or substrate ubiquitination .
we tested all of these possibilities using the isolated hect , which retains the ability to ubiquitinate itself as well as substrates , albeit with reduced efficiency ( not shown ) .
pull - down and spr assays showed that mutants have no significant impairment in binding with either the apo or the ubiquitin thioester - linked form of e2 enzyme ube2d3 ( fig 3a and data not shown ) .
indeed , the e2 binding is built on an adjacent but non - overlapping surface on the large subdomain of the n - lobe ( huang et al , 1999 ; fig 4c ) .
we then tested the importance of the ubiquitin - binding surface in the e2-to - hect transthiolation process by using a pulse - chase protocol ( fig 3b ) .
again , no appreciable transthiolation defects were observed for the mutants , supporting the notion that the ubiquitin - binding surface is not involved in the upstream steps of the enzymatic cascade . of note ,
the thioester hectubiquitin bond is unstable and the ubiquitin moiety is immediately transferred to the lysine / s of the hect , as demonstrated by the appearance of higher molecular - weight proteins that are resistant to dithiothreitol treatment ( fig 3b , lower panels ) .
these results led us to propose that the ubiquitin - binding surface on the hect might act to bind a ubiquitin moiety that is already conjugated to a substrate , thus promoting polyubiquitination .
indeed , when we assayed f707a and y605a in an in vitro ubiquitination reaction , we found that mutations in the ubiquitin - binding surface strongly impaired free - chain formation and ubiquitination of all the substrates tested ( fig 3c ; supplementary fig s5 online ) .
the mutant enzymes were efficient in the first cycle of substrate ubiquitination and in ubiquitin dimer formation , using free ubiquitin as a pseudosubstrate ( fig 3c ; supplementary fig s5 online ) .
this was confirmed by using a ubiquitin lys0 mutant and quantification of the results repeated as fold differences between wild - type hect and mutants ( supplementary fig s5a online ) .
interestingly , f707a and y605a mutations did not affect self - ubiquitination of nedd4 ( fig 3b , lower panels and fig 3d ) , indicating that this in cis reaction is a catalytically distinct process that can not be used as a surrogate assay for ligase activity on substrates . most hect e3s synthesize polyubiquitin chains with specific linkages ( wang et al , 2006 ; kim et al , 2007 ) . to gain insight into the type of chains synthesized by nedd4 , we tested substrate ubiquitination using ubiquitin - bearing individual lysine - to - arginine mutations ( kr mutants ) .
we found that nedd4 has a strong preference for building lys 63-chains on substrates , a feature retained by the f707a mutant ( fig 4a ) .
consistent with previous data , however , f707a has defective chain elongation on substrate and shorter free chains , regardless of the type of ubiquitin used ( fig 4a ) .
therefore , we conclude that the ubiquitin - binding surface on the hect acts to promote substrate polyubiquitination , but it does not dictate the preference for a specific lysine during elongation .
indeed , recent observations suggest that the c - lobe , rather than the ubiquitin - binding n - lobe , is the crucial determinant of lysine selection during elongation ( kim & huibregtse , 2009 ) .
collectively , our results support the hypothesis that the ubiquitin - binding surface is required for the processivity of the polyubiquitination reaction ( ogunjimi et al , 2010 ) , rather than for limiting chain elongation , as suggested previously ( french et al , 2009 ) .
how can this occur ? it is tempting to envision a model in which the distal ubiquitin on the substrate occupies this surface , promoting retention of the ubiquitinated substrate to the e3 , and also keeping the small subdomain of the n - lobe in a conformation that favours processive ubiquitin addition .
this could be achieved by either reducing the gap between the catalytic cysteine of the hect and the c - terminus of ubiquitin linked to the e2 enzyme ( kamadurai et al , 2009 ) or by facilitating the transfer of a subsequent ubiquitin to the hect - bound substrate . in support of this model
, we found that the non - covalent ubiquitin - binding surface that we mapped remains accessible in the complex of the hect domain of nedd4-like with the ubiquitin - loaded e2 ( kamadurai et al , 2009 ; fig 4c ) .
our data support the notion that nedd4 adopts a simple sequential - addition model to build a chain on a substrate ; after the first ubiquitin is attached , the chain is elongated through lys 63 linkage , because of the ability of the n - lobe to maintain the growing polyubiquitin chain in close proximity . a similar conclusion about the role of the hect ubiquitin - binding site in promoting chain elongation was reached in the accompanying study by kim et al ( 2011 ) on rsp5 . although the position of lys 63 on bound ubiquitin does not seem to be able to orient the growing chain towards the e2 catalytic cysteine ( fig 4b ) , the average b factors for the ubiquitin molecules are considerably higher than those of their hect counterparts ( approximately 76 a for ubiquitin molecules , approximately 48 a for hect domains ; supplementary table s1 online ) , suggesting freedom of movement of ubiquitin on its docking site .
this could imply that the binding of ubiquitin to the e3 is strong enough to promote polyubiquitination , yet loose enough to allow chain growth , possibly through a slippage mechanism by which the ubiquitin - binding surface specifically binds to the distal ubiquitin at the end of the chain . the moderate affinity and fast kinetic rates of the hect : ubiquitin interaction fit well with such a mechanism .
future structural studies with nedd4 in complex with a ubiquitinated substrate might be required to provide a definitive picture of this dynamic process .
the detailed molecular view provided by our structure allows the identification of the crucial residues required for binding ( fig 1e ) , which can be used to predict the hect e3 enzymes that are able to bind to ubiquitin .
it remains to be established whether the presence of this binding surface might determine the mechanism of chain synthesis adopted by the different hect ligases to become processive . at our level of understanding ,
generalizing the mechanisms that underlie polyubiquitination would be premature , but it is interesting to note from the comparison of the small nedd4 family of e3 , that nature has used a variety of protein architectures to ensure specificity .
crystallization and structure determination . crystals of hect and hect : ubiquitin complex were obtained by sitting - drop vapour diffusion at 20c , using a honeybee cartesian robot in 96-well plates .
diffraction - quality crystals were obtained by optimizing the initial conditions in 24-well plates , hanging drops at 20c .
the optimized conditions were 100 mm na - mes , ph 6.0 , 24% peg 400 or peg 600 , 3060 mm cacl2 or mgcl2 , 5 mm tris(2-carboxyethyl)phosphine , with protein concentration in the 2.55 mg / ml range .
crystals were cryoprotected in 100 mm na - mes , ph 6.0 , 4% peg 400 , supplemented with 20% ethylene glycol .
the structure was solved with a data set collected at the european synchrotron radiation facility ( esrf ) at beamline id14 - 2 . for the hect : ubiquitin complex
, initial crystals were obtained in 100 mm na - hepes , ph 78 , 1020% peg 2000 mme , 5 mm tris(2-carboxyethyl)phosphine , with proteins purified individually , then mixed in a 1:1 molar ratio and a concentration of approximately 30 mg / ml . to obtain good - quality diffraction and to overcome twinning ,
the complex was crystallized in the presence of excess ubiquitin ( 600900 m of complex , 1.2 2.3 ubiquitin molar excess ) , lower concentration of peg 2000 mme ( 210% ) , and the crystals were carefully frozen by equilibrating them into cryo - buffer ( 100 mm na - hepes , ph 7.5 , 10% peg 2000 mme ) with increasing concentrations of glycerol , reaching a final concentration of 20% .
the structure was solved with a data set collected at the esrf at beamline id14 - 1 on a crystal grown in a 1.9 ubiquitin molar excess .
x - ray diffraction data were processed with hkl2000 ( otwinowski & minor , 1997 ) or xds ( kabsch , 2010 ) .
both structures were solved by molecular replacement with phaser within the ccp4 suite ( ccp4 , 1994 ) , using as a search model the hect domain of nedd4-like ( protein data bank entry 2oni ) in the case of hect , and hect and a high - resolution structure of ubiquitin ( protein data bank entry 1ubi ) in the case of hect : ubiquitin complex .
initial models were refined with the cns suite ( brunger , 2007 ) , refmac ( murshudov et al , 1997 ) , the phenix suite ( adams et al , 2010 ) and manual building in coot ( emsley et al , 2010 ) .
for the hect : ubiquitin complex , non - crystallographic symmetry ( ncs ) restraints were used in refinement . in the first cycles of refinement carried out with refmac ,
hect molecules were divided into two ncs groups ( the n - lobe and the c - lobe ) , and ubiquitin molecules were the third ncs group . for further refinement cycles carried out with phenix.refine ,
five ncs groups were used : ubiquitin molecules and hect domain residues 522:699 , 724:778 , 785:828 and 850:891 , thus not subjecting hect domain loops to ncs restraints .
hect crystallized in spacegroup c2 , whereas hect : ubiquitin crystallized in spacegroup p21 , with two complexes per asymmetrical unit .
the two complexes differ slightly in the orientation of the hect c - lobe with respect to the n - lobe , and the relative orientation of hect with respect to ubiquitin , but the interactions discussed here are present in both complexes .
superpositions of pairs of domains of the asymmetrical unit indicate that they are almost identical ( root mean square deviations of n - lobes : 0.36 over 260 c ; c - lobes : 0.63 over 115 c ; ubs : 0.25 over 76 c ) .
ubiquitination assays were performed with hect domains produced as glutathione s - transferase ( gst ) fusion proteins and cleaved with prescission protease .
the e2 enzyme ube2d3 , was produced as a his6-fusion protein and eluted from ni - nta agarose beads ( qiagen ) .
reaction mixtures contained purified enzymes ( 20 nm e1 , 250 nm of purified his6-tagged ube2d3 , 250 nm hect ) , 300 nm of substrate ( epithelial na channel and lmp2a were produced as gst fusion proteins and used attached to glutathione beads ) and 1 m of ubiquitin in ubiquitination buffer ( 25 mm tris hcl , ph 7.6 , 5 mm mgcl2 , 100 mm nacl , 0.2 m dithiothreitol , 2 mm atp ) .
reactions were incubated at 37c . at the indicated time point , samples were centrifuged to separate the pellet containing the ubiquitinated substrates and the supernatant containing the enzymes and the soluble ubiquitin chains , if produced .
the pellet was washed four times in yy buffer ( 50 mm na - hepes ph 7.5 , 150 mm nacl , 1 mm edta , 10% glycerol , 1% triton x-100 ) before loading on sds polyacrylamide gel electrophoresis gel . for self - ubiquitination reaction ,
the mixtures contained 20 nm e1 , 250 nm of purified his6-tagged ube2d3 , 250 nm of gst - hect and 0.5 m of ubiquitin in ubiquitination buffer .
coomassie - stained membrane was used to show loading of gst - fusion protein after immunoblotting .
reagents and constructs , protein expression and purification , transthiolation assay , pull - down experiments , fluorescence polarization assay and spr are described in the supplementary methods online .
accession codes : coordinates for hect and the hect : ubiquitin complex have been deposited at the protein data bank under accession codes 2xbf and 2xbb , respectively . | structure of the hect : ubiquitin complex and its role in ubiquitin chain elongationanalysis of ubiquitin binding to the hect domain of nedd4 suggests that the ubiquitin chain being elongated is kept close to the catalytic cysteine to promote processivity . together with the accompanying paper by the huibregtse group , this study shows the catalysis of polyubiquitin chains by hect e3 ligases . | Introduction
Results and Discussion
Structure of HECT
Role of ubiquitin binding in Nedd4 activity
CONCLUSIONS
Methods
Supplementary Material | this class of e3 enzymes seems to use a sequential addition mechanism , by which ubiquitin molecules are added one at a time from the catalytic cysteine to the distal lysine of the growing chain ( kim & huibregtse , 2009 ) . this surface was proposed to have a role in regulating polyubiquitination , although opposite mechanisms were suggested by the groups , with the surface being required to either restrict the length of polyubiquitin chains synthesized by the hect domain ( french et al , 2009 ) or to promote polyubiquitination ( ogunjimi et al , 2010 ) . in this study , we show the crystal structure of the hect domain of nedd4 alone and in complex with ubiquitin , and we present molecular insights into the mechanism by which nedd4 catalyses polyubiquitination . the asn 628 , tyr 634 and glu 554 side chains on nedd4 form hydrogen bonds with the main chain nitrogen atoms of ubiquitin - leu 73 , arg74 and gly 75 , whereas the ubiquitin - leu 73 side chain is stacked between tyr 634 and tyr 605 of nedd4 . of note ,
the thioester hectubiquitin bond is unstable and the ubiquitin moiety is immediately transferred to the lysine / s of the hect , as demonstrated by the appearance of higher molecular - weight proteins that are resistant to dithiothreitol treatment ( fig 3b , lower panels ) . this could be achieved by either reducing the gap between the catalytic cysteine of the hect and the c - terminus of ubiquitin linked to the e2 enzyme ( kamadurai et al , 2009 ) or by facilitating the transfer of a subsequent ubiquitin to the hect - bound substrate . in support of this model , we found that the non - covalent ubiquitin - binding surface that we mapped remains accessible in the complex of the hect domain of nedd4-like with the ubiquitin - loaded e2 ( kamadurai et al , 2009 ; fig 4c ) . this could imply that the binding of ubiquitin to the e3 is strong enough to promote polyubiquitination , yet loose enough to allow chain growth , possibly through a slippage mechanism by which the ubiquitin - binding surface specifically binds to the distal ubiquitin at the end of the chain . the asn 628 , tyr 634 and glu 554 side chains on nedd4 form hydrogen bonds with the main chain nitrogen atoms of ubiquitin - leu 73 , arg74 and gly 75 , whereas the ubiquitin - leu 73 side chain is stacked between tyr 634 and tyr 605 of nedd4 . , the ubiquitin - binding surface might have a role at three stages of the e3 catalysis : binding to the e2 , transthiolation process from e2 to e3 or substrate ubiquitination . of note ,
the thioester hectubiquitin bond is unstable and the ubiquitin moiety is immediately transferred to the lysine / s of the hect , as demonstrated by the appearance of higher molecular - weight proteins that are resistant to dithiothreitol treatment ( fig 3b , lower panels ) . this could be achieved by either reducing the gap between the catalytic cysteine of the hect and the c - terminus of ubiquitin linked to the e2 enzyme ( kamadurai et al , 2009 ) or by facilitating the transfer of a subsequent ubiquitin to the hect - bound substrate . in support of this model
, we found that the non - covalent ubiquitin - binding surface that we mapped remains accessible in the complex of the hect domain of nedd4-like with the ubiquitin - loaded e2 ( kamadurai et al , 2009 ; fig 4c ) . this could imply that the binding of ubiquitin to the e3 is strong enough to promote polyubiquitination , yet loose enough to allow chain growth , possibly through a slippage mechanism by which the ubiquitin - binding surface specifically binds to the distal ubiquitin at the end of the chain . the moderate affinity and fast kinetic rates of the hect : ubiquitin interaction fit well with such a mechanism . both structures were solved by molecular replacement with phaser within the ccp4 suite ( ccp4 , 1994 ) , using as a search model the hect domain of nedd4-like ( protein data bank entry 2oni ) in the case of hect , and hect and a high - resolution structure of ubiquitin ( protein data bank entry 1ubi ) in the case of hect : ubiquitin complex . accession codes : coordinates for hect and the hect : ubiquitin complex have been deposited at the protein data bank under accession codes 2xbf and 2xbb , respectively . | [
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in mammals , sound waves stimulate the cochlea via the vibration of the ossicular chain .
the opposite vibrating phase between the round window and the oval window causes relative motion between the endolymph and perilymph and thus produces displacement waves travelling on the spirally basilar membrane . the motion of hair cell stereocilia created by basilar membrane ( bm ) vibration gates stereocilia transduction channels , leading to the generation of hair cell receptor potential and the excitation of afferent auditory nerve fibers .
changes in cochlear lymphatic fluid homeostasis may result in cochlear acoustic dysfunctions , like endolymphatic hydrops ( eh ) , semicircular canal dehiscence , labyrinthine fistulas , and so forth [ 2 , 3 ] . since hallpike and carins described the presence of eh in the temporal bones of patients with mnire 's disease ( md ) in 1938 , eh has generally been accepted as the basic histopathologic sign of this disease , which is an intractable disease that results in hearing loss that is often fluctuating and initially involves the low frequencies .
wu et al . reported that low - tone and middle - tone hearing thresholds were related to the severity of eh in the cochlea . in the study by lee
, spontaneous low frequency air - bone gaps in evaluating hearing sensitivity were found in approximately 13.9% of patients with mnire 's disease and may indirectly reflect aggravation of the eh in the cochlear and the vestibular compartments .
yoshida et al . suggested an association between endolymphatic hydrops and low frequency hearing loss in a 13-year - old girl with mutation of the slc26a4 gene .
endolymphatic hydropic condition was described in certain cases of sudden deafness [ 8 , 9 ] .
noguchi et al . assumed that eh give rise to the pathogenesis of acute low - tone sensorineural hearing loss ( alhl ) with little or no impairment of hair cells that resembles early - stage md .
it is widely hypothesized that the hydrops generates the clinical symptoms of this illness . yet
, there are few studies that have investigated how the dilated endolymph affects the middle ear acoustic transmission .
a laser doppler vibrometer ( ldv ) is a noncontact , established optical technique that can be used to measure the displacement of middle ear components in response to sound stimulation [ 1116 ] .
it uses the doppler - shift principle to determine the instantaneous velocity of a moving object by comparing the frequency of the laser 's emitted light with the frequency of the light reflected from the moving object .
this technique has been used to test the vibration of the round window membrane ( rwm ) , the tympanic membrane ( tm ) , and the stapes footplate in fresh and embalmed cadaveric human temporal bone and animal specimens . moreover ,
animal models of different diseases have been created to investigate vibration changes in the ossicular chain and rwm , to explore the potential mechanism underlying the clinical symptoms of diseases .
for instance , vibration of the rwm that is associated with acute otitis media is significantly decreased compared with those of the rwm from the ears of normal guinea pigs . moreover , middle ear effusion reduces the mobility of the tm , the incus tip , and the rwm at frequencies above 1 khz in guinea pigs .
the mechanical properties of the incudostapedial joint directly affect the stapes movement or the middle ear transfer function for sound transmission . however , no prior study has reported changes in the dynamic behavior of the umbo , the stapes head , and the rwm in association with eh . to better understand how eh affects the sound transmission process and hearing loss in low frequency
, we used guinea pigs to create eh models and measured the hydropic ratio ( hr ) and morphology in each turn , the vibration of the umbo , stapes head , and rwm , as well as the auditory - evoked brainstem response ( abr ) .
the primary objective of this study was to compare the different dynamic properties of the ossicular chain and the rwm between normal and eh guinea pigs and explore the potential pathogenic mechanism underlying the associated hearing loss at low frequencies .
all animal work conducted during the course of this study was approved by the institutional animal care and use committee at eye ear nose & throat hospital , fudan university , and conformed to the national institutes of health guide for care and use of laboratory animals .
eighteen healthy albino male guinea pigs with an initial weight of 250 g300 g and a positive preyer reflex were used in this study .
all animals were free of middle ear diseases , such as tympanic membrane perforation or otitis media , as evaluated by otoscopic examination .
identification of preexisting abnormalities of auditory function was made by a prerecruiting abr measurement in each animal .
guinea pigs were randomly allocated into two groups , the control group ( 9 guinea pigs with sham surgical procedure in the right ear ) and the eh group ( 9 guinea pigs with obliteration of the endolymphatic sac in the right ear ) .
specimens from each group ( control and eh ) were used for histologic observations of paraffin sections ( six ears ) and whole - mount immunostaining ( three ears ) .
the obliteration of the endolymphatic sac was performed surgically using an extradural posterior cranial fossa approach , under sterile conditions and under a surgical microscope ( 6030116204 , carl zeiss , jena , germany ) .
the right ears of the nine animals in the control group were subjected to sham operation .
exposure of the sigmoid sinus through the occipital bone was accomplished without obliteration of the endolymphatic sac .
the temperature was maintained stably to keep animals warm during surgery , by using an electric heating pad .
anesthesia was induced with a combination of ketamine hydrochloride ( 35 mg / kg , intramuscular injection ) and 1% xylazine hydrochloride ( 10 mg / kg , subcutaneous injection ) .
the right ears from six animals in the control group as well as those from the eh group were prepared .
after anesthesia ( as also described above ) , the right auricles were cut off and the dorsal auditory bulla was opened into a square block under a surgical microscope ( 6030116204 , carl zeiss , jena , germany ) , from which the incus - stapes complex , rwm , and the medial side of the umbo could be viewed clearly ( figure 1 ) .
a 0.250.5-mm square ( approximately 24 pieces , each piece with a mass of 40 g ) of laser reflective tape ( 3 m , maplewood , ms , usa ) was placed on the medial side of the umbo , the stapes head , and the middle of the rwm , as the laser - reflecting target ( figures 1(a)1(d ) ) .
abr measurements in the control group and in the 8-week eh group were performed before the surgery and prior to the ldv measurement .
after anesthesia , the abr ( bio - logic navpro , 580-navpr2 , natus medical incorporated , pleasanton , ca , usa ) were tested in a sound - proofed booth to assess the auditory threshold .
needle electrodes were placed subcutaneously at the vertex for recording and behind the bilateral ears as reference and ground electrodes .
stimulation was presented as tone bursts ( 5 ms duration , 0.5 ms rise - fall time , blackman envelope ) at a frequency of 0.5 , 1 , 2 , 4 , 6 , and 8 khz ; the sound - intensity level was decreased in 10 db steps from 80 to 20 db spl ; 500 responses at each sound level were recorded and averaged .
if i , iii , and v waves disappeared , we increased the tone bursts by 5 db repeatedly to judge the threshold by the waveform .
the measurement system included a compact laser vibrometer ( clv-2534 - 4 , polytec , wurzburg , germany ) coupled with a microscope ( opmi 1-fc , carl zeiss , jena , germany ) with a micromanipulator ( a - hlv - mm30 , polytec , wurzburg , germany ) , signal generator ( 33210a , agilent , santa clara , ca , usa ) , and power amplifier ( rmx 850 , qsc , costa mesa , ca , usa ) ( figure 1(a ) ) .
the intensity of each excitation frequency was calibrated to 85 db spl using a sound - level meter ( awa-5661 - 1b , aihua , yiyang city , china ) .
an earphone associated with the microphone ( er-4pt , er-7c , hlv - spec adapter , etymotic , elk grove village , il , usa ) was inserted into the osseous external auditory canal to give signal stimuli and monitor sound pressure .
the distance between the tympanic membrane and the earphone and microphone was maintained at 1 mm .
the vibration of the moving surface was acquired through the reflective bead by the system and recorded on computer software ( vibsoft-20 , polytec , wurzburg , germany ) for further analysis .
the vibration amplitude of the moving surface was calculated from the voltage output of the laser vibrometer 's velocity decoder .
appropriate anesthesia ( subcutaneous injection of 10 mg / kg xylazine hydrochloride 1 h after normal anesthesia described above ) was maintained to retain respiratory amplitude so as not to affect the test during the process .
testing was conducted in a sound - proofed booth to maintain a high signal - to - noise ratio . for each stimulus frequency
, sound stimuli were repeated three times with a good signal - to - noise ratio , after which all data were averaged .
six animals of each group were sacrificed by an overdose of anesthetic ; intracardiac perfusion was performed with 150 ml of 0.2 m pbs , followed by 4% polymerized formaldehyde , and the temporal bones were then removed and fixed in 4% polymerized formaldehyde ( ph = 7.4 ) for more than 24 h at 4c .
the temporal bones were decalcified in ethylenediaminetetraacetic acid , dehydrated in increasingly higher concentrations of alcohol , embedded in paraffin , and sectioned serially at 10 m in the plane parallel to the modiolus .
cochlear sections were stained with hematoxylin and eosin ( he ) and then observed under a light microscope ( 6030116204 , carl zeiss , jena , germany ) .
three guinea pigs of each group were killed by means of decapitation by overdose anesthesia . under magnification , the tympanic bullae were dissected from the surrounding tissues ; the bony wall of the cochlea was removed with a pick and forceps to expose the upper aspect of the organ of corti .
cochleae were fixed in a 4% paraformaldehyde ( pfa ) in phosphate - buffered saline ( pbs ) for 24 hours and then we dissected the cochlea and get the basilar membrane of the cochlea .
the whole - mount tissues were incubated with alexa fluor 488-conjugated phalloidin ( invitrogen , usa , 1 : 1000 ) for 30 minutes to stain for f - actin prior to mounting .
confocal fluorescent microscope ( zeiss , lsm800 , germany ) was used in scanning the surface image of corti stained for stereocilia with green phalloidin . in order to quantify the hydropic ratio ( hr ) of the eh models ,
areas of scala media ( sm ) and scala vestibule ( sv ) ( figure 3(a ) ) were first obtained by using photoshop cs6 and scala media area ratio ( smr ) was calculated in formula(1)smr = smareasmarea+svarea.as deviations in the plane of section and interanimal variability in anatomy can not be avoided exactly , smr is imprecise for representing hydropic degree of eh animals .
then we use the ratio of smr in the eh ear divided by smr in the contralateral ear to quantify the hydropic degree in the eh group , whose right ears were subject to obliteration of the endolymphatic sac . and
this ratio was named as hydropic ratio ( hr ) here . in formula(2)hr = smr in the eh earsmr in the contralateral eara hr value of one
suggests no hydrops in the given turn.this is when hydropic ratio is close to 1 ; that to say , there is no hydrops in the given turn .
two - tailed students ' t - tests were used to determine the confidence interval for comparison between two groups and p values 0.05 were considered significant .
spearman bivariate correlation analysis was taken to explore the relationship between abr threshold elevation and endolymphatic hydropic ratio .
the mid - modiolar he - stained section of the cochlea showed that there was no displacement of reissner 's membrane in the control ear and the angle between reissner 's membrane and the osseous spiral lamina was almost 45 , suggesting that there was no eh in the control group ( figure 2(a ) ) .
figure 2(b ) illustrates that reissner 's membrane of each turn bulged significantly towards the scala vestibule in the 8-week eh group .
reissner 's membrane was attached to the bony wall of the scala vestibule ( sv ) in the subapical turn of the cochlea .
a high level eh was observed in the subapical turn , while the eh in the basal turn was markedly more moderate .
these results agreed with those from a previous study by chi and liang and indicated that the chronic eh model was successfully created . in order to observe the extent of eh
, we had quantified the hr in each turn of the cochlea . when hydropic ratio is close to 1 , there is no labyrinthine hydrops in the given turn .
the hr is much larger according to the most serious hydrops in the scala media .
the results of the individual and average hr for each turn of all animals in the eh group are shown at table 1 and figure 3(b ) .
hr values for all turns of the six eh models were obviously larger than 1 ( p < 0.05 ) , demonstrating that conspicuous hydrops was induced .
the most serious labyrinthine hydrops is in the subapical turn , and the second is in the suprabasal turn .
the results of the abr threshold in the control group and eh group are listed in table 2 and figure 4(a ) . both the mean and standard deviation of each group were recorded .
the abr threshold was elevated in the eh group relative to the control group by more than 25 db at 2 , 4 , 6 , and 8 khz and by less than 25 db at 0.5 and 1 khz .
student 's t - tests revealed that the mean abr threshold of the eh cases was significantly higher than that of the control group at 0.5 , 1 , 2 , 4 , 6 , and 8 khz ( p < 0.05 ) .
spearman bivariate correlation analysis was taken to explore the relationship between abr threshold elevation and extent of the endolymphatic hydrops .
strong positive correlation was found between hr at apical turn and abr threshold elevation at 1000 hz ( r = 0.82 , correlation is significant at the 0.01 level ) , as well as between hr at subapical turn and abr threshold elevation at 2000 hz ( r = 0.88 , correlation is significant at the 0.05 level ) .
figures 5(a ) and 5(b ) illustrate the peak - to - peak displacement amplitude - frequency curves of the umbo from the six control ears and that of the six eh ears , respectively , over the 0.58-khz range in response to 85 db spl stimuli in the ear canal .
the two groups possessed similar displacement - frequency curves for the 0.58-khz range and the results of the eh ears were lower than that for the control ears overall ( figure 5(c ) ) . a maximum displacement amplitude , almost 14 nm , presented at 0.5 khz in the control group . in the eh group ,
peak displacement was found at 1 khz ( 11.48 nm ) and there was a sudden decrease in displacement amplitude between 1 and 2 khz frequencies in both groups .
mean sd ( n = 6 ) of the control and eh groups was compared in figure 5(c ) showing that there are no significant differences in each frequency ; particularly the biggest reduction of displacement amplitude among the two groups was present at 0.5 khz ( 6.05 nm , p = 0.12 ) and this reduction remained below 1 nm at frequencies above or equal to 1 khz .
figures 5(d ) and 5(e ) display the peak - to - peak displacement curves of the stapes head in two groups .
displacements in the eh group were generally lower than those in the control group ( figure 5(f ) ) .
the displacement amplitude reached a maximum of 12.3 nm at 500 hz and decreased gradually to 0.52 nm at 8 khz in normal ears , while in the eh group the maximal displacement amplitude was 7.6 nm at 1 khz .
a statistically significant difference ( p < 0.05 ) was found at 500 hz and the reduction reached almost 4.82 nm . figures 5(g ) and 5(h ) show the peak - to - peak displacement curves of the rwm in both groups .
each curve shows a prominent displacement peak at 0.5 , 1 , 2 , 4 , 6 , and 8 khz .
the displacement decreased along with the increase in frequency from 0.5 to 8 khz in the two groups in response to 85 db spl input at the ear canal .
the displacements of the eh group were generally lower than those in the control group ( figure 5(i ) ) .
there was a statistically significant difference ( p < 0.05 ) at 0.5 and 6 khz . for rmw , the best vibration response to displacement in the control ears was 20.79 nm at 500 hz , while peak displacement in the eh group was 12.43 nm at 1 khz . the displacement transmission ratio ( dtr ) of the stapes head to the umbo was used to represent the middle ear transfer function under normal and eh conditions in this study .
figure 6 shows the mean dtr values ( n = 6 ) at frequencies from 0.5 to 8 khz . in the control ears ,
the displacement of the stapes head was slightly less than that of the umbo by factors of 0.640.72 at frequencies above 2 khz and close to the umbo by factors of 0.91.06 at frequencies of 0.52 khz . when eh was present in the cochlea , the displacement of the stapes head was much lower than that in the umbo , by factors of 0.20.5 , at frequencies of 48 khz .
as for frequencies below 2 khz , a factor of 0.91 suggested nearly equal displacement between stapes head and umbo ( figure 6 ) .
the lower dtr of the stapes head to the umbo in the eh group than in the control group suggested that vibration of the stapes head was reduced more than that of the umbo by eh .
three ears of two groups were dissected and stained with alexa fluor 488-conjugated phalloidin for f - actin of stereocilia . the hair cell bundles in each turn
were shown to be normal in the control group 8 weeks later in figure 6 . in the eh group
, we could observe the accidental loss of the stereocilia of outer hair cells ( star in figure 7 ) in the suprabasal , subapical , and apical turn . in the basal turn of eh model ,
furthermore , the stereocilia in the third row of outer hair cells were sporadically collapsed and deranged in the suprabasal turn .
interestingly , the most obvious morphology change in the apical and subapical turn of the eh group compared to the control group , which showed the high level extent of the labyrinthine hydrops and low frequency abr threshold shift .
we reported the effect of endolymphatic hydrops of live guinea pigs on the abr threshold , morphology changes , and movements of the umbo , stapes head , and rwm under 85 db spl pure tone stimuli in the external auditory canal in this study .
we had explored the mechanism of the hearing loss in eh model , which showed displacements reduction of the umbo , stapes head , and rwm was greater at low frequency ( < 1 khz ) and the stereocilia of outer hair cell were missing in apical and subapical turn , which was associated with the extent of the endolymphatic hydrops . since the target location of laser beam along the tm affects data quality in terms of the signal - to - noise ratio and the tm is directly attached to the ossicular chain ( the umbo and the lateral process of the malleus ) , tm movement in this study was measured on the medial side of the umbo to minimize variability .
the umbo displacement was slightly decreased in the eh group compared with the control group at all measured frequencies and the differences were more notable at low frequencies .
the biggest reduction in umbo displacement was 5.01 db ( ref = 1 pm ) at 0.5 khz .
the control group shared a similar displacement amplitude - frequency curve of umbo with that reported by guan and gan ( figure 3a in their paper ) . but data in this study was generally lower .
this discrepancy may be explained by the different measurement sides and the subtle difference between angles of the laser beam in both studies . in the study by guan and gan ,
the target position was at the lateral side of umbo , while in this study the medial side of the umbo was taken as test point .
. suggested that a positive inner ear fluid pressure weakened the vibration of the umbo mainly at low frequencies ( 1 khz ) .
jang et al . reported that the endolymphatic pressure load caused greater reduction in the umbo velocity than did the perilymphatic pressure load at frequencies below 1 khz .
all these findings supported the result in this study . the displacement of the incus tip in guinea pigs under 80 db spl acoustic stimulation in the ear canal was reported by guan and gan and by chen et al . .
the measurement of the stapes head in this study was recorded at a nearby site compared with the incus tip reported previously , both around the incudostapedial joint .
the mean stapes head displacement of the control group in this study was in accordance with that reported by guan and gan ( figure 4a in their paper ) on the whole but was generally higher than that reported by chen et al . .
murakami et al . reported that the stapes velocity decreased over 0.45.0 khz in fresh human temporal bones with increased hydrostatic pressure of the inner ear under sound pressure of 114 db spl .
found that the stapes motion changed at low frequencies when inner ear fluid was drained . in this study ,
the mean displacement amplitude of the stapes in the eh group decreased at all measured frequencies , with profound reduction below 1 khz .
all those findings suggested that cochlear fluid pressure / volume can affect inner ear impedance and cause changes in stapes displacement under acoustic stimulation .
the round window is one of the two openings into the cochlea from the middle ear .
the rwm serves as a barrier between the middle ear cavity and the cochlea and plays an important role in the middle ear and cochlear mechanics .
the mechanical response of the rwm can be indirect detection of acoustic dysfunction . compared with literature published by guan and gan ( figure 5a in their paper )
, these two studies shared a parallel mean displacement amplitude - frequency curve and the data were in the same order of magnitude .
the maximum reduction of the rwm displacement was 13.01 nm ( 8.5 db ref = 1 pm ) at 500 hz .
the difference between 1 khz and 4 khz was about 11.8 db , which is relatively flat .
a second difference - peak was found at 6 khz ( 5 db ) .
those findings suggested that eh has an influence on the vibration of the rwm and that changes occur mainly at low frequencies .
studies of motion of the ossicular chain revealed that it was more complicated than a simple piston - like motion and that the complexities increased with frequency . in order to evaluate the transfer function of middle ear
approximately , dtr of single points ( the tm to the footplate / incus tip ) was calculated by researchers [ 17 , 26 ] .
nevertheless , the dtr of the stapes head to the umbo may not reveal the exact middle ear transfer function . through numerical conversion
, the results of the control group from guan and gan showed that the mean displacement of the incus tip was less than that of the tm by a factor of 0.20.34 at lower frequencies and low to 0.125 at higher frequencies .
results from this study do not correspond well with those reported by guan and gan . as described
before , the angle between the motion of the umbo and the laser beam in this study is different and this may be one reason for the discrepant findings between the two studies .
moreover , the complex motion of the stapes may increase the interindividual variations in the results of dtr and may underlie differences in the findings . when eh was present in the cochlea , the displacement of the stapes head was much lower than that of the umbo , by factors of 0.20.5 , at frequencies of 48 khz .
the lower dtr in the eh group than in the control group suggested that vibration of the stapes head was reduced more than that of the umbo by eh .
both the umbo and stapes head vibration decreased due to the increased inner ear impedance , decreasing the stapes footplate movement . as the ossicular rotation axis and incudostapedial joint
have some laxity , the increased inner ear impedance may not be wholly transmitted to the umbo . therefore , the reduction of stapes head displacement should be greater , as described by the dtr . since the milestone findings on the temporal bones of mnire 's disease ( md ) patients [ 4 , 27 , 28 ] , endolymphatic hydrops ( eh ) has been considered as the histopathological origin of md , as characteristic morphological changes were reported previously by surgical obstruction of the endolymphatic sac ( es ) in guinea pig . because surgical experiment induction procedure is still by far the most common method for producing experimental endolymphatic hydrops ( eh )
, it is particularly important to characterize the pathologic changes in this model so that their relevance to mnire 's disease can be fully appreciated .
the simplest explanation for the hearing loss in the endolymphatic hydrops is initial loss of cochlear sensitivity , the shift in the cochlear microphonics ( cm ) , and distortion product otoacoustic emission ( dpoae ) ; increased sp ( summating potential ) amplitude , with a reduction in ep ( endocochlear potential ) , is one which has been suggested by researchers who have performed acute endolymph injections .
many authors have demonstrated that the significant correlation between the degree of hydrops and a reduction in the low frequency cochlear microphonic change was found in short- and long - standing surgically induced hydrops guinea pigs [ 3133 ] . because high - frequency hearing is ultimately affected in both the human and the animal conditions ,
these same studies imply a poor relationship between hair cell loss and the degree and nature of hearing loss . in our experiment
the most serious labyrinthine hydrops is in the subapical turn , and the second is in the suprabasal turn .
meanwhile , the mean abr threshold of the eh cases was significantly higher than that of the control group at 0.5 , 1 , 2 , 4 , 6 , and 8 khz frequencies .
strong positive correlation was found between hr at apical turn and abr threshold elevation at 1 khz , as well as between hr at subapical turn and abr threshold elevation at 2 khz . in order to underline the morphology change of the hair cells stained with alexa fluor
488-conjugated phalloidin , we could observe the accidental loss of the outer hair cells and sporadic collapse of the stereocilia in the suprabasal , subapical , and apical turn .
in the basal turn of eh model , there were almost normal hair cell bundles .
interestingly , the most obvious morphology change in the apical and subapical turn of the eh group compared to the control group , which showed the high level extent of the labyrinthine hydrops and low frequency abr threshold shift .
we could not observe the serious hair cell damage in the basal and suprabasal turn with hydrops and higher frequency abr threshold elevation , which should be needed for long - time eh model observation .
overall , the hearing loss of the surgical eh model in our experiment could be explained for two reasons : one is that the higher inner ear impedance in eh affected the dynamic behavior of the two opening windows of the cochlea and then reduced the vibration of the ossicular chain by increasing the afterload , resulting in acoustic dysfunction .
the other is the morphological damage of the stereocilia of the outer hair cells especially in the apical and subapical turn with more serious labyrinthine hydrops .
in this study , an eh model was created in live guinea pigs by obliteration of the endolymphatic sac .
it is the first time to detect the dynamic behaviors of the ossicular chain and the two opening windows by ldv in vivo guinea pigs .
eh reduced the displacement of the umbo , stapes , and rwm mainly at lower frequencies .
the ldv provides an accurate assessment of the dynamic properties of the middle ear and cochlear mechanics in eh animal models .
the displacement of reissner 's membrane caused by eh indicates that the membranous cochlear duct reached a higher hydrostatic pressure .
the ldv results suggested that abnormal endolymphatic pressure affects the dynamic behaviors of the two opening windows of the cochlea and that this may be one of the major reasons for hearing loss at lower frequencies in eh - related conditions . |
objective . this study aimed at describing the mechanism of hearing loss in low frequency and the different dynamic behavior of the umbo , the stapes head , and the round window membrane ( rwm ) between normal guinea pigs and those with endolymphatic hydrops ( eh ) , using a laser doppler vibrometer ( ldv )
. methods .
cochlear sections were stained with hematoxylin and eosin ( he ) to evaluate the hydropic ratio ( hr ) .
auditory brainstem responses ( abr ) and whole - mount immunostaining were measured .
displacement of the umbo , stapes head , and rwm in response to ear - canal sound was evaluated using a ldv .
results .
mean hr values in eh model of all the turns are larger than the control group .
the abr threshold of the eh group was significantly higher than that of the control .
strong positive correlation was found between hr at apical turn and abr threshold elevation at 1000 hz and at subapical turn and abr threshold elevation at 2000 hz .
fitc - phalloidin immunostaining of the cochlear basilar membrane in the apical , subapical , and suprabasal turns showed missing and derangement stereocilia of third - row outer hair cells .
the umbo , stapes head , and rwm displacement in ears with eh was generally lower than that of normal ears .
the eh - induced differences in stapes head and rwm motion were significant at 0.5 khz .
conclusion .
the ldv results suggested that the higher inner ear impedance in eh affected the dynamic behavior of the two opening windows of the cochlea and then reduced the vibration of the ossicular chain by increasing the afterload , resulting in acoustic dysfunction .
the vibration reduction mainly occurred at low frequencies , which has related with the morphology changes of the apical and subapical turns in eh model . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions | this technique has been used to test the vibration of the round window membrane ( rwm ) , the tympanic membrane ( tm ) , and the stapes footplate in fresh and embalmed cadaveric human temporal bone and animal specimens . however , no prior study has reported changes in the dynamic behavior of the umbo , the stapes head , and the rwm in association with eh . to better understand how eh affects the sound transmission process and hearing loss in low frequency
, we used guinea pigs to create eh models and measured the hydropic ratio ( hr ) and morphology in each turn , the vibration of the umbo , stapes head , and rwm , as well as the auditory - evoked brainstem response ( abr ) . the primary objective of this study was to compare the different dynamic properties of the ossicular chain and the rwm between normal and eh guinea pigs and explore the potential pathogenic mechanism underlying the associated hearing loss at low frequencies . cochlear sections were stained with hematoxylin and eosin ( he ) and then observed under a light microscope ( 6030116204 , carl zeiss , jena , germany ) . student 's t - tests revealed that the mean abr threshold of the eh cases was significantly higher than that of the control group at 0.5 , 1 , 2 , 4 , 6 , and 8 khz ( p < 0.05 ) . strong positive correlation was found between hr at apical turn and abr threshold elevation at 1000 hz ( r = 0.82 , correlation is significant at the 0.01 level ) , as well as between hr at subapical turn and abr threshold elevation at 2000 hz ( r = 0.88 , correlation is significant at the 0.05 level ) . when eh was present in the cochlea , the displacement of the stapes head was much lower than that in the umbo , by factors of 0.20.5 , at frequencies of 48 khz . the lower dtr of the stapes head to the umbo in the eh group than in the control group suggested that vibration of the stapes head was reduced more than that of the umbo by eh . in the eh group
, we could observe the accidental loss of the stereocilia of outer hair cells ( star in figure 7 ) in the suprabasal , subapical , and apical turn . interestingly , the most obvious morphology change in the apical and subapical turn of the eh group compared to the control group , which showed the high level extent of the labyrinthine hydrops and low frequency abr threshold shift . we reported the effect of endolymphatic hydrops of live guinea pigs on the abr threshold , morphology changes , and movements of the umbo , stapes head , and rwm under 85 db spl pure tone stimuli in the external auditory canal in this study . we had explored the mechanism of the hearing loss in eh model , which showed displacements reduction of the umbo , stapes head , and rwm was greater at low frequency ( < 1 khz ) and the stereocilia of outer hair cell were missing in apical and subapical turn , which was associated with the extent of the endolymphatic hydrops . when eh was present in the cochlea , the displacement of the stapes head was much lower than that of the umbo , by factors of 0.20.5 , at frequencies of 48 khz . the lower dtr in the eh group than in the control group suggested that vibration of the stapes head was reduced more than that of the umbo by eh . meanwhile , the mean abr threshold of the eh cases was significantly higher than that of the control group at 0.5 , 1 , 2 , 4 , 6 , and 8 khz frequencies . strong positive correlation was found between hr at apical turn and abr threshold elevation at 1 khz , as well as between hr at subapical turn and abr threshold elevation at 2 khz . in order to underline the morphology change of the hair cells stained with alexa fluor
488-conjugated phalloidin , we could observe the accidental loss of the outer hair cells and sporadic collapse of the stereocilia in the suprabasal , subapical , and apical turn . interestingly , the most obvious morphology change in the apical and subapical turn of the eh group compared to the control group , which showed the high level extent of the labyrinthine hydrops and low frequency abr threshold shift . overall , the hearing loss of the surgical eh model in our experiment could be explained for two reasons : one is that the higher inner ear impedance in eh affected the dynamic behavior of the two opening windows of the cochlea and then reduced the vibration of the ossicular chain by increasing the afterload , resulting in acoustic dysfunction . the ldv results suggested that abnormal endolymphatic pressure affects the dynamic behaviors of the two opening windows of the cochlea and that this may be one of the major reasons for hearing loss at lower frequencies in eh - related conditions . | [
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] |
due to continuous material processing and 24 hours activity , shift work is common in many occupations .
consequently , 15 - 30% of the working population in industrialized countries is involved in various types of permanent night and rotating shift work , although this percentage is higher in developing countries , due to lack of job organization , irregular work hours and night working ( 1 - 3 ) . the term
shift working refers to a job , which is performed in unusual and unconventional times , and is one of the factors affecting workers ' health in many workplaces and industries . on the other hand ,
shift work is a well - recognized occupational health hazard in both industrialized and industrially developing countries ( 4 , 5 ) .
day / night shift rotation is one of the most important features of shift working , which has serious complications in terms of circadian rhythm and sleep disorders ( 6 - 8 ) .
in addition to several health problems , such as cardiovascular , gastrointestinal diseases and mental disorders , shift working has been shown to be associated with fatigue ( 9 , 10 ) . sleeplessness and exhaustion , caused by fatigue during shift working ,
can lead to decreased alertness and performance and to increased failure or incident rates , and this may have serious constraints for workers , their families and the society ( 11 - 13 ) .
fatigue , as a common symptom , can be defined as a mental sense of weakness , energy defect and burn out , which disrupts any physical and cognitive activity ( 13 - 15 ) .
fatigue is also one of the most important symptoms of distress , which is a common experience , caused by insufficient rest or sleep , heavy physical or mental activity and lack of motivation to begin any activity ( 16 ) .
evidence suggests that fatigue is a frequent complaint among different working groups ( 17 , 18 ) .
the findings from a study , conducted among the working population of 15 european countries , indicated that 5 - 56% of workers experienced fatigue ( 18 ) .
this is important , because the link between fatigue and accidents , in different occupational groups , has been well established ( 13 ) .
psychological distress is another important issue , which should be considered in shift work related studies .
several previous studies have evaluated fatigue and psychological distress , among shift workers ( 19 - 21 ) . in a study among industrial workers ,
fatigue was conceptualized as a decrease in energy and motivation , which was more likely to be experienced by night shift workers , compared to those working in other shifts ( 21 ) .
the authors noted that longer reaction times were associated with increased ratings of mental aspects of fatigue .
studies conducted among other working groups , such as anesthesiologists , have also reported a significant relationship between fatigue and psychological distress ( 17 , 22 ) .
there are also studies , which reported no relationship between psychological distress and shift working ( 19 , 20 ) , and , therefore , this is an area that needs further investigation .
petrochemical industries are one of the well - known and economically important continuous work areas , in which shift working is unavoidable .
it is , therefore , necessary to evaluate the adverse health effects of shift working , on workers in these industries .
a review of the literature shows that there are limited studies conducted among industrial shift workers , about the relationship between fatigue and psychological distress , particularly in petrochemical industries .
moreover , very little is known about the prevalence of fatigue and psychological distress of 12-hour shift workers , in this industry .
a systematic review study showed the fatigue and safety , as the main concerns of 12-hour shift working ( 23 ) .
several case studies , in the petrochemical industry , in north america , investigated the health problems among shift workers ( 24 ) .
in an attempt to address this issue , the present study was conducted to evaluate the prevalence of fatigue and psychological distress and their relationship in 12-hour rotating shift workers , in a petrochemical plant .
this cross - sectional field study was conducted between september and december 2013 , in a governmental petrochemical plant ( southern pars gas field ) , in southwest iran . due to the relatively hot and humid climate
the research sites included operational , maintenance , engineering , security and health , safety and environment ( hse ) departments , as well as administrative offices .
the company , as a non - referral field , had approximately 400 rotating male shift workers , at the time of study , and all of these workers were asked to participate in the study .
the shift work used here refers to a job , which is performed in unusual and unconventional times .
the work schedule time duration was from 7.00 am to 19.00 pm and from 19.00 pm to 7.00 am , and the shift schedule followed a 7 days - 7 nights - seven rests pattern , in a forward rotating program .
the ethical review committee of the tabriz university of medical sciences , tabriz , iran , reviewed and approved the study protocol .
all the 400 male shift workers of the petrochemical plant were included in this study , as a census method .
work experience of less than one year and , also , having physical disabilities or mental diseases were defined as the exclusion criteria .
all workers were familiarised with the study procedure and any questions were answered , by the investigator .
the participation was strictly on a voluntary basis and the workers were under no obligation to complete the study .
a written informed consent form was signed by each shift - worker , before participation in the study .
a questionnaire , consisting of three sections , including demographic characteristics , multidimensional fatigue and psychological distress status , was used for data collection .
demographic details included : age , weight , height , educational level , marital status , shift work tenure and job title .
the whole questionnaire took about 20 minutes to complete . the multidimensional fatigue inventory ( mfi20 ) ( 25 )
the mfi-20 is a reliable and validated tool , which is widely used for evaluating fatigue in both patients and healthy individuals ( 26 - 28 ) .
the mfi-20 is a 20 item self - report assessment tool , which assesses five dimensions of fatigue , including general fatigue , physical fatigue , reduced motivation , mental fatigue and reduced activity .
each dimension consists of four items : two indicative for fatigue , where a high score indicates a high fatigue level , and two contraindicative , where a high score indicates low fatigue level .
the respondents to the mfi-20 are asked to specify the extent to which the particular statements relate to them , on a 5-point scale .
the total possible score , for each subscale , ranges from 4 to 20 , with higher scores indicating a higher level of fatigue .
the english version of mfi-20 has been translated and revised into the persian language ( farsi ) and has an established validity and reliability ( 29 ) .
the 28 item general health questionnaire ( ghq-28 ) is generally used as a self - report assessment tool , to evaluate the status of psychological distress ( 30 ) , and is a well - tried and tested technique ( 31 ) .
the brevity , intelligibility , and psychometric properties of the ghq-28 are the most important reasons for using this tool ( 31 ) .
the ghq-28 has four subscales : somatic symptoms ( questions 1 to 7 ) , anxiety and insomnia ( questions 8 to 14 ) , social dysfunction ( questions 15 to 21 ) and severe depression ( questions 22 to 28 ) .
each item is accompanied by four possible responses , ranging from never true to always true , and scoring from 0 to 3 .
the total possible score for each subscale ranges from 0 to 21 , and for the ghq-28 ranges from 0 to 84 , with higher scores indicating more severe mental problems ( 30 ) .
the persian version of ghq-28 , which has an established validity and reliability ( 32 ) , was used in this study . moreover , a cut - off point of 27.38 was used for the ghq-28 ( e.g. those scoring 27.38 or above were designated as poor psychological distress ) in this study , which was based on the noorbala et al.s study ( 32 ) .
analysis of the data was performed , using spss version 11.5 ( spss inc . ,
chicago , il , usa ) . to compare the two questionnaires ( e.g. ghq-28 and mfi-20 ) ,
the range of all scores obtained form both questionnaires were transformed into 0 - 100 domain .
therefore , the cut - off point of ghq28 was changed from 23 to 27.38 , in the modified domain .
normality was tested using qq plots and all discriptive values were expressed as mean sd .
stepwise regression analysis was performed , to evaluate the relationship between the fatigue and psychological distress status .
the r square and pearson s correlation coefficients were also calculated , for these variables .
in addition , error - bar was used to show the relationship between the mean scores .
this cross - sectional field study was conducted between september and december 2013 , in a governmental petrochemical plant ( southern pars gas field ) , in southwest iran . due to the relatively hot and humid climate
the research sites included operational , maintenance , engineering , security and health , safety and environment ( hse ) departments , as well as administrative offices .
the company , as a non - referral field , had approximately 400 rotating male shift workers , at the time of study , and all of these workers were asked to participate in the study .
the shift work used here refers to a job , which is performed in unusual and unconventional times .
the work schedule time duration was from 7.00 am to 19.00 pm and from 19.00 pm to 7.00 am , and the shift schedule followed a 7 days - 7 nights - seven rests pattern , in a forward rotating program .
the ethical review committee of the tabriz university of medical sciences , tabriz , iran , reviewed and approved the study protocol .
all the 400 male shift workers of the petrochemical plant were included in this study , as a census method .
work experience of less than one year and , also , having physical disabilities or mental diseases were defined as the exclusion criteria .
all workers were familiarised with the study procedure and any questions were answered , by the investigator .
the participation was strictly on a voluntary basis and the workers were under no obligation to complete the study .
a written informed consent form was signed by each shift - worker , before participation in the study .
a questionnaire , consisting of three sections , including demographic characteristics , multidimensional fatigue and psychological distress status , was used for data collection .
demographic details included : age , weight , height , educational level , marital status , shift work tenure and job title .
the whole questionnaire took about 20 minutes to complete . the multidimensional fatigue inventory ( mfi20 ) ( 25 )
the mfi-20 is a reliable and validated tool , which is widely used for evaluating fatigue in both patients and healthy individuals ( 26 - 28 ) .
the mfi-20 is a 20 item self - report assessment tool , which assesses five dimensions of fatigue , including general fatigue , physical fatigue , reduced motivation , mental fatigue and reduced activity .
each dimension consists of four items : two indicative for fatigue , where a high score indicates a high fatigue level , and two contraindicative , where a high score indicates low fatigue level .
the respondents to the mfi-20 are asked to specify the extent to which the particular statements relate to them , on a 5-point scale .
the total possible score , for each subscale , ranges from 4 to 20 , with higher scores indicating a higher level of fatigue .
the english version of mfi-20 has been translated and revised into the persian language ( farsi ) and has an established validity and reliability ( 29 ) .
the 28 item general health questionnaire ( ghq-28 ) is generally used as a self - report assessment tool , to evaluate the status of psychological distress ( 30 ) , and is a well - tried and tested technique ( 31 ) .
the brevity , intelligibility , and psychometric properties of the ghq-28 are the most important reasons for using this tool ( 31 ) .
the ghq-28 has four subscales : somatic symptoms ( questions 1 to 7 ) , anxiety and insomnia ( questions 8 to 14 ) , social dysfunction ( questions 15 to 21 ) and severe depression ( questions 22 to 28 ) .
each item is accompanied by four possible responses , ranging from never true to always true , and scoring from 0 to 3 .
the total possible score for each subscale ranges from 0 to 21 , and for the ghq-28 ranges from 0 to 84 , with higher scores indicating more severe mental problems ( 30 ) . the persian version of ghq-28 , which has an established validity and reliability ( 32 ) , was used in this study . moreover , a cut - off point of 27.38 was used for the ghq-28 ( e.g. those scoring 27.38 or above were designated as poor psychological distress ) in this study , which was based on the noorbala et al.s study ( 32 ) .
analysis of the data was performed , using spss version 11.5 ( spss inc . ,
chicago , il , usa ) . to compare the two questionnaires ( e.g. ghq-28 and mfi-20 ) ,
the range of all scores obtained form both questionnaires were transformed into 0 - 100 domain .
therefore , the cut - off point of ghq28 was changed from 23 to 27.38 , in the modified domain .
normality was tested using qq plots and all discriptive values were expressed as mean sd .
stepwise regression analysis was performed , to evaluate the relationship between the fatigue and psychological distress status .
the r square and pearson s correlation coefficients were also calculated , for these variables .
in addition , error - bar was used to show the relationship between the mean scores .
the mean age and shift work experience of the participants were 31.5 ( range = 21 - 59 ; sd = 5.11 ) years and 5.83 ( range = 1 - 28 ; sd = 2.89 ) years , respectively .
most of the participants were married ( 77.2% ) and their educational level ranged from primary school ( 4.1% ) to master s degree ( 4.4% ) .
overall , in this study , a relatively weak impact of demographic characteristics was found on fatigue and psychological distress .
as shown in table 1 , the total fatigue score of the mfi20 , for the study population , was 42.68 ( range = 0 - 100 ; sd = 17.88 ) .
the general fatigue ( mean = 69.40 ; sd = 18.22 ) and reduced motivation ( mean = 30.90 ; sd = 18.73 ) were the subscales with the highest and lowest mean scores , respectively .
the pearson s correlation test showed a moderate positive correlation between the mfi20 subscales , with r values ranging from 0.41 to 0.67 ( as shown in table 2 ) .
the general fatigue and mental fatigue subscales had a strong positive correlation ( r = 0.67 ) , while there was a weak positive correlation ( r = 0.41 ) , between the general fatigue and reduced activity subscales .
the correlation coefficients for the total mfi and its subscales ranged from 0.71 to 0.86 .
the cronbach s for the mfi subscales were , as follows : general fatigue ( 0.72 ) , physical fatigue ( 0.68 ) , reduced activity ( 0.70 ) , reduced motivation ( 0.52 ) and mental fatigue ( 0.77 ) .
these values suggest good internal consistency for the scale , as a whole , and for the subscales , individually .
the proportions in this sample above the cut - point were : total ( 59.7% ) , social dysfunction ( 86.2% ) , anxiety and insomnia ( 71.7% ) , somatic symptoms ( 57.9% ) and severe depression ( 26.2% ) .
the total mean ghq score was 34.66 ( range = 0 - 100 ; sd = 18.56 ) .
the mean scores of the ghq subscales were : social dysfunction , 43.0 ( sd = 18.05 ) ; anxiety and insomnia , 42.17 ( sd = 24.52 ) ; somatic symptoms , 35.25 ( sd = 21.80 ) ; and severe depression , 18.19 ( sd = 20.44 ) . a moderate to high positive correlation
was found between the ghq subscales , with r values , ranging from 0.61 to 0.80 ( as shown in table 4 ) .
a strong correlation was also found between the total ghq and its subscales ( pearson s coefficients , ranging from 0.82 to 0.92 ) .
for the ghq subscales were as follows : somatic symptoms ( 0.88 ) , anxiety and insomnia ( 0.93 ) , social dysfunction ( 0.86 ) , severe depression ( 0.89 ) .
these values indicate good internal consistency for the scale , as a whole , and for the individual subscales .
levels of significance : r = 0.11 , 0.13 , 0.17 corresponds for p = 0.05 , 0.01 , 0.001 , respectively .
the results of stepwise regression analysis , which evaluated the relationship between psychological distress ( ghq total score ) and fatigue ( mfi subscales scores ) , indicated that only general fatigue , mental fatigue and reduced motivation remained in the model ( p < 0.05 ) ( as shown in table 5 ) .
therefore , the model was as follows : r square = 0.676 , adjusted r square = 0.451 . in addition , to diagnose the relationship between the ghq and mfi , a linear regression test was performed with r square = 0.626 and adjusted r square = 0.390 , which means that , per each unit score of mfi , 0.651 is added to the ghq score .
therefore , the final model was as follows : ghq = 6.89 + 0.651 ( mfi ) .
the results of pearson s correlation test between the ghq and mfi subscales , presented in table 2 , indicated that the most positive correlations were found between the total ghq with general fatigue ( r = 0.61 ) and mental fatigue ( r = 0.59 ) , while a weak positive correlation was found for reduced activity ( r = 0.30 ) .
finally , the pearson s correlation test showed a relatively high positive correlation , between the total ghq and mfi ( r = 0.62 ) ( table 2 ) .
the scores of ghq subscales were significantly higher for workers with moderate to high level of total fatigue ( 3 - 5 on the 5-point scale ) than those with a low level of total fatigue ( 1 - 2 on the 5-point scale ) ( figure 1 ) .
similarly , the scores of the mfi subscales were significantly higher in those workers with the total ghq score 27.38 than in those with a lower score ( total ghq score < 27.38 ) ( figure 2 ) .
the mean age and shift work experience of the participants were 31.5 ( range = 21 - 59 ; sd = 5.11 ) years and 5.83 ( range = 1 - 28 ; sd = 2.89 ) years , respectively .
most of the participants were married ( 77.2% ) and their educational level ranged from primary school ( 4.1% ) to master s degree ( 4.4% ) .
overall , in this study , a relatively weak impact of demographic characteristics was found on fatigue and psychological distress .
as shown in table 1 , the total fatigue score of the mfi20 , for the study population , was 42.68 ( range = 0 - 100 ; sd = 17.88 ) .
the general fatigue ( mean = 69.40 ; sd = 18.22 ) and reduced motivation ( mean = 30.90 ; sd = 18.73 ) were the subscales with the highest and lowest mean scores , respectively .
the pearson s correlation test showed a moderate positive correlation between the mfi20 subscales , with r values ranging from 0.41 to 0.67 ( as shown in table 2 ) .
the general fatigue and mental fatigue subscales had a strong positive correlation ( r = 0.67 ) , while there was a weak positive correlation ( r = 0.41 ) , between the general fatigue and reduced activity subscales .
the correlation coefficients for the total mfi and its subscales ranged from 0.71 to 0.86 .
the cronbach s for the mfi subscales were , as follows : general fatigue ( 0.72 ) , physical fatigue ( 0.68 ) , reduced activity ( 0.70 ) , reduced motivation ( 0.52 ) and mental fatigue ( 0.77 ) .
these values suggest good internal consistency for the scale , as a whole , and for the subscales , individually .
the proportions in this sample above the cut - point were : total ( 59.7% ) , social dysfunction ( 86.2% ) , anxiety and insomnia ( 71.7% ) , somatic symptoms ( 57.9% ) and severe depression ( 26.2% ) .
the total mean ghq score was 34.66 ( range = 0 - 100 ; sd = 18.56 ) .
the mean scores of the ghq subscales were : social dysfunction , 43.0 ( sd = 18.05 ) ; anxiety and insomnia , 42.17 ( sd = 24.52 ) ; somatic symptoms , 35.25 ( sd = 21.80 ) ; and severe depression , 18.19 ( sd = 20.44 ) . a moderate to high positive correlation
was found between the ghq subscales , with r values , ranging from 0.61 to 0.80 ( as shown in table 4 ) .
a strong correlation was also found between the total ghq and its subscales ( pearson s coefficients , ranging from 0.82 to 0.92 ) .
for the ghq subscales were as follows : somatic symptoms ( 0.88 ) , anxiety and insomnia ( 0.93 ) , social dysfunction ( 0.86 ) , severe depression ( 0.89 ) .
these values indicate good internal consistency for the scale , as a whole , and for the individual subscales .
levels of significance : r = 0.11 , 0.13 , 0.17 corresponds for p = 0.05 , 0.01 , 0.001 , respectively .
the results of stepwise regression analysis , which evaluated the relationship between psychological distress ( ghq total score ) and fatigue ( mfi subscales scores ) , indicated that only general fatigue , mental fatigue and reduced motivation remained in the model ( p < 0.05 ) ( as shown in table 5 ) .
therefore , the model was as follows : r square = 0.676 , adjusted r square = 0.451 . in addition , to diagnose the relationship between the ghq and mfi , a linear regression test was performed with r square = 0.626 and adjusted r square = 0.390 , which means that , per each unit score of mfi , 0.651 is added to the ghq score .
therefore , the final model was as follows : ghq = 6.89 + 0.651 ( mfi ) . the results of pearson s correlation test between the ghq and mfi subscales , presented in table 2 , indicated that the most positive correlations were found between the total ghq with general fatigue ( r = 0.61 ) and mental fatigue ( r = 0.59 ) , while a weak positive correlation was found for reduced activity ( r = 0.30 ) .
finally , the pearson s correlation test showed a relatively high positive correlation , between the total ghq and mfi ( r = 0.62 ) ( table 2 ) .
the scores of ghq subscales were significantly higher for workers with moderate to high level of total fatigue ( 3 - 5 on the 5-point scale ) than those with a low level of total fatigue ( 1 - 2 on the 5-point scale ) ( figure 1 ) .
similarly , the scores of the mfi subscales were significantly higher in those workers with the total ghq score 27.38 than in those with a lower score ( total ghq score < 27.38 ) ( figure 2 ) .
the findings of the present study provide an insight into the fatigue and psychological distress and their relationship among petrochemical workers , involved in rotating 12-hour shift work schedules .
the results confirm that the fatigue ( particularly general fatigue ) and poor psychological distress ( especially social dysfunction and anxiety and insomnia ) are frequent among 12-hour shift workers , which might reflect the working condition , nature of work and workload of this working group .
these findings suggest that 12-hour shift schedules should be appropriately managed to ensure that the health and safety so that the employees would not be compromised . as a corollary of this study
, a relatively weak impact of demographic characteristics was found on fatigue and psychological distress ( 33 ) .
one of the main findings of the study was that the total mean ghq score was relatively high , indicating distress among the study population .
it was also shown that the total mean ghq score was above the cut - point , for more than half of the study population , which highlights the high prevalence of poor psychological distress , in this group .
interestingly , the scores of the ghq subscales indicated that social dysfunction , anxiety and insomnia were more problematic than somatic symptoms and depression among 12-hour shift workers . although there is limited research to compare this result with , there is a certain amount of indirect evidence that this may be the case for shift workers in other industrial settings . in a study among machine operator shift workers , it was found that work / non - work conflict was positively related to psychological health ( 34 ) , and not to physical health .
other studies have also shown a positive association between poor sleep and shift work ( 4 , 35 ) .
this can possibly be explained by prolonged working hours , high workload and insufficient off the job social interactions , of this working group .
this is , also , in part consistent with the findings of a previous study , among general working population ( 36 ) .
the proportion of the sample with the total ghq and social dysfunction scores above the cut - point in this study is also much higher than those reported among the iranian general population ( 37 ) .
the results also provide further evidence that fatigue is a frequent complaint among shift workers , which is in line with findings from previous research ( 4 ) .
the scores of the mfi subscales indicated that the general fatigue is more problematic than other dimensions of fatigue , in these workers .
the mean score for subscale measuring general fatigue among the study participants was relatively high .
this was followed by the mean scores of mental fatigue and physical fatigue subscales , respectively .
it is also interesting to note that the mean score for the reduced motivation subscale was very low , among the respondents .
although there is no other similar study reported in the literature , with which to compare the results , these findings are not consistent with previous reports among general population , in other communities ( 28 , 38 , 39 ) .
for example , the mean score of 69.4 for general fatigue subscale , in our study , is much higher than those reported among the american ( 27.6 ) , danish ( 28.0 ) and colombian ( 30.0 ) population , which highlights the working condition of 12-hour industrial shift workers , compared to general population . on the other hand ,
the mean scores of all fatigue subscales in our study population are much lower than those reported for patient groups ( 40 ) .
these findings provide additional evidence that , in addition to the tools and methods used in different studies , several factors , such as sample community , cultural status and racial - ethnic differences can also affect the various aspects of fatigue , and , therefore , this can results in the contradictory findings . as shown in this study
, there was a relatively high positive correlation between fatigue and psychological distress , so that those participants , with poor mental health condition , experienced a higher level of fatigue .
( 17 ) , who found a fairly positive correlation between fatigue and psychological distress among british general population . according to the stepwise regression model ,
the psychological distress was only significantly related to general fatigue , mental fatigue and reduced motivation , while it did not relate to the physical fatigue and reduced activity .
this is of particular interest , since it highlights the importance of mental aspect of fatigue ( rather than physical aspect ) , in this working population .
this can possibly be attributed to the arrangement of shift work schedules , in the study region .
additionally , the poor psychological distress condition , in those in the group with moderate to high level of total fatigue ( figure 1 ) , and a high level of fatigue , in those in the group with the total ghq score 27.38 ( figure 2 ) , indicated a two - way relationship between the fatigue and psychological distress condition of this sample .
these findings may have significant implications for shift workers health and well - being and for the design of shift work systems , in industrial settings .
in this study , the rate of fatigue and psychological distress and their relationship were investigated in a petrochemical plant , with 12-hour shift system .
therefore , it was not the purpose of this study to investigate the causal effect of 12-hour shift on fatigue or psychological distress , because it can be influenced by various factors . in order to control the confounding factors , such as chemical , physical and other ergonomic risk factors ,
another study was designed and implemented in a second petrochemical plant , with 8-hour shift , which have relatively same chemical , physical and ergonomic exposure pattern .
the findings of the current study ( 12-hour ) will be discussed compared with 8-hour shift work study , as another article .
this study has limitations that need to be taken into account , when considering the implications of the findings .
the present study was cross - sectional in design , and , therefore , no causal inferences can be drawn .
the study was conducted in a workplace with special environmental conditions ( e.g. relatively high temperature and humidity ) , and , therefore , further research in other settings is required to have a better understanding and knowledge in this regard . due to limited access to various petrochemical plants , with different shift rotating systems ,
the relationship between shift rotating system and workers health status was not considered in the current study design .
lastly , as the mfi-20 has not been used for evaluation of fatigue , among industrial workers , it was difficult to compare the results with other studies .
however , the findings of this study confirm that mfi-20 is an instrument , on which the researchers can rely on , to assess fatigue in industrial workers .
it is therefore recommended that mfi-20 , which has been mainly used for non - industrial populations so far , can be used in other industrial and occupational groups . taken together , the results of the present study suggest that the prevalence of fatigue and poor psychological distress are relatively high among the workers , with 12-hour shift system . the relatively high mean ghq score , particularly in the form of social dysfunction , anxiety and insomnia , indicates distress among this working group
. the results also highlight that the mental aspect of fatigue is more problematic than the physical aspect , in this working group .
these findings emphasize the need for ergonomic interventions for improving the working conditions of 12-hour industrial shift workers .
the results also indicated that there is a relatively strong positive correlation between fatigue and psychological distress status of the study population , which may have possible implications for industrial workers health and well - being , and for the design of shift - work systems . | background : shift work is a well - recognized occupational health hazard in both industrialized and industrially developing countries . prolonged working time , day / night shift rotation , circadian rhythm and sleep disorders , family and social problems are the most important features of shift working , which have serious complications.objectives:the present study evaluated the fatigue and psychological distress and their relationship among shift workers , in a petrochemical plant ( southern pars gas field ) in southwest iran.materials and methods : in this cross - sectional field study , 400 shift workers from a plant were involved , with participation rate of 72.5% ( 290 persons ) .
the multidimensional fatigue inventory ( mfi-20 ) and general health questionnaire ( ghq-28 ) were used to evaluate the level of fatigue and psychological distress , respectively.results:the results showed that the fatigue and psychological distress ( particularly social dysfunction , anxiety and insomnia ) are frequent among 12-hour shift workers ( the total mfi and total ghq scores were 42.68 17.88 and 34.66 18.56 ) .
a relatively strong positive correlation was found between fatigue and psychological distress ( r = 0.62 ) .
the results of the stepwise regression model indicated that the psychological distress was significantly related only to general fatigue , mental fatigue and reduced motivation , whereas it was not to the physical fatigue and reduced activity.conclusions:the study findings highlight the importance of the mental aspect of fatigue in this working group .
these results have possible implications for workers health and well - being and for the design of shift work systems , for industrial workers . | 1. Background
2. Objectives
3. Materials and Methods
3.1. Study Design and Setting
3.2. Participants
3.3. Data Collection
3.4. Statistical Analysis
4. Results
4.1. Demographic Data
4.2. Fatigue
4.3. Psychological distress
4.4. Relationship Between Fatigue and Psychological Distress
5. Discussion | on the other hand ,
shift work is a well - recognized occupational health hazard in both industrialized and industrially developing countries ( 4 , 5 ) . day / night shift rotation is one of the most important features of shift working , which has serious complications in terms of circadian rhythm and sleep disorders ( 6 - 8 ) . in an attempt to address this issue , the present study was conducted to evaluate the prevalence of fatigue and psychological distress and their relationship in 12-hour rotating shift workers , in a petrochemical plant . this cross - sectional field study was conducted between september and december 2013 , in a governmental petrochemical plant ( southern pars gas field ) , in southwest iran . the mfi-20 is a 20 item self - report assessment tool , which assesses five dimensions of fatigue , including general fatigue , physical fatigue , reduced motivation , mental fatigue and reduced activity . the 28 item general health questionnaire ( ghq-28 ) is generally used as a self - report assessment tool , to evaluate the status of psychological distress ( 30 ) , and is a well - tried and tested technique ( 31 ) . this cross - sectional field study was conducted between september and december 2013 , in a governmental petrochemical plant ( southern pars gas field ) , in southwest iran . the mfi-20 is a 20 item self - report assessment tool , which assesses five dimensions of fatigue , including general fatigue , physical fatigue , reduced motivation , mental fatigue and reduced activity . the 28 item general health questionnaire ( ghq-28 ) is generally used as a self - report assessment tool , to evaluate the status of psychological distress ( 30 ) , and is a well - tried and tested technique ( 31 ) . the results of stepwise regression analysis , which evaluated the relationship between psychological distress ( ghq total score ) and fatigue ( mfi subscales scores ) , indicated that only general fatigue , mental fatigue and reduced motivation remained in the model ( p < 0.05 ) ( as shown in table 5 ) . the results of pearson s correlation test between the ghq and mfi subscales , presented in table 2 , indicated that the most positive correlations were found between the total ghq with general fatigue ( r = 0.61 ) and mental fatigue ( r = 0.59 ) , while a weak positive correlation was found for reduced activity ( r = 0.30 ) . the general fatigue and mental fatigue subscales had a strong positive correlation ( r = 0.67 ) , while there was a weak positive correlation ( r = 0.41 ) , between the general fatigue and reduced activity subscales . the results of stepwise regression analysis , which evaluated the relationship between psychological distress ( ghq total score ) and fatigue ( mfi subscales scores ) , indicated that only general fatigue , mental fatigue and reduced motivation remained in the model ( p < 0.05 ) ( as shown in table 5 ) . the results of pearson s correlation test between the ghq and mfi subscales , presented in table 2 , indicated that the most positive correlations were found between the total ghq with general fatigue ( r = 0.61 ) and mental fatigue ( r = 0.59 ) , while a weak positive correlation was found for reduced activity ( r = 0.30 ) . the findings of the present study provide an insight into the fatigue and psychological distress and their relationship among petrochemical workers , involved in rotating 12-hour shift work schedules . the results confirm that the fatigue ( particularly general fatigue ) and poor psychological distress ( especially social dysfunction and anxiety and insomnia ) are frequent among 12-hour shift workers , which might reflect the working condition , nature of work and workload of this working group . as shown in this study
, there was a relatively high positive correlation between fatigue and psychological distress , so that those participants , with poor mental health condition , experienced a higher level of fatigue . according to the stepwise regression model ,
the psychological distress was only significantly related to general fatigue , mental fatigue and reduced motivation , while it did not relate to the physical fatigue and reduced activity . additionally , the poor psychological distress condition , in those in the group with moderate to high level of total fatigue ( figure 1 ) , and a high level of fatigue , in those in the group with the total ghq score 27.38 ( figure 2 ) , indicated a two - way relationship between the fatigue and psychological distress condition of this sample . these findings may have significant implications for shift workers health and well - being and for the design of shift work systems , in industrial settings . in this study , the rate of fatigue and psychological distress and their relationship were investigated in a petrochemical plant , with 12-hour shift system . taken together , the results of the present study suggest that the prevalence of fatigue and poor psychological distress are relatively high among the workers , with 12-hour shift system . the results also highlight that the mental aspect of fatigue is more problematic than the physical aspect , in this working group . the results also indicated that there is a relatively strong positive correlation between fatigue and psychological distress status of the study population , which may have possible implications for industrial workers health and well - being , and for the design of shift - work systems . | [
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] |
the last decade has witnessed significant improvements in survival outcomes of patients with localized esophageal and gastric cancers , possibly related to the more frequent use of multimodal therapy .
however , the prognosis of metastatic esophago - gastric ( og ) cancer remains poor .
therapeutic strategies typically employed in this situation include systemic chemotherapy , palliative external - beam radiotherapy , high dose - rate brachytherapy ( hdr - bt ) , endoscopic therapies including endoluminal stenting ( els ) , argon plasma coagulation , photodynamic therapy , and occasionally surgery .
2.2013 recommend chemotherapy as a therapeutic option for palliating symptoms , improving quality of life ( qol ) , and prolonging survival in patients with metastatic og cancer incorporating esophageal , gastro - esophageal junction ( goj ) , and gastric tumours . published response rates of palliative chemotherapy ( pct ) in metastatic og cancer range from 40%-60% with median life expectancy ranging from 7 - 12 months [ 2 - 7 ] .
the chemotherapy protocols commonly employed include the use of doublet regimes incorporating platinum agent ( cisplatin or oxaliplatin ) combined with fluoropyrimidine ( 5 fluorouracil [ 5 fu ] or capecitabine ) , or triplet regimes including the use of a third drug , usually in the form of epirubicin , docetaxel , or mitomycin .
the recent trastuzumab for gastric cancer ( toga ) trial demonstrated a survival benefit from addition of trastuzumab to standard doublet regime of platinum / fluoropyrimidine ( fp ) chemotherapy in patients with her-2 positive metastatic goj and gastric adenocarcinomas ( ac ) .
in contrast to pct , palliative radiotherapy ( prt ) is primarily employed for symptom control in patients presenting with local symptoms of dysphagia or bleeding . in our centre ,
patients with metastatic og cancer with good performance status ( ps ) are treated with pct consistent with nccn recommendations and local guidelines .
subsequently , prt may be employed in those patients developing a good response to initial chemotherapy with the aim of optimising local control .
we have observed significantly improved outcomes in patients treated with prt in the presence of well - controlled metastatic disease after initial chemotherapy .
we report on the outcomes of such patients and compare them against a comparable favourable cohort of patients treated with pct alone .
before conducting the current study , a retrospective audit review protocol was designed and approved for evaluation of outcomes of patients with metastatic og cancer treated with pct . between january 2009 and june 2013 , 132 patients were identified with histopathologically confirmed diagnoses of metastatic og cancer who went on to receive pct .
the clinical evaluation of these patients included a complete medical history , physical examination , laboratory investigations , diagnostic endoscopy , and appropriate radiological imaging including computed tomography ( ct ) scan of the chest , abdomen and pelvis .
subsequently , all patients were discussed at the local multidisciplinary team ( mdt ) where the histopathology and radiological imaging were reviewed , and in a few instances further diagnostic investigations were requested ( positron - emission tomography , magnetic resonance imaging , and bone scan ) , as appropriate depending on the discretion of the mdt and the treating physician .
most patients commenced chemotherapy within six weeks of diagnosis and received standard platinum - based combination chemotherapy regimes for metastatic og cancer .
patients underwent regular clinical assessments and staging ct scan at appropriate time - intervals ( 3 - 4 months ) for objective assessment of response to chemotherapy . from the original 132 patients , 35 patients experienced rapid disease progression within the first 3 months characterised by rapid clinical deterioration and poor prognosis with median life - expectancy of less than 6 months .
in addition , there were 97 patients with stable or responding disease at 3 months after initial chemotherapy , of whom 53 patients received prt to primary tumour in the presence of stable and well - controlled metastatic disease ( group a , pct - rt ) .
in contrast , the remaining 44 patients were treated primarily with pct alone ( group b , pct ) ( fig .
most patients underwent virtual simulation for target definition and radiotherapy portals were defined to include the primary tumour ( entire stomach in gastric cancer ) and any adjacent areas of residual disease that could be safely encompassed in the radiotherapy field with 1.5- to 2-cm margin .
the radiotherapy dose was prescribed to midplane at the isocentre and treatment was delivered using parallelopposed fields and 6-mv photons with wedge compensation employed in selected cases for ensuring homogenous dose - distribution within the irradiated volume .
patients with gastric tumours treated with higher radiotherapy dosage ( 30 gy in 10 fractions [ n=10 ] , 45 gy in 25 fractions [ n=1 ] ) underwent ct planning for target definition to reduce dose to adjacent critical organs .
the planning target volume was defined to including the entire stomach and any adjacent areas of residual disease with 1.5-cm isotropic margin .
the target volume was treated using 3-dimensional - conformal radiotherapy with 3- to 4-field beam arrangement and 6-mv photons ( table 1 ) .
the evaluation included a comprehensive assessment of patient demographics , tumour - related characteristics , therapeutic modalities employed including chemotherapy ( regime , duration and number of cycles of 1st and subsequent lines of chemotherapy ) and radiotherapy ( dose - fractionation , technologies , planning and dosimetric characteristics ) .
furthermore , treatment - related outcomes were analysed , including progression - free survival ( pfs ) , overall survival ( os ) , and time to local progression ( ttlp ) .
descriptive statistics such as the meanstandard deviation ( parametric ) , median ( range ) ( nonparametric ) , and frequency ( percentage ) ( categorical ) were employed to summarize the distribution of various patient , tumour , and treatment - related variables .
further statistical analysis was performed using the chi - square test for categorical variables and the mann - whitney u - test for continuous nonparametric variables . for the analysis of os , disease - specific death was the only event noted , with surviving patients censored at the time of last visit .
the date of disease progression or recurrence , as demonstrated by clinical deterioration and radiological imaging , was defined as an event for the analysis of pfs .
the duration of pfs and os was estimated from date of diagnosis , and kaplan - meier technique was used to estimate the pfs probabilities , combined with the univariate and multivariate cox proportional hazards models to measure the association of various clinical parameters with pfs and os .
the log - rank test was used to assess the difference in pfs and os among different groups .
null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 .
before conducting the current study , a retrospective audit review protocol was designed and approved for evaluation of outcomes of patients with metastatic og cancer treated with pct . between january 2009 and june 2013 , 132 patients were identified with histopathologically confirmed diagnoses of metastatic og cancer who went on to receive pct .
the clinical evaluation of these patients included a complete medical history , physical examination , laboratory investigations , diagnostic endoscopy , and appropriate radiological imaging including computed tomography ( ct ) scan of the chest , abdomen and pelvis .
subsequently , all patients were discussed at the local multidisciplinary team ( mdt ) where the histopathology and radiological imaging were reviewed , and in a few instances further diagnostic investigations were requested ( positron - emission tomography , magnetic resonance imaging , and bone scan ) , as appropriate depending on the discretion of the mdt and the treating physician .
most patients commenced chemotherapy within six weeks of diagnosis and received standard platinum - based combination chemotherapy regimes for metastatic og cancer .
patients underwent regular clinical assessments and staging ct scan at appropriate time - intervals ( 3 - 4 months ) for objective assessment of response to chemotherapy . from the original 132 patients , 35 patients experienced rapid disease progression within the first 3 months characterised by rapid clinical deterioration and poor prognosis with median life - expectancy of less than 6 months .
in addition , there were 97 patients with stable or responding disease at 3 months after initial chemotherapy , of whom 53 patients received prt to primary tumour in the presence of stable and well - controlled metastatic disease ( group a , pct - rt ) .
in contrast , the remaining 44 patients were treated primarily with pct alone ( group b , pct ) ( fig .
most patients underwent virtual simulation for target definition and radiotherapy portals were defined to include the primary tumour ( entire stomach in gastric cancer ) and any adjacent areas of residual disease that could be safely encompassed in the radiotherapy field with 1.5- to 2-cm margin .
the radiotherapy dose was prescribed to midplane at the isocentre and treatment was delivered using parallelopposed fields and 6-mv photons with wedge compensation employed in selected cases for ensuring homogenous dose - distribution within the irradiated volume .
patients with gastric tumours treated with higher radiotherapy dosage ( 30 gy in 10 fractions [ n=10 ] , 45 gy in 25 fractions [ n=1 ] ) underwent ct planning for target definition to reduce dose to adjacent critical organs .
the planning target volume was defined to including the entire stomach and any adjacent areas of residual disease with 1.5-cm isotropic margin .
the target volume was treated using 3-dimensional - conformal radiotherapy with 3- to 4-field beam arrangement and 6-mv photons ( table 1 ) .
the evaluation included a comprehensive assessment of patient demographics , tumour - related characteristics , therapeutic modalities employed including chemotherapy ( regime , duration and number of cycles of 1st and subsequent lines of chemotherapy ) and radiotherapy ( dose - fractionation , technologies , planning and dosimetric characteristics ) .
furthermore , treatment - related outcomes were analysed , including progression - free survival ( pfs ) , overall survival ( os ) , and time to local progression ( ttlp ) .
descriptive statistics such as the meanstandard deviation ( parametric ) , median ( range ) ( nonparametric ) , and frequency ( percentage ) ( categorical ) were employed to summarize the distribution of various patient , tumour , and treatment - related variables .
further statistical analysis was performed using the chi - square test for categorical variables and the mann - whitney u - test for continuous nonparametric variables . for the analysis of os , disease - specific death was the only event noted , with surviving patients censored at the time of last visit .
the date of disease progression or recurrence , as demonstrated by clinical deterioration and radiological imaging , was defined as an event for the analysis of pfs .
the duration of pfs and os was estimated from date of diagnosis , and kaplan - meier technique was used to estimate the pfs probabilities , combined with the univariate and multivariate cox proportional hazards models to measure the association of various clinical parameters with pfs and os .
the log - rank test was used to assess the difference in pfs and os among different groups .
null hypotheses of no difference were rejected if p - values were less than 0.05 , or , equivalently , if the 95% confidence intervals ( cis ) of risk point estimates excluded 1 .
the study groups were well - matched , with male predominance and most patients falling within the 60- to 70-year age group .
the commonest tumour site was the oesophagus(n=38 ) followed by stomach ( n=30 ) and the gastrooesophageal junction ( n=29 ) .
most tumours were ac ( n=86 ) with the remaining representing squamous cell carcinomas ( n=10 ) .
there was evidence of positive her-2 expression in two patients with goj ac . the most common site of metastatic disease was nonregional lymphadenopathy followed by liver , peritoneum , lung , bone , and less common sites including adrenal glands , ovaries , and skin , with one instance of brain metastasis .
the proportion of patients presenting with more than one site of metastatic disease ranged between 30%-50% .
there was no significant difference in the frequency and distribution of different sites of metastatic disease between the two groups ( table 2 ) .
most patients ( n=86 ) received triplet combination chemotherapy employing ecx ( epirubicin , cisplatin , capecitabine ) , eox ( epirubicin , oxaliplatin , capecitabine ) regime and nine patients received doublet chemotherapy with a platinum / fp regime .
in addition , two patients with her-2 positive tumours received a combination regime of platinum / fp and trastuzumab .
the mean number of cycles received was similar in both groups and ranged from 5.84.1.62 in group a to 5.451.53 in group b. all patients had responding or stable disease upon completion of chemotherapy except two patients in group a who developed mild disease progression characterised by progressive unilateral supraclavicular lymphadenopathy ( vide infra ) .
the proportion of patients receiving second line chemotherapy ranged between 35%-50% ( table 2 ) .
most patients received prt to primary tumour as consolidation therapy within three months of completion of firstline pct ( n=44 ) with a median time - interval of 0.7 months ( range , 0 to 2.9 months ) .
in addition , nine patients received delayed prt at the time of developing local tumour progression and more than 6 months after completion of first - line pct with median time - interval of 10 months ( range , 6 to 26 months ) .
the palliative radiation dose - schedule of 30 gy in 10 fractions was the most commonly employed radiationtherapy protocol in 38 patients ( oesophagus and goj , n=28 ; gastric , n=10 ) followed by 20 gy in five fractions in 13 patients ( oesophagus and goj , n=9 ; gastric , n=4 ) .
in addition , two patients ( goj , 1 ; gastric , 1 ) received 45 gy in 25 fractions with concurrent chemotherapy .
all patients had stable and well - controlled metastatic disease at time of radiotherapy except two cases with progressive unilateral supraclavicular lymphadenopathy that was treated synchronously ( 20 gy in 5 fractions ) with prt to the primary tumour .
one patient with synchronous brain metastasis also received whole brain prt ( 20 gy in 5 fractions ) .
radiotherapy - induced esophagitis and nausea were common but no significant ( grades 3/4 ) toxicities were observed except in one patient who required admission postradiotherapy with severe esophagitis requiring parenteral analgesia and nutritional support . the median os for the entire group was 16.9 months ( range , 4.8 to 55.4 months ) .
after median follow - up of 16.1 months ( range , 4.8 to 55.4 months ) , 39/53 of patients ( 74% ) had died in group a ( pct - rt ) compared to 37/44 patients ( 84% ) in group b ( pct ) . the median os for patients treated with
pct - rt ( group a ) was 23.3 months ( 95% ci , 17.70 to 28.89 months ) compared to 14 months ( 95% ci , 10.91 to 17.08 months ) after pct ( group b ) ( p < 0.001 ) .
similarly , the median pfs was significantly increased in patients treated with pct - rt at 14 months ( 95% ci , 10.90 to 17.09 months ) compared to 9.5 months ( 95% ci , 7.23 to 11.76 months ) after pct ( p < 0.015 ) ( fig .
more specifically , the postradiotherapy os in group a was 11.8 months ( 95% ci , 7.4 to 16.2 months ) ( fig .
1 ) . we performed a multivariate analysis to assess the effect of different confounding variables on os using cox regression analysis and proportional hazard statistical model .
os was not influenced by the site of tumour and the nature or distribution of metastatic disease ( fig .
furthermore , stratification analysis based on the above covariates demonstrated the presence of therapeutic benefit in favour of pct - rt across all sites of tumour , including patients with gastric ac .
the therapeutic benefit was observed in all subgroups of metastatic disease , although this was nonsignificant in patients with nonregional lymphadenopathy and liver metastasis ( fig .
patients treated with higher biological equivalent dose ( bed ) dose - fractionation schedules demonstrated a trend towards improvement in os .
more specifically , patients treated with higher bed palliative regimes ( 30 gy in 10 fractions or 45 gy in 25 fractions ) improved , although nonsignificantly , in os , 26.2 months ( 95% ci , 21.5 to 30.9 months ) compared to 16.0 months ( 95% ci , 9.58 to 22.41 months ) in patients treated with lower bed regimes ( 20 gy in 5 fractions ) ( p=0.08 ) ( fig . 2c ) . local recurrence ( lr ) was observed in 12/53 patients ( 23% ) in group a ( pct - rt ) compared to 16/44 patients ( 36% ) in group b ( pct ) .
all patients developing recurrence after pct - rt underwent insertion of an oesophageal els as primary treatment for palliation of dysphagia . in comparison , 9/16 patients developing lr after pct underwent insertion of els and 8/16 patients received prt with one patient receiving combined stent and prt .
the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( range , 5.23 to 44.50 months ) compared to 8.3 months(range , 4.10 to 25.23 months ) in patients treated with pct ( p=0.006 ) .
the study groups were well - matched , with male predominance and most patients falling within the 60- to 70-year age group .
the commonest tumour site was the oesophagus(n=38 ) followed by stomach ( n=30 ) and the gastrooesophageal junction ( n=29 ) .
most tumours were ac ( n=86 ) with the remaining representing squamous cell carcinomas ( n=10 ) .
there was evidence of positive her-2 expression in two patients with goj ac . the most common site of metastatic disease was nonregional lymphadenopathy followed by liver , peritoneum , lung , bone , and less common sites including adrenal glands , ovaries , and skin , with one instance of brain metastasis .
the proportion of patients presenting with more than one site of metastatic disease ranged between 30%-50% .
there was no significant difference in the frequency and distribution of different sites of metastatic disease between the two groups ( table 2 ) .
most patients ( n=86 ) received triplet combination chemotherapy employing ecx ( epirubicin , cisplatin , capecitabine ) , eox ( epirubicin , oxaliplatin , capecitabine ) regime and nine patients received doublet chemotherapy with a platinum / fp regime .
in addition , two patients with her-2 positive tumours received a combination regime of platinum / fp and trastuzumab .
the mean number of cycles received was similar in both groups and ranged from 5.84.1.62 in group a to 5.451.53 in group b. all patients had responding or stable disease upon completion of chemotherapy except two patients in group a who developed mild disease progression characterised by progressive unilateral supraclavicular lymphadenopathy ( vide infra ) .
the proportion of patients receiving second line chemotherapy ranged between 35%-50% ( table 2 ) .
most patients received prt to primary tumour as consolidation therapy within three months of completion of firstline pct ( n=44 ) with a median time - interval of 0.7 months ( range , 0 to 2.9 months ) .
in addition , nine patients received delayed prt at the time of developing local tumour progression and more than 6 months after completion of first - line pct with median time - interval of 10 months ( range , 6 to 26 months ) .
the palliative radiation dose - schedule of 30 gy in 10 fractions was the most commonly employed radiationtherapy protocol in 38 patients ( oesophagus and goj , n=28 ; gastric , n=10 ) followed by 20 gy in five fractions in 13 patients ( oesophagus and goj , n=9 ; gastric , n=4 ) .
in addition , two patients ( goj , 1 ; gastric , 1 ) received 45 gy in 25 fractions with concurrent chemotherapy .
all patients had stable and well - controlled metastatic disease at time of radiotherapy except two cases with progressive unilateral supraclavicular lymphadenopathy that was treated synchronously ( 20 gy in 5 fractions ) with prt to the primary tumour .
one patient with synchronous brain metastasis also received whole brain prt ( 20 gy in 5 fractions ) .
radiotherapy - induced esophagitis and nausea were common but no significant ( grades 3/4 ) toxicities were observed except in one patient who required admission postradiotherapy with severe esophagitis requiring parenteral analgesia and nutritional support .
the median os for the entire group was 16.9 months ( range , 4.8 to 55.4 months ) .
after median follow - up of 16.1 months ( range , 4.8 to 55.4 months ) , 39/53 of patients ( 74% ) had died in group a ( pct - rt ) compared to 37/44 patients ( 84% ) in group b ( pct ) . the median os for patients treated with
pct - rt ( group a ) was 23.3 months ( 95% ci , 17.70 to 28.89 months ) compared to 14 months ( 95% ci , 10.91 to 17.08 months ) after pct ( group b ) ( p < 0.001 ) .
similarly , the median pfs was significantly increased in patients treated with pct - rt at 14 months ( 95% ci , 10.90 to 17.09 months ) compared to 9.5 months ( 95% ci , 7.23 to 11.76 months ) after pct ( p < 0.015 ) ( fig .
1 ) . more specifically , the postradiotherapy os in group a was 11.8 months ( 95% ci , 7.4 to 16.2 months ) ( fig .
1 ) . we performed a multivariate analysis to assess the effect of different confounding variables on os using cox regression analysis and proportional hazard statistical model .
os was not influenced by the site of tumour and the nature or distribution of metastatic disease ( fig .
furthermore , stratification analysis based on the above covariates demonstrated the presence of therapeutic benefit in favour of pct - rt across all sites of tumour , including patients with gastric ac .
the therapeutic benefit was observed in all subgroups of metastatic disease , although this was nonsignificant in patients with nonregional lymphadenopathy and liver metastasis ( fig .
patients treated with higher biological equivalent dose ( bed ) dose - fractionation schedules demonstrated a trend towards improvement in os .
more specifically , patients treated with higher bed palliative regimes ( 30 gy in 10 fractions or 45 gy in 25 fractions ) improved , although nonsignificantly , in os , 26.2 months ( 95% ci , 21.5 to 30.9 months ) compared to 16.0 months ( 95% ci , 9.58 to 22.41 months ) in patients treated with lower bed regimes ( 20 gy in 5 fractions ) ( p=0.08 ) ( fig . 2c ) .
local recurrence ( lr ) was observed in 12/53 patients ( 23% ) in group a ( pct - rt ) compared to 16/44 patients ( 36% ) in group b ( pct ) .
all patients developing recurrence after pct - rt underwent insertion of an oesophageal els as primary treatment for palliation of dysphagia . in comparison , 9/16 patients developing lr after pct underwent insertion of els and 8/16 patients received prt with one patient receiving combined stent and prt .
the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( range , 5.23 to 44.50 months ) compared to 8.3 months(range , 4.10 to 25.23 months ) in patients treated with pct ( p=0.006 ) .
the median os for the entire group was 16.9 months ( range , 4.8 to 55.4 months ) .
after median follow - up of 16.1 months ( range , 4.8 to 55.4 months ) , 39/53 of patients ( 74% ) had died in group a ( pct - rt ) compared to 37/44 patients ( 84% ) in group b ( pct ) . the median os for patients treated with
pct - rt ( group a ) was 23.3 months ( 95% ci , 17.70 to 28.89 months ) compared to 14 months ( 95% ci , 10.91 to 17.08 months ) after pct ( group b ) ( p < 0.001 ) .
similarly , the median pfs was significantly increased in patients treated with pct - rt at 14 months ( 95% ci , 10.90 to 17.09 months ) compared to 9.5 months ( 95% ci , 7.23 to 11.76 months ) after pct ( p < 0.015 ) ( fig .
1 ) . more specifically , the postradiotherapy os in group a was 11.8 months ( 95% ci , 7.4 to 16.2 months ) ( fig .
1 ) . we performed a multivariate analysis to assess the effect of different confounding variables on os using cox regression analysis and proportional hazard statistical model .
os was not influenced by the site of tumour and the nature or distribution of metastatic disease ( fig .
furthermore , stratification analysis based on the above covariates demonstrated the presence of therapeutic benefit in favour of pct - rt across all sites of tumour , including patients with gastric ac .
the therapeutic benefit was observed in all subgroups of metastatic disease , although this was nonsignificant in patients with nonregional lymphadenopathy and liver metastasis ( fig .
patients treated with higher biological equivalent dose ( bed ) dose - fractionation schedules demonstrated a trend towards improvement in os .
more specifically , patients treated with higher bed palliative regimes ( 30 gy in 10 fractions or 45 gy in 25 fractions ) improved , although nonsignificantly , in os , 26.2 months ( 95% ci , 21.5 to 30.9 months ) compared to 16.0 months ( 95% ci , 9.58 to 22.41 months ) in patients treated with lower bed regimes ( 20 gy in 5 fractions ) ( p=0.08 ) ( fig . 2c ) .
local recurrence ( lr ) was observed in 12/53 patients ( 23% ) in group a ( pct - rt ) compared to 16/44 patients ( 36% ) in group b ( pct ) .
all patients developing recurrence after pct - rt underwent insertion of an oesophageal els as primary treatment for palliation of dysphagia . in comparison , 9/16 patients developing lr after pct underwent insertion of els and 8/16 patients received prt with one patient receiving combined stent and prt .
the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( range , 5.23 to 44.50 months ) compared to 8.3 months(range , 4.10 to 25.23 months ) in patients treated with pct ( p=0.006 ) .
in this retrospective study , we presented the therapeutic outcomes in metastatic og cancer after a sequential multimodal approach in which patients responding well to initial chemotherapy were treated with prt to primary tumour ( figs . 3 and 4 ) .
the median os for the entire cohort of patients was 16.9 months , higher than previously reported historical controls .
however , patients receiving pct - rt improved significantly in pfs and os compared to a favourable cohort of patients treated with chemotherapy alone .
more specifically , patients treated with pct - rt had an overwhelming survival benefit of approximately nine months , a new finding .
the use of pct - rt was associated with favourable response in all subgroups of metastatic disease , but the benefit was reduced and nonsignificant in patients with nonregional lymphadenopathy and liver metastasis . however , the observed survival benefit was robust and significant in patients with other visceral metastasis including peritoneum , bone and lung .
there were no objective assessments of qol performed in the study , but patients treated with pct - rt had reduced probability of local progression and significantly increased median ttlp compared to patients treated with chemotherapy alone .
in addition , most patients maintained their nutritional status and general fitness / ps after pct - rt which may reflect the higher proportion of patients receiving subsequent second - line therapies on relapse .
most recent studies investigating prt in advanced og cancer reported its role for palliation of dysphagia in patients with or without els [ 8 - 13 ] .
all of these studies consistently report successful and durable palliation of dysphagia after prt , with a suggestion of superior outcomes in patients having combined els insertion and prt ( table 3 ) . however , the effects of prt on survival are poorly defined , with a few studies reporting survival outcomes that are potentially contradictory and conflicting . homs et al
. reported on a randomized phase iii study comparing the outcomes of hdr - bt and els in which 209 patients were randomised to els placement ( n=108 ) or single - dose ( 12 gy ) hdr - bt ( n=101 ) .
dysphagia improved more rapidly after els placement but long - term relief of dysphagia was better after hdr - bt combined with improved qol measures .
however , the use of hdr - bt was not associated with an improvement in os . in contrast , some recent studies have reported on probable survival benefit in favour of prt after els insertion compared to els alone [ 8 - 10 ] .
these studies are limited by their predominantly retrospective nature and small sample size combined with heterogeneities across the study populations and therapeutic protocols .
sreedharan et al . reported a large systematic review for the cochrane database incorporating 2,542 patients from 40 studies and recommended hdr - bt as an appropriate alternative to els for palliation of dysphagia that may provide additional survival benefit with a better qol .
however , the role of prt in metastatic gastric cancer is limited primarily to control of local bleeding complications there is limited evidence on the use of multi - modal therapy in metastatic og cancer .
most initial studies evaluated the role of palliative conformal radiotherapy ( crt ) primarily in patients with advanced oesophageal cancer , and demonstrated evidence of good local response ranging from 60%-90% but overall prognosis remained poor at approximately 6 months ( table 4 ) [ 14 - 20 ] .
more recently , ikeda et al . reported on a retrospective study of 40 patients with metastatic scc of the oesophagus treated with median four cycles ( range , 1 to 7 cycles ) of chemotherapy using 5 fu and cisplatin followed by concurrent crt with 40 gy in 20 fractions and two cycles of concurrent 5 fu and cisplatin .
the study reported excellent local control of the primary lesion in 95% of patients including 12 patients ( 30% ) who achieved a complete response .
in contrast to the above studies , the multimodal approach adopted in our study supports the use of a sequential strategy in which patients developing good response to initial pct received prt to the primary tumour , but in the context of well - controlled metastatic disease . the limitations of a retrospective analysis are well - recognised including the existence of possible disease and treatment - related heterogeneities and selection bias .
the observed heterogeneities in our study were not statistically significant and were not found to have any association with survival .
however , this does not exclude the possibility of a selection bias particularly as there were no predefined clinical criteria for recommending prt , which was essentially at the clinician s discretion .
approximately 90% of patients ( 86/97 ) in our series had ac , which are usually less sensitive to radiation than squamous cell cancers .
indeed , radical crt is the widely accepted definitive therapeutic modality for localised squamous cell cancer of oesophagus .
however , the effectiveness of combined - modality treatment in our cohort comprised primarily of ac reinforces the clinical importance of this therapeutic strategy and its possible application in the future .
furthermore , the benefit of palliative crt was also observed in gastric ac that has not been reported previously .
the observed benefit after pct - rt was more evident in patients receiving higher bed radiation dose - schedules indicative of possible dose - dependent effect .
this remains untenable in the absence of rigid prescribing criteria and possibility of selection bias in determining appropriate doseradiation schedules .
however , it does lend support for further investigation of alternative high - bed hypofractionated schedules ( e.g. , 40 gy in 15 fractions ; 36 gy in 12 fractions ) in future studies . the possible intriguing cellular mechanisms by which prt to primary tumour may influence metastatic disease remain unknown
. however , the immunological effects of radiation are well - recognised including the existence of abscopal bystander effects that may influence tumour growth outside of the irradiated region .
the development of bystander effect is proposed to be related to the activation of radiation - induced immunological cell - death mechanisms , as evident by an increase in levels of serum tumour necrosis factor after radiotherapy that can enhance the cytotoxic activity of natural killer cells .
the results from our study are encouraging and distinct , and provide indication of possible radiation - induced cellular mechanisms with systemic reverberations that may influence survival in patients with metastatic og cancer .
however , the true benefit of using this approach can be validated only in a randomized clinical trial that should also include investigation of alternative high - bed hypofractionated dose - schedules and objective assessment of other important parameters including qol .
in our study , the median os of patients who received prt after initial chemotherapy was 23.3 months , one of the highest reported survival outcomes in metastatic og cancer . improved outcomes were observed when prt was employed after optimum systemic dosage of chemotherapy and in the context of well - controlled metastatic disease .
furthermore , our study included patients with gastric cancer , and the use of multimodal therapy in metastatic gastric cancer has not been reported .
the interesting and provocative observations in our study present strong arguments for further investigation of this strategy in randomized clinical trials . | purposewe report the outcomes of patients treated with palliative radiotherapy ( prt ) to the primary tumour in the context of well - controlled metastatic disease after initial chemotherapy.materials and methodsclinical records of 132 patients with metastatic esophago - gastric ( og ) cancer treated with palliative chemotherapy ( pct ) between january 2009 and june 2013 were reviewed .
ninetyseven patients had responding or stable disease after 3 months of chemotherapy , of whom 53 patients received prt to the primary tumour after initial chemotherapy in the presence of well - controlled metastatic disease ( group a , pct - rt ) .
the remaining 44 patients were treated with pct alone ( group b , pct ) .
treatment - related outcomes were assessed in above groups including time to local progression ( ttlp ) , progression - free and overall survival.resultsthe median overall survival for patients treated with prt after initial chemotherapy ( group a ) was 23.3 months ( 95% confidence interval [ ci ] , 17.70 to 28.89 months ) and significantly higher than the 14 months ( 95% ci , 10.91 to 17.08 months ) in patients treated with pct alone ( group b ) ( p < 0.001 ) .
the use of pct - rt was an independent predictor of os in multivariate analysis .
local recurrence was observed in 12/53 of patients ( 23% ) in group a compared to 16/44 ( 36% ) in group b. the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( 5.23 months to 44.50 months ) compared to 8.3 months ( range , 4.10 to 25.23 months ) in patients treated with pct alone ( p=0.006).conclusionthe possibility of prt influencing systemic disease in advanced og cancer has not been reported , and results from the present study present strong arguments for investigation of this therapeutic strategy in a randomized trial . | Introduction
Materials and Methods
1. Study population
2. Palliative radiotherapy
Results
1. Patient and tumour characteristics
2. Palliative radiotherapy (group A: pCT-RT)
3. Treatment-related outcomes
1) OS and PFS
2) Local recurrence and TTLP
Discussion
Conclusion | in addition , there were 97 patients with stable or responding disease at 3 months after initial chemotherapy , of whom 53 patients received prt to primary tumour in the presence of stable and well - controlled metastatic disease ( group a , pct - rt ) . in addition , there were 97 patients with stable or responding disease at 3 months after initial chemotherapy , of whom 53 patients received prt to primary tumour in the presence of stable and well - controlled metastatic disease ( group a , pct - rt ) . after median follow - up of 16.1 months ( range , 4.8 to 55.4 months ) , 39/53 of patients ( 74% ) had died in group a ( pct - rt ) compared to 37/44 patients ( 84% ) in group b ( pct ) . the median os for patients treated with
pct - rt ( group a ) was 23.3 months ( 95% ci , 17.70 to 28.89 months ) compared to 14 months ( 95% ci , 10.91 to 17.08 months ) after pct ( group b ) ( p < 0.001 ) . similarly , the median pfs was significantly increased in patients treated with pct - rt at 14 months ( 95% ci , 10.90 to 17.09 months ) compared to 9.5 months ( 95% ci , 7.23 to 11.76 months ) after pct ( p < 0.015 ) ( fig . local recurrence ( lr ) was observed in 12/53 patients ( 23% ) in group a ( pct - rt ) compared to 16/44 patients ( 36% ) in group b ( pct ) . the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( range , 5.23 to 44.50 months ) compared to 8.3 months(range , 4.10 to 25.23 months ) in patients treated with pct ( p=0.006 ) . after median follow - up of 16.1 months ( range , 4.8 to 55.4 months ) , 39/53 of patients ( 74% ) had died in group a ( pct - rt ) compared to 37/44 patients ( 84% ) in group b ( pct ) . the median os for patients treated with
pct - rt ( group a ) was 23.3 months ( 95% ci , 17.70 to 28.89 months ) compared to 14 months ( 95% ci , 10.91 to 17.08 months ) after pct ( group b ) ( p < 0.001 ) . similarly , the median pfs was significantly increased in patients treated with pct - rt at 14 months ( 95% ci , 10.90 to 17.09 months ) compared to 9.5 months ( 95% ci , 7.23 to 11.76 months ) after pct ( p < 0.015 ) ( fig . local recurrence ( lr ) was observed in 12/53 patients ( 23% ) in group a ( pct - rt ) compared to 16/44 patients ( 36% ) in group b ( pct ) . the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( range , 5.23 to 44.50 months ) compared to 8.3 months(range , 4.10 to 25.23 months ) in patients treated with pct ( p=0.006 ) . after median follow - up of 16.1 months ( range , 4.8 to 55.4 months ) , 39/53 of patients ( 74% ) had died in group a ( pct - rt ) compared to 37/44 patients ( 84% ) in group b ( pct ) . the median os for patients treated with
pct - rt ( group a ) was 23.3 months ( 95% ci , 17.70 to 28.89 months ) compared to 14 months ( 95% ci , 10.91 to 17.08 months ) after pct ( group b ) ( p < 0.001 ) . similarly , the median pfs was significantly increased in patients treated with pct - rt at 14 months ( 95% ci , 10.90 to 17.09 months ) compared to 9.5 months ( 95% ci , 7.23 to 11.76 months ) after pct ( p < 0.015 ) ( fig . local recurrence ( lr ) was observed in 12/53 patients ( 23% ) in group a ( pct - rt ) compared to 16/44 patients ( 36% ) in group b ( pct ) . the median ttlp was significantly higher in patients after pct - rt at 17.3 months ( range , 5.23 to 44.50 months ) compared to 8.3 months(range , 4.10 to 25.23 months ) in patients treated with pct ( p=0.006 ) . | [
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] |
most of the critically ill patients in the intensive care units require the use of mechanical ventilation . these patients need endotracheal suctioning ( ets ) to prevent partial or complete endotracheal tube obstruction .
ets is one of the most common and important procedures performed for the patients requiring mechanical ventilation .
these patients are unable to spontaneously clear their airways , and ets clears the endotracheal tree from secretions , assures adequate oxygen supply , avoids obstruction of the tube lumen , decrease patients breathing work , and prevents atelectasis and pulmonary infection . although ets may be a necessary procedure , it can be associated with some potentially harmful effects including hypoxemia due to interruption of the mechanical ventilation and subsequently loss of lung volume , vasovagal response , arrhythmia , and hypotension , bleeding , and cross infection .
open system suctioning ( oss ) is the conventional suctioning technique , which requires disconnecting the patient from the ventilator during ets .
it involves suctioning the airway using a single - use catheter , and then reconnection of the patient to the ventilator and discarding the suctioning catheter .
in contrast , closed suctioning system ( css ) can remain in line for 24 h and , thus , can be used for multiple ets procedures .
this multiple - use catheter is enclosed in a plastic sheath ; therefore , the nurse is not exposed to patient 's airway secretions .
css has become very popular in the intensive care units as ventilation continues during the suctioning procedure and consequently decreases the loss of lung volume and avoids gas exchange impairment while ets is performed . maintaining a positive end - expiratory pressure ( peep ) , decreasing contamination of the environment , convenience of the system , saving time in equipment set - up and clean - up , and reducing the anxiety in patients are among the advantages of this system .
critical care nurses , as one of the important members of a multidisciplinary team , play the main role in care of critically ill patients .
in fact , these patients are highly dependent on skilled nurses throughout all aspects of their care , especially airway management and clearance .
clearing the mechanically ventilated patients airways by ets is an inevitable procedure , which is one of the concerns in nursing practice , as it is potentially a harmful intervention that can cause serious complications , when performed inappropriately or incorrectly .
have mentioned that nursing practice in ets of critically ill patients varies widely between institutions and practitioners . in their study , paul - allen and ostrow aimed to identify the closed - system ets practices of nurses and reported some variations in the suctioning procedure .
other studies have also demonstrated that nurses practice with ets is based on their personal experience and the ward routine over any other sources .
ets can cause life - threatening effects , and should be implemented based on standard protocols and be evidence - based as evidence - based care promotes quality outcome by decreasing the mortality , morbidity , costs , length of hospitalization , and improvement of the patients recovery . as css has recently been adopted in the hospitals in iran and is still new in most of the settings , an observational study was necessary to investigate nurses closed suctioning practice to assess an actual nursing practice .
the aim of this study was to investigate closed - system ets practices of critical care nurses and to compare their practice with standard recommendations .
a non - participant structured observational design was used in the present research to collect data concerning nurses practice prior to , during , and after endotracheal closed suctioning .
observational studies are comprehensive studies conducted to investigate practice as in this method , data are collected based on participants observations in a natural environment and real situation . after obtaining approval for the research in isfahan university of medical sciences and getting permission from the hospital nurse manager and the head nurse in the ward , the researcher has accessed the study setting , and amin hospital was selected . after sampling , the targeted population was informed about the study details and their right to withdraw from the study any time they liked .
the study was conducted for two consecutive months in two general adult icus on the patients requiring mechanical ventilation or high dependency care .
there were two wards with 40 nurses ( n = 20 in each ward ) .
the wards had adopted css for all intubated patients since 1 year prior to the study .
sampling was conducted in several shifts and two suctioning events were observed per nurse in one shift .
all 40 full - time icu nurses with a minimum of 1 year icu experience were considered eligible for the study .
one year is an appropriate time for all nurses to have the same practice and knowledge base .
data on the practice of nurses using the css were collected using a 23-item structured observational checklist .
this checklist was made after studying the standard suctioning guidelines in several references , and was distributed for content validity appraisal to a range of experts including faculty members of the medical - surgical and critical care nursing departments in isfahan nursing and midwifery school , as well as two senior nursing intensive care practitioners . in the pilot study ,
the observational checklist was checked for inter - rater reliability using split - half method , and the spearman correlation coefficient was 0.959 .
each item in the 23-item checklist was weighted with 0 and 1 , or 0 and 2 depending on the strength of its effects on ets , based on evidences .
nurses practice in using css was observed , and the checklist was ticked in three time points : prior to suctioning , during the suctioning event , and post - suctioning practice . recommended best practice was developed by calculating the highest score in each item , which was 44 representing perfect adherence to the best practice recommendations .
the checklist was also designed to elicit nurses working experience in icu , nurses level of education , and passing the educational course for css .
practice of all 40 nurses was observed and the checklists were ticked . at the end of sampling ,
the data were analyzed by descriptive statistics including frequency rating and percentage for nominal - level data .
spearman correlation coefficient was used for finding the correlation between baseline variables and suctioning practice scores .
ethical approval to conduct the study was obtained from the appropriate research committee , and all participants were informed that their participation was voluntary and that their right to withdraw the study would be respected at the all time .
ethical approval to conduct the study was obtained from the appropriate research committee , and all participants were informed that their participation was voluntary and that their right to withdraw the study would be respected at the all time .
during 1 month of the study , 80 checklists were ticked for 40 nurses working in two icus .
baseline variables such as nurses length of working in icu , passing their training course in css , and educational are presented in table 1 .
about 97.5% of nurses had a bachelor 's and 2.5% had a master 's degree in nursing .
thirteen nurses ( 32.5% ) had passed the css educational course . in accordance with the main data of the observational checklist
, the results were categorized into three sections : practices prior to suctioning , during the suctioning event , and post - suctioning practices .
baseline variables frequencies in study subjects nurses practice prior to suctioning is shown in table 2 . to assess the need for ets , 4 nurses ( 10% ) auscultated the patients chest , 11 nurses ( 27.5% ) prepared the patients and explained the necessities of the procedure in several forms , and more than half of the nurses ( 27 , 67.5% ) positioned the patients in a semi - fowler position if it was possible .
about 28 participants ( 70% ) performed hyperoxygenation for the patients prior to ets and 29 icu nurses ( 72.5% ) performed nacl instillation prior to suctioning to dilute the secretions . in relation to hand washing prior to the procedure , disparities in practice
were noted and only 7 nurses ( 17.5% ) washed their hands , 36 ( 90% ) did not wear goggles , but all the participants were fully compliant with the best practice in relation to wearing gloves .
practices prior to suctioning most of the participants ( 87.5% ) used a catheter that was larger than half of the internal diameter of endotracheal tube ( ett ) , which was against the recommended size for suctioning [ table 3 ] . also , 20% of the nurses suctioned patients more than the maximum number in each suctioning episode ( two times ) , but most of them ( 90% ) complied with the best practice recommendation in relation to suctioning time ( less than 10 s ) .
about 95% of the participants inserted the appropriate length of catheter , and applied negative pressure for less than 10 s based on continuous method , which is found in most of the guidelines .
one minute is the appropriate length of time between each suctioning episode , with which 35% of the participants were not compliant .
suctioning events practices two nurses ( 5% ) did not notice the black indicator when removing the suctioning catheter from the ett .
existence of this indicator at the end of catheter represents the complete withdrawal of suctioning catheter from the ets .
also , 13 nurses ( 32.5% ) failed to hyperoxygenate the patients at the end of the procedure .
similar to the first step , most of the participants ( 95% ) did not evaluate the effectiveness of suctioning by the chest auscultation .
six participants ( 15% ) left the suctioning port open , in contrast to css recommendations [ table 4 ] . in relation with infection control principles ,
all the participants complied with the best practice recommendations in relation with closed - suctioning catheter irrigation and procedure documentation .
frequency distribution was used to evaluate the suctioning practices scores and their quality with the best practice score [ table 5 ] . suctioning procedure scores ranged totally from 20 to 36 . in suctioning three sections ,
the highest mean score , in suctioning event practices , was 11.75 ( 1.51 ) and the lowest mean score , in pre - suctioning practices , was 7.5 ( 2.66 ) . results of the spearman correlation coefficient showed a statistically significant positive correlation between passing the suctioning education period and suctioning practices scores ( p = 0.00 ) and between the length of working in icu and suctioning practices scores ( p = 0.02 ) .
nurses practice prior to suctioning is shown in table 2 . to assess the need for ets , 4 nurses ( 10% ) auscultated the patients chest , 11 nurses ( 27.5% ) prepared the patients and explained the necessities of the procedure in several forms , and more than half of the nurses ( 27 , 67.5% ) positioned the patients in a semi - fowler position if it was possible .
about 28 participants ( 70% ) performed hyperoxygenation for the patients prior to ets and 29 icu nurses ( 72.5% ) performed nacl instillation prior to suctioning to dilute the secretions . in relation to hand washing prior to the procedure , disparities in practice
were noted and only 7 nurses ( 17.5% ) washed their hands , 36 ( 90% ) did not wear goggles , but all the participants were fully compliant with the best practice in relation to wearing gloves .
most of the participants ( 87.5% ) used a catheter that was larger than half of the internal diameter of endotracheal tube ( ett ) , which was against the recommended size for suctioning [ table 3 ] . also , 20% of the nurses suctioned patients more than the maximum number in each suctioning episode ( two times ) , but most of them ( 90% ) complied with the best practice recommendation in relation to suctioning time ( less than 10 s ) .
about 95% of the participants inserted the appropriate length of catheter , and applied negative pressure for less than 10 s based on continuous method , which is found in most of the guidelines .
one minute is the appropriate length of time between each suctioning episode , with which 35% of the participants were not compliant .
two nurses ( 5% ) did not notice the black indicator when removing the suctioning catheter from the ett .
existence of this indicator at the end of catheter represents the complete withdrawal of suctioning catheter from the ets .
also , 13 nurses ( 32.5% ) failed to hyperoxygenate the patients at the end of the procedure .
similar to the first step , most of the participants ( 95% ) did not evaluate the effectiveness of suctioning by the chest auscultation .
six participants ( 15% ) left the suctioning port open , in contrast to css recommendations [ table 4 ] . in relation with infection control principles ,
all the participants complied with the best practice recommendations in relation with closed - suctioning catheter irrigation and procedure documentation .
frequency distribution was used to evaluate the suctioning practices scores and their quality with the best practice score [ table 5 ] . suctioning procedure scores ranged totally from 20 to 36 . in suctioning three sections , the highest mean score , in suctioning event practices , was 11.75 ( 1.51 ) and the lowest mean score , in pre - suctioning practices , was 7.5 ( 2.66 )
. results of the spearman correlation coefficient showed a statistically significant positive correlation between passing the suctioning education period and suctioning practices scores ( p = 0.00 ) and between the length of working in icu and suctioning practices scores ( p = 0.02 ) .
this study compared the current ets procedure , conducted by the closed system , and the best practice recommendations . according to the best practice ets recommendations ,
as ets has several disadvantages , assessment of the patients concerning the presence of congestion by auscultation of their thorax is necessary to perform the procedure as needed .
the findings showed that 36 participants ( 90% ) did not follow standard recommendations , which is in line with the studies of kelleher and andrews , day et al . , and jansson et al .
in the studies by these authors , they found that only six , two , and two nurses performed ets assessment , respectively . in most cases ( 90% ) , the researcher observed that majority of nurses performed the procedure at the beginning of shift and at the time of delivery of the patients from the previous nurse .
it raised the issue that they routinely perform the intervention instead of performing it only when needed . on the other hand ,
several criteria describe the potential indications for ets , such as a raised respiratory rate , reduced spo2 levels , skin color , pattern of respiration , coughing , increased work of breathing , and other changes in clinical signs as well as patients behavior .
observational studies of clinical practices have suggested that the identification of the need for ets is a complex issue .
thus , it is hard to judge whether pre - suctioning assessment practice conducted by nurses was on a routine basis or based on other clinical signs and patients behavior .
about 29 nurses ( 72.5% ) did not communicate with the patients in any form and did not explain how the procedure would be performed .
a 72.5% failure in this practice indicates that the weakness in compliance with the standards is high because care providers should normally explain to the patient what they are going to do and why it is necessary , as it is the patients right to know , and on the other hand , informed patients are more cooperative .
similarly , 8 nurses ( 17% ) of general icu in the study of kelleher and andrews and 16 nurses ( 40% ) in the study of jansson et al . did not adhere to the best practice in patient preparation .
about 13 nurses ( 32.5% ) failed to assist patients into a semi - fowler position prior to suctioning when it was possible , which resulted in an uncomforted procedure for both the nurse and the patient , and not a lung expansion .
about 12 ( 30% ) and 13 ( 32.5% ) subjects failed to provide hyperoxygenation prior to and after suctioning , respectively .
demir and dramal 's study did not show desaturation when closed suction was used , even in the absence of hyperoxygenation .
many references firmly recommend hyperoxygenation prior to and after ets . in the studies of day
kelleher and andrew , and jansson et al.,2 ( 7% ) , 17 ( 17% ) , and 17 ( 42% ) participants were found not to perform hyperoxygenation in practice , respectively , which is in line with our obtained results .
although routine nacl instillation is not recommended for ets in literature , subjects still failed to adhere to best practice in this issue .
many studies documented that nacl instillation enhances secretion removal but may also create the sensation of dyspnea and even cause greater bacterial release in the respiratory system . all participants ( 100% ) in kelleher and andrew 's study refused saline instillation into the endotracheal tube , but in the study of jansson et al . ,
consistent with the present study , 25% of the nurses used saline instillation . in infection control aspects of ets , majority of the participants ( 33 , 82.5% ) did not wash their hands and also 36 ( 90% ) nurses did not wear goggles , although all of them were observed to wear gloves during ets .
ventilator - associated pneumonia is the first common nosocomial infection in patients receiving mechanical ventilation , which increases mortality and morbidity . because ets interferes with normal body defense mechanism , maintaining aseptic techniques including hand washing and gloves wear
according to standard precautions , because of secretion splashing risk , even during ccs , the eyes should be protected by goggles . meanwhile , 25 participants ( 62% ) in kelleher and andrew 's ( 2008 ) study and 14 participants in jansson et al .
also , all the subjects in both studies did not adhere to the best practice in eye protection , but wore gloves .
these findings empower the hypothesis that nurses suppose that hand washing can be replaced by wearing gloves and a 90% discrepancy in eye protection may suggest that nurses underestimate secretion splashing during css . in the care of a patient , consideration of universal standard precautions , hand washing before and after patient contact , and eye protection precautions are essential , in addition to the wearing gloves and regardless of the suctioning method ( open or closed ) . regarding the selection of an appropriate catheter size , 87.5% of the participants in the present study , like 40% participants in kelleher and 's study , chose an inappropriate catheter size . in this regard , the judgment can be uncertain as there were only 16 catheter sizes in the setting that might have affected the power of choice .
guidelines recommend that the suction catheter should occlude less than half of the internal lumen of ett , and a deficit in different catheter sizes affects adherence to recommendations . in the present study ,
32 and 36 participants adhered to the recommended practice with regard to the number of suctioning events ( <3 times ) and active suctioning time ( < 10 s ) , respectively , compared to the nurses in jansson et al .
's study ( 29 and 30 , respectively ) showing their moderate to high superiority in the practice .
about 34 subjects ( 84% ) used an appropriate negative pressure ( 80150 mmhg ) ; 38 subjects ( 95% ) inserted the catheter to an appropriate depth and applied continuous pressure while withdrawing the catheter , and at the end , guaranteed full exit of the catheter by observing the black indicator and locked the catheter . based on clinical experience and the results of studies , there are recommendations for using minimally invasive catheter insertion , continuous rather than intermittent suctioning during withdrawal of the catheter , and maintaining a patent ett by taking out the full length of catheter at the end of procedure using css . practices in relation with
as shown in the results , most discrepancies were observed in nurses practice in relation to the practices prior to ets .
our findings are in accordance with other previous researches on ets practices , especially in infection control practice .
another significant finding in our study was the positive relationship between passing the css educational course and nurses total ets practices score ( p = 0.00 ) .
this finding is supported by other studies which revealed that initial knowledge and practice of the nurses in relation to ets was poor and reported a significant improvement in both knowledge and practice after educating the nurses ( intervention ) .
there was a positive relationship between length of working in icu and ets practice ( p = 0.02 ) , which can be logically accepted .
the least practice score , attained in the present study , was for pre - suctioning aspects , which was a little different with open suctioning system .
findings showed that the mean scores were the minimum in pre - suctioning practices , especially in infection control aspects like hand washing , wearing goggles , and patient assessment and preparation .
although the ets protocol adopted in the present study was similar to the standard international guidelines and the related written policy and procedure that are available for all nurses in icus , current practice was not based on recommended best practice .
so , educational interventions are necessary for strengthening the nurses knowledge and practice . generally ,
because an observational method can not interpret subjective decisions , some aspect of ets practice , such as patient assessment for suctioning , may not be strictly evaluated except for chest auscultation . on the other hand
, the observer can not interpret whether the nurse 's decision for suctioning , based on auscultation , was correct or not .
also , interpretation of the length of catheter insertion , when the care provider acts quickly , may be difficult .
the present study was conducted in two general icus in a single university hospital ; therefore , the findings may not represent the practice of the general population of critical care units concerning ets .
in general , based on the findings of this study , it can be concluded that the critical care nurses do not fully adhere to the best practice recommendations in css .
this finding is consistent with the aforementioned previous studies on nurses practice in oss in other settings .
since previous studies have shown higher clinical values of closed - system ets , the researchers recommend that standard guidelines on ets practice be included in the current education of critical care nurses . maximum deviation in score from the standard practice
was observed in pre - suctioning practice , which is strictly associated with patients safety and infection control .
this highlights the need for more education in clinical setting and special practical educational interventions based on clinical standard guidelines .
also , continuing education , especially for icu staff who work with sophisticated and modern devices and tools is so necessary .
overall , continuous evaluation of nurses practice concerning implementation of the safe and correct procedures , based on best practice recommendations is so important . | background : endotracheal suctioning ( ets ) is an essential procedure performed for mechanically ventilated patients .
ets can be either performed by open or closed suctioning system ( css )
. there may be some concern on how closed - system ets is practiced by intensive care nurses .
this study was designed to investigate closed - system ets practices of critical care nurses and to compare their practice with standard recommendations.materials and methods : a prospective observational study was conducted during august and december 2012 to establish how critical care nurses ( n = 40 ) perform different steps in a typical ets practice and to compare it with the current best practice recommendations through a 23-item structured checklist .
the results were categorized into three sections : pre - suctioning , suctioning , and post - suctioning practices.results:pre-suctioning , suctioning , and post - suctioning practices mean scores were 7.5 , 11.75 , and 8.5 , respectively , out of 16 , 16 , and 12 , respectively .
the total suctioning practice score was 27.75 out of 44 .
most discrepancies were observed in the patients assessment and preparation , infection control practices , and use of an appropriate catheter .
spearman correlation coefficient indicated a significant statistical positive correlation between suctioning education period and suctioning practice score ( p < 0.0001 ) and between working experience and suctioning practice score ( p = 0.02).conclusions : the findings revealed that critical care nurses do not fully adhere to the best practice recommendation in css .
we recommend that standard guidelines on ets practice be included in the current education of critical care nurses . | I
M
Ethical considerations
R
Practices prior to suctioning
During the suctioning event
Post-suctioning practice
D
C | in contrast , closed suctioning system ( css ) can remain in line for 24 h and , thus , can be used for multiple ets procedures . in their study , paul - allen and ostrow aimed to identify the closed - system ets practices of nurses and reported some variations in the suctioning procedure . as css has recently been adopted in the hospitals in iran and is still new in most of the settings , an observational study was necessary to investigate nurses closed suctioning practice to assess an actual nursing practice . the aim of this study was to investigate closed - system ets practices of critical care nurses and to compare their practice with standard recommendations . nurses practice in using css was observed , and the checklist was ticked in three time points : prior to suctioning , during the suctioning event , and post - suctioning practice . in accordance with the main data of the observational checklist
, the results were categorized into three sections : practices prior to suctioning , during the suctioning event , and post - suctioning practices . to assess the need for ets , 4 nurses ( 10% ) auscultated the patients chest , 11 nurses ( 27.5% ) prepared the patients and explained the necessities of the procedure in several forms , and more than half of the nurses ( 27 , 67.5% ) positioned the patients in a semi - fowler position if it was possible . in relation to hand washing prior to the procedure , disparities in practice
were noted and only 7 nurses ( 17.5% ) washed their hands , 36 ( 90% ) did not wear goggles , but all the participants were fully compliant with the best practice in relation to wearing gloves . also , 20% of the nurses suctioned patients more than the maximum number in each suctioning episode ( two times ) , but most of them ( 90% ) complied with the best practice recommendation in relation to suctioning time ( less than 10 s ) . in relation with infection control principles ,
all the participants complied with the best practice recommendations in relation with closed - suctioning catheter irrigation and procedure documentation . frequency distribution was used to evaluate the suctioning practices scores and their quality with the best practice score [ table 5 ] . in suctioning three sections ,
the highest mean score , in suctioning event practices , was 11.75 ( 1.51 ) and the lowest mean score , in pre - suctioning practices , was 7.5 ( 2.66 ) . results of the spearman correlation coefficient showed a statistically significant positive correlation between passing the suctioning education period and suctioning practices scores ( p = 0.00 ) and between the length of working in icu and suctioning practices scores ( p = 0.02 ) . in relation to hand washing prior to the procedure , disparities in practice
were noted and only 7 nurses ( 17.5% ) washed their hands , 36 ( 90% ) did not wear goggles , but all the participants were fully compliant with the best practice in relation to wearing gloves . in relation with infection control principles ,
all the participants complied with the best practice recommendations in relation with closed - suctioning catheter irrigation and procedure documentation . in suctioning three sections , the highest mean score , in suctioning event practices , was 11.75 ( 1.51 ) and the lowest mean score , in pre - suctioning practices , was 7.5 ( 2.66 )
. results of the spearman correlation coefficient showed a statistically significant positive correlation between passing the suctioning education period and suctioning practices scores ( p = 0.00 ) and between the length of working in icu and suctioning practices scores ( p = 0.02 ) . this study compared the current ets procedure , conducted by the closed system , and the best practice recommendations . in the present study ,
32 and 36 participants adhered to the recommended practice with regard to the number of suctioning events ( <3 times ) and active suctioning time ( < 10 s ) , respectively , compared to the nurses in jansson et al . practices in relation with
as shown in the results , most discrepancies were observed in nurses practice in relation to the practices prior to ets . another significant finding in our study was the positive relationship between passing the css educational course and nurses total ets practices score ( p = 0.00 ) . findings showed that the mean scores were the minimum in pre - suctioning practices , especially in infection control aspects like hand washing , wearing goggles , and patient assessment and preparation . the present study was conducted in two general icus in a single university hospital ; therefore , the findings may not represent the practice of the general population of critical care units concerning ets . in general , based on the findings of this study , it can be concluded that the critical care nurses do not fully adhere to the best practice recommendations in css . since previous studies have shown higher clinical values of closed - system ets , the researchers recommend that standard guidelines on ets practice be included in the current education of critical care nurses . | [
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] |
proteins of this target class are
classified into readers , writers , and erasers of marks on histones
or other nuclear proteins and dna .
the
complex combinations of these posttranslational marks regulate gene
expression and have been termed the histone code .
bromodomains represent one of the readers
of these marks , specifically recognizing acetyl - lysine ( kac ) through
an architecturally conserved interaction module .
sixty - one unique bromodomains have been identified from
the human genome , each containing a conserved
tertiary structure as described by mutjaba et al .
, with the hydrophobic kac binding site at
one end formed between the z short helix , the za loop , and
the bc loop ( figure 1a ) .
this binding site
is primarily hydrophobic , with the carbonyl oxygen of the acetyl - group
forming two hydrogen bonds , one to a donor from either asparagine
or threonine and the other to a conserved water molecule at the base
of the pocket ( figure 1b , c ) .
( a ) conserved protein
fold of bromodomains comprising the four canonical helices z ,
a , b , and c .
all illustrated by falz ( pdb 3qzs ) . through discovery of potent small molecule inhibitors
( figure 2 ) , bet
family members
, a definition that will
be used throughout the paper : proteins able ( or predicted to be able )
to bind drug - like molecules ( not necessarily a drug ) .
a short hairpin rna screen
suggested that inhibition of the bet family may be a therapeutic strategy
for aml . through discovery of pan - bet
family inhibitor gsk1210151a from the isoxazole class ,
it has been
suggested that inhibition of the bet family may be a therapeutic strategy
for mll - fusion leukemia , and pan - bet family inhibitor gsk525762a ,
from the benzodiazepine class , has demonstrated anti - inflammatory
potential in mouse models of inflammatory disease and sepsis .
inhibitors of other bromodomains ( crebbp and pcaf ) have been found
( figure 2 ) , but
none show the submicromolar inhibition reported for bet family inhibitors
so far .
bromodomains are currently an underexplored protein family
in both basic biology and drug discovery , however , therapeutic potential
is becoming increasingly recognized . with many bromodomain structures
publicly available ,
this led us to investigate the structure - based
druggability across the protein family .
selected published bromodomain
inhibitors . from an initial inspection of various bromodomain
binding sites
, we hypothesized that not all bromodomains would be
as druggable as the bet family and a wide range of druggabilities
would be observed .
further , we wanted to identify variations in the
amino acids within the binding site that correlated with predicted
druggability .
prediction of the druggability of a novel protein
target allows realistic expectations of hit rates before any screening
effort is undertaken . for a less druggable target ,
the acceptable
potencies and associated ligand efficiencies are likely to be lower
than for a more druggable one and there is an associated risk of not
finding tractable hit matter . in this scenario , alternative strategies
may be sought such as higher screening concentrations , the use of
larger and more diverse libraries , or the choice of screening technique
employed .
one analysis of the druggability across a protein
family was performed by campagna - slater et al . on another epigenetic
target family , the histone methyltransferases . in this study ,
sitemap was used alongside the degree of
buried surface area of the bound cofactor to assess the druggability .
all of the histone methyltransferases were predicted to be druggable
with dscores from sitemap ranging from 0.96 to 1.13 but with the degree
of buried surface area of the cofactor showing some variability .
another study has also recently been published , performed by santiago
et al . primarily on methyl - lysine
sitemap
was also used in this work , and the authors suggest the methyl - lysine
binding proteins to be less druggable than bromodomains .
however ,
they only consider the eight members of the bet family that may not
be representative of the family as a whole .
many structure - based druggability
prediction methods have been published in recent years , these include
dlid , dogsitescorer , the ebi s drugebility , drugpred , fpocket , mappod , screen , and sitemap .
cover a number of these methods and some of
the challenges in computational druggability assessment .
to
assess the druggability of bromodomain proteins , we required a tool
that is readily available but more importantly allows water molecules
to be included in the analysis .
this is necessary as we have identified
five water molecules that appear to be conserved for most bromodomains
and reduce the overall volume of the pocket . to our knowledge ,
the
use of sitemap is consistent with the analyses of the histone methyltransferases
and methyl lysine binding proteins highlighted above .
a detailed validation
of this method has been published , with sitemap accurately identifying
86% of the ligand binding sites from a set of 538 complexes of the
pdbbind database as the top scoring site .
, demonstrating comparable performance of sitemap
to fpocket on their nonredundant data set ( nrdd ) .
sitemap uses the same definition of druggable as we are
using here and uses contributions from the volume of the pocket , the
enclosure , and the degree of hydrophobicity to assess druggability .
the main output from sitemap is two druggability assessment scores :
sitescore ( eq 1 ) and dscore ( eq 2 ) where n is the number of site points , e is the enclosure score , and p is the
hydrophilic score.12 both scores take contributions from
the same properties but with different coefficients .
both scores use
a cap of 100 for the number of site points ( for our analysis only
two structures reach this cap ) , and sitescore uses a cap of 1.0 for
the hydrophilic score , whereas dscore is not capped . for our data
set , the two scores have high correlation , with r equal to 0.92 . because of the high correlation with
sitescore and the suggestion that it is more discriminatory of druggable
and undruggable sites , dscore was selected
to be used alone in our analyses .
sitemap was applied to a filtered
set of the published bromodomain structures extracted from the pdb , and a wide range of predicted druggabilities
was observed , from difficult to druggable . from this initial druggability
assessment ,
the dscores were compared with the clustering generated
from whole sequence similarity of structure - based alignments by filippakopoulos
et al .
this analysis showed that whole
sequence similarity alone did not sufficiently explain the trends
in the druggability that were observed , therefore we inspected the
binding sites and identified unique amino acid signatures that showed
better correlation with observed druggabilities .
we propose that this
new classification is more relevant to small molecule binding than
whole sequence similarity due to its focus on the binding site residues .
it allows druggability prediction of bromodomains without structural
characterization and will aid the selection of templates for homology
models by comparison to members within the same classification .
crucially ,
it also enables the medicinal chemist to identify family members that
are likely to bind the same inhibitors as the targeted bromodomain ,
which can be explored either for lead hopping or selectivity screening .
many bromodomain - containing proteins possess
multiple bromodomains but also exist as different sequence isoforms .
when referring to a single bromodomain of one isoform , we have used
this format : bromodomain - containing protein name , followed by isoform
if present ( a / b / c if isoforms are identical ) , followed by the bromodomain
number . for example , the second bromodomain of the b isoform of brd8
would be shortened to brd8b(2 ) , and the single bromodomain of baz1a ,
which is identical between isoforms a and b , would be shortened to
baz1a(a / b ) .
when referring to different chains within a pdb
file , we have used this format : pdb code followed by a letter corresponding
to the number of the protein chain within the file .
for example , the
second chain of the protein brd1 in pdb 3rcw would be shortened to 3rcw_b .
protein chains within each pdb
file were separated , ligands and nonconserved water molecules removed ,
and protonation states assigned using protonate3d in moe .
forty - six chains from 14 pdb files with unresolved
binding site residues were filtered out ( supporting
information , table s1 ) .
bound state , resolution , presence of
unresolved side chains , and presence of conserved water molecules
were recorded for each chain . for taf1(a
/ b ) , whereby both bromodomains
have been crystallized within one peptide chain , these were separated
and treated individually .
individual chains were then preprocessed
using the protein preparation wizard in
maestro with
,
create disulfide bonds , and convert selenomethionines
to methionines options selected .
the preprocessed
chains were submitted to sitemap using default parameters and with
identify top - ranked potential receptor binding sites
to avoid any bias from using ligands / peptides to define pockets .
the
minimum number of site points per pocket identified needed to be reduced
to 14 from 15 for pb1(a / b / c)(1 ) .
kac binding sites were then selected
from all identified sites and all outputted values recorded .
module
and the blosum62 matrix with default settings . as used in the full sequence alignment by filippakopoulos
et al .
, we have also used brd4(a / b)(1 )
as a reference sequence for numbering of the residues .
surfaces are color - coded using the pocket
coloring from moe with green indicating enclosed surface of the protein
and white indicating exposed .
having selected sitemap to assess
druggability , the next step was to collect the available crystal structures
from the pdb .
this yielded 105 different pdb entries covering 33 of
the 61 unique human bromodomains .
these pdb entries were then separated
into the separate protein chains , as each protein chain within a crystal
structure can be of a different conformation , and any chains with
unresolved residues in the binding site were removed . through inspection
of available bromodomain structures , it was apparent that five water
molecules are conserved across most bromodomain kac binding sites .
no publicly available structures demonstrate the displacement of any
of these by a ligand ( figures 1a , 3 , and supporting information , figure s1 ) , suggesting that the water
molecules are an important feature of the binding pocket .
frequently ,
water molecules are removed prior to druggability assessment and we
decided to determine druggability in the presence and absence of these
conserved water molecules to assess their effect on the dscore .
all
five water molecules could be identified in structures of 23 of the
28 unique bromodomains passing the requirement of a structure without
unresolved binding site residues , although not all of the water molecules
were always present in the same structure due to limitations of protein
crystallography ( most frequently in low resolution structures ) . to
maximize our coverage of the observed protein conformations
while
ensuring that all assessed structures contained the same number of
water molecules , for structures with missing water molecules , structures
of the same protein with the missing water molecules were aligned
and the missing water molecules were included from the other structure(s ) .
for smarca4 , a high resolution ( 1.50 ) structure and
one bound
with nmp were available and both of these structures demonstrate only
four of the five water molecules present , so the druggability assessment
has been assessed as such , raising the number of bromodomains initially
considered to 24 .
details of all the water molecules included can
be found in supporting information , table s3 . conserved water molecules in the binding site of brd4(a / b)(1 ) ( pdb 3mxf ) .
the absence of these water molecules led to a larger
identified pocket and consequently a higher druggability score , with
most of the bromodomains classified in the druggable range ( dscore
> 0.85 ) .
crucially , without the water molecules , a smaller range
of scores was observed , making the assessment less discriminative
between sites ( figure 4 ) .
given that these water molecules enclose the pocket and
have a significant effect on the druggability , all subsequent analysis
was performed with the water molecules present .
( a ) plot
of dscores obtained when conserved water molecules were included in
analysis against the same structures with all water molecules removed .
linear line of best fit added to plot .
( b ) histogram of same data
showing distribution of scores with normal distribution fitted to
this data .
colors indicate druggability classification : red , druggable ;
yellow , intermediate ; white , difficult .
the 178 qualifying
protein chains ( 24 of the 61 unique human bromodomains ) were prepared
and submitted to sitemap druggability assessment .
a wide range of
druggabilities was observed for the bromodomains from difficult ( dscore
<
0.75 ) ( e.g. , baz2b pdb 3q2f , dscore = 0.52 ) to druggable ( dscore > 0.85 ) ( e.g. ,
pcaf pdb 3gg3_b , dscore = 1.08 ) . scores in between these two have been classified
as intermediate ( e.g. , crebbp pdb 3p1e_b , dscore = 0.82 ) ( figure 5 ) . details of all outputted
scores from this assessment can be found in supporting
information , table s3 .
box - plots showing range and distribution of
druggability for each bromodomain across available structures passing
imposed filters ( including presence of binding site water molecules )
colors indicate druggability classification :
red , druggable ; yellow , intermediate ; white , difficult .
( a ) four outliers
removed from druggability assessment ( see group 4 text ) .
most bromodomains contain a small and tight binding
site to recognize kac of the protein substrate .
this conveys a basic
level of druggability as demonstrated by baz2b ( pdb 3q2f , dscore 0.52 ) and
the potential to bind a small fragment with acceptable ligand efficiency ( nmp , baz2b le 0.29 ) .
the differences between the sites stem from the environments around
that small and tight pocket . for
the bromodomain family as a
whole , even the lowest scoring bromodomain ( pb1(a / b / c)(1 ) ) would be
classed as more druggable than a protein
protein interaction
with a large ( > 1000 ) and fairly featureless
interface .
sitemap would fail to identify binding sites such as this
( e.g. , siah1 pdb 2a25 ) . at the other end of the druggability scale ,
some bromodomains
have demonstrated comparable predicted druggability to what are currently
considered druggable targets such as protein kinases ( e.g. , aurora
a , pdb 1mq4 ,
dscore = 0.96 ) or hsp90 ( pdb 1am1 , dscore = 0.99 ) .
when a ligand or peptide binds , the observed conformation of the protein
may change to potentially induce a more druggable pocket .
this has
been seen for the bcl-2 family of proteins whereby small molecule
inhibitors bind to pockets not observed in the apo or peptide bound
structures and show improved potency .
this is in line with an increase in the sitemap predicted druggabilities
of the bcl - xl binding sites from apo ( pdb 1r2d_a , dscore = 0.76 ) to ligand bound ( abt-737 ,
pdb 2yxj , dscore
= 0.95 ) . to assess whether this could be the case for bromodomains ,
those with both apo and ligand or peptide bound ( holo ) structures
available were collated and the dscores from sitemap compared ( table 1 ) .
dscores after four outliers were
removed ( see group 4 text for details ) .
colors : white , bromodomains for which a more druggable
pocket was not observed for the holo structures ; green , bromodomains
for which a more druggable pocket was observed for the holo structures
but a comparably druggable pocket was observed in the ensemble of
apo structures ; red , bromodomains for which a more druggable pocket
was observed for the holo structures and a comparably druggable pocket
was not observed in the ensemble of apo structures . from table 1 , it can be seen
that only crebbp of the 12 bromodomains with apo and holo structures
available show evidence of a more druggable pocket ( 0.050.1
increase in median dscore ) in the presence of a ligand or peptide
that is not observed in the ensemble of apo structures .
the median
dscore for the entire ensemble of structures was classed as intermediate
at 0.75 , but the highest scoring structure was classed as druggable
at 0.89 ( pdb 3p1c_b ) when bound with kac .
three bromodomains ( brd2(2 ) , baz2b ,
and pb1(a / b / c)(5 ) ) unexpectedly show reduced median druggability for
the ligand or peptide bound structures when compared to the apo .
comparably
druggable conformations of the holo structures for brd2(2 ) and pb1(a / b / c)(5 )
within the range of the apo are observed in the full ensemble , but
this is not the case for baz2b .
these effects will be discussed later ,
in the context of the similarity of each bromodomain with other members
of the family .
other than for crebbp , in general it does not appear
that ligand binding is able to induce a significantly more druggable
structure than is observed in the apo ensemble for the bromodomain
family . having completed the initial computational druggability assessment ,
the next step was to identify trends within the data set . when compared
with the clustering generated from whole sequence similarity performed
by filippakopoulos et al .
, a lack of correlation
was observed such as the first and second bromodomains of taf1 being
placed in the same cluster despite dscores at either end of the scale .
it is not surprising to see a lack of correlation between druggability
and whole sequence similarity , as when dealing with a druggability
assessment it is the nature of the binding site that affects the score ,
not the rest of the domain .
for this reason , we decided to inspect
the structures for binding site for features that vary across the
bromodomain family and can be used to order the members of the family
into groups .
this led to the identification of eight groups
characterizing 49 of the 61 unique bromodomains .
each of these groups
is defined by the presence of a unique signature of up to three amino
acid residues that is shared by all members of that particular group
and gives a characteristic shape to the kac binding site ( table 2 , figure 9 , and supporting information , figure s1 ) .
taken together ,
the amino acid residues of all group signatures span seven residues
that enclose the kac binding site . these were position 81 , which is
a tryptophan in the bet family and forms
what has been termed the
za - channel , the two residues facing the
binding pocket on the za - loop , both leucine at position 92 and 94
in the bet family , the residue at position 140 , which is most commonly
asparagine and forms the key hydrogen bond donor interaction with
the kac carbonyl , residues 144 and 146 on the bc - loop and c - helix ,
which enclose the hydrophobic shelf in the bet family , and residue
149 , which although not enclosing the pocket does influence the position
of residue 81 , which can have a large effect on both the za - channel
and hydrophobic shelf .
residue 145 on the c - helix has also been included
in the analysis , which although not part of any of the signatures
has been used in further differentiating some of the bromodomains
within each of the groups .
thus , in total , eight residues have been
used to characterize the bromodomain binding sites ( figure 6 ) .
number of bromodomains with available
structures passing the filters out of the number of members in the
group .
eight residues around the binding site used in analysis ( brd4(a / b)(1 )
used as reference , pdb 3mxf ) .
( b ) binding site residues shown with transparent surface representation . to determine which residues were present at each
position ,
available bromodomain structures were overlaid with the
reference structure , brd4(a / b)(1 ) ( pdb 3mxf ) , and the eight binding site residues
were recorded that best aligned with the brd4(a / b)(1 ) residues ( supporting information , table s5 and figure s1 ) . for five of the eight identified residues ( 140 , 144 , 145 , 146 ,
and 149 ) , the spacial alignment corresponded with the sequence alignment ,
making the identification of the residues for the bromodomains without
a structure straightforward .
for the other three residues , due to
the variation in length and position of the za - loop , spatial alignment
did not always correspond with the sequence alignment .
here we have
used the residue that aligned best in space with the brd4(a / b)(1 )
structure , as this should be more relevant to the nature of the binding
pocket . for bromodomains without a structure , the matching residue
from a protein within the same grouping from the alignment of filippakopoulos
et al . was used ( e.g. , between brpf1b
and brpf3 ) . in a few cases ,
this was not always possible due to the
lack of a sufficiently homologous bromodomain with a structure being
available ( e.g. , mll1 or trim28 ) .
these bromodomains have been excluded
from groups that are characterized by the za - loop residues ( 81 , 92 ,
and 94 ) . using the binding site groupings obtained , a qualitative classification
tree was generated ( figure 7 ) .
this allowed plotting of the predicted druggabilities to
visualize where the most druggable groups are as well relationships
between the groups .
these included the relationship between crebbp
and ep300 with the bet family as they share the extended length of
the za - loop , the relationship between groups 2 and 3 ( y or f at position
146 ) , and between groups 5 and 6 ( y or f at position 94 ) ( see group
texts for more details ) .
bromodomain classification tree generated on
the basis of eight binding site amino acid signatures showing bromodomain
druggability .
druggabilities of ash1l , atad2b , brpf1b , pb1(a / b / c)(3 ) ,
pb1(a / b / c)(4 ) , and smarca2b assessed using structures failing imposed
filters included ( see group text ) .
furthermore , we further divided several groups
into subclassifications such as the separation of the baz family within
group 4 by exploring changes in whole sequence similarity that may
have an effect on the overall fold of the bromodomains such as the
za loop position or more subtle changes in the binding site that have
smaller effects on the pockets than the signature residues .
we believe that this grouping better explains the trend in druggability
assessment than whole sequence similarity , but also that this grouping
will predict small molecule selectivity patterns more accurately due
to the focus on the binding site .
this should prove useful when determining
selectivity of inhibitors and the potential to identify possibilities
to transfer hit matter from one bromodomain to another .
another potential
use of this grouping is for the building of a homology model .
if the
use of the model is to predict the binding mode of a small molecule
inhibitor or to select compounds in a virtual screening approach ,
then the choice of template is very important .
the bromodomains grouped
together here share binding site features , and thus members of the
same group should represent the best templates for building homology
models for binding mode prediction . when comparing the clustering based on whole sequence similarity
to the grouping performed here , differences can be observed ( figure 8) .
the two classifications
are not dramatically different ( 42/61 placed in the same group ) , which
is not surprising , but there is sufficient difference to suggest that
when dealing with small molecule inhibitors the binding site classification
may be more informative .
groups from left
to right in same number order as table 2 with
the same coloring .
whole sequence classification colors generated
from binding site classification group which shares highest percentage
similarity .
an example of the differences is baz1a(a / b ) that
shares whole sequence similarity with the bet family but does not
share binding site similarity with this family . it is therefore unlikely
to bind similar ligands and likely possesses druggability similar
to that of the group it has been placed in by binding site classification
( group 4 ) .
another example is that atad2a and atad2b are placed in
the same cluster as group 3 by whole sequence similarity but do not
share binding site similarity with this group or any other bromodomain .
each of the binding site classification groups will now be discussed
individually , commenting on their druggability , but also any trends
or inconsistencies within the groups .
the bet family of bromodomains was classified as druggable , which
correlates with the fact that a number of potent small molecule inhibitors
have been found .
the comparatively high
druggability for this family can be explained by a more enclosed upper
part of the pocket and thus additional surface area for interaction
with small molecules not present in other less druggable bromodomains .
this is predominantly due to the presence of a tryptophan residue
at position 81 and a methionine residue at position 149 that influences
the position of the tryptophan residue , forming the za channel .
on
the other side of the pocket , the za loop is longer than most other
bromodomains , providing additional surface that can be utilized for
interaction with small molecules .
these features result in above - average
druggability of the sites ( figure 9a ) .
bromodomains aligned with
brd4(a / b)(1 ) pdb 3mxf and colors generated using moe pocket coloring : green = enclosed
and white = exposed .
pocket colors are used to highlight binding sites
and does not represent pockets identified by sitemap used for druggability
assessment .
images captured from the same viewpoint except image g
at the same orientation as figure 6 . ( a )
( g ) pb1(a / b / c)(1 ) pdb 3iu5 structure representative of group 8 .
( h ) crebbp pdb 3p1c_b structure . given the high similarity of the pockets , it is
not surprising that nonselective bet family inhibitors have been found ,
although there are subtle differences between the first and second
bromodomains of the bet family that could be exploited for selectivity .
the entire bet family has the same za loop residues facing the pocket ,
but the first bromodomains possess an aspartate at position 144 whereas
the second bromodomains possess a histidine . at position 145
, the
entire bet family has an acidic residue but this changes between aspartate
and glutamate .
we have separated this group in the classification
tree into the first and second bromodomains to reflect these small
changes .
an outlier in the druggability assessment was seen
for a peptide bound structure of brd2(2 ) ( pdb 2e3k_b , dscore = 0.64 ) .
the reason for this low score was the position of the tryptophan at
position 81 .
for the rest of the bet family ( and other brd2(2 ) structures ) ,
this residue is directed toward the binding site creating the za channel
( figure 10a ) . in
this outlier ,
the tryptophan residue is directed away from the pocket ,
opening the pocket significantly ( figure 10b ) and inducing a pocket more like crebbp or ep300 ( leucine at position
81 ) ( figure 10c ) , greatly reducing the druggability .
for the 56 bet family structures passing the initial filters ,
this
is the only one for which the tryptophan is oriented away from the
binding site , suggesting that this is an unusual conformation and
does not appear relevant to small molecule inhibitor binding .
( c ) crebbp
pdb 3dwy_a structure
showing similarity to brd2(2 ) atypical conformation .
four of these ( cecr2 , falz(a / b ) , gcn5l2 , and pcaf ) were classified
at the high end of the druggability scale by sitemap and are within
the same cluster by whole sequence similarity .
the key features of
these pockets are the aromatic residue at position 146 compared with
a small hydrophobic residue in many other bromodomains and a tryptophan
at position 81 . together , these signature residues provide a significant
amount of hydrophobic surface on this side of the pocket .
the za loop
is two amino acids shorter than the bet family , but this part of the
pocket is still sufficiently enclosed to provide high druggability
( figure 9b ) .
these bromodomains represent a
family that demonstrate high predicted druggability but to date have
not been exploited with high affinity compounds .
two outliers
were seen for the bromodomain falz(a / b ) ( pdb 2f6n_a and 2fsa_c ) , which both scored
significantly less than the other nine structures passing the imposed
filters of this bromodomain .
when inspecting the structures and comparing
them with more druggable conformations of falz(a / b ) , no obvious changes
could be seen , as was the case with brd2(2 ) . however , when examined
more closely , it could be seen that both the za loop and the bc loop
are moved slightly away from the pocket , inducing a more open conformation
and thus reducing the druggability .
the four structures that are peptide
bound do not demonstrate this more open conformation and may be stabilized
in the closed conformation by the presence of the peptide .
interestingly ,
the remaining two members of group 2 ( taf1(a / b)(2 ) and taf1l(2 ) ) possess
the same signature residues but are not present in the same whole
sequence classification as the other members of group 2 .
taf1(a / b)(2 )
also scored in the druggable range ( dscore = 0.89 ) , however , taf1l(2 )
scored in the difficult range ( dscore = 0.73 ) , possibly due to the
za loop and tryptophan 81 positions opening the binding site . with
only one structure available , this could be an example of a false
negative , with an effect similar to the outliers of falz(a / b ) ( pdb 2f6n_a and 2fsa_c ) , and with further
conformational sampling of the za loop and tryptophan 81 , a more druggable
conformation could be observed .
taf1(a / b)(2 ) and taf1l(2 ) differ to
the other members of group 2 by the residues at position 94 , 145 ,
and 149 within the binding site as well as having reduced sequence
similarity ; for these reasons they have been given their own subclassification
in the classification tree .
group 3 contains six bromodomains ,
which all fall into the same classification from the whole sequence
similarity and are related to the group 2 by the presence of the aromatic
residue at position 146 , enclosing this part of the pocket .
they differ
by the lack of the tryptophan at position 81 opening the za channel ,
so the druggability scores are somewhat lower , placing them in the
intermediate category ( figure 9c ) . within the
group , brd7 and brd9
have been given their own subclassification due
to the changes in za loop residues and having tyrosine rather than
phenylalanine at position 146 .
although a crystal structure
for brpf1b was not available , an nmr structure ( pdb 2d9e ) was and passed
the filters other than the presence of the conserved water molecules .
when sitemap druggability assessment was applied to the ensemble of
structures , a median dscore of 1.04 was obtained ( supporting information , table s4 ) and is slightly higher but
in a similar range to the dscore values obtained from the other members
of the group without water present . using the lines of best fit from
figure 4a and a subset of the values from this
group ,
estimates of the dscore with water molecules were achieved
of 0.91 and 0.97 respectively .
these values are higher than other
members of the group and places this bromodomain in the druggable
category .
the four members of the baz family
cluster together by binding site similarity within group 4 , unlike
the whole sequence similarity classification .
the group is characterized
by a shorter za loop than the bet family , with no residue overlapping
with leucine 92 from brd4(a / b)(1 ) in space , making the pocket fairly
open and reducing druggability .
the baz family share the tryptophan
at position 81 with the bet family , but this does not form the same
za channel due to the change in residue at position 149 ( figure 9d ) . for the bet family , this is a methionine , which
restricts the movement of the tryptophan forming the za channel , but
in the baz family , this residue is small ( alanine or cysteine ) , which
results in movement of the tryptophan toward residue 149 , removing
the za channel and hydrophobic shelf present in the bet family and
heavily reducing the druggability into the difficult category .
the baz family is joined by trim24 , trim33a , and trim66 in this group ,
and although these do not possess a tryptophan at position 81 , they
share a very similar za loop , with no residue overlapping with the
leu92 from the bet family .
this open part of the pocket , and the lack
of a za channel , give these bromodomains similar pockets to the baz
family .
they have been given their own subclassification due to this
change in position 81 from tryptophan to a leucine or valine .
four structures of trim24 score highly in the druggability assessment
and appear to be outliers ( supporting information ,
table s4 ) .
when inspecting the sites identified by sitemap ,
it was apparent that the favorable score is not solely due to the
kac binding site but also an extended site ranging from the kac binding
site to the interface between the bromodomain and the adjacent phd .
for this reason , the analysis performed here has excluded these data
points .
the kac binding site is better assessed by the other generated
scores , placing it in the difficult range , but there may be small
pockets close to the kac binding site which could be exploited by
using fragments followed by a linking effort .
baz2b surprisingly
indicated reduced druggability of the ligand bound structure when
compared to the apo ( 0.18 reduction in dscore ) . from the initial definition
of druggability
, it would be expected that in general , holo structures
should be as druggable if not more so than their apo counterparts .
when comparing the two baz2b structures ( pdb 3g0l and 3q2f ) , there are only
subtle differences between the two conformations of the binding site ,
namely that for the ligand bound structure the pocket is slightly
narrower due to movements of the za loop and bc loop , increasing the
enclosure score ( 0.610.69 ) .
this narrowing most likely occurs
to maximize contact with the flat heteroaromatic part of the ligand ,
but in doing this reduces the volume of the most enclosed part of
the pocket ( 105 to 92 ) . for
sitemap druggability assessment ( particularly for low druggability
sites )
, the reduction in volume counts more toward the dscore than
the increase in enclosure , so the overall effect is to reduce the
predicted druggability of the ligand bound structure relative to the
apo .
group 5 is characterized
by the presence of an aromatic residue at position 94 , the effect
of this is to provide a lid to the pocket , thus increasing
the enclosure and therefore the druggability ( figure 9e ) .
structures of pb1(a / b / c)(3 ) and pb1(a / b / c)(4 ) were
available ( pdb 3k2j and 3tlp ) ,
but these structures were excluded from the initial analysis due to
them missing some of the conserved water molecules . to include them
in the analysis and to allow direct comparison of the dscores , water
molecules from the highly similar pb1(a / b / c)(2 ) were included through
alignment of the structures .
this yielded median druggabilities for
the two bromodomains of 0.57 and 0.70 , respectively , placing them
in the difficult category ( supporting information ,
table s4 ) .
phip(2 ) scored highest and was placed in the
druggable range . the second , third , fourth , and sixth bromodomains
of pb1 also fall into this grouping but none show as high a druggability
as phip(2 ) and also show less whole - sequence similarity , and this
has been indicated with a different subclassification with pb1a(6 )
given its own subclassification due to a four - residue shorter za loop .
the phip(2 ) structure does have a ligand bound , so this reduced druggability
of the pb1 members could either be due to lack of protein conformational
sampling ( za loop position ) and be a false negative or could be due
to more subtle effects influencing the overall conformation of the
protein and therefore the druggability .
the members of group
6 also share this aromatic residue at position 94 , but without any
available structures it is difficult to say whether these would be
druggable like phip(2 ) or more challenging like many of the pb1 bromodomains .
unlike group 5 , group 6 members cluster together by whole sequence
similarity , however , there are six other bromodomains that share whole
sequence similarity with group 6 but do not appear to share binding
site similarity .
the four proteins in this group
all fall into the same classification by whole sequence similarity .
by whole sequence similarity , group 7 is joined by four other bromodomains
( pb1(a / b / c)(24 ) and pb1a(6 ) ) but do not share the signature
residues and do not fall into this group .
pb1(a / b / c)(5 ) was classified
in the intermediate druggability range but smarca4 as difficult .
all
of these proteins possess a shorter za loop than the bet family , reducing
the surface available for interaction with small molecules .
the shape
of the kac binding pockets are also different to those of the bet
family in the available structures , with the location of the aromatic
residue at position 139 moved toward the pocket and leucine at position
87 rather than the valine present in most other bromodomains .
this
induces a wider entrance , opening the tight binding pocket at the
base of the binding site ( figures 9f and 11a ) . for this reason
,
it is expected that this group could bind ligands differently to the
other groups , as the tightest , most conserved part of the binding
site is significantly different .
( a ) pb1(a / b / c)(5 ) structure representative
of group 7 .
( c ) overlaid backbones of usual conformation ( pdb 3g0j_a ) in green and
unusual ( pdb 3g0j_b ) in blue . orientation and coloring consistent with figure 9 .
one structure of pb1(a / b / c)(5 ) that failed the
original filters on the presence of the conserved water molecules
( pdb 3g0j_b )
showed a particularly unusual conformation that is unlike any conformation
of this bromodomain or any other bromodomain ( figure 11b ) .
the za loop is moved toward the z helix , which
is not possible in many other bromodomains due to the presence of
alanine at position 81 ( figure 11c ) but may
also be allowed due to the change in shape of the binding site discussed
previously .
the effect of this is to close this part of the pocket ,
which for many of the other bromodomains is the location of the za
channel .
this results in an increase in hydrophobicity of the remaining
pocket and reduced preference for the conserved water molecules . when
sitemap druggability assessment was applied to this structure ( with
a single water molecule at the base of the pocket ) a dscore of 0.87
was achieved , which is significantly higher than any other conformation
of this protein and places it in the druggable range .
this unusual
conformation of the protein may represent an opportunity for inhibiting
this bromodomain selectively over any other , as this unusual conformation
is not expected to be common and may be unique to pb1(a / b / c)(5 ) .
this
conformation may also allow for substitution of the water molecules ,
which appear to be highly conserved for most other bromodomains . as was the case for brpf1b ,
no crystal structure was available for
smarca2b , but an nmr structure was available that passed the filters
imposed apart from the presence of the conserved water molecules ( pdb 2dat ) . when sitemap druggability
assessment was applied to the ensemble of structures , a median dscore
of 0.81 was achieved ( supporting information ,
table s4 ) , which is higher than the dscore for smarca4 without
water present but lower than the dscore for pb1(a / b / c)(5 ) .
when converted ,
as with brpf1b , using the dscore values with and without water molecules
of the other bromodomains and the other members of the group from
figure 4a , estimates of the dscore with water
molecules of 0.53 and 0.64 were obtained .
this places smarca2b in
the difficult range with comparable predicted druggability to smarca4 ,
which shares high whole sequence similarity . for the binding of kac to bromodomains
,
a key hydrogen bond is formed between the carbonyl of the acetyl group
and a donor from either an asparagine or threonine residue at position
140 .
for group 8 , this key residue is
replaced with tyrosine ( eight bromodomains ) or aspartic acid ( mll1 :
although protein construct has been engineered and may not be true
for full length protein ) .
this changes the nature of the pocket significantly ,
and it has been suggested that these domains may not bind kac , or
if they do , the manner in which they do would be unlike most other
bromodomains .
mll1 has been given its
own subclassification due to it possessing an aspartate at position
140 as have the sp family due to their high sequence similarity to
each other over the other members .
a structure for pb1(a / b / c)(1 )
is available which shows how tyrosine 140 reduces the size of the
pocket ( figure 9 g ) , and sitemap assessed this
site as the least druggable of the bromodomains with only a very small
pocket being identified .
with such a low assessed druggability , the
only opportunity to target this site with a small molecule inhibitor
would be to displace the conserved water molecules .
but , even with
the water molecules removed , the site only achieves a dscore in the
low end of the intermediate range suggesting that pb1(1 ) would be
very challenging to bind small molecules to the equivalent of the
kac binding site of other bromodomains .
a structure for ash1l
( pdb 3mqm ) ,
another member of this group , is also available , but the conserved
water molecules are not present as is the case for pb1(a / b / c)(1 ) ,
so it was removed by the initial filter .
the site scored comparably
to pb1(a / b / c)(1 ) without water molecules with a median dscore of 0.74 ,
suggesting that this bromodomain would be similarly difficult to target
with a small molecule inhibitor .
the remaining bromodomains
failed to be placed into any groups larger than two , with little similarity
to any of the other groupings described here .
of those with available
structures , none showed any particular druggability as assessed by
sitemap , except crebbp , which was classified in the intermediate range .
crebbp is interesting as it possesses the same longer za loop as the
bet family , with similar residues facing the binding site that provide
similar interaction potential .
however , the tryptophan at position
81 in the bet family , which forms the za channel and hydrophobic shelf ,
is a considerably smaller leucine , resulting in a loss of these features
and a decrease in predicted druggability ( figure 9h ) .
another unusual feature is the presence of an arginine
at position 145 , which provides the potential to form charged interactions
with this strongly basic center .
flexibility of both the za loop and
the unusual arginine could explain the large changes in predicted
druggability of crebbp , with the highest scoring protein conformation
being placed in the druggable category and the lowest in the difficult .
with high sequence similarity and binding site similarity
, the bromodomain
of ep300 would be expected to bind similar ligands to crebbp and have
similar potential for a more druggable pocket to be induced . similarly to pb1(a / b / c)(3 ) and pb1(a / b / c)(4 ) , a structure of atad2b
( pdb 3lxj_d )
was available that was filtered out due to missing two of the conserved
water molecules .
all five water molecules were present in a structure
of the similar atad2a ( pdb 3dai ) , and through aligning the two structures , the missing
water molecules of atad2b were included .
sitemap druggability assessment
yielded a score of 0.64 , placing this bromodomain in the difficult
category . when targeting any protein with small molecule inhibitors , selectivity
for the bromodomains , the highly conserved small
and tight binding site ( binding acetyl part of kac ) at the base of
the pocket makes prediction of selectivity for a low molecular weight
( < 200 da ) fragment challenging as it is the environment around
this site which will determine the selectivity for larger molecules .
from this analysis ,
the first proteins that should be tested for selectivity
issues would be those within the same group ( figure 7 ) .
there are , however , some similarities between groups that
may give rise to comparable binding of small molecules .
the groups
that are related to each other that have been previously discussed
( groups 2 and 3 and groups 5 and 6 ) may bind similar ligands , but
there are differences that may be exploited for selectivity . also
as discussed , crebbp and ep300 show some similarity through the za
loop to the bet family , but also phip(2 ) and wdr9(a / b)(2 ) show some
similarity in the shape of the binding site with the same hydrophobic
shelf and above average druggability .
other than these , selectivity
would be expected between groups for molecules larger than small fragments . adequately assessing the full ensemble of protein conformations
is an issue that affects any prediction that uses crystal structures ,
as by their nature a static image is observed . to address this issue ,
we have used as many experimentally observed conformations of the
bromodomains as possible , and scores generated by the druggability
assessment do vary between conformations of the same protein , including
different protein chains within the same crystal structure . for this
reason , we can not rule out that some proteins that were classified
as difficult or intermediate may be false negatives and may have the
potential to be druggable with additional conformational sampling .
however , the range of scores observed for different conformations
of the same bromodomain appear to be less than those between different
bromodomains due to changes in the eight selected residues identified
here ( figure 5 ) . for this reason ,
it is possible
that new bromodomain conformers may show slightly increased druggability
than predicted from the currently available structures ( e.g. , intermediate
instead of difficult or druggable instead of intermediate ) .
however ,
it is unlikely that large leaps will occur ( e.g. , for a protein classified
as difficult to move into the druggable category ) , and other than
a few special cases discussed in the text ( brd2(2 ) , falz(a / b ) and
crebbp ) , these have not so far been observed .
predicted druggabilities of available bromodomain
structures were assessed and a range of scores observed .
the bet family
members were predicted to be druggable , consistent with literature
evidence .
one group ( group 2 ) showed comparable or increased predicted
druggability relative to the bet family and represents a currently
unexplored group of proteins that may have relevance in drug discovery
as their biology is revealed .
many of the other bromodomains showed
lower predicted druggability and some of these were classed as difficult
based on their dscore .
the comparatively low score suggests that these
will show lower hit rates in screening efforts and that it will be
more challenging to identify and optimize hit matter .
however , it
should be noted that even these bromodomains are far more druggable
than featureless protein protein interactions .
trends
within the data set were then sought and rationalized by unique signatures
characterizing the binding pockets , leading to a new classification
of the bromodomains into groups with similar amino acids in key positions
and similar predicted druggabilities .
this classification showed significant
differences to the whole sequence classification , suggesting that
it may prove more useful to drug discovery directed toward the acetyl - lysine
binding site .
our proposed classification also allows medicinal
chemists who work on a particular bromodomain to identify other family
members that are likely to bind similar inhibitors .
this information
can be explored to select proteins for counterscreening or to identify
bromodomain inhibitors that can be explored in a target hopping approach .
furthermore , our results highlight the significance of water molecules
in the computational analysis of bromodomain binding sites .
a number
of conserved water molecules occupy the base of the pocket and so
far no example has been reported in which these have been replaced
by small molecules . for this reason , all bromodomains have been treated
equally with all of these water molecules kept as part of the binding
site and the druggability assessment performed as such . the corresponding
assessment without the water molecules present
has also been performed ,
which places more of the bromodomains in the druggable category and ,
crucially , appears to increase the observed score more for the less
druggable sites , making it less discriminatory between druggable and
difficult sites .
this work represents the first analysis of
this type for the bromodomain family and will prove useful for drug
discovery projects aiming to identify inhibitors of the acetyl - lysine
binding site of bromodomains . | bromodomains are readers of the epigenetic code that
specifically bind acetyl - lysine containing recognition sites on proteins .
recently the bet family of bromodomains has been demonstrated to be
druggable through the discovery of potent inhibitors , sparking an
interest in protein
protein interaction inhibitors that directly
target gene transcription . here
, we assess the druggability of diverse
members of the bromodomain family using sitemap and show that there
are significant differences in predicted druggability .
furthermore ,
we trace these differences in druggability back to unique amino acid
signatures in the bromodomain acetyl - lysine binding sites . these signatures
were then used to generate a new classification of the bromodomain
family , visualized as a classification tree .
this represents the first
analysis of this type for the bromodomain family and can prove useful
in the discovery of inhibitors , particularly for anticipating screening
hit rates , identifying inhibitors that can be explored for lead hopping
approaches , and selecting proteins for selectivity screening . | Introduction
Materials and Methods
Results and Discussion
Conclusion | through discovery of pan - bet
family inhibitor gsk1210151a from the isoxazole class ,
it has been
suggested that inhibition of the bet family may be a therapeutic strategy
for mll - fusion leukemia , and pan - bet family inhibitor gsk525762a ,
from the benzodiazepine class , has demonstrated anti - inflammatory
potential in mouse models of inflammatory disease and sepsis . from an initial inspection of various bromodomain
binding sites
, we hypothesized that not all bromodomains would be
as druggable as the bet family and a wide range of druggabilities
would be observed . to
assess the druggability of bromodomain proteins , we required a tool
that is readily available but more importantly allows water molecules
to be included in the analysis . this analysis showed that whole
sequence similarity alone did not sufficiently explain the trends
in the druggability that were observed , therefore we inspected the
binding sites and identified unique amino acid signatures that showed
better correlation with observed druggabilities . crucially ,
it also enables the medicinal chemist to identify family members that
are likely to bind the same inhibitors as the targeted bromodomain ,
which can be explored either for lead hopping or selectivity screening . for
the bromodomain family as a
whole , even the lowest scoring bromodomain ( pb1(a / b / c)(1 ) ) would be
classed as more druggable than a protein
protein interaction
with a large ( > 1000 ) and fairly featureless
interface . for this reason , we decided to inspect
the structures for binding site for features that vary across the
bromodomain family and can be used to order the members of the family
into groups . these were position 81 , which is
a tryptophan in the bet family and forms
what has been termed the
za - channel , the two residues facing the
binding pocket on the za - loop , both leucine at position 92 and 94
in the bet family , the residue at position 140 , which is most commonly
asparagine and forms the key hydrogen bond donor interaction with
the kac carbonyl , residues 144 and 146 on the bc - loop and c - helix ,
which enclose the hydrophobic shelf in the bet family , and residue
149 , which although not enclosing the pocket does influence the position
of residue 81 , which can have a large effect on both the za - channel
and hydrophobic shelf . the bet family of bromodomains was classified as druggable , which
correlates with the fact that a number of potent small molecule inhibitors
have been found . given the high similarity of the pockets , it is
not surprising that nonselective bet family inhibitors have been found ,
although there are subtle differences between the first and second
bromodomains of the bet family that could be exploited for selectivity . for the bet family , this is a methionine , which
restricts the movement of the tryptophan forming the za channel , but
in the baz family , this residue is small ( alanine or cysteine ) , which
results in movement of the tryptophan toward residue 149 , removing
the za channel and hydrophobic shelf present in the bet family and
heavily reducing the druggability into the difficult category . however , the tryptophan at position
81 in the bet family , which forms the za channel and hydrophobic shelf ,
is a considerably smaller leucine , resulting in a loss of these features
and a decrease in predicted druggability ( figure 9h ) . also
as discussed , crebbp and ep300 show some similarity through the za
loop to the bet family , but also phip(2 ) and wdr9(a / b)(2 ) show some
similarity in the shape of the binding site with the same hydrophobic
shelf and above average druggability . trends
within the data set were then sought and rationalized by unique signatures
characterizing the binding pockets , leading to a new classification
of the bromodomains into groups with similar amino acids in key positions
and similar predicted druggabilities . this information
can be explored to select proteins for counterscreening or to identify
bromodomain inhibitors that can be explored in a target hopping approach . this work represents the first analysis of
this type for the bromodomain family and will prove useful for drug
discovery projects aiming to identify inhibitors of the acetyl - lysine
binding site of bromodomains . | [
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according to world health organization global status reports , 80% of the 347 million people with diabetes globally will die of cardiovascular disease , and it will be the 7th leading cause of death in 2030 . moreover , international diabetes federation ( idf ) predicts that people with metabolic syndrome have a fivefold greater risk of developing type 2 diabetes mellitus ( t2 dm ) . until now a quarter of the world 's adults has metabolic syndrome , and it is becoming more common due to a rise in obesity . in the future , it may overtake smoking as the leading risk factor for heart disease .
to define , metabolic syndrome is a cluster of metabolic abnormalities which includes hyperlipidemia ( elevated triglycerides ( tg ) , low serum high - density lipoprotein ( hdl ) cholesterol ) , hypertension , central obesity , and elevated blood glucose .
however , the risk of developing metabolic syndrome is closely linked to overweight , obesity , and lack of physical activity . furthermore , insulin resistance also may raise the risk for metabolic syndrome .
accumulating data reveals that the prevalence of this syndrome within individual cohorts varies with age , gender , and ethnicity .
the pathological factors responsible for this syndrome can be high waist measurement of 35 inches or more for women or 40 inches or more for men , a high triglyceride level of 150 mg / dl or higher ( the mg / dl is milligrams per deciliter the units used to measure triglycerides , cholesterol , and blood sugar . ) , and a low hdl cholesterol level sometimes called as good cholesterol because it helps to remove cholesterol from arteries . an hdl cholesterol level of less than 50 mg / dl for women and less than 40
mg / dl for men is a risk factor , and also high blood pressure of 130/85 mmhg or ( the mmhg is millimeters of mercury the units used to measure blood pressure ) high fasting blood sugar level between 100 and 125 mg / dl is considered prediabetes . a fasting blood sugar level of 126 mg / dl or higher is considered diabetes .
a fasting blood sugar level of 100 mg / dl or higher ( or being on medicine to treat high blood sugar ) is a metabolic risk factor . from investigations ,
metabolic syndrome has been found to be associated with a greater risk of coronary heart disease , stroke , diabetes , and cardiovascular mortality than the risk conferred by each one of its individual components . even though the etiology of metabolic syndrome has not yet established definitely , but growing incidences and pathetic states raise an alert to reduce the risk factors of metabolic syndrome through better lifestyle and the treatment with better therapeutics which are the cornerstones for the management of metabolic syndrome . during the last decade , it has been shown that pharmacological activations of peroxisome proliferator - activated receptors ( ppars ) are effective therapeutic approaches to correct some aspects of metabolic syndrome mainly hypertriglyceridemia ( fibrates ) and type 2 diabetes mellitus ( thiazolidinediones ) .
peroxisome proliferator - activated receptor ( ppar ) is a subfamily of nuclear hormone receptors [ 68 ] , that function as ligand - activated transcription factors to regulate various biological processes .
later on , a number of compounds were discovered which share the same characteristic of inducing peroxisome proliferation .
thus , they were named as peroxisome proliferators . in 1990 , the first receptor for these compounds was cloned from mouse liver and named it as peroxisome proliferator - activated receptor ( ppar ) .
shortly , it was realized that ppars in fact represent a group of three receptors ppar- , ppar-/ , and ppar- .
they control and regulate the expression of large number of genes involved in regulating the intermediary metabolism of glucose and lipids , homeostasis , adipogenesis , insulin sensitivity , immune response , cell growth , and differentiation [ 6 , 1215 ] .
a variety of endogenous and synthetic ligands for ppars have been identified , activation of ppars by a suitable ligand will result in the recruitment of coactivators and loss of co - repressors that remodel chromatin and activate gene transcription . furthermore its diverse distribution in tissue has been shown to have multiple functions upon activation such as adipogenesis , fatty acid oxidation , and anti - inflammation . during the last decade it has been extensively demonstrated that risk factors of metabolic syndrome often associated with obesity , characterized by macrophage infiltration and activation in adipose tissue and liver can be treated by ppars targets .
in fact , inflammation a major determinant of health complications seen in overweight and obesity , which underscores the link between nutrition , metabolic organs , and the immune system , can be regulated by ppars targets [ 1921 ] .
this review focuses on the characterization of ppars family , mechanism of action , ligand selectivity , and physiologic role of the ppar family and then discusses the understanding of the pathogenic roles of metabolic syndrome and its treatment with ppars agonists , with particular focus on the therapeutic potentials of ppar modulators in the treatment of thrombosis .
principally , four functional domains have been identified in ppars , called a / b , c , d , and e / f ( figure 1 ) .
the n - terminal a / b domain contains a ligand - independent activation function 1 ( af-1 ) , responsible for the phosphorylation of ppar .
the dna binding domain ( dbd ) or c domain promotes the binding of ppar to the peroxisome proliferation response element ( ppre ) in the promoter region of target genes . the d domain or co -
the e / f domain or ligand - binding domain ( lbd ) is responsible for ligand specificity and activation of ppar binding to the ppre , which increases the expression of targeted genes .
recruitment of ppar cofactors is to assist the gene transcription processes carried out by the ligand - dependent activation function 2 ( af-2 ) , which is located in the e / f domain .
substantial progress has been made to delineate the molecular mechanisms that mediate ppar - regulated gene expression and associated cellular functions . in the classical model of ppar activation ,
ppar with rxr nuclear receptor is heterodimerized with ppre termed dr-1 , which consists of direct repeats of aggtca separated by a single intervening nucleotide [ 25 , 26 ] .
activation of transcription through this dimer is blocked by associated corepressor proteins , such as nuclear receptor corepressors ( ncor ) , histone deacetylases ( hdac ) , and g - protein pathway suppressor 2 ( gps2 ) [ 27 , 28 ] .
the addition of ligand causes dissociation of the corepressor proteins followed by the recruitment of coactivators such as ppar coactivator ( pgc-1 ) , the histone acetyltransferase p300 , creb binding protein ( cbp ) , and steroid receptor coactivator ( src)-1
. formation of the ppar activation complex leads to histone modification ( e.g. , through acetylation ) and altered expression of genes involved in fatty acid metabolism , lipid homeostasis , and adipocyte differentiation [ 29 , 30 ] . like ppars ,
the formation of different heterodimers seems to influence promoter recognition on the target gene sequences resulting in various metabolic processes .
furthermore , activation of ppars by natural and synthetic ligands , other factors such as rxr , ppres and cofactors also play a fundamental role in the process of desired transcription .
although ppar is commonly parked on dr-1 with rxr waiting for a ligand to activate it , there appear to be other modes of ppar action . as with other nuclear receptors , heat shock proteins ( hsp )
the association of ppar with hsps , and perhaps other proteins , may keep ppar in the cytoplasm until the appropriate ligand binds the ppar in lbd , leading to protein dissociation and nuclear import of ppar . in an even simpler scenario , ppar may remain soluble , presumably in the nucleus .
another pattern for ppar action involves its heterodimerization with a nuclear receptor other than rxr , such as the thyroid hormone receptor ( thr ) . in these cases , the dna pattern recognized by the heterodimer may vary from the usual dr-1 pattern .
finally , activation of ppar by its ligand may allow it to associate with other transcription factors , such as p65 or c - jun .
the binding of ppar with p65 will prevent completion of signaling through the nf-b pathway , and binding with c - jun will interfere with ap-1 signaling pathway .
from elucidated crystal structure studies of ppar , the divergent amino acid sequence in the lbd of the three ppar isoforms is thought to provide the molecular basis for ligand selectivity . and
a large ligand - binding pocket ( 1300 ) is found to exist in all three ppar isoforms , allowing diverse and structurally distinct compounds access to the lbd and enabling ppar to sense a broad range of endogenous and exogenous compounds .
a variety of endogenous and exogenous compounds , including fatty acids and their metabolites , industrial chemicals such as herbicides and plasticizers as well as synthetic pharmaceutical agents have been shown to bind to activate ppar .
these ligand activated ppars regulate metabolic activities leading to fa catabolism , lipid storage , glucose metabolism , cardiovascular risks and other effects , such as those affecting inflammation [ 3436 ] .
a variety of ligands , including n-3 and n-6 fatty acids ( fas ) , eicosanoids , and a few endocannabinoids and phospholipids , have been identified as ppar endogenous ligands .
although many fatty acids are capable of activating all three ppar isoforms [ 37 , 38 ] , some preference for specific fatty acids by each ppar has been demonstrated .
table 1 shows fatty acids and their derivatives , including 8-hydroxyeicosatetraenoic acid , the arachidonic acid lipoxygenase metabolite ltb4 , and arachidonate monooxygenase metabolite epoxyeicosatrienoic acids which have been shown to potently activate ppar- [ 33 , 37 , 39 ] .
synthetic lipid - lowering drug fibrates and clofibrates are also potent ligands for activating ppar- and are clinically proved to be lipid lowering drugs [ 4042 ] .
endogenous arachidonic acid cyclooxygenase metabolite prostacyclin , the linoleic acid 15-lipoxygenase-1 product 13-s - hydroxyoctadecadienoic acid and synthetic compounds including l-165041 and gw2433 have been found to be selective ppar-/- ligands [ 43 , 44 ] . naturally occurring ppar- ligands including 15-deoxy-(12,14)-prostaglandin j2 and
oxidized metabolites of linoleic acid 9-hydroxy- and 13-hydroxy - octadecadienoic acids have been identified [ 46 , 47 ] .
furthermore , synthetic antidiabetic tzd including rosiglitazone ( avandia ) and pioglitazone ( actos ) are potent ppar- selective agonists and have been very effective in improving glycemic control via insulin sensitization ( see table 1 ) .
furthermore , the most crucial factor noticed is that the activity of these ligands depends on their presence in cells or tissues enriched in ppars , their binding specificity toward the different ppars and the availability of coregulators that can act either as coactivators or corepressors of transcription . given the variety and anatomic distribution of endogenous ppar ligands , and the combinations in which they occur depending on physiological ( e.g. , abundance and composition of food , physical activity ) and pathophysiological conditions ( e.g. , hyperlipidemia , hypertension , diabetes , chronic inflammation , cancer , and atherosclerosis ) , and it is difficult to carefully evaluate the roles of each ppar ligand in a given cell at a fixed time - point , and this remains a major challenge in the field of investigation .
but it is tempting to speculate that the diversity of ppar functions has been acquired in association with the rich variety of ligands . with the development and clinical use of ppar ligands in the past decade
have greatly advanced our understanding of the physiological and pathophysiological roles of ppar and therapeutic implications of modulating these receptors ( see table 1 ) .
ppar- is most widely expressed in adipose tissue but is also expressed in immune / inflammatory cells ( e.g. , monocytes , macrophages ) , mucosa of the colon and cecum , and the placenta .
ppar- is essential for the differentiation and functioning of brown and white adipocytes and promotes the accumulation of lipids in the adipocytes .
there are three isoforms of ppar- well identified , namely , ppar-1 , ppar-2 , and ppar-3 and are derived from the same gene by the use of alternative promoters [ 50 , 51 ] .
ppar-1 differs by the presence of 30 amino acids extras in the n - terminus region .
ppar-1 and 3 rna transcripts translate into the identical ppar-1 protein . from animal studies , in knock - out mice
it was evident that ppars- is specific for adipocytes by expressing hypocellular adipocytes , developing insulin resistance in liver but not in muscles .
however , since ppar- expression is tissue dependent , ppar-1 is found in a broad range of tissues ; whereas ppar-2 is restricted to adipose tissue and ppar-3 is abundant in macrophages , large intestine , and white adipose tissue [ 5154 ] .
several investigations have elucidated that adipogenesis , glucose homeostasis , and lipid metabolisms are the major mechanisms of ppars- , and it is also involved in the improvement of insulin resistance and plays a key role in inflammation and neoplastic growth [ 5559 ] .
thiazolidinediones ( tzds ) are known for anti - diabetics ( troglitazone , pioglitazone , ciglitazona , and englitazone ) ( see table 1 ) .
these are a class of synthetic agonists that activate ppar- , whose properties are to improve insulin resistance and lower blood glucose levels in patients with t2dm60 . even with low concentration , ppar- it exhibits its effect in adipose tissue , liver , and muscle
several clinical studies have evaluated the efficacy of ppar- agonists ( troglitazone , pioglitazone , and rosiglitazone ) in the management of insulin resistance and t2 dm [ 6165 ] .
a number of studies have assessed the effects of ppar- agonists in the prevention of onset of t2 dm .
genomics studies have shown that ppar- is associated with several genes that affect insulin action .
in particular , ppar- appears to be associated with genes involved in fa transport , lipid droplet formation , and tag synthesis and breakdown .
furthermore , activation of ppar- increases adipocyte insulin sensitivity and this may be mediated in part by direct activation of genes encoding factors of the insulin signaling pathway [ 66 , 67 ] .
synthetic agonists are successful in insulin sensitivity by blocking the interaction of ppar- with co - repressors as a result there is an increase in the accumulation of lipids in the adipose tissue and a decrease in the release of free fatty acids .
the benefits of tzds are attributed to direct effects on lipid metabolism in adipose tissue and secondary effects on lipid and glucose metabolism in liver and skeletal muscle .
as elucidated , tzd treatment leads to adipocyte remodeling as a result of selective preadipocyte differentiation in subcutaneous depots and apoptosis of older and larger insulin - resistant visceral adipocytes .
furthermore activation of ppar- by tzds enhances lipolysis of circulating tgs and their storage in adipose tissue .
in addition , there are also numerous studies that show a direct effect of these drugs on the pancreatic -cells through a reduction of the lipotoxicity on the islets of pancreas , and the mechanism of action is not yet clear , but the transcriptional repressor b - cell lymphoma-6 ( bcl-6 ) was found to play a crucial role in reducing lipotoxicity [ 71 , 72 ] . however , ppar- activation by tzds agonists modulates the insulin signal transduction pathway by increasing the expression of intracellular proteins such as c - cbl - associated protein ( cap ) , which is predominant in insulin - sensitive tissues and correlates well with insulin sensitvity .
moreover , ppar- triggers an increase in plasma concentrations of adiponectin , a hormone secreted from adipose tissue that is found at low levels in plasma of patients with t2 dm .
overall functions of adiponectin are to increase fa oxidation in liver and skeletal muscle , improve insulin sensitivity in skeletal muscle and liver , and decrease glucose production in liver , resulting in decreased circulating ffas and tg and glucose levels in liver .
in addition , ppar- affects insulin sensitivity by regulating adipocyte hormones , cytokines , and proteins that are involved in insulin resistance .
in fact , the tzds agonists are well known to have a greater effect on the secretion of adipokines . where the action of insulin is suppressed in the adipocytes and macrophages ,
both produce inflammatory cytokines such as tnf- and il-6 , and express tlrs . since free fa levels are elevated in obesity , their effects may be mediated by tlrs which are thought to connect metabolism to innate immunity by secretion of inflammatory cytokines and chemokines .
other adipokines are overregulated , particularly adiponectin and resistin which are known to be potential insulin sensitizers in liver and skeletal muscle [ 75 , 76 ] .
a recent study showed that metabolic syndrome is associated with an increased risk of all - cause cancer mortality in men . a possible explanation for the increased cancer risk connected to metabolic syndrome , elevated nocturnal free - fatty acids and hyperinsulinemia , which were found in obese animals .
additional pathophysiological mechanisms , which link metabolic syndrome to cancer , are elevated adipokines levels , igf , the mitogenic action of insulin , and increased levels of reactive oxygen species .
this pathological state can be regulated with tzds agonists ( see table 1 ) .
as well known insulin is the most potent physiological anabolic agent , promoting the storage and synthesis of lipids , protein , and carbohydrates and inhibiting their breakdown and release into the circulation . the first step by which insulin increases energy storage or utilization involves the regulated transport of glucose into the cell , mediated by the facilitative glucose transporter glut4 .
insulin increases glucose uptake mainly by enriching the concentration of glut4 proteins at the plasma membrane , rather than by increasing the intrinsic activity of the transporter [ 81 , 82 ] .
ppar- activation by rosiglitazones was found to increase the expression and translocation of the glucose transporters glut1 and glut4 to the cell surface , thus increasing glucose uptake in adipocytes and muscle cells and reducing glucose plasma levels [ 83 , 84 ] .
activation of ppar- by tzds decreases glycated hemoglobin ( hba1c ) and fasting and postprandial glucose and lowers circulating insulin levels in patients with t2 dm , largely as a consequence of the improvement in insulin sensitivity .
furthermore , tzds stimulate the use of glycerol for tg production , thereby reducing ffa release from adipocytes and this reduction in ffas alleviates lipotoxicity in skeletal muscle , liver , and pancreas , leading to a reduction in hepatic glucose production and improved glucose utilization in skeletal muscles , resulting in the hypoglycemic effects of tzds .
the changes in glucose homeostasis can also be partly attributed to a direct action of ppar- activation on insulin - stimulated glucose disposal . moreover ,
t2 dm is associated with a state of chronic inflammation of fat cells that secrete elevated levels of cytokines ; agonists of ppar- were shown to inhibit the expression of cytokines such as resistin , tumor necrosis factor a ( tnf ) , and interleukin-6 which promote insulin resistance .
ppar- agonists trigger an increase in plasma concentrations of adiponectin , a hormone secreted from adipose tissue that is found at low levels in plasma of patients with t2 dm . adiponectin increases fa oxidation in liver and skeletal muscle .
overall , adiponectin improves insulin sensitivity in skeletal muscle and liver and decreases glucose production in liver , resulting in decreased circulating ffas and tg and glucose levels .
interestingly ppar- was the first reported to undergo agonist - dependent simulation , which promotes binding to nuclear receptor co - repressor-1 protein ( ncor ) and stabilizes association with promoter - bound nf-b , thereby leading to the transrepression of inflammatory genes in macrophages [ 8688 ] .
other beneficial and inhibitory effects of ppar- agonists on inflammation were reduction in the production of proinflammatory molecules in t lymphocytes , promotion of the expression of anti - inflammatory mediators in the innate immune system , reduce cytokines ( tnf- , il-1 , and il-6 ) production by inhibition of genes encoding pro - inflammatory molecules , and reduction of transcriptional activities nuclear factor-b ( nf-b ) , ap-1 , and stat [ 89 , 90 ] .
ppar- also reduces vascular smooth muscle cell proliferation , increases monocyte apoptosis , and suppresses metalloproteinase-9 expression in atherotic plaques [ 9194 ] . during atherogenesis ppar-
first , monocytes are attracted to the vascular wall of large arteries by adhering to them through integrins l and integrins endothelial cell activation .
monocytes infiltrate to the subendothelial space by a chemokine gradients , such as il-8 , which originates from the source of infection , where they will be differentiated to macrophages .
this key step is already altered in response to activation of ppar-. thus , troglitazone ( tzd ) inhibits the early formation of injury atherosclerotic , decreasing the accumulation of macrophages in the intimate through the inhibition of monocyte transendothelial migration .
in addition , ppar- agonists may indirectly suppress systemic production of a proinflammatory milieu mainly by inhibiting tnf- , plasminogen activator inhibitor-1 , and il-6 expression in adipose tissue [ 95 , 96 ] .
elevated levels of hdl cholesterol and reduced triglyceride levels may also contribute to the beneficial effect of ppar- agonists in atherosclerosis .
on the basis of these data , it seems likely that tzd ppar- agonists will have beneficial effects on atherosclerosis and provide a promising therapy for the metabolic syndrome and its cardiovascular complications .
novel antagonist and partial agonists of ppar- have recently been identified ; triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid ( cddo ) is a partial agonist with anti - inflammatory properties and bisphenol diglycidyl ether ( badge ) and lg-100641 have been identified as antagonists for ppar- ( see table 1 ) [ 99 , 100 ] .
even though these compounds have little clinical significance , they can be used to understand the physiology of the ppar- and for the identification of new ligands .
in addition to synthetic chemical methods , research in natural products has also yielded potent ppar- agonists from several medicinal plants . saurufuran a from saururus chinensis ( saururaceae ) , flavonoids such as chrysin , apigenin and kaempferol , and phenolic compounds from glycyrrhiza uralensis ( fabaceae ) are recently identified ppar- agonists to treat risk factors of metabolic syndrome .
ppar- belongs to the nuclear receptor superfamily and was the first to be identified as ppar receptor .
it was named based on the ability to be activated by substances leading to the proliferation of peroxisomes in rodents .
ppar- is expressed in numerous tissues , both in rodents and in humans , for example , in liver , kidneys , heart , skeletal muscle , and brown fat [ 104 , 105 ] , and in a wide range of vascular cells such as endothelial cells , vascular smooth muscle cells ( vsmcs ) , and monocytes / macrophages [ 106109 ] .
accumulating evidence demonstrates that ppar- is an important modulator of the metabolic syndrome and may be a therapeutic target for treating some of its features , especially cardiovascular complications .
ppar- has been identified as a key regulator of the genes involved in fatty acid oxidation , which occurs in mitochondria , peroxisomes , and microsomes in the liver .
transcription and protein levels of critical enzymes in -oxidation and -oxidation pathways are direct targets of ppar- , including acyl coa oxidase , carnitine palmitoyl transferase i , mitochondrial hydroxymethylglutaryl coa synthase , and cytochrome p450 4a enzymes ( cyp4a ) . by increasing the expression of these genes , ppar- ligands significantly activate hepatic fatty acid oxidation , whereas genetic inactivation of the ppar- gene results in massive accumulation of lipids in the liver , severe hypoketonemia , hypoglycemia , hypothermia and elevated plasma free fatty acid levels . these data clearly indicate that ppar- is a key factor in governing metabolic adaptation to increased fatty acids .
ppar- is also known to regulate the expression of genes for the transport of proteins and enzymes that are involved in the processes of inflammation .
many lines of study show that ppar- regulates lipid homeostasis . by increasing -oxidation of fatty acids and providing energy to the cell
, it also cuts the long - chain fatty acids , thereby preventing the accumulation and toxicity of lipids in cells .
in addition , they also stimulate the cellular uptake of fatty acids by increasing the expression of the fatty acid transport protein ( fatp ) and fatty acid translocase ( fat ) .
its known target genes are involved in almost all aspects of lipid metabolism , including uptake , binding , and oxidation of fatty acids ; lipoprotein assembly ; and lipid transport [ 115 , 116 ] .
synthetic ppar- ligands , such as gemfibrozil , fenofibrate , and clofibrate ( see table 1 ) , have been used in clinical practice as hypolipidemic agents for more than 3 decades .
notably , activation of ppar- with synthetic agonists not only increases hepatic fao , but it also increases lipogenesis and fa chain elongation , in a sterol regulatory element binding protein ( srebp)-1c dependent manner .
this indicates that ppar- induces the entire fa handling regimen in liver to either catabolize it or store it , thereby diminishing cytotoxic effects of free fa . as evident ppar-
is highly expressed in liver and activation of this receptor promotes the expression of cytochrome p4504a ( cyp4a ) .
the cyp4a is a subclass of cytochrome p450 enzyme that catalyzes the -hydroxylation of fatty acids which is beneficial in reducing the synthesis of triglycerides ( tgs ) .
in addition , ppar- agonists are used for the treatment of dyslipidemia , which is characterized by decreased triglycerides levels and increased hdl - c levels in plasma .
this state can be achieved by increasing the production of the major component of hdl - c , called apolipoprotein a - i and a - ii ( apo a - i & apo a - ii ) [ 118 , 119 ] which plays a vital role in reverse cholesterol transport ( rct ) pathway from peripheral cells . moreover , ppar- ppar - activation further decreases tg levels by amplifying the expression of lipoprotein lipase ( lpl ) and inhibiting apo c - iii in the liver .
the biological mechanism of ppar- is activated under nutrient - deficient conditions and is necessary for the process of ketogenesis , a key adaptive response to prolonged fasting . in an experiment that pharmacologically blocked transport of long - chain fatty acids into mitochondria , knockout mice developed hepatic steatosis , severe hypoketonemia , hypoglycemia , and hypertriglyceridemia .
this indicates that genetic inactivation of ppar- gene results in massive accumulation of lipids in the liver .
metformin is most commonly used in the initial management of t2 dm , but its mechanisms by which it lowers glucose levels are not completely known .
t2 dm patients with metformin treatment reduce the production of hepatic glucose via gluconeogenesis decrease .
it is believed that metformin exerts its action via incretins , increasing levels of glp-1 ( glucagon - like peptides ) via ppar- dependent or independent mechanism .
activation of ppar- reduces weight gain in rodents , and inactivation of ppar- results in a late onset of obese phenotype [ 124 , 125 ] .
treatment of ppar- null mice with a high - fat diet leads to a more dramatic increase in body weight , further suggesting that ppar- may be involved in the genesis of obesity .
treatment with ppar- activators dramatically improved insulin resistance and glycemic control in type 2 diabetic db / db mice and oletf rats [ 127129 ] .
similarly , the ppar- agonist bezafibrate markedly improved glucose intolerance and insulin resistance in a lipoatrophic diabetic patient .
more importantly , recently it was reported that the ppar- agonist fenofibrate prevents the development of diabetes in insulin - resistant obese oletf rats . although the downstream mechanisms underlying these observations are not clear , they are consistent with the idea that ppar- plays a critical role in regulating insulin sensitivity in vivo and that its activation may lead to the delay of onset of type 2 diabetes .
activation of ppar- in endothelial cell receptor will block the cellular adhesion induced by cytokines and increase the expression of the hdl in cla-1/sr - bi receptor and the cholesterol efflux pump atp binding cassette a-1 ( abca1 ) transporter in macrophages .
some studies suggest that activation of ppar- increased the expression of abca1 by enhancing the expression of the liver x receptor , lxr- .
finally , fibrate ppar- activators have been reported to potently reduce atherosclerosis both in apoe mice and in human apoai transgenic apoe mice . in heart , ppar- supplies energy to the myocardium by regulating the genes responsible for fatty acid uptake and -oxidation .
this function is achieved by decreasing fatty acid oxidation and inhibiting lipoprotein lipase ( lpl ) .
consequently all these mechanisms have been reducing progression of atherosclerosis and decreasing the incidence of coronary events in several clinical studies .
more important , fibrate treatment of patients who exhibit more than three features characteristic of the metabolic syndrome ( diabetes , glucose intolerance or high fasting insulin , hypertension , obesity and high triglycerides or low hdl cholesterol ) was associated with a significant 35% risk reduction in the rate of coronary artery disease death , nonfatal myocardial infarction , or definite stroke [ 137139 ] .
these data support the concept that fibrate ppar- agonists may be particularly effective agents for the cardiovascular complications of the metabolic syndrome .
ppar- agonist 's exhibit anti - inflammatory effect in vascular cells by inhibiting the production of some inflammatory cytokines such as tnf- , il-6 in blood , and decreased the expression of cyclooxygenase-2 in vsmcs by nf-b signaling repression in cardiac myositis and induced expression of vacam-1 and increased expression of endothelial nitric oxide synthase ( enos ) .
moreover , wy14.643 , a potent agonist of ppar- , can directly increase the expression of adiponectins , antidiabetics , antiatherosclerosis , and anti - inflammatory effects .
ppar-/- has important functions in the skin , gut , placenta , skeletal and heart muscles , adipose tissue , and brain .
contrary to ppar- and ppar- , ppar- is expressed all over the body , but its pharmacology is less understood than the other subtypes .
it is the most abundant isoform among the three ppars in skeletal muscle . since skeletal muscle accounts for about 50% of whole body mass , and more
therefore , activities involved in muscle contraction may significantly increase energy expenditure and result in the usage of glucose or breakdown of fat as fuel .
it can be greatly beneficial because it decreases triglycerides and ldl - cholesterol ( bad cholesterol ) levels and increases insulin sensitivity and hdl - cholesterol ( good cholesterol ) levels .
since metabolic syndrome is a problem caused by too much fat stored in the body , ppar- has been recognized to be a possible solution because it makes the body burn more fat .
fat is the main source of fuel for endurance exercise ; therefore , ppar- is produced for increasing the breakdown of body fat to generate energy .
indeed , the increase of ppar- helps to lose fat and exercise longer because it facilitates the use of fat .
so far reported candidates for endogenous activators of ppar-/- are fatty acids , triglycerides , and prostacyclin . in addition a number of synthetic compounds including l-165,041 , gw501516 , gw0742 , and gw610742 ( see table 1 ) [ 144 , 146149 ] have also been designed as selective ppar-/- ligands . unlike ppar- ( fibrates ) or - , ( glitazones ) there are no ppar-/- drugs in clinical use yet , though lead compounds such as gw501516 are in phase ii clinical trials for dyslipidaemia . as
yet only one selective ppar-/- antagonist has been described gsk0660 . in skeletal muscle myoblast cells in culture
, gsk0660 inhibits gw0742 induction of established ppar-/- target genes ( carnitine palmitoyltransferase 1a , angiopoietin - like 4 protein , and pyruvate dehydrogenase kinase-4 ) , along with the concurrent ppar-/- induced increase in fatty acid oxidation . as
yet this is the only report on gsk0660 , so still nothing is known regarding its long - term effects in vivo .
ppar-/- is the most abundant isoform among the three ppars in skeletal muscle ; it acts as a central regulator of fatty acid catabolism in skeletal muscle by controlling expression of proteins directly implicated in this metabolic pathway and also by increasing the intrinsic oxidative capability of the tissue . since skeletal muscle accounts for about 50% of whole body mass and more than 50% of metabolism occurs in it .
therefore , activities involved in muscle contraction may significantly increase energy expenditure and result in the usage of glucose or breakdown of fat as fuel .
this is greatly beneficial because it decreases triglycerides and ldl - cholesterol ( bad cholesterol ) levels and increases insulin sensitivity and hdl - cholesterol ( good cholesterol ) levels .
since metabolic syndrome is a problem caused by too much fat stored in the body , ppar-/- has been recognized to be a possible solution because it makes the body burn more fat . since fat is the main source of fuel for endurance exercise ; therefore , ppar-/- is produced for increasing the breakdown of body fat to generate energy .
indeed , the increase of ppar-/- helps to lose fat and exercise longer because it facilitates the use of fat .
an experimental study using obese diabetic db / db mice as a model was examined to see the effect of a ppar-/- specific agonist l-165041 on plasma lipid profile .
l-165041 treatment significantly increases hdl cholesterol levels , possibly associated with a decreased lipoprotein lipase activity in the white adipose tissue .
confirmatory results were recently obtained with a more potent and selective ppar-/- agonist , gw501516 ( ki = 1.1 0.1 nm ) in insulin - resistant middle - aged obese rhesus monkeys .
these results showed that gw501516 causes a dramatic dose dependent increase in serum hdl cholesterol and a reduction in ldl and triglyceride , suggesting that activation of ppar-/- is associated with a less atherogenic lipid profile and antidiabetic action [ 152154 ] .
ppar-/- has also exhibited a potential role in placentation , adiposity , colorectal cancer , and diabetic factors .
gw0742 is a closely related analog of gw501516 and shows equivalent potency and selectivity for ppar-/- .
the role of ppar-/- in the regulation of glucose homeostasis has emerged with the findings that ppar-/- agonists reduce adiposity and improve glucose tolerance and insulin sensitivity in different mouse models of obesity . in addition , ppar-/- null mice display glucose intolerance when fed a chow diet .
the use of ppar-/- tissue - specific transgenic mice , in adipose tissue or skeletal muscle , has shown that activated ppar-/- induces the expression of genes involved in fa oxidation and in energy expenditure through the induction of uncoupling proteins in brown adipose tissue and in skeletal muscle [ 154156 ] . as a consequence ,
substrate supply for lipid storage in white adipose tissue is decreased , resulting in the reduction of adiposity .
it is also believed that ppar-/- induces fat burning in muscle , which together with an overall improvement in systemic lipid metabolism is responsible for lowering fat overload in insulin - sensitive tissues , thereby reducing insulin resistance . in a primate model of the metabolic syndrome ,
the ppar-/- selective agonist gw501516 dose - dependently lowered plasma insulin levels , without adverse effects on glycemic control .
similarly , in ob / ob mice , a model of the metabolic syndrome , a ppar-/- specific agonist markedly improved glucose tolerance and insulin resistance .
although the underlying mechanism is unclear , activation of ppar-/- in skeletal muscle , which has a significant role in insulin sensitivity , has been proposed to account for the beneficial metabolic effects of ppar-/- agonists on lipid profile and insulin resistance , possibly as a result of increased fatty acid catabolism , cholesterol efflux , energy expenditure [ 153157 ] , and oxidative capability in the muscle .
recently it was described that lipid peroxidation and the consequent production of 4-hne in -cells stimulate the secretion of insulin via a dependent mechanism , and these results were confirmed by treating these cells with an antagonist of this nuclear factor that resulted in blocking of the effect .
activation of ppar-/- with agonist prevents induction of the transcription factor stat3 by inhibiting the activation of erk and inhibiting the interaction of stat3 and hsp90 ; this translates into the prevention of insulin resistance in adipose tissue . in ppar-/- knockout mice it showed an obese phenotype when they were fed a diet rich in fats .
the overexpression of ppar-/- or activation by the ligand gw501516 shows induction of muscle fiber type i , cell type rich in mitochondria that allow mice to undertake large periods of aerobic activity , and for this reason they are called mouse marathon runner .
finally , with the availability of three synthetic ligands ( gw501516 , gw0742 , and l-165041 ) that activate ppar-/- at very low concentrations both in vivo and in vitro with high selectivity over other ppar isotypes had led to a huge increase in experimental studies on the role of ppar-/- in cellular processes .
the ic50 for these compounds assessed with recombinant human ppar-/- were 1.0 nm for gw0742 , 1.1 nm for gw501516 , and 50 nm for l-165041 [ 161 , 162 ] .
few studies have shown that ppar-/- ligands have the potential to inhibit cardiac hypertrophy due to their inhibitory activity on nf-b transcription factor which produces inflammatory cytokines such as tnf- , mcp-1 , and il-6 , and these are secreted by cardiac cells under various pathophysiological stimuli which may participate in myocardial inflammation . activated ppar-/- also dampens lps - induced tnf- inflammation signaling in cultured cardiomyocytes and blocks palmitate - induced inflammatory pathways in mouse heart and human cardiac cells through protein - protein interaction between ppar-/- and p65 , suggesting inhibition of nf-b [ 164 , 165 ] . from these biological effects ( figure 3 )
, ppar-/- may serve as a potential therapeutic target to prevent cardiac hypertrophy and heart failure in metabolic disorders .
ppar-/- also attenuates progressive cardiac fibrosis occurring in diabetic cardiomyopathy . in null mice , ppar-/- specific cardiomyocyte and macrophage
were infused with angiotensin ii to trigger cardiac fibrosis , and then treated with pioglitazone ; it is observed that the macrophage and not cardiomyocyte ppar-/- that attenuates fibrosis .
finally , ppar-/- has recently been proposed as a potential target for modulating foam cell and macrophage activation in atherosclerosis . in vitro studies suggested that ppar-/- activation in cultured macrophage results in increased expression of the reverse cholesterol transporter atp - binding cassette a1 and enhances efflux of cholesterol .
ppar-/- also participates in cellular vldl sensing and mediates vldl - triglyceride - driven transcription events in macrophage .
vldl - triglyceride treatment results in triglyceride accumulation and the induction of adipocyte phenotype , which can be blocked by disruption of the ppar-/- gene .
its synthetic ligands gw0742 and gw501516 reduce atherosclerosis in low - density lipoprotein receptor ( ldlr ) null mice , possibly by decreasing monocyte chemotactic protein-1 ( mcp-1 ) , intercellular adhesion molecule-1 , and tnf- expression [ 166 , 167 ] .
these new observations suggest that agonists for par-/- may be effective agents to reverse cholesterol deposition in foam cells in atherotic lesions and therefore decrease cardiovascular disease associated with the metabolic syndrome .
taken together , ppar-/- is a critical player in the pathogenesis of the metabolic syndrome , and its ligands may provide useful agents for treating dyslipidemia , obesity , insulin resistance , and atherosclerosis .
the role of ppar ligands has been well established in some very important therapeutic areas such as diabetes , obesity , cardiovascular diseases , and inflammation .
but , more recently , it is becoming clear that they are also involved in antithrombosis .
diabetes mellitus is associated with a heightened risk of developing atherosclerotic vascular disease and its acute thrombotic complications , such as myocardial infarction .
interestingly platelets play an important role in hemostasis and thrombosis , and there is an increasing evidence showing they are involved in mechanisms of inflammation and host defense , contributing to the pathogenesis and progression of vascular complications in the t2 dm .
therefore , treatment with antiplatelet agents may be a beneficial strategy to prevent and improve thrombosis - related cardiovascular diseases .
platelets are enucleated cells derived from megakaryocytes ; they contain transcription factors , notably the peroxisome proliferator - activated receptors ( ppars ) .
so far , three ppar isoforms ( ppar- , ppar-/- , and ppar- ) have been found in human platelets , and upregulation of ppars inhibits platelet activation through a nongenomic mechanism .
platelet activation is associated with signaling that affects cell shape and spreading , secretion , and the release of multiple prothrombotic factors ; all through the binding of plasma fibrinogen and von willebrand factor ( vwf ) to integrin iib3 , this leads to the formation of a stable platelet thrombus [ 172174 ] .
many findings suggest that agents with ppars - activating effect may exert an anti - platelet activity .
a recent study was done by ching - yu shih and group to prove that ppars - mediated pathways contribute to the anti - platelet activity .
they used a natural product , magnolol , extracted from chinese medicinal herbs , which has demonstrated multiple pharmacological effects , including antiatherosclerosis , antioxidative , anti - inflammatory , and anti - bacterial , even anti - platelet activity [ 176 , 177 ] .
magnolol is a ppar- agonist through direct binding to the ppar- ligand binding domain it exhibit the anti - platelet activity and inhibits various important mediator formation and signaling pathways related to platelet activation . in the presence of selective ppar- antagonist ( gsk0660 ) or ppar- antagonist ( gw9662 ) , the inhibition of magnolol on collagen - induced platelet aggregation and intracellular ca mobilization was significantly reversed .
these show that the excellent anti - platelet and antithrombotic activities of magnolol are modulated by upregulation of ppar-/--dependent pathways .
other findings of anti - platelet activity are lipid - lowering agents such as fibrates and statins that reduce thrombotic and cardiovascular risk .
these are hypolipidemic drugs which decrease cardiac events in individuals without raised levels of cholesterol . in platelets ,
simvastatin and fenofibrate drugs inhibit platelet activation by inhibiting pkc which are associated with ppars .
selective ppar- antagonist gw9662 , and ppar- antagonist gw6471 showed inhibition on effects of simvastatin and fenofibrate on platelet function which are mediated by ppar- and ppar- , respectively , and the aggregation effects of -lipoic acid can be attributed to the activation of ppar-/- . in another study
they demonstrate the ability of ppar- ligands to modulate collagen stimulated platelet function and suppress activation of the glycoprotein vi ( gpvi ) signaling pathway .
ppar- ligands inhibited collagen - stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and p - selectin exposure .
the incorporation of gw9662 antagonists reversed the inhibitory actions of ppar- agonists , implicating ppar- as modulator .
furthermore , ppar- ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the gpvi signaling pathway .
all this association was prevented by ppar- agonists , indicating a potential mechanism for ppar- function in collagen - stimulated platelet activation .
treatment and prevention of metabolic syndrome require lifestyle changes , including weight reduction , increased physical activity , and better diet .
however , as many patients can not control the pathology with lifestyle modification , there is a need for drugs to manage the metabolic syndrome . as discussed in this paper , that ppars are transcription regulators which are involved in different metabolic pathways , they are able to interfere with many normal cellular processes as well as in altered processes that ultimately lead to pathology . this nuclear hormone receptor is believed to originate from a common ancestral receptor of 600 million years old .
furthermore it has managed to differentiate into several subtypes that can adapt to different ligands . its large capacity and strict control location allow these nuclear regulators to control complex processes such as inflammation or control energy homeostasis .
they are also attributed for their ability to regulate cellular differentiation in numerous cell lines such as keratinocytes , adipocytes , or immune system cells [ 182 , 183 ] .
another factor which makes them particularly interesting is its ability to be activated or repressed by internal or natural ligands and synthetic or exogenous ligand ; this capability opens thousands of possibilities in the treatment of various pathologies by numerous processes that are capable of controlling genes [ 184186 ] .
the pharmacological race generated new and increasingly potent agonists or antagonists , and it has prompted pharmaceutical companies to invest large sums of money on science , but nothing had been positive ; unfortunately adverse effects have been reported for many synthetic compounds .
for instance , one of the most important problems of ppar agonists is cardiac toxicity which changes in the qt segment of the ecg , although the results obtained for this were controversially fair .
now recently it was confirmed that therapeutic concentrations of aleglitazar ( dual agonist ) show no evidence of changes in the qt segment of ecg .
consequently , the new dual agonist of ppar alpha and gamma shows great advantages over its predecessors because the results obtained in phase i and ii show a clear balance between patient safety and efficacy in improving metabolic parameters [ 187191 ] and are extremely encouraging to start phase iii .
clinical and basic sciences are on a quest to find double or triple agonists ( pan ) in which the unwanted effects of agonists can be decreased significantly , and as a result individuals get a better quality of life with more potent drugs that result in increasingly lower doses ( ic50 under toxic levels ) .
furthermore , with the development of ppar delta targets , it has been shown that this nuclear regulator has become a therapeutic target in combating cardiovascular diseases that is more common in industrialized countries .
ppar delta targets will regulate the level of lipid oxidation in skeletal muscle and regulate the ratio of ldl and high density lipids [ 192 , 193 ] ; it is even able to inhibit the formation of foam cells induced by the small , dense ldl .
recent studies have used gw501516 ( a potent agonist of ppar delta ) which show a change in the lipoprotein profile , and highly atherogenic profile was passed to onefold less , proving to be a powerful cardiovascular protector for individuals with ms .
it is evident that platelets are extremely important in ischemic cardiovascular diseases , and it has been demonstrated that the greater number of active circulating platelets or a greater number of platelet - leukocyte complexes predict larger plates with greater lipid accumulation .
this fact evidences that rosiglitazone is capable of reducing the amount of circulating activated platelets and makes these compounds an ideal therapeutic target to control atherogenesis [ 196198 ] . above all
it is tempting to speculate that the diversity of ppar functions has been acquired in association with the rich variety of ligands . with the development and clinical use of ppar ligands in the past decade
have greatly advanced our understanding of the physiological and pathophysiological roles of ppar and therapeutic implications of modulating these receptors .
surely the solution to metabolic diseases can be found in a drug response with super powers or with a combination of drugs that individually present positive effects and enhance the whole colud open pandora 's box , which could result in a real treatment . | metabolic syndrome is estimated to affect more than one in five adults , and its prevalence is growing in the adult and pediatric populations .
the most widely recognized metabolic risk factors are atherogenic dyslipidemia , elevated blood pressure , and elevated plasma glucose .
individuals with these characteristics commonly manifest a prothrombotic state and a proinflammatory state as well .
peroxisome proliferator - activated receptors ( ppars ) may serve as potential therapeutic targets for treating the metabolic syndrome and its related risk factors .
the ppars are transcriptional factors belonging to the ligand - activated nuclear receptor superfamily .
so far , three isoforms of ppars have been identified , namely , ppar- , ppar-/ , and ppar-. various endogenous and exogenous ligands of ppars have been identified .
ppar- and ppar- are mainly involved in regulating lipid metabolism , insulin sensitivity , and glucose homeostasis , and their agonists are used in the treatment of hyperlipidemia and t2 dm . whereas ppar-/ function is to regulate lipid metabolism , glucose homeostasis , anti - inflammation , and fatty acid oxidation and its agonists
are used in the treatment of metabolic syndrome and cardiovascular diseases .
this review mainly focuses on the biological role of ppars in gene regulation and metabolic diseases , with particular focus on the therapeutic potential of ppar modulators in the treatment of thrombosis . | 1. Introduction
2. Peroxisome Proliferator-Activated Receptors (PPARs)
3. Ligands for PPAR Isoforms
4. PPAR-
5. PPAR-
6. PPAR-
7. Other Biological Mechanisms
8. Conclusions and Future Prospects | to define , metabolic syndrome is a cluster of metabolic abnormalities which includes hyperlipidemia ( elevated triglycerides ( tg ) , low serum high - density lipoprotein ( hdl ) cholesterol ) , hypertension , central obesity , and elevated blood glucose . during the last decade , it has been shown that pharmacological activations of peroxisome proliferator - activated receptors ( ppars ) are effective therapeutic approaches to correct some aspects of metabolic syndrome mainly hypertriglyceridemia ( fibrates ) and type 2 diabetes mellitus ( thiazolidinediones ) . shortly , it was realized that ppars in fact represent a group of three receptors ppar- , ppar-/ , and ppar- . they control and regulate the expression of large number of genes involved in regulating the intermediary metabolism of glucose and lipids , homeostasis , adipogenesis , insulin sensitivity , immune response , cell growth , and differentiation [ 6 , 1215 ] . a variety of endogenous and synthetic ligands for ppars have been identified , activation of ppars by a suitable ligand will result in the recruitment of coactivators and loss of co - repressors that remodel chromatin and activate gene transcription . this review focuses on the characterization of ppars family , mechanism of action , ligand selectivity , and physiologic role of the ppar family and then discusses the understanding of the pathogenic roles of metabolic syndrome and its treatment with ppars agonists , with particular focus on the therapeutic potentials of ppar modulators in the treatment of thrombosis . , through acetylation ) and altered expression of genes involved in fatty acid metabolism , lipid homeostasis , and adipocyte differentiation [ 29 , 30 ] . a variety of endogenous and exogenous compounds , including fatty acids and their metabolites , industrial chemicals such as herbicides and plasticizers as well as synthetic pharmaceutical agents have been shown to bind to activate ppar . there are three isoforms of ppar- well identified , namely , ppar-1 , ppar-2 , and ppar-3 and are derived from the same gene by the use of alternative promoters [ 50 , 51 ] . several investigations have elucidated that adipogenesis , glucose homeostasis , and lipid metabolisms are the major mechanisms of ppars- , and it is also involved in the improvement of insulin resistance and plays a key role in inflammation and neoplastic growth [ 5559 ] . on the basis of these data , it seems likely that tzd ppar- agonists will have beneficial effects on atherosclerosis and provide a promising therapy for the metabolic syndrome and its cardiovascular complications . saurufuran a from saururus chinensis ( saururaceae ) , flavonoids such as chrysin , apigenin and kaempferol , and phenolic compounds from glycyrrhiza uralensis ( fabaceae ) are recently identified ppar- agonists to treat risk factors of metabolic syndrome . ppar- has been identified as a key regulator of the genes involved in fatty acid oxidation , which occurs in mitochondria , peroxisomes , and microsomes in the liver . by increasing the expression of these genes , ppar- ligands significantly activate hepatic fatty acid oxidation , whereas genetic inactivation of the ppar- gene results in massive accumulation of lipids in the liver , severe hypoketonemia , hypoglycemia , hypothermia and elevated plasma free fatty acid levels . in addition , ppar- agonists are used for the treatment of dyslipidemia , which is characterized by decreased triglycerides levels and increased hdl - c levels in plasma . more important , fibrate treatment of patients who exhibit more than three features characteristic of the metabolic syndrome ( diabetes , glucose intolerance or high fasting insulin , hypertension , obesity and high triglycerides or low hdl cholesterol ) was associated with a significant 35% risk reduction in the rate of coronary artery disease death , nonfatal myocardial infarction , or definite stroke [ 137139 ] . taken together , ppar-/- is a critical player in the pathogenesis of the metabolic syndrome , and its ligands may provide useful agents for treating dyslipidemia , obesity , insulin resistance , and atherosclerosis . the role of ppar ligands has been well established in some very important therapeutic areas such as diabetes , obesity , cardiovascular diseases , and inflammation . platelets are enucleated cells derived from megakaryocytes ; they contain transcription factors , notably the peroxisome proliferator - activated receptors ( ppars ) . so far , three ppar isoforms ( ppar- , ppar-/- , and ppar- ) have been found in human platelets , and upregulation of ppars inhibits platelet activation through a nongenomic mechanism . selective ppar- antagonist gw9662 , and ppar- antagonist gw6471 showed inhibition on effects of simvastatin and fenofibrate on platelet function which are mediated by ppar- and ppar- , respectively , and the aggregation effects of -lipoic acid can be attributed to the activation of ppar-/- . | [
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molecular docking has had important recent successes and is now widely used in industry and academia . but whereas other techniques in computational biology such as homology modeling(13 ) and sequence database searching(14 ) have been successfully deployed on a proteomic scale , docking has remained manually intensive .
docking programs are challenging to use , with many parameters to be chosen , file formats to be manipulated , and decisions to be made at both the preparation and analysis stages .
even in expert hands , there are targets for which docking simply fails to recapitulate experimentally known binding information .
these barriers to entry have diminished the impact of the technique by making it less accessible to biologically oriented nonexperts and challenging even for specialists to deploy on a large scale .
one approach to make docking accessible to more investigators , and to make it more systematic even for experts , is to automate it .
we have therefore investigated an expert system , dock blaster , which aims to emulate experts at all stages of the docking process .
ideally , dock blaster could start from as little as a pdbaaabbreviations : rmsd , root mean squared deviation ; sar , structureactivity relationship ; pdb , protein data bank ; roc , receiver operator characteristic .
code and from that launch a full screen of a large compound library to find novel ligands . to do
so it must overcome substantial challenges in preparing a target site for docking , it must explore variation in the sampling and often scoring , and it must conduct control calculations to judge the quality of the screen .
for instance , the automated procedure must recognize common cofactors , metals , post - translational modifications , and solutes to identify the ligand and separate it from the receptor .
second , hot spots for docking must be identified for a wide range of binding site sizes and shapes .
third , parameters must be assigned to receptor atoms in a robust way that can cope not only with cofactors , metals , and post - translational modifications but also with unforeseen moieties for which no dictionary is available . because an expert would normally experiment with several variations in sampling and scoring ,
an automated system should do so also , picking the best parameters to use for a full database screen . finally , the entire docking process should be integrated from end to end so as to recover from simple problems , continue as far as possible , and end gracefully should an unrecoverable error occur .
abbreviations : rmsd , root mean squared deviation ; sar , structureactivity relationship ; pdb , protein data bank ; roc , receiver operator characteristic . here
the method is tested for pose - fidelity , the ability to reproduce experimentally observed poses within some tolerance limit , and enrichment , the ability to enrich actives from among a database of decoys , where a decoy is a member of the database that does not bind to the target .
we have used three of the most common benchmarking sets : the astex-85 set,(7 ) having 85 high - quality crystal structures of therapeutically relevant targets and drug - like ligands , the gold-114 benchmark,(15 ) derived from the most widely used docking benchmarks , and the dud set,(27 ) 38 protein targets for which sets of annotated actives and corresponding property - matched decoys are available for each target .
property - matched decoys have similar physical properties but different topologies that one would not expect should be recognized by the protein , a key requirement for a high score . whereas we find that the automated method is typically outperformed by an expert , its performance is nevertheless respectable . in principle
, such an automated procedure could bring docking to a much larger community and could be used to explore targets on a proteomic scale . for the first goal to provide more good than harm , it is important to provide what may often be nave users with an automated self - assessment of the docking results .
we investigate methods to do so using pose fidelity and enrichment based on a single observed crystallographic ligand .
to investigate the plausibility of the second goal , we describe here docking screens on 7755 protein targets where each screen is performed against a feasibility library of 100 molecules .
dock blaster is composed of an expert system engine and a web - enabled user interface ( figure 1 ) .
the dock blaster pipeline is composed of six modules ( figure 2 ) : ( a ) the parser , which identifies the receptor and ligand from a pdb file , ( b ) the scrutinizer , which attempts to correct for problems , such as incomplete or disordered residues on the receptor , ( c ) the preparer , which protonates the receptor , calculates hot spots and scoring grids , assigns atomic parameters , including these for cofactors , post - translational modifications and metals , and prepares the ligand , decoys , and any actives and inactives for docking , ( d ) the calibrator , which uses supplied data to assess docking performance and suggests optimal docking parameters , ( e ) the docker , which manages a full database screen on the computer cluster , and ( f ) the assessor , which prepares reports to interpret database screening results .
the parser uses dictionaries of common cofactors , ions , post - translational modifications , and solutes to identify and separate the ligand from the receptor .
if the ligand itself is a common solute or cofactor , a three letter code of the ligand must be specified .
if more than one ligand is available , the parser asks the user to pick one . in fully automated screens , such ambiguity stops the procedure .
if no ligand can be identified , the parser also halts the calculation with an error message .
a future version may use automatic binding site identification software to identify the binding site in this case .
the parser produces files that are ready to be used by the scrutinizer in the next step and may be accessed online at http://blaster.docking.org/parser.shtml .
the scrutinizer takes as input a target structure and a specification of the binding site , which may be either a docked ligand in mol2 format or atoms in or near the binding site in pdb format .
the scrutinizer checks that the receptor and ligand are properly formatted , and that at least one atom of the ligand is within a binding site on the protein .
this step also attempts to flesh out incomplete residues and pick the first of any disorder models present .
the scrutinizer can take input directly from the parser ( module a , above ) or from the job preparation page , http://blaster.docking.org/start.shtml .
this includes maturing the receptor model by removal of ordered water molecules , protonation of receptor atoms to a united atom model , and assignment of amber atom types(33 ) including to metals , cofactors , and post - translational modifications that are effectively part of the receptor .
subsequently docking hot spots are calculated by sphgen,(34 ) while van der waals and electrostatics scoring grids are calculated by chemgrid(35 ) and delphi,(36 ) respectively . to correct for ligand desolvation , solvmap(37 )
is used to calculate a solvent occlusion grid . to prepare a dockable database of the ligand and any actives and inactive controls ,
the pdb format ligand is converted to smiles using openeye s oechem(38 ) to eliminate bias .
the molecule is then processed using the standard zinc protocol,(39 ) which aims to enumerate all physiologically relevant protonated and tautomeric forms of the molecule . in parallel ,
property - matched decoys for the ligand are found from zinc using the dud protocols for enrichment and ranking .
four parameter sets are evaluated by performing sampling and scoring each in two different ways .
dock blaster uses two different sampling schemes called coarser and finer to sample fewer or more ligand orientations by adjusting the bins used to generate initial orientations .
the coarser scheme uses 45 hot spots and wider bins and generally runs somewhat faster , whereas finer uses 55 hot spots , narrower bins , and is often slower .
scheme uses standard amber 94 partial atomic charges on the protein , while polarized increases the dipoles on selected polar atoms in residues within 3.5 of the crystallographic ligand without changing the net charge . in prospective studies involving experimental testing of novel inhibitors ,
each ligand configuration that passes a rapid steric fit filter is scored for electrostatic and van der waals complementarity and adjusted for partial ligand desolvation due to solvent occlusion.(37 ) the high - scoring ligand conformation is minimized with 100 steps of simplex rigid - body minimization .
more details of these schemes and other technical details may be found on the dock blaster documentation web site , http://disi.docking.org/dock_blaster:technical_details .
this subsystem evaluates how well docking works as judged by pose fidelity to the crystal structure and by enrichment versus decoys . in doing so
it evaluates pose fidelity using the rmsd of all non - h atoms to the crystallographic pose and calculates the rank of the redocked ligand among the 100 property - matched decoys , which are also docked using all four docking parameter sets . if actives and inactives were supplied , as is the case for the dud benchmark , these are also docked and roc plots are calculated .
a concise calibration docking report allows users to judge which parametrization , if any , is best for prospective docking ( figure 3 ) .
outcomes are color coded to indicate successful ( in green ) , unsuccessful ( in red ) , and borderline ( in yellow ) results . particularly for borderline cases ,
the user may wish to inspect the scores and poses of each ligand and decoy before selecting the best parametrization for prospective docking .
calibration docking report , containing pose fidelity ( , rmsd ) and enrichment ( % rank ) of the redocked crystallographic ligand compared to 100 property matched decoys using four parametrizations , separated by a forward slash ( / ) in each cell .
successful runs are in green , unsuccessful ones in red , and marginal ones in yellow .
the most successful set of docking parameters from the calibration phase is selected and used .
the docker manages the screen across multiple cpus , combining the results when the entire library has been searched .
when database docking is complete , the assessor prepares reports including full purchasing information , annotation of physical properties , annotation of known activities derived from public data sources , and similarity to annotated compounds , other top hits , and other purchasable compounds . docked structures
may be viewed in the context of the binding site using pymol,(45 ) chimera(46 ) or jmol.(47 ) these reports may be browsed online or downloaded in tab - delimited format for processing by analysis software such as excel .
dock blaster is accessible via a web - based interface at http://blaster.docking.org ( figure 1 ) .
it is hosted on a cluster of 700 cpu cores managed using the sun grid engine with access to 30 terabytes of raid-6 storage .
dock blaster is integrated with zinc,(39 ) a public access database of commercially available compounds for library screening , and the dud decoy maker , which are also accessible separately .
we do preserve the results of some screens , which we may use for methods development and analyses .
dock blaster was tested using ligand - bound crystal structures from the pdb,(48 ) including some of the most widely used benchmarking standards : astex-85,(7 ) gold-114(15 ) , and dud-38(27 ) ( release 2 , oct 2006 ) .
we have used only the 38 structures in dud for which a ligand - bound crystal structure is available .
we have used only postremediation(49 ) pdb structures for which a single small ( < 500 da ) organic ligand is bound , having 2.5 resolution or better , and no polynucleotides .
dock blaster is a new tool for automatic docking and is available for free anonymous public access at http://blaster.docking.org .
dock blaster can start from the structure of a target and a specification of the binding site or simply from a pdb code in many cases . to assess the performance of our automatic high throughput docking procedure
retrospectively , we turned to common benchmarks in the field : astex-85,(7 ) gold-114,(15 ) and dud-38.(27 ) we began by retrieving the files directly from the remediated(49 ) pdb and used the parser to automatically separate the ligand from the receptor . in all but a few cases , the parser succeeded well enough in this task to proceed to docking ( ) . occasionally the ligand could not be identified automatically .
these could be rescued by simply specifying the three letter code of the ligand to the parser .
the average total cpu time per target , from preparation to docking the ligand and its 100 decoys to final analysis of the results , all automated , was 92 min .
we began by using dock blaster to automatically dock to targets in the astex-85 set.(7 ) in all but one case , the ligand was automatically separated from the protein starting from the pdb code alone ( ) .
for 1lf7 , we were required to specify the ligand ( citrate , cit ) because it is a common solute .
of the 84 jobs started automatically , 83 finished normally and produced a docked pose and score for the ligand and its decoys ( ) .
we asked how well automatic docking could recapitulate the crystal structure using each of four docking parameter sets .
of the 83 proteinligand pairs that yielded a docked ligand score , 51 were within 2 rmsd of the crystal structure ( , and http://data.docking.org/2009/astextables.doc ) .
coarser / polarized with 37 , and the least successful being coarser / normal with 34 successes .
the polarized dipoles in the electrostatics of polarized were slightly more successful than the
for the 51 proteinligand complexes docked within 2 rmsd of the crystal pose , we then asked how well the redocked crystallographic ligand ranked compared to around 100 property - matched decoys ( tables 1 and 2 and http://data.docking.org/2009/astextables.doc ) .
the well - posed ligand also ranked in the top 5% of the property matched decoys in 29 cases , suggesting that these are suitable for automatic prospective docking . just beyond the 2 cutoff , there were 11 cases in which the ligand posed close , within 3 , and a further 20 cases where the ligand never got even as close as 3 rmsd to the crystallographic ligand .
number of targets where fully automatic docking with dock blaster is successful , as judged by both good pose fidelity ( < 2.0 rmsd ) and rank ( top 5% ) versus about 100 property - matched decoys .
success is defined as ligand within 2 rmsd of the crystal pose and rank in the top 5% of about 100 property - matched decoys .
for instance , whereas 29 targets succeeded for pose fidelity and rank using any of four parameter sets and the standard dock scoring function , only 18 of these succeeded with all parameter sets . for 18 targets ,
the results were excellent regardless of which docking parameter set was chosen ; we could find no common theme among the targets to which to attribute this good fortune ( figure 4 ) .
there were three nuclear receptors ( 1m2z , 1n46 , 1sqn ) , six kinases ( 1ke5 , 1opk , 1ywr , 1v4s , 1pmn , 1t46 ) , and seven other enzymes ( 1hwi , 1of6 , 1lpz , 1q4 g , 1jla , 1r9o , 1vcj ) .
there was no obvious pattern in the physical or chemical properties of the ligands to explain why these targets worked so reliably .
ligands from the astex-85 benchmarks that redock with good pose fidelity and good rank ( top 5% of property matched decoys ) with all parameter sets used .
( a ) nuclear hormone receptors ( b ) kinases ( c ) other enzymes . five targets achieved good poses with all parametrizations , yet the ligand never ranked well ( figure 5 and http://data.docking.org/2009/astextables.doc , class 4 ) : 1ia1 , 1j3j , 1tz8 , 1xm6 , and 1xoq .
these cases are interesting because pose fidelity is often used as a metric for docking success .
poor ranks despite good poses can arise when either the ligand scores unexpectedly poorly or the decoys score unexpectedly well .
for instance , many decoys for 1ia1 , dihydrofolate reductase ( dhfr ) , contained guanidinium and other groups that might well bind to dhfr and in turn received excellent scores . because the ligands looked competitive to us
, we attribute the failure to achieve a good rank as being due to highly competitive decoys rather than a failure of the ligand itself , as it also received a good score .
future work will investigate whether the decoy selection procedure might be improved to remove viable - looking ligands that are not currently eliminated by our fingerprint - based dissimilarity metric .
ligands from the astex-85 benchmarks that redock with good pose fidelity but poor rank compared to property - matched decoys .
failure to achieve even a good pose against perhaps 2030% of targets is common in docking studies , so we were not much surprised to note 31 targets where the ligand did not dock even close ( within 3 rmsd ) to a satisfactory pose ( figure 6 and http://data.docking.org/2009/astextables.doc , class 6 ) .
many of these pose fidelity failures could be attributed to one of four common causes : ( a ) insufficient conformational sampling of the ligand , particularly of aliphatic ring puckering , ( b ) symmetric or pseudosymmetric molecule , ( c ) critical missing water molecules , and ( d ) ligand pose dominated by electronic ( orbital ) effects .
these issues are common to all docking methods and protocols and are therefore expected for this study . in the
scripted , automatic context of dock blaster , some of these problems could be addressed , for instance by using a rescoring method to allow for relaxation of ring puckering or an approach that samples explicit water molecule positions.(50 ) other failures , such as the plausible reversed docking pose of a pseudosymmetrical molecule in 1yv3 , could be better addressed using a density - based pose - assessment method.(51 ) some failures are probably simply beyond the capabilities of an orbital - nave method like dock , such as 1p2y , a p450 , where the ligand pose appears to be under electronic rather than steric control . whereas some of these failures may be addressed by improvements in the protocol
, we worry about a tendency to build in too much expertise that leads to over fitting . some of these problem targets will likely remain beyond the capability of full automation , at least in the near term .
ligands from the astex-85 benchmark that do not achieve good pose fidelity under any circumstances . with four parameter sets to choose from
, we wondered whether one of them was more successful at achieving good pose fidelity than the others . to answer this
, we plotted the frequency and cumulative frequency of pose fidelity for the best parameter set for each case ( figure 7 ) and also for each parameter set separately ( ) .
we found that all parameter sets achieved a pose within 2 rmsd at least 50% of the time .
these graphs confirm that all parameter sets are roughly comparable at achieving good ligand poses , that each is best some of the time , and none of the parametrizations is either much better or much worse than average .
histogram and cumulative frequency of pose fidelity ( , rmsd ) using the best parameter set .
if it were , good pose fidelity as measured by rmsd could be used to select the best docking parameters .
we plotted pose fidelity ( , rmsd ) versus rank using the parameters that led to the best pose fidelity in each case ( figure 8) and also for each parameter set separately ( ) .
poor pose fidelity was generally associated with poorly ranked compounds , although there was one notable exception , beta ii tryptase ( 2bm2 ) , where the ligand docked backward but still managed to get a good score compared to its decoys .
however , good pose fidelity was entirely uncorrelated with rank , regardless of which parameter set was used .
it would thus appear that pose fidelity can not be used to predict rank and thus whether the model is suitable for prospective docking .
plot of pose fidelity ( , rmsd ) versus % rank compared to about 100 property matched decoys ( log scale ) . for astex-85 benchmark . in each case , the parameter set that gave the best pose fidelity was used .
we were curious about whether ligand rank was sensitive to the scoring function used . to explore this , we rescored the ligand and all its decoys as docked using dock with four widely used , different scoring functions ( table 2 and http://data.docking.org/2009/rescoringtables.doc ) , in each case without any change to the receptor or ligand structure .
three of the scoring functions ranked the ligand substantially higher compared to the decoys , resulting in between 4 and 9 more successes .
only pmf resulted in fewer successes than dock . because we used the astex-85 set to tune our protocol and parameter choices ,
we worried about overfitting.(52 ) to control for protocol bias , we turned to the gold-114 benchmark(15 ) for which we did not allow ourselves to adjust the automatic procedure or its parameters in any way .
a total of 75 targets were successfully separated into ligand and receptor automatically from the pdb code alone ( ) , and a further 19 cases ( 94 total ) could be run if the ligand three letter code was also specified . of the 94 calculations that started automatically ,
of the 20 targets that failed to produce a docked ligand , three failed during ligand identification and separation , five failed to prepare the ligand for docking , and 12 tried but failed to dock and score the ligand .
we asked how well automatic docking could recapitulate the crystal structure when the docking protocol could not be modified .
of the 92 that yielded a docked and scored ligand , 58 posed within 2 rmsd of the crystal structure ( and http://data.docking.org/2009/goldtables.doc ) .
all parameter sets performed nearly equivalently , and no single one was as successful as combining the best result from all four .
when the docked pose was within 2 rmsd , 27 of these also managed to rank in the top 5% of about 100 property matched decoys ( table 1 and http://data.docking.org/2009/goldtables.doc ) .
worrying about depending on a single positive control as the sole basis for judging docking performance , we turned to a benchmark for which more actives were available .
we used dock blaster to dock to targets in the dud-38 benchmark(27 ) for which the ligand pose was crystallographically observed .
all targets could be handled by the parser and submitted to the dock blaster queue .
one ligand failed to be converted to smiles automatically ( 1ckp ) , but the rest of the 37 targets completed normally yielding a redocked and scored ligand .
we began by assessing performance as before using a single crystallographic ligand and its automatically generated decoys , asking how well automatic docking could recapitulate the crystal structure .
of the 37 proteinligand pairs that yielded a docked ligand score , 23 posed within 2 rmsd of the crystallographic ligand ( and http://data.docking.org/2009/dudtables.doc ) .
when the docked pose was within 2 rmsd , 15 of these also ranked the ligand in the top 5% of about 100 property matched decoys ( table 1 and http://data.docking.org/2009/dudtables.doc ) .
as part of dud , these targets benefited from between 20 and 600 additional annotated actives and their corresponding property - matched decoys with which to test dock blaster .
we began by checking that the fully automatic docking results using dock blaster were at least comparable to the expert docking reported in the dud paper ( figure 9 and ) .
if automatic docking were consistently different from an expert , the conclusions of this study could be undermined .
for instance , an expert can experiment with the placement of specific water molecules , modification of receptor parameters , or docking parameters and do so repeatedly until the results converge . whereas fully automated docking often produced results somewhat inferior to an expert , we thought it was close in all but five cases .
receiver operator characteristic ( roc ) plots comparing enrichment by dock blaster ( magenta ) versus an expert ( dark blue ) against four targets from the dud benchmark .
( a ) cox-2 ( b ) rxr alpha ( c ) er - agonist ( d ) sahh . random enrichment shown by a thin gray line .
a critical question was how predictive the single molecule metric used thus far was of the multimolecule dud metric ( table 3 and http://data.docking.org/2009/ ) .
bad to indicate whether the docking results looked compelling enough to use for a discovery project .
good meant both pose fidelity within 2 rmsd and rank strictly in the top 5% of the property matched decoys . for the multiligand ( dud )
metric , good meant that either the adjusted logauc(53 ) was greater than 10 , or that ef1 , the fraction of ligands found by the first percentile , was greater than 10% , or that ef5 was greater than 20% .
our results show that in 26 of 36 or 72% of cases where automatic docking completed normally , the single ligand metric of docking success predicted the multiligand assessment .
this result allows us to deploy the single ligand metric as a reasonable predictor of overall enrichment .
success for the single molecule metric has pose fidelity within 2 rmsd and rank in the top 5% of property - matched decoys .
success for the dud metric is adjusted logauc > 10 or ef1 > 10% or ef5 > 20% .
a common criticism of docking is that it often seems to encounter unforeseen problems with each new target , and correspondingly one never knows in advance whether docking will
equipped with a fully automated system and single - ligand based assessment tools , we turned to a larger set of targets to investigate the prevalence of situations that are not well handled by our scripts .
of the 9050 eligible pdb targets submitted to the parser , 7755 produced a docked ligand structure that could be scored and ranked ( table 4 ) .
of these , 3056 had the ligand redock within 2 rmsd of the crystal structure ( http://data.docking.org/2009/pdb3056.xls ) .
about 100 property - matched decoys were generated automatically for each ligand , docked , and used to calculate the rank of the redocked ligand using each of four parameter sets . in 1398 of these cases
, the ligand also scored in the top 5% , representing 18% of the 7755 targets that were viable for automatic docking .
aug 1 , 2008 version , of the pdb having 52000 x - ray crystal structures in total .
recognizing the potential benefit of automated high throughput docking to interpret the growing number of protein structures , we have developed an automatic docking system and assessed its performance in retrospective tests .
first , dock blaster , an automatic docking system , is now available for free public use and can produce useful results starting with as little as a pdb code .
second , useful results as judged by good pose fidelity and rank compared to property matched decoys were achieved in around 30% of cases against common benchmarks .
third , surprisingly , pose fidelity as measured by rmsd is not predictive of rank and is therefore not a useful metric of docking success by itself.(54 ) fourth , a single ligand metric using the crystallographic ligand is predictive of the multiligand enrichment over property - matched decoys 72% of the time .
finally , dock blaster has been deployed on a large scale and produced screening results that deserve further consideration for experimental testing in 1398 of 7755 cases drawn from the pdb . the result that will have
perhaps the greatest pragmatic impact is the creation of the dock blaster server itself . because dock blaster can start from as little as a pdb code , can produce results like an expert in some cases , and can self - evaluate , it is suitable for use by nonexperts and for large - scale experiments .
when docking performs poorly or fails completely , this can usually be deduced automatically from failures in pose fidelity and rank - to - decoys statistics .
thus the prospective user of this system can estimate whether the docking results are likely to be useful for discovery , should be ignored completely , or perhaps fall somewhere in between .
of course , any system of this complexity is bound to have flaws , and some projects will simply not work .
still , it is our hope that dock blaster will be useful for nonexperts , lowering barriers to entry to the field .
pose fidelity using dock blaster in retrospective studies is not quite as good as when performed by an expert .
our black - box system achieves pose fidelity within 2 rmsd for about 5060% of targets , compared to 7080% for expert - guided docking .
perhaps surprisingly , pose fidelity as measured by rmsd deviation did not correlate at all with rank . for enrichment ,
this should be unsurprising , however , because experts are at liberty to manually curate sites , ligands , and protocols to maximize performance .
useful results , i.e. , good pose fidelity and good enrichment , were obtained in 2540% of benchmark cases .
the success rates for astex-85 was 29 out of 85 , for gold-114 it was 27 out of 114 , and for dud-38 15 out of 38 were successful for both pose - fidelity and enrichment .
whereas this might not seem a very high success rate , no study has ever held itself to such a stringent standard before , and this was only possible with an automated program . as a basis for performance assessment ,
a single ligand with generated property - matched decoys predicts the multiligand assessment three times out of four .
because only one ligand is needed , this has significant advantages for automation , enabling automatic self - assessment of docking results , which would be impractical if a list of actives had to be assembled to benchmark each target .
the correlation was largely insensitive to the precise cutoffs used to characterize docking success or to which scoring function was used .
of course , this result is provisional , as it is based on the dud-38 benchmark and may be revised as larger benchmarks appear .
dock blaster has been deployed on a near - proteome scale , and produced useful results for 1398 of 7755 targets or 18% of the pdb structures that were amenable to this method .
because docking can recapitulate what is already known about the site in these cases , there is a reasonable hope for prospective docking to suggest ligands that might actually bind .
many of these problems were previously known ( conformational sampling , ligand representation , structural waters ) , but their prevalence is now better quantified . there is a critical need to find new small molecule reagents for biology . at the same time , a rapidly growing backlog of uninterpreted structures has accumulated in the pdb . yet in an age of plentiful target structures , freely available small molecule databases , many sophisticated docking programs , and fast computers to run them on , many biologically oriented investigators still find docking for ligand discovery daunting . we have developed dock blaster to help nonspecialists find new reagents for biology without the need for an expert . as for experts , dock blaster does not produce compelling results for all targets .
but for the nearly 20% of targets we tried where it does produce results deserving of further investigation , dock blaster offers a way to automatically leverage structure for ligand discovery . | molecular docking is the most practical approach to leverage protein structure for ligand discovery , but the technique retains important liabilities that make it challenging to deploy on a large scale .
we have therefore created an expert system , dock blaster , to investigate the feasibility of full automation .
the method requires a pdb code , sometimes with a ligand structure , and from that alone can launch a full screen of large libraries .
a critical feature is self - assessment , which estimates the anticipated reliability of the automated screening results using pose fidelity and enrichment . against common benchmarks ,
dock blaster recapitulates the crystal ligand pose within 2 rmsd 5060% of the time ; inferior to an expert , but respectrable . half the time the ligand also ranked among the top 5% of 100 physically matched decoys chosen on the fly .
further tests were undertaken culminating in a study of 7755 eligible pdb structures . in 1398 cases ,
the redocked ligand ranked in the top 5% of 100 property - matched decoys while also posing within 2 rmsd , suggesting that unsupervised prospective docking is viable .
dock blaster is available at http://blaster.docking.org . | Introduction
Methods
Results
Discussion | these barriers to entry have diminished the impact of the technique by making it less accessible to biologically oriented nonexperts and challenging even for specialists to deploy on a large scale . we have therefore investigated an expert system , dock blaster , which aims to emulate experts at all stages of the docking process . code and from that launch a full screen of a large compound library to find novel ligands . we have used three of the most common benchmarking sets : the astex-85 set,(7 ) having 85 high - quality crystal structures of therapeutically relevant targets and drug - like ligands , the gold-114 benchmark,(15 ) derived from the most widely used docking benchmarks , and the dud set,(27 ) 38 protein targets for which sets of annotated actives and corresponding property - matched decoys are available for each target . the dock blaster pipeline is composed of six modules ( figure 2 ) : ( a ) the parser , which identifies the receptor and ligand from a pdb file , ( b ) the scrutinizer , which attempts to correct for problems , such as incomplete or disordered residues on the receptor , ( c ) the preparer , which protonates the receptor , calculates hot spots and scoring grids , assigns atomic parameters , including these for cofactors , post - translational modifications and metals , and prepares the ligand , decoys , and any actives and inactives for docking , ( d ) the calibrator , which uses supplied data to assess docking performance and suggests optimal docking parameters , ( e ) the docker , which manages a full database screen on the computer cluster , and ( f ) the assessor , which prepares reports to interpret database screening results . in doing so
it evaluates pose fidelity using the rmsd of all non - h atoms to the crystallographic pose and calculates the rank of the redocked ligand among the 100 property - matched decoys , which are also docked using all four docking parameter sets . calibration docking report , containing pose fidelity ( , rmsd ) and enrichment ( % rank ) of the redocked crystallographic ligand compared to 100 property matched decoys using four parametrizations , separated by a forward slash ( / ) in each cell . dock blaster is a new tool for automatic docking and is available for free anonymous public access at http://blaster.docking.org . the polarized dipoles in the electrostatics of polarized were slightly more successful than the
for the 51 proteinligand complexes docked within 2 rmsd of the crystal pose , we then asked how well the redocked crystallographic ligand ranked compared to around 100 property - matched decoys ( tables 1 and 2 and http://data.docking.org/2009/astextables.doc ) . the well - posed ligand also ranked in the top 5% of the property matched decoys in 29 cases , suggesting that these are suitable for automatic prospective docking . number of targets where fully automatic docking with dock blaster is successful , as judged by both good pose fidelity ( < 2.0 rmsd ) and rank ( top 5% ) versus about 100 property - matched decoys . success is defined as ligand within 2 rmsd of the crystal pose and rank in the top 5% of about 100 property - matched decoys . when the docked pose was within 2 rmsd , 27 of these also managed to rank in the top 5% of about 100 property matched decoys ( table 1 and http://data.docking.org/2009/goldtables.doc ) . when the docked pose was within 2 rmsd , 15 of these also ranked the ligand in the top 5% of about 100 property matched decoys ( table 1 and http://data.docking.org/2009/dudtables.doc ) . good meant both pose fidelity within 2 rmsd and rank strictly in the top 5% of the property matched decoys . success for the single molecule metric has pose fidelity within 2 rmsd and rank in the top 5% of property - matched decoys . about 100 property - matched decoys were generated automatically for each ligand , docked , and used to calculate the rank of the redocked ligand using each of four parameter sets . in 1398 of these cases
, the ligand also scored in the top 5% , representing 18% of the 7755 targets that were viable for automatic docking . first , dock blaster , an automatic docking system , is now available for free public use and can produce useful results starting with as little as a pdb code . finally , dock blaster has been deployed on a large scale and produced screening results that deserve further consideration for experimental testing in 1398 of 7755 cases drawn from the pdb . because dock blaster can start from as little as a pdb code , can produce results like an expert in some cases , and can self - evaluate , it is suitable for use by nonexperts and for large - scale experiments . dock blaster has been deployed on a near - proteome scale , and produced useful results for 1398 of 7755 targets or 18% of the pdb structures that were amenable to this method . | [
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although pregnancy and childbirth are often viewed as periods of emotional well - being , the perinatal period is a time of substantial vulnerability to affective illness .
pregnancy is a period of great disruption and adjustment of biological , psychological and social aspects , which can be a risk factor for the development , occurrence or recurrence of mental disorders ( 3 ) .
research from the past two decades has suggested a link between prenatal depression and anxiety and adverse obstetric and neonatal outcomes , such as infant s low birth weight ( lbw ) ( 4 , 5 ) .
prevalence rates of depressive symptoms , among women of childbearing age , range from 10% to 50% ( 6 - 10 ) , depending on the instrument used and the demographic characteristics of the study population .
previous studies from greece have indicated that rates of prenatal depressive symptoms range from 11.5% to 47% ( 11 - 13 ) .
firm estimates for prenatal anxiety symptoms do not exist , although past studies suggest that prevalence rates of prenatal anxiety symptoms are similar to those in the general population , affecting 6.6% - 52.9% ( 14 - 17 ) .
according to the world health organization ( who ) , elevated levels of prenatal anxiety are observed during the first and third trimesters of pregnancy ( 18 ) .
it therefore , appears that the estimates of prenatal depressive and anxiety symptoms prevalence rates vary .
comparisons across studies are complicated due to differences in study methodology and design , including use of various depression and anxiety screening instruments , variable time of depression and anxiety screening in pregnancy and differences in composition of the sample and sample sizes . as an example , in the study of kurki et al .
( 14 ) , prenatal anxiety symptomatology in 10 - 17 weeks of gestation was assessed by one question only ( are you tense or distressed ? ) and was observed in 16% of the study sample . on the other hand , in the study by nasreen et al .
( 17 ) , prenatal anxiety symptoms were estimated by a psychometric scale ( stai ) , during the third trimester of pregnancy and were observed in 26% ( 17 ) .
research findings are inconsistent regarding the association between prenatal depression anxiety symptomatology and adverse neonatal outcomes , such as the lbw of the newborn , the low apgar score and the newborn s admission in neonatal intensive care unit ( nicu ) .
the allegations of possible association of psychological factors with lbw have been expressed for decades ( 4 ) .
prenatal depression has been associated with lbw ( 17 , 19 , 20 ) . however , contradicting findings have also been reported ( 21 - 25 ) , possibly due to differences in study methodology and design ( e.g. different psychometric tools , diverse study samples ) . in two of the studies ( 19 , 20 ) confirming the association of prenatal depressive symptoms and lbw , the study samples recruited individuals from disadvantaged ( low socioeconomic status or minority groups ) populations .
anxiety symptoms during pregnancy have been reported to be associated with lbw in several studies ( 16 , 17 , 26 ) , while others have not confirmed this association ( 22 , 23 , 27 - 29 ) .
a number of studies indicate an association between low apgar score of the newborn and prenatal depression and anxiety ( 9 , 22 ) , while , in other studies , this association does not exist ( 22 , 24 , 25 ) .
the main aim of this investigation was to examine the association between prenatal depressive and anxiety symptoms with neonatal outcomes .
part of the aim of this study was the assessment of prenatal depressive and anxiety symptomatology in the third trimester of pregnancy , in a sample of pregnant women from greece .
the findings of this study will contribute to this research area , where there are conflicting research views , and will help even more health professionals in shaping a more comprehensive view on the issue , also in other countries besides greece .
during the study period , of all the pregnant women we encountered , 117 agreed to participate in this exploratory , longitudinal study .
all of the subjects were recruited from the private medical sector and were in the third trimester of pregnancy .
inclusion criteria were pregnant women between 32 to 35 weeks of gestation , which received routine prenatal care throughout pregnancy and were either of greek origin or fluent in greek language .
we collected socio - demographic and obstetric data using a demographic questionnaire , which was administered to the women .
the questionnaire included questions relating to age , ethnicity , education level , professional and economic status , number of pregnancies and children , and the health status of women .
prenatal depressive symptoms were assessed with the beck depression inventory ( bdi ) and the edinburg postnatal depression scale ( epds ) .
the bdi is a self - report rating scale , consisting of 21 categories of symptoms and behaviors .
each category describes a specific behavioral manifestation of depression and consists of 4 - 5 self - evaluation statements , sequentially classified .
these statements are classified to reflect the variation in severity of symptoms from none to maximum importance .
numeric values from 0 to 3 were set for each statement , to determine the degree of importance .
items are scored on a 0 - 3 scale , yielding a score range of 0 - 63 , where higher scores indicate greater depression severity .
scores in the range of 0 - 13 indicate minimal depression , 14 - 19 mild depression , 20 - 28 moderate depression and 29 - 63 severe depression ( 30 ) . in the greek version of the bdi ,
the internal consistency reliability is satisfactory and the cronbach s index is = 0.84 ( 31 ) .
it is a widely used tool , with satisfactory validity and reliability , both in prenatal and postnatal period populations ( 32 ) .
the ten - topic version of epds scale consists of statements describing depressive symptoms and have four possible answers , each graded according to the complaint s severity or duration .
the answers are graded from 0 to 3 , while several of them are graded conversely and their total sum is calculated at the end . the instrument has been validated for greece , with cronbach s index being = 0.9 and cut off point 11 ( 33 ) .
the bai is a self - administered scale consisting of 21 entries of anxiety symptoms .
the score of each entry ranges on a scale from 0 to 3 , with an overall score range of 0 - 63 , and the final rating scale consists of the sum of the degree of distress for each symptom .
scores of 0 - 7 represent minimal anxiety , 8 - 15 mild anxiety , 16 - 25 moderate anxiety and 26 - 63 severe anxiety ( 34 ) .
finally , a questionnaire containing questions about neonatal parameters ( e.g. birth weight , admission to the nicu and apgar score ) was administered to the women to record neonatal outcomes .
the participants were recruited during their routine follow up in the private medical sector , in the time period between march to april 2012 .
the first sampling was carried out in the third trimester of pregnancy during 32 to 35 week of pregnancy .
the recording of demographic data and the completion of the bdi , epds and bai were performed in this sampling .
the second sampling was carried out on the first postpartum week , where the recording of neonatal parameters was performed and the sample consisted of 93 women .
the reason of attrition probably could be attributed to the fact that the second phase of the study was taking place during the postpartum period .
the requirements of this particularly sensitive period , such as the care of the newborn , may have played the main reason of attrition .
the study protocol was approved by the assembly of athens university medical school , athens , greece , medical school , approval number 6761 , for scientific and ethical standards .
information about the study protocol and the confidentiality of the data and the identity of each participant were written to the informed consent form that was signed by each participant .
descriptive statistics were calculated for initial data analysis . because of failure of all continuous variables in kolmogorov - smirnov normality tests , spearman s rho coefficients were calculated to define correlations between continuous variables and non - parametric tests ( mann - whitney u and kruskal - wallis ) were applied in order to define significant relationships between the continuous and categorical variables .
all association testing was conducted assuming a 5% significance level and a two - sided alternative hypothesis , while statistical analyses were performed using spss software package version 17.0 ( spss inc . ,
during the study period , of all the pregnant women we encountered , 117 agreed to participate in this exploratory , longitudinal study .
all of the subjects were recruited from the private medical sector and were in the third trimester of pregnancy .
inclusion criteria were pregnant women between 32 to 35 weeks of gestation , which received routine prenatal care throughout pregnancy and were either of greek origin or fluent in greek language .
we collected socio - demographic and obstetric data using a demographic questionnaire , which was administered to the women .
the questionnaire included questions relating to age , ethnicity , education level , professional and economic status , number of pregnancies and children , and the health status of women .
prenatal depressive symptoms were assessed with the beck depression inventory ( bdi ) and the edinburg postnatal depression scale ( epds ) .
the bdi is a self - report rating scale , consisting of 21 categories of symptoms and behaviors .
each category describes a specific behavioral manifestation of depression and consists of 4 - 5 self - evaluation statements , sequentially classified .
these statements are classified to reflect the variation in severity of symptoms from none to maximum importance .
numeric values from 0 to 3 were set for each statement , to determine the degree of importance .
items are scored on a 0 - 3 scale , yielding a score range of 0 - 63 , where higher scores indicate greater depression severity .
scores in the range of 0 - 13 indicate minimal depression , 14 - 19 mild depression , 20 - 28 moderate depression and 29 - 63 severe depression ( 30 ) . in the greek version of the bdi ,
the internal consistency reliability is satisfactory and the cronbach s index is = 0.84 ( 31 ) .
it is a widely used tool , with satisfactory validity and reliability , both in prenatal and postnatal period populations ( 32 ) .
the ten - topic version of epds scale consists of statements describing depressive symptoms and have four possible answers , each graded according to the complaint s severity or duration .
the answers are graded from 0 to 3 , while several of them are graded conversely and their total sum is calculated at the end . the instrument has been validated for greece , with cronbach s index being = 0.9 and cut off point 11 ( 33 ) .
the bai is a self - administered scale consisting of 21 entries of anxiety symptoms .
the score of each entry ranges on a scale from 0 to 3 , with an overall score range of 0 - 63 , and the final rating scale consists of the sum of the degree of distress for each symptom .
scores of 0 - 7 represent minimal anxiety , 8 - 15 mild anxiety , 16 - 25 moderate anxiety and 26 - 63 severe anxiety ( 34 ) .
finally , a questionnaire containing questions about neonatal parameters ( e.g. birth weight , admission to the nicu and apgar score ) was administered to the women to record neonatal outcomes .
the participants were recruited during their routine follow up in the private medical sector , in the time period between march to april 2012 .
the first sampling was carried out in the third trimester of pregnancy during 32 to 35 week of pregnancy .
the recording of demographic data and the completion of the bdi , epds and bai were performed in this sampling .
the second sampling was carried out on the first postpartum week , where the recording of neonatal parameters was performed and the sample consisted of 93 women .
the reason of attrition probably could be attributed to the fact that the second phase of the study was taking place during the postpartum period .
the requirements of this particularly sensitive period , such as the care of the newborn , may have played the main reason of attrition .
the study protocol was approved by the assembly of athens university medical school , athens , greece , medical school , approval number 6761 , for scientific and ethical standards .
information about the study protocol and the confidentiality of the data and the identity of each participant were written to the informed consent form that was signed by each participant .
descriptive statistics were calculated for initial data analysis . because of failure of all continuous variables in kolmogorov - smirnov normality tests , spearman s rho coefficients were calculated to define correlations between continuous variables and non - parametric tests ( mann - whitney u and kruskal - wallis ) were applied in order to define significant relationships between the continuous and categorical variables .
all association testing was conducted assuming a 5% significance level and a two - sided alternative hypothesis , while statistical analyses were performed using spss software package version 17.0 ( spss inc . ,
the initial sample of this study comprised 117 women from 32 to 35 week of pregnancy .
their average age was 32.61 4.06 years and the vast majorities of them were greek ( 93.2% ) , graduates of higher - education ( 65.0% ) and married ( 95.7% ) ( table 1 ) .
furthermore , in a proportion of 83.8% they were employed , with most of them ( 56.4% ) having a monthly family income from 1000 to 3000 . also , for 77 ( 65.8% ) of the women in the sample this pregnancy was planned , while for 10 ( 8.5% ) of them , in vitro fertilization was employed .
the largest relative rate of the women ( 47.0% ) in the sample in the initial phase of this study was at the 32 week of pregnancy , which was the first for most of them ( 57.3% ) , while only 36 ( 30.8% ) and seven ( 6.0% ) of these women had one and two children , respectively .
with regard to health problems experienced by women during pregnancy , hyperemesis was recorded at a rate of 13.7% , hypertension in 3.4% , diabetes mellitus in 14.5% and placental abruption in 12.0% .
similarly , bleeding disorders were experienced by 15.4% of the pregnant women , in the first trimester of pregnancy , 0.9% in the second and 2.6% in the third trimester .
an important parameter of this study was the exploration of pregnant women s mental health .
therefore , 8.5% of the pregnant women reported a positive past history of depression , with three ( 2.6% ) of them appearing to have received antidepressant treatment . similarly
, 9.4% of the pregnant women reported a history of anxiety disorder , with three ( 2.6% ) of them reporting having followed anxiolytic treatment ( table 1 ) .
ninety - five ( 81.1% ) participants were identified as experiencing minimal depressive symptoms , while 18 ( 15.4% ) women showed mild , three ( 2.6% ) moderate and one ( 0.9% ) , severe symptoms , respectively ( table 2 ) .
abbreviations : bai , beck anxiety inventory ; bdi , beck depression inventory ; epds , edinburg postnatal depression scale .
ninety - four ( 80.3% ) women showed absence of depression , while 23 ( 19.7% ) indicated a likely diagnosis of depression ( table 2 ) .
fifty - one ( 43.6% ) participants showed minimal anxiety symptoms , while 50 ( 42.7% ) women showed mild , 12 ( 10.3% ) moderate and four ( 3.4% ) , severe , anxiety symptoms ( table 2 ) .
participation in the second phase of the study ( after childbirth ) included 93 ( 79.48% ) of the initial 117 women .
thereby , the average pregnancy week was 38 1.35 weeks , for these women ( n = 93 ) , while births took place between the 34 and 40 week .
fifty - three ( 57.0% ) of them had a natural childbirth and 40 ( 43.0% ) gave birth by cesarean section .
of the puerperants , who had natural birth ( = 53 ) , 27 ( 50.9% ) received analgesics and 43 ( 81.1% ) received epidural anesthesia . respectively , two ( 5.0% ) of the women with cesarean section ( n = 40 ) received general anesthesia , during childbirth , and 38 ( 95.0% ) epidural anesthesia . at the same time , the average weight of the newborns ( = 93 ) was 3090.32 456.13 gram .
one ( 1.2% ) newborn had apgar score 7 , 19 ( 23.2% ) had 8 , 33 ( 40.2% ) had 9 and 29 ( 35.4% ) had apgar score 10 .
according to bdi score , non - significant associations were found with the newborn s birth weight ( spearman s rho = -0.052 , p = 0.623 ) , with its admission in nicu ( mann - whitney u test , p = 0.992 ) and the apgar score ( kruskal - wallis test , p = 0.307 ) ( table 3 ) .
abbreviations : bai , beck anxiety inventory ; bdi , beck depression inventory ; epds , edinburg postnatal depression scale ; nicu , neonatal intensive care unit .
the values are presented as mean sd . according to epds scores , non - significant associations were found with the newborn s birth weight ( spearman s rho = 0.017 , p = 0.872 ) , with its admission in nicu ( mann - whitney u test , p = 0.918 ) , and the apgar score ( kruskal - wallis test , p = 0.434 ) ( table 3 ) .
non - significant associations were found between prenatal anxiety symptoms , as assessed by bai scale and the newborn s birth weight ( spearman s rho = -0.108 , p = 0.302 ) , its admission in nicu ( mann - whitney u test , p = 0.375 ) , and its apgar score ( kruskal - wallis test , p = 0.542 ) ( table 3 ) .
according to bdi score , non - significant associations were found with the newborn s birth weight ( spearman s rho = -0.052 , p = 0.623 ) , with its admission in nicu ( mann - whitney u test , p = 0.992 ) and the apgar score ( kruskal - wallis test , p = 0.307 ) ( table 3 ) .
abbreviations : bai , beck anxiety inventory ; bdi , beck depression inventory ; epds , edinburg postnatal depression scale ; nicu , neonatal intensive care unit .
the values are presented as mean sd . according to epds scores , non - significant associations were found with the newborn s birth weight ( spearman s rho = 0.017 , p = 0.872 ) , with its admission in nicu ( mann - whitney u test , p = 0.918 ) , and the apgar score ( kruskal - wallis test , p = 0.434 ) ( table 3 ) .
non - significant associations were found between prenatal anxiety symptoms , as assessed by bai scale and the newborn s birth weight ( spearman s rho = -0.108 , p = 0.302 ) , its admission in nicu ( mann - whitney u test , p = 0.375 ) , and its apgar score ( kruskal - wallis test , p = 0.542 ) ( table 3 ) .
our findings suggest that prenatal depressive and anxiety symptoms were not associated with neonatal outcomes , such as birth weight of the newborn , apgar score and the newborn s admission in nicu .
therefore , in regard to the association of prenatal depression and anxiety symptoms with birth weight , our results mirror a number of previous studies ( 21 - 23 , 25 , 27 - 29 ) .
other authors have found different results ( 16 , 17 , 19 , 20 , 26 ) .
more specifically , several of these studies include women who are of low income ( 16 , 17 , 20 ) or belong to minority groups ( 19 ) , not all are of adult age and their level of education is basic ( 16 ) .
as we have already reported , the status of our sample was not low socioeconomic , all the participants were adults , the majority were graduates of higher education and received regular prenatal care .
probably , the difference in socioeconomic status , in health care systems and in maternal and neonatal profiles , may explain the adverse results .
another finding that we consider important in our study is that prenatal depressive and anxiety symptoms were not associated with apgar score .
this result is in agreement with previous studies ( 21 , 22 , 24 , 25 ) , although not all ( 9 , 22 ) . several factors that may explain the inconsistency with our findings are the different moment at which took place the assessment of prenatal depression and anxiety symptomatology , the different screening instruments , as well as the different cultural background . in this study , we also found no association between prenatal depressive and/or anxiety symptoms and the newborn s admission in nicu and we did not find any relationship .
( 2009 ) ( 10 ) , although is contradicted by the study of chung et al .
the sample of their study was 959 women from hong kong and they found that prenatal depressive symptoms were associated with increased risk of newborn s admission in nicu .
authors reported that it is unclear how a high score in a scale can interact with this indication and suggest further investigation .
the findings of our study should be interpreted with regard to a series of limitations .
the sample size was not large enough , as well as the fact that , in the second phase , i.e. the period in which the recording of neonatal outcomes was performed , not all the initial sample participated .
in addition , there was no selection in the population involved , i.e. no women were excluded from the study due to certain characteristics , such as pathological obstetric history .
also , our sample comes from a large urban center and the majority of the women had a high educational level .
furthermore , it should be mentioned that the majority of our sample reported the occurrence of limited depressive and anxiety symptoms that did not reach the cut - off point of a mood disorder .
, the evidence from our study suggests that limited depressive and anxiety symptoms during the third trimester of pregnancy do not appear to affect neonatal outcomes , such as the newborn s birth weight , the apgar score and the newborn s admission in the nicu .
this could be reassuring in clinical practice for pregnant women who suffer prenatally from low to moderate levels of prenatal depressive and anxiety symptoms .
the present study confirms previous findings and contributes additional evidence in this field of research .
one of the strengths of the study is that the assessment of prenatal depressive symptoms was based on two different psychometric scales , with similar results , therefore strengthening our conclusions .
we hope our findings will stimulate further research that would yield evidence in antepartum depressive and anxiety symptomatology and its possible association with neonatal outcomes
. further research might assess prenatal depressive and anxiety symptoms using a combination of self - administrated scales and clinical interviews , in all trimesters of pregnancy .
another interesting area of future research would be to investigate possible associations between prenatal psychopathology with other neonatal and obstetric parameters , such as neonate small for gestational age ( sga ) , preterm birth and mode of delivery . | background : prior studies have reported inconsistent findings regarding the link between antenatal depressive and anxiety symptomatology , with neonatal outcomes.objectives:the aim of the present study was to assess the possible association of prenatal depressive and anxiety symptoms , in the third trimester of pregnancy , with perinatal outcomes ( birth weight of the newborn , apgar score and the newborn s admission in neonatal intensive care unit ) in a sample of pregnant women , in greece.patients and methods : a total of 117 women from athens , during the 32nd to 35th week of pregnancy , participated in the study . demographic and obstetric history data , as well as neonatal outcomes , were recorded .
three self - administered psychometric scales ( beck depression inventory ( bdi ) , edinburg postnatal depression scale ( epds ) and beck anxiety inventory ( bai ) ) were used to evaluate in detail the prenatal depressive and anxiety symptoms .
descriptive statistics , spearman s rho coefficients , mann - whitney u and kruskal - wallis testes were applied to analyze the data.results:on the basis of bdi , 81.1% of the sample showed minimal , 15.4% mild , 2.6% moderate and 0.9% severe depressive symptoms , respectively . furthermore , 80.3% of the participants , scored on epds below the cut - off point for a likely diagnosis of depression . according to bai scale ,
43.6% showed minimal , 42.7% women mild , 10.3% moderate and 3.4% severe anxiety symptoms .
no statistically significant correlations were found between depressive and anxiety symptoms and neonatal outcomes ( birth weight , apgar score and admission in neonatal intensive care unit).conclusions : limited levels of prenatal depressive and anxiety symptoms do not seem to be associated with neonatal outcomes . in clinical practice , pregnant women , who suffer from low levels of prenatal depressive and anxiety symptoms ,
may be reassured , in respect of the adverse outcomes of these mood symptoms on the neonate . | 1. Background
2. Objectives
3. Patients and Methods
3.1. Study Design and Participants
3.2. Data Collection
3.3. Ethical Considerations
3.4. Data Analysis
4. Results
4.1. Associations of Prenatal Depressive and Anxiety Symptoms With Neonatal Parameters
5. Discussion | research findings are inconsistent regarding the association between prenatal depression anxiety symptomatology and adverse neonatal outcomes , such as the lbw of the newborn , the low apgar score and the newborn s admission in neonatal intensive care unit ( nicu ) . part of the aim of this study was the assessment of prenatal depressive and anxiety symptomatology in the third trimester of pregnancy , in a sample of pregnant women from greece . prenatal depressive symptoms were assessed with the beck depression inventory ( bdi ) and the edinburg postnatal depression scale ( epds ) . because of failure of all continuous variables in kolmogorov - smirnov normality tests , spearman s rho coefficients were calculated to define correlations between continuous variables and non - parametric tests ( mann - whitney u and kruskal - wallis ) were applied in order to define significant relationships between the continuous and categorical variables . prenatal depressive symptoms were assessed with the beck depression inventory ( bdi ) and the edinburg postnatal depression scale ( epds ) . because of failure of all continuous variables in kolmogorov - smirnov normality tests , spearman s rho coefficients were calculated to define correlations between continuous variables and non - parametric tests ( mann - whitney u and kruskal - wallis ) were applied in order to define significant relationships between the continuous and categorical variables . the largest relative rate of the women ( 47.0% ) in the sample in the initial phase of this study was at the 32 week of pregnancy , which was the first for most of them ( 57.3% ) , while only 36 ( 30.8% ) and seven ( 6.0% ) of these women had one and two children , respectively . similarly , bleeding disorders were experienced by 15.4% of the pregnant women , in the first trimester of pregnancy , 0.9% in the second and 2.6% in the third trimester . according to bdi score , non - significant associations were found with the newborn s birth weight ( spearman s rho = -0.052 , p = 0.623 ) , with its admission in nicu ( mann - whitney u test , p = 0.992 ) and the apgar score ( kruskal - wallis test , p = 0.307 ) ( table 3 ) . abbreviations : bai , beck anxiety inventory ; bdi , beck depression inventory ; epds , edinburg postnatal depression scale ; nicu , neonatal intensive care unit . according to epds scores , non - significant associations were found with the newborn s birth weight ( spearman s rho = 0.017 , p = 0.872 ) , with its admission in nicu ( mann - whitney u test , p = 0.918 ) , and the apgar score ( kruskal - wallis test , p = 0.434 ) ( table 3 ) . non - significant associations were found between prenatal anxiety symptoms , as assessed by bai scale and the newborn s birth weight ( spearman s rho = -0.108 , p = 0.302 ) , its admission in nicu ( mann - whitney u test , p = 0.375 ) , and its apgar score ( kruskal - wallis test , p = 0.542 ) ( table 3 ) . according to bdi score , non - significant associations were found with the newborn s birth weight ( spearman s rho = -0.052 , p = 0.623 ) , with its admission in nicu ( mann - whitney u test , p = 0.992 ) and the apgar score ( kruskal - wallis test , p = 0.307 ) ( table 3 ) . according to epds scores , non - significant associations were found with the newborn s birth weight ( spearman s rho = 0.017 , p = 0.872 ) , with its admission in nicu ( mann - whitney u test , p = 0.918 ) , and the apgar score ( kruskal - wallis test , p = 0.434 ) ( table 3 ) . non - significant associations were found between prenatal anxiety symptoms , as assessed by bai scale and the newborn s birth weight ( spearman s rho = -0.108 , p = 0.302 ) , its admission in nicu ( mann - whitney u test , p = 0.375 ) , and its apgar score ( kruskal - wallis test , p = 0.542 ) ( table 3 ) . our findings suggest that prenatal depressive and anxiety symptoms were not associated with neonatal outcomes , such as birth weight of the newborn , apgar score and the newborn s admission in nicu . , the evidence from our study suggests that limited depressive and anxiety symptoms during the third trimester of pregnancy do not appear to affect neonatal outcomes , such as the newborn s birth weight , the apgar score and the newborn s admission in the nicu . | [
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] |
construction of vectors complementary dnas encoding
c - myc epitope - tagged human plc1 ( 1291 aa , accession
number abb84466 ) and human plc2 ( 1265 aa , accession number
np_002652 ) were inserted into pcdna3.1(- ) and pvl1393 or pcdna3.1(+ ) and
pvl1392 , respectively .
the epitope was attached to the carboxyl termini
( ( l / s)eqkliseedl , carboxyl - terminal residues of
plc1 and plc2 underlined ) . in our discussion of the chimeric versions of plc1 and
plc2
, the following nomenclature will be used :
plcw - xyz , where w refers to the plc isoform backbone ; x , the
amino - terminal ph domain ; y , the n - terminal portion of spph ; and z , the
c - terminal portion of spph . according to this designation , for example ,
construct plc1 - 122 corresponds to plc1 with the
amino - terminal ph domain from plc1 , the n - terminal portion
of spph from plc2 , and the c - terminal portion of spph from
plc2 ( fig .
for construction of the cdnas encoding the chimeric ,
c - myc epitope - tagged plc enzymes plc1 - 211 and
plc2 - 122 , two separate cdna fragments , one encoding the amino - terminal
ph domain of either plc1 ( aa 1 - 144 ) or
plc2 ( aa 1 - 133 ) and the other encoding the remainder of
plc2 or plc1 followed by the epitope tag ,
were obtained by pcr and joined together .
the cdna of c - myc
epitope - tagged plc1-11 was constructed using the pcr overlap
extension method ( 28 ) to join
the cdnas encoding the amino - terminal ph domain of human plc2
( aa 1 - 137 ) to the cdna encoding plc1 without its
amino - terminal ph domain ( aa 145 - 1291 ) .
the cdnas of the chimeric ,
c - myc epitope - tagged plc isozymes plc1 - 112 ,
plc1 - 121 , plc1 - 122 , plc2 - 212 , plc2 - 221 , and
plc2 - 211 , in which one or both portions of the split ph domain of one
isozyme ( aa 482 - 527 and 872 - 937 of plc1 ; aa 468 - 513 and
849 - 914 of plc2 ) were replaced by the corresponding regions
of the other , were constructed using a two - step megaprimer pcr protocol
( 29 ) .
the cdna of a
deletion mutant ( 1 - 188 ) of human vav1 was amplified by pcr and ligated
into pcdna3.1(+ ) already containing a dna sequence encoding the 12ca5
hemagglutinin epitope tag ( mgypydvpdyaggsm ; hemagglutinin
epitope underlined and met of vav1 shown in italic type ) . to prepare a baculovirus encoding gst - tagged rac2
, the cdna of human rac2
was inserted into the baculovirus transfer vector pac2gt ( pharmingen ) . for
expression of proteins in escherichia coli ( plc1
spph , aa 485 - 936 , 530 - 864 ; plc2 spph , aa 471 - 913 ,
516 - 841 , wild type , and mutants k862i , v893q , and f897q ; and
rac2 , aa 2 - 177 )
, the particular cdnas were cloned into the
ptriex4 vector ( novagen ) using the ek / lic methodology following the
manufacturer 's instructions .
all expression constructs were pcr - amplified with
a tev protease recognition sequence followed by a ggsggs linker followed by
the domain open reading frame .
expression and purification of proteins for production of
recombinant isoprenylated rac2 , baculovirus - infected insect ( sf9 ) cells
( invitrogen ) were grown at 27 c in suspension culture in tnm - fh medium
containing 10% ( v / v ) fetal calf serum ( catalog number p04 - 83500 ; pan biotech ,
aidenbach , germany ) supplemented with 0.2% ( w / v ) pluronic
f-68
( invitrogen ) , 50 g / ml gentamicin ( paa laboratories ) , and 2.5 g / ml
amphotericin b ( fungizone ; invitrogen ) in a 1800-ml fernbach culture
flask . cells ( 10 cells / flask ) were incubated at 27 c with
recombinant baculovirus in 400 ml of medium at 80 rpm on a rotary shaker with
an amplitude of 25 mm .
three days after infection , the cells were harvested at
room temperature by centrifugation at 300 g for 5 min and
washed once with 100 ml of buffer a ( 10 mm
na2hpo4 , 1.8 mm kh2po4 ,
140 mm nacl , 2.7 mm kcl , ph 7.4 ) per 10
intact cells at the time of cell harvesting . to obtain detergent - solubilized
rho gtpases ,
the cells were resuspended in 15 ml per 10 intact
cells of ice - cold buffer b containing 20 mm tris / hcl , ph 8.0 , 1
mm edta , 1 mm dithiothreitol , 100 mm nacl ,
3.75 mm mgcl2 , 0.1 mm phenylmethylsulfonyl
fluoride , 1 g / ml leupeptin , 1 g / ml aprotinin , and 3 m
gdp and homogenized using a precooled 5-ml teflon - glass homogenizer . nuclei
and unbroken cells were removed by centrifugation at 300 g
for 10 min at 4 c .
the membrane fraction was collected from the resulting
supernatant by centrifugation at 12,000 g for 15 min at 4
c .
rho gtpases were solubilized by resuspending the membranes in 2 ml per
10 intact cells at the time of cell harvesting of ice - cold buffer
b supplemented with 23 mm sodium cholate and incubating this
mixture for 90 min at 4 c with vigorous vortexing every 10 min .
insoluble
material was removed from this suspension by centrifugation at 12,000
g for 15 min at 4 c .
the resulting detergent extract was
aliquoted , snap - frozen in liquid n2 , and stored at -80 c .
for production of recombinant plc isozymes , sf9 cells were grown at
27 c in adherent culture in 75-cm flasks in tnm - fh medium
( catalog number t3285 ; sigma ) supplemented with 10% ( v / v ) fetal calf serum
( catalog number f7524 ; sigma ) and 50 g
cells ( 20
10 cells / flask ) were incubated with recombinant baculovirus at 27
c in 10 ml of medium / flask .
three days after infection , the cells were
detached from the plastic surface , harvested by centrifugation at 300
g for 5 min at room temperature , washed once at room temperature with
1 ml / flask of buffer a , and then resuspended in 100 l / flask of ice - cold
buffer c containing 20 mm tris / hcl , ph 7.5 , 2 mm edta , 2
g / ml soybean trypsin inhibitor , 3 mm benzamidine , 0.1
mm phenylmethylsulfonyl fluoride , 1 m pepstatin , 1
m leupeptin , and 1 g / ml aprotinin .
the cells were
homogenized by forcing the suspension ten times through a 0.45 25-mm
needle attached to a disposable syringe .
the homogenate was centrifuged at
100,000 g for 1 h at 4 c , and the resulting supernatant
was aliquoted , snap - frozen in liquid n2 , and stored at -80
c .
c - myc epitope - tagged plc1 - 111 , plc2 - 222 , and
plc1 - 122 were purified from soluble fractions of baculovirus - infected
insect cells grown in suspension culture by sequential chromatography on
hitrap heparin hp and monoq ( ge healthcare ) as described for
plc2 in ref .
30 . for purification of
posttranslationally modified rac2 , the protein was expressed as a glutathione
s - transferase fusion protein in baculovirus - infected insect cells and
solubilized from the particulate fraction as described for wild - type rac2
( 12 ) .
the protein was purified
from the detergent extract by batch adsorption to glutathione - sepharose
4b ( ge healthcare ) ; cleavage of the rac2 portion from the resin by proteolysis
with thrombin ( 22.5 units / ml 75% ( v / v ) slurry ) in buffer d containing 50
mm tris / hcl , ph 7.5 , 50 mm nacl , 2 mm
mgcl2 , 1 mm dithiothreitol , and 0.1% ( v / v ) triton x-100 ;
and removal of the protease by batch adsorption to
p - aminobenzamidine - agarose ( sigma ) .
recombinant proteins were
expressed from ptriex4 vectors in e. coli overnight at 25 c in
the presence of 100 m isopropyl
1-thio--d - galactopyranoside ( induction was carried out when
the bacterial culture attained an a600 of between 0.4 and
1.0 ) .
first ,
ni - chelating chromatography utilizing 5-ml histrap columns ( ge
healthcare ) and wash buffer e ( 25 mm tris / cl , 500 mm
nacl , 40 mm imidazole , and 1 mm
tris(2-carboxyethyl)phosphine hydrochloride , ph 8.0 ) and eluting buffer f ( 25
mm tris / cl , 500 mm nacl , 500 mm imidazole ,
and 1 mm tris(2-carboxyethyl ) phosphine hydrochloride , ph 8.0 ) .
second , the his and s - tags were proteolytically cleaved overnight by tev
protease in cleavage and dialysis buffer g ( 25 mm tris / cl , 150
mm nacl , 1 mm tris(2-carboxyethyl ) phosphine
hydrochloride , ph 8.0 ) at 4 c .
third , the cleaved protein mix was passed
over a ni - loaded 5-ml hitrap chelating column ( ge healthcare ) in
buffer g , and the flow - through was collected .
last , the flow - through fractions
were loaded on a superdex 75 26/60 gel filtration column ( ge healthcare ) in
buffer g , and fractions of monomeric protein were collected and concentrated .
proteins were either used immediately or stored by snap freezing in liquid
n2 and transfer to -80 c . labeled proteins for nmr studies
were expressed essentially as outlined in ref .
phospholipase c
activity was determined as described
( 30 ,
34 ) with minor modifications .
in brief , aliquots ( 10 l ) of the soluble fraction of
plc-baculovirus - infected insect cells appropriately diluted in buffer
h , containing 60 mm tris / maleate , ph 7.3 ,
84 mm kcl , 3.6
mm egta , 2.4 mm dithiothreitol , 2 mg / ml bovine serum
albumin , were incubated for 45 min at 30 c in a volume of 60 l
containing 50 mm tris / maleate , ph 7.3 , 70 mm kcl , 3
mm egta , 2 mm dithiothreitol , 536 m
phosphatidylethanolamine , 33.4 m
[ h]phosphatidylinositol ( 4,5)-bisphosphate ( 185 gbq / mmol ) , 0.33
mg / ml bovine serum albumin , and the concentrations of sodium deoxycholate and
free ca specified in the figure legends . for reconstitution of
wild - type and mutant plc isozymes with rac2 , the diluted soluble
fraction containing the plc or purified plc2 was
reconstituted with 5 l of detergent extract containing crude or purified
isoprenylated rac2 and incubated with the phospholipid substrate as described
above .
fifty mm hepes / naoh , ph 7.2 , was present in the incubation
medium instead of 50 mm tris / maleate , ph 7.3 , when purified
proteins were reconstituted . the concentration of cacl2 required to
adjust the concentration of free ca to the desired value was
calculated using the program eqcal for windows ( biosoft , ferguson , mo ) .
the
reaction was terminated , and the samples were analyzed for inositol
phosphates , as described
( 30 ) .
cos-7 cells were maintained at
37 c in a humidified atmosphere of 90% air and 10% co2 in
dulbecco 's modified eagle 's medium ( catalog number 41965 - 039 ; invitrogen )
supplemented with 10% ( v / v ) fetal calf serum ( catalog number 10270 - 106 ;
invitrogen ) , 2 mm glutamine , 100 units / ml penicillin , and 100
g / ml streptomycin ( all from paa laboratories , clbe , germany ) .
prior
to transfection , the cells were seeded into 12-well plates at densities of 1
10 cells / well , respectively , and grown for 24 h in 1
ml / well of the same medium .
one hour before transfection , the medium was
replaced with 1 ml / well of fresh medium .
for transfection of cos-7 cells ,
plasmid dna ( 1.0 g dna / well ) was mixed with 2.0 l lipofectamine
2000 reagent ( invitrogen ) in 0.2 ml of opti - mem i ( invitrogen ) according
to the manufacturer 's instructions .
after the addition of the
dna - lipofectamine 2000-complexes to the dishes , the cells were
incubated for a further 24 h at 37 c and 10% co2 without
changing the medium .
twenty - four hours after transfection , the cells were washed
once with 0.5 ml / well of buffer a and then supplied with 0.4 ml / well of
dulbecco 's modified eagle 's medium containing fetal calf serum and supplements
as specified above , 10 ci / ml myo-[2-h]inositol
( catalog number trk911 ; ge healthcare ) , and 10 mm licl .
the cells
were incubated in this medium for 20 h , washed once with 0.4 ml / well of buffer
a , and then lysed by the addition of 0.2 ml / well of 10 mm ice - cold
formic acid ( 35 ) .
after
keeping the samples on ice for 30 min , 0.3 ml / well of 10 mm
nh4oh was added for neutralization , and the sample was centrifuged
for 5 min at 15,000 g. the supernatant was loaded onto a
column containing 0.25 ml of dowex 18 - 200 ion exchange resin
( catalog number 217425 ; sigma ) that had been converted to the formate form and
equilibrated with h2o as described
( 34 ) .
the columns were washed
once with 3 ml of h2o and then twice with 3.5 ml each of 60
mm sodium formate and 5 mm sodium tetraborate , and
inositol phosphates were eluted with 3 ml of 1 m ammonium formate
and 100 mm formic acid .
the eluate was supplemented with 15 ml of
scintillation fluid ( ultima gold ; perkinelmer life sciences ) , and the
radioactivity was quantified by liquid scintillation counting . the columns
were reused after regeneration , as described
( 34 ) .
figure 1.plc2 is activated by rac2 . a ,
domain organization of plc isoforms and their chimera .
the common core
domains ( n - ph , ef , catalytic , and c2 ) and unique regions are shown .
the
plcw - xyz nomenclature , used throughout , refers to w ( plc isoform
backbone ) , amino - terminal ph domain ( x ) , n - terminal portion of spph ( y ) , and
c - terminal portion of spph ( z ) . according to this designation , for example ,
construct plc1 - 122 corresponds to plc1 with the
amino - terminal ph domain from plc1 , the n - terminal portion
of spph from plc2 , and c - terminal portion of spph from
plc2 .
b , the activation of purified
plc2 by purified rac2 can be reconstituted in vitro .
recombinant rac2 and plc2 were purified from
baculovirus - infected insect cells and reconstituted in the presence of 100
m gdp or 100 m gtps with phospholipid
vesicles containing phosphatidylinositol ( 4,5)-bisphosphate ( left ) .
the purity of the preparations is also shown ( analysis by sds - page and
coomassie blue staining ) ( right ) .
plc2 is activated by rac2 . a ,
domain organization of plc isoforms and their chimera .
the common core
domains ( n - ph , ef , catalytic , and c2 ) and unique regions are shown .
the
plcw - xyz nomenclature , used throughout , refers to w ( plc isoform
backbone ) , amino - terminal ph domain ( x ) , n - terminal portion of spph ( y ) , and
c - terminal portion of spph ( z ) . according to this designation , for example ,
construct plc1 - 122 corresponds to plc1 with the
amino - terminal ph domain from plc1 , the n - terminal portion
of spph from plc2 , and c - terminal portion of spph from
plc2 .
b , the activation of purified
plc2 by purified rac2 can be reconstituted in vitro .
recombinant rac2 and plc2 were purified from
baculovirus - infected insect cells and reconstituted in the presence of 100
m gdp or 100 m gtps with phospholipid
vesicles containing phosphatidylinositol ( 4,5)-bisphosphate ( left ) .
the purity of the preparations is also shown ( analysis by sds - page and
coomassie blue staining ) ( right ) .
nmr spectra were acquired at 298 k on a
varian unityplus ( 500 mhz ) , varian inova ( 600 and 800 mhz ) , or bruker avance
iii spectrometer ( 700 mhz ) equipped with either a triple resonance probe or a
cryogenically cooled triple resonance probe , including z axis pulse
field gradient coil .
sequence - specific resonance assignments were obtained
using standard triple resonance nmr spectroscopy , namely
h - n hsqc , h - c hsqc , hnca ,
hn(co)ca , hnco , hncacb , cbca(co)nh , h - n tocsy - hsqc ,
hc(c)h - tocsy .
distance restraints were derived from three - dimensional
n- and c - edited noesy - hsqc spectra with a mixing
time of 100 ms .
all nmr spectra were processed using nmrpipe / nmrdraw
( 36 ) and analyzed using ansig
for opengl version 1.0.3 ( 37 ) .
h , c , and n chemical shifts were
referenced indirectly to sodium 2,2-dimethyl-2-silanepentane-5-sulfonate ,
using absolute frequency ratios for the h signals
( 38 ) .
the interaction of the plc2 spph with gppnhp - loaded
rac2 ( aa 2 - 177 ) was performed at constant concentration of spph
protein using the method previously described
( 39 ) , ranging from spph / rac2
molar ratios of 1:0 to 1:1.1 .
protein concentrations were estimated by using
predicted extinction coefficients based upon amino acid composition .
any
changes in the spectrum of labeled component during the titration can be
attributed directly to an intermolecular interaction , since in each experiment
both proteins are pre - exchanged into the same buffer .
interproton distance restraints were
derived from the ansig cross - peaks file of three - dimensional n
noesy - hsqc and c noesy - hsqc spectra for the
plc2 spph domain .
a proportion of the resonances were
successfully assigned in a manual fashion without ambiguity .
the remaining
cross - peaks appearing at positions in the spectrum with overlapping resonances
were labeled with ambiguous assignments by reference to the chemical shift
list obtained with through - bond correlation spectra , using the
connect module from the program azara
( 40 ) .
the cross - peaks were
grouped into five categories according to their relative peak intensities
( strong , medium , weak , very weak , and very , very weak ) and were designated
with the corresponding interproton distance restraint limit of 1.8 - 2.5 ,
1.8 - 3.0 , 1.8 - 3.5 , 1.8 - 4.0 , and 1.8 - 5.0 , respectively .
a distance of
0.5 per methyl group was added to the upper bound of the distance
restraint for noe cross - peaks that involved methyl groups .
all structures for spph were calculated using an ab initio
simulated annealing protocol within the cns program
( 41 ) , with parallhdg version
5.3 force field and prolsq nonbonded energy function
( 42 ) .
the protocol adopts a
mixture of cartesian molecular dynamics and torsion angle dynamics simulated
annealing to refine structures starting from random generated conformers with
good local geometry .
a total of 2487 noe - derived interproton distance restraints for spph were
included in the final iterations of the structure calculations ( see
table 1 ) .
backbone torsion
angle restraints for and were derived from analysis of
h , c , c ,
c , and nh chemical shift data bases as
implemented in the program talos
( 43 ) .
hydrogen bond restraints
for amide protons were derived from an assessment of the regular secondary
structure elements .
this analysis included the overall and local patterns of
noes and the pattern of amide proton solvent exchange rates .
a total of 114
dihedral angle and 70 hydrogen bond ( 35 hydrogen bonds ; two distance
restraints per hydrogen bond ) interatomic distance restraints were used for
spph .
table 1kinetic parameters and derived dissociation constants for the
interaction between rac2 and plc isozymes determined by surface plasmon
resonance measurementskd values were derived from the ratio
ka / kd .
wt , wild type.analyteligandakakdkdm ss m plc2 - 222 ( wt )
his - rac2 ( gtps )
806
3.1 10 3.9
plc1 - 111 ( wt )
his - rac2 ( gtps )
noneb plc1 - 122
his - rac2 ( gtps )
434
2.5 10 5.8
plc2 ( ph - c2 )
his - rac2 ( gtps )
385
2.3 10 6.0
2spph ( wt )
his - rac2 ( gtps )
93.8
1.6 10 17
2spph ( wt )
his - rac2 ( gdps )
none
1spph ( wt )
his - rac2 ( gtps )
none
arac2 protein designated as his - rac2 contains the sequence
his6-s - tag - tev - ggs - ggs- at the n terminus.bnone , no significant association signal was detected .
kinetic parameters and derived dissociation constants for the
interaction between rac2 and plc isozymes determined by surface plasmon
resonance measurements kd values were derived from the ratio
ka / kd .
rac2 protein designated as his - rac2 contains the sequence
his6-s - tag - tev - ggs - ggs- at the n terminus .
biosensor measurements the biosensor measurements were
carried out on the biacore 3000 system ( ge healthcare ) at 25 c . the
sensor chip nta was utilized and loaded with ni according to the
manufacturer 's instructions .
purified , hexahistidine - tagged
rac2 ( aa 2 - 177 ) was loaded with gtps or gdps and
immobilized in biosensor buffer ( 10 mm hepes / naoh , ph 8.0 , 150
mm nacl , 1 mm mgcl2 , 5% ( w / v ) cm - dextran , and
0.01% ( v / v ) nonidet p-40 ) at a flow rate of 5 l / min for 5 min , which
resulted in a deposition of 300 response units .
next , the purified
analytes ( full - length plc molecules or their isolated spphs ) were
injected at varying concentrations .
the evaluation of kinetic
parameters was performed by nonlinear fitting of binding data using
biaevaluation 2.1 software .
the apparent association ( ka )
and dissociation rate ( kd ) constants were evaluated from
the differential binding curves ( fc2 - fc1 ) assuming an a + b = ab association
type for the protein - protein interaction .
the dissociation constant
kd was calculated from the equation ,
kd = kd / ka .
figure 2.the split ph domain of plc2 is required for its
regulation by rac2 in vitro .
a - c , left , soluble
fractions of sf9 cells infected with baculoviruses encoding
-galactosidase ( control ) , wild - type plc isoforms ( plc1 - 111
and plc2 - 222 ) , and their chimeras ( plc1 - 211 , plc2 - 122 ,
plc1 - 121 , plc1 - 112 , plc1 - 122 , plc2 - 212 ,
plc2 - 221 , and plc2 - 211 ) were diluted with buffer and incubated
at increasing protein concentrations for 45 min at 30 c with phospholipid
vesicles containing phosphatidylinositol ( 4,5)-bisphosphate .
the incubation
was performed in the presence of 10 m free ca and
2.5 mm sodium deoxycholate .
a - c , right , the soluble
fractions of sf9 cells infected with baculoviruses encoding the indicated
wild - type and mutant plc isozymes were adjusted by dilution with buffer
to contain similar basal plc activity according to the results shown in the
left panel .
the soluble fraction of sf9 cells infected with
baculovirus encoding -galactosidase ( control ) was used at the maximal
protein concentration among the plc-containing fractions , 1.4 mg
protein / ml .
aliquots ( 10 l ) of these samples were reconstituted with
aliquots of detergent extracts prepared from membranes of sf9 cells infected
with baculoviruses encoding -galactosidase ( no bracket ) or rac2
( brackets ) and incubated for 2 h at 30 c in the presence of 100
mm gdp or gtps with phospholipid vesicles containing
phosphatidylinositol ( 4,5)-bisphosphate .
the incubation was performed in the
presence of 30 nm free ca and 1 mm sodium
deoxycholate .
inset , aliquots ( 10 l ) of the samples were
subjected to sds - page , and immunoblotting was performed using an antibody
reactive against the c - myc epitope . in a , there are five
lanes in the inset but six samples in the corresponding
bar chart .
the lane corresponding to plc2 - 222 without
rac2 is not shown in the inset .
lanes 1 - 5 , control , plc1 - 111 ,
plc2 - 222 , plc1 - 211 , and plc2 - 122 , respectively ( all with
rac2 ) .
the split ph domain of plc2 is required for its
regulation by rac2 in vitro .
a - c , left , soluble
fractions of sf9 cells infected with baculoviruses encoding
-galactosidase ( control ) , wild - type plc isoforms ( plc1 - 111
and plc2 - 222 ) , and their chimeras ( plc1 - 211 , plc2 - 122 ,
plc1 - 121 , plc1 - 112 , plc1 - 122 , plc2 - 212 ,
plc2 - 221 , and plc2 - 211 ) were diluted with buffer and incubated
at increasing protein concentrations for 45 min at 30 c with phospholipid
vesicles containing phosphatidylinositol ( 4,5)-bisphosphate .
the incubation
was performed in the presence of 10 m free ca and
2.5 mm sodium deoxycholate .
a - c , right , the soluble
fractions of sf9 cells infected with baculoviruses encoding the indicated
wild - type and mutant plc isozymes were adjusted by dilution with buffer
to contain similar basal plc activity according to the results shown in the
left panel .
the soluble fraction of sf9 cells infected with
baculovirus encoding -galactosidase ( control ) was used at the maximal
protein concentration among the plc-containing fractions , 1.4 mg
protein / ml . aliquots ( 10 l ) of these samples were reconstituted with
aliquots of detergent extracts prepared from membranes of sf9 cells infected
with baculoviruses encoding -galactosidase ( no bracket ) or rac2
( brackets ) and incubated for 2 h at 30 c in the presence of 100
mm gdp or gtps with phospholipid vesicles containing
phosphatidylinositol ( 4,5)-bisphosphate .
the incubation was performed in the
presence of 30 nm free ca and 1 mm sodium
deoxycholate .
inset , aliquots ( 10 l ) of the samples were
subjected to sds - page , and immunoblotting was performed using an antibody
reactive against the c - myc epitope . in a , there are five
lanes in the inset but six samples in the corresponding
bar chart .
the lane corresponding to plc2 - 222 without
rac2 is not shown in the inset .
lanes 1 - 5 , control , plc1 - 111 ,
plc2 - 222 , plc1 - 211 , and plc2 - 122 , respectively ( all with
rac2 ) . miscellaneous methods
the mouse monoclonal antibody 9b11 reactive against the c - myc epitope
eqkliseedl was obtained from cell signaling technology .
the sources of all
other reagents and recombinant dnas as well as all other experimental
protocols have been described
( 12 ) .
the plc2 split ph domain is required for
isoform - specific regulation by rac2it has previously been shown
with reconstitution experiments that rac gtpases activate
plc2 in the presence of gtps
( 12 ) .
these experiments were
conducted with cell extracts enriched in recombinant plc2
and rac2 that had been separately expressed in baculovirus - infected sf9 cells .
to exclude the possibility that other signaling proteins could mediate
activation of plc2 by rac , we set out to extend these data
with purified components ( fig .
1b ) . we noted a 7-fold activation of
plc2 by gtps - loaded rac2 .
this result strongly
suggests that rac2 interacts directly with plc2 and that the
presence of rac2 is both necessary and sufficient for guanine nucleoside
triphosphate - dependent plc2 activation .
next , we prepared a
number of chimeric proteins where the n - terminal ph domains of the
rac - responsive plc2 and the rac - nonresponsive
plc1 were exchanged .
the specific plc activities of each of
these chimeras were tested in the presence of 10 m
ca and shown to be comparable
( fig .
however , when introduced into the reconstitution assay , it was
evident that the n - terminal ph domain of plc2 does not
impart rac2 activation on plc1 ( variant plc1 - 211 )
( fig .
similarly , the exchange of the plc1
n - terminal ph domain into plc2 ( variant plc2 - 122 ) did
not significantly alter the propensity of this chimera to be activated by
rac2 .
these in vitro observations were further supported by
experiments in intact cos-7 cells co - transfected with cdnas encoding rac2 and
plc isozymes to display the same basal plc activities
( fig .
we
confirmed that rac2 activates plc2 but not
plc1 and that the n - terminal ph domain is not involved in
the rac2-mediated activation .
in addition , we showed that the rac - binding
n - terminal ph domain of plc2 is functionally interchangeable
by constructing a plc1 chimera that incorporates this domain
( variant plc1-11 ) and showing its activation by
rac2 ( fig .
together , these experiments suggest that , unlike
with plc2 , the n - terminal ph domain of plc2
is not involved in the observed interaction of rac2 .
furthermore , the findings
support the idea that rho family gtpase effector interaction sites are not
conserved and can not easily be predicted
( 45 ) .
figure 3.the role of the n - terminal and split ph domains of
plc2 in cellular activation by rac2
.
left , cos-7 cells were transfected with increasing amounts per well
of vector encoding wild - type or mutant plc isozymes .
the total amount
of dna was maintained constant in each transfection by adding empty vector .
the empty vector ( control ) ( a and b ) and the vectors
encoding plc2 - 222 , plc2 - 212 , plc2 - 221 , and
plc2 - 211 ( b ) were used only at 1000 ng / well , since there were
only minimal changes in inositol phosphate production even at this high amount
of vector dna . under these conditions ,
the inositol phosphate formation in
b was as follows : control , 223 30 cpm ; plc2 - 222 , 436
67 cpm ; plc2 - 212 , 390 59 cpm ; plc2 - 221 , 348
54 cpm ; plc2 - 211 , 360 6 cpm ( mean s.d . of
triplicate determinations ) .
right , cos-7 cells were cotransfected as indicated with empty vector
( control ) and/or vectors encoding rac2 , rac2 , or either
wild - type or mutant plc isozymes .
the amounts of vectors encoding the
plc isozymes were adjusted according to their basal activities shown in
the left panels ( plc1 - 111 and plc1 - 211 , 300 ng / well ;
plc1 - 112 , 100 ng / well ; plc1 - 121 and plc1 - 122 , 10 ng / well ;
all other vectors , 1000 ng per well ) .
the total amount of dna was maintained
constant in each transfection by adding empty vector . in additional
experiments
( results not shown ) , we found that expression of
rac2 also caused only a minor ( 1.9-fold ) stimulation of
inositol phosphate formation in cells cotransfected with 1000 ng / well of
the role of the n - terminal and split ph domains of
plc2 in cellular activation by rac2 .
left , cos-7 cells were transfected with increasing amounts per well
of vector encoding wild - type or mutant plc isozymes .
the total amount
of dna was maintained constant in each transfection by adding empty vector .
the empty vector ( control ) ( a and b ) and the vectors
encoding plc2 - 222 , plc2 - 212 , plc2 - 221 , and
plc2 - 211 ( b ) were used only at 1000 ng / well , since there were
only minimal changes in inositol phosphate production even at this high amount
of vector dna . under these conditions ,
the inositol phosphate formation in
b was as follows : control , 223 30 cpm ; plc2 - 222 , 436
67 cpm ; plc2 - 212 , 390 59 cpm ; plc2 - 221 , 348
54 cpm ; plc2 - 211 , 360 6 cpm ( mean s.d . of
triplicate determinations ) .
right , cos-7 cells were cotransfected as indicated with empty vector
( control ) and/or vectors encoding rac2 , rac2 , or either
wild - type or mutant plc isozymes .
the amounts of vectors encoding the
plc isozymes were adjusted according to their basal activities shown in
the left panels ( plc1 - 111 and plc1 - 211 , 300 ng / well ;
plc1 - 112 , 100 ng / well ; plc1 - 121 and plc1 - 122 , 10 ng / well ;
all other vectors , 1000 ng per well ) .
the total amount of dna was maintained
constant in each transfection by adding empty vector . in additional
experiments
( results not shown ) , we found that expression of
rac2 also caused only a minor ( 1.9-fold ) stimulation of
inositol phosphate formation in cells cotransfected with 1000 ng / well of
vector encoding plc1 - 111 or plc1 - 211 .
the plc isoforms contain a second ph domain within the specific
array ( sa ) region located between the x and y domains of the catalytic barrel .
this spph consists of two parts separated by the tandem array insert of two
sh2 domains and an sh3 domain .
although the two halves of
plc1 spph can form a contiguous fold when expressed without
the other domains ( 4 ) , it is
not known whether , in the context of the full - length plc molecules ,
these two sections also form a contiguous ph domain or are spatially
separated .
recently , there have been reports that attribute different
functions to spphs ( 46 ,
47 ) with the insertion of
other domains between either -strands 6 and 7 or -strands 3 and 4 ,
as in the case of the plc1 isoform
( 4 ) .
accordingly , we prepared
chimeric plc1 proteins that contained either one or both of
the plc2 spph sections .
the swapping of either the n- or
c - terminal spph subdomains ( see fig .
1a ) from plc2 did not confer rac
activation on plc1 ( fig .
2b , right ) . however , the insertion of both
partial spph subdomains from plc2 produced a
plc1 chimera that was stimulated 5.4-fold in activity by
recombinant rac2 .
the
plc1 spph subdomains were engineered into the
plc2 polypeptide chain both as individual partial domains
and as both halves together ( fig .
the exchange of either or both of
the partial domains abolished activation by rac2 in reconstitution
experiments .
therefore , both spph subdomains of plc2 are
necessary and sufficient to impart rac2-dependent plc activation . this
conclusion is supported by experiments that tested these plc chimeras
in transfected cos-7 cells ( fig .
, the two spph subdomains of
plc2 also imparted on plc1 a marked
sensitivity to activation by exogenous vav1 and endogenous rac gtpases present
in cos-7 cells , whereas the presence of the two spph subdomains of
plc1 within the context of plc2 rendered
the chimeric enzyme indistinguishable in this regard from wild - type
plc1 ( fig .
the data obtained from the analysis of plc spph ( figs .
2 , b and c ,
and 3b ) , suggest that
either the site of rac2 interaction is distributed over both halves of spph or
that correct folding of each half requires the presence of the other from the
same isoform . since our further studies suggest that the first scenario is
unlikely ( see fig .
7 ) , the
incorrect folding of each spph half could be the reason for the loss of
interaction with rac . indeed , recent studies of plc1 have
shown that the construct of the isolated second half of its spph was unfolded
but that the interaction with the complementary half induces the correct
folding ( 4 ) .
since the sequence
identity ( 29% ) of the spph regions of the plc isoforms is low , it is
likely that their subdomains can not be interchanged without losing correct
spph folding .
a , heteronuclear nmr spectroscopy of
plc2 spph ( 471 - 913 , 516 - 841 ) .
the two - dimensional
h - n hsqc spectrum of
c / n - labeled plc2 spph ( 471 - 913 ,
516 - 841 ) , recorded on a 600-mhz varian inova spectrometer at 298 k. the
resonance assignments for the backbone and side chain nh group cross - peaks are
included .
c , ribbon representation of the lowest
energy plc2 spph conformer with secondary structure elements
labeled .
structural elements derived from the n - terminal spph region ( aa
471 - 515 ) are depicted in red , and those from the c - terminal spph
region ( aa 842 - 913 ) are shown in orange .
d , superposition of the
backbone c trace of the mean solution structures of the
plc2 and plc1 spphs .
plc2 ( protein data bank code 2k2j ; red / orange ) and
plc1 ( protein data bank code 2fjl ; blue / cyan )
spphs .
a , heteronuclear nmr spectroscopy of
plc2 spph ( 471 - 913 , 516 - 841 ) . the two - dimensional
h - n hsqc spectrum of
c / n - labeled plc2 spph ( 471 - 913 ,
516 - 841 ) , recorded on a 600-mhz varian inova spectrometer at 298 k. the
resonance assignments for the backbone and side chain nh group cross - peaks are
included .
c , ribbon representation of the lowest
energy plc2 spph conformer with secondary structure elements
labeled .
structural elements derived from the n - terminal spph region ( aa
471 - 515 ) are depicted in red , and those from the c - terminal spph
region ( aa 842 - 913 ) are shown in orange .
d , superposition of the
backbone c trace of the mean solution structures of the
plc2 and plc1 spphs .
plc2 ( protein data bank code 2k2j ; red / orange ) and
plc1 ( protein data bank code 2fjl ; blue / cyan )
spphs .
the isolated split ph domain from plc2
directly binds rac to evaluate the role of plc2
spph as the site of rac interaction , we purified a number of
plc2 and plc1 variants ( including the
full - length and isolated spphs ) in order to carry out interaction studies
in vitro . for comprehensive , quantitative analysis we used surface
plasmon resonance ( table 1 ) .
consistent with the data where we analyzed the requirements for activation of
plc2 by rac2 ( figs .
1 ,
2 ,
3 ) , plc2 , but
not plc1 , selectively bound gtps - activated rac2
( table 1 ) .
furthermore , the
plc1 chimera incorporating both halves of the
plc2 spph ( plc1 - 122 ) was fully functional in
rac2-gtps binding .
the strength of the binding of
plc2 and plc1 - 122 ( kd = 3.9 and
5.8 m , respectively ) was similar to that determined in this and
a previous study for plc2 ( kd = 6.0 and
7.0 m , respectively )
( 26 ) .
these data confirm
plc2 as a direct effector of rac and show that its spph
determines the isoform specificity for this interaction .
we also assessed whether plc2 spph in isolation can bind
gtps - activated rac2 .
based on the recent structural characterization of
plc1 spph
( 4 ) , we designed a contiguous
plc2 spph construct lacking the intervening sh2 and sh3
domains and being replaced by a linker consisting of the remaining natural
loop regions .
the corresponding domain from
plc1 was also constructed . both plc2 and
plc1 spphs
selectively bound rac2-gtps , similar to the
full - length protein ; importantly , for plc1 spph , no
interaction with rac2 could be detected
( table 1 ) . the binding strength
for plc2 spph with rac2 ( kd = 17
m ) is in close agreement with previously reported affinities of
rac2 for the isolated ph domain of plc2
( 26 ) .
subsequent structural
studies have shown that the plc2 isoform contacts rac solely
through its ph domain
( 23 ) .
the strengths of interaction between plc2 and rac2 shown
here ( table 1 ) , in the
micromolar range for kd , are generally consistent with
values obtained for plc2-rac
( 26 ) and plc-ras
( 22 ) complexes and more
broadly with a number of other small gtpase - effector interactions
( 48 ) measured in
vitro .
there are , however , instances of rac- and cdc42-effector
interactions with dissociation constants in the nanomolar range
( 49 ,
50 ) .
however , in a cellular
setting , the posttranslationally modified c terminus of rac2 or the plasma
membrane could be involved in stabilizing the interaction between activated
rac2 and full - length plc2 .
it is important to note that the
spphs of plc1 and -2 are unlikely to bind
to membrane lipids directly .
experimental
( 4 ) and molecular modeling
( 51 ) studies agree that these
domains do not possess the amino acid sequence motifs typical of lipid binding
modules .
accordingly , although some changes in the subcellular distribution do
occur with some of the chimeras ( plc1 - 121 , plc1 - 112 , and
plc2 - 221 ) ( fig .
s2 ) , these effects do not correlate with the variation
in activity observed in fig .
3b ( left ) , which are also evident in the
cell - free system ( fig .
2b , right ) .
structural analysis of the plc2 split ph
domain interaction interface with rac2to provide a basis for
further analysis of the interaction between the plc2 spph
and rac2 , we determined the three - dimensional solution structure of the spph
by heteronuclear nmr spectroscopy .
single ( n ) and double
( c , n ) isotope - labeled samples of
plc2 spph were prepared , and nearly complete resonance
assignments were obtained using standard triple resonance nmr experiments
( fig .
4a ) . on the
basis of the analysis of three - dimensional n- and
c - edited h noesy spectra , 2487 interproton distance
restraints were obtained and used in structure calculations along with 70
hydrogen bond restraints and 114 dihedral angle restraints .
table 2 shows the structural
statistics for the bundle of 20 lowest energy conformers , each of which
displays low restraint violations and good stereochemical and nonbonded
interaction scores .
the best fit superposition of the backbone atoms of the
conformer set is shown in fig .
the lowest energy structure is shown in a ribbon
representation in fig .
4c , demonstrating the conserved core structure of a
partially open two - sheet -barrel with one end capped by the c - terminal
helix .
as predicted , the plc2 spph structure conforms well
with the canonical ph domain architecture with seven -strands and one
-helix : residues 478 - 485 ( 1 ) ; 490 - 499 ( 2 ) and 502 - 506
( 3 ) from the n - terminal spph subdomain ; 851 - 853 ( 4 ) ; 860 - 863
( 5 ) ; 873 - 876 ( 6 ) ; 886 - 889 ( 7 ) ; and 893 - 906 ( 1 ) from
the c - terminal spph subdomain . omitting the loop between 3 and 4 ,
which contains the linker between the spph subdomains , and a small number of
apparently flexible residues at the n and c termini , the refined conformer
bundle provides a well defined model for the plc2 spph with
coordinate root mean square difference values of 0.41 0.05
for the backbone and 0.76 0.06 for all heavy atoms ( residues
478 - 508 and 849 - 908 ) .
table 2summary of structure statistics for plc2 spph < sa > represents the set of 20 selected lowest energy conformers
obtained by restrained dynamical simulated annealing in cns .
there
were no noe ( > 0.4 ) or dihedral ( > 5 ) violations for any of
the lowest energy conformers.<sa > salowestexperimental
restraintsa all ( ) ( 2487 )
0.018 0.002
0.014
intraresidue ( 786 )
0.014 0.003
0.010
sequential ( 553 )
0.014 0.005
0.010
short ( 373 )
0.023 0.003
0.020
long ( 766 )
0.018 0.001
0.015
ambiguous ( 9 )
0.009 0.005
0.009
hydrogen bond restraints ( ) ( 70 )
0.031 0.004
0.029
dihedral angle restraints ( degrees ) ( 114 )
0.26 0.03
0.236
deviations from idealized covalent
geometryb bonds ( ) ( 1930 )
0.0013 0.0001
0.0012
angles ( degrees ) ( 3490 )
0.31 0.004
0.31
improper dihedrals ( degrees ) ( 1020 )
0.2 0.01
0.2
structural statistics for the
ensemblec procheck parameters
most favored region ( % )
73.1 2.7
74.3
additionally allowed ( % )
22.5 2.7
23.8
generously allowed ( % )
3.0 1.6
1.0
disallowed ( % )
1.5 0.7
1.0
number of bad contacts
3 2
1
root mean square difference from the average
structured backbone ( n , c , c ) ( )
0.41 0.05
0.34
heavy atoms ( )
0.76 0.06
0.62
asum averaging of noe distance restraints was used for groups with
degenerate proton chemical shifts .
the interproton unambiguous distance
restraint list comprised 786 intraresidue , 553 sequential ( |i -
j| = 1| ) , 373 short range ( 1 < |i -
j| < 5 ) , and 776 long range ( |i - j|
> 5 ) . hydrogen bond restraints were applied as pairs of distance
restraints : ho , 1.2 - 2.2 ;
no , 1.2 - 3.2 .
the final values for the
respective force constants were as follows : noe , 30 kcal
mol ; hydrogen bonds , 50 kcal
mol ; dihedral angles , 200 kcal
mol rad.bthe final values for the respective force constants were as follows : bond
lengths , 1000 kcal mol ; angles
and improper torsions , 500 kcal mol rad ;
the improper torsion angle restraints serve to maintain planarity and
chirality.cthe program procheck ( 64 )
was used to assess the stereochemical parameters of the family of conformers
for the spph .
the figures indicate the percentage of residues with backbone
and angles in separate regions of the ramachandran plot , defined
in the program .
the number of bad contacts per 100 residues is expected to be
in the range 0 - 30 for protein crystal structures of better than 3.0
resolution.dthe precision of the atomic coordinates is defined as the average pairwise
root mean square difference between each of the 20 conformers and a mean
coordinate structure sa generated by iterative best fit of the backbone atoms
( n , c , and c ) over residues 478 - 508 and 849 - 908 of
plc2spph ( comprising the core secondary structure elements
and omitting the flexible n and c termini and the disordered loop between
3 and 4 ) , followed by coordinate averaging .
summary of structure statistics for plc2 spph < sa > represents the set of 20 selected lowest energy conformers
obtained by restrained dynamical simulated annealing in cns .
there
were no noe ( > 0.4 ) or dihedral ( > 5 ) violations for any of
the lowest energy conformers .
sum averaging of noe distance restraints was used for groups with
degenerate proton chemical shifts .
the interproton unambiguous distance
restraint list comprised 786 intraresidue , 553 sequential ( |i -
j| = 1| ) , 373 short range ( 1 < |i -
j| < 5 ) , and 776 long range ( |i - j|
> 5 ) . hydrogen bond restraints were applied as pairs of distance
restraints : ho , 1.2 - 2.2 ;
no , 1.2 - 3.2 .
the final values for the
respective force constants were as follows : noe , 30 kcal
mol ; hydrogen bonds , 50 kcal
mol ; dihedral angles , 200 kcal
mol rad .
the final values for the respective force constants were as follows : bond
lengths , 1000 kcal mol ; angles
and improper torsions , 500 kcal mol rad ;
the improper torsion angle restraints serve to maintain planarity and
chirality .
the program procheck ( 64 )
was used to assess the stereochemical parameters of the family of conformers
for the spph .
the figures indicate the percentage of residues with backbone
and angles in separate regions of the ramachandran plot , defined
in the program .
the number of bad contacts per 100 residues is expected to be
in the range 0 - 30 for protein crystal structures of better than 3.0
resolution .
the precision of the atomic coordinates is defined as the average pairwise
root mean square difference between each of the 20 conformers and a mean
coordinate structure sa generated by iterative best fit of the backbone atoms
( n , c , and c ) over residues 478 - 508 and 849 - 908 of
plc2spph ( comprising the core secondary structure elements
and omitting the flexible n and c termini and the disordered loop between
3 and 4 ) , followed by coordinate averaging .
the secondary structure elements of the spphs from plc1
and plc2 align reasonably well
( fig .
4d ) with a
backbone root mean square difference of 2.9 over 65 core region
c atoms .
it is noteworthy that the sequence identity of the two spphs
is considerably lower ( 29% ) than for the intact plc1 and
plc2 proteins ( 49.5% ) or the respective n - terminal ph
domains ( 48% ) . despite the relatively low sequence identity , when surface
representations of the plc2 and plc1 spph
structures are compared ( fig .
5 )
overall , there is a strong
correspondence of the surface distribution of charge and hydrophobicity .
notably , a patch of negative charge visible at the lower region of the n view
is common between the two domains
. given this apparent global homology , it
seems likely that the differences in rac2 interaction with these two spphs
must reside in rather specific variation in surface side chain distribution .
to probe this hypothesis ,
the residues important for the specific rac2-binding
interface were identified through nmr titration experiments and subsequent
site - directed mutagenesis .
surface electrostatic potentials
representations of these two spphs were computed with pymol ( top ,
plc2 spph ; bottom , plc1 spph ) .
electrostatic potentials are represented as positive ( blue ) , negative
( red ) , and neutral ( white ) charges .
the large loop that
links the two parts of the spphs ( which is present in the published nmr
structure of the plc1 spph ) is not shown .
the n view
notation refers to the surface derived from the amino acid residues from the
n - terminal half of the domain , and the c view refers to those residues derived
from the c - terminal part .
surface electrostatic potentials
representations of these two spphs were computed with pymol ( top ,
plc2 spph ; bottom , plc1 spph ) .
electrostatic potentials are represented as positive ( blue ) , negative
( red ) , and neutral ( white ) charges .
the large loop that
links the two parts of the spphs ( which is present in the published nmr
structure of the plc1 spph ) is not shown .
the n view
notation refers to the surface derived from the amino acid residues from the
n - terminal half of the domain , and the c view refers to those residues derived
from the c - terminal part .
overlay of
h - n hsqc spectra of plc2 spph in
the absence ( blue ) and presence ( red ) of gppnhp - loaded
rac2 ( aa 2 - 177 ) at a 1:1 molar ratio . binding of gppnhp - loaded
rac2 ( aa 2 - 177 ) to plc2 spph leads to a
generalized broadening of the spectrum consistent with complex formation .
the
spectrum of the mixed proteins was plotted at slightly lower contour levels
( by a factor of 0.67 ) in order to emphasize those spph peaks that are
differentially perturbed , thereby highlighting the specific binding site but
masking the overall broadening effect .
b , surface representation of
the amino acid residues of plc2 spph at the interaction
surface with rac2 .
amino acid residues on the surface of
plc2 spph that were perturbed by the titration of rac2 are
labeled and highlighted in green .
amino acid
residues that are underlined are those proposed to be important for
the binding to rac2 , as presented in fig .
overlay of
h - n hsqc spectra of plc2 spph in
the absence ( blue ) and presence ( red ) of gppnhp - loaded
rac2 ( aa 2 - 177 ) at a 1:1 molar ratio . binding of gppnhp - loaded
rac2 ( aa 2 - 177 ) to plc2 spph leads to a
generalized broadening of the spectrum consistent with complex formation .
the
spectrum of the mixed proteins was plotted at slightly lower contour levels
( by a factor of 0.67 ) in order to emphasize those spph peaks that are
differentially perturbed , thereby highlighting the specific binding site but
masking the overall broadening effect .
b , surface representation of
the amino acid residues of plc2 spph at the interaction
surface with rac2 .
amino acid residues on the surface of
plc2 spph that were perturbed by the titration of rac2 are
labeled and highlighted in green .
amino acid
residues that are underlined are those proposed to be important for
the binding to rac2 , as presented in fig .
comparison of the h - n hsqc spectra of
n - labeled plc2 spph in the presence of
increasing concentrations of unlabeled gppnhp - loaded rac2 ( aa
2 - 177 ) reveals complex formation characterized by differential broadening and
some shifting of a distinct subset of the plc2 spph
cross - peaks ( fig .
we observed 20 backbone nh cross - peaks for plc2 spph that
shift or disappear completely upon rac titration .
the corresponding residues
are highlighted in the amino acid sequence of spph and cluster on the protein
surface within the -helix and around -strand 5 ( figs .
interestingly ,
several of these residues , including lys , ala ,
ile , val , and phe , are not conserved
in plc1 spph ( fig .
, we assign the site of rac
interaction to the -strand 5 and -helix regions in the c - terminal
plc2 spph subdomain .
to define more rigorously the amino acid residues in plc2
that are important for interaction with rac , we carried out site - directed
mutagenesis experiments and activity assays .
a number of full - length
plc2 mutants were prepared with amino acid substitutions in
its spph subdomains .
the specific mutations were designed so as to swap the
plc2 residue for the amino acid type in the equivalent
position in plc1 residue
( fig .
specifically , full - length k862i , v893q , f897q , q901k / s902k , and k909 t
plc2 variants were constructed .
the wild - type and mutant
plc2 constructs were each co - expressed in cos-7 cells with
rac2 , and the plc activities were assessed
( fig .
we
identified three residues as important for activation of the enzyme by rac2 .
in the -strand 5 region ,
the v893q and f897q mutations , bordering the
-helix , also yielded a substantially lower activation by rac2 .
the
remaining mutants demonstrated either a small reduction or even an enhancement
of rac2-dependent activation .
we ruled out the possibility that the mutations
perturb the spph fold ; the corresponding substitutions were introduced into
the isolated spph construct , and these variants were assessed by
one - dimensional h nmr .
, it is also unlikely that these
mutations have a direct impact on the structure and function of the catalytic
domain ( 3 ) .
these data support
the conclusion that these mutated residues ( underlined in
fig .
figure 7.analysis of plc2 mutants that are insensitive
to rac activation . a , alignment of the primary structures of the
plc1 and plc2 split ph domains .
amino acid
residues in plc2 whose nmr resonances were perturbed in the
rac2 titration are labeled in green .
cos-7
cells were cotransfected as indicated with empty vector ( control ) , vector
encoding rac2 , and vector encoding either wild - type or mutant
plc2 isozymes ( k862i , v893q , f897q , q901k / s902k , and k909 t ) .
the total amount of dna was maintained constant in each transfection by adding
empty vector .
twenty - four h after transfection , the cells were incubated for
24 h in the presence of [ h]inositol ( 1.5 ci / ml ) and 10
mm licl , and the levels of inositol phosphates were then
determined .
c , one - dimensional nmr spectra of wild - type and mutant
plc2 spph proteins .
the indicated substitutions were
introduced into the isolated spph construct , and the corresponding encoded
proteins were assessed and compared with their wild - type counterparts by
one - dimensional h nmr spectroscopy .
the downfield region
encompassing resonances from the backbone nh and aromatic side chain ch
protons is depicted for each variant .
the maintenance of the overall chemical
shift dispersion indicates that the mutants adopt a globular structure highly
similar to the wild type .
analysis of plc2 mutants that are insensitive
to rac activation . a , alignment of the primary structures of the
plc1 and plc2 split ph domains .
amino acid
residues in plc2 whose nmr resonances were perturbed in the
rac2 titration are labeled in green .
cos-7
cells were cotransfected as indicated with empty vector ( control ) , vector
encoding rac2 , and vector encoding either wild - type or mutant
plc2 isozymes ( k862i , v893q , f897q , q901k / s902k , and k909 t ) .
the total amount of dna was maintained constant in each transfection by adding
empty vector .
twenty - four h after transfection , the cells were incubated for
24 h in the presence of [ h]inositol ( 1.5 ci / ml ) and 10
mm licl , and the levels of inositol phosphates were then
determined .
c , one - dimensional nmr spectra of wild - type and mutant
plc2 spph proteins .
the indicated substitutions were
introduced into the isolated spph construct , and the corresponding encoded
proteins were assessed and compared with their wild - type counterparts by
one - dimensional h nmr spectroscopy .
the downfield region
encompassing resonances from the backbone nh and aromatic side chain ch
protons is depicted for each variant .
the maintenance of the overall chemical
shift dispersion indicates that the mutants adopt a globular structure highly
similar to the wild type .
interestingly , our data also show that the surface of
plc2 spph involved in the interaction with rac2
( fig .
6 ) is quite different
from that described for the n - terminal plc2 ph domain
involved in binding of rac1
( 23 ) , where the main contact
region is located in -strand 1 and loop regions that align with this
strand .
this variance is generally consistent with the observed diversity of
binding sites for rho family gtpases
( 45 ) and further shows that
even when the same fold ( i.e. aph domain ) is involved , the
interaction surfaces engaged in rac binding can be different .
implications for regulation of plc
isoforms in conjunction with the conclusions drawn from our
previous study ( 12 ) , the
results described here reveal several aspects of the regulation of
plc2 activity by rac . first , in reconstitution assays , rac2
is sufficient to activate plc2 in the absence of other
protein components ( fig .
1 ) .
second , when the proteins are co - expressed in cos-7 cells , rac2 mediates
translocation of the plc2 isoform to cellular membranes
( 12 ) .
third , in transfected
cells , rac2-mediated plc2 activation is not dependent upon
the phosphorylation of critical tyrosine residues
( 12 ) previously linked to
plc2 regulation in b - cells
( 52 ,
53 ) .
taken together , these
results could be taken to imply that substantial activation of
plc2 in vivo can be achieved through interaction
with rac alone .
however , such a view potentially ignores complexity in the
regulation of plc isoforms that is suggested by other studies that
implicate synergy of signaling inputs .
most notably , recent studies of
activation of plc1 by growth factor receptors have shown
that tyr phosphorylation is not sufficient for full plc
activation ( 6 ) .
the concurrent
production of phosphatidylinositol 3,4,5-trisphosphate ( pip3 ) , via
epidermal growth factor - stimulated phosphatidylinositol 3-kinase activity , was
reported to contribute to the activation of phosphorylated
plc1 .
similarly , in b - cells , where signaling via
plc2 has been best documented , it is possible that rac2 can
contribute to full activation of this isoform together with a set of specific
adapter proteins and tyrosine kinases
( 54 - 56 ) .
in the b - cell system
, the evidence suggests that rac proteins and their
activators ( such as the rho guanine nucleotide exchange factors vav )
contribute to production of higher levels of ip3 and greater
calcium responses rather then being essential for plc2
activation ( 57 ,
58 ) .
however , the role and
relative contribution of rac gtpases in the regulation of
plc2 could vary between cell types .
importantly , the
identification of plc2 residues critical for rac binding
described here ( figs .
6 ,
7 , and s1 ) provides a solid
basis for the design of a rac - insensitive plc2 variant that
could be exploited to dissect the roles and assess the relative importance of
the rac - dependent and other modes of plc2 regulation in
different cell types .
it is also interesting to ponder the mechanism by which rac might regulate
plc2 activity .
here we have identified the spph as the
binding site for rac , the domain within the -sa region and thus in
proximity to critical tyrosine residues and the sh2 and sh3 domains that
mediate interactions with a number of binding partners linked to activation
( 59 ) .
some reports suggest
that the unliganded -sa region may have an autoinhibitory role , since
removal of this region appears to enhance enzymatic activity
( 60 ,
61 ) .
the observation that some
of the chimeric plc1 spph mutants displayed enhanced basal
activity is consistent with this concept ( cf .
based on
the published initial observations , it has been proposed that the -sa
region acts as a hinged lid that can adopt either a closed or
open state , thereby occluding or exposing the active site
( 62 ) , respectively , depending
upon the occupancy of the various ligand binding sites in the -sa
region .
it has recently been suggested that the inactive conformation is
characterized by an intramolecular association between the n - terminal half of
the split ph domain and the c - terminal sh2 domain
( 63 ) . in the context of this
model , the role of rac could be to , on its own or together with other
regulatory inputs , mediate the release of intramolecular constraints
. however ,
it seems clear that more experimental data , including a greater understanding
of the three - dimensional structures of holo - plc enzymes , are required
to critically evaluate such models for autoinhibition and activation
mechanisms . | several isoforms of phospholipase c ( plc ) are regulated through
interactions with ras superfamily gtpases , including rac proteins .
interestingly , of two closely related plc isoforms , only
plc2 has previously been shown to be activated by rac . here ,
we explore the molecular basis of this interaction as well as the structural
properties of plc2 required for activation .
based on
reconstitution experiments with isolated plc variants and rac2 , we show
that an unusual pleckstrin homology ( ph ) domain , designated as the split ph
domain ( spph ) , is both necessary and sufficient to effect activation of
plc2 by rac2 .
we also demonstrate that rac2 directly binds
to plc2 as well as to the isolated spph of this isoform .
furthermore , through the use of nmr spectroscopy and mutational analysis , we
determine the structure of spph , define the structural features of spph
required for rac interaction , and identify critical amino acid residues at the
interaction interface .
we further discuss parallels and differences between
plc1 and plc2 and the implications of our
findings for their respective signaling roles . | EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
Supplementary Material | the cdnas of the chimeric ,
c - myc epitope - tagged plc isozymes plc1 - 112 ,
plc1 - 121 , plc1 - 122 , plc2 - 212 , plc2 - 221 , and
plc2 - 211 , in which one or both portions of the split ph domain of one
isozyme ( aa 482 - 527 and 872 - 937 of plc1 ; aa 468 - 513 and
849 - 914 of plc2 ) were replaced by the corresponding regions
of the other , were constructed using a two - step megaprimer pcr protocol
( 29 ) . for
expression of proteins in escherichia coli ( plc1
spph , aa 485 - 936 , 530 - 864 ; plc2 spph , aa 471 - 913 ,
516 - 841 , wild type , and mutants k862i , v893q , and f897q ; and
rac2 , aa 2 - 177 )
, the particular cdnas were cloned into the
ptriex4 vector ( novagen ) using the ek / lic methodology following the
manufacturer 's instructions . the
plcw - xyz nomenclature , used throughout , refers to w ( plc isoform
backbone ) , amino - terminal ph domain ( x ) , n - terminal portion of spph ( y ) , and
c - terminal portion of spph ( z ) . the
plcw - xyz nomenclature , used throughout , refers to w ( plc isoform
backbone ) , amino - terminal ph domain ( x ) , n - terminal portion of spph ( y ) , and
c - terminal portion of spph ( z ) . figure 2.the split ph domain of plc2 is required for its
regulation by rac2 in vitro . the split ph domain of plc2 is required for its
regulation by rac2 in vitro . the plc2 split ph domain is required for
isoform - specific regulation by rac2it has previously been shown
with reconstitution experiments that rac gtpases activate
plc2 in the presence of gtps
( 12 ) . to exclude the possibility that other signaling proteins could mediate
activation of plc2 by rac , we set out to extend these data
with purified components ( fig . this result strongly
suggests that rac2 interacts directly with plc2 and that the
presence of rac2 is both necessary and sufficient for guanine nucleoside
triphosphate - dependent plc2 activation . similarly , the exchange of the plc1
n - terminal ph domain into plc2 ( variant plc2 - 122 ) did
not significantly alter the propensity of this chimera to be activated by
rac2 . in addition , we showed that the rac - binding
n - terminal ph domain of plc2 is functionally interchangeable
by constructing a plc1 chimera that incorporates this domain
( variant plc1-11 ) and showing its activation by
rac2 ( fig . the isolated split ph domain from plc2
directly binds rac to evaluate the role of plc2
spph as the site of rac interaction , we purified a number of
plc2 and plc1 variants ( including the
full - length and isolated spphs ) in order to carry out interaction studies
in vitro . structural analysis of the plc2 split ph
domain interaction interface with rac2to provide a basis for
further analysis of the interaction between the plc2 spph
and rac2 , we determined the three - dimensional solution structure of the spph
by heteronuclear nmr spectroscopy . the n view
notation refers to the surface derived from the amino acid residues from the
n - terminal half of the domain , and the c view refers to those residues derived
from the c - terminal part . the n view
notation refers to the surface derived from the amino acid residues from the
n - terminal half of the domain , and the c view refers to those residues derived
from the c - terminal part . the indicated substitutions were
introduced into the isolated spph construct , and the corresponding encoded
proteins were assessed and compared with their wild - type counterparts by
one - dimensional h nmr spectroscopy . | [
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participants were recruited in january 2008 by 48 general practitioners from a cooperation of 14 primary care practices in eindhoven and five surrounding villages .
a dutch translation of the finnish diabetes risk score ( findrisc ) ( 13 ) was sent to general practitioner patients aged 40 and 70 years ( n = 16,032 ) .
all individuals with a score of 13 points were invited to participate in the program .
random assignment was performed on the level of the individual , by assigning every second individual contacting the general practitioner assistant to schedule an admission interview to the usual - care group .
the aphrodite intervention was based on the stages - of - change model by prochaska and diclemente ( 15 ) . for the aphrodite intervention ,
five objectives of behavioral change were specified as follows : a weight reduction of 5% if overweight ; physical exercise of moderate or high intensity for at least 30 min at least 5 days a week ; intake of dietary fat < 30% of total energy intake ; intake of saturated fat < 10% of total energy intake ; and intake of dietary fiber of at least 3.4 g per megajoule ( mj ) .
after the admission interview with the general practitioner ( details have been described previously ) , 11 consultations of 20 min over 2.5 years were scheduled alternately with the nurse practitioner and the general practitioner .
although the general practitioners oversaw guarding the participants progress , nurse practitioners intensively guided the behavioral - change process . before the start of the project , all nurse practitioners underwent a 5-evening course in motivational interviewing ( 16 ) .
individual consultations were supported by five group meetings to give more detailed information on diet and exercise .
these 1-h meetings were conducted by trained dietitians ( meetings 1 , 2 , 4 , and 5 ) and physiotherapists ( meeting 3 ) .
in addition , all individuals in the intervention group were invited for a 1-h personal consultation with the dietitian . during this consultation , dietary intake according to a 3-day food record was discussed , and suggestions were given for improvement . during the admission interview with the general practitioner , participants in the usual - care group received oral and written information about type 2 diabetes , their risk for developing the disease , and the benefits of exercise and a healthy diet .
after this first meeting , the participants visited the nurse practitioner only for measurements at baseline and after 6 and 18 months ( 10-min visits ) .
apart from the admission interview , participants did not have study - related encounters with the general practitioner .
primary outcome measures were fasting and 2-h plasma glucose values , waist circumference , and bmi .
diagnosis of type 2 diabetes was based on one oral glucose tolerance test according to the 2006 world health organization diagnostic criteria ( 18 ) .
individuals with glucose values in the diabetic range were excluded from the study and were referred to the general practitioner for a second blood test to confirm the diagnosis and for additional care .
measurements of height , weight , and waist circumference were performed by the nurse practitioner in accordance with the standards of the dutch society of general practitioners .
secondary outcome measures were physical activity , physical activity of moderate to high intensity , and intakes of energy , total fat , saturated fat , and dietary fiber .
activity measures were estimated from the short questionnaire to assess health - enhancing physical activity ( squash ) ( 19 ) .
dietary intakes of the previous 4 weeks , as a proxy for usual intake , were estimated from a validated food - frequency questionnaire ( 20 ) . before data entry ,
all filled out food questionnaires were checked by trained dietitians for missing information and for inconsistencies .
differences in baseline characteristics between the two study groups and differences between participants who were successful or unsuccessful in losing weight or keeping a stable weight over 1.5 years were evaluated with either an independent - samples t test or a test . for individuals with a healthy weight at baseline ( bmi : 25 kg / m ) ,
( n = 80 , 17.4% in the intervention group ; n = 81 , 19.4% in the usual - care group ) . differences between practices were evaluated using ancova adjusted for baseline differences .
differences over time between and within the study groups were evaluated using multilevel analysis ( level 1 = time point ; level 2 = participant ; level 3 = nurse practitioner ) . for the between - group analyses ,
mixed models were estimated for each outcome variable , with study group , sex , age , smoking , time point , and group time point as fixed variables . in the within - group analyses , sex , age , smoking , and time point
the random part of both between - group and within - group models consisted of an adjustment for repeated measurements with an unstructured covariance matrix ( also accounting for baseline differences in outcome variables between subjects ) and an adjustment for nurse practitioner by allowing a random intercept on level 3 .
all analyses were performed without data from dropouts ( n = 46 , 9.6% in the intervention group ; n = 36 , 8.1% in the usual - care group ) and patients with diabetes ( n = 32 , 6.8% in the intervention group ; n = 32 , 7.3% in the usual - care group ) .
all other analyses were performed using spss version 18.0 . a p value < 0.05 was considered to be significant .
the aphrodite intervention was based on the stages - of - change model by prochaska and diclemente ( 15 ) . for the aphrodite intervention ,
five objectives of behavioral change were specified as follows : a weight reduction of 5% if overweight ; physical exercise of moderate or high intensity for at least 30 min at least 5 days a week ; intake of dietary fat < 30% of total energy intake ; intake of saturated fat < 10% of total energy intake ; and intake of dietary fiber of at least 3.4 g per megajoule ( mj ) .
after the admission interview with the general practitioner ( details have been described previously ) , 11 consultations of 20 min over 2.5 years were scheduled alternately with the nurse practitioner and the general practitioner . each participant in the intervention group , therefore , had encounters with both professionals .
although the general practitioners oversaw guarding the participants progress , nurse practitioners intensively guided the behavioral - change process . before the start of the project , all nurse practitioners underwent a 5-evening course in motivational interviewing ( 16 ) .
individual consultations were supported by five group meetings to give more detailed information on diet and exercise .
these 1-h meetings were conducted by trained dietitians ( meetings 1 , 2 , 4 , and 5 ) and physiotherapists ( meeting 3 ) .
in addition , all individuals in the intervention group were invited for a 1-h personal consultation with the dietitian . during this consultation , dietary intake according to a 3-day food record was discussed , and suggestions were given for improvement .
during the admission interview with the general practitioner , participants in the usual - care group received oral and written information about type 2 diabetes , their risk for developing the disease , and the benefits of exercise and a healthy diet .
after this first meeting , the participants visited the nurse practitioner only for measurements at baseline and after 6 and 18 months ( 10-min visits ) .
apart from the admission interview , participants did not have study - related encounters with the general practitioner .
primary outcome measures were fasting and 2-h plasma glucose values , waist circumference , and bmi .
diagnosis of type 2 diabetes was based on one oral glucose tolerance test according to the 2006 world health organization diagnostic criteria ( 18 ) .
individuals with glucose values in the diabetic range were excluded from the study and were referred to the general practitioner for a second blood test to confirm the diagnosis and for additional care .
measurements of height , weight , and waist circumference were performed by the nurse practitioner in accordance with the standards of the dutch society of general practitioners .
secondary outcome measures were physical activity , physical activity of moderate to high intensity , and intakes of energy , total fat , saturated fat , and dietary fiber .
activity measures were estimated from the short questionnaire to assess health - enhancing physical activity ( squash ) ( 19 ) .
dietary intakes of the previous 4 weeks , as a proxy for usual intake , were estimated from a validated food - frequency questionnaire ( 20 ) . before data entry ,
all filled out food questionnaires were checked by trained dietitians for missing information and for inconsistencies .
differences in baseline characteristics between the two study groups and differences between participants who were successful or unsuccessful in losing weight or keeping a stable weight over 1.5 years were evaluated with either an independent - samples t test or a test . for individuals with a healthy weight at baseline ( bmi : 25 kg / m ) ,
( n = 80 , 17.4% in the intervention group ; n = 81 , 19.4% in the usual - care group ) . differences between practices were evaluated using ancova adjusted for baseline differences .
differences over time between and within the study groups were evaluated using multilevel analysis ( level 1 = time point ; level 2 = participant ; level 3 = nurse practitioner ) . for the between - group analyses ,
mixed models were estimated for each outcome variable , with study group , sex , age , smoking , time point , and group time point as fixed variables . in the within - group analyses , sex , age , smoking , and time point were entered as fixed variables .
the random part of both between - group and within - group models consisted of an adjustment for repeated measurements with an unstructured covariance matrix ( also accounting for baseline differences in outcome variables between subjects ) and an adjustment for nurse practitioner by allowing a random intercept on level 3 .
all analyses were performed without data from dropouts ( n = 46 , 9.6% in the intervention group ; n = 36 , 8.1% in the usual - care group ) and patients with diabetes ( n = 32 , 6.8% in the intervention group ; n = 32 , 7.3% in the usual - care group ) .
all other analyses were performed using spss version 18.0 . a p value < 0.05 was considered to be significant .
table 1 shows baseline characteristics and mean changes in clinical measures , physical activity pattern , and diet after 0.5 and 1.5 years of all participants who completed the intervention period . at baseline , weight was higher in the intervention group than in the usual - care group ( p = 0.03 ) . for all clinical measures ,
changes over time were modest in both groups . fasting plasma glucose significantly decreased by 0.10
improvements in fasting plasma glucose were comparable between the two study groups ( p = 0.77 ) . in both groups , 2-h plasma glucose improved over the first half - year ( 0.05 mmol / l in the intervention group ; 0.15
mmol / l in the usual - care group ) but increased to levels higher than at baseline in the next year ( 0.13 mmol / l in the intervention group ; 0.18
changes over time were significant within the usual - care group but not within the intervention group ( p = 0.09 ) or between groups ( p = 0.49 ) .
changes in clinical measures , physical activity patterns , and diet after 0.5 and 1.5 years within and between study groups of participants who completed the intervention data are means sd or n ( % ) .
p int = p value for the analysis regarding change over time within the intervention group ( n = 393 ) .
p uc = p value for the analysis regarding change over time within the usual - care group ( n = 371 ) .
p bet = p value for the analysis regarding differences in change over time between the groups ( group * measurement point interaction ) . *
both groups showed significant reductions in bmi that were less pronounced after 1.5 years than after 0.5 years .
however , improvements after 1.5 years were comparable between groups ( 0.2 kg / m in the intervention group ; 0.1 kg / m in the usual - care group ; p = 0.66 ) .
waist circumference decreased by 0.4 cm in the intervention group ( p = 0.008 ) but nonsignificantly increased by 0.3 cm in the usual - care group ( p = 0.26 ) .
changes in waist circumference also were not significant between groups ( p = 0.35 ) ( table 1 ) . compared with baseline ,
participants in both groups reported significantly lower levels of total physical activity after 1.5 years ( 84 min / week in the intervention group ; 290 min / week in the usual - care group ) .
however , the decrease after 1.5 years was smaller in the intervention group than in the usual - care group ( p = 0.02 ) .
increases in physical activity of moderate to high intensity ( 70 min / week in the intervention group ; 29 min / week in the usual - care group ) were not significant within groups or between groups ( p = 0.34 ) .
although there was a significant decrease in total energy intake in both the intervention group ( 279 kcal ) and the usual - care group ( 197 kcal ) , differences between groups were not significant ( p = 0.11 ) . in both study groups ,
total fat intake and total saturated fat intake did not significantly change over time . however , unlike total fat intake , changes in total saturated fat intake over time significantly differed between groups ( p = 0.03 ) . in both the intervention group ( 0.1 g / mj ) and the usual - care group ( 0.3 g / mj )
, less dietary fiber was consumed after 1.5 years compared with baseline , although reductions were more pronounced in the usual - care group ( p = 0.01 ) .
figure 1 shows between - practice variation for changes in bmi , 2-h plasma glucose , energy intake , and physical activity after 1.5 years in both study groups .
mean change in bmi ranged from 1.60 to 0.37 kg / m in the intervention group and from 0.61 to 0.95 kg / m in the usual - care group . for 2-h plasma glucose ,
mean change ranged from 0.74 to 1.11 mmol / l in the intervention group and from 0.25 to 1.12 mmol / l in the usual - care group .
mean change in energy intake ranged from 415 to 99 kcal / day in the intervention group and from 336 to 27 kcal / day in the usual - care group .
for total physical activity , mean change ranged from 551 to 346 min / week in the intervention group and from 860 to 30 min / week in the usual - care group .
changes over time in the intervention group significantly differed between practices for bmi and 2-h plasma glucose ( p = 0.01 and p < 0.0001 ) but not for change in total energy intake and total physical activity ( p = 0.48 and p = 0.78 ) ( table 2 ) .
variability between practices regarding change in weight , 2-h plasma glucose , energy intake , and physical activity after 1.5 years in both study groups .
p = 0.01 ( bmi ) , p 0.0001 ( 2-h plasma glucose [ 2-h pg ] ) , p = 0.48 ( energy intake ) , and p = 0.78 ( physical activity ) for the ancova analyses for differences between practices of individuals in the intervention group completing the 1.5-year intervention period ( n = 393 ) .
. differences between participants who were overweight at baseline and who were either successful or unsuccessful in losing weight or maintaining a stable weight over 1.5 years in both study groups data are means sd , unless otherwise indicated .
significant differences between groups as tested by either anova or tests . to investigate factors that may facilitate success , several participant and professional characteristics
were compared between individuals who were losing weight or those maintaining a stable weight and individuals who were gaining weight over 1.5 years .
the outcome variable weight was chosen because of the significant interpractice variation in bmi . in the intervention group ,
successful individuals more often were married or were in a stable relationship ( p = 0.02 ) .
furthermore , in this group the mean work experience of nurse practitioners was more than in the unsuccessful individuals ( p = 0.04 ) . in the usual - care group ,
unsuccessful individuals more often had normal instead of prediabetic baseline glucose values ( p = 0.03 ) .
in this article , we reported the overall effectiveness of the aphrodite intervention program . risk factors for type 2 diabetes could significantly be reduced by lifestyle counseling in dutch primary care .
however , differences in changes over time between the two study groups were small and mostly not significant .
differences between general practices were significant for clinical measures investigated but not for lifestyle measures .
differences in the characteristics of both health care providers and participants may underlie this interpractice variation .
for all clinical measures , changes over time were modest , a pattern that also was found in other diabetes - prevention studies in daily - life settings ( 7,11,12 ) .
three other studies in the real world showed larger effects on risk factors for type 2 diabetes ( 810 ) .
however , in these studies the intensity of the interventions was higher than in our study . only in the greater green triangle study ( 10 ) was risk reduction larger despite relatively low program intensity . risk - factor reduction also may have been larger in these three studies because of the less favorable initial risk profile of the participants . mean baseline bmi , for example , ranged between 31.4 and 33.5 kg / m ( 810 ) in these studies compared with 28.7 kg / m in our study .
a less favorable initial profile leaves more room for improvement and may , moreover , increase participant motivation ( 5,11 ) .
however , in other studies results were comparable despite a higher initial bmi and findrisc score ( 7,11,12 ) .
risk - factor profile in our study may seem more favorable than in previous studies because of the exclusion of individuals who developed type 2 diabetes . in the aphrodite study , participation and follow - up
were ended when blood glucose values in the diabetic range were measured , reflecting a real - life situation . in our study ,
mean findrisc score indeed was nearly one point higher in participants who developed diabetes within 1.5 years than in other participants .
although results were modest , changes over time were more pronounced in the intervention group than in the usual - care group for all lifestyle measures .
intervention group levels of total physical activity were lower after 1.5 years than at baseline despite project recommendations .
comparable with the finnish diabetes prevention study ( dps ) ( 21 ) , improvements in leisure - time physical activity over time were not significant within or between groups ( intervention group mean = 34 min / week , p = 0.50 ; usual - care group mean = 13 min / week , p = 0.82 ; p between groups = 0.96 ) .
this pattern differed from that in the dps ( 21 ) and the dutch study on lifestyle intervention and impaired glucose tolerance maastricht ( slim ) ( 22 ) , where dietary fiber intake was modestly increased over time . with the exception oftotal energy intake in the dps study ,
improvements in total energy intake , total fat intake , and saturated fat intake were much larger in the dps and the slim studies than in our study .
however , both of these studies were performed in an experimental setting , and extensive individual dietary counseling was offered to each participant .
it must be noted that the self - reported dietary and activity measures in our study may have been subject to a differential hawthorne effect because participants in the intervention group may have been more aware of the rationale and objectives of the study than participants in the usual - care group .
furthermore , in both the food - frequency questionnaire and the squash individuals were asked about their lifestyle during the previous 4 weeks .
this 1-month reference period may not be representative for the usual activity pattern or dietary intake .
in addition , correct quantification of behavioral change using questionnaires relies on the memory of the participants .
individuals may have had difficulties recalling their eating and activity patterns . however , despite these constraints validity and reliability of both questionnaires was reasonable ( 19,20 ) . moreover , using questionnaires may be unavoidable when measuring lifestyle change in large populations . with the exceptions of fasting and 2-h plasma glucose ,
the effects were more pronounced in the intervention group . however , the usual - care group showed significant improvements for several measures as well .
furthermore , differences between the two study groups in changes over time were small and were significant only for total physical activity and fiber intake .
these observations suggest that the additional effect of the aphrodite intervention program above the effect attributable to usual preventive care in dutch general practice is modest .
the beneficial changes in the usual - care group may be explained by several factors .
first , the prospect of the annual check - up with the nurse practitioner and annual blood sampling may have motivated individuals to lose weight , eat healthier , and exercise more .
second , as a part of daily general - practice care , participants may have been enrolled in other behavioral - change programs as well , for example , for high blood pressure or cardiovascular disease .
third , during the study several campaigns were held by the government and by the dutch nutrition council to promote a healthy lifestyle . in the aphrodite study ,
general practices significantly differed in change in bmi and 2-h plasma glucose in intervention - group participants completing the 1.5-year period .
however , as discussed above , difficulties quantifying behavioral change may have influenced accuracy of lifestyle measurements .
variability between general practices may be partly caused by differences in characteristics of both the patient populations and the health care providers in the practices ( 5,6 ) .
although in the intervention group mean work experience of nurse practitioners was higher for the successful individuals than for the unsuccessful individuals , there was no difference in work experience of general practitioners .
this could first be explained by the different roles of the nurse practitioners and general practitioners in the aphrodite intervention program .
although the nurse practitioners intensively guided the behavioral - change process , the general practitioners oversaw guarding the participants progress .
second , nearly all general practitioners already were working in a practice for over 10 years and may therefore all be considered experienced .
it is important to note that the mean experience of nurse practitioners was lower in the usual - care group ( 4.1 years ) than in the intervention group ( 5.2 years ) ( p = < 0.001 ) .
this difference in nurse practitioner experience may be caused by a differential drop - out rate between study groups , which may have influenced the results of the between - group analyses of risk - factor reductions over time .
however , although the mean experience of nurse practitioners indeed was larger in dropouts in the usual - care group ( 4.5 years ) compared with dropouts in the intervention group ( 4.0 years ) , this result was not significant ( p = 0.523 ) . the relatively low amount of dropouts in both groups ( n = 29 in the intervention group and n = 19 in the usual - care group ) may , however , have led to a lack of statistical power to detect the differential drop - out rates . in our study , individuals who were successful in losing weight more often were married or were in a stable relationship .
in addition , they may contribute to the action or coping self - efficacy necessary to change .
in line with this hypothesis , lack of family support was found to be a barrier for both achieving ( 6 ) and maintaining ( 23 ) behavioral change in interventions for type 2 diabetes prevention .
furthermore , the use of social support was found to increase the effectiveness of weight - loss interventions at 1 year of follow - up ( 24 ) . in the usual - care group ,
unsuccessful individuals more often had normal baseline glucose values . knowing that glucose values still are within the normal range
however , in the intervention group , the proportion of individuals with normal glucose values was similar between successful and unsuccessful participants .
this may be explained by the efforts of the general practitioner and/or nurse practitioner in the intervention group to convince individuals of the importance of weight maintenance or loss despite normal glucose values . in conclusion , although low - intensity and low - cost interventions like the aphrodite program are particularly suitable for implementation in real - life settings , they may come with the price of lower intervention effectiveness ( 5,7,11 ) . in our study , the additional effect of the aphrodite intervention above the effect attributable to usual preventive efforts in dutch general practice was modest .
furthermore , it may prove useful to health policymakers to investigate the conditions under which interventions are most likely to be successful ( 5 ) . in our study
, the level of nurse practitioner experience and the availability of direct social support particularly seemed to facilitate intervention success .
program effectiveness may furthermore be increased by inclusion of participants with a higher initial risk and/or a higher motivation to change behavior ( 5,11 ) or by quality - based financial incentives ( 25 ) .
in addition to studying effectiveness , process evaluations are needed to identify organizational or motivational barriers to the implementation of diabetes prevention initiatives in the real world .
for all clinical measures , changes over time were modest , a pattern that also was found in other diabetes - prevention studies in daily - life settings ( 7,11,12 ) .
three other studies in the real world showed larger effects on risk factors for type 2 diabetes ( 810 ) .
however , in these studies the intensity of the interventions was higher than in our study . only in the greater green triangle study ( 10 ) was risk reduction larger despite relatively low program intensity . risk - factor reduction also may have been larger in these three studies because of the less favorable initial risk profile of the participants . mean baseline bmi , for example , ranged between 31.4 and 33.5 kg / m ( 810 ) in these studies compared with 28.7 kg / m in our study .
a less favorable initial profile leaves more room for improvement and may , moreover , increase participant motivation ( 5,11 ) .
however , in other studies results were comparable despite a higher initial bmi and findrisc score ( 7,11,12 ) .
risk - factor profile in our study may seem more favorable than in previous studies because of the exclusion of individuals who developed type 2 diabetes . in the aphrodite study , participation and follow - up
were ended when blood glucose values in the diabetic range were measured , reflecting a real - life situation . in our study ,
mean findrisc score indeed was nearly one point higher in participants who developed diabetes within 1.5 years than in other participants .
although results were modest , changes over time were more pronounced in the intervention group than in the usual - care group for all lifestyle measures .
intervention group levels of total physical activity were lower after 1.5 years than at baseline despite project recommendations .
comparable with the finnish diabetes prevention study ( dps ) ( 21 ) , improvements in leisure - time physical activity over time were not significant within or between groups ( intervention group mean = 34 min / week , p = 0.50 ; usual - care group mean = 13 min / week , p = 0.82 ; p between groups = 0.96 ) . dietary fiber intake also was reduced in the intervention group despite project recommendations .
this pattern differed from that in the dps ( 21 ) and the dutch study on lifestyle intervention and impaired glucose tolerance maastricht ( slim ) ( 22 ) , where dietary fiber intake was modestly increased over time . with the exception oftotal energy intake in the dps study , improvements in total energy intake , total fat intake , and saturated fat intake were much larger in the dps and the slim studies than in our study .
however , both of these studies were performed in an experimental setting , and extensive individual dietary counseling was offered to each participant .
it must be noted that the self - reported dietary and activity measures in our study may have been subject to a differential hawthorne effect because participants in the intervention group may have been more aware of the rationale and objectives of the study than participants in the usual - care group .
furthermore , in both the food - frequency questionnaire and the squash individuals were asked about their lifestyle during the previous 4 weeks .
this 1-month reference period may not be representative for the usual activity pattern or dietary intake .
in addition , correct quantification of behavioral change using questionnaires relies on the memory of the participants .
individuals may have had difficulties recalling their eating and activity patterns . however , despite these constraints validity and reliability of both questionnaires was reasonable ( 19,20 ) . moreover , using questionnaires may be unavoidable when measuring lifestyle change in large populations .
with the exceptions of fasting and 2-h plasma glucose , the effects were more pronounced in the intervention group . however , the usual - care group showed significant improvements for several measures as well .
furthermore , differences between the two study groups in changes over time were small and were significant only for total physical activity and fiber intake .
these observations suggest that the additional effect of the aphrodite intervention program above the effect attributable to usual preventive care in dutch general practice is modest .
the beneficial changes in the usual - care group may be explained by several factors .
first , the prospect of the annual check - up with the nurse practitioner and annual blood sampling may have motivated individuals to lose weight , eat healthier , and exercise more .
second , as a part of daily general - practice care , participants may have been enrolled in other behavioral - change programs as well , for example , for high blood pressure or cardiovascular disease .
third , during the study several campaigns were held by the government and by the dutch nutrition council to promote a healthy lifestyle .
in the aphrodite study , general practices significantly differed in change in bmi and 2-h plasma glucose in intervention - group participants completing the 1.5-year period .
however , as discussed above , difficulties quantifying behavioral change may have influenced accuracy of lifestyle measurements .
variability between general practices may be partly caused by differences in characteristics of both the patient populations and the health care providers in the practices ( 5,6 ) .
although in the intervention group mean work experience of nurse practitioners was higher for the successful individuals than for the unsuccessful individuals , there was no difference in work experience of general practitioners .
this could first be explained by the different roles of the nurse practitioners and general practitioners in the aphrodite intervention program .
although the nurse practitioners intensively guided the behavioral - change process , the general practitioners oversaw guarding the participants progress .
second , nearly all general practitioners already were working in a practice for over 10 years and may therefore all be considered experienced .
it is important to note that the mean experience of nurse practitioners was lower in the usual - care group ( 4.1 years ) than in the intervention group ( 5.2 years ) ( p = <
this difference in nurse practitioner experience may be caused by a differential drop - out rate between study groups , which may have influenced the results of the between - group analyses of risk - factor reductions over time .
however , although the mean experience of nurse practitioners indeed was larger in dropouts in the usual - care group ( 4.5 years ) compared with dropouts in the intervention group ( 4.0 years ) , this result was not significant ( p = 0.523 ) .
the relatively low amount of dropouts in both groups ( n = 29 in the intervention group and n = 19 in the usual - care group ) may , however , have led to a lack of statistical power to detect the differential drop - out rates . in our study , individuals who were successful in losing weight more often were married or were in a stable relationship .
in addition , they may contribute to the action or coping self - efficacy necessary to change .
in line with this hypothesis , lack of family support was found to be a barrier for both achieving ( 6 ) and maintaining ( 23 ) behavioral change in interventions for type 2 diabetes prevention .
furthermore , the use of social support was found to increase the effectiveness of weight - loss interventions at 1 year of follow - up ( 24 ) . in the usual - care group ,
knowing that glucose values still are within the normal range may limit motivation to change unhealthy behavior .
however , in the intervention group , the proportion of individuals with normal glucose values was similar between successful and unsuccessful participants .
this may be explained by the efforts of the general practitioner and/or nurse practitioner in the intervention group to convince individuals of the importance of weight maintenance or loss despite normal glucose values . in conclusion , although low - intensity and low - cost interventions like the aphrodite program are particularly suitable for implementation in real - life settings , they may come with the price of lower intervention effectiveness ( 5,7,11 ) . in our study , the additional effect of the aphrodite intervention above the effect attributable to usual preventive efforts in dutch general practice was modest .
furthermore , it may prove useful to health policymakers to investigate the conditions under which interventions are most likely to be successful ( 5 ) . in our study
, the level of nurse practitioner experience and the availability of direct social support particularly seemed to facilitate intervention success .
program effectiveness may furthermore be increased by inclusion of participants with a higher initial risk and/or a higher motivation to change behavior ( 5,11 ) or by quality - based financial incentives ( 25 ) .
in addition to studying effectiveness , process evaluations are needed to identify organizational or motivational barriers to the implementation of diabetes prevention initiatives in the real world . | objectiveto study the overall effect of the active prevention in high - risk individuals of diabetes type 2 in and around eindhoven ( aphrodite ) lifestyle intervention on type 2 diabetes risk reduction in dutch primary care after 0.5 and 1.5 years and to evaluate the variability between general practices.research design and methodsindividuals at high risk for type 2 diabetes ( finnish diabetes risk score 13 ) were randomly assigned into an intervention group ( n = 479 ) or a usual - care group ( n = 446 ) .
comparisons were made between study groups and between general practices regarding changes in clinical and lifestyle measures over 1.5 years .
participant , general practitioner , and nurse practitioner characteristics were compared between individuals who lost weight or maintained a stable weight and individuals who gained weight.resultsboth groups showed modest changes in glucose values , weight measures , physical activity , energy intake , and fiber intake .
differences between groups were significant only for total physical activity , saturated fat intake , and fiber intake .
differences between general practices were significant for bmi and 2-h glucose but not for energy intake and physical activity . in the intervention group ,
the nurse practitioners mean years of work experience was significantly longer in individuals who were successful at losing weight or maintaining a stable weight compared with unsuccessful individuals . furthermore , successful individuals more often had a partner.conclusionsrisk factors for type 2 diabetes could be significantly reduced by lifestyle counseling in dutch primary care .
the small differences in changes over time between the two study groups suggest that additional intervention effects are modest . in particular , the level of experience of the nurse practitioner and the availability of partner support seem to facilitate intervention success . | RESEARCH DESIGN AND METHODS
Theoretical framework and objectives
Planning and intensity of the intervention
Usual-care group
Outcome measures
Statistical analysis
RESULTS
CONCLUSIONS
Changes in clinical measures
Changes in lifestyle measures
Intervention effectiveness
Variability between practices
Supplementary Material | during the admission interview with the general practitioner , participants in the usual - care group received oral and written information about type 2 diabetes , their risk for developing the disease , and the benefits of exercise and a healthy diet . differences in baseline characteristics between the two study groups and differences between participants who were successful or unsuccessful in losing weight or keeping a stable weight over 1.5 years were evaluated with either an independent - samples t test or a test . during the admission interview with the general practitioner , participants in the usual - care group received oral and written information about type 2 diabetes , their risk for developing the disease , and the benefits of exercise and a healthy diet . differences in baseline characteristics between the two study groups and differences between participants who were successful or unsuccessful in losing weight or keeping a stable weight over 1.5 years were evaluated with either an independent - samples t test or a test . table 1 shows baseline characteristics and mean changes in clinical measures , physical activity pattern , and diet after 0.5 and 1.5 years of all participants who completed the intervention period . in both groups , 2-h plasma glucose improved over the first half - year ( 0.05 mmol / l in the intervention group ; 0.15
mmol / l in the usual - care group ) but increased to levels higher than at baseline in the next year ( 0.13 mmol / l in the intervention group ; 0.18
changes over time were significant within the usual - care group but not within the intervention group ( p = 0.09 ) or between groups ( p = 0.49 ) . changes in clinical measures , physical activity patterns , and diet after 0.5 and 1.5 years within and between study groups of participants who completed the intervention data are means sd or n ( % ) . compared with baseline ,
participants in both groups reported significantly lower levels of total physical activity after 1.5 years ( 84 min / week in the intervention group ; 290 min / week in the usual - care group ) . although there was a significant decrease in total energy intake in both the intervention group ( 279 kcal ) and the usual - care group ( 197 kcal ) , differences between groups were not significant ( p = 0.11 ) . in both the intervention group ( 0.1 g / mj ) and the usual - care group ( 0.3 g / mj )
, less dietary fiber was consumed after 1.5 years compared with baseline , although reductions were more pronounced in the usual - care group ( p = 0.01 ) . figure 1 shows between - practice variation for changes in bmi , 2-h plasma glucose , energy intake , and physical activity after 1.5 years in both study groups . changes over time in the intervention group significantly differed between practices for bmi and 2-h plasma glucose ( p = 0.01 and p < 0.0001 ) but not for change in total energy intake and total physical activity ( p = 0.48 and p = 0.78 ) ( table 2 ) . variability between practices regarding change in weight , 2-h plasma glucose , energy intake , and physical activity after 1.5 years in both study groups . differences between participants who were overweight at baseline and who were either successful or unsuccessful in losing weight or maintaining a stable weight over 1.5 years in both study groups data are means sd , unless otherwise indicated . to investigate factors that may facilitate success , several participant and professional characteristics
were compared between individuals who were losing weight or those maintaining a stable weight and individuals who were gaining weight over 1.5 years . risk factors for type 2 diabetes could significantly be reduced by lifestyle counseling in dutch primary care . furthermore , differences between the two study groups in changes over time were small and were significant only for total physical activity and fiber intake . however , although the mean experience of nurse practitioners indeed was larger in dropouts in the usual - care group ( 4.5 years ) compared with dropouts in the intervention group ( 4.0 years ) , this result was not significant ( p = 0.523 ) . in our study
, the level of nurse practitioner experience and the availability of direct social support particularly seemed to facilitate intervention success . furthermore , differences between the two study groups in changes over time were small and were significant only for total physical activity and fiber intake . it is important to note that the mean experience of nurse practitioners was lower in the usual - care group ( 4.1 years ) than in the intervention group ( 5.2 years ) ( p = <
this difference in nurse practitioner experience may be caused by a differential drop - out rate between study groups , which may have influenced the results of the between - group analyses of risk - factor reductions over time . however , although the mean experience of nurse practitioners indeed was larger in dropouts in the usual - care group ( 4.5 years ) compared with dropouts in the intervention group ( 4.0 years ) , this result was not significant ( p = 0.523 ) . in our study
, the level of nurse practitioner experience and the availability of direct social support particularly seemed to facilitate intervention success . | [
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] |
age - related declines in muscle mass and strength , known as sarcopenia , are often an important antecedent of the onset of disability in older adulthood .
although the term is applied clinically to denote loss of muscle mass , sarcopenia is often used to describe both a set of cellular processes ( denervation , mitochondrial dysfunction , and inflammatory and hormonal changes ) and a set of outcomes such as decreased muscle strength , decreased mobility and function , increased fatigue , a greater risk of falls , and reduced energy needs .
lean muscle mass generally contributes up to ~50% of total body weight in young adults but declines with aging to be 25% at 7580 years old [ 3 , 4 ] .
the loss is most notable in the lower limb muscle groups , with the cross - sectional area of the vastus lateralis being reduced by as much as 40% between the ages of 20 and 80 yr .
several possible mechanisms for age - related muscle atrophy have been described ; however , the precise contribution of each is unknown .
age - related muscle loss is a result of reductions in the size and number of muscle fibers possibly due to a multifactorial process that involves physical activity , nutritional intake , oxidative stress , and hormonal changes [ 2 , 7 ] .
the specific contribution of each of these factors is unknown , but there is emerging evidence that the disruption of several positive regulators ( akt and serum response factor ) of muscle hypertrophy with age is an important feature in the progression of sarcopenia [ 810 ] .
in contrast , many investigators have failed to demonstrate an age - related enhancement in levels of common negative regulators ( atrogin-1 , myostatin , and calpain ) in senescent mammalian muscles .
several lines of evidence point to inflammation being associated with loss of muscle strength and mass with aging .
animal studies have shown that the administration of interleukin ( il)-6 or tumor necrosis factor ( tnf)- increases skeletal muscle breakdown , decreases the rate of protein synthesis , and reduces plasma concentrations of insulin - like growth factor [ 12 , 13 ] . in older men and women , higher levels of il-6 and c - reactive protein ( crp ) were associated with a two- to threefold greater risk of losing more than 40% of grip strength over 3 years . on the other hand ,
several studies have indicated age - related endocrine defects such as decreases in anabolic hormones ( testosterone , estrogen , growth hormone ( gh ) , and insulin - like growth factor - i ( igf - i ) ) [ 1518 ] . although hormonal supplementation for the elderly has been conducted on a large scale , it was found not to be effective against sarcopenia and to have minor side effects [ 9 , 10 , 15 , 16 , 19 , 20 ] . in this paper
, we summarize the current understanding of the endocrine contribution to sarcopenia and provide an update on practical hormonal intervention for the elderly .
inflammation may negatively influence skeletal muscle through direct catabolic effects or through indirect mechanisms ( i.e. , decreases in gh and igf - i concentrations , induction of anorexia , etc . ) .
there is growing evidence that higher levels of inflammatory markers are associated with physical decline in older individuals , possibly through the catabolic effects of these markers on muscle . in an observational study of more than 2000
men and women , tnf- showed a consistent association with declines in muscle mass and strength .
the impact of inflammation on the development of sarcopenia is further supported by a recently published animal study showing that a reduction in low - grade inflammation by ibuprofen in old ( 20 months ) rats resulted in a significant decrease in muscle mass loss .
an age - related disruption of the intracellular redox balance appears to be a primary causal factor for a chronic state of low - grade inflammation .
more recently , chung et al . hypothesized that abundant nuclear factor-b ( nf-b ) protein induced age - related increases in il-6 and tnf-. moreover , reactive oxygen species ( ros ) also appear to function as second messengers for tnf- in skeletal muscle , activating nf-b either directly or indirectly .
indeed , marked production of ros has been documented in muscle of the elderly [ 26 , 27 ] .
despite some evidence supporting enhanced nf-b signaling in type i fibers of aged skeletal muscle , direct evidence for increased activation and dna binding of nf-b is lacking [ 28 , 29 ] .
for example , phillips and leeuwenburgh found that neither p65 protein expression nor the binding activity of nf-b was significantly altered in the vastus lateralis muscles of 26-month - old rats despite the marked upregulation of tnf- expression in both blood and muscle .
upregulated tnf- expression in serum and muscle seems to enhance apoptosis through increased mitochondrial defects resulting in a loss of muscle fibers [ 2931 ] .
it has been shown that tnf- is one of the primary signals inducing apoptosis in muscle .
myostatin was first discovered during screening for new members of the transforming growth factor- ( tgf- ) superfamily and shown to be a potent negative regulator of muscle growth .
like other family members , myostatin is synthesized as a precursor protein that is cleaved by furin proteases to generate the active c - terminal dimer .
most , if not all , of the myostatin protein that circulates in blood also appears to exist in an inactive complex with a variety of proteins , including the propeptide .
the latent form of myostatin seems to be activated in vitro by dissociation from the complex with either acid or heat treatment [ 33 , 34 ] or by proteolytic cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid family of metalloproteases .
studies indicate that myostatin inhibits cell cycle progression and reduces levels of myogenic regulatory factors ( mrfs ) , thereby controlling myoblastic proliferation and differentiation during developmental myogenesis [ 3537 ] .
myostatin binds to and signals through a combination of actriia / b receptors on the cell membrane but has higher affinity for actriib . on binding to actriib , myostatin forms a complex with either activin receptor - like kinase ( alk ) 4 or alk5 to activate ( phosphorylate ) the smad2/3 transcription factors .
then smad2/3 are translocated and modulate the nuclear transcription of genes such as myod via a tgf--like mechanism .
more recently , the igf - i - akt - mtor ( mammalian target of rapamycin ) pathway , which mediates both differentiation in myoblasts and hypertrophy in myotubes , has been shown to inhibit myostatin - dependent signaling .
blockade of the akt - mtor pathway , using sirna to raptor , a component of torc1 ( tor signaling complex 1 ) , facilitates myostatin 's inhibition of muscle differentiation because of an increase in smad2 phosphorylation .
several researchers have investigated the effect of inhibiting myostatin to counteract sarcopenia using animals [ 41 , 42 ] .
lebrasseur et al . found that treatment with a mouse chimera of antihuman myostatin antibody ( 24 mg / kg , 4 weeks ) , a drug for inhibiting myostatin , elicited a significant increase in muscle mass and in running performance probably due to decreased levels of phosphorylated smad3 and muscle ring finger-1 ( murf-1 ) .
more recently , murphy et al . showed , by way of once weekly injections , that a lower dose of this anti - human myostatin antibody ( 10 mg / kg ) significantly increased the fiber cross - sectional area ( by 12% ) and in situ muscle force ( by 35% ) of aged mice ( 21 mo old ) .
these findings highlight the therapeutic potential of antibody - directed myostatin inhibition for sarcopenia by inhibiting protein degradation .
although many researchers expect myostatin levels to be increased not only in muscle but also in serum , blood myostatin levels have not been shown to increase with age .
glucocorticoid - associated atrophy appears to be specific to type ii or phasic muscle fibers . in a study of controlled hypercortisolaemia in healthy men ,
experimental inactivity increased the catabolic effect of glucocorticoids , suggesting that an absence of mechanical signals potentiates the effect .
the mechanism of glucocorticoid - induced atrophy may involve upregulated expression of myostatin and glutamine synthetase , the latter via the glucocorticoid receptor 's interaction with the glutamine synthetase promoter .
glucocorticoids inhibit the physiological secretion of gh and appear to induce igf - i activity in target organs .
changes in steroid - induced glutamine synthetase represent a potential mechanism of action , and dose - dependent inhibition of glutamine synthetase by igf - i was observed in rat l6 cells .
the increased incidence of various diseased states during aging is associated with the hypersecretion of glucocorticoids [ 47 , 48 ] .
in addition , when adult ( 7-month - old ) and aged ( 22-month - old ) rats received dexamethasone ( approximately 500 g / kg body weight / day ) in their drinking water for 5 - 6 days , muscle wasting was much more rapid in aged animals .
furthermore , glucocorticoids induced prolonged leucine resistance to muscle protein synthesis in old rats . still , it remains to be directly elucidated , using pharmacological inhibitors for glucocorticoids , whether age - related increases in serum glucocorticoid levels actually inhibit protein synthesis and/or enhance protein degradation .
il-6 and crp , known as geriatric cytokines , are multifunctional cytokine produced in situations of trauma , stress , and infection . during the aging process ,
the natural production of cytokines is likely beneficial during inflammation , but overproduction and the maintaining of an inflammatory state for long periods of time , as seen in elderly individuals , are detrimental [ 50 , 51 ] .
a number of authors have demonstrated that a rise in plasma levels of proinflammatory cytokines , especially il-6 , and proteins under acute conditions is associated with a reduction in mobility as well as a reduced capacity to perform daily activities , the development of fragility syndrome , and increased mortality rates [ 5052 ] . in older men and women , higher levels of il-6 and crp
were associated with a two- to threefold greater risk of losing more than 40% of grip strength over 3 years .
in contrast , there were no longitudinal associations between inflammatory markers and changes in grip strength among high functioning elderly participants from the macarthur study of successful ageing .
more recently , hamer and molloy demonstrated , in a large representative community - based cohort of older adults ( 1,926 men and 2,260 women ( aged 65.3 9.0 years ) ) , that crp was associated with poorer hand grip strength and chair stand performance in women but only chair stand performance in men .
in addition , haddad et al . demonstrated atrophy in the tibialis anterior muscle of mice following the injection of relatively low doses of il-6 . in a recent randomized trial that employed aerobic and strength training in a group of elderly participants , significant reductions in various inflammatory markers
( il-6 , crp , and il-18 ) were observed for aerobic but not strength training .
in contrast , combined resistance and aerobic training that increased strength by 38% resulted in significant reductions in crp .
more descriptive data appears to be needed whether il-6 and crp have an actual catabolic effect in sarcopenic muscle .
in males , levels of testosterone decrease by 1% per year , and those of bioavailable testosterone by 2% per year from age 30 [ 16 , 58 , 59 ] . in women ,
testosterone increases muscle protein synthesis , and its effects on muscle are modulated by several factors including genetic background , nutrition , and exercise .
numerous studies of treatment with testosterone in the elderly have been performed over the past few years [ 6366 ] . in 1999 , snyder et al . suggested that increasing the level of testosterone in old men to that seen in young men increased muscle mass but did not result in functional gains in strength .
systemic reviews of the literature have concluded that testosterone supplementation attenuates several sarcopenic symptoms including decreases in muscle mass [ 6466 ] and grip strength .
for instance , a recent study of 6 months of supraphysiological dosage of testosterone in a randomized placebo - controlled trial reported increased leg lean body mass and leg and arm strength .
although there are significant increases in strength among elderly males given high doses of testosterone , the potential risks may outweigh the benefits .
risks associated with testosterone therapy in older men include sleep apnea , thrombotic complications , and the increased risk of prostate cancer .
novel , nonsteroidal compounds , called selective androgen receptor modulators , have shown tissue - selective activity and improved pharmacokinetic properties .
dehydroepiandrosterone ( dhea ) is marketed as a nutritional supplement in the usa and is available over the counter .
unlike testosterone and estrogen , dhea is a hormone precursor which is converted into sex hormones in specific target tissues .
however , supplementation of dhea in aged men and women resulted in an increase in bone density and testosterone and estradiol levels , but no changes in muscle size , strength , or function [ 72 , 73 ] .
it has been hypothesized that menopause transition and the subsequent decline in estrogen may play a role in muscle mass loss [ 7 , 18 ] .
van geel et al . reported a positive relationship between lean body mass and estrogen levels .
observed that muscle mass is correlated significantly with plasma estrone and estradiol levels in women .
however , baumgartner et al . reported that estrogen levels were not associated with muscle mass in women aged 65 years and older .
the mechanisms by which decrease in estrogen levels may have a negative effect on muscle mass are not well understood but may be associated with an increase in proinflammatory cytokines , such as tnf- and il-6 , which might be implicated in the apparition of sarcopenia .
furthermore , estrogen could have a direct effect on muscle mass since it has been shown that skeletal muscle has estrogen beta - receptors on the cell membrane . therefore , a direct potential mechanistic link could exist between low estrogen levels and a decrease in protein synthesis .
further studies are needed to investigate this hypothesis . nevertheless , before reaching a conclusion on the contribution of estrogens to the onset of sarcopenia
, it would be important to measure urinary estrogen metabolites since a relationship between breast cancer and urinary estrogen metabolites has been shown .
growth hormone ( gh ) is a single - chain peptide of 191 amino acids produced and secreted mainly by the somatotrophs of the anterior pituitary gland .
gh secretion occurs in a pulsatile manner with a major surge at the onset of slow - wave sleep and less conspicuous secretory episodes a few hours after meals and is controlled by the actions of two hypothalamic factors , gh - releasing hormone ( ghrh ) , which stimulates gh secretion , and somatostatin , which inhibits gh secretion .
the secretion of gh is maximal at puberty accompanied by very high circulating igf - i levels , with a gradual decline during adulthood .
indeed , circulating gh levels decline progressively after 30 years of age at a rate of ~1% per year . in aged men ,
the age - dependent decline in gh secretion is secondary to a decrease in ghrh and to an increase in somatostatin secretion . with respect to the somatomedin hypothesis ,
the growth - promoting actions of gh are mediated by circulating or locally produced igf - i .
gh - induced muscle growth may be mediated in an endocrine manner by circulating igf - i derived from liver and/or in an autocrine / paracrine manner by direct expression of igf - i from target muscle via gh receptors on muscle membranes .
the effects of gh administration on muscle mass , strength and physical performance are still under debate . in animal models ,
gh treatment is very effective at inhibiting sarcopenic symptoms such as muscle atrophy and decreases in protein synthesis particularly in combination with exercise training .
some groups demonstrated an improvement in strength after long - term administration ( 311 months ) of gh .
in contrast , many researchers have found that muscle strength or muscle mass did not improve on supplementation with gh [ 19 , 89 ] .
one recent study reported a positive effect for counteracting sarcopenia after the administration of both gh and testosterone .
several reasons may underlie the ineffectiveness of gh treatment in improving muscle mass and strength in the elderly , such as a failure of exogeneous gh to mimic the pulsatile pattern of natural gh secretion or the induction of gh - related insulin resistance .
in addition , reduced mrna levels of the gh receptor in skeletal muscle have been observed in older versus younger healthy men , exhibiting a significant negative relationship with myostatin levels .
it should also be considered that the majority of the trials conducted on gh supplementation have reported a high incidence of side effects , including soft tissue edema , carpal tunnel syndrome , arthralgias , and gynecomastica , which pose serious concerns especially in older adults .
therefore , one should pay very careful attention when administering gh to the elderly .
there is evidence that the age - associated decline in gh levels in combination with lower igf - i levels contributes to the development of sarcopenia .
igf - i is perhaps the most important mediator of muscle growth and repair possibly by utilizing akt - mtor - p70s6k ( p70 ribosomal protein s6 kinase ) signaling .
although the transgenic approach of upregulating igf - i expression in skeletal muscle would be appropriate for inhibiting sarcopenia , the administration of igf - i to the elderly has resulted in controversial findings on muscle strength and function .
the ineffectiveness may be attributable to age - related insulin resistance to amino acid transport and protein synthesis or a marked decrease in igf - i receptors [ 96 , 97 ] and receptor affinity for igf - i in muscle with age .
. demonstrated a reduced effect of insulin on protein breakdown in the legs in older versus younger subjects probably due to the blunted activation of akt by insulin .
ghrelin is a 28-amino - acid peptide mainly produced by cells in the stomach , intestines , and hypothalamus .
ghrelin is a natural ligand for the gh - secretagogue receptor ( ghs - r ) , which possesses a unique fatty acid modification , an n - octanoylation , at ser 3 .
ghrelin plays a critical role in a variety of physiological processes , including the stimulation of gh secretion and regulation of energy homeostasis by stimulating food intake and promoting adiposity via a gh - independent mechanism .
in contrast , ghrelin inhibits the production of anorectic proinflammatory cytokines , including il-1 , il-6 , and tnf- .
because of their combined anabolic effects on skeletal muscle and appetite , ghrelin and low - molecular - weight agonists of the ghrelin receptor are considered attractive candidates for the treatment of cachexia . for example , nagaya et al
. gave human ghrelin ( 2 g / kg twice daily intravenously ) for 3 weeks to cachexic patients with chronic obstructive pulmonary disease in an open - label study .
after ghrelin therapy , significant increases from baseline measurements were observed for body weight , lean body mass , food intake , hand grip strength , maximal inspiratory pressure , and karnofsky performance score . in another unblinded study , the same group demonstrated that treatment with human ghrelin ( 2 g / kg twice daily intravenously , 3 weeks ) significantly improved several parameters ( eg . , lean body mass measured by dual - energy x - ray absorption and left ventricular ejection fraction ) in 10 patients with chronic heart failure . in a 1-year placebo - controlled study in healthy older adults over the age of 60 years given an oral ghrelin - mimetic ( mk-677 ) , an increase in appetite was observed .
the study did not show a significant increase in strength or function in the ghrelin - mimetic treatment group , when compared to the placebo group ; however , a tendency was observed .
as pointed out in a recent review by nass et al . , the use of this compound induces the potential deterioration of insulin sensitivity and development of diabetes mellitus in older adults with impaired glucose tolerance .
figure 1 provides an overview of several regulators for muscle mass in both young and sarcopenic mammalian muscles .
vitamin d has been traditionally considered a key regulator of bone metabolism and calcium and phosphorus homeostasis through negative feedback with the parathyroid hormone [ 107 , 108 ] .
it is also well established that vitamin d deficiency causes rickets in children and osteomalacia and osteoporosis in adults .
a large and growing body of evidence suggests that vitamin d is not only necessary for bone tissue and calcium metabolism but may also represent a crucial determinant for the development of major ( sub)clinical conditions and health - related events [ 107 , 109 ] .
today , approximately 1 billion , mostly elderly people , worldwide have vitamin d deficiency . the prevalence of low vitamin d concentrations in subjects older than 65 years of age has been estimated at approximately 50% [ 110112 ] , but this figure is highly variable because it is influenced by sociodemographic , clinical , therapeutic , and environmental factors .
similarly there is an age - dependent reduction in vitamin d receptor expression in skeletal muscle .
prolonged vitamin d deficiency has been associated with severe muscle weakness , which improves with vitamin d supplementation .
the histological examination of muscle tissue from subjects with osteomalacia is characterized by increased interfibrillar space , intramuscular adipose tissue infiltrates , and fibrosis .
interestingly , muscle biopsies performed before and after vitamin d supplementation have documented an increased number and sectional area of type ii ( or fast ) muscle fibers [ 113 , 116 ] .
a large body of evidence currently demonstrates that low vitamin d concentrations represent an independent risk factor for falls in the elderly [ 117119 ] .
supplementation with vitamin d in double - blind randomized - controlled trials has been shown to increase muscle strength and performance and reduce the risk of falling in community - living elderly and nursing home residents with low vitamin d levels [ 120124 ] .
in contrast , several groups found no positive effect of vitamin d supplementation on fall event outcomes [ 125127 ] .
cesari et al . attributed these contradictory findings to the selection criteria adopted to recruit study populations , adherence to the intervention , or the extreme heterogeneity of cut - points defining the status of deficiency
. a more comprehensive knowledge on vitamin - d - related mechanisms may provide a very useful tool preventing muscle atrophy for older persons ( sarcopenia ) .
given the current and future demographic age shift in the world 's population , intense research in this area is imperative .
decreases in muscle mass have been shown to be a key element in the development of frailty . currently , resistance training combined with amino - acid - containing supplements would be the best way to prevent age - related muscle wasting and weakness .
comprehensive trials have demonstrated that supplementation with gh , igf - i , or estrogen has a minor sarcopenia - inhibiting effect .
testosterone supplementation in large amounts improves muscle defects with aging but has several side effects .
ghrelin - mimetics which have the ability to increase caloric intake as well as to increase lean body mass in the older population could be potentially beneficial and reverse the catabolic state associated with sarcopenia .
myostatin inhibition seems to be an intriguing strategy for attenuating sarcopenia as well as muscular dystrophy . | sarcopenia , the age - related loss of skeletal muscle , is characterized by a deterioration of muscle quantity and quality leading to a gradual slowing of movement , a decline in strength and power , and an increased risk of fall - related injuries .
since sarcopenia is largely attributed to various molecular mediators affecting fiber size , mitochondrial homeostasis , and apoptosis , numerous targets exist for drug discovery . in this paper ,
we summarize the current understanding of the endocrine contribution to sarcopenia and provide an update on hormonal intervention to try to improve endocrine defects .
myostatin inhibition seems to be the most interesting strategy for attenuating sarcopenia other than resistance training with amino acid supplementation .
testosterone supplementation in large amounts and at low frequency improves muscle defects with aging but has several side effects .
although igf - i is a potent regulator of muscle mass , its therapeutic use has not had a positive effect probably due to local igf - i resistance .
treatment with ghrelin may ameliorate the muscle atrophy elicited by age - dependent decreases in growth hormone .
ghrelin is an interesting candidate because it is orally active , avoiding the need for injections . a more comprehensive knowledge of vitamin - d - related mechanisms is needed to utilize this nutrient to prevent sarcopenia . | 1. Introduction
2. The Adaptative Changes in Catabolic Mediators
3. Anabolic Hormones in Sarcopenic Muscle
4. Conclusion | age - related declines in muscle mass and strength , known as sarcopenia , are often an important antecedent of the onset of disability in older adulthood . although the term is applied clinically to denote loss of muscle mass , sarcopenia is often used to describe both a set of cellular processes ( denervation , mitochondrial dysfunction , and inflammatory and hormonal changes ) and a set of outcomes such as decreased muscle strength , decreased mobility and function , increased fatigue , a greater risk of falls , and reduced energy needs . several possible mechanisms for age - related muscle atrophy have been described ; however , the precise contribution of each is unknown . age - related muscle loss is a result of reductions in the size and number of muscle fibers possibly due to a multifactorial process that involves physical activity , nutritional intake , oxidative stress , and hormonal changes [ 2 , 7 ] . several lines of evidence point to inflammation being associated with loss of muscle strength and mass with aging . on the other hand ,
several studies have indicated age - related endocrine defects such as decreases in anabolic hormones ( testosterone , estrogen , growth hormone ( gh ) , and insulin - like growth factor - i ( igf - i ) ) [ 1518 ] . in this paper
, we summarize the current understanding of the endocrine contribution to sarcopenia and provide an update on practical hormonal intervention for the elderly . an age - related disruption of the intracellular redox balance appears to be a primary causal factor for a chronic state of low - grade inflammation . myostatin was first discovered during screening for new members of the transforming growth factor- ( tgf- ) superfamily and shown to be a potent negative regulator of muscle growth . more recently , the igf - i - akt - mtor ( mammalian target of rapamycin ) pathway , which mediates both differentiation in myoblasts and hypertrophy in myotubes , has been shown to inhibit myostatin - dependent signaling . blockade of the akt - mtor pathway , using sirna to raptor , a component of torc1 ( tor signaling complex 1 ) , facilitates myostatin 's inhibition of muscle differentiation because of an increase in smad2 phosphorylation . found that treatment with a mouse chimera of antihuman myostatin antibody ( 24 mg / kg , 4 weeks ) , a drug for inhibiting myostatin , elicited a significant increase in muscle mass and in running performance probably due to decreased levels of phosphorylated smad3 and muscle ring finger-1 ( murf-1 ) . changes in steroid - induced glutamine synthetase represent a potential mechanism of action , and dose - dependent inhibition of glutamine synthetase by igf - i was observed in rat l6 cells . still , it remains to be directly elucidated , using pharmacological inhibitors for glucocorticoids , whether age - related increases in serum glucocorticoid levels actually inhibit protein synthesis and/or enhance protein degradation . systemic reviews of the literature have concluded that testosterone supplementation attenuates several sarcopenic symptoms including decreases in muscle mass [ 6466 ] and grip strength . risks associated with testosterone therapy in older men include sleep apnea , thrombotic complications , and the increased risk of prostate cancer . in aged men ,
the age - dependent decline in gh secretion is secondary to a decrease in ghrh and to an increase in somatostatin secretion . with respect to the somatomedin hypothesis ,
the growth - promoting actions of gh are mediated by circulating or locally produced igf - i . there is evidence that the age - associated decline in gh levels in combination with lower igf - i levels contributes to the development of sarcopenia . igf - i is perhaps the most important mediator of muscle growth and repair possibly by utilizing akt - mtor - p70s6k ( p70 ribosomal protein s6 kinase ) signaling . although the transgenic approach of upregulating igf - i expression in skeletal muscle would be appropriate for inhibiting sarcopenia , the administration of igf - i to the elderly has resulted in controversial findings on muscle strength and function . the ineffectiveness may be attributable to age - related insulin resistance to amino acid transport and protein synthesis or a marked decrease in igf - i receptors [ 96 , 97 ] and receptor affinity for igf - i in muscle with age . similarly there is an age - dependent reduction in vitamin d receptor expression in skeletal muscle . the histological examination of muscle tissue from subjects with osteomalacia is characterized by increased interfibrillar space , intramuscular adipose tissue infiltrates , and fibrosis . a more comprehensive knowledge on vitamin - d - related mechanisms may provide a very useful tool preventing muscle atrophy for older persons ( sarcopenia ) . currently , resistance training combined with amino - acid - containing supplements would be the best way to prevent age - related muscle wasting and weakness . testosterone supplementation in large amounts improves muscle defects with aging but has several side effects . myostatin inhibition seems to be an intriguing strategy for attenuating sarcopenia as well as muscular dystrophy . | [
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often , the frontal chest radiograph provides the first clue to the presence of an abnormal pulmonary artery ( fig .
if the pulmonary artery is enlarged , it presents with an enlarged contour of the vessel below the aortopulmonary window .
transverse diameter of the normal right interlobar artery from its lateral aspect to the intermediate bronchus is 15 mm in women and 16 mm in men .
1b ) provides more detail of the lumen , vessel wall and adjacent mediastinal structures .
greater anatomical detail is obtained with magnetic resonance ( mr ) imaging , allowing for improved evaluation of the vessel wall and quantification of flow ( fig .
it also allows for pulmonary artery maximal and minimal cross - sectional area measurement to be made perpendicular to the axis of blood flow , useful in identifying distensibility ( fig .
1d ) . positron emission tomography ( pet)-ct is useful to evaluate for malignancy and arteritis
. more invasive methods of imaging the pulmonary artery include intravascular ultrasound and catheter angiography.fig .
1a frontal radiograph of a patient with sinus venosus atrial septal defect demonstrates a dilated pulmonary artery , pulmonary oedema , left pleural effusion and cardiomegaly . the right lower lobe pulmonary artery measures 26 mm .
b contrast - enhanced axial ct image demonstrates an enlarged main pulmonary artery in the same patient .
c axial steady state free precession images in the same patient demonstrate enlarged pulmonary artery ( arrow ) .
d pulmonary artery distensibility ( 1.8 % ) can be calculated using cine mr , by measuring the diastolic minimal ( 10.9 cm ) and systolic maximal ( 11.1 cm ) cross - sectional area obtained perpendicular to direction of blood flow .
e average velocity obtained with phase contrast mr in the main pulmonary artery is 11.6 cm / s a frontal radiograph of a patient with sinus venosus atrial septal defect demonstrates a dilated pulmonary artery , pulmonary oedema , left pleural effusion and cardiomegaly . the right lower lobe pulmonary artery measures 26 mm .
b contrast - enhanced axial ct image demonstrates an enlarged main pulmonary artery in the same patient . the pulmonary artery to aorta ratio is 1.9 .
c axial steady state free precession images in the same patient demonstrate enlarged pulmonary artery ( arrow ) .
d pulmonary artery distensibility ( 1.8 % ) can be calculated using cine mr , by measuring the diastolic minimal ( 10.9 cm ) and systolic maximal ( 11.1 cm ) cross - sectional area obtained perpendicular to direction of blood flow .
e average velocity obtained with phase contrast mr in the main pulmonary artery is 11.6 cm / s in this article , we will briefly review the embryology and anatomy of the pulmonary arteries , followed by a discussion of the ct appearance of the common congenital anomalies and acquired conditions affecting the pulmonary arteries .
for ease of discussion , the acquired entities will be categorised as those affecting the vessel wall , intraluminal abnormalities and extraluminal abnormalities .
in addition , a brief discussion of imaging appearance in patients with repaired congenital heart diseases affecting the pulmonary arteries is also included .
during the 4th-5th week of embryogenesis , the aortic sac gives rise to six paired arteries called the aortic arches , which will eventually develop into the mature aortic arch and other major vessels ( fig .
the arches originate from the aortic sac and terminate in the right and left dorsal aorta .
the right sixth aortic arch persists as the proximal right pulmonary and the distal main pulmonary artery .
the left pulmonary artery and the distal right pulmonary artery develop from arteries arising from the adjacent lung buds and surrounding mesoderm .fig .
2a illustrations depicting the developing six paired aortic arches with the left and right dorsal aorta during early embryogenesis .
b further development leads to formation of right and left pulmonary arteries from the sixth aortic arches , primitive truncus arteriosus and adjacent arteries .
arrest in this normal development can lead to agenesis , partial agenesis , pulmonary sling , etc .
a illustrations depicting the developing six paired aortic arches with the left and right dorsal aorta during early embryogenesis .
b further development leads to formation of right and left pulmonary arteries from the sixth aortic arches , primitive truncus arteriosus and adjacent arteries .
arrest in this normal development can lead to agenesis , partial agenesis , pulmonary sling , etc .
the bronchial circulation draws 1 % of systemic cardiac output and normally only supplies nutrients to the lungs .
the primary circulation is the pulmonary arteries , which convey venous blood to the lungs from the heart .
a pulmonary artery branch accompanies the bronchial tree and ends in capillary network within the alveolar wall . the normal main pulmonary artery ( mpa )
divides into the right and left branches before it exits the pericardium . the left pulmonary artery ( lpa )
travels over the left mainstem bronchus before dividing into its two branches at the root of the left lung .
the right pulmonary artery ( rpa ) continues from the mpa before diving into its two branches , the superior and inferior ( interlobar ) trunk , at the root of the right lung .
the superior trunk supplies the right upper lobe with the interlobar trunk supplying the middle and lower lobes .
right middle lobe medial and lateral segmental arteries may arise as a common trunk from the interlobar artery or as separate branches .
the right lower lobe artery first gives off an apical segmental branch and distal to this the right lower lobe artery is called the basal trunk .
lower lobe artery gives off the medial basal and anterior basal followed by the lateral and posterior basal segmental arteries .
on the left , there is no truncus anterior , and the segmental branches originate directly from the lpa . for the left upper lobe and lingual arteries
the superior segmental artery of the lower lobe arises from the left interlobar artery above the origin of lingular branches .
caudal to this , the left interlobar artery becomes basal trunk giving rise to lower lobe segmental branches .
on ct , the main pulmonary artery measures up to 28 mm , some studies have found 29 mm in men and 27 mm in women to be the upper limit for normal [ 3 , 6 ] .
a convenient method to evaluate for pulmonary artery enlargement is to determine whether the ratio of the main pulmonary artery to the ascending aorta ( fig .
normal main pulmonary artery pressure ranges from 8 to 20 mmhg . in pulmonary hypertension ( intraluminal pressure exceeding 25 mmhg at rest or 30 mmhg with exercise ) , frontal chest radiograph demonstrates a prominent pulmonary artery silhouette with dilated hilar vessels and diminished peripheral vascularity ( fig .
phase - contrast mr - derived mean average velocity < 11.7 cm / s can help in detection of pulmonary hypertension ( sensitivity 92.9 % and specificity 82.4 % ) .
pulmonary arterial transit times measured using time - resolved mr angiography can be used as a simple , non - invasive metric for detection of altered haemodynamics in pulmonary arterial hypertension .
cine mr derived pulmonary artery distensibility of > 10 % ( systolic pulmonary artery area - diastolic pulmonary artery area systolic pulmonary artery area 100 ) is useful to evaluate pulmonary hypertensive patients who would respond to vasodilator therapy . in patients with fontan circulation ,
pulmonary perfusion ratios are more accurately evaluated with phase contrast mr compared with lung perfusion scintigraphy .
adequate contrast opacification is critical for diagnostic quality , which depends upon patient weight , cardiac output , scan duration and contrast delivery protocol [ 11 , 12 ] .
arterial enhancement depends on the amount of contrast delivered per unit of time ( injection flow rate ) and the injection duration , measured in seconds . suggested minimal luminal attenuation to see all acute and chronic pulmonary venous emboli ( pe ) is 93 and 211 hu respectively . on a 64-detector ct
, a mean pulmonary artery opacification of 250 hu could be achieved with 1.2 ml / kg of 350 mg i / ml injected at 4 ml / s .
iodine flow rate of 1.6 g i / s has been suggested as optimal to reach the pulmonary artery enhancement of 300 hu .
the scan duration depends upon the scanner ( 16 , 64 , dual source , dual source high pitch , 256 , 320 slice multidetector [ md ] ct ) , which on a high pitch scanner this may be less than 2 seconds . with a faster scanner , contrast volume
, a region of interest can be placed in the main pulmonary artery and a timing bolus or bolus tracking can be utilised to determine the time it takes for intravenously injected contrast to reach the pulmonary arteries .
contrast flow rate of at least 3 ml / s is associated with lower frequency of insufficient contrast enhancement during chest ct .
flow rate of more than 4 ml / s using an 18-g cannula has been suggested for pe exams [ 20 , 21 ] a lower volume of contrast and iodine dose can be administered using a higher concentration ( 350 mg iodine / ml vs 300 mg / ml ) .
wu et al . have described a low contrast dose ( 30 ml ) pulmonary 64-detector ct angiography technique without compromising diagnostic image quality .
the duration of contrast administration is calculated as scan duration plus additional few seconds ( 68 s ) .
this delay accounts for the interval between the scan trigger and the start of acquisition . when evaluating for fontan circulation , park et al
. found that a 3-min delay time from the time of injection to be optimal for enhancement of the pulmonary arteries , irrespective of the intravenous route used for administration .
bolus tracking demonstrated a high failure rate in providing homogenous enhancement of the fontan circulation and of the pulmonary arteries .
for all pulmonary ct angiography studies , a caudocranial direction of acquisition is recommended as it reduces the chances of having respiratory motion related artefacts . at our institution , in - patients with normal ( stage 1 , glomerular filtration rate [ gfr ml / min/1.73 m2 ] = 90 + ) and mildly reduced renal function ( stage 2 , gfr = 6089 ) and no contraindication to ct contrast agent , contrast volume is determined from patient height , weight , age , sex , heart rate and scan duration using vendor - specified protocol ( medrad ) with a timing bolus ( test bolus of 20 ml contrast and 50 ml saline at 4 ml / s to find the time to peak in the main pulmonary artery is used to determine the scan delay , scan delay = time to peak in pulmonary artery + 9 s ) .
the maximum allowed injection flow rate is 6 ml / s . in patients with moderately impaired renal function ( stage 3 a , gfr = 4560 ) , bolus tracking with 75 ml of contrast at 45
moderately reduced renal function ( stage 3 b , gfr = 3044 ) 30 ml of contrast with bolus tracking from svc , preferably on the 256 slice mdct is used .
any contrast injection is avoided in patients with gfr less than 29 unless they are on haemodialysis .
ct angiography protocol used at our institution is presented in table 1.table 1pulmonary ct angiography protocol used at our institutionindicationcontrast , flow ratekvpmas(ap scout)reconstructionscommentscongenitalpower or hand injection 3 ml / s , 50 ml contrast ( 300 mg i / ml ) , no saline chasersmall = 80 , medium = 100 , large = 120 80120tube current modulationaxial : 3 2 mm , 2 1 mm coronal : 3 2 axial mips : 8 mm25 s delay , complete thoraxpulmonary embolismdual head power injector 45 ml / s ( 350 mg / ml ) , + 50 ml saline chaser80140tube current modulationaxial : 3 2 mm , 2 1 mm coronal : 32 axial mips : 8 mmweight - based contrast , bolus track or timing bolus , minimal post threshold delaypulmonary hypertensionpower or hand injection 23 ml / s , no saline chaser80140tube current modulationaxial : 1 0.5 mm , 3 2 coronal : 3 2 axial mips : 8 mmlow kvp , 5075 ml contrast , additional expiratory scans , hrct reconspregnant patientdual head power injector 45 ml / s , + 50 ml saline chaser80100tube current modulationaxial : 3 2 mm coronal : 3 2 axial mips : 8 mmlow kvp , max . 75 ml contrast , z - axis coverage : aortic arch - diaphragmrenal dysfunctiondual head power injector 34 ml / s , + 50 ml saline chaser80120tube current modulationaxial : 3 2 mm coronal : 3 2 axial mips : 8 mm3075 ml contrast , preferably on 256 mdct , trigger from svcthe kvp used depends on patient size : small = 80 ( body mass index ( bmi ) < 20 kg / m ) , medium = 100 ( bmi = 2025 ) , large = 120 ( bmi = 2530 ) .
scan parameters based on scouts including kvp > 140 pulmonary ct angiography protocol used at our institution the kvp used depends on patient size : small = 80 ( body mass index ( bmi ) < 20 kg / m ) , medium = 100 ( bmi = 2025 ) , large = 120 ( bmi = 2530 ) .
scan parameters based on scouts including kvp > 140 mr imaging for the diagnosis of pulmonary artery disease can be performed using high - field mr scanners ( > 1.5 t ) .
it is indicated when cardiac function and flow needs to be evaluated , such as congenital heart disease , calculating intra / extra - cardiac shunts , right ventricle strain in pe and pulmonary hypertension .
non - contrast sequences used include a bright blood steady state free precession ( ssfp ) , t2-weighted inversion recovery and t1 gre ( gradient echo ) .
post - contrast mr angiography is performed with extracellular gadolinium contrast agent injected at 0.10.2 mmol / kg .
when evaluating for pe , a combination of mr angiography gre and ssfp images have the highest sensitivity .
mr is the imaging modality of choice for evaluating the right ventricle size and function .
contrast - enhanced mr angiography with gadolinium - based mri contrast agent , using both high spatial - resolution and high temporal - resolution protocols ( high spatial - resolution contrast - enhanced mr angiography and time - resolved contrast - enhanced mr angiography ) , is an excellent non - invasive imaging tool for the evaluation of surgical cavopulmonary connections .
pulmonary mr angiography should be considered as an alternative to ct angiography when iodine contrast injection or radiation is a significant matter .
it has been proposed that electrocardiograph ( ecg)-gated and respiratory navigator - gated mr angiography at 3 t using a blood - pool contrast agent at 0.3 mmol / kg can deliver better image quality and vessel sharpness . although , gadolinium - based contrast agents are not recommended in patients with a gfr less than 30 or acute renal failure in patients with hepatorenal syndrome unless essential due to risk for nephrogenic systemic sclerosis .
pulmonary mr angiography protocol used at our institution is presented in table 2.table 2pulmonary mr angiography protocol used at our institution on a 1.5-t magnetsequence typeorientationslice thickness / gap ( mm)te / tr ( msec)flip angle ( degrees)matrixfield of view ( mm)bandwidth ( khz)nexinformation acquirednon - contrastssfpaxial , coronal , ventricle short axis4/01.4/3.445200 1603504201250.75morphology , ventricle functiont1axial , short axis6/042902563862.51morphology , characterise mass lesions , oedemat2axial , short axis6/041/1,79190256 25635062.51phase contrastperpendicular to pulmonary flow82.7/5.625192 12835031.251quantify pulmonary flow volume , peak - mean velocity , regurgitationcontrast - enhanced mr angiographymr angiographycoronal2.01.4/3.930224 22432042062.5.5luminal assessmenttime resolvedcoronal2.61.2/3.238256 1924062.50.50.753d greaxial4/21.9/3.912320 16032042083.30.75delayed enhancedaxial , short axis8/01.3/5.320224 1923522.71thrombus , vessel wall , inflammation / scar pulmonary mr angiography protocol used at our institution on a 1.5-t magnet f-18 fluorodeoxyglucose ( fdg ) pet / ct is useful in identifying a pulmonary artery lesion as malignant if the luminal lesion has high fdg uptake and is useful in preoperative evaluation .
it is also very useful in identifying active vasculitis in patients with pulmonary vasculitis such as takayasu s arteritis and monitoring response to immunosuppressive treatment . at our institution ,
a pet - ct for these indications is combined with a contrast - enhanced ct angiography of pulmonary arteries to better depict the vascular anatomy rather than a non - contrast ct for attenuation correction .
adequate contrast opacification is critical for diagnostic quality , which depends upon patient weight , cardiac output , scan duration and contrast delivery protocol [ 11 , 12 ] .
arterial enhancement depends on the amount of contrast delivered per unit of time ( injection flow rate ) and the injection duration , measured in seconds . suggested minimal luminal attenuation to see all acute and chronic pulmonary venous emboli ( pe ) is 93 and 211 hu respectively . on a 64-detector ct
, a mean pulmonary artery opacification of 250 hu could be achieved with 1.2 ml / kg of 350 mg i / ml injected at 4 ml / s .
iodine flow rate of 1.6 g i / s has been suggested as optimal to reach the pulmonary artery enhancement of 300 hu .
the scan duration depends upon the scanner ( 16 , 64 , dual source , dual source high pitch , 256 , 320 slice multidetector [ md ] ct ) , which on a high pitch scanner this may be less than 2 seconds . with a faster scanner , contrast volume
, a region of interest can be placed in the main pulmonary artery and a timing bolus or bolus tracking can be utilised to determine the time it takes for intravenously injected contrast to reach the pulmonary arteries .
contrast flow rate of at least 3 ml / s is associated with lower frequency of insufficient contrast enhancement during chest ct .
flow rate of more than 4 ml / s using an 18-g cannula has been suggested for pe exams [ 20 , 21 ] a lower volume of contrast and iodine dose can be administered using a higher concentration ( 350 mg iodine / ml vs 300 mg / ml ) .
wu et al . have described a low contrast dose ( 30 ml ) pulmonary 64-detector ct angiography technique without compromising diagnostic image quality .
the duration of contrast administration is calculated as scan duration plus additional few seconds ( 68 s ) .
this delay accounts for the interval between the scan trigger and the start of acquisition . when evaluating for fontan circulation , park et al
. found that a 3-min delay time from the time of injection to be optimal for enhancement of the pulmonary arteries , irrespective of the intravenous route used for administration .
bolus tracking demonstrated a high failure rate in providing homogenous enhancement of the fontan circulation and of the pulmonary arteries .
for all pulmonary ct angiography studies , a caudocranial direction of acquisition is recommended as it reduces the chances of having respiratory motion related artefacts . at our institution , in - patients with normal ( stage 1 , glomerular filtration rate [ gfr ml / min/1.73 m2 ] = 90 + ) and mildly reduced renal function ( stage 2 , gfr = 6089 ) and no contraindication to ct contrast agent , contrast volume is determined from patient height , weight , age , sex , heart rate and scan duration using vendor - specified protocol ( medrad ) with a timing bolus ( test bolus of 20 ml contrast and 50 ml saline at 4 ml / s to find the time to peak in the main pulmonary artery is used to determine the scan delay , scan delay = time to peak in pulmonary artery + 9 s ) .
the maximum allowed injection flow rate is 6 ml / s . in patients with moderately impaired renal function ( stage 3 a , gfr = 4560 ) , bolus tracking with 75 ml of contrast at 45
moderately reduced renal function ( stage 3 b , gfr = 3044 ) 30 ml of contrast with bolus tracking from svc , preferably on the 256 slice mdct is used .
any contrast injection is avoided in patients with gfr less than 29 unless they are on haemodialysis .
ct angiography protocol used at our institution is presented in table 1.table 1pulmonary ct angiography protocol used at our institutionindicationcontrast , flow ratekvpmas(ap scout)reconstructionscommentscongenitalpower or hand injection 3 ml / s , 50 ml contrast ( 300 mg i / ml ) , no saline chasersmall = 80 , medium = 100 , large = 120 80120tube current modulationaxial : 3 2 mm , 2 1 mm coronal : 3 2 axial mips : 8 mm25 s delay , complete thoraxpulmonary embolismdual head power injector 45 ml / s ( 350 mg / ml ) , + 50 ml saline chaser80140tube current modulationaxial : 3 2 mm , 2 1 mm coronal : 32 axial mips : 8 mmweight - based contrast , bolus track or timing bolus , minimal post threshold delaypulmonary hypertensionpower or hand injection 23 ml / s , no saline chaser80140tube current modulationaxial : 1 0.5 mm , 3 2 coronal : 3 2 axial mips : 8 mmlow kvp , 5075 ml contrast , additional expiratory scans , hrct reconspregnant patientdual head power injector 45 ml / s , + 50 ml saline chaser80100tube current modulationaxial : 3 2 mm coronal : 3 2 axial mips : 8 mmlow kvp , max . 75 ml contrast , z - axis coverage : aortic arch - diaphragmrenal dysfunctiondual head power injector 34 ml / s , + 50 ml saline chaser80120tube current modulationaxial : 3 2 mm coronal : 3 2 axial mips : 8 mm3075 ml contrast , preferably on 256 mdct , trigger from svcthe kvp used depends on patient size : small = 80 ( body mass index ( bmi ) < 20 kg / m ) , medium = 100 ( bmi = 2025 ) , large = 120 ( bmi = 2530 ) .
scan parameters based on scouts including kvp > 140 pulmonary ct angiography protocol used at our institution the kvp used depends on patient size : small = 80 ( body mass index ( bmi ) < 20 kg / m ) , medium = 100 ( bmi = 2025 ) , large = 120 ( bmi = 2530 ) .
mr imaging for the diagnosis of pulmonary artery disease can be performed using high - field mr scanners ( > 1.5 t ) .
it is indicated when cardiac function and flow needs to be evaluated , such as congenital heart disease , calculating intra / extra - cardiac shunts , right ventricle strain in pe and pulmonary hypertension .
non - contrast sequences used include a bright blood steady state free precession ( ssfp ) , t2-weighted inversion recovery and t1 gre ( gradient echo ) .
post - contrast mr angiography is performed with extracellular gadolinium contrast agent injected at 0.10.2 mmol / kg .
when evaluating for pe , a combination of mr angiography gre and ssfp images have the highest sensitivity .
mr is the imaging modality of choice for evaluating the right ventricle size and function .
contrast - enhanced mr angiography with gadolinium - based mri contrast agent , using both high spatial - resolution and high temporal - resolution protocols ( high spatial - resolution contrast - enhanced mr angiography and time - resolved contrast - enhanced mr angiography ) , is an excellent non - invasive imaging tool for the evaluation of surgical cavopulmonary connections .
pulmonary mr angiography should be considered as an alternative to ct angiography when iodine contrast injection or radiation is a significant matter .
it has been proposed that electrocardiograph ( ecg)-gated and respiratory navigator - gated mr angiography at 3 t using a blood - pool contrast agent at 0.3 mmol / kg can deliver better image quality and vessel sharpness . although , gadolinium - based contrast agents are not recommended in patients with a gfr less than 30 or acute renal failure in patients with hepatorenal syndrome unless essential due to risk for nephrogenic systemic sclerosis .
pulmonary mr angiography protocol used at our institution is presented in table 2.table 2pulmonary mr angiography protocol used at our institution on a 1.5-t magnetsequence typeorientationslice thickness / gap ( mm)te / tr ( msec)flip angle ( degrees)matrixfield of view ( mm)bandwidth ( khz)nexinformation acquirednon - contrastssfpaxial , coronal , ventricle short axis4/01.4/3.445200 1603504201250.75morphology , ventricle functiont1axial , short axis6/042902563862.51morphology , characterise mass lesions , oedemat2axial , short axis6/041/1,79190256 25635062.51phase contrastperpendicular to pulmonary flow82.7/5.625192 12835031.251quantify pulmonary flow volume , peak - mean velocity , regurgitationcontrast - enhanced mr angiographymr angiographycoronal2.01.4/3.930224 22432042062.5.5luminal assessmenttime resolvedcoronal2.61.2/3.238256 1924062.50.50.753d greaxial4/21.9/3.912320 16032042083.30.75delayed enhancedaxial , short axis8/01.3/5.320224 1923522.71thrombus , vessel wall , inflammation / scar pulmonary mr angiography protocol used at our institution on a 1.5-t magnet
f-18 fluorodeoxyglucose ( fdg ) pet / ct is useful in identifying a pulmonary artery lesion as malignant if the luminal lesion has high fdg uptake and is useful in preoperative evaluation .
it is also very useful in identifying active vasculitis in patients with pulmonary vasculitis such as takayasu s arteritis and monitoring response to immunosuppressive treatment . at our institution ,
a pet - ct for these indications is combined with a contrast - enhanced ct angiography of pulmonary arteries to better depict the vascular anatomy rather than a non - contrast ct for attenuation correction .
unilateral pulmonary agenesis presents with unilateral absence of the lung and absence of the ipsilateral pulmonary artery and veins ( fig .
the aetiology is unknown , although genetic factors , viral infections , folate and vitamin a deficiencies have been proposed as possible causes .
newborns with this abnormality typically do not present with respiratory distress , but are likely to have other anomalies associated with the cardiovascular , musculoskeletal or gastrointestinal system . later in life
ct demonstrates decreased volume in the ipsilateral hemithorax , complete absence of lung parenchyma , agenesis of pulmonary artery and veins .
there is elevation of hemidiaphragm and mediastinal shift to the affected side [ 38 , 39].fig .
3contrast - enhanced ct image ( a ) shows complete agenesis of left lung and left pulmonary artery .
the left hemithorax is smaller with mediastinal shift toward the left and the elevation of the left hemidiaphragm .
the abdominal contents are seen in the left hemithorax . contrast - enhanced axial ct image ( b )
demonstrates partial agenesis of the left pulmonary artery ( arrow ) with hypoplasia of left lung .
there is no mediastinal shift , but the abdominal organs extend into the thorax contrast - enhanced ct image ( a ) shows complete agenesis of left lung and left pulmonary artery .
the left hemithorax is smaller with mediastinal shift toward the left and the elevation of the left hemidiaphragm .
contrast - enhanced axial ct image ( b ) demonstrates partial agenesis of the left pulmonary artery ( arrow ) with hypoplasia of left lung .
there is no mediastinal shift , but the abdominal organs extend into the thorax partial pulmonary artery agenesis involves an absence of the proximal portion or a rudimentary pulmonary artery .
blood flow to the ipsalateral lung is achieved through collaterals provided from the brachial arteries and transpleural branches of the thoracic arteries .
patients with the anomaly show an increased predisposition to dyspnea , recurrent respiratory infections and pulmonary haemorrhage .
transpleural collaterals can be seen as pleural thickening and subpleural parenchymal bands on the ct [ 3 , 40 ] .
the primitive left sixth aortic arch gives rise to the ductus arteriosus , which connects the descending thoracic aorta to the left pulmonary artery .
patent ductus arteriosus anomaly arises with persistent postnatal hypoxia , leading to failure of contraction of the ductus with formation of a continuous left to right shunt forms .
a small shunt predisposes to endocarditis and a larger shunt causes haemodynamic derangement , eventually leading to eisenmenger syndrome .
symptomatic patients may present with dyspnea , tachycardia , a widened pulse pressure and a machinery - like continuous murmur .
ct demonstrates dilated pulmonary artery , pruning of the peripheral compared with central pulmonary vasculature .
contrast - enhanced ct will identify the patent communication between the descending thoracic aorta and the pulmonary artery .
cardiac mr can be used to quantitate the left to right shunt ( fig .
4 ) [ 41 , 42].fig . 4contrast - enhanced axial ct image ( a ) and a volume rendered image ( b ) in a patient with patent ductus arteriosus ( pda ) depicting the persistent communication between the pulmonary artery and descending aorta ( arrow ) .
the flow direction in the post - natal period is aorta to pulmonary artery as the pulmonary pressures decrease .
this can lead to pulmonary hypertension , which on ct will present as enlarged pulmonary trunk as seen on the volume rendered image contrast - enhanced axial ct image ( a ) and a volume rendered image ( b ) in a patient with patent ductus arteriosus ( pda ) depicting the persistent communication between the pulmonary artery and descending aorta ( arrow ) . the flow direction in the post - natal period is aorta to pulmonary artery as the pulmonary pressures decrease .
this can lead to pulmonary hypertension , which on ct will present as enlarged pulmonary trunk as seen on the volume rendered image pulmonary artery sling presents when the left pulmonary artery arises from the posterior aspect of the right pulmonary artery before coursing between the trachea and oesophagus to reach the left hilum ( fig .
the sling around the distal trachea and right mainstem bronchus causes a variable amount of compression of these structures and may lead to stenosis of a long segment of the trachea .
the amount of upper airway stenosis correlates to the degree of the patient s symptoms .
in addition , phase contrast mr may be used for quantification of pulmonary blood flow [ 3 , 43 ] .
flow measurements are calculated from single slice phase contrast mr obtained perpendicular to mpa , rpa and lpa.fig .
5illustration ( a ) and contrast - enhanced axial ct image ( b ) depicting the left pulmonary artery coursing between the trachea and oesophagus to reach the left pulmonary hilum .
patient s symptoms correlate with the degree of upper airway obstruction present from narrowing of the trachea illustration ( a ) and contrast - enhanced axial ct image ( b ) depicting the left pulmonary artery coursing between the trachea and oesophagus to reach the left pulmonary hilum .
patient s symptoms correlate with the degree of upper airway obstruction present from narrowing of the trachea pulmonary artery stenosis leads to right ventricular outflow tract obstruction and can be secondary to a variety of congenital or acquired aetiologies . in tetralogy of fallot ( tof ) , hemodynamic consequences depend largely on the degree of right ventricular outflow tract obstruction , including supravalvular narrowing , which has been reported in up to 50 % of patients . other congenital aetiologies for pulmonary artery stenosis include williams syndrome , alagille syndrome and congenital rubella .
affected regions of the vessel demonstrate fibrous intimal proliferation with loss of elastic fibres , leading to varying degrees of stenosis .
post - stenotic segments may be dilated or aneurysmal and often is the first clue on radiographs .
a pulmonary artery aneurysm is commonly defined as the pulmonary trunk measuring more than 4.5 cm and the right or left pulmonary artery measuring greater than 3 cm .
ct can demonstrate stenosis in the main and branch pulmonary arteries with dilated post - stenotic segment ( fig .
6contrast - enhanced axial ct image ( a ) from a patient with tetralogy of fallot ( tof ) and a prosthetic pulmonic valve demonstrates severe stenosis of the left and mild stenosis of the right pulmonary artery ( arrows ) .
in addition there is an ascending aortic aneurysm . in a different patient ( b ) with an unrepaired tof , an aneurysm of the pulmonary trunk ( arrow ) formed with a chronic thrombus in the right and left pulmonary arteries .
ct angiogram c performed with 30 ml of contrast in a patient with chronic renal failure and a prosthetic pulmonary valve demonstrates a main pulmonary artery aneurysm ( measuring 44 mm ) contrast - enhanced axial ct image ( a ) from a patient with tetralogy of fallot ( tof ) and a prosthetic pulmonic valve demonstrates severe stenosis of the left and mild stenosis of the right pulmonary artery ( arrows ) . in addition there is an ascending aortic aneurysm . in a different patient ( b ) with an unrepaired tof , an aneurysm of the pulmonary trunk ( arrow ) formed with a chronic thrombus in the right and left pulmonary arteries .
ct angiogram c performed with 30 ml of contrast in a patient with chronic renal failure and a prosthetic pulmonary valve demonstrates a main pulmonary artery aneurysm ( measuring 44 mm ) coronary to pulmonary artery fistula is an anomaly that accounts for 1530 % of all coronary artery fistulas .
the fistulous communication can either be congenital or acquired , as in the case of trauma , endovascular procedures and cardiac transplantation .
in a few patients , a significant shunt can form , leading to congestive heart failure from volume overload or angina .
most reported cases have been incidentally detected during catheter angiography , but more recently ct angiography has been used to describe the features of the fistula .
if the ct images are acquired in the systemic arterial phase , the only finding will be a contrast blush within the pulmonary artery ( fig .
. 7contrast - enhanced axial ct image in systemic arterial phase demonstrates contrast blush within the pulmonary trunk emanating from a tubular enhancing structure along the left anterior descending coronary artery .
communication is noted between this and the pulmonary artery , suggesting a coronary to pulmonary artery fistula ( arrow ) contrast - enhanced axial ct image in systemic arterial phase demonstrates contrast blush within the pulmonary trunk emanating from a tubular enhancing structure along the left anterior descending coronary artery .
communication is noted between this and the pulmonary artery , suggesting a coronary to pulmonary artery fistula ( arrow )
it involves the pulmonary artery is 5080 % of cases . in early disease ,
the vessel wall may demonstrate enhancement and thickening , and in advanced disease , may demonstrate stenosis or occlusion [ 3 , 49 ] .
behcet disease is a chronic multisystem small vessel vasculitis that can cause aneurysmal dilatation of the pulmonary artery ( fig .
8contrast - enhanced axial ct image ( a ) in a 16-year - old patient with progressive dyspnea and absent left upper extremity pulse shows a focus of smooth narrowing and aneurysmal dilatation of the left main pulmonary artery ( arrow ) .
late venous phase axial mr image from a 3d gre acquisition ( b ) shows delayed enhancement of an aneurysmal left pulmonary artery branch ( arrow ) .
also note the wall enhancement of descending thoracic aorta ( arrowhead ) consistent with vasculitis .
9contrast - enhanced axial ct ( a ) in a patient with bechet s disease demonstrate a focal aneurysm of the right lower lobe pulmonary artery with eccentric mural thrombus ( arrow ) .
volume rendered image ( b ) better depicts the eccentric saccular aneurysm ( arrow ) .
this patient underwent right lower lobectomy for recurrent haemoptysis contrast - enhanced axial ct image ( a ) in a 16-year - old patient with progressive dyspnea and absent left upper extremity pulse shows a focus of smooth narrowing and aneurysmal dilatation of the left main pulmonary artery ( arrow ) .
late venous phase axial mr image from a 3d gre acquisition ( b ) shows delayed enhancement of an aneurysmal left pulmonary artery branch ( arrow ) .
also note the wall enhancement of descending thoracic aorta ( arrowhead ) consistent with vasculitis .
these findings are suggestive of takayasu arteritis contrast - enhanced axial ct ( a ) in a patient with bechet s disease demonstrate a focal aneurysm of the right lower lobe pulmonary artery with eccentric mural thrombus ( arrow ) .
volume rendered image ( b ) better depicts the eccentric saccular aneurysm ( arrow ) .
this patient underwent right lower lobectomy for recurrent haemoptysis infected ( mycotic ) aneurysm of the pulmonary artery can develop from haematogenous seeding of the infectious agent or continuous involvement from an adjacent source .
10 ) is the modality of choice for evaluation of the infected aneurysm and demonstrates a lobulated vascular mass with an irregular wall arising from the vessel in question .
10contrast - enhanced axial ct image demonstrates aneurysmal formation with irregular thick walls in the segmental branches of right and left lower lobe pulmonary arteries ( arrow ) in this patient with a known infected aneurysm .
these findings were new compared with prior chest ct contrast - enhanced axial ct image demonstrates aneurysmal formation with irregular thick walls in the segmental branches of right and left lower lobe pulmonary arteries ( arrow ) in this patient with a known infected aneurysm .
these findings were new compared with prior chest ct pulmonary artery sarcoma arises from the mesenchymal cells of the intima . on initial evaluation , the entity
the two entities can be differentiated using a contrast - enhanced ct by evaluating for a low - attenuation filling defect occupying the entire lumen and leading to expansion of the artery or with extraluminal tumour extension ( fig .
fdg - pet shows the sarcoma to have higher metabolic activity than blood pool ( fig . 11b).fig .
11contrast - enhanced axial ct image ( a ) demonstrates a large filling defect in the left pulmonary artery ( arrow ) . the lesion remained stable after a course of anticoagulation , which raised the suspicion for a malignancy .
subsequently obtained fdg - pet ( b ) demonstrated the central part of this filling defect to be hypermetabolic , consistent with a primary pulmonary artery sarcoma .
gadolinium - enhanced cardiac mr ( c ) performed 60 s post contrast for preoperative evaluation demonstrates a lesion in the left pulmonary artery with an non - enhancing central portion , consistent with a bland thrombus , and an enhancing component in the pulmonary arteries and left lower lobe , suggestive of a tumour contrast - enhanced axial ct image ( a ) demonstrates a large filling defect in the left pulmonary artery ( arrow ) .
the lesion remained stable after a course of anticoagulation , which raised the suspicion for a malignancy .
subsequently obtained fdg - pet ( b ) demonstrated the central part of this filling defect to be hypermetabolic , consistent with a primary pulmonary artery sarcoma .
gadolinium - enhanced cardiac mr ( c ) performed 60 s post contrast for preoperative evaluation demonstrates a lesion in the left pulmonary artery with an non - enhancing central portion , consistent with a bland thrombus , and an enhancing component in the pulmonary arteries and left lower lobe , suggestive of a tumour the majority of intraluminal filling defects of the pulmonary artery are secondary to pulmonary thromboembolism .
several malignancies , including breast and colorectal carcinoma , metastasise to the lungs by the way of the pulmonary arteries .
in addition , the pulmonary arteries maybe the site for non - thrombotic emboli , such as non - target embolisation of intravascular glue , broken embolised fragments of an ivc filter or vertebroplasty cement ( fig .
12different patients with non - thrombotic emboli to the pulmonary arteries : catheter fragment ( a ) , non - target emboli from n - butyl-2-cyanoacrylate injection of gastric varices ( b ) , inferior vena cava filter prong ( c ) and bone cement for vertebropasty ( d ) different patients with non - thrombotic emboli to the pulmonary arteries : catheter fragment ( a ) , non - target emboli from n - butyl-2-cyanoacrylate injection of gastric varices ( b ) , inferior vena cava filter prong ( c ) and bone cement for vertebropasty ( d ) the pulmonary artery can also be affected by extrinsic processes .
luminal narrowing of the pulmonary artery may be due to extrinsic compression from bronchogenic carcinoma ( fig .
pulmonary artery dilatation can be seen with pulmonary hypertension , which can be secondary to a pulmonary parenchymal disease .
ct is essential in evaluating the lung parenchyma and , in addition , will demonstrate pulmonary artery diameter greater than 28 mm or a pulmonary artery to ascending aorta transverse diameter ratio greater than 0.9 [ 6 , 51 ] .
granulomatous fibrosing mediastinitis is an infiltrative disorder that results from excessive fibrosis in the mediastinum , usually a sequela of histoplasmosis ( fig .
. it can result in encasement of the mediastinal viscera with narrowing of the vessels , airway , and other mediastinal structures .fig .
13contrast - enhanced axial ct image in a patient with left hilar lung cancer demonstrates the left main pulmonary artery being completely encased and narrowed by the left upper lobe mass ( arrow ) , which also extends into the mediastinumfig .
14axial ct images in a patient with prior histoplasmosis demonstrates an enlarged pulmonary trunk ( 36 mm ) .
the proximal right and left pulmonary arteries are normal in calibre but taper and are severely narrowed at the level of hila .
in addition , there are calcified mediastinal lymph nodes , calcified pulmonary granulomas and interlobular interstitial thickening .
these findings represent fibrosing mediastinitis contrast - enhanced axial ct image in a patient with left hilar lung cancer demonstrates the left main pulmonary artery being completely encased and narrowed by the left upper lobe mass ( arrow ) , which also extends into the mediastinum axial ct images in a patient with prior histoplasmosis demonstrates an enlarged pulmonary trunk ( 36 mm ) .
the proximal right and left pulmonary arteries are normal in calibre but taper and are severely narrowed at the level of hila .
in addition , there are calcified mediastinal lymph nodes , calcified pulmonary granulomas and interlobular interstitial thickening .
these findings represent fibrosing mediastinitis corrective surgical procedures for congenital cardiovascular diseases which affect the pulmonary arteries result in a characteristic appearance .
cavopulmonary shunts or fontan circulation are used to treat infants with single effective ventricle ( tricuspid / pulmonary atresia , hypoplastic left heart / hypoplastic right heart syndrome ) .
the norwood procedure is used to correct hypoplastic left heart syndrome , which is frequently associated with hypoplasia of the ascending aorta .
stage 1 involves creating a neoaorta from the proximal main pulmonary artery , which is connected to the ascending aorta ( figs .
is then connected to the right pulmonary artery to provide blood flow to the lungs .
stage 2 of the procedure creates a glenn shunt , a superior cavopulmonary shunt from an end - to - end anastomosis between the superior vena cava and right pulmonary artery , thus directing systemic venous flow directly to the lungs .
stage 3 creates a total cavopulmonary connection by attaching the inferior vena cava to the right pulmonary artery , referred to as a fontan procedure [ 53 , 54 ] ( figs .
contrast timing during pulmonary ct angiography is critical in such patients to when evaluating for a suspected stenosis or pe.fig .
15illustration ( a ) demonstrates stage 1 , the norwood procedure , for correcting hypoplastic left heart syndrome with the creation of a neoaorta from the pulmonary artery .
post - repair images ( b ) have a characteristic appearance with a rudimentary proximal ascending aorta and the proximal main pulmonary artery ( arrow ) reconstituting flow to the distal ascending aorta ( arrowhead ) . during the procedure ,
the pulmonary trunk is ligated and the pulmonary arterial flow is re - established from either the subclavian artery or the brachiocephalic trunk .
after completion of stage 3 ( c , d ) , by attaching the inferior vena cava to the right pulmonary artery , the fontan procedure , complete systemic venous flow is directed through the right pulmonary artery into the lungs .
note the right pulmonary artery ( arrow ) shows higher attenuation secondary to the contrast injection from the right arm veins compared with the left pulmonary artery ( arrowhead ) which has lower attenuation due to blood flow from the inferior vena cava illustration ( a ) demonstrates stage 1 , the norwood procedure , for correcting hypoplastic left heart syndrome with the creation of a neoaorta from the pulmonary artery .
post - repair images ( b ) have a characteristic appearance with a rudimentary proximal ascending aorta and the proximal main pulmonary artery ( arrow ) reconstituting flow to the distal ascending aorta ( arrowhead ) . during the procedure ,
the pulmonary trunk is ligated and the pulmonary arterial flow is re - established from either the subclavian artery or the brachiocephalic trunk .
after completion of stage 3 ( c , d ) , by attaching the inferior vena cava to the right pulmonary artery , the fontan procedure , complete systemic venous flow is directed through the right pulmonary artery into the lungs .
note the right pulmonary artery ( arrow ) shows higher attenuation secondary to the contrast injection from the right arm veins compared with the left pulmonary artery ( arrowhead ) which has lower attenuation due to blood flow from the inferior vena cava an arterial switch is performed for treating transposition of great arteries .
it results in a characteristic appearance of the main pulmonary artery situated anterior to the ascending aorta with the right and left pulmonary arteries draped around the aorta .
16contrast enhanced axial ct images in a patient with transposition of great vessels demonstrates the characteristic appearance post - arterial switch .
the pulmonary arteries ( arrow ) are positioned anterior to the aorta with the left and right main branches draping around the aorta .
there is a higher incidence of pulmonary artery stenosis in these patients contrast enhanced axial ct images in a patient with transposition of great vessels demonstrates the characteristic appearance post - arterial switch .
the pulmonary arteries ( arrow ) are positioned anterior to the aorta with the left and right main branches draping around the aorta .
congenital and acquired pulmonary artery anomalies have a characteristic appearance on a variety of imaging modalities .
even though imaging findings on ct were mainly discussed , the interpreting radiologist needs to be familiar with findings of these entities on a spectrum of imaging modalities to avoid misinterpretation and reach the correct diagnosis .
table 3maximum contrast volume for ct angiography using power injector is based on patient weight ( iodine concentration of 350 mg / ml)weight ( kg)contrast volume ( ml)406945775086559560103651127012075129801378514390143 maximum contrast volume for ct angiography using power injector is based on patient weight ( iodine concentration of 350 mg / ml ) | backgroundpulmonary arteries are not just affected by thrombus .
congenital and acquired conditions can also involve the pulmonary arteries .
an awareness of these conditions is important for the radiologist interpreting chest computed tomography ( ct).methodsthe anatomy of the pulmonary arteries was reviewed .
ct and magnetic resonance ( mr ) acquisition protocols for imaging the pulmonary arteries were discussed .
the imaging appearances of congenital and acquired anomalies involving the pulmonary arteries , using ct and other modalities , were presented.resultsimaging features of congenital anomalies presented include pulmonary agenesis , partial pulmonary artery agenesis , patent ductus arteriosus , pulmonary artery sling , congenital pulmonary artery stenosis and coronary to pulmonary artery fistula .
acquired pulmonary artery anomalies discussed include arteritis , infected aneurysm and sarcoma .
pulmonary artery filling defects besides thromboembolism are also discussed , including foreign body emboli .
imaging features of bronchogenic carcinoma and mediastinal fibrosis demonstrating compression of the pulmonary arteries are presented , followed by a brief discussion of post repair appearance of the pulmonary arteries for congenital heart disease.conclusionscongenital and acquired pulmonary artery anomalies have a characteristic appearance on a variety of imaging modalities .
an acquaintance with the imaging features of these anomalies is needed to avoid misinterpretation and reach the correct diagnosis.teaching points discuss a variety of congenital and acquired anomalies of the pulmonary arteries. discuss the imaging appearance of the presented congenital or acquired pulmonary artery anomalies. describe ct and mr acquisition protocols for imaging the pulmonary arteries. review the anatomy of the pulmonary arteries . | Introduction
Embryology
Anatomy
Acquisition protocols
CT
MR
PET-CT
Congenital
Acquired
Conclusion
Appendix
Conflict of interest | greater anatomical detail is obtained with magnetic resonance ( mr ) imaging , allowing for improved evaluation of the vessel wall and quantification of flow ( fig . e average velocity obtained with phase contrast mr in the main pulmonary artery is 11.6 cm / s in this article , we will briefly review the embryology and anatomy of the pulmonary arteries , followed by a discussion of the ct appearance of the common congenital anomalies and acquired conditions affecting the pulmonary arteries . in addition , a brief discussion of imaging appearance in patients with repaired congenital heart diseases affecting the pulmonary arteries is also included . arrest in this normal development can lead to agenesis , partial agenesis , pulmonary sling , etc . with a faster scanner , contrast volume
, a region of interest can be placed in the main pulmonary artery and a timing bolus or bolus tracking can be utilised to determine the time it takes for intravenously injected contrast to reach the pulmonary arteries . with a faster scanner , contrast volume
, a region of interest can be placed in the main pulmonary artery and a timing bolus or bolus tracking can be utilised to determine the time it takes for intravenously injected contrast to reach the pulmonary arteries . 4contrast - enhanced axial ct image ( a ) and a volume rendered image ( b ) in a patient with patent ductus arteriosus ( pda ) depicting the persistent communication between the pulmonary artery and descending aorta ( arrow ) . this can lead to pulmonary hypertension , which on ct will present as enlarged pulmonary trunk as seen on the volume rendered image contrast - enhanced axial ct image ( a ) and a volume rendered image ( b ) in a patient with patent ductus arteriosus ( pda ) depicting the persistent communication between the pulmonary artery and descending aorta ( arrow ) . this can lead to pulmonary hypertension , which on ct will present as enlarged pulmonary trunk as seen on the volume rendered image pulmonary artery sling presents when the left pulmonary artery arises from the posterior aspect of the right pulmonary artery before coursing between the trachea and oesophagus to reach the left hilum ( fig . patient s symptoms correlate with the degree of upper airway obstruction present from narrowing of the trachea illustration ( a ) and contrast - enhanced axial ct image ( b ) depicting the left pulmonary artery coursing between the trachea and oesophagus to reach the left pulmonary hilum . patient s symptoms correlate with the degree of upper airway obstruction present from narrowing of the trachea pulmonary artery stenosis leads to right ventricular outflow tract obstruction and can be secondary to a variety of congenital or acquired aetiologies . communication is noted between this and the pulmonary artery , suggesting a coronary to pulmonary artery fistula ( arrow ) contrast - enhanced axial ct image in systemic arterial phase demonstrates contrast blush within the pulmonary trunk emanating from a tubular enhancing structure along the left anterior descending coronary artery . communication is noted between this and the pulmonary artery , suggesting a coronary to pulmonary artery fistula ( arrow )
it involves the pulmonary artery is 5080 % of cases . gadolinium - enhanced cardiac mr ( c ) performed 60 s post contrast for preoperative evaluation demonstrates a lesion in the left pulmonary artery with an non - enhancing central portion , consistent with a bland thrombus , and an enhancing component in the pulmonary arteries and left lower lobe , suggestive of a tumour the majority of intraluminal filling defects of the pulmonary artery are secondary to pulmonary thromboembolism . 12different patients with non - thrombotic emboli to the pulmonary arteries : catheter fragment ( a ) , non - target emboli from n - butyl-2-cyanoacrylate injection of gastric varices ( b ) , inferior vena cava filter prong ( c ) and bone cement for vertebropasty ( d ) different patients with non - thrombotic emboli to the pulmonary arteries : catheter fragment ( a ) , non - target emboli from n - butyl-2-cyanoacrylate injection of gastric varices ( b ) , inferior vena cava filter prong ( c ) and bone cement for vertebropasty ( d ) the pulmonary artery can also be affected by extrinsic processes . luminal narrowing of the pulmonary artery may be due to extrinsic compression from bronchogenic carcinoma ( fig . these findings represent fibrosing mediastinitis corrective surgical procedures for congenital cardiovascular diseases which affect the pulmonary arteries result in a characteristic appearance . it results in a characteristic appearance of the main pulmonary artery situated anterior to the ascending aorta with the right and left pulmonary arteries draped around the aorta . congenital and acquired pulmonary artery anomalies have a characteristic appearance on a variety of imaging modalities . even though imaging findings on ct were mainly discussed , the interpreting radiologist needs to be familiar with findings of these entities on a spectrum of imaging modalities to avoid misinterpretation and reach the correct diagnosis . | [
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when attempting to measure potential health benefits , a number of methods are available including cost benefit analysis , cost - effectiveness analysis , and cost - of - illness analysis .
the selection of an appropriate method should be consistent with the available data and the phenomenon being studied .
benefit analysis and cost - effectiveness analysis are both used for evaluation of two or more alternative treatments ; these techniques require the costs associated with both the treatment and the alternatives to be well - known .
the purpose of this research is to determine the potential benefits from allowing plant sterol - enriched foods to be sold into a market where they are currently not available , not to compare plant sterol - enriched foods to alternative treatments .
additionally , it is not possible to estimate the cost premium that would be attached to foods enriched with plant sterols in canada as this market would need to evolve in order to establish a long - term price . as such , neither cost - effectiveness nor cost benefit analysis is an appropriate tool for this analysis .
a cost - of - illness analysis measures the direct and indirect costs of a specific disease in this case chd .
a variation of a cost - of - illness analysis is to calculate the dollar savings associated with preventing or avoiding an illness .
this variation has been used to calculate the economic benefit of drug abuse interventions ( 10 ) , preventable drug - related medical complications ( 11 ) , and trans - fat - free canola oil ( 9 ) . a similar approach to the technique employed here was used previously by malla , hobbs , and perger to estimate health care savings from trans - fat - free canola oil in canada ( 12 ) .
the economic benefit of consumption of plant sterol - containing foods is evaluated using an economic simulation involving four steps , described below . in order to realize health benefits from plant sterols
the proportion of the canadian population that will consume a sufficient quantity of plant sterol - enriched foods to reduce their cholesterol level is defined as the success rate .
plant sterols can feasibly be added to a number of food products such as orange juice , cheese , milk , yogurt , salad dressing , cereal products , cooking oils , and margarine . according to statistics canada , the average canadian consumption of these food categories is around nine servings per day ( 13 ) .
it is thus reasonable to conclude that there is sufficient scope within the canadian diet to accommodate an adequate dose of plant sterols without having to drastically alter food consumption patterns .
it should be noted that all of the foods listed above may not be equally effective as delivery vehicles for plant sterols ; it may also be the case that claims will be allowed for different foods containing plant sterols along different timelines . since the market for plant sterol - enriched foods has not until very recently existed in canada , estimating its size and characteristics is difficult .
the overall canadian functional food market , of which phytosterol fortified foods will become a subset , is still evolving and changing rapidly and thus available data are relatively sparse . given the lack of available information ,
the best method for predicting how canadians would respond to plant sterol - enriched food products is to examine the markets for similar functional foods in canada and plant sterol - enriched foods in other countries .
an activities , interests , and opinions ( aio ) framework and cluster analysis was used to determine the potential markets for functional foods in canada ( 14 ) .
survey information from across canada enabled a cluster analysis to determine attitudes , knowledge , and motivations combining to identify consumers likely to buy functional foods .
it was discovered that a cluster representing approximately 47% of the adult canadian population possesses the characteristics that would make them likely to purchase functional food products .
canadian survey data available to determine the acceptance of specific functional foods are limited when compared to larger markets ; however , the acceptance of conjugated linoleic acid ( cla ) fortified foods has been examined in western canada ( 15 ) . it was found that the high percentage of survey respondents likely to purchase cla - enriched dairy products , if available where they normally shop , ranged from 4% to 13% .
since many of these dairy products are similar to potential phytosterol - fortified foods , it seems reasonable to extend these results to such products . in finland , a country which has allowed phytosterol / stanol - fortified foods in the marketplace for quite some time ,
a survey found 17% of adults reported daily use of plant sterol - enriched margarine , with another 20% reporting monthly use ( 16 ) .
an additional 7% indicated having occasionally used the product . for the purpose of this economic evaluation ,
the success rates shown in table 1 were used in the various sensitivity analysis scenarios as an estimate of the success ratio .
estimated plant sterol success rates the primary health benefit of consuming foods fortified with phytosterols is a reduction in serum cholesterol levels . approximating this reduction
requires extrapolation of results of meta - analyses of available human phytosterol - feeding studies .
since plant sterols are relatively well - established as functional food additives , several thorough meta - analyses exist ( 25 ) .
one of these was limited in scope to plant sterol - enriched spreads and found a reduction in ldl - cholesterol of 8.4% ( 95% ci 6.610.0% ) ( 3 ) .
another included 23 plant sterol and stanol trials and found a serum cholesterol reduction ranging between 4.6 and 24.3% with a mean ldl - cholesterol reduction of 11.0% ( 4 ) .
yet another used 21 published plant sterol studies found that average dose of 2.3 g of plant sterols per day yielded an average cholesterol reduction of 9.7% ( 95% ci 8.510.8% ) ( 2 ) .
the most recent meta - analysis examined the results of 59 studies and found that plant sterol containing products reduced ldl - cholesterol by 0.31
the results from the meta - analysis based on 21 published plant sterol studies ( 2 ) were adopted for the majority of the scenarios in the sensitivity analysis .
this work included the largest number of trials limited exclusively to plant sterol - enriched foods and did not include plant stanol - enriched foods .
as a result , this dataset was judged as best representing the relationship between plant sterol consumption and serum ldl - cholesterol level reduction in humans . since
no long - term studies have directly tracked the relationship between phytosterol consumption and heart disease in humans , it was necessary to make assumptions regarding the rate at which lower cholesterol levels due to plant sterol consumption will reduce the incidence of chd .
a meta - analysis of cholesterol - reducing treatment studies found a 1314% reduction in chd mortality for every 10-percentage - point net reduction in serum cholesterol levels ( 17 ) . in a similar examination of studies concerning the relationship between cholesterol levels and ischemic heart disease
, it was found that a 10% cholesterol reduction would lead to a 2530% reduction in mortality from ischemic heart disease over the long - term ( 18 ) . as both those studies focused on reductions in chd mortality
, it seems likely that the non - fatal incidence of chd would decrease in a similar fashion if serum cholesterol was reduced across the population . in an economic evaluation of the health benefits associated with trans - fat - free canola oil , a ratio of a 1% reduction of cholesterol corresponding to 23% reduction in the incidence of chd
this result is consistent with the findings of previous research ( 17 , 18 ) and was therefore used as the assumption regarding the reduction in the incidence of chd due to decreased blood cholesterol levels resulting from plant sterol consumption in the majority of the sensitivity analysis scenarios .
the final step in the procedure is to quantify the economic benefit resulting from introducing plant sterol - enriched food to the canadian market .
health canada has estimated the cost of chd in canada ( 7 ) ; even though the data are more than a decade old , they are still the most recent and comprehensive available .
since the 1998 data are in nominal dollar amounts but health care costs are subject to inflation , the 1998 amounts require adjustment to more current monetary terms .
this adjustment is made using statistics canada 's consumer price index for health and personal care ( 19 ) .
adjustment of the 1998 estimate of $ 18.472 billion to 2007 levels yields a new approximation of $ 21.176 billion as the assumed economic cost of chd in canada .
the economic cost of disease is generally broken down into direct and indirect components ; the former are incurred directly by the health care system with the goal of improving and/or preventing a patient 's health status from deteriorating . in the case of heart disease ,
direct costs include hospital care , drug costs , physician expenses , and other miscellaneous items .
indirect costs are those incurred due to the loss of production arising from disease and include loss of production due to mortality and morbidity costs arising from long- and short - term disability . table 2 provides a summary of direct and indirect costs attributable to chd in canada in both 1998 and inflation - adjusted 2007 dollars . while it seems obvious that a reduction in the level of chd will lower related costs , is the extent to which these costs will be lowered is less clear .
previous work utilized a directly proportional relationship and assumed that a 1% reduction in chd would result in a 1% reduction in chd - related costs ( 9 ) .
this type of assumption may oversimplify the health care process and could thus result in an overstatement of the potential cost reduction .
summary of costs attributed to coronary heart disease in canada ( $ millions ) source : health canada ( 7 ) . rather than assuming a proportional cost reduction , it is appropriate to more
for example , hospitalization expenditures related to chd include a sizeable fixed cost component the costs of operating a hospital will be incurred regardless of the number of heart attack patients treated at the hospital .
research conducted with respect to the fixed cost variable cost breakdown in hospitals found that hospital costs are approximately 84% fixed and 16% variable ( 20 ) .
accordingly , it is assumed that a reduction in chd will not result in a reduction in fixed costs of hospitalization , but will cause a proportional reduction in variable hospitalization costs . approximating the reduction in drug costs due
to chd assumes that fewer individuals with chd will require less medication to treat chd .
since 49.6% of drug costs resulting from chd are for the treatment of hypertension ( 7 ) , a condition not normally improved as a result of cholesterol reduction , these costs are not reduced in the calculations .
the remaining 50.4% of drug costs associated with chd are reduced proportionally to the reduction in the overall rate of chd .
physician care costs associated with chd are based on physician billings which in turn are derived from patient visits to doctors offices .
it is assumed that if fewer people are afflicted with chd , fewer visits to physicians for chd treatment will occur , resulting in less numerous billings and lower health care costs . as such , a 1% reduction in chd is assumed to lead to a 1% reduction in chd - related physician costs .
a large component of the miscellaneous chd - related expenditures has been categorized by health canada ( 7 ) as services provided by other health providers. for this analysis ,
the cost of chiropractors ( 0.8% ) , dental ( 26.2% ) , and vision ( 9.6% ) health professionals were excluded , as such costs are unlikely to be reduced due to a reduction in heart disease .
however , the cost of physiotherapists ( 1% ) and all other health professionals ( 4.7% ) should be reduced in proportion to the overall chd reduction .
also ignored in this analysis are costs which are largely fixed and therefore unlikely to decrease with the rate of chd : expenditures on health research ( 4.4% ) , administration ( 6.5% ) , and public health ( 20.2% ) . by contrast , costs that will likely be reduced by a reduction in chd include ambulance ( 4.2% ) , home care ( 6.2% ) , and all other health expenditures ( 7.2% ) .
the final result is that a 1% decrease in the incidence of chd is expected to result in a 0.23% reduction in miscellaneous chd - related costs .
mortality , short - term disability , and long - term disability costs were taken to have a directly proportional chd reduction to reduction in cost relationship .
since the incidence of chd will decline , it was deemed reasonable to assume the number of people who will die or become disabled as a result of chd will decline proportionately . as a consequence ,
the loss of human capital that would ordinarily be incurred as a result of chd - related death and disability , would not take place ; this reduction results in an economic saving .
table 3 summarizes the proportion of cost reductions that could be achieved by a reduction in the incidence of chd .
summary of coronary heart disease cost reduction corresponding to a 1% decrease in incidence of coronary heart disease a number of assumptions are required to approximate the potential reduction in the cost of chd from introducing phytosterol - fortified foods to the canadian market .
though these assumptions are based on a review of the peer - reviewed scientific literature , it could be misleading to suggest a single number as best representing the exact economic cost savings .
as such , results are reported as a sensitivity analysis ; as this approach allows the effects of varying model assumptions within the simulation to be gauged . to make the numbers as robust as possible , four scenarios were examined ; a description of the sensitivity analysis scenarios is provided below with a summary of the assumptions shown in table 4 .
in order to realize health benefits from plant sterols , an individual must consume between 1 and 3 g / day .
the proportion of the canadian population that will consume a sufficient quantity of plant sterol - enriched foods to reduce their cholesterol level is defined as the success rate .
plant sterols can feasibly be added to a number of food products such as orange juice , cheese , milk , yogurt , salad dressing , cereal products , cooking oils , and margarine . according to statistics canada , the average canadian consumption of these food categories is around nine servings per day ( 13 ) .
it is thus reasonable to conclude that there is sufficient scope within the canadian diet to accommodate an adequate dose of plant sterols without having to drastically alter food consumption patterns .
it should be noted that all of the foods listed above may not be equally effective as delivery vehicles for plant sterols ; it may also be the case that claims will be allowed for different foods containing plant sterols along different timelines . since the market for plant sterol - enriched foods has not until very recently existed in canada , estimating its size and characteristics is difficult .
the overall canadian functional food market , of which phytosterol fortified foods will become a subset , is still evolving and changing rapidly and thus available data are relatively sparse . given the lack of available information ,
the best method for predicting how canadians would respond to plant sterol - enriched food products is to examine the markets for similar functional foods in canada and plant sterol - enriched foods in other countries .
an activities , interests , and opinions ( aio ) framework and cluster analysis was used to determine the potential markets for functional foods in canada ( 14 ) .
survey information from across canada enabled a cluster analysis to determine attitudes , knowledge , and motivations combining to identify consumers likely to buy functional foods .
it was discovered that a cluster representing approximately 47% of the adult canadian population possesses the characteristics that would make them likely to purchase functional food products .
canadian survey data available to determine the acceptance of specific functional foods are limited when compared to larger markets ; however , the acceptance of conjugated linoleic acid ( cla ) fortified foods has been examined in western canada ( 15 ) .
it was found that the high percentage of survey respondents likely to purchase cla - enriched dairy products , if available where they normally shop , ranged from 4% to 13% . since many of these dairy products are similar to potential phytosterol - fortified foods , it seems reasonable to extend these results to such products . in finland , a country which has allowed phytosterol / stanol - fortified foods in the marketplace for quite some time ,
a survey found 17% of adults reported daily use of plant sterol - enriched margarine , with another 20% reporting monthly use ( 16 ) .
an additional 7% indicated having occasionally used the product . for the purpose of this economic evaluation ,
the success rates shown in table 1 were used in the various sensitivity analysis scenarios as an estimate of the success ratio .
the primary health benefit of consuming foods fortified with phytosterols is a reduction in serum cholesterol levels .
approximating this reduction requires extrapolation of results of meta - analyses of available human phytosterol - feeding studies .
since plant sterols are relatively well - established as functional food additives , several thorough meta - analyses exist ( 25 ) .
one of these was limited in scope to plant sterol - enriched spreads and found a reduction in ldl - cholesterol of 8.4% ( 95% ci 6.610.0% ) ( 3 ) .
another included 23 plant sterol and stanol trials and found a serum cholesterol reduction ranging between 4.6 and 24.3% with a mean ldl - cholesterol reduction of 11.0% ( 4 ) .
yet another used 21 published plant sterol studies found that average dose of 2.3 g of plant sterols per day yielded an average cholesterol reduction of 9.7% ( 95% ci 8.510.8% ) ( 2 ) .
the most recent meta - analysis examined the results of 59 studies and found that plant sterol containing products reduced ldl - cholesterol by 0.31
the results from the meta - analysis based on 21 published plant sterol studies ( 2 ) were adopted for the majority of the scenarios in the sensitivity analysis .
this work included the largest number of trials limited exclusively to plant sterol - enriched foods and did not include plant stanol - enriched foods .
as a result , this dataset was judged as best representing the relationship between plant sterol consumption and serum ldl - cholesterol level reduction in humans .
since no long - term studies have directly tracked the relationship between phytosterol consumption and heart disease in humans , it was necessary to make assumptions regarding the rate at which lower cholesterol levels due to plant sterol consumption will reduce the incidence of chd .
a meta - analysis of cholesterol - reducing treatment studies found a 1314% reduction in chd mortality for every 10-percentage - point net reduction in serum cholesterol levels ( 17 ) . in a similar examination of studies concerning the relationship between cholesterol levels and ischemic heart disease
, it was found that a 10% cholesterol reduction would lead to a 2530% reduction in mortality from ischemic heart disease over the long - term ( 18 ) . as both those studies focused on reductions in chd mortality
, it seems likely that the non - fatal incidence of chd would decrease in a similar fashion if serum cholesterol was reduced across the population . in an economic evaluation of the health benefits associated with trans - fat - free canola oil , a ratio of a 1% reduction of cholesterol corresponding to 23% reduction in the incidence of chd
this result is consistent with the findings of previous research ( 17 , 18 ) and was therefore used as the assumption regarding the reduction in the incidence of chd due to decreased blood cholesterol levels resulting from plant sterol consumption in the majority of the sensitivity analysis scenarios .
the final step in the procedure is to quantify the economic benefit resulting from introducing plant sterol - enriched food to the canadian market .
health canada has estimated the cost of chd in canada ( 7 ) ; even though the data are more than a decade old , they are still the most recent and comprehensive available .
since the 1998 data are in nominal dollar amounts but health care costs are subject to inflation , the 1998 amounts require adjustment to more current monetary terms .
this adjustment is made using statistics canada 's consumer price index for health and personal care ( 19 ) .
adjustment of the 1998 estimate of $ 18.472 billion to 2007 levels yields a new approximation of $ 21.176 billion as the assumed economic cost of chd in canada .
the economic cost of disease is generally broken down into direct and indirect components ; the former are incurred directly by the health care system with the goal of improving and/or preventing a patient 's health status from deteriorating . in the case of heart disease ,
direct costs include hospital care , drug costs , physician expenses , and other miscellaneous items .
indirect costs are those incurred due to the loss of production arising from disease and include loss of production due to mortality and morbidity costs arising from long- and short - term disability .
table 2 provides a summary of direct and indirect costs attributable to chd in canada in both 1998 and inflation - adjusted 2007 dollars . while it seems obvious that a reduction in the level of chd will lower related costs ,
previous work utilized a directly proportional relationship and assumed that a 1% reduction in chd would result in a 1% reduction in chd - related costs ( 9 ) .
this type of assumption may oversimplify the health care process and could thus result in an overstatement of the potential cost reduction .
summary of costs attributed to coronary heart disease in canada ( $ millions ) source : health canada ( 7 ) . rather than assuming a proportional cost reduction , it is appropriate to more closely examine the nature of specific cost categories .
for example , hospitalization expenditures related to chd include a sizeable fixed cost component the costs of operating a hospital will be incurred regardless of the number of heart attack patients treated at the hospital .
research conducted with respect to the fixed cost variable cost breakdown in hospitals found that hospital costs are approximately 84% fixed and 16% variable ( 20 ) .
accordingly , it is assumed that a reduction in chd will not result in a reduction in fixed costs of hospitalization , but will cause a proportional reduction in variable hospitalization costs . approximating the reduction in drug costs due
to chd assumes that fewer individuals with chd will require less medication to treat chd .
since 49.6% of drug costs resulting from chd are for the treatment of hypertension ( 7 ) , a condition not normally improved as a result of cholesterol reduction , these costs are not reduced in the calculations .
the remaining 50.4% of drug costs associated with chd are reduced proportionally to the reduction in the overall rate of chd .
physician care costs associated with chd are based on physician billings which in turn are derived from patient visits to doctors offices .
it is assumed that if fewer people are afflicted with chd , fewer visits to physicians for chd treatment will occur , resulting in less numerous billings and lower health care costs . as such , a 1% reduction in chd is assumed to lead to a 1% reduction in chd - related physician costs .
a large component of the miscellaneous chd - related expenditures has been categorized by health canada ( 7 ) as services provided by other health providers. for this analysis ,
the cost of chiropractors ( 0.8% ) , dental ( 26.2% ) , and vision ( 9.6% ) health professionals were excluded , as such costs are unlikely to be reduced due to a reduction in heart disease .
however , the cost of physiotherapists ( 1% ) and all other health professionals ( 4.7% ) should be reduced in proportion to the overall chd reduction .
also ignored in this analysis are costs which are largely fixed and therefore unlikely to decrease with the rate of chd : expenditures on health research ( 4.4% ) , administration ( 6.5% ) , and public health ( 20.2% ) . by contrast , costs that will likely be reduced by a reduction in chd include ambulance ( 4.2% ) , home care ( 6.2% ) , and all other health expenditures ( 7.2% ) .
the final result is that a 1% decrease in the incidence of chd is expected to result in a 0.23% reduction in miscellaneous chd - related costs .
mortality , short - term disability , and long - term disability costs were taken to have a directly proportional chd reduction to reduction in cost relationship .
since the incidence of chd will decline , it was deemed reasonable to assume the number of people who will die or become disabled as a result of chd will decline proportionately . as a consequence ,
the loss of human capital that would ordinarily be incurred as a result of chd - related death and disability , would not take place ; this reduction results in an economic saving .
table 3 summarizes the proportion of cost reductions that could be achieved by a reduction in the incidence of chd .
summary of coronary heart disease cost reduction corresponding to a 1% decrease in incidence of coronary heart disease a number of assumptions are required to approximate the potential reduction in the cost of chd from introducing phytosterol - fortified foods to the canadian market .
though these assumptions are based on a review of the peer - reviewed scientific literature , it could be misleading to suggest a single number as best representing the exact economic cost savings .
as such , results are reported as a sensitivity analysis ; as this approach allows the effects of varying model assumptions within the simulation to be gauged . to make the numbers as robust as possible , four scenarios were examined ; a description of the sensitivity analysis scenarios is provided below with a summary of the assumptions shown in table 4 .
the ideal scenario is one in which the most positive assumptions are used to calculate the greatest possible economic benefit of allowing consumption of plant sterol - enriched food in canada .
it is unlikely that this scenario is achievable in the short - term ; however , over the long - term this estimate provides an index of the full potential of the economic savings resulting from the consumption of plant sterol - enriched foods in canada .
the optimistic and pessimistic scenarios make more reasonable assumptions and represent a medium - to - short - term estimate of the potential economic savings possible through plant sterol consumption .
very pessimistic scenario is included to determine the impact on the cost estimations under a worst - case scenario .
this scenario could be considered appropriate if the acceptance of phytosterol fortified foods is very low and the efficacy of plant sterols in reducing cholesterol and the health benefits of cholesterol reduction are considerably below expectations .
table 5 shows the range of values calculated to be representative of the economic savings to canada from allowing plant sterols in the diet of canadians .
each of the direct and indirect cost components is represented for the ideal , optimistic , pessimistic , and very pessimistic scenarios described above . under even the most pessimistic set of assumptions , substantial cost savings would be realized to canada 's publicly funded single - payer health system . under more reasonable assumptions ,
although it is unlikely the ideal scenario would be achieved in the short term , it is evident that under a very optimistic set of assumptions , the potential savings become substantial .
reduction in coronary heart disease cost due to consumption of plant sterol enriched foods ( $ millions )
denotes non - zero amounts too small for inclusion .
the dynamic process from the point in time when plant sterol - enriched foods are introduced to the market to the point in time at which the savings predicted in the various scenarios will occur is difficult to model with certainty .
the scenarios presented in this paper represent a static picture at some point in the future .
how quickly the product is adopted will determine the speed with which that point is reached .
the ideal scenario with a high success rate is more likely to occur at a point in time that is further into the future than the optimistic or pessimistic scenarios which have lower success rates built into their models .
while a specific timeline can not be predicted with certainty , the optimistic scenario requires that canadians adopt phytosterol - fortified foods in a manner similar to that of finland , which does not seem unreasonable .
based on the introduction pattern of plant sterol - enriched foods in finland over the 1990s of 17% , it would be reasonable to conclude that the impact of introducing plant sterol fortified foods into the canadian market could be realized in a period of 510 years .
the results of the simulation indicate that substantial economic savings can result from introducing plant sterol - enriched foods into the canadian market .
the rising costs of health care exist as a growing concern globally ; especially in countries such as canada where direct costs of treating disease are borne largely by the public health care systems .
it has also been found that the cholesterol - lowering effects of plant sterols are additive to other cholesterol lowering strategies such as diet ( 21 ) and statin treatment ( 22 ) . as such
, phytosterol enhanced foods can work in concert with lifestyle changes and medication to lower the economic cost of chd . a possible direction for
future research would be to explore the cost effectiveness of reducing chd through plant sterols in food products compared to statins , which currently enjoy a cost advantage over sterols . when considering the cost savings presented in this analysis
it should be noted that the cost premiums associated with plant sterol - enriched foods have not been accounted for in this valuation .
the costs of phytosterol fortified foods will fall outside of the medical system in canada and thus represent a benefit to the publicly funded health care system .
an excessive price premium would result in a lower economic benefit of plant sterol - enriched foods .
as the market for phytosterol fortified foods develops and more is known about possible price premiums , this issue should be revisited with a cost benefit or cost - effectiveness study .
chd is a substantial component of the economic burden of illness in canada and high serum cholesterol is a major risk factor in developing chd .
functional foods such as those which contain plant sterols have the potential to demedicalize and reduce significant portion of the economic costs associated with chd in canada , which in turn would allow health care funds to be allocated more efficiently .
this research was supported by the coalition for advancement of plant sterols in canada ( capsic ) . | background increased consumption of foods containing plant sterols has the potential to reduce the incidence of coronary heart disease ( chd ) and thus reduce costs associated with treating that disease in a significant way .
this paper reports the results of an investigation of the potential monetary benefits of allowing foods enriched with plant sterols to be marketed in canada.objective the objective of this research was to estimate the annual savings that would accrue to canada 's single - payer publicly funded health care system if plant sterols were approved for use .
if foods containing plant sterols are consumed at a sufficient rate , a reduction in chd should follow .
given the significant costs associated with chd , approval of plant sterols in canada has important public policy implications.design this research employs a variation of traditional cost - of - illness analysis entailing four steps : ( 1 ) estimation of a success rate
( proportion of persons who would consume plant sterols at the necessary rate ) ; ( 2 ) presumption of blood cholesterol reduction due to plant sterol consumption ; ( 3 ) assumption of reduction in chd that follows from blood cholesterol reduction ; and ( 4 ) calculation of cost savings associated with reduced incidence of chd.results calculations were carried out for four scenarios : ideal , optimistic , pessimistic , and very pessimistic .
it was estimated that between $ 38 million ( very pessimistic scenario ) and $ 2.45 billion ( ideal scenario ) could be saved annually by canada 's health care system with plant sterol - enriched food products being made available for sale.conclusion significant expenditure reductions within canada 's publicly funded health care system could be realized with plant sterols approved for sale . reduced chd resulting from lower blood cholesterol levels would lessen the financial burden of disease in canada . | Methods
Step 1: assessment of a success rate
Step 2: presumption of a possible blood cholesterol reduction due to consumption of foods containing plant sterols
Step 3: assumption of the reduction in the incidence of CHD due to decrease in blood cholesterol levels
Step 4: calculation of the reduction in costs associated with CHD due to a reduction in the overall incidence of CHD
Results and discussion
General discussion and conclusions
Conflicts of interest and funding | the purpose of this research is to determine the potential benefits from allowing plant sterol - enriched foods to be sold into a market where they are currently not available , not to compare plant sterol - enriched foods to alternative treatments . additionally , it is not possible to estimate the cost premium that would be attached to foods enriched with plant sterols in canada as this market would need to evolve in order to establish a long - term price . a cost - of - illness analysis measures the direct and indirect costs of a specific disease in this case chd . a variation of a cost - of - illness analysis is to calculate the dollar savings associated with preventing or avoiding an illness . in order to realize health benefits from plant sterols
the proportion of the canadian population that will consume a sufficient quantity of plant sterol - enriched foods to reduce their cholesterol level is defined as the success rate . given the lack of available information ,
the best method for predicting how canadians would respond to plant sterol - enriched food products is to examine the markets for similar functional foods in canada and plant sterol - enriched foods in other countries . one of these was limited in scope to plant sterol - enriched spreads and found a reduction in ldl - cholesterol of 8.4% ( 95% ci 6.610.0% ) ( 3 ) . since
no long - term studies have directly tracked the relationship between phytosterol consumption and heart disease in humans , it was necessary to make assumptions regarding the rate at which lower cholesterol levels due to plant sterol consumption will reduce the incidence of chd . in an economic evaluation of the health benefits associated with trans - fat - free canola oil , a ratio of a 1% reduction of cholesterol corresponding to 23% reduction in the incidence of chd
this result is consistent with the findings of previous research ( 17 , 18 ) and was therefore used as the assumption regarding the reduction in the incidence of chd due to decreased blood cholesterol levels resulting from plant sterol consumption in the majority of the sensitivity analysis scenarios . table 3 summarizes the proportion of cost reductions that could be achieved by a reduction in the incidence of chd . the proportion of the canadian population that will consume a sufficient quantity of plant sterol - enriched foods to reduce their cholesterol level is defined as the success rate . given the lack of available information ,
the best method for predicting how canadians would respond to plant sterol - enriched food products is to examine the markets for similar functional foods in canada and plant sterol - enriched foods in other countries . one of these was limited in scope to plant sterol - enriched spreads and found a reduction in ldl - cholesterol of 8.4% ( 95% ci 6.610.0% ) ( 3 ) . since no long - term studies have directly tracked the relationship between phytosterol consumption and heart disease in humans , it was necessary to make assumptions regarding the rate at which lower cholesterol levels due to plant sterol consumption will reduce the incidence of chd . in an economic evaluation of the health benefits associated with trans - fat - free canola oil , a ratio of a 1% reduction of cholesterol corresponding to 23% reduction in the incidence of chd
this result is consistent with the findings of previous research ( 17 , 18 ) and was therefore used as the assumption regarding the reduction in the incidence of chd due to decreased blood cholesterol levels resulting from plant sterol consumption in the majority of the sensitivity analysis scenarios . table 3 summarizes the proportion of cost reductions that could be achieved by a reduction in the incidence of chd . the ideal scenario is one in which the most positive assumptions are used to calculate the greatest possible economic benefit of allowing consumption of plant sterol - enriched food in canada . it is unlikely that this scenario is achievable in the short - term ; however , over the long - term this estimate provides an index of the full potential of the economic savings resulting from the consumption of plant sterol - enriched foods in canada . each of the direct and indirect cost components is represented for the ideal , optimistic , pessimistic , and very pessimistic scenarios described above . under even the most pessimistic set of assumptions , substantial cost savings would be realized to canada 's publicly funded single - payer health system . reduction in coronary heart disease cost due to consumption of plant sterol enriched foods ( $ millions )
denotes non - zero amounts too small for inclusion . based on the introduction pattern of plant sterol - enriched foods in finland over the 1990s of 17% , it would be reasonable to conclude that the impact of introducing plant sterol fortified foods into the canadian market could be realized in a period of 510 years . the costs of phytosterol fortified foods will fall outside of the medical system in canada and thus represent a benefit to the publicly funded health care system . functional foods such as those which contain plant sterols have the potential to demedicalize and reduce significant portion of the economic costs associated with chd in canada , which in turn would allow health care funds to be allocated more efficiently . | [
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all mice were housed in microisolator cages under specific pathogen - free conditions at the harvard school of public health and the la jolla institute for allergy and immunology .
all animal studies were performed according to institutional and national institutes of health guidelines for animal use and care .
blood glucose levels were monitored weekly using onetouch ultra ( lifescan ) or ascensia contour glucometers ( bayer ) .
diabetes in nod mice was defined as two consecutive blood glucose values > 250 mg / dl .
il-21 receptor knockout mice were generated by homologous recombination as previously described ( 11 ) .
nod / ltj mice were purchased from the jackson laboratories , and the il-21 receptor null allele was backcrossed to the nod background for 10 generations .
the il-21 transgenic ( il-21tg ) construct was generated by cloning the full - length murine il-21 cdna into a transgenic expression vector between the 5 human insulin promoter and 3 hepatitis virus b terminator sequence ( 21 ) .
the purified plasmid was linearized using the saci and hindiii restriction sites , injected into c57bl/6 fertilized embryos and implanted into pseudopregnant females .
pancreata were harvested from il-21r and il-21r nod mice , immersed in oct compound ( tissue - tek , sakura ) and quick - frozen on dry ice .
the 6-m sections were cut at three nonoverlapping levels ( 200 m apart ) and fixed in acetone for 10 min at room temperature .
sections were incubated for 1 h at room temperature with guinea pig anti - swine insulin ( dako , 1:500 ) , biotin anti - mouse cd8 ( bd clone 536.7 , 1:50 ) , and biotin anti - mouse cd4 ( bd , clone rm4 - 5 , 1:50 ) .
next , goat anti guinea pig alkaline phosphatase ( sigma , 1:50 ) and avidin - hrp ( vector , 1:2,000 ) were incubated for 45 min at room temperature . alkaline phosphatase or horseradish peroxidase ( hrp ) activity was visualized using vector blue alkaline phosphatase iii ( blue signal ) and aec substrate (
islets were scored visually by light microscopy and categorized as no insulitis , peri - insulitis , mild infiltration ( < 25% ) , and heavy infiltration and scars .
pancreata were harvested from il-21tg and littermate controls and fixed overnight with 4% paraformaldehyde ( sigma - aldrich ) before routine paraffin embedding .
after dewaxing , 6-m sections were cut and treated with 3% h2o2 in meoh ( 20 min at room temperature ) to quench endogenous peroxidase activity .
next , slides were blocked in 1% bsa and 3% normal serum in pbs for 30 min at room temperature .
primary antibodies were incubated overnight at 4c at the following concentrations : insulin 1:100 ( # a0564 , dako ) , il-21 1:1,200 ( # af594 r&d systems ) , cd4 1:100 ( biolegend ) , b220 1:100 ( # 550286 , bd biosciences ) , f4/80 1:100 ( bd biosciences ) , cd11c 1:100 ( # 553800 , bd biosciences ) , and lgl-1 1:50 ( # 555314 , bd biosciences ) .
primary antibodies were visualized using sequential detection with hrp - conjugated secondary antibodies ( jackson immunoresearch ) , tyramide signal amplification ( perkin - elmer ) , streptavidin - hrp ( jackson immunoresearch ) , and diaminobenzidine ( sigma - aldrich ) .
specific islet infiltration was scored using an arbitrary scale ranging from 0 to 4 : 0 , no islet inflammation ; 1 , scattered cells surrounding islets ; 2 , foci of cells surrounding islets ; 3 , foci of cellular infiltrates surrounding and cells within islets ; and 4 , dense foci of cellular infiltrates surrounding and within islets .
single - cell suspensions were prepared from spleen and peripheral lymph nodes by mechanical disruption , filtration through a 70-m cell strainer ( bd biosciences ) , erythrocyte lysis using ack buffer , and two washes infacs buffer ( pbs/0.5% bsa/0.01% nan3 ) .
splenocyte transfers were performed from newly diabetic il-21rnod mice or age - matched il-21rnod mice .
the 2 10 splenocytes were transferred intravenously into 6-week - old female nod / scid mice .
fitc- , pe- , percp- , apc- , cascade blue , and cascade orange conjugated monoclonal antibodies to cd3 , cd4 , cd8 , cd21 , cd23 , b220 , igm , and igd ( all bd ) were used according to the manufacturer 's instructions .
mice were killed using co2 narcosis , and harvested tissues were snap - frozen in liquid nitrogen .
total rna was purified using trizol reagents , and 2 g was used for cdna synthesis .
real - time quantitative pcr was performed using a stratagene m3005p qpcr system using -actin as an internal reference control .
levels of autoantibodies to murine insulin ( miaa ) were determined by a radioactive assay as described ( 23 ) .
the limit of normal ( 0.010 ) was chosen based on historical data ( 23 ) .
for diabetes incidence , significance was calculated using the log - rank test or one - way anova followed by a bonferroni 's post test . for all other parameters ,
significance was calculated by student 's t test , indicated as follows in the figures : * p < 0.05 , * * p < 0.01 , and * * * p < 0.005 .
all mice were housed in microisolator cages under specific pathogen - free conditions at the harvard school of public health and the la jolla institute for allergy and immunology .
all animal studies were performed according to institutional and national institutes of health guidelines for animal use and care .
blood glucose levels were monitored weekly using onetouch ultra ( lifescan ) or ascensia contour glucometers ( bayer ) .
diabetes in nod mice was defined as two consecutive blood glucose values > 250 mg / dl .
il-21 receptor knockout mice were generated by homologous recombination as previously described ( 11 ) .
nod / ltj mice were purchased from the jackson laboratories , and the il-21 receptor null allele was backcrossed to the nod background for 10 generations .
the il-21 transgenic ( il-21tg ) construct was generated by cloning the full - length murine il-21 cdna into a transgenic expression vector between the 5 human insulin promoter and 3 hepatitis virus b terminator sequence ( 21 ) .
the purified plasmid was linearized using the saci and hindiii restriction sites , injected into c57bl/6 fertilized embryos and implanted into pseudopregnant females .
pancreata were harvested from il-21r and il-21r nod mice , immersed in oct compound ( tissue - tek , sakura ) and quick - frozen on dry ice .
the 6-m sections were cut at three nonoverlapping levels ( 200 m apart ) and fixed in acetone for 10 min at room temperature .
sections were incubated for 1 h at room temperature with guinea pig anti - swine insulin ( dako , 1:500 ) , biotin anti - mouse cd8 ( bd clone 536.7 , 1:50 ) , and biotin anti - mouse cd4 ( bd , clone rm4 - 5 , 1:50 ) .
next , goat anti guinea pig alkaline phosphatase ( sigma , 1:50 ) and avidin - hrp ( vector , 1:2,000 ) were incubated for 45 min at room temperature .
alkaline phosphatase or horseradish peroxidase ( hrp ) activity was visualized using vector blue alkaline phosphatase iii ( blue signal ) and aec substrate ( red precipitates ) .
islets were scored visually by light microscopy and categorized as no insulitis , peri - insulitis , mild infiltration ( < 25% ) , and heavy infiltration and scars .
pancreata were harvested from il-21tg and littermate controls and fixed overnight with 4% paraformaldehyde ( sigma - aldrich ) before routine paraffin embedding .
after dewaxing , 6-m sections were cut and treated with 3% h2o2 in meoh ( 20 min at room temperature ) to quench endogenous peroxidase activity .
next , slides were blocked in 1% bsa and 3% normal serum in pbs for 30 min at room temperature .
primary antibodies were incubated overnight at 4c at the following concentrations : insulin 1:100 ( # a0564 , dako ) , il-21 1:1,200 ( # af594 r&d systems ) , cd4 1:100 ( biolegend ) , b220 1:100 ( # 550286 , bd biosciences ) , f4/80 1:100 ( bd biosciences ) , cd11c 1:100 ( # 553800 , bd biosciences ) , and lgl-1 1:50 ( # 555314 , bd biosciences ) .
primary antibodies were visualized using sequential detection with hrp - conjugated secondary antibodies ( jackson immunoresearch ) , tyramide signal amplification ( perkin - elmer ) , streptavidin - hrp ( jackson immunoresearch ) , and diaminobenzidine ( sigma - aldrich ) .
specific islet infiltration was scored using an arbitrary scale ranging from 0 to 4 : 0 , no islet inflammation ; 1 , scattered cells surrounding islets ; 2 , foci of cells surrounding islets ; 3 , foci of cellular infiltrates surrounding and cells within islets ; and 4 , dense foci of cellular infiltrates surrounding and within islets .
single - cell suspensions were prepared from spleen and peripheral lymph nodes by mechanical disruption , filtration through a 70-m cell strainer ( bd biosciences ) , erythrocyte lysis using ack buffer , and two washes infacs buffer ( pbs/0.5% bsa/0.01% nan3 ) .
splenocyte transfers were performed from newly diabetic il-21rnod mice or age - matched il-21rnod mice .
the 2 10 splenocytes were transferred intravenously into 6-week - old female nod / scid mice .
fitc- , pe- , percp- , apc- , cascade blue , and cascade orange conjugated monoclonal antibodies to cd3 , cd4 , cd8 , cd21 , cd23 , b220 , igm , and igd ( all bd ) were used according to the manufacturer 's instructions .
mice were killed using co2 narcosis , and harvested tissues were snap - frozen in liquid nitrogen .
total rna was purified using trizol reagents , and 2 g was used for cdna synthesis .
real - time quantitative pcr was performed using a stratagene m3005p qpcr system using -actin as an internal reference control .
levels of autoantibodies to murine insulin ( miaa ) were determined by a radioactive assay as described ( 23 ) .
the limit of normal ( 0.010 ) was chosen based on historical data ( 23 ) .
for diabetes incidence , significance was calculated using the log - rank test or one - way anova followed by a bonferroni 's post test . for all other parameters ,
significance was calculated by student 's t test , indicated as follows in the figures : * p < 0.05 , * * p < 0.01 , and * * * p < 0.005 .
genetic and cellular studies have suggested that il-21 could be important for the pathogenesis of type 1 diabetes in the nod mouse model ( 3,4 ) . to begin , we defined the expression patterns of il-21 and il-21r mrna in the pancreas and pancreatic draining lymph nodes in pre - diabetic and diabetic nod mice .
il-21 mrna levels , essentially unaltered in pancreas draining lymph nodes , showed an upward trend in the pancreas as diabetes developed in the nod ( fig .
levels of il-21r mrna remained unchanged in both pancreas and associated draining lymph nodes as diabetes develops ( fig .
il-21r is clearly detectable on the surface of pancreatic cd4 and cd8 t - cells ( fig .
1c ) , including diabetogenic nrp - v7 tetramer cd8 t - cells ( data not shown ) , at levels comparable to splenic cd4 and cd8 t - cells from nod ( data not shown ) and other strains ( 24 ) .
these data indicate that increased pancreatic il-21 production correlates with diabetes onset in nod mice and that t - cells infiltrating the pancreas express il-21 receptor ( il-21r ) .
expression of il-21 and il-21r in pancreas and pancreatic lymph nodes of nod mice . a : il-21 mrna analyzed in pancreas and pancreatic lymph node at indicated ages by quantitative pcr ( n = 6 per group ) .
b : il-21r mrna analyzed in pancreas and pancreatic lymph node at indicated ages by quantitative pcr ( n = 6 per group ) .
c : il-21r expression on cd4 ( left panel ) and cd8 t - cells ( right panel ) from pancreas of pre - diabetic nod mice was determined by flow cytometry using the three - step staining protocol described before ( 24 ) .
specific and control staining are represented by the solid line and tinted area , respectively .
au , arbitrary units . to assess the importance of signaling through the il-21r during spontaneous type 1 diabetes development
, we generated a colony of il-21r deficient nod mice and compared disease parameters with littermate control animals .
the il-21rnod littermates developed type 1 diabetes beginning at week 11 , with a median onset at 19 weeks and > 90% penetrance of disease ( fig .
, il-21r nod animals were completely protected from type 1 diabetes development up to 60 weeks of age ( fig .
heterozygotes displayed an intermediate phenotype with delayed onset ( median onset 29 weeks ) and reduced penetrance of disease ( 50% ) .
the effect of il-21r deficiency on mononuclear cell infiltration in the pancreas was determined by immunohistochemistry on pancreatic tissue sections .
the observed infiltrate was composed predominantly of cd4 t - cells that preferentially resided in the islet zones where -cell destruction had occurred ( fig .
cd8 t - cells were found scattered throughout the islet ( fig . 2c ) .
in contrast , we observed minimal mononuclear cell infiltration in islets of il-21rnod mice up to 40 weeks of age . in keeping with the lack of insulitis , autoimmunity to islet antigens
quantitation of serum insulin autoantibodies revealed seropositivity in 10/27 il-21rnod mice ( 812 weeks old ) , in contrast to only 1/20 il-21rnod mice ( fig .
thus , loss of il-21 signaling protects nod mice from diabetes , islet inflammation , and the generation of islet autoantibodies .
a : diabetes incidence per group per week was determined from blood glucose levels of il-21r ( n = 35 ) , il-21r ( n = 17 ) , and il-21rnod ( n = 16 until week 40 , n = 8 until week 60 ) .
onset was dated on the first of two consecutive readings of blood glucose levels > 250 mg / dl .
data shown are significantly different in one - way anova analysis ( p < 0.0001 ) and bonferonni 's multiple comparison post - test ( p < 0.001 for all comparisons ) .
b : mononuclear infiltration was scored in pancreatic islets from il-21r and il-21rnod mice at the indicated ages .
c : absence of islet infiltration by cd4 and cd8 t - cells in il-21rnod mice .
frozen tissue sections ( 6 microns ) from 15-week - old female il-21r or il-21rnod mice were stained for insulin ( blue ) and cellular infiltration by cd4 or cd8 t - cells ( red ) .
d : insulin autoantibodies ( iaa ) in serum of il-21rnod and il-21rnod mice at 812 weeks of age .
( a high - quality digital representation of this figure is available in the online issue . )
we found roughly equal splenocyte numbers in il-21rnod , il-21rnod , and il-21rnod at both early ( 79 weeks ) and late pre - diabetic stages ( 1215 weeks ) ( data not shown ) .
the proportion of cd4 and cd8 t - cells within the lymphocyte population in spleen and the pancreas draining lymph node was not significantly influenced by il-21r deficiency ( fig .
moreover , the fraction of b - cells and nk cells at 1215 weeks of age was similar between all genotypes ( data not shown ) .
enumeration of pancreatic cd4 and cd8 t - cells corroborated the insulitis index scores ( fig .
2b ) as cd4 and cd8 t - cell numbers increased from early to late pre - diabetic stage in il-21rnod but were significantly reduced in il-21rnod pancreata ( fig . 3a and b , lower panels ) .
we hypothesized that an increased regulatory compartment could explain the observed diabetes resistance of il-21rnod mice . whereas no significant differences in cd4foxp3 tregs were observed in the spleen of late - stage pre - diabetic mice ( fig .
3c ) , the treg fraction in the pancreatic lymph nodes of il-21rnod mice was reduced ( 50% ) compared with controls .
this may represent a relative reduction of tregs in il-21rnod mice or an increase in il-21rnod related to disease onset ( 25 ) .
regardless , we conclude that the peripheral lymphoid compartment in il-21rnod is essentially normal , with the unexpected exception of reduced treg numbers in the pancreatic lymph nodes , which suggests that diabetes resistance is not due to an increased regulatory compartment .
il-21rnod mice have a normal peripheral lymphoid compartment but less t - cells in the pancreas .
cd4 ( a ) and cd8 ( b ) t - cells were measured in spleen , pancreas - draining lymph node , and pancreas of il-21r , il-21r , and il-21rnod mice at 79 weeks ( early pre - diabetic ) or 1215 weeks ( late pre - diabetic ) of age .
indicated percentages are fractions of cd4 or cd8 t - cells in the live lymphocyte gate .
c : flow cytometric determination of the proportion of cd4foxp3 t - cells in the lymphocyte gate of il-21r , il-21r , and il-21rnod spleens ( left panel ) and pancreatic lymph nodes ( right panel ) at 1215 weeks of age .
given the absence of obvious cellular defects , we reasoned that modulation of th effector responses from pathogenic ( th1 , th17 ) to protective ( th2 ) may account for diabetes protection in il-21rnod mice .
lymphocytes from spleens and pancreatic lymph nodes of 8- to 9-week - old il-21rnod and il-21rnod mice were restimulated in vitro with phorbol 12-myristate 13-acetate / ionomycin for 3 h for intracellular cytokine detection by flow cytometry .
we found a slight increase in the proportion of cd4 t - cells that produce il-17 or interferon ( ifn)- in the splenic and pancreatic lymph node cells of il-21rnod mice ( fig .
splenocytes from il-21rnod and il-21rnod mice were stimulated for 72 h with anti - cd3/anti - cd28 under nonpolarizing conditions .
we observed significantly increased numbers of il-17 and il-4producing cells in il-21rnod splenocytes compared with controls .
we also found a trend toward increased ifn- and il-10producing cells that failed to reach statistical significance .
thus , whereas there are increases in il-4 production , concomitant increases in il-17 and possibly ifn- make it unlikely that skewing toward protective th2 response explains the diabetes resistance in il-21rnod mice .
il-21rnod mice display altered cytokine production profiles . a : ifn- and il-17 production by cd4 t - cells upon 3 h phorbol 12-myristate 13-acetate / ionomycin stimulation of freshly isolated il-21r and il-21rnod splenocytes .
b : enzyme - linked immunosorbent spot analysis of ifn- , il-17 , il-4 , and il-10 production upon 72 h of in vitro unpolarized anti - cd3/28 stimulation of freshly isolated il-21r and il-21rnod splenocytes . to decipher
whether an il-21rnod environment was sufficient to restore the diabetogenic potential of il-21rnod lymphocytes , we performed parallel transfers of il-21rnod and il-21rnod splenocytes into lymphopenic nod / scid recipients .
as previously published , splenocytes from recently diabetic il-21rnod mice induced diabetes upon transfer to nod / scid mice starting at 3 weeks post - transfer ( fig .
5a ) . in contrast , transfer of age - matched il-21rnod splenocytes could not induce diabetes in nod / scid mice ( fig .
immunohistochemistry on pancreatic sections revealed limited islet infiltration by cd4 and cd8 t - cells in nod / scid recipients of il-21rnod splenocytes , but abundant infiltration by transferred il-21rnod splenocytes .
defective reconstitution of lymphoid space by il-21rnod lymphocytes could not explain these observations , as we found equivalent numbers of lymphoid cells in spleen or pancreatic lymph nodes of nod / scid mice receiving either il-21rnod or il-21rnod splenocytes ( fig .
these observations indicate that il-21r nod mice lack auto - aggressive splenocytes compared with their wild - type littermates and that lymphopenia - induced proliferation of il-21rnod lymphocytes does not confer them with diabetogenic properties .
a : splenocytes ( 2 10 ) from diabetic il-21rnod mice ( ) or age - matched il-21rnod mice ( ) were transferred into 6-week - old nod / scid mice .
the diabetes incidence in recipient mice is shown ( il-21rnod , n = 5 ; il-21rnod , n = 6 ) .
b : insulitis scores for cd4 and cd8 infiltration in nod / scid recipients of il-21rnod ( left ) and il-21rnod ( right ) splenocytes .
c : representative picture of cd4 ( red ) and insulin ( blue ) staining of pancreas sections from nod / scid after transfer of il-21rnod ( left ) or il-21rnod ( right ) splenocytes .
d : splenic and pancreatic lymph nodes cell numbers in nod / scid after transfer of il-21rnod ( ) or il-21rnod ( ) splenocytes .
( a high - quality digital representation of this figure is available in the online issue . )
we showed that pancreatic levels of il-21 increase during diabetes development in nod ( fig .
1a ) and that loss of il-21 signaling protects nod mice from islet infiltration and diabetes development ( fig .
we therefore hypothesized that elevated levels of il-21 would exacerbate disease pathogenesis . to test this
, we generated transgenic c57bl/6 mice in which il-21 is under the control of the human insulin promoter , resulting in pancreatic -cell specific overexpression of il-21 ( fig .
next , we measured il-21 levels by quantitative rt - pcr ( fig . 6b ) and by immunohistochemistry using a polyclonal anti - mouse il-21 antibody ( fig .
these data revealed distinct overexpression of il-21 mrna and protein in pancreatic islets of il-21 transgenic animals .
expression of il-21 in pancreatic islets through an insulin promoter transgenic construct leads to increased cellularity of lymphoid organs and altered expression of b - cell maturation markers in il-21tg mice .
a : murine il-21 was cloned into the transgenic construct under the control of the human insulin promoter and hbs ( hepatitis b virus ) terminator sequences .
b : il-21 mrna levels were measured in pancreatic tissue from il-21tg and littermate controls by quantitative rt - pcr .
c : immunohistochemical staining for paraffin embedded pancreatic tissue with an il-21specific polyclonal antibody ( left panel tg , right panel tg , a representative islet is marked with an arrow ) .
total cell numbers in spleen ( d ) and pancreatic draining lymph nodes ( e ) ( * p < 0.05 ) and igd vs. igm staining ( f ) of b220 cells ( top panels ) and frequencies of mature ( igm igd ) and marginal zone / transitional ( igm igd ) b - cells are shown .
cd23 vs. cd21 staining of igm cells ( bottom panels ) and mean fluorescence intensity for each marker is shown on the relevant axis ( n = 4 for all experiments shown ) .
( a high - quality digital representation of this figure is available in the online issue . ) analysis of lymphoid compartments revealed splenomegaly and lymphadenopathy in il-21tg mice .
6e ) resultant from expansion of both the t - cell ( cd3 ) and b - cell ( b220 ) compartments ( data not shown ) .
most b - cells in our il21tg mice displayed a mature phenotype , while expressing reduced levels of cd21 and cd23 ( igd , igm , cd21 , cd23 ) ( fig .
other studies have shown that il-21 can downregulate surface cd21 and cd23 on b - cells , and expansion of igdigmcd21cd23 b - cells was also observed in other il-21tg mouse lines driven by ubiquitous promoters ( 10 ) .
thus , these data suggest that bioactive il-21 , expressed specifically by pancreatic -cells , is released systemically from the endocrine pancreas to mediate effects in peripheral lymphoid compartments . to determine whether il-21 overexpression resulted in diabetes onset , we monitored blood glucose levels of il-21tg mice and wild - type littermate controls weekly from the time of weaning until 40 weeks of age . as expected , wild - type animals ( c57bl/6 ) remained normoglycemic over the duration of the study ( fig .
in contrast , il-21tg animals started developing diabetes from 8 weeks of age ( fig .
7a ) , with a median onset at 22 weeks and 80% penetrance in both sexes of the experimental population . to prove specificity of the il-21 effect and to exclude a transgenesis artifact , we crossed il-21tgb6 to il-21rb6 mice .
these data show that -cell specific overexpression can induce type 1 diabetes on the diabetes - resistant c57bl/6 background .
il-21tg mice develop spontaneous type 1 diabetes on the c57bl/6 background , the severity of which is increased in the context of nod alleles . a : il-21tg mice and wild - type littermate controls .
diabetic incidence was calculated per group as two consecutive readings over 250 mg / dl and displayed as percentage of diabetic mice per group ( tg , n = 24 ; tg , n = 25 ) .
diabetes incidence was scored as survival curve data and is different by log - rank test ( p < 0.0001 ) .
b : weekly measurements of blood glucose were performed on il-21tg mice after crossing to the il-21r knockout ( c57bl/6 ) , and diabetes incidence was calculated ( all mice tg , il-21r , n = 7 ;
paraffin - embedded pancreatic tissue from wild - type and il-21tg mice was sectioned , stained with an anti - insulin polyclonal antibody , and counterstained with hematoxylin .
d : individual islets were scored for the presence of peri- and intra - islet infiltration and displayed as percentage of infiltrated islets per group ( < 10 weeks , tg , n = 3 ; tg , n = 8 ; 1016 weeks , tg , n = 12 ; tg , n = 14 ; > 16 weeks , tg , n = 7 ; tg , n = 12 ) .
e : il-21tg mice on the c57bl/6 background were crossed with the nod strain to generate a b6nod f1 mixed background .
weekly measurements of blood glucose were performed on il-21tg littermate controls , and diabetes incidence was calculated ( tg , n = 19 ; tg , n = 16 ) .
diabetes incidence was scored as survival curve data and is different by logrank test ( p < 0.0001 ) .
paraffin - embedded pancreatic tissue from this cross was quantitated for total number of insulin - positive islets per visual field ( f ) and individual islets scored for the presence of peri- and intra - islet infiltration and displayed as percentage of infiltrated islets per group ( g ) ( 2 weeks , tg , n = 11 ; tg , n = 12 ; 3 weeks , tg , n = 15 ; tg , n = 7 ) .
pancreatic sections stained for insulin and insulin - positive islets were quantified per visual field .
islet inflammation was scored based on the presence of peri- and intra - islet cellular infiltration .
the number of islets in il-21tg mice was significantly reduced at all time points compared with controls ( fig .
in addition , 50% of the islets in pre - diabetic ( 10 weeks of age ) il-21tg mice were infiltrated ( fig .
the severity of islet inflammation increased with age and , at 16 weeks of age , 90% of the islets revealed some level of cellular infiltration .
7a ) . to test whether the presence of diabetes susceptibility alleles from nod influences disease onset , we crossed il-21tg mice ( c57bl/6 ) to nod mice . we found that il-21tg f1 ( b6xnod ) mice developed diabetes as early as 3 weeks of age , with a median onset at 4 weeks and 100% penetrance of disease at 6 weeks ( fig .
this represents a striking acceleration of diabetes onset in il-21tg on the mixed b6nod versus the b6 background ( median onset 4 vs. 22 weeks , respectively ; fig .
we determined -cell mass and pancreatic islet infiltration by immunohistochemistry and found a reduced amount of islets and distinct infiltration of the remaining islets between 2 and 3 weeks of age in the il-21tg b6nod f1 compared with wild - type b6nod littermates ( fig . 7f and g ) .
our data show that one dose of nod - derived alleles exacerbates diabetes in il-21tg c57bl/6 mice .
next , we used immunohistochemistry to determine which cell subsets infiltrate the islets in il-21tg c57bl/6 mice .
we analyzed the presence of b - cells ( b220 ) , cd4 cells ( cd4 ) , nk cells ( lgl-1 ) , macrophages ( f4/80 ) , and dendritic cells ( cd11c ) in islet infiltrates from pre - diabetic ( 810 weeks ; fig .
we observed more severe infiltration by all cell types in il-21tg versus littermate controls , and in diabetic versus pre - diabetic mice ( fig .
the infiltrates in the pre - diabetic il-21tg cohort predominantly contained f4/80 macrophages but also cd4 and dendritic cells .
in diabetic il-21tg mice , the infiltrates consisted mostly of macrophages and contained focal accumulation of cd4 cells , b - cells , dendritic cells , and nk cells .
we reasoned that the distinct pattern of infiltration could result in part from the production of cytokines and chemokines .
therefore , we performed quantitative rt - pcr on pancreatic tissue from il-21tg and littermate controls , which revealed significantly increased production of ifn- , il-17a , and il-17f in the pancreas of il-21tg mice ( fig .
in addition , we found a significant increase in monocyte chemoattractant protein ( mcp)-1 , mcp-2 , and ifn - inducible protein ( ip)-10 production ( fig .
thus , pancreatic -cell specific overexpression of il-21 results in the production of inflammatory cytokines and chemokines and predominant infiltration of the islets by macrophages and cd4 t - cells .
spontaneous type 1 diabetes in il-21tg mice is associated with a macrophage - rich islet infiltrate and the expression of inflammatory cytokines and chemokines in the pancreas .
a : paraffin - embedded pancreatic tissue was sectioned and stained with anti - b220 , cd4 , lgl-1 , f4/80 , and cd11c antibodies .
representative sections from 8-week - old ( top panels ) and 24-week - old mice ( bottom panels ) are shown ( a representative islet is marked with an arrow ) .
all sections were scored by visual inspection on an arbitary scale of 04 ( no infiltrate to dense , severe infiltrate ) and plotted in b ( < 12 weeks , tg , n = 3 ; tg , n = 5 ; > 12 weeks , tg , n = 4 ; tg , n = 7 ) .
total rna was extracted from pancreatic tissue harvested from a cohort of il-21tg mice and wild - type controls .
levels of transcripts for a panel of cytokines ( c ) and chemokines ( d ) was measured using quantitative rt - pcr ( 2430 weeks , tg , n = 3 ; tg , n = 5 , * p < 0.05 ) .
( a high - quality digital representation of this figure is available in the online issue . )
in this study , we demonstrate a causal relationship between il-21 production and type 1 diabetes .
third , -cell specific overexpression of il-21 precipitates diabetes in diabetes - resistant c57bl/6 mice .
initially , islet antigens are released during postnatal remodeling of the pancreas and captured by migratory and resident antigen - presenting cell that prime anti - islet t - cells in the pancreatic draining lymph nodes ( 20,2628 ) . at an early stage ,
macrophages are recruited to the islets ( 29 ) and are a necessary cellular component of diabetes pathogenesis ( 30 ) .
next , chemotactic factors , produced by -cells in response to inflammatory stimuli , attract mononuclear cells to the islets , particularly cd4 and cd8 effector t - cells .
the transition from nondestructive islet inflammation to a -cell destructive state is a key event that precipitates type 1 diabetes ( 18,31 ) .
because il-21r is broadly expressed throughout the immune system and other nonhematopoeitic lineages ( 6,3235 ) , there are multiple time points and sites of action for il-21 during the pathogenesis of type 1 diabetes .
we show here that il-21 levels are increased in the pancreas as nod mice develop diabetes and that cd4 and cd8 t - cells infiltrating the pancreas can respond to local il-21 as they express il-21r .
our data are consistent with recent studies by the labs of leonard and sarvetnick ( 36,37 ) showing that il-21rnod mice are protected from insulitis and type 1 diabetes .
( 37 ) , we find unaltered numbers of t - cells , b - cells , and nk cells in il-21rnod lymphoid organs ( fig . 3 and data not shown ) .
in contrast to ours and other studies , datta and sarvetnick detected higher lymphocyte numbers in il-21rnod mice , interpreting this as a normalization of il-21induced , type 1 diabetes promoting lymphopenia ( 4,36 ) .
we see no differences in the expansion of il-21rnod and il-21rnod splenocytes when transferred to lymphopenic nod / scid recipients ( fig .
we therefore think it unlikely that il-21 catalyzes diabetes development by regulating homeostatic proliferation . given that t - cell numbers and responses are intact in il-21r mice ( 8,11 )
, we hypothesized that altered cytokine production may partially account for the protection from type 1 diabetes .
our analyses show that production of various effector cytokines was not impaired in il-21rnod mice ( fig .
one of these cytokines , il-17 , has recently been shown to modulate some aspects of the type 1 diabetes pathogenesis in nod ( 38 ) , and recent studies identify il-21 as an amplifying factor for th17 responses ( 39,40 ) .
( 37 ) identify defective polarization toward the th17 lineage in il-21rnod lymphocytes and reason that defective il-17 production may explain diabetes resistance in il-21rnod mice .
we ( data not shown ) and others ( 39 ) find similarly defective in vitro th17 polarization using il-21r t - cells .
moreover , our data show increased il-17 production in the pancreas of -cell specific il-21 overexpressing mice ( fig .
however , we show increased numbers of il-17producing cells in il-21rnod mice when cells are restimulated directly ex vivo , which is likely to be more reflective of the in vivo context .
thus , we conclude that reduced il-17 production in il-21rnod mice is unlikely to be the mechanism for the protection from type 1 diabetes . the reduced frequency of insulin autoantibodies and insulitis in il-21rnod mice ( fig .
2b and d ) shows that the anti - islet response is impaired at multiple levels .
reduced autoantibody levels may reflect impairments in cd4 t helper cell function or antibody production in the absence of il-21r ( 9,41,42 ) .
anti - islet il-21r t - cells may be primed ineffectively or possess inherent defects in migration to islet tissue . since il-21rnod mice have normal or fewer numbers of regulatory t - cells ( fig .
3c ) , and the function of these cells is not altered ( 37 ) , it is unlikely that increased regulatory function explains the reductions in autoimmunity .
transfer experiments using diabetogenic t - cell receptor transgenic t - cells may elucidate the existence of defects in priming or trafficking and are the subject of ongoing studies .
although il-21r deficiency protects diabetes - prone nod mice from type 1 diabetes , -cell specific overexpression of il-21 causes severe diabetes in otherwise diabetes - resistant c57bl/6 mice .
few other models of cytokine overexpression in pancreatic islets cause diabetes of similar severity ( 43,44 ) .
the phenotype of il-21tg mice most closely resembles that of ifn-tg mice in terms of onset and severity of disease . the ifn-tg model is both t - cell and macrophage dependent ( 21,45,46 )
similar to the ifn-tg model , the high numbers of macrophages in the islet infiltrates of il-21tg mice suggest an important role for macrophages , since macrophage - derived inflammatory cytokines and reactive oxygen species are directly toxic to -cells ( 47 ) . in vitro stimulation of macrophages with il-21
enhances their t - cell priming capacity ( 48 ) ; thus , phagocytosis of damaged islets and presentation of -cell antigens to cd4 t - cells may cause enhanced killing of islets in the il-21tg model . in il-21tg pancreatic tissue , we showed upregulation of chemokine transcripts such as mcp-1 , mcp-2 , and ip-10 , which recruit inflammatory cells such as macrophages and cxcr3 t - cells ( fig .
previous studies identified -cells as an important source of chemokines during diabetes pathogenesis , but our experiments have failed to identify il-21r expression on -cells ( supplementary fig .
, we believe that il-21dependent inflammatory chemokine production could be an important element of the pathogenesis of type 1 diabetes and partially explain the protection afforded by il-21r deficiency in the nod model ( 33,50,51 ) . in conclusion , we demonstrate a critical role of il-21 for diabetes pathogenesis in animal models .
the disease - promoting activities of il-21 involve the recruitment of cd4 cells and macrophages to inflamed islets and may reflect events that occur in response to il-21 production by infiltrating cells .
in addition , the partial protection from diabetes in il-21rnod mice shows the sensitivity of the diabetogenic response to alterations in il-21 signaling and , by inference , il-21 levels .
thus , both of our experimental models suggest that the use of il-21 blocking agents , antibodies , or il-21r - fc fusion proteins has potential therapeutic value for the prevention or treatment of human type 1 diabetes . | objectiveinterleukin ( il)-21 is a type 1 cytokine that has been implicated in the pathogenesis of type 1 diabetes via the unique biology of the nonobese diabetic ( nod ) mouse strain . the aim of this study was to investigate a causal role for il-21 in type 1 diabetes.research design and methodswe generated il-21r
deficient nod mice and c57bl/6 mice expressing il-21 in pancreatic -cells , allowing the determination of the role of insufficient and excessive il-21 signaling in type 1 diabetes.resultsdeficiency in il-21r expression renders nod mice resistant to insulitis , production of insulin autoantibodies , and onset of type 1 diabetes .
the lymphoid compartment in il-21r/ nod is normal and does not contain an increased regulatory t - cell fraction or diminished effector cytokine responses .
however , we observed a clear defect in autoreactive effector t - cells in il-21r/ nod by transfer experiments .
conversely , overexpression of il-21 in pancreatic -cells induced inflammatory cytokine and chemokines , including il-17a , il17f , ifn- , monocyte chemoattractant protein ( mcp)-1 , mcp-2 , and interferon - inducible protein-10 in the pancreas .
the ensuing leukocytic infiltration in the islets resulted in destruction of -cells and spontaneous type 1 diabetes in the normally diabetes - resistant c57bl/6 and nod c57bl/6
backgrounds.conclusionsthis work provides demonstration of the essential prodiabetogenic activities of il-21 on diverse genetic backgrounds ( nod and c57bl/6 ) and indicates that il-21 blockade could be a promising strategy for interventions in human type 1 diabetes . | RESEARCH DESIGN AND METHODS
Mice.
Tissue isolation, fixation, and immunohistochemical staining.
Lymphocyte preparations, transfers, and flow cytometry.
Tissue collection and quantitative RT-PCR.
Insulin autoantibody assay.
Statistical analysis.
RESULTS
DISCUSSION
Supplementary Material | genetic and cellular studies have suggested that il-21 could be important for the pathogenesis of type 1 diabetes in the nod mouse model ( 3,4 ) . to begin , we defined the expression patterns of il-21 and il-21r mrna in the pancreas and pancreatic draining lymph nodes in pre - diabetic and diabetic nod mice . these data indicate that increased pancreatic il-21 production correlates with diabetes onset in nod mice and that t - cells infiltrating the pancreas express il-21 receptor ( il-21r ) . c : il-21r expression on cd4 ( left panel ) and cd8 t - cells ( right panel ) from pancreas of pre - diabetic nod mice was determined by flow cytometry using the three - step staining protocol described before ( 24 ) . to assess the importance of signaling through the il-21r during spontaneous type 1 diabetes development
, we generated a colony of il-21r deficient nod mice and compared disease parameters with littermate control animals . regardless , we conclude that the peripheral lymphoid compartment in il-21rnod is essentially normal , with the unexpected exception of reduced treg numbers in the pancreatic lymph nodes , which suggests that diabetes resistance is not due to an increased regulatory compartment . il-21rnod mice have a normal peripheral lymphoid compartment but less t - cells in the pancreas . c : flow cytometric determination of the proportion of cd4foxp3 t - cells in the lymphocyte gate of il-21r , il-21r , and il-21rnod spleens ( left panel ) and pancreatic lymph nodes ( right panel ) at 1215 weeks of age . to test this
, we generated transgenic c57bl/6 mice in which il-21 is under the control of the human insulin promoter , resulting in pancreatic -cell specific overexpression of il-21 ( fig . these data show that -cell specific overexpression can induce type 1 diabetes on the diabetes - resistant c57bl/6 background . therefore , we performed quantitative rt - pcr on pancreatic tissue from il-21tg and littermate controls , which revealed significantly increased production of ifn- , il-17a , and il-17f in the pancreas of il-21tg mice ( fig . in addition , we found a significant increase in monocyte chemoattractant protein ( mcp)-1 , mcp-2 , and ifn - inducible protein ( ip)-10 production ( fig . thus , pancreatic -cell specific overexpression of il-21 results in the production of inflammatory cytokines and chemokines and predominant infiltration of the islets by macrophages and cd4 t - cells . spontaneous type 1 diabetes in il-21tg mice is associated with a macrophage - rich islet infiltrate and the expression of inflammatory cytokines and chemokines in the pancreas . in this study , we demonstrate a causal relationship between il-21 production and type 1 diabetes . third , -cell specific overexpression of il-21 precipitates diabetes in diabetes - resistant c57bl/6 mice . initially , islet antigens are released during postnatal remodeling of the pancreas and captured by migratory and resident antigen - presenting cell that prime anti - islet t - cells in the pancreatic draining lymph nodes ( 20,2628 ) . because il-21r is broadly expressed throughout the immune system and other nonhematopoeitic lineages ( 6,3235 ) , there are multiple time points and sites of action for il-21 during the pathogenesis of type 1 diabetes . we show here that il-21 levels are increased in the pancreas as nod mice develop diabetes and that cd4 and cd8 t - cells infiltrating the pancreas can respond to local il-21 as they express il-21r . ( 37 ) , we find unaltered numbers of t - cells , b - cells , and nk cells in il-21rnod lymphoid organs ( fig . given that t - cell numbers and responses are intact in il-21r mice ( 8,11 )
, we hypothesized that altered cytokine production may partially account for the protection from type 1 diabetes . although il-21r deficiency protects diabetes - prone nod mice from type 1 diabetes , -cell specific overexpression of il-21 causes severe diabetes in otherwise diabetes - resistant c57bl/6 mice . in il-21tg pancreatic tissue , we showed upregulation of chemokine transcripts such as mcp-1 , mcp-2 , and ip-10 , which recruit inflammatory cells such as macrophages and cxcr3 t - cells ( fig . , we believe that il-21dependent inflammatory chemokine production could be an important element of the pathogenesis of type 1 diabetes and partially explain the protection afforded by il-21r deficiency in the nod model ( 33,50,51 ) . | [
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atherosclerosis is a multifactorial , inflammatory disease characterized by the presence of t - cells , macrophages , and dendritic cells ( dcs ) in the arterial intima in response to injury caused by classical atherosclerosis risk factors .
adhesion molecules and chemotactic factors mediate migration of cells of the immune system into the arterial wall .
leukocyte adhesion and rolling depend on p- and e - selectins and intercellular and vascular cell adhesion molecules 1 ( icam-1 and vcam-1 ) , while monocyte migration is mediated by monocyte chemotactic protein ( mcp-1 ) and its receptor ccr2 . under physiological conditions ,
the stress protein heat shock protein 60 ( hsp60 ) functions as a chaperone known to assist protein folding and intracellular transport .
hsp60 is typically expressed in the mitochondria and is highly conserved with 97% homology between bacterial species and more than 50% homology between microbial and human molecules .
hsp60 is a strongly immunogenic microbial antigen able to induce protective humoral and cellular immune responses .
exposed to classical atherosclerotic risk factors , ecs simultaneously express hsp60 and adhesion molecules on their surface .
surface expressions have been shown in stressed human umbilical venous endothelial cells ( huvecs ) and aortic endothelial cells ( ecs ) [ 79 ] and also in bacterially infected huvecs .
hsp60-expressing ecs can then become target cells for pre - existing cellular and humoral immunity against this ubiquitous protein leading to the formation of inflammatory lesions in the intima . in humans ,
t - cells isolated from surgically removed advanced ( late ) atherosclerotic lesions ( ll = plaques ) which recognize hsp60 have a restricted t - cell receptor ( tcr)/ repertoire and predominantly produce th1 cytokines .
it has also been shown that increased levels of anti - hsp60 autoantibodies correlate with the severity of ll .
furthermore , studies performed in animals following immunization with hsp60 have shown excessive plaque formation under normo and hypercholesterolemic conditions [ 1517 ] .
these and other data demonstrate the importance of immunity against hsp60 in the development and progression of atherosclerosis .
however , the role of hsp60 in the initiation of atherosclerosis is still unclear . to our knowledge
, the present study is the first to investigate phenotypic and functional characteristic of t - cells isolated from early , clinically still inapparent human atherosclerotic lesions ( el ) and to compare these with data on ll by evaluating their reactivity to human hsp60 ( hhsp60 ) and hhsp60-derived peptides .
the study was approved by the ethics committee of the innsbruck medical university ( # un2670 ) .
iliac arteries with clinically and macroscopically inapparent early atherosclerotic lesions at the known predilections sites at arterial branching points as well as blood samples from 7 healthy transplant organ donors ( 5 male , 2 female , age : 54 5.4 years ) who died due to cerebrovascular incidents ( brain death ) were obtained .
( el 2 , 4 , and 5 ) , pathohistological data were not available . in these instances ,
macroscopically healthy , very small arterial tissue specimens were taken from the known predilection sites at the branching point of the iliac artery and immediately processed in toto for cell cultures .
the higher serum values of c - reactive protein ( crp ) in el donors as compared to ll patients was due to the fact that these brain death individuals were kept under intensive care conditions known to be associated with an
. pathological classification of atherosclerotic lesions ( score i vi , see table 1 ) was performed according to the criteria recommended by the american heart association .
austria is part of eurotransplant and complete laboratory values are often not available from donors the organs of whom are transported to the local transplantation unit .
internal carotid arteries with advanced atherosclerotic lesions were obtained from 8 patients undergoing surgery either for symptomatic or high - grade asymptomatic stenosis with written informed consent ( 6 male , 2 female , age : 74.2 3.1 years ) . for more detailed clinical information see table 1 .
pbmcs were obtained by density gradient centrifugation ( ge healthcare bio - sciences ab ; sweden ) from freshly drawn venous blood .
after washing , pbmc were resuspended in rpmi 1640 ( lonza , belgium ) supplemented with 1% penicillin / streptomycin , 1% glutamine , and 10% fetal calf serum ( fcs ; serum supreme , lonza , belgium ) .
plasma samples were obtained from all 7 donors with el and 8 patients with ll and two healthy age- and sex - matched control groups , one young control group ( control y ; age : 33 4 years ; n = 26 ) and one elderly control group ( control e ; age : 74 4 years ; n = 14 ) .
the presence of cd1a , cd3 , cd4 , cd8 , cd40 , cd68 , tryptase mast cells , cd106 ( vcam-1 ) , and cd62e ( e - selectin ) molecules were analyzed by immunohistochemistry in formalin - fixed , paraffin - embedded arterial specimens from 4 el and 8 ll donors ( see supplementary table 1 ) .
briefly , eight-m - thick tissue slides were treated with xylene ( 3 for 3 min ) , 100% ethanol ( 2 for 2 min ) , 96% ethanol ( 2 for 3 min ) , 70% ethanol ( 1 for 3 min ) , tbs ( ph = 7.2 ) ( 1 for 3 min ) , and finally washed in water .
antigen retrieval was achieved either with 1 epitope retrieval solution , ph = 9 , ( eubio , austria ) or 0.5 m edta for 10 min at 750 w in a microwave oven .
slides were cooled , rinsed with tbs , and blocked either for 6 min with ultra - v block solution ( thermo scientific , usa ) or overnight with blocking buffer ( 5% in tbs ) , ( roche diagnostics , germany ) containing 10% fcs . following the blocking step ,
slides were washed ( 3 for 5 min ) with tbs at room temperature ( rt ) .
antibodies and corresponding controls ( supplementary figure 1 ) were added to the tissue sections as shown in supplementary table 1 and incubated for 1 h at rt . following 3 further washing steps and addition of primary antibody enhancer ( thermo scientific , usa ) the sections were first incubated for 10 min at rt with the primary antibody enhancer and then treated with ap - polymer ( thermo scientific , usa ) for 15 min at rt . for visualization , either fast red ( sigma aldrich , usa ) or 3,3 diaminobenzidine ( dab ) ( sigma aldrich , usa ) was used when ap - anti - mouse or hrp - anti - mouse antibody was applied , respectively . staining was monitored microscopically and color development was stopped with water .
nuclear staining was performed by incubation of the slides with mayer 's hemalum solution ( 1:10 ) for 10 min . to identify hsp60-expressing cells in atherosclerotic lesions , triple
the sections were treated with either monoclonal anti - human antibodies for ecs ( vwf ) , cd40 , cd106 or anti - hla - dr , dp , dq and corresponding controls ( supplementary figure 1 ) incubated for 1 h at rt .
slides were washed ( 3 for 3 min ) in tbs to remove excess antibody . for visualization , goat alexa 488 labeled anti - mouse igg ( invitrogen , usa ) was applied to the sections and incubated for 1 h in the dark at rt .
hsp60 expression , in combination with the markers indicated above , was determined first by incubation with polyclonal rabbit anti - human hsp60 igg ( santa cruz biotechnology , usa ) for 1 h and then by incubation with goat alexa 568-labeled anti - rabbit igg ( invitrogen , usa ) for 1 h at rt in the dark .
nuclear staining was achieved by incubation with hoechst stain ( 1:40,000 ) for 10 min .
microscopic analyses and documentation were carried out at room temperature with a nikon eclipse e800 microscope connected to digital sight ds-5 m with a ds - l1 control unit ( nikon , japan ) .
el and ll from 7 to 8 donors respectively , were cut into small pieces and cultured in rpmi 1640 medium ( lonza , belgium ) supplemented with 1% penicillin / streptomycin , 1% glutamine ( lonza , belgium ) , 7% human serum ( type ab ; lonza , belgium ) and recombinant human interleukin ( rhil)-2 ( 20 u / ml ) for 7 days in a petri dish ( sarstedt , usa ) .
t - cells migrating out of the tissue fragments were harvested and cell suspensions were filtered through a cell strainer ( 100 m ; becton dickinson , usa ) . the t - cell suspension was then transferred to a 24-well plate ( sarstedt , usa ) .
these t - cells were then expanded by stimulation with 1 g / ml each of anti - human anti - cd3/anti - cd28 antibodies ( ebioscience , austria ) in the presence of autologous irradiated pbmcs and rhil-2 ( 10 u / ml ) for 7 days . after expansion
, intralesional hhsp60-specific t - cell blasts were stimulated with recombinant human hsp60 ( rhhsp60 ) protein ( 10 g / ml ) together with autologous feeder cells in the presence of rhil-2 ( 10 u / ml ) once a week for two weeks .
cell culture medium and rhil-2 ( 10 u / ml ) were replaced every third day .
finally , t - cells were harvested , counted , and used for phenotypic and functional studies .
phenotypic characterization of intralesional hhsp60-specific t - cells from 7 el and 8 ll donors was performed using monoclonal antibodies specific for cd3 , cd4 , cd8 , cd45ra , cd45ro , cd28 , and cd25 ( all purchased from bd pharmingen , usa ) labeled with fluorescein isothiocyanate ( fitc ) , phycoerythrin ( pe ) , peridinin chlorophyll protein ( percp ) , or allophycocyanin ( apc ) for 30 min at 4 c .
subsequently , cells were washed twice with facs buffer ( 0.5% bsa in pbs ) and measured by four - color flow cytometry .
mouse igg isotype controls and unstained cells were used to define the level of non - specific background signal ( supplementary figure 2 ) .
all analyses were performed using a facscalibur flow cytometer utilizing cellquest software ( bd pharmingen , usa ) .
interferon ( ifn) , il-4 , il-10 ( bd pharmingen , usa ) , il-17 ( peprotech , usa ) , and transforming growth factor ( tgf) ( r&d systems , usa ) production was determined in intralesional hhsp60-specific t - cells from 7 el and 8 ll donors by intracellular staining .
t - cells were stimulated with 30 ng / ml phorbol myristate acetate ( pma ) and 5 g / ml ionomycin / ml in the presence of 10 g of brefeldin a / ml for 4 h at 37 c ( sigma aldrich , usa ) .
after washing with pbs , stimulated t - cells were resuspended in facs buffer and incubated with monoclonal antibodies for cd4-fitc and cd8-percp for 30 min at 4 c . following two washing steps
, cells were resuspended in cytofix - cytoperm ( bd pharmingen , usa ) according to the specifications of the manufacturer .
monoclonal antibodies against ifn , il-4 , il-10 , il-17 , and tgf were added to the stimulated cells resuspended in perm - wash buffer ( bd pharmingen , usa ) for 30 min at 4 c .
mouse igg isotype controls and unstained cells were used to define the level of non - specific background signal ( supplementary figure 2 ) .
all analyses were performed in a facscalibur flow cytometer utilizing cellquest software . to span the 573-long amino acid sequence of the hhsp60 protein , 113 15-mer
synthetic peptides overlapping 10 amino acids were produced ( peptides and elephants , germany ) .
twelve peptide pools containing equal amounts of 10 overlapping peptides were used in each pool ( supplementary table 2 ) .
t - cells ( 1 10 ) were co - cultured with 5 10 autologous irradiated ( 30gy ) pbmcs and stimulated with either 10 g / ml rhhsp60 or peptide pools in which each peptide component was present at 10 g / ml , in a 96-well plate ( sarstedt , usa ) .
anti - human anti - cd3/anti - cd28 antibodies ( 1 g / ml each ) and pha ( 5 g / ml ) ( sigma aldrich , usa ) were used as positive controls .
after 3 days of incubation , cells were pulsed for the last 10 h with 1 ci of h - thymidine ( icn biomedicals inc .
the response to a given peptide pool was considered positive when the stimulation index was higher than two . to identify hsp60 peptide - specific responses ,
1 10 intralesional atherosclerotic t - cells from 7 el and 8 ll donors were co - cultured with 5 10 autologous irradiated pbmcs and stimulated with 10 individual peptides ( 10 g / ml ) contained in the corresponding positive responder pool . all 7 el and 8 ll were used individually in these experiments . as indicated before , monoclonal anti - human anti - cd3/anti - cd28 antibodies and pha were used as positive controls .
a non - related peptide ( pepc , aaaaeeeee , 10 g / ml ; bachem , germany ) was used as a negative peptide control .
after 3 days of incubation , cells were pulsed with 1 ci of h - thymidine .
the response to a given peptide was considered positive when the stimulation index was higher than two .
the concentration of shsp60 was assessed in plasma samples from persons with 7 early and 8 late lesions and healthy ( young : control y , n = 26 and elderly : control e , n = 14 ) controls by a sandwich elisa as described previously .
the levels of anti - hhsp60 autoantibodies present in plasma / serum from donors with 7 el and 8 ll and healthy ( young : control y , n = 26 and elderly : control e , n = 14 ) controls were determined by elisa as described .
hsp60 was expressed by ecs at arterial branching sites stressed by turbulent blood flow conditions ( fig .
1a ) ; simultaneous expression of adhesion molecules , such as vcam-1 ( cd106 ) ( fig .
hsp60 expression was also observed in intralesional cells co - expressing cd40 ( fig .
1e ) , an inflammatory marker and co - stimulatory molecule , which plays an important role in atherosclerosis .
surface expression of adhesion molecules provides the prerequisites for adhesion and subsequent transmigration of circulating t - cells ( cd3 ) ( fig .
1j ) into the intima . both , cd4 ( fig . 1k ) and cd8 ( fig .
1l ) t - cells were identified demonstrating that a specific immunologic - inflammatory process was taking place intima . in ll ( plaques ) , cd3 ( fig . 1 m ) , cd4 ( fig . 1n ) , cd8 ( fig .
cd3 t - cells ( cd4 > cd8 ) prevailed over cd68 macrophages in el , the opposite was true in ll .
t - cells isolated from el were mostly cd4 t - cells with high expression of the memory cell marker cd45ro and almost complete down - regulation of cd45ra ( fig .
a subset of these cells co - expressed cd25 and showed a modest accumulation of cells lacking the expression of the co - stimulatory molecule cd28 .
thus , the dominant phenotype of t - cells from el is the cd4cd45rocd25cd28 effector memory type ( fig .
although t - cells isolated from ll also showed an effector memory phenotype ( fig .
2b ) , there was an increase in the number of cd8 t - cells at this stage of the disease .
both cd4 and cd8 t - cells produced higher levels of ifn than il-10 , il-17 , and tgf ( fig .
interestingly , in el , cd4 t - cells significantly contributed to the increased levels of ifn , il-4 ( p < 0.01 ) , and il-17 as compared to cd8 t - cells ( p < 0.05 ) whereas , there was a significant increase ( p < 0.05 ) in the percentage of ifn-producing cd8 t - cells isolated from ll ( fig .
furthermore , a th1-driven cytokine production by ll - derived cells is confirmed by the reduction of il-4-producing cd4 t - cells at late stages of the disease ( fig .
autoreactive t - cells with high proliferative capacity against the whole hhsp60 protein ( s.i .
stimulation with specific regions of the protein represented in the different peptide pools resulted in proliferation of these t - cells ( fig . 4 , supplementary figure 3 ) .
peptide pools iii ( positive / total donors : 2/7 ) , v ( 2/7 ) , vi ( 3/7 ) , vii ( 3/7 ) , viii ( 2/7 ) , ix ( 4/7 ) , x ( 2/7 ) , and xii ( 5/7 ) ( fig . 4 , table 2 ) induced proliferative responses by t - cells from el from multiple donors suggesting the presence of atherogenic hsp60 peptides involved in the initiation of the disease .
t - cells from ll also showed proliferation in response to stimulation with whole rhhsp60 ( s.i .
= 2.3 0.1 ) ( fig . 5 , supplementary figure 4 ) which
, interestingly , is restricted to a particular region of the protein ( amino acids 401510 ) represented by pools ix and x ( fig . 5 , supplementary figure 4 ) .
these findings are in agreement with earlier results demonstrating that t - cells from ll display a highly monoclonal and oligoclonal tcr/ repertoire .
autoreactive intralesional atherosclerotic t - cells with a positive proliferative response to hhsp60 peptide pools were then screened against each of the individual overlapping peptides forming the respective pool ( supplementary table 2 ) .
t - cells from el recognized a total of 24 hhsp60 peptides ( supplementary figure 4 , supplementary table 3 ) eight of which induced proliferation of t - cells ( table 3 ) , suggesting that these peptides may be involved in the initiation of the disease .
peptide 111 ( 551565 ) was recognized by 71.4% of t - cells from el while peptide 54 ( 266280 ) , 57 ( 281295 ) , 66 ( 326340 ) , and 84 ( 416430 ) were recognized by 42.9% of t - cells .
peptides 50 ( 246260 ) , 85 ( 421435 ) , and 88 ( 436450 ) were recognized by 28.6% of t - cells from el ( table 3 ) .
in contrast , of the 19 peptides recognized by t - cells from ll , peptides 85 ( 421435 ) and 95 ( 471485 ) induced proliferative response in 75% of t - cells , while overlapping peptides 84 ( 416430 ) and 96 ( 476490 ) were recognized by 50% of t - cells ( table 3 , supplementary figure 5 , supplementary table 4 ) .
no reactivity was observed against a non - related control peptide ( pepc ) . taken together ,
these results demonstrate a highly specific reactivity of t - cells from el to hhsp60 peptides .
additionally , the cellular immune response to the hsp60 epitopes in ll was increasingly restricted .
levels of circulating shsp60 present in plasma of ll donors ( 310 57.8 ) was almost double of that found in plasma of el donors or in the control groups ( fig .
6a ) , ( el : 78.6 25.2 ; control y : 167 24 ; control e : 131 35.7 ) .
the levels of anti - hsp60 autoantibodies ( expressed as titer ) in the four study groups were determined .
again , significantly higher titers of autoantibodies were detected in ll ( 1070 213.6 ) as compared to the el ( 134.5 28.2 ) donors and the control groups ( control y : 364.8 73 ; control e : 416 112.8 ) ( fig .
these results confirm earlier findings from our group that patients with ll display increased amounts of circulating shsp60 and anti - hsp60 autoantibodies .
however , no difference in these humoral atherosclerosis biomarkers emerged in persons with el compared to healthy controls .
hsp60 have been shown to play an important role in the generation and progression of atherogenesis .
specific t - cells and antibodies generated against hsp60 following recurrent microbial infections carry the danger of a cross - reaction between pro and eukaryotic ( microbial and human ) hsp60 .
biochemically altered autologous hsp60 can also lead to the formation of bona fide autoreactive hsp60-specific t - cells and autoantibodies . in humans , peripheral hsp60-reactive t - cells from young clinically healthy persons
correlate with increased intima - media thickness ( imt ) at different vascular territories . however
, anti - hhsp60 reactivity of peripheral t - cells from elderly persons did not correlate with an increased imt , suggesting that hsp60-specific cellular immunity plays an important role at very early stages of the disease , while anti - hhsp60 antibodies seem to have an accelerating and perpetuating effect .
furthermore , t - cells isolated from ll show reactivity to bacterial and human hsp60 , have a restricted t - cell repertoire , and recognize hsp60-derived peptides .
despite the obviously important role of hsp60 in the progression of atherosclerosis , the immune response of t - cells from human el , clinically still inapparent atherosclerotic lesions has not been analyzed so far . in the present study , hsp60 expression on ecs from el and simultaneous up - regulation of adhesion molecules , such as vcam-1 and e - selectin ( fig .
this allows for interaction of hsp60-specific t - cells with stressed ecs under arterial blood flow conditions .
intralesional activated cd40/hla - dr mononuclear cells also displayed hsp60 expression , suggesting that hsp60 may be implicated in the activation and maturation of dcs as observed in hodgkin 's disease .
in fact , hsp60 may act as a danger signal for pre - existing adaptive immunity . cd4 and
cd8 t - cells as well as macrophages and dcs are present within the intima providing the appropriate microenvironment for an initial antigen - driven t - cell - mediated inflammatory process .
t - cells from el are predominantly cd4 with a memory effector phenotype characterized by the expression of cd25 . here
we demonstrate , albeit on morphological evidence , that hsp60-specific cd4cd25cd45ro t - cells interact with ecs that express hsp60 and adhesion molecules on their surface after being exposed to various risk factors at sites with predilection for later development of atherosclerotic lesions . if such risk factors persist , the inflammatory process progresses within the intima where it is continuously stimulated by intralesional hsp60-expressing cells .
t - cells from early lesions produced high amounts of the pro - inflammatory cytokine ifn while the th2 cytokine il-4 was detected only at low levels ( fig .
it has been demonstrated in several studies that atherosclerosis can be attenuated via th1 cytokine inhibition [ 3234 ] and/or with stimulation of th2 cytokine production .
the accumulation of ifn-producing cd8 t - cells in atherosclerotic plaques may contribute to a general increase in the serum level of the ifn , as reflected by high plasma levels of neopterin in patients with complex carotid plaques , acute coronary syndrome ( acs ) [ 3840 ] , and chronic cmv infections .
this study provides functional proof for the presence of autoreactive hhsp60-specific t - cells in early stages of the disease .
proliferation of t - cells from in response to hhsp60 , and their recognition of hhsp60-derived peptide pools ( fig . 4 , table 2 )
it is of interest to compare our observations on t - cell lines from both el and ll with those of benagiano et al . on t - cell clones from ll only .
furthermore , while we assessed t - cell reactivity exclusively to hhsp60-derived t - cell peptides , benagiano et al . also investigated cross - reactivity against chlamydia pneumoniae hsp60 ( chsp60 ) .
with respect to ll , we confirmed the data of benagiano et al . since all four epitopes of hhsp60 ( table 3 ) were also identified by these authors , two specific for hhsp60 ( 471485 and 476490 ) and two cross - reactive between hhsp60 and chsp60 ( 416430 and 421435 ) .
more importantly , el , not yet been studied by other authors so far , contain t - cells that show interesting parallels to the findings of benagiano et al . as well to our own data on ll t - cells .
seven of the eight el - associated hhsp60 epitopes described by us ( table 3 ) were also identified by these authors studying ll t - cells for reactivity to either hhsp60 and/or chsp60 . in the present study ,
two of the eight el hhsp60 epitopes were also recognized by ll t - cells ( 416430 and 421435 ) .
one el hsp60 epitope ( 551565 ) recognized by t - cells derived from more than 70% of el appeared neither in the investigation of ll by benagiano et al . nor in our present study . except for the latter epitope ,
this congruence is a strong indication that these hhsp60 epitopes recognized already by el t - cells play a pathogenic role throughout atherogenesis and may represent interesting early targets for prevention and therapy .
however , taking into account the fact that such an elaborate investigation so far could , of course , only be performed in a limited number of subjects with inapparent lesions , the issue of private vs common , i.e. shared potentially atherogenic , hhsp60 epitopes has to be considered in the context of personalized medicine . stressed cells up - regulate mitochondrial production of hsp60 that are then translocated into the cytosol , transported to the cell surface , and released into the microenvironment by mechanisms that are still not well understood .
ll donors had significantly higher serum levels of shsp60 than donors with el or healthy controls ( fig .
however , no difference in serum levels of shsp60 was found between el donors and healthy controls .
these findings corroborate our previous notion that levels of circulating shsp60 may be an indicator of cardiovascular diseases in general and advanced degree of atherosclerosis in particular .
in fact , increased levels of circulating shsp60 may reflect the inflammatory processes occurring in the arterial wall as well as the release from necrotic cells within the plaque .
extracellular hsp60 can interact with endothelial surface receptors such as cd14 , cd40 and toll - like receptors ( tlrs ) , and activate antigen - presenting cells .
finally , we determined the levels of anti - hhsp60 autoantibodies in the plasma of individuals with early and late lesions , as well as two age- and sex - matched healthy control groups .
the results revealed a significant increase in the levels of anti - hhsp60 autoantibodies in patients with ll in comparison to donors with el and healthy controls ( fig .
interestingly , the group with early lesions did not show a significant difference in the anti - hhsp60 autoantibody titers compared with age - matched controls . taken together , both low levels of shsp60 and low titers of anti - hhsp60 autoantibodies in el donors suggest that the humoral immune response is not involved in the initiation of the disease , but rather in its progression to advanced stages .
this is comparable to what have earlier been described in a large population - based prospective study involving an elderly cohort , which showed a good correlation between serum antibodies to mhsp65 and atherosclerosis , while only a weak or no such correlation emerged in two younger groups .
furthermore , high levels of antibodies against mhsp65 that cross - react with the hhsp60 are not only a predictor of morbidity but also of mortality from cardiovascular disease .
these parameters have been shown by us and other laboratories to be biomarkers for atherosclerosis independent of other classical disease biomarkers .
if anti - hsp60 antibodies are injected into mice directly they do of course act as endothelial stressors entailing intimal arterial infiltration mediated by hsp60-reactive t - cells . in conclusion
, this is the first report determining the reactivity of t - cells isolated from human early clinically still inapparent atherosclerotic lesions reacting to potentially atherogenic peptides from hhsp60 .
furthermore , these t - cells produced abundant levels of the pro - inflammatory cytokine ifn and induced activation and differentiation of macrophages , contributing to the thickening of the intima at the known arterial predisposed sites .
t - cells initiate the inflammatory processes in the intima , and anti - hhsp60 autoantibodies accelerate and perpetuate the disease . the existence of atherogenic private and common , i.e. shared hhsp60 peptides makes these epitopes attractive diagnostic and therapeutic targets .
this work is supported by the austrian science fund ( fwf ; p19881-b05 ) to gw .
the authors declare that they have no competing interests as defined by the journal of autoimmunity , or other interests that might be perceived to influence the results and discussion in this manuscript . | atherosclerosis is a multifactorial chronic inflammatory disease characterized by the presence of t - cells , macrophages , and dendritic cells in the arterial intima .
classical risk factors lead to over - expression of stress proteins , especially heat shock protein 60 ( hsp60 ) .
hsp60 on the surface of arterial endothelial cells ( ecs ) then becomes a target for pre - existing adaptive anti - hsp60 immunity resulting in infiltration of the intima by mononuclear cells . in the present study , t - cells derived from early ,
clinically still inapparent human atherosclerotic lesions were analyzed phenotypically and for their reactivity against hsp60 and hsp60-derived peptides .
hsp60 was detected in ecs and cd40- and hla class ii - positive cells within the intima .
effector memory cd4 + t - cells producing high amounts of interferon- and low levels of interleukin-4 were the dominant subpopulation .
t - cells derived from late lesions displayed a more restricted t - cell receptor repertoire to hsp60-derived peptides than those isolated from early lesions . increased levels of soluble hsp60 and circulating anti - human hsp60 autoantibodies
were found in donors with late but not early lesions .
this is the first functional study of t - cells derived from early human atherosclerotic lesions that supports the previously proposed concept that hsp60-reactive t - cells initiate atherosclerosis by recognition of atherogenic hsp60 epitopes . | Introduction
Material and methods
Generation of T-cell lines from early atherosclerotic lesions
Results
Discussion
Funding sources
Disclosures | atherosclerosis is a multifactorial , inflammatory disease characterized by the presence of t - cells , macrophages , and dendritic cells ( dcs ) in the arterial intima in response to injury caused by classical atherosclerosis risk factors . under physiological conditions ,
the stress protein heat shock protein 60 ( hsp60 ) functions as a chaperone known to assist protein folding and intracellular transport . in humans ,
t - cells isolated from surgically removed advanced ( late ) atherosclerotic lesions ( ll = plaques ) which recognize hsp60 have a restricted t - cell receptor ( tcr)/ repertoire and predominantly produce th1 cytokines . to our knowledge
, the present study is the first to investigate phenotypic and functional characteristic of t - cells isolated from early , clinically still inapparent human atherosclerotic lesions ( el ) and to compare these with data on ll by evaluating their reactivity to human hsp60 ( hhsp60 ) and hhsp60-derived peptides . these t - cells were then expanded by stimulation with 1 g / ml each of anti - human anti - cd3/anti - cd28 antibodies ( ebioscience , austria ) in the presence of autologous irradiated pbmcs and rhil-2 ( 10 u / ml ) for 7 days . after expansion
, intralesional hhsp60-specific t - cell blasts were stimulated with recombinant human hsp60 ( rhhsp60 ) protein ( 10 g / ml ) together with autologous feeder cells in the presence of rhil-2 ( 10 u / ml ) once a week for two weeks . thus , the dominant phenotype of t - cells from el is the cd4cd45rocd25cd28 effector memory type ( fig . interestingly , in el , cd4 t - cells significantly contributed to the increased levels of ifn , il-4 ( p < 0.01 ) , and il-17 as compared to cd8 t - cells ( p < 0.05 ) whereas , there was a significant increase ( p < 0.05 ) in the percentage of ifn-producing cd8 t - cells isolated from ll ( fig . 4 , table 2 ) induced proliferative responses by t - cells from el from multiple donors suggesting the presence of atherogenic hsp60 peptides involved in the initiation of the disease . t - cells from el recognized a total of 24 hhsp60 peptides ( supplementary figure 4 , supplementary table 3 ) eight of which induced proliferation of t - cells ( table 3 ) , suggesting that these peptides may be involved in the initiation of the disease . in contrast , of the 19 peptides recognized by t - cells from ll , peptides 85 ( 421435 ) and 95 ( 471485 ) induced proliferative response in 75% of t - cells , while overlapping peptides 84 ( 416430 ) and 96 ( 476490 ) were recognized by 50% of t - cells ( table 3 , supplementary figure 5 , supplementary table 4 ) . furthermore , t - cells isolated from ll show reactivity to bacterial and human hsp60 , have a restricted t - cell repertoire , and recognize hsp60-derived peptides . despite the obviously important role of hsp60 in the progression of atherosclerosis , the immune response of t - cells from human el , clinically still inapparent atherosclerotic lesions has not been analyzed so far . t - cells from early lesions produced high amounts of the pro - inflammatory cytokine ifn while the th2 cytokine il-4 was detected only at low levels ( fig . the accumulation of ifn-producing cd8 t - cells in atherosclerotic plaques may contribute to a general increase in the serum level of the ifn , as reflected by high plasma levels of neopterin in patients with complex carotid plaques , acute coronary syndrome ( acs ) [ 3840 ] , and chronic cmv infections . in the present study ,
two of the eight el hhsp60 epitopes were also recognized by ll t - cells ( 416430 and 421435 ) . taken together , both low levels of shsp60 and low titers of anti - hhsp60 autoantibodies in el donors suggest that the humoral immune response is not involved in the initiation of the disease , but rather in its progression to advanced stages . in conclusion
, this is the first report determining the reactivity of t - cells isolated from human early clinically still inapparent atherosclerotic lesions reacting to potentially atherogenic peptides from hhsp60 . furthermore , these t - cells produced abundant levels of the pro - inflammatory cytokine ifn and induced activation and differentiation of macrophages , contributing to the thickening of the intima at the known arterial predisposed sites . t - cells initiate the inflammatory processes in the intima , and anti - hhsp60 autoantibodies accelerate and perpetuate the disease . | [
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esophageal cancer ( ec ) is ranked as the eighth common cancer worldwide , with an estimated 456,000 new cases in 2012 ( 3.2% of the total ) , and the sixth cause of cancer - related death with an estimated 400,000 deaths ( 4.9% of the total ) ( 1 ) .
more than 90% of the malignancy are esophageal squamous cell carcinomas ( escc ) especially in high risk area of central asian esophageal cancer belt , which extended from northern iran through the central asian republics to north - central china ( 2 ) .
a wide incidence had been shown for ec by nearly 16-folds . in that context , while southern , eastern africa and eastern asia have the highest rate ; western , middle africa and central america are among the lowest ( 1 ) .
escc is the second most common cancer in iran with incidence rates of 17.6 and 14.4 per 10 in males and females , respectively .
these rates are higher than world standards , but has decreased dramatically compared with the data of 30 years ago ( 3 ) .
this decrease can be attributed to better economic status , improved personal health , nutrition habits , and change in high - risk behavior ( 4 ) . in northeastern iran ,
there are high incidence areas , including golestan and khorasan provinces with an age - standardized incidence rate ( asr ) of 43.4 and 36.3 per 100,000 for men and women , respectively ( 4 ) .
escc prognosis is very poor and the patients are diagnosed in advanced stages of the disease .
although surgery and other therapeutic intervene - tions such as chemo- and radiotherapy help to suppress tumor growth and development , most of patients show tumor metastasis through the body ( due to aggressiveness of ec ) leading to increased rate of mortality and decreased rate of patients 5 year survival ( 5 ) . compared to other common human malignancies , little
alterations of microsatellite dna are one of the major factors which may induce immortal and neoplastic transformation of normal cell ( 6 ) .
microsatellites are repeated dna sequences widely scattered within the genome , exhibiting length polymorphisms and variations among individuals ( 7 , 8) . due to widespread application and clinical relationship of microsatellite with the malignant phenotypes
, their variation seems clinically impor - tant ( 9 ) . any change in length of microsatellite sequences , as a result of base deletion or insertion ,
msi is an indicator of deficient mismatch repair ( mmr ) system , which is a multi - protein complex responsible for correction of errors arising during dna replication and cell division ( 10 ) . as well as msi ,
this process includes either mutation of one allele , followed by the deletion of the second allele , which called loss of heterozygosity ( loh ) , or homozygous deletion of both alleles ( 10 ) .
analysis of microsatellite using pcr admits elucidation of cancer specific dna alterations such as loh and msi ( 11 ) . a wide range of msi frequency ( from 2 to 67% ) was reported in escc .
it has been shown that msi status of long arm of chromosome 17 is associated with escc invasion ( 12 ) .
furthermore , loh in long arm of chromosome 5 containing tumor suppressor genes can affect escc development ( 13 ) .
interestingly , significant correlations were reported between loh of mmr system genes and general loh , as well as msi status in escc patients ( 14 , 15 ) .
therefore , in this study we aimed to analyze a panel of 6 microsatellite markers , based on involved chromosomal locations in escc tumorigenesis developing a molecular approach for escc detection and elucidating possible indicators for patient s prognosis .
fifty tumor and margin normal formalin fixed paraffin embedded ( ffpe ) tissues with histologically confirmed as escc were collected from imam reza hospital , mashhad , iran .
first , all samples were new escc cases without any history of any other malignancy .
second , all recruited samples did not receive any chemo- and radiotherapy treatment before surgery . and finally hematoxylin and eosin ( h&e ) staining were performed to assure a tumor cell content of more than 70% .
the ethic committee of mashhad university of medical sciences ( mums ) approved the study ( no .
dna was extracted from tumor and related tumor free tissues using proteinase k digestion method .
about five to ten sections ( 5 m ) of ffpes were deparaffinized by adding 1ml of xylene ( merck , germany ) and washed two times by 500 l ethanol 96% ( merck , germany ) .
digestion buffer contains 50 mm tris ph 8.5 , 1 mm edta and 0.5% tween 20 ( merck , germany ) .
20 mg / ml proteinase k ( fermentas , lithuania ) was added to digestion buffer followed by overnight incubation at 37 c . for any 10 mg tissue , 100 l digestion buffer and 200 g proteinase k were used .
the concentration of extracted dna was measured using a uv spectrophotometer ( uv 1101 , biotech photometer ) .
the selected markers , their chromosome locations and primer sequences are shown in table 1 ( 16 - 18 ) .
pcr reaction mixture was consisted of 1x cinnagen pcr buffer , 500 nm of each pcr primer , 1.5 mmol / l mgcl2 , 200 mol / l dntps and 1u of taq dna polymerase ( cinnagen , tehran , iran ) .
200 - 300 ng of dna was used in a reaction volume of 25 l .
thermal profile of pcr was as follows : 5 min at 95 c followed by 40 cycles of 50sec at 95 c , 1 min at annealing temperature ( 49 c -64 c ; depending on the primer sequences ; table 1 ) , and 1 min at 72 c followed by 30 min at 72 c as final extension , with maximum heating and cooling settings in techne thermal cycler ( techgene , techne , uk ) . for selecting the appropriate amount of pcr product to load on denaturing gel
, 4 microliters of pcr product were electrophoresed through the 2% agarose gel and stained with ethidium bromide .
pcr products were diluted in formamide loading buffer containing 95% formamide , 0.05% bromophenol blue , 0.05% xylene cyanol and 20 mm edta according to the intensity of bands on agarose bands , in 10 to 20 folds ; denatured at 95 c for 10 min and chilled on ice for at least 10 min .
the volume of 8 l of diluted pcr products were electrophoresed on 6% denaturing polyacrylamide gel containing 7 m urea , at constant 60 w , 1200 v , 50 c , in tbe 0.5x ( 44.5 mmol / l tris - base ; 1 mmol / l edta ; 44.5 mmol / l boric acid ) for 1 - 2 hr using high voltage power supply ( ec600 - 90 , usa ) , and vertical electrophoresis system ( veu-7703 , iran ) .
characteristics of microsatellite markers and the related sequence of used primer sets a tumor was classified as having undergone loh at a particular locus only if the predominant band(s ) of one allele showed a decreased intensity in the tumor dna relative to corresponding normal dna .
microsatellite instability was scored when there was appearance of new bands in the tumor as compared to the normal dna ( 19 - 21 ) .
it was considered low - level ( msi - l ) when 1 of 6 markers was altered and high ( msi - h ) when equal or more than 2 markers were changed .
if no difference was revealed in electrophoretic banding patterns of tumor dna in comparison with related margin normal tissues , tumor dna was considered as microsatellite - stable ( mss ) ( figure 1 ) .
pathological data and patient demographics were obtained from the patients medical history to determine any correlation with their microsatellite alterations .
examples of loh , msi and mss in paired tumor ( t ) and normal ( n ) tissues on 6% denaturing polyacrylamide gel .
arrows show lost or reduced alleles of the pcr products for loh and appearance new bands at tumor cells for msi .
[ loh : loss of heterozygosity ; msi : microsatellite instability ; mss : microsatellite stability ] the data were analyzed by spss statistical software version 11.5 .
the chi - square , kruskal valis , and spearman tests were used to evaluate the frequency of microsatellite alterations and relationship between alterations of given markers with clinical / pathological parameters . a p - value equal or less than 0.05 was considered statistically significant .
fifty tumor and margin normal formalin fixed paraffin embedded ( ffpe ) tissues with histologically confirmed as escc were collected from imam reza hospital , mashhad , iran .
first , all samples were new escc cases without any history of any other malignancy .
second , all recruited samples did not receive any chemo- and radiotherapy treatment before surgery . and finally hematoxylin and eosin ( h&e ) staining were performed to assure a tumor cell content of more than 70% .
the ethic committee of mashhad university of medical sciences ( mums ) approved the study ( no .
dna was extracted from tumor and related tumor free tissues using proteinase k digestion method .
about five to ten sections ( 5 m ) of ffpes were deparaffinized by adding 1ml of xylene ( merck , germany ) and washed two times by 500 l ethanol 96% ( merck , germany ) .
digestion buffer contains 50 mm tris ph 8.5 , 1 mm edta and 0.5% tween 20 ( merck , germany ) .
20 mg / ml proteinase k ( fermentas , lithuania ) was added to digestion buffer followed by overnight incubation at 37 c . for any 10 mg tissue , 100 l digestion buffer and 200 g proteinase k were used .
the concentration of extracted dna was measured using a uv spectrophotometer ( uv 1101 , biotech photometer ) .
the selected markers , their chromosome locations and primer sequences are shown in table 1 ( 16 - 18 ) .
pcr reaction mixture was consisted of 1x cinnagen pcr buffer , 500 nm of each pcr primer , 1.5 mmol / l mgcl2 , 200 mol / l dntps and 1u of taq dna polymerase ( cinnagen , tehran , iran ) .
200 - 300 ng of dna was used in a reaction volume of 25 l .
thermal profile of pcr was as follows : 5 min at 95 c followed by 40 cycles of 50sec at 95 c , 1 min at annealing temperature ( 49 c -64 c ; depending on the primer sequences ; table 1 ) , and 1 min at 72 c followed by 30 min at 72 c as final extension , with maximum heating and cooling settings in techne thermal cycler ( techgene , techne , uk ) . for selecting the appropriate amount of pcr product to load on denaturing gel
, 4 microliters of pcr product were electrophoresed through the 2% agarose gel and stained with ethidium bromide .
pcr products were diluted in formamide loading buffer containing 95% formamide , 0.05% bromophenol blue , 0.05% xylene cyanol and 20 mm edta according to the intensity of bands on agarose bands , in 10 to 20 folds ; denatured at 95 c for 10 min and chilled on ice for at least 10 min .
the volume of 8 l of diluted pcr products were electrophoresed on 6% denaturing polyacrylamide gel containing 7 m urea , at constant 60 w , 1200 v , 50 c , in tbe 0.5x ( 44.5 mmol / l tris - base ; 1 mmol / l edta ; 44.5 mmol / l boric acid ) for 1 - 2 hr using high voltage power supply ( ec600 - 90 , usa ) , and vertical electrophoresis system ( veu-7703 , iran ) .
characteristics of microsatellite markers and the related sequence of used primer sets a tumor was classified as having undergone loh at a particular locus only if the predominant band(s ) of one allele showed a decreased intensity in the tumor dna relative to corresponding normal dna .
microsatellite instability was scored when there was appearance of new bands in the tumor as compared to the normal dna ( 19 - 21 ) .
it was considered low - level ( msi - l ) when 1 of 6 markers was altered and high ( msi - h ) when equal or more than 2 markers were changed .
if no difference was revealed in electrophoretic banding patterns of tumor dna in comparison with related margin normal tissues , tumor dna was considered as microsatellite - stable ( mss ) ( figure 1 ) .
pathological data and patient demographics were obtained from the patients medical history to determine any correlation with their microsatellite alterations .
examples of loh , msi and mss in paired tumor ( t ) and normal ( n ) tissues on 6% denaturing polyacrylamide gel .
arrows show lost or reduced alleles of the pcr products for loh and appearance new bands at tumor cells for msi .
[ loh : loss of heterozygosity ; msi : microsatellite instability ; mss : microsatellite stability ] the data were analyzed by spss statistical software version 11.5 .
the chi - square , kruskal valis , and spearman tests were used to evaluate the frequency of microsatellite alterations and relationship between alterations of given markers with clinical / pathological parameters . a p - value equal or less than 0.05 was considered statistically significant .
fifty tumor and margin normal formalin fixed paraffin embedded ( ffpe ) tissues with histologically confirmed as escc were collected from imam reza hospital , mashhad , iran .
first , all samples were new escc cases without any history of any other malignancy .
second , all recruited samples did not receive any chemo- and radiotherapy treatment before surgery . and finally hematoxylin and eosin ( h&e ) staining were performed to assure a tumor cell content of more than 70% .
the ethic committee of mashhad university of medical sciences ( mums ) approved the study ( no .
dna was extracted from tumor and related tumor free tissues using proteinase k digestion method .
about five to ten sections ( 5 m ) of ffpes were deparaffinized by adding 1ml of xylene ( merck , germany ) and washed two times by 500 l ethanol 96% ( merck , germany ) .
digestion buffer contains 50 mm tris ph 8.5 , 1 mm edta and 0.5% tween 20 ( merck , germany ) .
20 mg / ml proteinase k ( fermentas , lithuania ) was added to digestion buffer followed by overnight incubation at 37 c . for any 10 mg tissue
the concentration of extracted dna was measured using a uv spectrophotometer ( uv 1101 , biotech photometer ) .
the selected markers , their chromosome locations and primer sequences are shown in table 1 ( 16 - 18 ) .
pcr reaction mixture was consisted of 1x cinnagen pcr buffer , 500 nm of each pcr primer , 1.5 mmol / l mgcl2 , 200 mol / l dntps and 1u of taq dna polymerase ( cinnagen , tehran , iran ) .
200 - 300 ng of dna was used in a reaction volume of 25 l . thermal profile of pcr
was as follows : 5 min at 95 c followed by 40 cycles of 50sec at 95 c , 1 min at annealing temperature ( 49 c -64 c ; depending on the primer sequences ; table 1 ) , and 1 min at 72 c followed by 30 min at 72 c as final extension , with maximum heating and cooling settings in techne thermal cycler ( techgene , techne , uk ) . for selecting the appropriate amount of pcr product to load on denaturing gel
, 4 microliters of pcr product were electrophoresed through the 2% agarose gel and stained with ethidium bromide .
pcr products were diluted in formamide loading buffer containing 95% formamide , 0.05% bromophenol blue , 0.05% xylene cyanol and 20 mm edta according to the intensity of bands on agarose bands , in 10 to 20 folds ; denatured at 95 c for 10 min and chilled on ice for at least 10 min .
the volume of 8 l of diluted pcr products were electrophoresed on 6% denaturing polyacrylamide gel containing 7 m urea , at constant 60 w , 1200 v , 50 c , in tbe 0.5x ( 44.5 mmol / l tris - base ; 1 mmol / l edta ; 44.5 mmol / l boric acid ) for 1 - 2 hr using high voltage power supply ( ec600 - 90 , usa ) , and vertical electrophoresis system ( veu-7703 , iran ) .
a tumor was classified as having undergone loh at a particular locus only if the predominant band(s ) of one allele showed a decreased intensity in the tumor dna relative to corresponding normal dna .
microsatellite instability was scored when there was appearance of new bands in the tumor as compared to the normal dna ( 19 - 21 ) .
it was considered low - level ( msi - l ) when 1 of 6 markers was altered and high ( msi - h ) when equal or more than 2 markers were changed .
if no difference was revealed in electrophoretic banding patterns of tumor dna in comparison with related margin normal tissues , tumor dna was considered as microsatellite - stable ( mss ) ( figure 1 ) .
pathological data and patient demographics were obtained from the patients medical history to determine any correlation with their microsatellite alterations .
examples of loh , msi and mss in paired tumor ( t ) and normal ( n ) tissues on 6% denaturing polyacrylamide gel .
arrows show lost or reduced alleles of the pcr products for loh and appearance new bands at tumor cells for msi .
[ loh : loss of heterozygosity ; msi : microsatellite instability ; mss : microsatellite stability ]
the chi - square , kruskal valis , and spearman tests were used to evaluate the frequency of microsatellite alterations and relationship between alterations of given markers with clinical / pathological parameters . a p - value equal or less than 0.05 was considered statistically significant .
fifty tumors and their corresponded normal tissue samples of patients with escc were recruited in this study .
male to female ratio was 1.04 ( 51/49 ) . mean age ( sd ) of patients were 61.9 ( 11.5 ) years .
for the samples with known pathologic data , 72% ( 18/25 ) were located at the middle of esophagus , and 24% ( 6/25 ) were located in the lower part of esophagus ; an estimate of 43.5% ( 20/46 ) of tumors were moderately differentiated ; in 81% ( 17/21 ) of tumors , the invasion progressed to adventitia ( t3 ) ; stage iii was observed in 63.1% ( 12/19 ) of cases and stage iia for 36.8% ( 7/19 ) ; regional lymph node metastasis was presented in 33.3% ( 7/21 ) of tumors ; addiction was reported for 60.9% ( 14/23 ) of patients which consisted each of cigarette , opium , water pipe , or alcohol .
clinicopathological characteristics of 50 patients ( the percentages were calculated among the available data ) loss of heterozygosity was shown in 66% ( 33/50 ) cases , and a total of 40% ( 20/50 ) of cases were microsatellite instable including 30% ( 15/50 ) low msi and 10% ( 5/50 ) high msi .
the marker clarified the most loss of heterozygosity was d13s260 26% ( 13/50 ) , and the most microsatellite instable marker was d5s2501 , 20% ( 10/50 ) .
some markers showed similar instability ; d9s171 and tp53 with 18% ( 9/50 ) , and d13s260 and d13s267 with 10% ( 5/50 ) ( table 3 ) .
the least frequent loh and msi marker were d9s171 ( 2% , 1/50 ) and d2s123 ( 8% , 4/50 ) respectively .
frequency and percentage of microsatellite alterations ( loh and msi ) in 50 escc tumor some samples had more than one loh+ and/or instable marker , therefore the total number is less than the addition of values for each marker ( loh : loss of heterozygosity , msi : microsatellite instability ) association of loh and msi with clinical , histological , and pathological parameters .
statistical analysis indicated a near significant reverse correlation between grade and loh ( p=0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly to well differentiated tumors ( p=0.002 and p=0.016 respectively ) .
higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p=0.026 , correlation coefficient= -0.485 ) .
it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci .
there was a positive correlation between addiction and msi ( p=0.026 , correlation coefficient= 0.465 ) , therefore any kind of addiction including opium , cigarette , water pipe and alcohol can be a susceptibility factor for microsatellite instability .
fifty tumors and their corresponded normal tissue samples of patients with escc were recruited in this study .
male to female ratio was 1.04 ( 51/49 ) . mean age ( sd ) of patients were 61.9 ( 11.5 ) years .
for the samples with known pathologic data , 72% ( 18/25 ) were located at the middle of esophagus , and 24% ( 6/25 ) were located in the lower part of esophagus ; an estimate of 43.5% ( 20/46 ) of tumors were moderately differentiated ; in 81% ( 17/21 ) of tumors , the invasion progressed to adventitia ( t3 ) ; stage iii was observed in 63.1% ( 12/19 ) of cases and stage iia for 36.8% ( 7/19 ) ; regional lymph node metastasis was presented in 33.3% ( 7/21 ) of tumors ; addiction was reported for 60.9% ( 14/23 ) of patients which consisted each of cigarette , opium , water pipe , or alcohol .
clinicopathological characteristics of 50 patients ( the percentages were calculated among the available data ) loss of heterozygosity was shown in 66% ( 33/50 ) cases , and a total of 40% ( 20/50 ) of cases were microsatellite instable including 30% ( 15/50 ) low msi and 10% ( 5/50 ) high msi .
the marker clarified the most loss of heterozygosity was d13s260 26% ( 13/50 ) , and the most microsatellite instable marker was d5s2501 , 20% ( 10/50 ) .
some markers showed similar instability ; d9s171 and tp53 with 18% ( 9/50 ) , and d13s260 and d13s267 with 10% ( 5/50 ) ( table 3 ) .
the least frequent loh and msi marker were d9s171 ( 2% , 1/50 ) and d2s123 ( 8% , 4/50 ) respectively .
frequency and percentage of microsatellite alterations ( loh and msi ) in 50 escc tumor some samples had more than one loh+ and/or instable marker , therefore the total number is less than the addition of values for each marker ( loh : loss of heterozygosity , msi : microsatellite instability ) association of loh and msi with clinical , histological , and pathological parameters .
statistical analysis indicated a near significant reverse correlation between grade and loh ( p=0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly to well differentiated tumors ( p=0.002 and p=0.016 respectively ) .
higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p=0.026 , correlation coefficient= -0.485 ) .
it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci .
there was a positive correlation between addiction and msi ( p=0.026 , correlation coefficient= 0.465 ) , therefore any kind of addiction including opium , cigarette , water pipe and alcohol can be a susceptibility factor for microsatellite instability .
fifty tumors and their corresponded normal tissue samples of patients with escc were recruited in this study .
male to female ratio was 1.04 ( 51/49 ) . mean age ( sd ) of patients were 61.9 ( 11.5 ) years .
for the samples with known pathologic data , 72% ( 18/25 ) were located at the middle of esophagus , and 24% ( 6/25 ) were located in the lower part of esophagus ; an estimate of 43.5% ( 20/46 ) of tumors were moderately differentiated ; in 81% ( 17/21 ) of tumors , the invasion progressed to adventitia ( t3 ) ; stage iii was observed in 63.1% ( 12/19 ) of cases and stage iia for 36.8% ( 7/19 ) ; regional lymph node metastasis was presented in 33.3% ( 7/21 ) of tumors ; addiction was reported for 60.9% ( 14/23 ) of patients which consisted each of cigarette , opium , water pipe , or alcohol .
loss of heterozygosity was shown in 66% ( 33/50 ) cases , and a total of 40% ( 20/50 ) of cases were microsatellite instable including 30% ( 15/50 ) low msi and 10% ( 5/50 ) high msi .
the marker clarified the most loss of heterozygosity was d13s260 26% ( 13/50 ) , and the most microsatellite instable marker was d5s2501 , 20% ( 10/50 ) .
some markers showed similar instability ; d9s171 and tp53 with 18% ( 9/50 ) , and d13s260 and d13s267 with 10% ( 5/50 ) ( table 3 ) .
the least frequent loh and msi marker were d9s171 ( 2% , 1/50 ) and d2s123 ( 8% , 4/50 ) respectively .
frequency and percentage of microsatellite alterations ( loh and msi ) in 50 escc tumor some samples had more than one loh+ and/or instable marker , therefore the total number is less than the addition of values for each marker ( loh : loss of heterozygosity , msi : microsatellite instability ) association of loh and msi with clinical , histological , and pathological parameters .
statistical analysis indicated a near significant reverse correlation between grade and loh ( p=0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly to well differentiated tumors ( p=0.002 and p=0.016 respectively ) .
higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p=0.026 , correlation coefficient= -0.485 ) .
it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci .
there was a positive correlation between addiction and msi ( p=0.026 , correlation coefficient= 0.465 ) , therefore any kind of addiction including opium , cigarette , water pipe and alcohol can be a susceptibility factor for microsatellite instability .
chromosomal regions which include frequent allelic loss may most likely point to main susceptibility genes which help to understand the involved molecular pathways in esophageal carcinogenesis .
these regions may be a probable potential for the development of markers for genetic susceptibility testing and screening for early identification of this type of cancer . while msi is linked to defects in the dna mismatch repair system and occurs in hereditary non - polyposis colon cancer (
22 ) and other malig - nancies ( 21 , 23 ) , our knowledge regarding the role of msi in esophageal carcinogenesis is too limited . in escc , msi has not been considered as a major event in tumorigenesis process . according to the relatively small studies ,
the frequency of msi in escc is ranged from 2 to 66.7% ( 14 , 23 - 28 ) . although criteria to define mss , msi - l and msi - h for colorectal cancer is accepted , for other human solid tumors the standard microsatellite markers , the required number of altered loci , and the degree of shift relative to normal , remained controversial . considering this fact , we reviewed previous articles about msi in escc and aimed to identify the chromosomal regions which were more informative for escc .
it is known that alterations in repeated sequences ( microsatellites ) are linked to the defects in some critical genes such as mismatch repair genes and tumor suppressor genes ( 29 ) . according to previous results ( 30 ) and frequency percentage of microsatellite alterations
, these microsatellite markers may be useful as predictive markers in detection of escc ( 17 ) . in this study , we have investigated two major types of changes including microsatellite instability and loss of heterozygosity in microsatellite sequences .
loh at the specific chromosomal regions was a strong indicator of tumor suppressor genes at the relevant segment ( e.g. p16 on chromosome 9 , p53 gene on chromosome 17p13.1 , brca2 gene on chromosome 13q , etc . ) .
the alterations of brca2 , p16 and p53 genes are common molecular events in esophageal carcinogenesis , and our results showed loh in d13s260 and tp53 markers can be common events in escc patients ( 31 ) .
a high frequency of replication error ( rer ) and loh involving 3p loci in the present study suggests that microsatellite alterations involving 3p loci may be early and frequent events and that multiple tumor suppressor genes harboring these loci may be implicated in esophageal tumorigenesis .
msi is reported as a frequent event in esophageal adenocarcinoma , but not in escc ( 24 , 32 ) . the results
suggest that msi could be an important event in the development of a subset of escc . a variation ( from 0 to 20% ) of msi frequency
this result suggests msi is not accumulated uniformly through the genome , and certain loci are more susceptible to genetic alterations .
one of the most frequent loh loci observed was d9s171 ( 9p21 ) , where p15ink4b and p16 reside .
with d9s171 , loh was previously reported to be 82% ( 14/17 ) in escc ( 32 ) .
furthermore , using the same microsatellite marker d9s171 , loh was observed in 1 out of 10 informative cases ( 14 ) .
in addition , we found frequent genetic alteration events within locus 9p21 ( 33 ) .
although loh at d9s171 was found to be associated with frequent homozygous deletion at p15 ink4b while not showing the same association at p16 ink4a , in our study this loh was only found in 18% of patients and this may confirm that such alterations are random .
however , inactivation of p16 gene by hypermethylation of its promoter is a frequent event in escc as reported by taghavi et al ( 34 ) . in a chinese study of d5s2501 and d2s123 markers , the highest level of msi
these results appear to differ from some previous studies in which very low frequency of msi ( 2/29 ) was found in escc ( 35 , 36 ) .
d13s260 marker showed the highest frequency of loh among the 6 analyzed microsatellite loci ( 26% , 13/33 ) ( figure 2 ) , which is similar to the results of previous studies ( 6 , 31 ) .
our finding regarding frequency of loh in d9s171 and d13s267 markers was different from previous investigations ( 6 , 37 ) .
this difference may in part occurred due to the more advanced stages of the resection specimens , which may have allowed them to accumulate a greater number of genetic alterations .
comparison of the frequency of microsatellite alterations in distinct markers lichun et al have not detected any micro - satellite instability in 34 patients with esophageal cancer .
this group also reported 0% msi for chromosomes 3 , 5 , 17 , 18 ( 0/40 patient ) and 5% msi for chromosome 11 ( 2/38 patients ) ( 38 ) . according to these results
, they claimed that msi does not play an important role in the development of this type of cancer .
differences in msi frequencies in the present and previous studies are possibly due to chance , however , there are alternative explanations that include the knowledge behind choosing microsatellite markers , differences in the type of samples studied ( endoscopic biopsies here vs. resection specimens before ) , microdissection alterations , potential differences in tumor stage , or simply the fact that different population behave differently in a random way .
several studies detected pcr artifacts in using paraffin - embedded tissues as the source of dna , especially when a small number of cells is analyzed ( 39 ) .
reported prevalence of msi in escc largely varies between various studies , ranging from 265% ( 24 - 27 ) .
this wide variation may reflect differences in msi criteria and in markers tested . in most studies , however , the msi - h frequency in escc was low nearly 10% , and the msi pathway was considered unlikely to be involved in carcinogenesis in escc ( 40 ) .
hayashi et al reported a msi frequency in escc of 40% ( 12 of 30 ) , but 11 of 12 tumors were categorized as msi - l leading to the conclusion that msi in esophageal tumors resulted from random replication error and not from mismatch repair defects ( 41 ) .
our study also showed that msi - h incidence of escc patients was relatively low ; 14% ( 6 of 42 ) , and it may confirm that msi pathway was not involved in escc carcinogenesis .
shimada et al found a msi frequency in 33 ecopc ( esophageal carcinoma with other primary carcinoma ) patients of only 3% ( 1 patient ) ( 42 ) .
in contrast to our microsatellite instability studies in colorectal , and endometrial cancer in iranian population ( 43 ) , there was no correlation between the age and msi status for escc patients .
this may have occurred due to the differences in the microsatellite markers used in these studies .
some other researchers suggested that there is no relation between msi status and the age of escc patients ( 26 ) .
in this study we showed significant association of loh and msi with different clinicopathological features of escc patients , suggesting that development of escc is correlated with genetic instability including loh and msi .
analysis of these abnormalities can be a useful method for cancer screening and may have potential prognostic value . | objective(s):variation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis .
our aim in this study was to illustrate microsatellite dna alteration in esophageal cancer.materials and methods : dna was extracted from formalin fixed paraffin embedded ( ffpe ) tissues from surgical and matched margin - normal samples .
microsatellite instability ( msi ) and loss of heterozygosity ( loh ) were studied in 50 cases of esophageal squamous cell carcinoma ( escc ) by amplifying six microsatellite markers : d13s260 ( 13q12.3 ) , d13s267 ( 13q12.3 ) , d9s171 ( 9p21 ) , d2s123 ( 2p ) , d5s2501 ( 5q21 ) and tp53 ( 17p13.1 ) analyzed on 6% denaturing polyacrylamide gel electrophoresis.results:statistical analysis indicated a near significant reverse correlation between grade and loh ( p= 0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly differentiated to well differentiated tumors ( p= 0.002 and p= 0.016 respectively ) .
in addition , higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p= 0.026 , correlation coefficient= -0.485 ) .
furthermore , there was a positive correlation between addiction and msi ( p= 0.026 , correlation coefficient= 0.465).conclusion : microsatellite dna alterations may be a prognostic tool for detection and the evolution of prognosis in patients with scc of esophagus .
it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci and addiction with any of opium , cigarette , water pipe or alcohol can be a susceptibility factor(s ) for msi . | Introduction
Materials and Methods
None
Study population
DNA extraction
Microsatellite amplification
Microsatellite analysis and scoring system
Statistical analysis
Results
None
Clinicopathological data
LOH and MSI analysis
Discussion
Conclusion | for the samples with known pathologic data , 72% ( 18/25 ) were located at the middle of esophagus , and 24% ( 6/25 ) were located in the lower part of esophagus ; an estimate of 43.5% ( 20/46 ) of tumors were moderately differentiated ; in 81% ( 17/21 ) of tumors , the invasion progressed to adventitia ( t3 ) ; stage iii was observed in 63.1% ( 12/19 ) of cases and stage iia for 36.8% ( 7/19 ) ; regional lymph node metastasis was presented in 33.3% ( 7/21 ) of tumors ; addiction was reported for 60.9% ( 14/23 ) of patients which consisted each of cigarette , opium , water pipe , or alcohol . statistical analysis indicated a near significant reverse correlation between grade and loh ( p=0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly to well differentiated tumors ( p=0.002 and p=0.016 respectively ) . higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p=0.026 , correlation coefficient= -0.485 ) . it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci . there was a positive correlation between addiction and msi ( p=0.026 , correlation coefficient= 0.465 ) , therefore any kind of addiction including opium , cigarette , water pipe and alcohol can be a susceptibility factor for microsatellite instability . for the samples with known pathologic data , 72% ( 18/25 ) were located at the middle of esophagus , and 24% ( 6/25 ) were located in the lower part of esophagus ; an estimate of 43.5% ( 20/46 ) of tumors were moderately differentiated ; in 81% ( 17/21 ) of tumors , the invasion progressed to adventitia ( t3 ) ; stage iii was observed in 63.1% ( 12/19 ) of cases and stage iia for 36.8% ( 7/19 ) ; regional lymph node metastasis was presented in 33.3% ( 7/21 ) of tumors ; addiction was reported for 60.9% ( 14/23 ) of patients which consisted each of cigarette , opium , water pipe , or alcohol . statistical analysis indicated a near significant reverse correlation between grade and loh ( p=0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly to well differentiated tumors ( p=0.002 and p=0.016 respectively ) . higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p=0.026 , correlation coefficient= -0.485 ) . it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci . there was a positive correlation between addiction and msi ( p=0.026 , correlation coefficient= 0.465 ) , therefore any kind of addiction including opium , cigarette , water pipe and alcohol can be a susceptibility factor for microsatellite instability . for the samples with known pathologic data , 72% ( 18/25 ) were located at the middle of esophagus , and 24% ( 6/25 ) were located in the lower part of esophagus ; an estimate of 43.5% ( 20/46 ) of tumors were moderately differentiated ; in 81% ( 17/21 ) of tumors , the invasion progressed to adventitia ( t3 ) ; stage iii was observed in 63.1% ( 12/19 ) of cases and stage iia for 36.8% ( 7/19 ) ; regional lymph node metastasis was presented in 33.3% ( 7/21 ) of tumors ; addiction was reported for 60.9% ( 14/23 ) of patients which consisted each of cigarette , opium , water pipe , or alcohol . statistical analysis indicated a near significant reverse correlation between grade and loh ( p=0.068 , correlation coefficient= -0.272 ) . specifically , increased loh in tumor dna
has a significant correlation with increased differentiation from poorly to well differentiated tumors ( p=0.002 and p=0.016 respectively ) . higher number of chromosomal loci with loh showed a reverse correlation with lymph node metastasis ( p=0.026 , correlation coefficient= -0.485 ) . it can be concluded that regional lymph node metastasis would be less likely with increased heterozygote loci . there was a positive correlation between addiction and msi ( p=0.026 , correlation coefficient= 0.465 ) , therefore any kind of addiction including opium , cigarette , water pipe and alcohol can be a susceptibility factor for microsatellite instability . | [
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the tendency and motivation to progress and achieve the ideal position have always persuaded people towards acquiring the required education . however , motivation is not the only prerequisite to get the intended situation but intelligence , ability , skill , knowledge , and so on are also the important factors .
for example , a student who is worried and doubtful about his or her self - efficiency , ability , intelligence in the class climate , and feels an anxiety state . during the time of exams , especially the most important ones ( e.g. , entrance exam and final exams ) ,
a little anxiety is natural and desirable and leads people to increase their effort and struggle ; however , if it exceeds the average level , it may reduce academic achievement .
a student who is anxious at test session feels that his or her mind is not working and has forgotten whatever learned so far .
such a person gives irrelevant , wrong , or imperfect answers to the questions and is involved in the consequences of the test with irrelevant and unwanted thoughts ( 1 ) .
some of these anxious states are related to the mentioned test and the others are irrelevant . if the level of the anxiety relates to the test content , the test s self - efficiency increases while the irrelevant anxiety with the assigned duty decreases the self - efficiency level ( 2 ) .
he defines self - efficiency as the person s judgment about his or her skills and capabilities for doing tasks in emergency situations when required .
self - efficiency affects someone s function only when he or she has the necessary skills for doing social job , and is excited enough for doing that job . on one hand
, bandura considers the feeling of self - efficiency as one of the most important effective constituents in self - regulation ( apart from considering as a goal ) ( 3 ) .
elias asserts academic self - efficiency as the student s trusting on his or her ability to succeed in difficult tasks ( 4 ) .
arbona linked high level of academic self - efficiency with variables such as adjustment , success in school , and seeking help from others in academic problems ( 5 ) .
crain also suggested that abilities are judged based on the physiological symbols and signs ( 6 ) .
kim states that a significant relationship exists between psychological variables and mental well - being of the people ( 7 ) .
social anxiety is an obvious and continuous fear of social situations . or it is a function that may lead to embarrassment and being exposed to some immediate social and functional situations resulting in the reaction of anxiety ( 8) . on one side , the mental and social climate of the students classrooms and the degree of its relationship with the academic achievement are of the main concerns of the teachers , parents , and people involved in education . the class as a small and minor community consists of different people distinguished based on their experiences , cultures , and personality traits .
these people bring their characteristics into the class and as a result , a different social and mental climate dominates the class ( 1 ) . accordingly , those who create such social and mental climate in the classroom are indeed the teacher and students whose purpose is teaching and learning . thus , for teaching and learning , it is required to let desirable and favorable climate dominate the classrooms ( 9 ) .
the present society is going forward with a rapid change and failure to adapt with its changes causes lack of compatibility in humans . in order to adapt oneself to the environment , both the environment and the self
if we consider such changes in the process of knowledge , the institute , school , and university should take these changes into account .
today , one of the issues studied widely in psychology is the role of excitements and emotions .
any intelligent being has anxiety , which is a natural state of the body . various kinds of life aspects such as individual , social , and physical ones require our continuous adjustments .
generally , it can be said that 10% of anxieties for a normal human is necessary but unfortunately this percentage is not always low ( 10 ) .
school is one of the most important formal , social , and organized structures that flourishes children s body and mind in the society and makes them committed to and responsible for oneself , family , and the society by providing a healthy environment . in this
regard , school setting creates a structure influencing a range of different emotions and in fact playing an effective role in creating different emotions ( 11 , 12 ) . during the last two decades , self - efficiency , which was focused on teachers , trainers , and parents ,
this new element as an effective predictor in learning and motivating students can have an effective and significant role .
if the students utilize the processes of self - efficiency , they can study more effectively and monitor how well they study ( 13 ) .
ability and self - efficiency have direct and intense impact on the function and self - efficiency .
they also have a direct and severe impact on self - efficiency that can modify the indirect effect of ability and the level on the function .
the image we have of our self - efficiency has a direct and intense impact on the levels of our anxiety .
for example , in the experiments conducted by zimmerman as the boys and girls were different in terms of self - efficiency , girls reported more anxiety ( 14 ) . to
what extent a person accurately estimates his or her behavior criteria , determines his or her personal self - efficiency . in the system of bandura
, the personal self - efficiency means the feelings of competence , efficiency , and capability to cope with life .
fulfilling and maintaining function s criteria increases personal self - efficiency and failure in realizing and sustaining those criteria leads to lowered personal self - efficiency ( 15 ) .
everybody would be anxiety - stricken , which is a pervasive , unpleasant , and vague agitation accompanied with the signs of the autonomic nervous system such as headache , perspiration , heartbeat , dyspnea , and mild stomachache .
an anxious person might feel restless which presents as inability to sit and stand in one place for a long time .
it seems that in this so called civilized era , the problems of human are always complicating ( 1 ) .
different researchers have noted that self - efficiency can be considered as a cognitive factor having a meditative role in the genesis of emotional problems ( anxiety , and depression ) .
it means that children and teenagers facing some negative and threatening events can manage them with their high self - efficiency that can protect them against anxiety .
in other words , feeling low self - efficiency inhibits effective opposing or confronting skills and exposes the children and teenagers to the risk of developing anxiety signs and other emotional issues ( 17 ) . according to bandura
, self - efficiency plays a pivotal role in self - regulating emotional states . in this sense ,
the received inability affects events and social conditions that influence the life of person significantly and helps to form futility and disappointment in the form of anxiety ( 17 ) .
furthermore , the students classroom mental and social climate and its degree of relationship with academic achievement is one of the important topics that has been constantly be paid attention to by teachers , managers , and educational authorities .
understanding classroom mental and social climate can provide valuable feedback for teachers ( 18 ) . because classroom mental and social climate is effective in quitting school or studying , skipping classes , sadness and depression , students resisting against teachers , lack of comradeship among students , lack of satisfaction and interest in studying and learning ( 19 ) .
many of the human behaviors are excited and controlled by the measures and functions of self - influence . among the self - influence mechanisms ,
none of them is more important and pervasive than the belief in personal self - efficiency ( 1 ) .
proposed the topic of self - efficiency , he defines self - efficiency as a person s judgment about ones skills and capabilities for doing tasks in the emergency situations that he requires .
self - efficiency affects someone s function only when he or she has the necessary skills for doing social task , and is excited enough for doing that task ( 1 ) .
the concept of believing self - efficiency was first used by bandura to clarify human s behavior and it was defined as the belief in individual abilities for organizing and performing operational units in order to achieve certain goals .
the concept of self - efficiency includes individual s effective , functional competence , and believing that successful activities will lead to certain results .
belief in self - efficiency is dynamic and changeable and the basis of individual differences when people match in terms of their knowledge .
most of the time , fear and anxiety are considered with each other , though they are distinct .
fear depends on a known stimulus while anxiety is related to an unknown and indefinite stimulus .
as for fear , the threatening stimulus is considerable and observable while in anxiety , the reason is unknown ( 1 ) .
fear means a reaction against a real danger but anxiety means reaction against a danger that does not exist ( 10 ) .
building climate at school is one of the important facilitating or preventive factors in human relationships . in this field ,
the dominant enclosed climate or an inapt one provides the reasons to be afraid of , including lack of trust , not expressing needs and problems , which lead to blocking the channels of communication and curbing talents and creativity . on the contrary , in an environment where there is a healthy climate on which people feel logical freedom , trust , possession and identity , and undoubtedly the grounds to exchange thoughts , the transfer of emotions and talents blooming are provided ( 20 ) . in the classroom
, there is a set of different factors that go hand in hand and create an especial mental climate . if we take two adjacent classes or schools or almost close to each other in one academic level , being completely similar with respect to economic , social , and cultural conditions , we will understand that the dominating climate on them is different . in this case , although the physical condition of the classroom and the level of mental and emotional motivation and states of the learners are in turn effective in forming the classroom mental climate , the role of the teacher in making such climate is more important and determining . because of this , the teacher himself should try to build a healthy mental climate in the classroom and to achieve this purpose , he should seek help from all factors involved in this issue ( 20 ) .
the presence and interaction of the students with their peers at this setting manifest their talents , problems , or their weaknesses . also , their perception will decrease or increase .
. considered students perceptions of their peers and the kind of existing relationship in the classroom as a social aspect of class environment and defined it as social climate referred to the relationships quality in the classroom ( 22 ) .
the interaction of class members with one another is mostly considered to be affected by the climate dominating the classroom . if the mental - social climate governing the classroom is filled with coherence and correlation , it may indicate the presence of discipline and assignment - orientation causing a confrontational and frictional climate that turns out to be like a competition ( 21 ) .
based on the above mentioned issues , a research about self - efficiency , social anxiety , and mental and social climate of the classroom , which simultaneously investigate the effect of the main dimensions and factors of social anxiety and the classroom social and mental climate on self - efficiency could be a very valuable achievement in order to propose a suitable strategy to increase self - efficiency and as a result academic achievement .
investigating the classroom mental and social climate , social anxiety , and the academic self - efficiency can be a great help for teachers , parents , and educational authorities .
the present research analyzes the relationships students understand their classroom dominating climate , social anxiety degree with students academic self - efficiency level , and along with that the research predicts their academic self - efficiency .
we also intend to determine which one of the social anxiety and the classroom mental and social climate variables plays a major role in predicting the academic self - efficiency .
the present research statistical population consists of all students of qaemshahr first grade high school .
the sample size was calculated according to the cochran s formula , based on which for 3600 subject population with as 0.05 and error of estimation ( d ) at 0.05 , a sample consisting of 350 people was suggested .
so , a total of 350 students were selected ( 172 girls and 178 boys ) . in order to choose the subjects ,
first of all , there are 76 high schools in qaemshahr , iran ( 52 public schools and 24 non - public schools ; 35 boys schools and 41 girls schools ) .
the number of the students in the first grade of high school was 3613 out of whom 1838 were boys and 1775 were girls .
twelve high schools were studied by the researcher ( 6 girls and 6 boys ) and in each school ; all first grade classes were chosen and evaluated . the social anxiety scale designed by la gierca was used to evaluate the variable of social anxiety , which includes 18 items and 3 sub - scales of fear of negative evaluation , social anxiety and distress - new , social anxiety , and distress - general ( 1 ) , and its validity was acceptable . to evaluate students educational self - efficiency the morgan - jinks efficiency scale ( 1999 ) was used .
this scale has 30 questions and includes 3 sub - scales of talent , effort , and context .
the creator of the scale declared the degree of inner homogeneity as 0.82 through the cronbach method .
also , the cronbach s alpha coefficients of the 3 sub - scales of talent , effort , and context were reported at 0.70 , 0.66 , and 0.78 , respectively ( 20 ) . to examine the mental - social climate of a class ,
this scale includes 4 secondary dimensions or scales of friction , dependence , regularity , and competition .
hosseinchari et al . , reported the validity and reliability of this scale as suitable ( 23 ) .
so in order to measure the questionnaires reliability before conducting the research , 20 students , including 10 boys and 10 girls were selected as the primary sample .
the cronbach coefficient for each scale is presented in table 1 . in order to analyze the collected data from the descriptive statistical indexes such as calculating the mean , standard deviation , and the like . also , to analyze correlation , and answer the research questions regression , and independent t test have been used . meanwhile , in order to investigate the probability behind the role of mediating social anxiety between two concepts of mental - social climate and academic achievement , multivariate regression method has been applied .
the social anxiety scale designed by la gierca was used to evaluate the variable of social anxiety , which includes 18 items and 3 sub - scales of fear of negative evaluation , social anxiety and distress - new , social anxiety , and distress - general ( 1 ) , and its validity was acceptable . to evaluate students educational self - efficiency the morgan - jinks efficiency scale ( 1999 )
this scale has 30 questions and includes 3 sub - scales of talent , effort , and context .
the creator of the scale declared the degree of inner homogeneity as 0.82 through the cronbach method .
also , the cronbach s alpha coefficients of the 3 sub - scales of talent , effort , and context were reported at 0.70 , 0.66 , and 0.78 , respectively ( 20 ) . to examine the mental - social climate of a class ,
this scale includes 4 secondary dimensions or scales of friction , dependence , regularity , and competition .
hosseinchari et al . , reported the validity and reliability of this scale as suitable ( 23 ) .
so in order to measure the questionnaires reliability before conducting the research , 20 students , including 10 boys and 10 girls were selected as the primary sample .
the cronbach coefficient for each scale is presented in table 1 . in order to analyze the collected data from the descriptive statistical indexes such as calculating the mean , standard deviation , and the like . also , to analyze correlation , and answer the research questions regression , and independent t test have been used . meanwhile , in order to investigate the probability behind the role of mediating social anxiety between two concepts of mental - social climate and academic achievement , multivariate regression method has been applied .
as it can be seen from table 2 , there are significant correlations between the variables of classroom mental - social climate , friction , coherence , discipline and competition , with the students self - efficiency with the correlation coefficients of -0.33 , -0.042 , -0.31 , 0.05 , and -0.07 , respectively , which indicates negative relationships . in this regard , there are significant negative relationships between variables of social anxiety, fear of negative evaluation, avoiding new situations and then
general situations with academic self - efficiency with values of -0.21 , -0.033 , -0.29 , and -0.0 , respectively , which means that whenever social anxiety increases , students self - efficiency decreases . as it can be seen in table 3
, there is a significant relationship between the variables of classroom mental - social climate , friction , coherence , discipline and competition with social anxiety and the correlation coefficients are 0.33 , 0.37 , 0.06 , 0.02 , and 0.24 , respectively .
now we try to answer this question : which of the social anxiety and the classroom mental - social climate variables play a more important role in predicting self - efficiency dimensions and generally self - efficiency ? to this effect , the regression analysis was used . in this analysis
, all dimensions of social anxiety and the classroom mental - social climate were included in the regression model and based on the standardized regression coefficient , table 4 shows that which variable plays a more important role in predicting self - efficiency and its dimensions .
so as you can see , the variables of fear of negative evaluation and
friction are respectively the first and second priority and avoiding general situations variable is the last priority . in order to study the difference between the means of the research , the main variables , and the dimensions of these variables between boys and girls , we used the independent t test .
as it can be seen in table 5 , the mean discipline score is 3.02 in boys and 3.94 in girls where there is a significant difference ( p < 0.05 ) .
the mean fear of negative evaluation score is 11.06 in boys and 15.20 in girls where there is significant difference ( p < 0.05 ) .
endeavor score is 20.41 in boys and 22.20 in girls , which has a significant difference ( p < 0.05 ) .
and the mean self - efficiency score is 50.45 in boys and 52.37 in girls where there is significant difference ( p < 0.05 ) .
values are presented as mean sd . values less than 0.05 are considered as significant .
the present study shows that there is a significant negative relationship between the general classroom mental - social climate and the students self - efficiency . in other words , increasing the classroom mental - social climate decreases the degree of students self - efficiency . after studying
the association of self - efficiency and its dimensions with social anxiety , the results suggest that there is a significant negative relationship between self - efficiency and social anxiety in general sense . with regard to the relationship between social anxiety and the classroom mental - social climate ,
the results indicate that there is a significant positive relationship between social anxiety and the classroom mental - social climate .
finally , regression analysis results demonstrated that the dimensions of fear of negative evaluation, friction, and avoiding new situations have the biggest share and avoiding general situations and competition have the smallest share in predicting the students self - efficiency .
the results suggest that there is a significant difference among the acquired means in dimensions of
discipline, fear of negative evaluation, avoiding new situations, endeavor and self - efficiency between boys and girls , and there was no significant difference in other dimensions .
discipline, fear of negative evaluation, endeavor and self - efficiency among girls are higher than those in boys and avoiding new situations in boys is higher than that in girls .
research findings highlighted that there is a significant negative relationship between the classroom mental - social climate and the students self - efficiency .
khaje and hosseinchari ( 1 ) and dadsetan ( 2 ) obtained the same results in their studies . to justify this finding
, it can be said that in classes with a more suitable mental climate students believe more in themselves and accordingly their academic function improves .
findings also revealed that there is a reverse relationship between social - anxiety and self - efficiency , which is generally due to effects of social anxiety on the students ; these effects destroy self - confidence and lower the person s self - efficiency .
it is worth mentioning that khaje and hosseinchari ( 1 ) got the same results .
all these findings are highly compatible with the conducted research cases in this field , which other studies confirmed them ( 1 , 2 ) . findings proved that there is a negative relationship between social anxiety and the classroom mental - social climate and an increase in social anxiety and its dimensions decreases the effective factors on the classroom mental - social climate .
fraser obtained the same results in his study ( 21 ) . to explain this finding
, it can be said that the main factors in creating the mental - social climate in the classroom are closely related to the social anxiety factors . in studying the existing difference between boys and girls
, the results showed that there is a significant relationship between boys and girls in terms of the mean of self - efficiency , and the mean of self - efficiency in girls is more than that in boys and among the dimensions of self - efficiency , the dimension of
endeavor in girls is also more than that in boys and this difference is significant .
, it can be said that girls usually try more in achieving their goals and by taking this fact into consideration that girls have more mental maturity than boys in the first grade , such results seem natural . among the dimensions of mental - social climate of the classroom , just in the dimension of
discipline, there is a meaningful relationship between boys and girls and it is more among girls than the boys .
fear of negative evaluation and avoiding new situations from the dimensions of the social anxiety . and the fear of negative evaluation is higher in girls than the boys , which is justifiable based on this fact that girls are more sensitive in being judged by others than boys and they like to attract more attention .
this result , which is to some extent acceptable , is based on boys more tendency towards friends and being among their acquaintances and friends . | background : the tendency and motivation to progress and achieve the ideal position have always encouraged people towards acquiring the required education.objectives:the present research aimed to investigate the association of mental - social climate and social anxiety with self - efficiency and also predict the academic self - efficiency of first grade high school students based on social anxiety and the mental - social climate of the classroom.materials and methods : a total of 350 subjects ( 172 girls and 178 boys ) have been chosen by a random clustering sampling form the first grade high school students of qaemshahr , iran .
the academic self - efficiency questionnaire , the social anxiety scale for teenagers and the classroom mental climate scale were used to collect the required data . for data analysis , the statistical method of correlation analysis , independent t test , and
multivariate regression have been used.results:the research findings showed that there was a significant negative relationship between mental - social climate of the classroom and students self - efficiency .
in addition , social anxiety has been a significant negative relationship with self - efficiency .
furthermore , a significant positive relationship exists between mental - social climate and social anxiety.conclusions:in order to develop students self - efficacy , there should be appropriate psychosocial climate .
therefore , teachers and administrators of education must provide all necessary arrangements to improve psychosocial climate classes . | 1. Background
2. Objectives
3. Materials and Methods
3.1. Instruments
3.2. The Procedure
4. Results
5. Discussion | the tendency and motivation to progress and achieve the ideal position have always persuaded people towards acquiring the required education . on one side , the mental and social climate of the students classrooms and the degree of its relationship with the academic achievement are of the main concerns of the teachers , parents , and people involved in education . furthermore , the students classroom mental and social climate and its degree of relationship with academic achievement is one of the important topics that has been constantly be paid attention to by teachers , managers , and educational authorities . in this case , although the physical condition of the classroom and the level of mental and emotional motivation and states of the learners are in turn effective in forming the classroom mental climate , the role of the teacher in making such climate is more important and determining . based on the above mentioned issues , a research about self - efficiency , social anxiety , and mental and social climate of the classroom , which simultaneously investigate the effect of the main dimensions and factors of social anxiety and the classroom social and mental climate on self - efficiency could be a very valuable achievement in order to propose a suitable strategy to increase self - efficiency and as a result academic achievement . investigating the classroom mental and social climate , social anxiety , and the academic self - efficiency can be a great help for teachers , parents , and educational authorities . we also intend to determine which one of the social anxiety and the classroom mental and social climate variables plays a major role in predicting the academic self - efficiency . so , a total of 350 students were selected ( 172 girls and 178 boys ) . meanwhile , in order to investigate the probability behind the role of mediating social anxiety between two concepts of mental - social climate and academic achievement , multivariate regression method has been applied . meanwhile , in order to investigate the probability behind the role of mediating social anxiety between two concepts of mental - social climate and academic achievement , multivariate regression method has been applied . as it can be seen from table 2 , there are significant correlations between the variables of classroom mental - social climate , friction , coherence , discipline and competition , with the students self - efficiency with the correlation coefficients of -0.33 , -0.042 , -0.31 , 0.05 , and -0.07 , respectively , which indicates negative relationships . in this regard , there are significant negative relationships between variables of social anxiety, fear of negative evaluation, avoiding new situations and then
general situations with academic self - efficiency with values of -0.21 , -0.033 , -0.29 , and -0.0 , respectively , which means that whenever social anxiety increases , students self - efficiency decreases . as it can be seen in table 3
, there is a significant relationship between the variables of classroom mental - social climate , friction , coherence , discipline and competition with social anxiety and the correlation coefficients are 0.33 , 0.37 , 0.06 , 0.02 , and 0.24 , respectively . now we try to answer this question : which of the social anxiety and the classroom mental - social climate variables play a more important role in predicting self - efficiency dimensions and generally self - efficiency ? in this analysis
, all dimensions of social anxiety and the classroom mental - social climate were included in the regression model and based on the standardized regression coefficient , table 4 shows that which variable plays a more important role in predicting self - efficiency and its dimensions . in order to study the difference between the means of the research , the main variables , and the dimensions of these variables between boys and girls , we used the independent t test . the present study shows that there is a significant negative relationship between the general classroom mental - social climate and the students self - efficiency . in other words , increasing the classroom mental - social climate decreases the degree of students self - efficiency . after studying
the association of self - efficiency and its dimensions with social anxiety , the results suggest that there is a significant negative relationship between self - efficiency and social anxiety in general sense . with regard to the relationship between social anxiety and the classroom mental - social climate ,
the results indicate that there is a significant positive relationship between social anxiety and the classroom mental - social climate . research findings highlighted that there is a significant negative relationship between the classroom mental - social climate and the students self - efficiency . findings proved that there is a negative relationship between social anxiety and the classroom mental - social climate and an increase in social anxiety and its dimensions decreases the effective factors on the classroom mental - social climate . in studying the existing difference between boys and girls
, the results showed that there is a significant relationship between boys and girls in terms of the mean of self - efficiency , and the mean of self - efficiency in girls is more than that in boys and among the dimensions of self - efficiency , the dimension of
endeavor in girls is also more than that in boys and this difference is significant . among the dimensions of mental - social climate of the classroom , just in the dimension of
discipline, there is a meaningful relationship between boys and girls and it is more among girls than the boys . | [
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highproducing dairy cows require diets that contain sufficient nutrients to support the desired levels of milk production and body maintenance . producing forage crops that meet nutrient requirements is a challenge especially in regions of northern latitudes with restricted growing season limiting the range of cropping alternatives .
cereals such as wheat , barley , and oats are well suited for production in cooler shorter growing seasons found in sweden.1 , 2 production of cereals in cooler environments can be challenging since the goal is to maximize total nutritive value.3 cell wall fractions ( fiber ) increase and generally have decreasing nutritive value due to decreased digestibility while continued development results in starch production that generally increases nutritive value . with increasing maturity total biomass increases but
digestibility decreases and is most often associated with increased levels of lignin.4 this relationship holds within a given species , but may not work so well across species even when comparing similar stages of development.5 in the poaceae family ( grass family ) the hydroxycinnamates , pcoumaric acid ( pca ) and ferulic acid ( fa ) influence digestibility . for pca
they are esterified primarily to sinapyl alcohol but also to coniferyl alcohol and become part of the lignin structure as the monolignols undergo radical mediated polymerization.6 the pca does not become incorporated but remains simply esterified to the growing lignin polymer.7 to a lesser extent pca can also be incorporated ester linked to arabinosyl ( ara ) units of arabinoxylans.8 ferulates are incorporated into the cell wall esterified to arabinosyl residues of arabinoxylans9 and form crosslinks with other ferulates10 as well as incorporation into growing lignin polymers.11 in grasses the digestibility of cell walls is dependent not only on the amount of lignin but also the degree of crosslinking .
crosslinking can be between arabinoxylans as well as arabinoxylans and lignin polymers both having a negative impact upon digestibility.12 , 13
the detergent system was developed as a rapid method of estimating nutritive value of forages and other feedstuffs and works well for this purpose.14 , 15 however , measuring lignin with the detergent system can be a challenge .
it has been clearly demonstrated that the typical detergent method , acid detergent lignin ( adl ) , can lead to underestimation of lignin in grass cell walls .
hot detergent solutions , especially acid detergent , solubilizes lignin from the cell wall matrix of grasses.16 , 17 the detergent system may not reveal sufficient detail about the chemical make up of certain types of forages to provide a clear picture of how chemical composition is related to animal performance .
this study was undertaken to determine the chemical composition obtained from a complete cell wall analysis of three different whole cereal crop silages ( wccs ) .
a total of 27 samples of nine different wccs from two feeding experiments with dairy heifers were analyzed.1 , 2 , 18 the wccs used were oats ( avena sativa l. ) , sixrowed barley ( hordeum vulgare l. ) and wheat ( triticum aestivum l. ) ( all swedish varieties ) harvested at the heading , early milk and early dough stages of maturity .
multiple cored subsamples were taken from each bale and combined to create the replicate sample for analyses.1 , 18 , 19
approximately 1.51.7 g of sample was accurately weighed into 40ml oakridge tubes on a dry matter ( dm ) basis ( 55 c ) .
all samples were extracted as outlined in the cell wall extraction flow chart ( scheme 1 ) .
final cell wall concentration was determined after drying at 55 c for at least 24 h and was expressed on an ashfree basis ( cwom ) .
cell wall residues were ovendried at 55 c for 24 h before weighing subsamples for the different cell wall analytical procedures .
comparison of the extraction methods to produce ( a ) total cell wall and ( b ) ndf components from cereal whole crop silages .
neutral sugar components were determined as alditol acetates of the released sugars following acid hydrolysis .
cell wall residues were hydrolyzed using the saeman method20 as modified by hatfield et al .
17 accurately weighed ( 100 mg ) samples were subjected to a twostage hydrolysis : stage 1 , 12 mol l h2so4 , ( 2 h , 2224 c ) ; stage 2 , acid was diluted to 1.5 mol l with dh2o , capped tightly and placed in a 100 c forced air oven for 3 h. after hydrolysis , samples were cooled in an ice waterbath , centrifuged ( 900 g ) for 10 min and 200 l removed from each for total uronosyls determination and inositol was added as internal standard .
subsamples were neutralized with barium carbonate , clarified by centrifuging ( 3200 g , 15 min ) and filtered through a glass fiber filter ( 0.2 m , acrodisc ) .
subsamples were dried and sugars converted to alditol acetate derivatives using the procedure of blakeney et al .
21 and analyzed by fidglc ( supelco , bellefonte , pa usa ; spb225 column 30 m 0.25 mm with 0.25 m film thickness ) using a temperature program of 215 c initial for 2 min , 4 c min to 230 c and hold for 11.25 min .
total uronosyls in the cell wall hydrolyzates were determined by colorimetric assay following the method of blumenkrantz and asboehansen.22 the 200l aliquots removed from the cell wall hydrolyzate were individually diluted to 2 ml using dh2o and this diluted sample was used in the assay .
acetyl bromide lignin ( absl ) was measured following the procedure of morrison23 as modified by hatfield et al .
24 dry cell wall samples of 2025 mg were weighed into pyrex tube ( 16 mm 200 mm ) fitted with a teflon lined cap , suspended in 2.5 ml of a 25% acetic bromide in glacial acetic acid and heated for 2 h in a heating block at 50 c . the samples were mixed every 20 min during heating .
the absorbance maximum between 275 nm and 280 nm was determined by evaluating spectral scans ( 250350 nm ) for each sample .
ester and ether linked ferulic acid and pcoumaric acid , were analyzed using the sequential method.25 phenolics were identified and quantified as trimethylsilane derivatives ( 40 l tmsi ; thermo scientfic , rockford , il , usa and 10 l pyridine ) using glcfid on a zb5 ms column ( zebron ; 30 m 0.25 mm , 0.25 m film ) .
the glc conditions were injector 315 c , detector 300 c , and a temperature program of 150 c for 5 min , 4 c min to 200 , 10 c min to 240 c , 30cmin to 300 c and hold for 10 min .
all cell wall samples were analyzed for total n using a combustion assay ( leco fp2000 n analyzer ; leco instruments , inc .
, st . joseph , mi , usa ) and they were also analyzed for ash by combustion at 500 c .
detergent fiber fraction information ( ndfom , adf , adl ) was complied from previous work on the same cereal silages.1 , 2 , 18
the statistical analysis was done with the mixed procedure in sas version 9.2 ( sas institute , cary , nc , usa ) .
the initial model for all chemical components was :
yijk = ci+mj+c*mij+eijk
where yijk is the general mean , ci is the fixed factor of cereal species , mj is the fixed factor of maturity stage at harvest , c*mij is the crop ( c ) maturity ( m ) fixed interaction factor of cereal species and maturity stage at harvest , and eijk is the random error .
if the c*m interaction factor was significant ( p < 0.05 ) , ls means for c*m was tested with the pdiff statement in sas , and the difference between the ls means was adjusted with tukey 's test . if the c*m interaction effect was near significance ( 0.10 > p > 0.05 ) it was kept in the model , but not evaluated and if it was not significant ( ns , p > 0.10 ) it was removed from the model .
the ls means of significant single factors c and m were evaluated with the pdiff statement in sas and the differences were corrected with tukey 's adjustment . in all situations differences between ls means was considered significant at p
tukey 's < 0.05 .
linear regressions and correlations between chemical fractions were analyzed with the regression procedure and the corr option in sas version 9.2 and correlations were considered significant at p < 0.05 .
a total of 27 samples of nine different wccs from two feeding experiments with dairy heifers were analyzed.1 , 2 , 18 the wccs used were oats ( avena sativa l. ) , sixrowed barley ( hordeum vulgare l. ) and wheat ( triticum aestivum l. ) ( all swedish varieties ) harvested at the heading , early milk and early dough stages of maturity .
multiple cored subsamples were taken from each bale and combined to create the replicate sample for analyses.1 , 18 , 19
approximately 1.51.7 g of sample was accurately weighed into 40ml oakridge tubes on a dry matter ( dm ) basis ( 55 c ) .
all samples were extracted as outlined in the cell wall extraction flow chart ( scheme 1 ) .
final cell wall concentration was determined after drying at 55 c for at least 24 h and was expressed on an ashfree basis ( cwom ) .
cell wall residues were ovendried at 55 c for 24 h before weighing subsamples for the different cell wall analytical procedures .
comparison of the extraction methods to produce ( a ) total cell wall and ( b ) ndf components from cereal whole crop silages .
neutral sugar components were determined as alditol acetates of the released sugars following acid hydrolysis .
cell wall residues were hydrolyzed using the saeman method20 as modified by hatfield et al .
17 accurately weighed ( 100 mg ) samples were subjected to a twostage hydrolysis : stage 1 , 12 mol l h2so4 , ( 2 h , 2224 c ) ; stage 2 , acid was diluted to 1.5 mol l with dh2o , capped tightly and placed in a 100 c forced air oven for 3 h. after hydrolysis , samples were cooled in an ice waterbath , centrifuged ( 900 g ) for 10 min and 200 l removed from each for total uronosyls determination and inositol was added as internal standard .
subsamples were neutralized with barium carbonate , clarified by centrifuging ( 3200 g , 15 min ) and filtered through a glass fiber filter ( 0.2 m , acrodisc ) .
subsamples were dried and sugars converted to alditol acetate derivatives using the procedure of blakeney et al .
21 and analyzed by fidglc ( supelco , bellefonte , pa usa ; spb225 column 30 m 0.25 mm with 0.25 m film thickness ) using a temperature program of 215 c initial for 2 min , 4 c min to 230 c and hold for 11.25 min .
total uronosyls in the cell wall hydrolyzates were determined by colorimetric assay following the method of blumenkrantz and asboehansen.22 the 200l aliquots removed from the cell wall hydrolyzate were individually diluted to 2 ml using dh2o and this diluted sample was used in the assay .
acetyl bromide lignin ( absl ) was measured following the procedure of morrison23 as modified by hatfield et al .
24 dry cell wall samples of 2025 mg were weighed into pyrex tube ( 16 mm 200 mm ) fitted with a teflon lined cap , suspended in 2.5 ml of a 25% acetic bromide in glacial acetic acid and heated for 2 h in a heating block at 50 c . the samples were mixed every 20 min during heating . the absorbance maximum between 275 nm and 280 nm was determined by evaluating spectral scans ( 250350 nm ) for each sample .
ester and ether linked ferulic acid and pcoumaric acid , were analyzed using the sequential method.25 phenolics were identified and quantified as trimethylsilane derivatives ( 40 l tmsi ; thermo scientfic , rockford , il , usa and 10 l pyridine ) using glcfid on a zb5 ms column ( zebron ; 30 m 0.25 mm , 0.25 m film ) .
the glc conditions were injector 315 c , detector 300 c , and a temperature program of 150 c for 5 min , 4 c min to 200 , 10 c min to 240 c , 30cmin to 300 c and hold for 10 min .
all cell wall samples were analyzed for total n using a combustion assay ( leco fp2000 n analyzer ; leco instruments , inc . , st .
joseph , mi , usa ) and they were also analyzed for ash by combustion at 500 c .
detergent fiber fraction information ( ndfom , adf , adl ) was complied from previous work on the same cereal silages.1 , 2 , 18
approximately 1.51.7 g of sample was accurately weighed into 40ml oakridge tubes on a dry matter ( dm ) basis ( 55 c ) .
all samples were extracted as outlined in the cell wall extraction flow chart ( scheme 1 ) .
final cell wall concentration was determined after drying at 55 c for at least 24 h and was expressed on an ashfree basis ( cwom ) .
cell wall residues were ovendried at 55 c for 24 h before weighing subsamples for the different cell wall analytical procedures .
comparison of the extraction methods to produce ( a ) total cell wall and ( b ) ndf components from cereal whole crop silages .
neutral sugar components were determined as alditol acetates of the released sugars following acid hydrolysis .
cell wall residues were hydrolyzed using the saeman method20 as modified by hatfield et al .
17 accurately weighed ( 100 mg ) samples were subjected to a twostage hydrolysis : stage 1 , 12 mol l h2so4 , ( 2 h , 2224 c ) ; stage 2 , acid was diluted to 1.5 mol l with dh2o , capped tightly and placed in a 100 c forced air oven for 3 h. after hydrolysis , samples were cooled in an ice waterbath , centrifuged ( 900 g ) for 10 min and 200 l removed from each for total uronosyls determination and inositol was added as internal standard .
subsamples were neutralized with barium carbonate , clarified by centrifuging ( 3200 g , 15 min ) and filtered through a glass fiber filter ( 0.2 m , acrodisc ) .
subsamples were dried and sugars converted to alditol acetate derivatives using the procedure of blakeney et al .
21 and analyzed by fidglc ( supelco , bellefonte , pa usa ; spb225 column 30 m 0.25 mm with 0.25 m film thickness ) using a temperature program of 215 c initial for 2 min , 4 c min to 230 c and hold for 11.25 min .
total uronosyls in the cell wall hydrolyzates were determined by colorimetric assay following the method of blumenkrantz and asboehansen.22 the 200l aliquots removed from the cell wall hydrolyzate were individually diluted to 2 ml using dh2o and this diluted sample was used in the assay .
acetyl bromide lignin ( absl ) was measured following the procedure of morrison23 as modified by hatfield et al .
24 dry cell wall samples of 2025 mg were weighed into pyrex tube ( 16 mm 200 mm ) fitted with a teflon lined cap , suspended in 2.5 ml of a 25% acetic bromide in glacial acetic acid and heated for 2 h in a heating block at 50 c . the samples were mixed every 20 min during heating . the absorbance maximum between 275 nm and 280 nm was determined by evaluating spectral scans ( 250350 nm ) for each sample .
ester and ether linked ferulic acid and pcoumaric acid , were analyzed using the sequential method.25 phenolics were identified and quantified as trimethylsilane derivatives ( 40 l tmsi ; thermo scientfic , rockford , il , usa and 10 l pyridine ) using glcfid on a zb5 ms column ( zebron ; 30 m 0.25 mm , 0.25 m film ) .
the glc conditions were injector 315 c , detector 300 c , and a temperature program of 150 c for 5 min , 4 c min to 200 , 10 c min to 240 c , 30cmin to 300 c and hold for 10 min .
all cell wall samples were analyzed for total n using a combustion assay ( leco fp2000 n analyzer ; leco instruments , inc . , st .
joseph , mi , usa ) and they were also analyzed for ash by combustion at 500 c .
detergent fiber fraction information ( ndfom , adf , adl ) was complied from previous work on the same cereal silages.1 , 2 , 18
the statistical analysis was done with the mixed procedure in sas version 9.2 ( sas institute , cary , nc , usa ) .
the initial model for all chemical components was :
yijk = ci+mj+c*mij+eijk
where yijk is the general mean , ci is the fixed factor of cereal species , mj is the fixed factor of maturity stage at harvest , c*mij is the crop ( c ) maturity ( m ) fixed interaction factor of cereal species and maturity stage at harvest , and eijk is the random error . if the c*m interaction factor was significant ( p < 0.05 ) , ls means for c*m was tested with the pdiff statement in sas , and the difference between the ls means was adjusted with tukey 's test . if the c*m interaction effect was near significance ( 0.10 > p > 0.05 ) it was kept in the model , but not evaluated and if it was not significant ( ns , p > 0.10 ) it was removed from the model .
the ls means of significant single factors c and m were evaluated with the pdiff statement in sas and the differences were corrected with tukey 's adjustment . in all situations differences between ls means was considered significant at p
tukey 's < 0.05 .
linear regressions and correlations between chemical fractions were analyzed with the regression procedure and the corr option in sas version 9.2 and correlations were considered significant at p < 0.05 .
table 1 provides the fermentation characteristics of the individual silages chemically characterized in this study .
all silage samples were dried at 60 c and ground in a hammer mill with 1mm sieve.1 , 2 , 18 more detailed information about the experiment with the oat and sixrowed barley can be found in wallsten et al.,1 , 19 and about the experiment with the wheat silages in rustas et al .
18 the average detergent composition and lignin concentration for nine silages can be found in table 2 .
fermentation characteristics of silages evaluated in this study data complied from previous work , with permission ( rustas et al .
18 and wallsten et al
.
1 )
all values are on a g kg dm basis except for the nh3n .
detergent composition and lignin content measured in nine different wholecrop cereal silages data are summarized from previously published work , with permission ( rustas et al .
. dm = dry matter , ndfom = ashfree neutral detergent fiber , adf = ashfree acid detergent fiber , pmlignin = permanganate lignin , hemicellulose = ndfomadf , cellulose = adfpmlignin .
the cwom concentrations were higher in oats than in barley and wheat at all maturity stages ( table 3 and table 4 ) .
total protein retained in the cell wall fraction ( cp g kg ) was highest in barley , intermediate in oats and lowest in wheat ( table 4 ) corresponding to wheat having the lowest cp .
permanganate lignin ( pmlignin ) performed on adf residues resulted in lower total lignin values compared to ablignin ( table 2 and table 3 ) .
as a consequence pmlignin represented a fraction of the total lignin as measured by ablignin in all the silages with oats having the lowest fraction of lignin ( tables 3 and 4 ) .
ashfree cell wall ( cwom ) concentration ( g kg dry matter ) and composition ( g kg cwom ) of three cereals harvested and stored as wholecrop silage at three maturity stages ( n = 9 ) cp , crude protein ; ablignin , acetyl bromide lignin ; pca , pcoumeric acid ; fa , ferulic acid ; ua , uronic acid ; hc sugars is the sum of xylose , arabinose , fucose , galactose , mannose and rhamnose .
ash and cp are the amounts found in the cw isolates and the ash ( g kg of total ash ) and cp ( g kg of the total cp ) are the totals found in the dm that was retained in the cw isolation .
values on the same row within cereal or maturity stage with different superscripts are significantly different ( p
tukey < 0.05 ) .
evaluation of the significant cereal*maturity stage interaction for nine different whole crop cereal silages ( n = 3 ) all values are in g kg cw .
cp , crude protein ; ablignin , acetyl bromide lignin ; pca , pcoumeric acid ; fa , ferulic acid ; ua , uronic acid ; hc sugars is the sum of xylose , arabinose , fucose , galactose , mannose and rhamnose .
ash and cp are the amounts found in the cw isolates and the ash ( g kgof total ash ) and cp ( g kg of the total cp ) are the % of the totals found in the dm that was retained in the cw isolation .
values on the same row within heading / milk / dough / barley / oats or wheat with different superscripts are significantly different ( p
tukey < 0.05 ) .
concentrations of pca were nearly double in oats compared to wheat or barley at all maturity stages ( tables 3 and 4 ) . the difference in total esterlinked fa ( monomer and dimer ) concentration among cereals varied with maturity stage , with highest concentration in barley at heading
. concentrations of total fa were higher in oats than barley at dough stage ( table 4 ) .
etherlinked fa varied from 5.8 to 8.3 g kg cwom ( monomer + dimer ) , but there were no significant differences among cereal species at any maturity stage ( table 4 ) .
cereals and grasses in general have low amounts of pectins ( < 2% ) with the bulk of cell wall carbohydrates distributed between hemicelluloses and cellulose.26 , 27 total uronic acids composed of both galacturonosyls and glucuronosyls were higher in wheat than in oats and tended ( p
tukey = 0.071 ) to be higher in wheat than in barley ( table 3 ) though these did not account for a major portion of the cell wall carbohydrates .
hemicellulosic sugars ( primarily arabinoxylans ) concentrations were higher in barley than in oats and wheat .
glucose concentrations in wheat tended ( p
tukey = 0.066 ) to be higher than barley but not significantly different from oat silage cell walls ( table 3 ) .
cwom concentrations were higher at the heading stage for all threecereal species ( tables 3 and 4 ) .
differences between milk and dough stage cwom were not large enough to be significant . the higher cell wall residual crude protein ( rcp )
concentration at dough stage was only evident for barley and oats ( tables 3 and 4 ) .
for wheat there was a marginal decrease in rcp concentration with later maturity stage ( table 4 ) .
the cp at dough stage was more difficult to remove during the washes and the percentage of rcp retained was higher at dough stage for all cereal species ( tables 3 and 4 ) .
moreover , the pmlignin at milk stage represented , and at dough stage tended to represent ( p
tukey = 0.069 ) , a higher portion of the corresponding ablignin , than pmlignin at heading .
the pca concentration decreased with maturity for barley and oats , but for wheat the concentrations were similar and the highest value was at milk stage ( tables 3 and 4 ) .
the concentration of esterlinked fa in barley decreased with maturity stage and a similar trend might be suggested for oats , though the numerical difference was small ( 7.2 to 6.7 g kg ) .
the hc sugars decreased with later maturity stage , but glucose actually increased between milk and dough stage ( table 3 ) .
wheat at milk stage was the only silage where ndfom and cwom values were similar , and excluding it increased the positive correlation between the fiber fractions from 0.63 to 0.76 .
the correlation between ndfom and cwom increased when splitting the dataset on cereal species , whereas splitting it on maturity stages increased the correlation only for the samples harvested at milk stage ( table 5 ) . generally , as cw increased , ndf increased across all types of silages ( fig .
the correlation between hc ns sugars and hcndf was significant , but the variation in hc ns values among the replicates was large for most of the silages ( fig .
glucose and cellulose was not significantly correlated for the whole dataset or for the cereal species ( table 5 ) . however , when splitting the data on maturity stages the correlation was significant for heading and milk stage .
there was a large variation in glucose concentration among replicates that was not evident for corresponding cellulose concentrations when considering the mean of replicates ( fig .
the pmlignin was not significantly correlated to the ablignin , when looking at the whole dataset . splitting it on cereal species resulted in positive correlation for oats , but negative correlation of similar magnitude for barley . for barley
correlations between chemical fractions in barley , oats , and wheat harvested at heading , milk stage , and dough stage of maturity .
all comparisons are between results of the detergent analysis system and the cell wall isolation system on an ash free basis .
( a ) total ndf versus total cell wall organic mater ( cw ) ; ( b ) detergent permanganate lignin ( pmlignin ) versus acetyl bromide lignin ( ablignin ) determination in cell wall isolates ; ( c ) ndfadf hemicellulose versus cell wall hemicellulose based on neutral sugar analysis(hc ns ) ; ( d ) adflignin cellulose versus total glucose from neural sugar analysis of cell walls .
error bars indicate the sd of the mean and all values are on a ash free dry matter basis .
oh , oats at heading ; om , oats at milk stage ; od , oats at dough stage ; bh , barley at heading ; bm , barley at milk stage ; bd , barley at dough stage ; wh , wheat at heading ; wm , wheat at milk stage ; wd , wheat at dough stage . correlation and linear regression statistics for different chemical fractions in nine different whole crop cereal silages total observations n = 27 , total silages three species three maturities , n = 9 . ndfom , ashfree neutral detergent fiber ; adf , ashfree acid detergent fiber ; ablignin , acetyl bromide lignin ; pmlignin , permanganate lignin ; hc ( hemicellulose ) = ndfomadf ; hc sugars = sum of xylose , arabinose , fucose , galactose , mannose and rhamnose ; cellulose = adfpmlignin .
table 1 provides the fermentation characteristics of the individual silages chemically characterized in this study .
all silage samples were dried at 60 c and ground in a hammer mill with 1mm sieve.1 , 2 , 18 more detailed information about the experiment with the oat and sixrowed barley can be found in wallsten et al.,1 , 19 and about the experiment with the wheat silages in rustas et al .
18 the average detergent composition and lignin concentration for nine silages can be found in table 2 .
fermentation characteristics of silages evaluated in this study data complied from previous work , with permission ( rustas et al .
18 and wallsten et al
.
1 )
all values are on a g kg dm basis except for the nh3n .
detergent composition and lignin content measured in nine different wholecrop cereal silages data are summarized from previously published work , with permission ( rustas et al .
. dm = dry matter , ndfom = ashfree neutral detergent fiber , adf = ashfree acid detergent fiber , pmlignin = permanganate lignin , hemicellulose = ndfomadf , cellulose = adfpmlignin .
the cwom concentrations were higher in oats than in barley and wheat at all maturity stages ( table 3 and table 4 ) .
total protein retained in the cell wall fraction ( cp g kg ) was highest in barley , intermediate in oats and lowest in wheat ( table 4 ) corresponding to wheat having the lowest cp .
permanganate lignin ( pmlignin ) performed on adf residues resulted in lower total lignin values compared to ablignin ( table 2 and table 3 ) .
as a consequence pmlignin represented a fraction of the total lignin as measured by ablignin in all the silages with oats having the lowest fraction of lignin ( tables 3 and 4 ) .
ashfree cell wall ( cwom ) concentration ( g kg dry matter ) and composition ( g kg cwom ) of three cereals harvested and stored as wholecrop silage at three maturity stages ( n = 9 ) cp , crude protein ; ablignin , acetyl bromide lignin ; pca , pcoumeric acid ; fa , ferulic acid ; ua , uronic acid ; hc sugars is the sum of xylose , arabinose , fucose , galactose , mannose and rhamnose .
ash and cp are the amounts found in the cw isolates and the ash ( g kg of total ash ) and cp ( g kg of the total cp ) are the totals found in the dm that was retained in the cw isolation .
values on the same row within cereal or maturity stage with different superscripts are significantly different ( p
tukey < 0.05 ) .
evaluation of the significant cereal*maturity stage interaction for nine different whole crop cereal silages ( n = 3 ) all values are in g kg cw .
cp , crude protein ; ablignin , acetyl bromide lignin ; pca , pcoumeric acid ; fa , ferulic acid ; ua , uronic acid ; hc sugars is the sum of xylose , arabinose , fucose , galactose , mannose and rhamnose .
ash and cp are the amounts found in the cw isolates and the ash ( g kgof total ash ) and cp ( g kg of the total cp ) are the % of the totals found in the dm that was retained in the cw isolation .
values on the same row within heading / milk / dough / barley / oats or wheat with different superscripts are significantly different ( p
tukey < 0.05 ) .
concentrations of pca were nearly double in oats compared to wheat or barley at all maturity stages ( tables 3 and 4 ) . the difference in total esterlinked fa ( monomer and dimer ) concentration among cereals varied with maturity stage , with highest concentration in barley at heading
. concentrations of total fa were higher in oats than barley at dough stage ( table 4 ) .
etherlinked fa varied from 5.8 to 8.3 g kg cwom ( monomer + dimer ) , but there were no significant differences among cereal species at any maturity stage ( table 4 ) .
cereals and grasses in general have low amounts of pectins ( < 2% ) with the bulk of cell wall carbohydrates distributed between hemicelluloses and cellulose.26 , 27 total uronic acids composed of both galacturonosyls and glucuronosyls were higher in wheat than in oats and tended ( p
tukey = 0.071 ) to be higher in wheat than in barley ( table 3 ) though these did not account for a major portion of the cell wall carbohydrates .
hemicellulosic sugars ( primarily arabinoxylans ) concentrations were higher in barley than in oats and wheat .
glucose concentrations in wheat tended ( p
tukey = 0.066 ) to be higher than barley but not significantly different from oat silage cell walls ( table 3 ) .
cwom concentrations were higher at the heading stage for all threecereal species ( tables 3 and 4 ) .
differences between milk and dough stage cwom were not large enough to be significant . the higher cell wall residual crude protein ( rcp )
concentration at dough stage was only evident for barley and oats ( tables 3 and 4 ) . for wheat
there was a marginal decrease in rcp concentration with later maturity stage ( table 4 ) .
the cp at dough stage was more difficult to remove during the washes and the percentage of rcp retained was higher at dough stage for all cereal species ( tables 3 and 4 ) .
moreover , the pmlignin at milk stage represented , and at dough stage tended to represent ( p
tukey = 0.069 ) , a higher portion of the corresponding ablignin , than pmlignin at heading .
the pca concentration decreased with maturity for barley and oats , but for wheat the concentrations were similar and the highest value was at milk stage ( tables 3 and 4 ) .
the concentration of esterlinked fa in barley decreased with maturity stage and a similar trend might be suggested for oats , though the numerical difference was small ( 7.2 to 6.7 g kg ) .
the hc sugars decreased with later maturity stage , but glucose actually increased between milk and dough stage ( table 3 ) .
wheat at milk stage was the only silage where ndfom and cwom values were similar , and excluding it increased the positive correlation between the fiber fractions from 0.63 to 0.76 .
the correlation between ndfom and cwom increased when splitting the dataset on cereal species , whereas splitting it on maturity stages increased the correlation only for the samples harvested at milk stage ( table 5 ) . generally , as cw increased , ndf increased across all types of silages ( fig .
the correlation between hc ns sugars and hcndf was significant , but the variation in hc ns values among the replicates was large for most of the silages ( fig .
glucose and cellulose was not significantly correlated for the whole dataset or for the cereal species ( table 5 ) . however ,
when splitting the data on maturity stages the correlation was significant for heading and milk stage .
there was a large variation in glucose concentration among replicates that was not evident for corresponding cellulose concentrations when considering the mean of replicates ( fig .
the pmlignin was not significantly correlated to the ablignin , when looking at the whole dataset . splitting it on cereal species
resulted in positive correlation for oats , but negative correlation of similar magnitude for barley . for barley
correlations between chemical fractions in barley , oats , and wheat harvested at heading , milk stage , and dough stage of maturity .
all comparisons are between results of the detergent analysis system and the cell wall isolation system on an ash free basis .
( a ) total ndf versus total cell wall organic mater ( cw ) ; ( b ) detergent permanganate lignin ( pmlignin ) versus acetyl bromide lignin ( ablignin ) determination in cell wall isolates ; ( c ) ndfadf hemicellulose versus cell wall hemicellulose based on neutral sugar analysis(hc ns ) ; ( d ) adflignin cellulose versus total glucose from neural sugar analysis of cell walls .
error bars indicate the sd of the mean and all values are on a ash free dry matter basis .
oh , oats at heading ; om , oats at milk stage ; od , oats at dough stage ; bh , barley at heading ; bm , barley at milk stage ; bd , barley at dough stage ; wh , wheat at heading ; wm , wheat at milk stage ; wd , wheat at dough stage . correlation and linear regression statistics for different chemical fractions in nine different whole crop cereal silages total observations n = 27 , total silages three species three maturities , n = 9 .
ndfom , ashfree neutral detergent fiber ; adf , ashfree acid detergent fiber ; ablignin , acetyl bromide lignin ; pmlignin , permanganate lignin ; hc ( hemicellulose ) = ndfomadf ; hc sugars = sum of xylose , arabinose , fucose , galactose , mannose and rhamnose ; cellulose = adfpmlignin .
based on the information complied in table 1 all the silages appeared to ferment reasonably well providing a good source of animal feed .
it is also clear that fermentation at the later maturities was not as robust as earlier stages most likely due to a decrease in readily available soluble sugars.1 , 3 the cw analysis system provides more detailed chemical information about the fiber fraction of plants .
neutral detergent fiber ( ndf ) method was developed to rapidly estimate the nutrient value of forages and other feedstuff .
it is intended to represent the more slowly digested cw material in a forage sample . typically for grasses
there is a good correlation between ndf ( table 2 ) and the cwom ( tables 3 and 4 ) .
grasses tend to produce ndf values aligned with cwom compared to legumes primarily due to the low levels of pectins in grasses.27 as forages mature there is an increase in the cwom as a proportion of the total dm . however , in the case of cereals advanced maturity results in the formation a grain head that contributes to a significant increase in overall dm content . for this study wccs cwom and consequentially ndf decreases as a portion of the dm with increased maturity due to rapid accumulation of starch during grain head development boosting the overall dm content .
differences among the types of wccs for cwom are due to compositional and structural changes during maturation.28 for this work the noncellulosic sugars were combined into one group referred to as the hemicellulosic sugars .
although the detergent system creates an ndf fraction that does a reasonable job of representing the cw fractions there are significant differences between the two methods . the cw isolation method ( scheme 1a ) is designed to preserve all the cw components in the final insoluble residue while removing as much of the noncw components as possible . with no detergent being used in the extraction protein removal
is less efficient than with the ndf method ( scheme 1b ) . the cp recovery in the cwom in the present study ranged from 190 to 420 g kg ( table 4 ) of the original cp in the dm .
29 reported a 113157 g kg recovery in barley silage harvested at the boot and the dough stages .
reported levels of 250320 g kg ( in ndf)30 for wheat and oat whole crops harvested between the heading and the dough stages but not ensiled .
higher cp was recovered in the ndf fraction at later maturity stages as was seen for cp in cwom in this study ( table 4).29 increases in cell wall associated protein might be related to a reduction in apparent cp digestibility at later maturity stages probably due to the slower degradation of mature cw.1 , 18 , 30
differences between ndf ( table 2 ) and total cwom ( table 4 ) may also reflect losses of cell wall material during the ndf procedure , due to solubility in the hot detergent16 and to particle losses during filtering.31 at early stages of development grass arabinoxylans may be highly branched32 and therefore more susceptible to solubilization in hot detergent solution .
arabinose side chains , which are typically in the furanose form , are susceptible to weak acid hydrolysis such as the low ph conditions produced during ensiling and could be sufficient to cleave some of theses residues.33 larger differences were seen in measuring the lignin fraction .
ablignin used in the cwom method accounts for all the lignin whereas the pmlignin method is measuring only a fraction of the total .
differences in the chemical / physical makeup of the cereal lignins could alter their solubility in hot detergent .
lignin , especially in grasses , can be soluble in both neutral16 and acid detergent16 , 34 resulting in much lower lignin values.17 acetyl bromide lignin method was originally proposed as a rapid method for woody samples and adapted by morrison for forages.35 the method effectively solubilizes the lignin from the cell wall matrix in an acidic medium leaving protein and complex carbohydrates insoluble .
these insoluble materials are removed with centrifugation to prevent light scattering that would alter the true lignin value . isolated and purified lignins can be used as standards to allow quantification of lignin in unknown samples.36 in this study all the wccs had higher lignin values compared to the detergent pmlignin in the earlier studies ( table 2 ) .
oat silages tended to have the highest ablignin whereas wheat tends to be higher for pmlignin .
the observation that cereals with the highest ablignin did not also give the highest pmlignin values suggests there are structural and possibly compositional differences among the different lignins .
these differences are due to variable solubility in hot detergent solutions especially the ad treatment and could have implications on how cell wall materials can be degraded by microbes .
lignin content does not appear to change significantly ( table 3 ) within individual silages during development when expressed on a cw basis .
1b ) due to the accumulation of starch as a significant portion of the dm .
the most significant changes occur in the forage stems as they transition from vegetative to reproductive stages .
continued development of the grain head in cereals could account for increased accumulation of cell walls associated with the grain but contain low levels of lignin .
this may be particularly true for oats and barley that accumulate large amounts of mixed linked glucans37 that would remain as cell wall components during the cw extraction process . to clarify this relationship
it would be necessary to evaluate the individual cereals as they stand in the field and separated into major tissue types ( e.g. leaves , stems , sheaths , and reproductive parts ) compared to what is available after ensiling . however , this was beyond the scope of this work and is a subject for future research .
grass pca is primarily incorporated into cw as an ester linked conjugate with monolignols.38 , 39 in this study oats had the highest pca and ablignin , while wheat and barley lignin concentrations were less ( 90 and 120 g kg , respectively ) .
previous studies indicated that across species there is not a good correlation between total lignin and pca.38 it remains unclear as to the role of pca in cw development .
it has been suggested that the formation of pcoumaroylsinapyl alcohol conjugates help in the formation of syringyl type lignins.40 recently , the gene for the pcatransferase was down regulated in two different plant systems , bracchypodium41 and corn.42 in both cases the proportion of syringyl units incorporated into lignin decreased , but the total lignin content did not decrease .
it may be the formation of pcasinapyl alcohol conjugates aids in the incorporation of syringyl units in lignin , but it does not control overall lignin formation . among the hyrdoxycinnamates ,
fa and fadimers can also be coupled with lignin forming crosslinked networks of arabinoxylans and lignin.10 for barley and oats ether linked fa increased or remained constant while wheat appeared to decline during maturation ( table 4 ) .
linkage of ferulates , both monomers and dimers , to lignin is not always through an ether type linkage43 but only the ether linkages can be hydrolyzed and accounted for in this analysis.44 therefore not all crosslinks between lignin and xylans can be accounted for in the typical analysis procedure . as already discussed barley and oat cereals incorporate relatively high levels of mixed linked glucans during grain development.37 the glucans are a part of the grain cw but would not be retained during hot detergent extraction .
their rapid accumulation during grain development would contribute to an overall increase in the cw fraction without increasing fa content making it seem like it is decreasing during this stage of cereal development .
the hemicellulosic ( hc ) fraction is typically composed of all the nonglucose sugars , mainly arabinose and xylose with contributions from galactose , mannose , rhamnose and fucose .
exceptions are oat and barley at the grain filling stage where part of the glucose is from mixed linked glucans .
the correlation between cwom hc and the ndfadf hc ( table 5 ) decreased due to a large variation between replicates .
replicates were from different bales and may be expected to have some difference in amounts .
however , since the hc sugars did not differ in the same way it may instead have been a result of determining the ndf and the adf on different subsamples instead of doing a sequential ndfadf analysis .
it is also possible for the hot detergent reagent for ndf to solubilize parts of the hemicellulosic fraction in these cereals resulting in variable recoveries.27 although there is variability in the individual samples a plot of the means for wccs samples by species and maturity stage shows a good relationship between cw hcns ( based on a sum of the nonglucose sugars ) and hc based ndfadf ( fig .
when calculated on a dm basis there is a consistent decrease in hemicellulosic sugars as the cereals mature .
the use of permanganate to degrade the lignin ( pmlignin ) leaving a cellulose residue may actually help remove some of the variability as a result of nd analysis ( fig .
permanganate treatment can degrade some of the noncellulosic carbohydrates in the cw that may remain in the adf residue .
the larger variation in the glucose content ( based on cw isolation ) is most likely due to the formation of mixed linked glucans in the developing grain in oat and barley .
depending upon the stage of grain fill the quantity of glucans could be variable as well as the solubility during the cw isolation procedure.45 wheat has low levels of glucans in the grain , but has arabinoxylans containing some ferulates.46 loss of lignin during the ndf and adf analyses16 , 47 could result in cellulose concentration being overestimated in these samples . the improved correlations ( table 5 ) when the dataset was split on maturity stages
could be explained by the presence of glucans at dough stage , which is present in high amounts in the grains of barley and oats , but in lower amounts in wheat.48 the presence of glucans might explain why the variation in glucose concentration among replicates was so much larger compared to both cellulose concentration and hc sugars .
differences between the cw and ndad method were due to solubilized cw fractions in hot detergents.differences were observed in cw composition between cereals and among maturity stages that could explain differences in animal performance.more research is warranted in order to further elucidate details of cw composition related to maturity cereal type .
differences between the cw and ndad method were due to solubilized cw fractions in hot detergents .
differences were observed in cw composition between cereals and among maturity stages that could explain differences in animal performance .
more research is warranted in order to further elucidate details of cw composition related to maturity cereal type . | abstractbackgroundin cooler climates such as found in scandinavian countries cereals are important feedstuffs for ruminants often ensiled as wholecrop cereal silages ( wccs ) to preserve nutrients .
animal performance varies with the type of cereal forage and stage of cereal development being ensiled .
cell wall isolation and analysis was undertaken to determine differences among cereal silages harvested at different stages of maturity.resultsa set of 27 wccs samples of barley , wheat and oats harvested at heading , early milk , and dough stages of maturity were analyzed for cell wall ( cw ) composition and compared to previous ndf analyses .
total cw concentrations of the wccs were higher than the ndf concentration .
the lignin concentration was higher ( p < 0.001 ) in oats ( 111 g kg1
dm ) than in barley ( 88 g kg1
dm ) and wheat ( 91 g kg1
dm ) .
ferulates ( ester and ether linked ) ranged from 12.2 to 14.9 g kg1 across forage types and maturity stages . the correlation between total cell wall xylose and hc concentrations ( ndfadf ) was lower than expected in all forages ( r = 0.63).conclusionthe more comprehensive analyses of cell walls provide detailed composition of the different wccs that vary due to the maturity and type of cereal .
2016 the authors .
journal of the science of food and agriculture published by john wiley & sons ltd on behalf of society of chemical industry . | INTRODUCTION
MATERIAL AND METHODS
Silages
Chemical analyses
Statistical analysis
RESULTS
Cereal species
Effect of maturity stages
Correlation of
DISCUSSION
CONCLUSIONS | cereals such as wheat , barley , and oats are well suited for production in cooler shorter growing seasons found in sweden.1 , 2 production of cereals in cooler environments can be challenging since the goal is to maximize total nutritive value.3 cell wall fractions ( fiber ) increase and generally have decreasing nutritive value due to decreased digestibility while continued development results in starch production that generally increases nutritive value . for pca
they are esterified primarily to sinapyl alcohol but also to coniferyl alcohol and become part of the lignin structure as the monolignols undergo radical mediated polymerization.6 the pca does not become incorporated but remains simply esterified to the growing lignin polymer.7 to a lesser extent pca can also be incorporated ester linked to arabinosyl ( ara ) units of arabinoxylans.8 ferulates are incorporated into the cell wall esterified to arabinosyl residues of arabinoxylans9 and form crosslinks with other ferulates10 as well as incorporation into growing lignin polymers.11 in grasses the digestibility of cell walls is dependent not only on the amount of lignin but also the degree of crosslinking . this study was undertaken to determine the chemical composition obtained from a complete cell wall analysis of three different whole cereal crop silages ( wccs ) . a total of 27 samples of nine different wccs from two feeding experiments with dairy heifers were analyzed.1 , 2 , 18 the wccs used were oats ( avena sativa l. ) , sixrowed barley ( hordeum vulgare l. ) and wheat ( triticum aestivum l. ) ( all swedish varieties ) harvested at the heading , early milk and early dough stages of maturity . a total of 27 samples of nine different wccs from two feeding experiments with dairy heifers were analyzed.1 , 2 , 18 the wccs used were oats ( avena sativa l. ) , sixrowed barley ( hordeum vulgare l. ) and wheat ( triticum aestivum l. ) ( all swedish varieties ) harvested at the heading , early milk and early dough stages of maturity . the cwom concentrations were higher in oats than in barley and wheat at all maturity stages ( table 3 and table 4 ) . cereals and grasses in general have low amounts of pectins ( < 2% ) with the bulk of cell wall carbohydrates distributed between hemicelluloses and cellulose.26 , 27 total uronic acids composed of both galacturonosyls and glucuronosyls were higher in wheat than in oats and tended ( p
tukey = 0.071 ) to be higher in wheat than in barley ( table 3 ) though these did not account for a major portion of the cell wall carbohydrates . for barley
correlations between chemical fractions in barley , oats , and wheat harvested at heading , milk stage , and dough stage of maturity . ( a ) total ndf versus total cell wall organic mater ( cw ) ; ( b ) detergent permanganate lignin ( pmlignin ) versus acetyl bromide lignin ( ablignin ) determination in cell wall isolates ; ( c ) ndfadf hemicellulose versus cell wall hemicellulose based on neutral sugar analysis(hc ns ) ; ( d ) adflignin cellulose versus total glucose from neural sugar analysis of cell walls . cereals and grasses in general have low amounts of pectins ( < 2% ) with the bulk of cell wall carbohydrates distributed between hemicelluloses and cellulose.26 , 27 total uronic acids composed of both galacturonosyls and glucuronosyls were higher in wheat than in oats and tended ( p
tukey = 0.071 ) to be higher in wheat than in barley ( table 3 ) though these did not account for a major portion of the cell wall carbohydrates . for barley
correlations between chemical fractions in barley , oats , and wheat harvested at heading , milk stage , and dough stage of maturity . higher cp was recovered in the ndf fraction at later maturity stages as was seen for cp in cwom in this study ( table 4).29 increases in cell wall associated protein might be related to a reduction in apparent cp digestibility at later maturity stages probably due to the slower degradation of mature cw.1 , 18 , 30
differences between ndf ( table 2 ) and total cwom ( table 4 ) may also reflect losses of cell wall material during the ndf procedure , due to solubility in the hot detergent16 and to particle losses during filtering.31 at early stages of development grass arabinoxylans may be highly branched32 and therefore more susceptible to solubilization in hot detergent solution . | [
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cushing 's syndrome ( cs ) is a rare but severe clinical condition caused by cortisol excess of various etiologies .
it is associated with significant morbidity and mortality and leads to metabolic , cardiovascular , infectious , psychiatric , and gonadal complications ( table 1 ) .
this paper will focus on traditional and novel medical therapy for hypercortisolism secondary to acth - secreting pituitary macroadenoma or carcinoma ( cushing 's disease , cd ) or to ectopic acth secretion .
the natural history of pituitary adenomas varies widely . in the majority of cases , acth - secreting pituitary adenomas are small ( < 1 cm in diameter ) and confined within the sella turcica .
pituitary microadenomas have a typically indolent growth rate , and clinically significant invasion and malignant transformation remain uncommon . however , 410% of patients present with larger tumors ( > 1 cm in diameter ) .
moreover , they are more refractory to surgical treatment and show a more unfavorable prognosis than microadenomas . for their behavior , presentation , and outcome , acth secreting macroadenomas present a distinct profile compared with microadenomas , although they probably represent one end of a spectrum of tumor autonomy , with specific growth and biochemical characteristics .
the 2004 who classification of pituitary adenomas now includes an atypical variant , defined as an mib-1 proliferative index greater than 3% , excessive p53 immunoreactivity and increased mitotic activity . in the absence of metastases , however , invasive or aggressive pituitary tumors are not considered malignant .
pituitary carcinomas , defined as primary tumors with intra- or extracranial metastases , are rare , encountered in less than 1% of all hypophyseal tumors .
ectopic acth secretion ( eas ) accounts for 1520% of cases of cushing 's syndrome and covers a spectrum of tumors from undetectable isolated lesions to extensive metastatic and aggressive malignancies .
eas is often associated with severe hypercortisolemia causing hypokalemia , diabetes , generalized infections , hypertension , and psychotic reactions .
eas is defined as overt when the tumor source is easily detected during the initial endocrine and radiological investigations , covert in patients presenting with hypercortisolemia where the ectopic source is not detected during initial tests but is discovered on subsequent evaluation or during prolonged followup , and occult when the patient 's clinical features suggest cs and all tests indicate an ectopic source , but the primary lesion is not identified even after prolonged and repeated followup .
occult eas is one of the most intriguing challenges for the clinical endocrinologist , as in some cases no tumor is found even after long - term followup or on autopsy .
the overall prognosis of patients with ectopic acth secretion is primarily determined by the nature of the underlying malignancy and the tumor stage on diagnosis .
management of patients with cs requires a major effort to understand the etiology and to control hypercortisolemia as soon as the diagnosis is established .
the most appropriate management of acth - dependent cs derives from a multidisciplinary approach that includes endocrinologists , neurosurgeons , oncologists , and radiotherapists .
the definitive treatment of cs consists in surgical resection of the tumor secreting acth . when the source of the excessive secretion is the pituitary gland , the standard approach is to perform an endoscopic endonasal trans - sphenoidal exploration , with excision of the tumor , if found .
this surgical procedure is demanding and should only be performed in centers with extensive experience , to minimize operative risks , reduce the possibility of remission , and maintain other pituitary functions .
it is successful in about 70% of cases ( defined by suppressed plasma cortisol levels and normal 24 h urinary free cortisol ) .
success rates can reach 90% in selective adenectomy of microadenomas ( < 10 mm in diameter ) , but decrease to 65% for macroadenomas .
about 20% of tumors recur , and recurrence is more likely ( and quicker ) in larger than in smaller tumors .
pituitary irradiation achieves eucortisolism in 5060% of cases , albeit after 35 years , and patients can develop pituitary insufficiency , brain vascular morbidity or secondary neoplasms .
stereotactic radiosurgery ( rs ) proved less effective results in macroadenomas , especially if they had already infiltrate the cavernous sinus . to obtain optimal efficacy , rs
the management of aggressive adenomas invading adjacent structures is a real challenge , as they rarely respond to any treatment . in the presence of ectopic secretion of acth , surgical resection of the primary tumor
often , however , the tumor may already have metastasized , it may not be resectable , or it may not be identified despite extensive investigation ( occult ) .
bilateral adrenalectomy can be chosen as a final approach , reserved for patients who do not respond to surgical exploration of the hypophysis or radiation therapy , or when the source of ectopic acth is not found .
adrenalectomy necessarily requires steroid replacement therapy for the rest of the patient 's life , as with primary adrenocortical insufficiency .
there is also a significant risk of developing nelson 's syndrome , which occurs in 510% of the patients , likely a subset with an aggressive phenotype , after adrenalectomy for cushing 's syndrome [ 4 , 6 ] .
it has been demonstrated that patients with invasive corticotrophinomas have a greater risk of subsequent ( and earlier ) development of nelson 's syndrome compared with less aggressive forms .
prophylactic , conventional 3-field radiotherapy can be used to reduce the incidence of subsequent nelson 's and it should always be considered in the management of these patients .
when these approaches can not be applied , a treatment is needed that has fewer side effects and can quickly reduce symptoms , and severe complications of hypercortisolism , aiming for the normalization of acth and serum cortisol values .
the therapeutic goal in the treatment of patients with acth - dependent cushing 's syndrome is normalization of plasma acth and serum cortisol values , tumor shrinkage and preservation of anterior pituitary function , in cases of pituitary acth - secreting tumor .
medical treatment can improve the clinical condition of patients with severe hypercortisolism pending surgery , during acute diseases ( infections , psychosis , etc . ) , or in patients undergoing radiotherapy while awaiting the effects of the radiotherapy itself .
in addition , patients with ectopic secretion of acth may be treated while expecting confirmation of the source , in the presence of metastatic cancer , or in patients who are not candidates for surgery for some reason .
current drug - based therapy for cs includes drugs that act on the adrenal glands to reduce steroid synthesis , which therefore do not treat the underlying cause of the disease , and neuromodulators acting at the hypothalamic - pituitary level .
the existing treatments can be divided according to the site of action into adrenal acting drugs and in centrally acting drugs ( table 2 ) .
adrenal function must be carefully monitored , as excessive inhibition of steroidogenesis may cause adrenal insufficiency and may require the administration of small doses of glucocorticoids .
it is a synthetic antifungal drug that works principally by inhibiting the cytochrome p450 system and 17,20-lyase , which are involved in the synthesis and degradation of steroids .
it has also been suggested that this drug may directly inhibit the pituitary corticotroph function , inhibiting acth secretion [ 1113 ] .
this is a fast - acting drug that quickly reduces urinary free cortisol ( ufc ) levels .
its use has been reported as effective in 50% of patients with ectopic acth secretion .
the most common side effects include gynecomastia , hypogonadism , gastrointestinal symptoms and reversible increases in liver enzymes .
metyrapone predominantly inhibits 11 hydroxylase and has been used either as a monotherapy , leading to a normalization of cortisol levels in 7580% of patients , or in combination with other steroidogenesis inhibitors or with radiation therapy , achieving even higher efficacy [ 15 , 16 ] .
it is able to reduce cortisol production in patients with ectopic acth production and cushing 's disease .
side effects are dose - dependent , with the most common being hypertension , edema , increased acne and hirsutism in women due to its ability to inhibit the synthesis of aldosterone , resulting in an accumulation of its precursors with mineralocorticoid and weak androgen activity .
however , when combined with ketoconazole , it offers a valuable and safe adjunct to control hypercortisolism .
recently , lci699 , a novel orally active drug that inhibits at high doses the 11-beta hydroxylase activity ( as well as aldosterone synthase ) is under phase 2 evaluation for the management of hypercortisolism ( http://clinicaltrial.gov/ identifier nct01331239 ) .
side effects are skin rash , headache , a generalized pruritic rash , hypothyroidism , and goiter , and because of its toxicity is reserved for adrenal cancer .
a study of 177 patients showed a significant increase in the recurrence - free interval after radical surgery followed by mitotane when compared to surgery alone .
mitotane blocks several steroidogenic enzymes , thus altering peripheral steroid metabolism , directly suppressing the adrenal cortex and altering cortisone metabolism .
its adrenolytic function appears at high doses ( > 4 g / day ) .
it is effective in reducing ufc levels in 83% of treated patients [ 19 , 20 ] .
a 2006 study confirmed that most patients under mitotane treatment in a dose ranging from 4 to 6.5 g daily had dramatic increase in cbg levels , and serum cortisol levels can be elevated even when the circulating free cortisol level is not , thus making difficult to control its biochemical effect [ 21 , 22 ] .
its main use is in patients with persistent disease despite surgical resection , those who are not candidates for surgery , and patients with metastatic disease .
the compound is distributed in the adipose tissue and has a long half - life .
it suppresses corticosteroid synthesis in the adrenal cortex by reversibly inhibiting 11--hydroxylase and 17,20 lyase at non - hypnotic doses .
it has a very rapid onset of action and can be used in acute settings in patients with cs . in addition
, its intravenous administration makes it easily used in patients with no oral or enteral access .
chronic therapeutic use of ethyl - alcohol - containing etomidate was effective for 8 weeks in a patient with ectopic cs and peritonitis . in a 2001 case report ,
etomidate was administered over 5.5 months , with daily dose modulation on the basis of serum cortisol levels .
it is a progesterone receptor antagonist and a powerful type-2 glucocorticoid receptor ( gr ) antagonist .
it binds to human gr with an affinity three to four times higher than that of dexamethasone and about 18 times higher than that of cortisol .
mifepristone affects both the central actions of cortisol ( negative feedback on crh / acth secretion ) and its peripheral actions and increases plasma acth and cortisol levels due to the loss of negative feedback of cortisol .
this drug , currently used in the interruption of early pregnancy , was recently approved in patients with hyperglycemia induced by cs who are not candidates for surgery or where surgery has failed .
medical literature suggests that mifepristone can improve clinical symptoms in 7380% of patients within one month after starting treatment .
reviewed the data of 37 treated cs patients ( 12 with eas , 5 with cushing 's disease , the others affected by other causes of cs ) .
it was suggested that this resulted from cortisol stimulation of the mineralocorticoid receptor , while grs were blocked by mifepristone .
followup of efficacy and the onset of adrenal insufficiency ( reported in 16% of 37 patients treated with mifepristone ) should only be clinical ( weight , blood pressure , skin lesions ) and biological ( regular blood potassium sampling ) .
mifepristone is often associated with the development of endometrial hyperplasia , so regular vaginal ultrasound is recommended in long - term treatment . in the last years
several novel therapies have been studied with a view to the potential biochemical control and inhibition of pituitary tumor growth .
da receptors have been found in a variety of organs ( pituitary , adrenals , brain , kidney , gastrointestinal tract , cardiovascular system ) , and possibly exert an inhibitory effect when activated .
d2-receptor agonists inhibit pituitary hormone secretion , particularly prl and proopiomelanocortin - derived hormones , and drugs such as cabergoline and bromocriptine effectively inhibit prl secretion in prolactinomas .
studies on corticotroph adenomas have shown that 80% of these tumors express d2 receptors [ 30 , 31 ] . in recent decades ,
published case reports and case series have demonstrated the effective use of da agonists in persistent or recurrent cushing 's disease .
the efficacy of bromocriptine in shrinking pituitary tumors was first reported in nelson 's syndrome and in the short - term treatment of cd [ 3234 ] . however , the effect was not very strong , and response to long - term treatment was < 30% .
cabergoline has a higher affinity for d2 receptors and a longer half - life compared to bromocriptine . in the short term
ufc levels normalized ( 40% ) or decreased ( 20% ) in a total cohort of 20 patients , 10 of whom underwent remission during long - term treatment ( 1224 months ) .
more recently a study demonstrated a 25% complete response to cabergoline in 12 patients with a followup of 6 months [ 36 , 37 ] and confirmed that short - term treatment of cd with cabergoline improves cortisol secretion in half the cohort studied ( 30 patients ) , while long - term followup ( 37 months ) demonstrated sustained effectiveness of cabergoline in 30% of subjects .
there are a few documented cases of use of da agonists in ectopic acth secretion .
a study describes 6 cases of ectopic tumors , three of which were not cured by surgery .
a prospective study evaluated the efficacy of cabergoline in monotherapy in patients with uncured cd , using sleeping midnight serum cortisol and the standard low dose dexamethasone suppression test ( lddst ) cut - off value as the response criteria .
this drug is generally well tolerated by most patients , and none of the subjects treated in these clinical trials showed signs of secondary heart dysfunction or valvulopathy , except a patient with a history of tricuspid regurgitation .
cabergoline has also been described as having potential positive metabolic effects ( pressure lowering , improvement of glucose tolerance ) , independently of its cortisol lowering effect .
these findings renew interest in the potential use of dopamine agonists in cushing 's disease .
peroxisome proliferative - activated receptor- ( ppar- ) , a member of the nuclear receptor superfamily , functions as a transcription factor mediating ligand - dependent transcriptional regulation .
ppar- is expressed in several organs , and its administration is reported to inhibit tumor cell growth in the prostate and colon [ 42 , 43 ] .
heaney et al . documented the abundant expression of ppar- in a series of acth - secreting tumor samples compared with aactivate ppar- receptors , might be effective as a treatment for cushing 's disease .
the literature evidence [ 44 , 45 ] does not support this treatment , due to the lack of long - term benefit . despite the finding of an initial reduction of acth and cortisol levels in a subset of patients with cd , clinical symptoms and biochemical parameters subsequently relapsed in this group of subjects .
the administration of thiazolidinediones does not seem to be more effective than other currently available neuromodulators .
it is a somatostatin receptor ( ssr ) ligand with high binding affinity for multiple receptor isoforms ( sst1 - 3 and sst5 ) .
sst5 and sst2 are highly expressed in acth pituitary adenomas , and animal studies documented that ssr mediates inhibition of camp and regulation of acth secretion .
a phase 2 trial suggested that administration of pasireotide for a 2-week period provoked a reduction in ufc in 76% of 29 patients affected by newly diagnosed , persistent or recurrent acth - dependent cushing 's disease . in a double blind , phase 3 study ,
162 patients were randomly assigned to receive 600 mcg or 900 mcg subcutaneously twice daily . at 12 months ,
26% and 15% of patients receiving , respectively , the higher and lower pasireotide dose showed normalization of ufc levels .
serum and salivary cortisol and plasma acth decreased , and clinical features of hypercortisolism diminished .
side effects of this therapy included hyperglycemia ( 73% ) and diabetes in 34% of patients , requiring treatment with glucose lowering medications in 45% .
the other common symptoms were gastrointestinal disorders ( diarrhea , abdominal pain , vomiting ) .
the significant results described in this 12-month phase 3 study support the use of pasireotide as a targeted therapy for acth - secreting tumors .
octreotide , which acts predominantly on sstr2 receptors , has not proven effective in inhibiting acth secretion in patients with cushing 's disease . in most cases ,
. however , their rate of growth can be fast and they can be resistant to standard medical , surgical and radiation treatment , especially acth macroadenomas .
the crooke 's cell variant of corticotroph adenoma has been described to be more aggressive and refractory to therapy , with a predisposition to malignant transformation [ 5052 ] . when invasive tumors recur repeatedly despite radical surgery and postoperative radiotherapy , with widespread extrasellar extension , proximity to cranial nerves and critical blood vessels , combined cytotoxic therapy may be useful .
it has also been suggested that early application of chemotherapy may be useful in patients who have already exhausted all surgical and radiotherapy options and are at high risk of malignant transformation [ 53 , 54 ] .
kaiser et al . reported a good response to cyclophosphamide , doxorubicin and 5-fluorouracil ( 5fu ) in a patient with adrenocorticotroph tumor , with regression of the metastases .
kaltsas et al . recommended the use of ccnu/5fu for relatively indolent tumor in the first instance .
there have been partial and short - lasting responses to other combinations of chemotherapy agents , such as paclitaxel and etoposide in ectopic cushing 's syndrome . in animal studies ,
cytotoxic hybrid compounds between the somatostatin analog vapreotide ( no longer commercially available ) and doxorubicin increased the effects of doxorubicin without increasing its toxicity .
combined with radiotherapy , it is known to be effective in some patients with glioblastoma multiforme and cerebral metastases of malignant melanoma .
it is administered orally , does not require hepatic metabolism for activation , and is able to cross the blood - brain barrier .
tmz promotes apoptosis of target cells and induces massive cell shrinkage and necrosis , depleting the dna repair enzyme o6-methylguanine - dna - methyl transferase ( mgmt ) in various cell types .
its use was firstly described in 2006 for the treatment of a pituitary carcinoma , and the first corticotroph adenoma was treated in 2007 . since then , more than 30 case reports on its use in acth - secreting pituitary tumors have been published , and on the whole described some type of positive response .
described four patients with acth tumors with 50% positive response after only four cycles , in terms of marked shrinkage of the pituitary tumor together with a markedly reduced extension of the vertebral metastases , and a drop in acth levels with clinical improvement .
published a case report of a patient with a corticotroph carcinoma in whom a 90% reduction in the size of the tumor , and a stabilization of the metastases volume was documented after four cycles of tmz .
dillard et al . described a case of an aggressive 3 cm corticotroph adenoma refractory to multiple surgery and radiotherapy which showed a 60% regression in size after tmz administration .
it has been reported that the initial response does not always correlate with long - term control of the disease and that the absence of mgmt expression may be associated with a better response .
tumor stabilization or reduction of tumor size can improve clinical outcomes , and it remains a last - line defense for life - threatening pituitary tumors .
retinoids are a family of signaling molecules that are related to vitamin a ( retinol ) in terms of their chemical structure .
the cell cycle is driven by complexes of cyclin - dependent kinases ( cdks ) and cyclins .
there is abundant evidence that retinoids , via various signaling pathways , inhibit cell - cycle progression in a variety of human cancer cells by directly or indirectly modulating cyclins , cdks , and cell - cycle inhibitors .
ra inhibited acth biosynthesis only in tumorous corticotroph cells , while normal cells were unaffected .
the authors concluded that ra inhibits acth synthesis by inhibiting pomc transcription through its activity on ap-1 and nur77/nurr1 and reduces the proliferation and survival of the corticotroph adenoma .
castillo et al . published an in vivo animal study in which retinoic acid or ketoconazole was administered to 42 dogs with cushing 's canine syndrome .
a reduction in acth and alpha - msh levels and pituitary adenoma volume was noted after 180 days of therapy with retinoic acid or ketoconazole , with similar results for both treatments
. mammalian target of rapamycin ( mtor ) functions as a central element in a signaling pathway involved in the control of cell growth and proliferation .
everolimus is an mtor inhibitor , and recent studies have demonstrated its antineoplastic activity in several human cancers , mostly when associated with the long - acting repeatable ( lar ) formulation of octreotide in neuroendocrine tumors .
the authors described the effects of a combination therapy with everolimus ( 5 mg / day ) and octreotide ( 30 mg / months ) and studied mtor expression in 1 pituitary carcinoma against 17 acth adenomas .
combined therapy did not control pituitary tumor growth or acth secretion , but the authors are waiting for more clinical cases before drawing any conclusions on this combined treatment .
epidermal growth factor receptor ( egfr ) activation , due to either mutation or ligand or receptor overexpression , is associated with a variety of human cancers .
approximately 60% of pituitary tumors , including acth - secreting adenomas , express egfr . in pituitary corticotroph tumors expressing egfr , p27 , a cyclin - dependent kinase inhibitor ,
, the authors hypothesized that the receptor could be a novel target for treatment of cushing 's disease , suppressing acth in corticotroph adenomas . in human acth - secreting tumors
gefitinib ( a tyrosine kinase inhibitor targeting egfr ) was found to suppress in vitro pomc expression by approximately 95% .
gefitinib effectively suppressed acth secretion and inhibited tumor growth in egfr - expressing tumors in vivo ( mouse model ) , in support of the in vitro results . since corticotroph adenomas express da and sst receptors simultaneously , some authors hypothesized the use of da agonists with ss analogues to reach a synergic effect in the treatment of acth - dependent cs .
recent studies evaluated the association of cabergoline and ketoconazole , which normalized ufc in approximately 2/3 of patients not achieving a full response to cabergoline alone . in a prospective open - label , multicenter trial , feelders et al .
administered pasireotide in monotherapy followed by sequential addition of cabergoline and ketoconazole if ufc remained high after 28 and 60 days of treatment , respectively . at the end of
an innovative chimeric molecule that acts simultaneously on sst and da receptors was created with a view to treatment of pituitary tumors .
this compound ( bim-23a760 ) has been tested in a phase 1 and phase 2a study in 11 patients affected by acromegaly from gh - secreting pituitary adenoma , but the weak evidence of somatostatinergic activity led to the discontinuation of its development .
management of persistent or recurrent cs is a challenge and medical therapy plays a critical role in the control of hypercortisolemia and associated symptoms .
should thus be adopted , including chemotherapy , radiotherapy , neuromodulatory drugs , and hormone analogs to control tumor growth and associated symptoms .
ideally , management should be commenced in centers with appropriate experience and knowledge and involve a multidisciplinary team , including endocrinologists , neuroradiologists , dedicated neurosurgeons with expertise in pituitary tumor surgery ( or general surgeons in cases of ectopic tumors ) , nuclear medicine physicians and oncologists .
several studies have demonstrated that early application of medical treatment , possibly incorporating new therapeutic developments , may have improved effectiveness and led to more acceptable side effects . nowadays
, combination of tumor debulking , radiotherapy , medical treatment , and chemotherapy , appropriately and timely used , can avoid progression of an otherwise lethal condition .
a better understanding of the pathogenesis of tumors underlying this puzzling syndrome is needed in order to help identify more effective and safe medical therapies .
control of hypercortisolemia should be obtained whenever possible , even in rapidly progressive disease , as small cell lung carcinomas , to reduce the associated complications ( generalized infections , hypokalemia , diabetes , hypertension , psychotic reactions , and reduction of quality of life ) .
total bilateral adrenalectomy induces a rapid resolution of the clinical features . with low morbidity associated with laparoscopic adrenal surgery
, this approach has been considered more frequently , and possibly even as main treatment in some individuals with cushing 's disease , especially when disease is severe or because of patient preference .
unfortunately the poor prognosis of this subgroup of eas often makes the physician give up any drug control of the disease .
we hope that the several offered noninvasive medical strategies can serve as a guide for the oncologists to improve the quality of life of their patients . | cushing 's syndrome ( cs ) is a rare but severe clinical condition represented by an excessive endogenous cortisol secretion and hence excess circulating free cortisol , characterized by loss of the normal feedback regulation and circadian rhythm of the hypothalamic - pituitary axis due to inappropriate secretion of acth from a pituitary tumor ( cushing 's disease , cd ) or an ectopic source ( ectopic acth secretion , eas ) .
the remaining causes ( 20% ) are acth independent .
as soon as the diagnosis is established , the therapeutic goal is the removal of the tumor . whenever surgery is not curative , management of patients with cs requires a major effort to control hypercortisolemia and associated symptoms . a multidisciplinary approach that includes endocrinologists , neurosurgeons , oncologists , and radiotherapists should be adopted .
this paper will focus on traditional and novel medical therapy for aggressive acth - dependent cs .
several drugs are able to reduce cortisol levels .
their mechanism of action involves blocking adrenal steroidogenesis ( ketoconazole , metyrapone , aminoglutethimide , mitotane , etomidate ) or inhibiting the peripheral action of cortisol through blocking its receptors ( mifepristone ru-486 ) .
other drugs include centrally acting agents ( dopamine agonists , somatostatin receptor agonists , retinoic acid , peroxisome proliferator - activated receptor ppar- ligands ) and novel chemotherapeutic agents ( temozolomide and tyrosine kinase inhibitors ) which have a significant activity against aggressive pituitary or ectopic tumors . | 1. Introduction
2. Management of Cushing's Syndrome
3. Medical Treatments
4. Conclusions | cushing 's syndrome ( cs ) is a rare but severe clinical condition caused by cortisol excess of various etiologies . this paper will focus on traditional and novel medical therapy for hypercortisolism secondary to acth - secreting pituitary macroadenoma or carcinoma ( cushing 's disease , cd ) or to ectopic acth secretion . ectopic acth secretion ( eas ) accounts for 1520% of cases of cushing 's syndrome and covers a spectrum of tumors from undetectable isolated lesions to extensive metastatic and aggressive malignancies . eas is defined as overt when the tumor source is easily detected during the initial endocrine and radiological investigations , covert in patients presenting with hypercortisolemia where the ectopic source is not detected during initial tests but is discovered on subsequent evaluation or during prolonged followup , and occult when the patient 's clinical features suggest cs and all tests indicate an ectopic source , but the primary lesion is not identified even after prolonged and repeated followup . the overall prognosis of patients with ectopic acth secretion is primarily determined by the nature of the underlying malignancy and the tumor stage on diagnosis . management of patients with cs requires a major effort to understand the etiology and to control hypercortisolemia as soon as the diagnosis is established . the most appropriate management of acth - dependent cs derives from a multidisciplinary approach that includes endocrinologists , neurosurgeons , oncologists , and radiotherapists . when the source of the excessive secretion is the pituitary gland , the standard approach is to perform an endoscopic endonasal trans - sphenoidal exploration , with excision of the tumor , if found . in the presence of ectopic secretion of acth , surgical resection of the primary tumor
often , however , the tumor may already have metastasized , it may not be resectable , or it may not be identified despite extensive investigation ( occult ) . there is also a significant risk of developing nelson 's syndrome , which occurs in 510% of the patients , likely a subset with an aggressive phenotype , after adrenalectomy for cushing 's syndrome [ 4 , 6 ] . the therapeutic goal in the treatment of patients with acth - dependent cushing 's syndrome is normalization of plasma acth and serum cortisol values , tumor shrinkage and preservation of anterior pituitary function , in cases of pituitary acth - secreting tumor . current drug - based therapy for cs includes drugs that act on the adrenal glands to reduce steroid synthesis , which therefore do not treat the underlying cause of the disease , and neuromodulators acting at the hypothalamic - pituitary level . it is able to reduce cortisol production in patients with ectopic acth production and cushing 's disease . a 2006 study confirmed that most patients under mitotane treatment in a dose ranging from 4 to 6.5 g daily had dramatic increase in cbg levels , and serum cortisol levels can be elevated even when the circulating free cortisol level is not , thus making difficult to control its biochemical effect [ 21 , 22 ] . mifepristone affects both the central actions of cortisol ( negative feedback on crh / acth secretion ) and its peripheral actions and increases plasma acth and cortisol levels due to the loss of negative feedback of cortisol . reviewed the data of 37 treated cs patients ( 12 with eas , 5 with cushing 's disease , the others affected by other causes of cs ) . a phase 2 trial suggested that administration of pasireotide for a 2-week period provoked a reduction in ufc in 76% of 29 patients affected by newly diagnosed , persistent or recurrent acth - dependent cushing 's disease . described four patients with acth tumors with 50% positive response after only four cycles , in terms of marked shrinkage of the pituitary tumor together with a markedly reduced extension of the vertebral metastases , and a drop in acth levels with clinical improvement . in pituitary corticotroph tumors expressing egfr , p27 , a cyclin - dependent kinase inhibitor ,
, the authors hypothesized that the receptor could be a novel target for treatment of cushing 's disease , suppressing acth in corticotroph adenomas . management of persistent or recurrent cs is a challenge and medical therapy plays a critical role in the control of hypercortisolemia and associated symptoms . should thus be adopted , including chemotherapy , radiotherapy , neuromodulatory drugs , and hormone analogs to control tumor growth and associated symptoms . ideally , management should be commenced in centers with appropriate experience and knowledge and involve a multidisciplinary team , including endocrinologists , neuroradiologists , dedicated neurosurgeons with expertise in pituitary tumor surgery ( or general surgeons in cases of ectopic tumors ) , nuclear medicine physicians and oncologists . control of hypercortisolemia should be obtained whenever possible , even in rapidly progressive disease , as small cell lung carcinomas , to reduce the associated complications ( generalized infections , hypokalemia , diabetes , hypertension , psychotic reactions , and reduction of quality of life ) . | [
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take generic initial data in general relativity , adjust any one parameter p of the initial data to the threshold of black hole formation , and compare the resulting spacetimes as a function of p. in many situations , the following critical phenomena are then observed :
near the threshold , black holes with arbitrarily small masses can be created , and the black hole mass scales as 1\documentclass[12pt]{minimal }
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\begin{document}$$m \propto { ( p - { p_\ast})^\gamma},$$\end{document } where p parameterises the initial data and black holes form for p > p*.the critical exponent is universal with respect to initial data , that is , independent of the particular 1-parameter family , although it depends on the type of collapsing matter.in the region of large curvature before black hole formation , the spacetime approaches a self - similar solution which is also universal with respect to initial data , the critical solution . near the threshold ,
black holes with arbitrarily small masses can be created , and the black hole mass scales as 1\documentclass[12pt]{minimal }
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\begin{document}$$m \propto { ( p - { p_\ast})^\gamma},$$\end{document } where p parameterises the initial data and black holes form for p > p*. the critical exponent is universal with respect to initial data , that is , independent of the particular 1-parameter family , although it depends on the type of collapsing matter . in the region of large curvature before black hole formation , the spacetime approaches a self - similar solution which is also universal with respect to initial data , the critical solution .
critical phenomena were discovered by choptuik in numerical simulations of a spherical scalar field . they have been found in numerous other numerical and analytical studies in spherical symmetry , and a few in axisymmetry ; in particular critical phenomena have been seen in the collapse of axisymmetric gravitational waves in vacuum . scaling laws similar to the one for black hole mass
it is still unclear how universal critical phenomena in collapse are with respect to matter types and beyond spherical symmetry , in particular for vacuum collapse .
progress is likely to be made over the next few years with better numerical simulations .
critical phenomena can usefully be described in dynamical systems terms . a critical solution is then characterised as an attracting fixed point within a surface that divides two basins of attraction , a critical surface in phase space
. such a fixed point can be either a stationary spacetime , or one that is scale - invariant and self - similar .
the latter is relevant for the ( type ii ) critical phenomena sketched above .
we shall also see how the dynamical systems approach establishes a connection with critical phenomena in statistical mechanics .
therefore , we could define the field of critical phenomena in gravitational collapse as the study of the boundaries among the basins of attraction of different end states of self - gravitating systems , such as black hole formation or dispersion .
in our view , the main physical motivation for this study is that those critical solutions which are self - similar provide a way of achieving arbitrarily large spacetime curvature outside a black hole , and in the limit a naked singularity , by fine - tuning generic initial data for generic matter to the black hole threshold .
faced with more material to review , we have attempted to make this update shorter and more systematic than the original 1999 version .
since 1999 , progress on the theory side has mainly been made on the global spacetime structure of the critical solution and cosmic censorship in the spherical scalar field model .
we have included this new material in an enlarged section 3 on the spherical scalar field , although we hope it will turn out to be sufficiently generic to merit inclusion in section 2 .
nonspherical perturbations of the spherical scalar field are also discussed in section 3 . in section 4
we review the rich phenomenology that has been found in many other systems restricted to spherical symmetry .
numerical work in spherical symmetry has proliferated since 1999 , but we have tried to keep this section as short as possible .
there has been less progress in going beyond spherical symmetry than we anticipated in 1999 , even though we continue to believe that important results await there .
the reader unfamiliar with the topic is advised to begin with either sections 2.1 , 2.2 , and 2.3 , which give the key theory of universality , self - similarity and scaling , or sections 3.1 and 3.2 , which describe the classic example , the massless scalar field .
this review is limited to numerical and theoretical work on phenomena at the threshold of black hole formation in 3 + 1-dimensional general relativity .
we report only briefly on work in higher and lower spacetime dimensions and non - gravity systems that may be relevant as toy models for general relativity .
we exclude other work on self - similarity in general relativity and work on critical phenomena in other areas of physics .
the 2002 review by gundlach gives more detailed explanations on some of the basic aspects of the theory .
a review of the role of self - similarity in the formation of singularities in evolutionary pdes in general is .
in this section we describe the basic theory underlying critical collapse of the type that forms arbitrarily small black holes ( later called type ii , see also section 2.4 ) .
we begin with the mathematical origin of its three main characteristics , which were already summarised in the introduction :
universality with respect to initial data;scale - invariance of the critical solution;black hole mass scaling .
universality with respect to initial data ; scale - invariance of the critical solution ; black hole mass scaling .
points in the phase space are initial data sets ( 3-metric , extrinsic curvature , and suitable matter variables , which together obey the einstein constraints ) .
we evolve with the einstein equations in a suitable gauge ( see section 2.5 ) .
solution curves of the dynamical system are spacetimes obeying the einstein - matter equations , sliced by specific cauchy surfaces of constant time t. an isolated system in gr can end up in qualitatively different stable end states .
two possibilities are the formation of a single black hole in collapse , or complete dispersion of the mass - energy to infinity . for a massless scalar field in spherical symmetry ,
any point in phase space can be classified as ending up in one or the other type of end state .
the entire phase space therefore splits into two halves , separated by a critical surface . a phase space trajectory that starts on a critical surface by definition
a critical surface is therefore a dynamical system in its own right , with one dimension fewer than the full system .
if it has an attracting fixed point , such a point is called a critical point .
it is an attractor of codimension one in the full system , and the critical surface is its attracting manifold .
the fact that the critical solution is an attractor of codimension one is visible in its linear perturbations : it has an infinite number of decaying perturbation modes spanning the tangent plane to the critical surface , and a single growing mode not tangential to it .
as illustrated in figures 1 and 2 , any trajectory beginning near the critical surface , but not necessarily near the critical point , moves almost parallel to the critical surface towards the critical point .
near the critical point the evolution slows down , and eventually moves away from the critical point in the direction of the growing mode .
all details of the initial data have been forgotten , except for the distance from the black hole threshold .
the closer the initial phase point is to the critical surface , the more the solution curve approaches the critical point , and the longer it will remain close to it . we should stress that this phase picture is extremely simplified . some of the problems associated with this simplification are discussed in section 2.5 .
figure 1the phase space picture for the black hole threshold in the presence of a critical point .
every point correspond to an initial data set , that is , a 3-metric , extrinsic curvature , and matter fields .
the arrow lines are solution curves , corresponding to spacetimes , but the critical solution , which is stationary ( type i ) or self - similar ( type ii ) is represented by a point .
the line without an arrow is not a time evolution , but a 1-parameter family of initial data that crosses the black hole threshold at p = p*. the 2-dimensional plane represents an ( 1)-dimensional hypersurface , but the third dimension represents really only one dimension .
figure 2a different phase space picture , specifically for type ii critical collapse , and two 2-dimensional projections of the same picture .
in contrast with figure 1 , one dimension of the two representing the ( infinitely many ) decaying modes has been suppressed .
the additional axis now represents a global scale which was suppressed in figure 1 , so that the scale - invariant critical solution cs is now represented as a straight line ( in red ) .
several members of a family of initial conditions ( in blue ) are attracted by the critical solution and then depart from it towards black hole formation ( a or b ) or dispersion ( d ) .
initial conditions starting closer to perfect fine tuning produce smaller black holes , such that the parameter along the line of black hole end states is ln mbh .
the horizontal projection of this figure is the same as the vertical projection of figure 1 .
the phase space picture for the black hole threshold in the presence of a critical point .
every point correspond to an initial data set , that is , a 3-metric , extrinsic curvature , and matter fields .
. the arrow lines are solution curves , corresponding to spacetimes , but the critical solution , which is stationary ( type i ) or self - similar ( type ii ) is represented by a point .
the line without an arrow is not a time evolution , but a 1-parameter family of initial data that crosses the black hole threshold at p = p*. the 2-dimensional plane represents an ( 1)-dimensional hypersurface , but the third dimension represents really only one dimension . a different phase space picture , specifically for type ii critical collapse , and two 2-dimensional projections of the same picture .
in contrast with figure 1 , one dimension of the two representing the ( infinitely many ) decaying modes has been suppressed .
the additional axis now represents a global scale which was suppressed in figure 1 , so that the scale - invariant critical solution cs is now represented as a straight line ( in red ) .
several members of a family of initial conditions ( in blue ) are attracted by the critical solution and then depart from it towards black hole formation ( a or b ) or dispersion ( d ) .
initial conditions starting closer to perfect fine tuning produce smaller black holes , such that the parameter along the line of black hole end states is ln mbh .
the horizontal projection of this figure is the same as the vertical projection of figure 1 .
fixed points of dynamical systems often have additional symmetries . in the case of type ii
critical phenomena , the critical point is a spacetime that is self - similar , or scale - invariant . these symmetries can be discrete or continuous .
the critical solution of a spherically symmetric perfect fluid ( see section 4.2 ) , has continuous self - similarity ( css ) .
a css spacetime is one that admits a homothetic vector field , defined by : 2\documentclass[12pt]{minimal }
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\begin{document}$${{\mathcal l}_\xi}{g_{ab } } = 2{g_{ab}}.$$\end{document } in coordinates x = ( , x ) adapted to the symmetry , so that 3\documentclass[12pt]{minimal }
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\begin{document}$$\xi = - { \partial \over { \partial \tau}},$$\end{document } the metric coefficients are of the form 4\documentclass[12pt]{minimal }
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\begin{document}$${g_{\mu \nu}}(\tau , { x^i } ) = { e^{- 2\tau}}{\tilde{g}_{\mu \nu}}({x^i}),$$\end{document } where the coordinate is the negative logarithm of a spacetime scale , and the remaining three coordinates x can be thought of angles around the singular spacetime point = ( see section 3.3 ) .
the critical solution of other systems , in particular the spherical scalar field ( see section 3 ) and axisymmetric gravitational waves ( see section 5.2 ) , show discrete self - similarity ( dss ) .
the simplest way of defining dss is in adapted coordinates , where 5\documentclass[12pt]{minimal }
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\begin{document}$${g_{\mu \nu}}(\tau , { x^i } ) = { e^{- 2\tau}}{\tilde{g}_{\mu \nu}}(\tau , { x^i}),$$\end{document } such that g( , x ) is periodic in with period . more formally , dss can be defined as a discrete conformal isometry . using the gauge freedom of general relativity ,
the lapse and shift in the adm formalism can be chosen ( non - uniquely ) so that the coordinates become adapted coordinates if and when the solution becomes self - similar ( see section 2.5 ) .
is then both a time coordinate ( in the usual sense that surfaces of constant time are cauchy surfaces ) , and the logarithm of overall scale at constant x. the minus sign in equation ( 3 ) and hence equations ( 4 ) and ( 5 ) , is a convention assuming that smaller scales are in the future .
let z stand for a set of scale - invariant variables of the problem , such as \documentclass[12pt]{minimal }
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\begin{document}${\tilde g_{\mu v}}$\end{document } and suitably rescaled matter variables .
if the dynamics is scale - invariant ( this is the case exactly for example for the scalar field , and approximately for other systems , see section 2.6 ) , then z(x ) is an element of the phase space factored by overall scale , and z(x , ) a solution .
note that z(x ) is an initial data set for gr only up to scale .
the overall scale is supplied by . for simplicity , assume that the critical solution is css .
to linear order , the solution near the critical point must be of the form 6\documentclass[12pt]{minimal }
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\begin{document}$$z(x,\tau ) \simeq { z_\ast}(x)\sum\limits_{i = 1}^\infty { { c_i}(p){e^{{\lambda _ i}\tau}}{z_i}(x)}.$$\end{document } the perturbation amplitudes ci depend on the initial data , and hence on p. as z * is a critical solution , by definition there is exactly one i with positive real part ( in fact it is purely real ) , say 0 . as ,
all other perturbations vanish . in the following we consider this limit , and retain only the one growing perturbation . from our phase space picture
, the evolution ends at the critical solution for p = p * , so we must have c0(p * ) = 0 .
linearising in p around p * , we obtain 7\documentclass[12pt]{minimal }
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\begin{document}$$\mathop { \lim}\limits_{\tau \rightarrow \infty } z(x,\tau ) \simeq { z_\ast}(x ) + { { d{c_0 } } \over { dp}}(p - { p_\ast}){e^{{\lambda _ 0}\tau}}{z_0}(x).$$\end{document } for p p * , but close to it , the solution has the approximate form ( 7 ) over a range of . now we extract cauchy data at one particular p - dependent value of within that range , namely * defined by 8\documentclass[12pt]{minimal }
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\begin{document}$${{d{c_0 } } \over { dp}}(p - { p_\ast}){e^{- { \lambda _ 0}{\tau _ 0 } } } \equiv \epsilon,$$\end{document } where is some constant 1 such that at this the linear approximation is still valid . at sufficiently large
, the linear perturbation has grown so much that the linear approximation breaks down , and for c0 > 0 a black hole forms while for c0 < 0 the solution disperses .
the crucial point is that we need not follow this evolution in detail , nor does the precise value of matter .
it is sufficient to note that the cauchy data at = * are 9\documentclass[12pt]{minimal }
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\begin{document}$$z(x,{\tau _ \ast } ) \simeq { z_\ast}(x ) + \epsilon { z_0}(x).$$\end{document } due to the funnelling effect of the critical solution , the data at * is always the same , except for an overall scale , which is given by \documentclass[12pt]{minimal }
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\begin{document}${e^{- { \tau _ \ast}}}$\end{document}. for example , the physical spacetime metric , with dimension ( length ) is given by \documentclass[12pt]{minimal }
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\begin{document}${g_{\mu v } } = { e^{- 2\tau}}{{\tilde g}_{\mu v}}$\end{document } , and similar scalings hold for the matter variables according to their dimension . in particular , as \documentclass[12pt]{minimal }
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\begin{document}${e^{- { \tau _ \ast}}}$\end{document } is the only scale in the initial data ( 9 ) , the mass of the final black hole must be proportional to that scale .
therefore 10\documentclass[12pt]{minimal }
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\begin{document}$$m \propto { e^{- { \tau _ \ast } } } \propto { ( p - { p_\ast})^{1/{\lambda _ 0}}},$$\end{document } and , comparing with equation ( 1 ) , we have found the critical exponent = 1/0 . when the critical solution is dss , a periodic or fine structure of small amplitude
is superimposed on this basic power law [ 98 , 127 ] : 11\documentclass[12pt]{minimal }
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\begin{document}$$\ln m = \gamma \ln ( p - { p_\ast } ) + c + f(\gamma \ln ( p - { p_\ast } ) + c),$$\end{document } where f(z ) has period and is universal , and only c depends on the initial data . as the critical solution is periodic in with period , the number n of scaling echos is approximated by 12\documentclass[12pt]{minimal }
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\begin{document}$$n \simeq { \delta ^{- 1}}\gamma \ln \vert p - { p_\ast}\vert + { \rm{constant}}.$$\end{document } note that this holds for both supercritical and subcritical solutions . in type i critical phenomena , the same phase space picture as in section 2.1 applies , but the critical solution is now stationary or time - periodic instead of self - similar or scale - periodic .
it also has a finite mass and can be thought of as a metastable star .
( type i and ii were so named after first and second order phase transitions in statistical mechanics , in which the order parameter is discontinuous and continuous , respectively . )
universality in this context implies that the black hole mass near the threshold is independent of the initial data , namely a certain fraction of the mass of the stationary critical solution .
the dimensionful quantity that scales is not the black hole mass , but the lifetime tp of the intermediate state where the solution is approximated by the critical solution .
this is clearly 13\documentclass[12pt]{minimal }
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\begin{document}$${t_p } = - { 1 \over { { \lambda _ 0}}}\ln \vert p - { p_\ast}\vert + { \rm{constant}}.$$\end{document } type i critical phenomena occur when a mass scale in the field equations becomes dynamically relevant .
( this scale does not necessarily set the mass of the critical solution absolutely : there could be a family of critical solutions selected by the initial conditions . )
conversely , as the type ii power law is scale - invariant , type ii phenomena occur in situations where either the field equations do not contain a scale , or this scale is dynamically irrelevant .
many systems , such as the massive scalar field , show both type i and type ii critical phenomena , in different regions of the space of initial data . the time evolution of cauchy data in gr can only be considered as a dynamical system if the adm evolution equations are complemented by a prescription for the lapse and shift . to realise the phase space picture of section 2.1 , the critical solution must be a fixed point or limit cycle .
we have seen how coordinates adapted to the self - similarity can be constructed , but is there a prescription of the lapse and shift for arbitrary initial data , such that , given initial data for the critical solution , the resulting time evolution actively drives the metric to a form ( 5 ) that explicitly displays the self - similarity ?
garfinkle and gundlach have suggested several combinations of lapse and shift conditions that leave css spacetimes invariant and turn the choptuik dss spacetime into a limit cycle ( see [ 91 , 81 ] for partial successes ) . among these , the combination of maximal slicing with minimal strain shift has been suggested in a different context but for related reasons .
maximal slicing requires the initial data slice to be maximal ( \documentclass[12pt]{minimal }
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\begin{document}$${k_a}^a = 0$$\end{document } ) , but other prescriptions , such as freezing the trace of k together with minimal distortion , allow for an arbitrary initial slice with arbitrary spatial coordinates .
all these coordinate conditions are elliptic equations that require boundary conditions , and will turn css spacetimes into fixed points ( or dss into limit cycles ) only given correct boundary conditions . roughly speaking ,
these boundary conditions require a guess of how far the slice is from the accumulation point
t = t * , and answers to this problem only exist in spherical symmetry .
appropriate boundary conditions are also needed if the dynamical system is extended to include the lapse and shift as evolved variables , turning the elliptic equations for the lapse and shift into hyperbolic or parabolic equations . turning a css or stationary spacetime into a fixed point of the dynamical system
also requires an appropriate choice of the phase space variables z(x ) . to capture css ( or dss ) solutions
the coordinates x and are dimensionless , le has dimension length , and g has dimension l. the scaling for the adm and any matter variables follows . even with a prescription for the lapse and shift in place , a given spacetime does not correspond to a unique trajectory in phase space .
rather , for each initial slice through the same spacetime one obtains a different slicing of the entire spacetime . a possibility for avoiding
this ambiguity would be to restrict the phase space further , for example by restricting possible data sets to maximal or constant extrinsic curvature slices .
another open problem is that in order to talk about attractors and repellers on the phase space we need to define a norm on a suitable function space which includes both asymptotically flat data and data for the exact critical solution .
the norm itself must favour the central region and ignore what is further out and asymptotically flat if all black holes of the same mass are to be considered as the same end state .
the field equations for the massless scalar field coupled to the einstein equations are scale - free .
realistic matter models introduce length scales , and the field equations then do not allow for exactly self - similar solutions
. they may however admit solutions which are css or dss asymptotically on small spacetime scales as the dimensionful parameters become irrelevant , including type ii critical solutions [ 49 , 34 , 52 ] .
this can be explored by a formal expansion in powers of the small parameter le , where l is a parameter with dimensions length in the evolution equations .
the zeroth order of the expansion is the self - similar critical solution of the system with l = 0 .
the zeroth order of the background expansion determines exactly and independently of l , and the zeroth order term of the linear perturbation expansion determines the critical exponent 1/0 exactly , so that there is no need in practice to calculate any higher orders in to make predictions for type ii critical phenomena where they occur .
( with l = 0 , the basin of attraction of the type ii critical solution will depend on l , and type i critical phenomena may also occur ; see section 2.4 . ) a priori , there could also be more than one type ii critical solution for l = 0 , although this has not been observed . )
this procedure has been carried out for the einstein - yang - mills system and for massless scalar electrodynamics .
both systems have a single length scale 1/e ( in geometric units c = g = 1 ) , where e is the gauge coupling constant .
all values of e can be said to form one universality class of field equations represented by e = 0 .
other examples include modifications to the perfect fluid equation of state ( eos ) that do not affect the limit of high density .
a simple example is that any scalar field potential v( ) becomes dynamically irrelevant compared to the kinetic energy || in a self - similar solution , so that all scalar fields with potentials are in the universality class of the free massless scalar field .
surprisingly , even two different models like the su(2 ) yang - mills and su(2 ) skyrme models in spherical symmetry are members of the same universality class .
present in the problem , a possible approach is to set the arbitrary scale in equation ( 29 ) equal to one of them , say l0 , and define the dimensionless constants li = li / l0 from the others .
the scope of the universality classes depends on where the li appear in the field equations .
if a particular li appears in the field equations only in positive integer powers , the corresponding li appears only multiplied by e , and will be irrelevant in the scaling limit .
all values of this li therefore belong to the same universality class . from the example above , adding a quartic self - interaction to the massive scalar field gives rise to the dimensionless number /m but its value is an irrelevant ( in the language of renormalisation group theory ) parameter .
contrary to the statement in , we conjecture that massive scalar electrodynamics , for any values of e and m , is in the universality class of the massless uncharged scalar field in a region of phase space where type ii critical phenomena occur .
examples of dimensionless parameters which do change the universality class are the k of the perfect fluid , the of the 2-dimensional sigma model or , probably , a conformal coupling of the scalar field ( the numerical evidence is weak but a dependence should be expected ) .
some basic aspects of critical phenomena in gravitational collapse , such as fine - tuning , universality , scale - invariant physics , and critical exponents for dimensionful quantities , can also be identified in critical phase transitions in statistical mechanics ( see for an introductory textbook ) . from an abstract point of view , the objective of statistical mechanics is to derive relations between macroscopic observables a of the system and macroscopic external forces f acting on it , by considering ensembles of microscopic states of the system .
the expectation values a can be generated as partial derivatives of the partition function 14\documentclass[12pt]{minimal }
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\begin{document}$$z(\mu , f ) = \sum\limits_{{\rm{microstates } } } { { e^{- h({\rm{microstate,}}\mu , f)}}}$$\end{document } here the are parameters of the hamiltonian such as the strength of intermolecular forces , and f are macroscopic quantities which are being controlled , such as the temperature or magnetic field .
phase transitions in thermodynamics are thresholds in the space of external forces f at which the macroscopic observables a , or one of their derivatives , change discontinuously .
we consider two examples : the liquid - gas transition in a fluid , and the ferromagnetic phase transition .
the liquid - gas phase transition in a fluid occurs at the boiling curve p = pb(t ) . in crossing this curve , the fluid density changes discontinuously
however , with increasing temperature , the difference between the liquid and gas density on the boiling curve decreases , and at the critical point ( p * = pb(t * ) , t * ) it vanishes as a non - integer power : 15\documentclass[12pt]{minimal }
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\begin{document}$${\rho _ { { \rm{liquid } } } } - { \rho _ { { \rm{gas } } } } \sim { ( { t_\ast } - t)^\gamma}.$$\end{document } at the critical point an otherwise clear fluid becomes opaque , due to density fluctuations appearing on all scales up to scales much larger than the underlying atomic scale , and including the wavelength of light .
this indicates that the fluid near its critical point is approximately scale - invariant ( for some range of scales between the size of molecules and the size of the container ) . in a ferromagnetic material at high temperatures , the magnetisation m of the material ( alignment of atomic spins ) is determined by the external magnetic field b. at low temperatures , the material shows a spontaneous magnetisation even at zero external field . in the absence of an external field
this breaks rotational symmetry : the system makes a random choice of direction . with increasing temperature , the spontaneous magnetisation
m decreases and vanishes at the curie temperature t * as 16\documentclass[12pt]{minimal }
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\begin{document}$$\vert m\vert \sim{({t_\ast } - t)^\gamma}.$$\end{document } again , the correlation length , or length scale of a typical fluctuation , diverges at the critical point , indicating scale - invariant physics .
quantities such as |m| or liquid gas are called order parameters . in statistical mechanics ,
one distinguishes between first - order phase transitions , where the order parameter changes discontinuously , and second - order , or critical , ones , where it goes to zero continuously .
one should think of a critical phase transition as the critical point where a line of first - order phase transitions ends as the order parameter vanishes .
this is already clear in the fluid example . in the ferromagnet example , at first one seems to have only the one parameter t to adjust .
but in the presence of a very weak external field , the spontaneous magnetisation aligns itself with the external field b , while its strength is to leading order independent of b. the function m(b , t ) therefore changes discontinuously at b = 0 .
the line b = 0 for t < t * is therefore a line of first order phase transitions between directions ( if we consider one spatial dimension only , between m up and m down ) .
this line ends at the critical point ( b = 0 , t = t * ) where the order parameter |m| vanishes .
the critical value b = 0 of b is determined by symmetry ; by contrast p * depends on microscopic properties of the material .
in particular , the atomic scale , and any dimensionful parameters associated with that scale , must become irrelevant at the critical point .
this is taken as the starting point for obtaining properties of the system at the critical point .
one first defines a semi - group acting on micro - states : the renormalisation group .
its action is to group together a small number of adjacent particles as a single particle of a fictitious new system by using some averaging procedure .
one then defines a dual action of the renormalisation group on the space of hamiltonians by demanding that the partition function is invariant under the renormalisation group action : 17\documentclass[12pt]{minimal }
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\begin{document}$$\sum\limits_{{\rm{microstates } } } { { e^{- h } } } = \sum\limits_{{\rm{microstates^{\prime } } } } { { e^{- h^{\prime}}}.}$$\end{document } the renormalised hamiltonian is in general more complicated than the original one , but it can be approximated by the same hamiltonian with new values of the parameters and external forces f. ( at this stage it is common to drop the distinction between and f , as the new and f depend on both and f. ) fixed points of the renormalisation group correspond to hamiltonians with the parameters at their critical values .
the critical values of many of these parameters will be 0 or , meaning that the dimensionful parameters they were originally associated with are irrelevant . because a fixed point of the renormalisation group can not have a preferred length scale , the only parameters that can have nontrivial values are dimensionless .
the behaviour of thermodynamical quantities at the critical point is in general not trivial to calculate .
but the action of the renormalisation group on length scales is given by its definition .
the blowup of the correlation length at the critical point is therefore the easiest critical exponent to calculate .
the same is true for the black hole mass , which is just a length scale .
we can immediately reinterpret the mathematics of section 2.3 as a calculation of the critical exponent for , by substituting the correlation length for the black hole mass m , t * t for p p * , and taking into account that the -evolution in critical collapse is towards smaller scales , while the renormalisation group flow goes towards larger scales : therefore diverges at the critical point , while m vanishes . in type ii critical phenomena in gravitational collapse
, we should think of the black hole mass as being controlled by the functions p and q on phase space defined by equation ( 27 ) .
clearly , p is the equivalent of the reduced temperature t t*. gundlach has suggested that the angular momentum of the initial data can play the role of b , and the final black hole angular momentum the role of m. like the magnetic field , angular momentum is a vector , with a critical value that must be zero because all other values break rotational symmetry .
critical phenomena in gravitational collapse were first discovered by choptuik [ 47 , 48 , 49 ] in the model of a spherically symmetric , massless scalar field minimally coupled to general relativity .
the scalar field matter is both simple , and acts as a toy model in spherical symmetry for the effects of gravitational radiation . given that it is still the best - studied model in spherical symmetry , we review it here as a case study . for other numerical work on this model , see [ 109 , 80 , 111 , 77 , 182 , 212 ] . important analytical studies of gravitational collapse in this model have been carried out by christodoulou [ 57 , 58 , 59 , 60 , 61 , 62 , 63 ] .
we first review the field equations and choptuik s observations at the black hole threshold , mainly as a concrete example for the general ideas discussed above .
we then summarise more recent work on the global structure of choptuik s critical solution , which throws an interesting light on cosmic censorship . in particular
, the exact critical solution contains a curvature singularity that is locally and globally naked , and any critical solution obtained in the limit of perfect fine - tuning of asymptotically flat initial data is at least locally naked . by perturbing around spherical symmetry , the stability of the choptuik solution in the full phase space can be investigated , and the scaling of black hole angular momentum can be predicted . by embedding the real scalar field in scalar electrodynamics and perturbing around the choptuik solution , the scaling of black hole charge can be predicted .
the einstein equations are 18\documentclass[12pt]{minimal }
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\begin{document}$${g_{ab } } = 8\pi \left({{\nabla _ a}\phi { \nabla _ b}\phi - { 1 \over 2}{g_{ab}}{\nabla _ c}\phi
{ \nabla ^c}\phi } \right),$$\end{document } and the matter equation is 19\documentclass[12pt]{minimal }
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\begin{document}$${\nabla _ a}{\nabla ^a}\phi = 0.$$\end{document } note that the matter equation of motion is contained within the contracted bianchi identities .
choptuik chose schwarzschild - like coordinates , 20\documentclass[12pt]{minimal }
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\begin{document}$$d{s^2 } = - { \alpha ^2}(r , t)d{t^2 } + { a^2}(r , t)d{r^2 } + { r^2}d{\omega ^2},$$\end{document } where d = d + sin
d is the metric on the unit 2-sphere .
this choice of coordinates is defined by the radius r giving the surface area of 2-spheres as 4r , and by t being orthogonal to r ( polar - radial coordinates ) .
choptuik chose = 1 at r = 0 , so that t is the proper time of the central observer . in the auxiliary variables 21\documentclass[12pt]{minimal }
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\begin{document}$$\phi = { \phi _ { , r}},\quad \prod = { a \over \alpha}{\phi _ { , t}},$$\end{document } the wave equation becomes a first - order system , 22\documentclass[12pt]{minimal }
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\begin{document}$${\phi _ { , t } } = { \left({{\alpha \over a}\prod } \right)_{,r}},$$\end{document }
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\begin{document}$$\prod\nolimits_{,t } { = { 1 \over { { r^2 } } } } { \left({{r^2}{\alpha
\over a}\phi } \right)_{,r}}.$$\end{document } in spherical symmetry there are four algebraically independent components of the einstein equations . of these , one is a linear combination of derivatives of the other and can be disregarded .
the other three contain only first derivatives of the metric , namely a , t , a , r , and ,r , and are 24\documentclass[12pt]{minimal }
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\begin{document}$${{{a_{,r } } } \over a } + { { { a^2 } - 1 } \over { 2r } } = 2\pi r({\prod ^2 } + { \phi ^2}),$$\end{document }
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\begin{document}$${{{\alpha _ { , r } } } \over \alpha } - { { { a_{,r } } } \over a } - { { { a^2 } - 1 } \over r } = 0,$$\end{document }
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\begin{document}$${{{a_{,t } } } \over \alpha } = 4\pi r\phi \prod.$$\end{document } because of spherical symmetry , the only dynamics is in the scalar field equations ( 22 , 23 ) .
the metric can be found by integrating the odes ( 24 ) and ( 25 ) for a and at any fixed t , given and . equation ( 26 ) can be ignored in this fully constrained evolution scheme .
the free data for the system are the two functions (r , 0 ) and (r , 0 ) .
choptuik investigated several 1-parameter families of such data by evolving the data for many different values of the parameter .
simple examples of such families are (r , 0 ) = 0 and a gaussian for (r , 0 ) , with the parameter p taken to be either the amplitude of the gaussian , with the width and centre fixed , or the width , with position and amplitude fixed , or the position , with width and amplitude fixed . for sufficiently small amplitude ( or the peak sufficiently wide ) , the scalar field will disperse , and for sufficiently large amplitude it will form a black hole .
generic 1-parameter families behave in this way , but this is difficult to prove in generality .
christodoulou showed for the spherically symmetric scalar field system that data sufficiently weak in a well - defined way evolve to a minkowski - like spacetime [ 58 , 61 ] , and that a class of sufficiently strong data forms a black hole .
choptuik found that in all 1-parameter families of initial data he investigated he could make arbitrarily small black holes by fine - tuning the parameter p close to the black hole threshold .
an important fact is that there is nothing visibly special to the black hole threshold .
one can not tell that one given data set will form a black hole and another one infinitesimally close will not , short of evolving both for a sufficiently long time . as p p * along the family ,
choptuik developed numerical techniques that recursively refine the numerical grid in spacetime regions where details arise on scales too small to be resolved properly . in the end , he could determine p * up to a relative precision of 10 , and make black holes as small as 10 times the adm mass of the spacetime .
the power - law scaling ( 10 ) was obeyed from those smallest masses up to black hole masses of , for some families , 0.9 of the adm mass , that is , over six orders of magnitude .
there were no families of initial data which did not show the universal critical solution and critical exponent .
choptuik therefore conjectured that is the same for all 1-parameter families of smooth , asymptotically flat initial data that depend smoothly on the parameter , and that the approximate scaling law holds ever better for arbitrarily small p p*. it is an empirical fact that typical 1-parameter families cross the threshold only once , so that there is every indication that it is a smooth submanifold , as we assumed in the phase space picture . taking into account the discussion of mass scaling above , we can formally write the black hole mass as a functional of the initial data z = ( (r , 0 ) , (r , 0 ) ) exactly as 27\documentclass[12pt]{minimal }
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\begin{document}$$m[z ] = q[z]h(p[z]){(p[z])^\gamma},$$\end{document } where p and q are smooth functions on phase space and h is the heaviside function .
( q could be absorbed into p. ) in hindsight , polar - radial gauge is well - adapted to self - similarity . in this gauge , dss corresponds to 28\documentclass[12pt]{minimal }
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\begin{document}$$z(r , t ) = z({e^{n\delta}}r,{e^{n\delta}}t)$$\end{document } for any integer n , where z stands for any one of the dimensionless quantities a , or ( and therefore also for r or r ) .
with 29\documentclass[12pt]{minimal }
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\begin{document}$$x = - { r \over { t - { t_\ast}}},\quad \tau = - \ln \left({- { { t - { t_\ast } } \over l } }
\right),\quad t < { t_\ast},$$\end{document } dss is 30\documentclass[12pt]{minimal }
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\begin{document}$$z(x,\tau + \delta ) = z(x,\tau).$$\end{document } the dimensionful constants t * and l depend on the particular 1-parameter family of solutions , but the dimensionless critical fields a * , * and * , and in particular their dimensionless period , are universal . empirically , 3.44 for the scalar field in numerical time evolutions , and = 3.445452402(3 ) from a numerical construction of the critical solution based on exact self - similarity and analyticity . in adapted coordinates ,
the metric of the critical spacetime is of the form \documentclass[12pt]{minimal }
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\begin{document}${e^{- 2\tau}}$\end{document } times a regular metric . from this general form alone
, one can conclude that = is a curvature singularity , where riemann and ricci invariants blow up like \documentclass[12pt]{minimal }
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\begin{document}${e^{4\tau}}$\end{document } ( unless the spacetime is flat ) , and which is at finite proper time from regular points in its past .
the weyl tensor with index position c is conformally invariant , so that components with this index position remain finite as . this type of singularity is called conformally compactifiable or
for a classification of all possible global structures of spherically symmetric self - similar spacetimes see .
the global structure of the scalar field critical solution was determined accurately in by assuming analyticity at the centre of spherical symmetry and at the past light cone of the singularity ( the self - similarity horizon , or ssh ) .
the critical solution is then analytic up to the future lightcone of the singularity ( the cauchy horizon , or ch ) .
global adapted coordinates x and can be chosen so that the regular centre r = 0 , the ssh and the ch are all lines of constant x , and surfaces of constant are never tangent to x lines .
approaching the ch , the scalar field oscillates an infinite number of times but with the amplitude of the oscillations decaying to zero .
the scalar field in regular adapted coordinates ( x , ) is of the form 31\documentclass[12pt]{minimal }
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\begin{document}$$\phi ( x,\tau ) \simeq { f_{{\rm{reg}}}}(\tau ) + \vert x\vert^{\varepsilon}{f_{{\rm{sing}}}}[\tau + h(\tau ) + k\ln \vert x\vert ] , $ $ \end{document } where freg( ) , fsing( ) and h( ) are periodic with period , and the ssh is at x = 0 .
these functions have been computed numerically to high accuracy , together with the constants k and . the scalar field itself is smooth with respect to , and as > 0 , it is continuous but not differentiable with respect to x on the ch itself .
surprisingly , the ratio m / r of the hawking mass over the area radius on the ch is of order 10 but not zero ( the value is known to eight significant figures ) .
as the ch itself is regular with smooth null data except for the singular point at its base , it is not intuitively clear why the continuation is not unique .
within a dss ansatz , the solution just to the future of the ch has the same form as equation ( 31 ) .
freg( ) is the same on both sides , but fsing( ) can be chosen freely on the future side of the ch .
within the restriction to dss this function can be taken to parameterise the information that comes out of the naked singularity .
there is precisely one choice of fsing( ) on the future side that gives a regular centre to the future of the ch , with the exception of the naked singularity itself , which is then a point .
this continuation was calculated numerically , and is almost but not quite minkowski in the sense that m / r remains small everywhere to the future of the ssh .
all other dss continuations have a naked , timelike central curvature singularity with negative mass .
more exotic continuations including further chs would be allowed kinematically but are not achieved dynamically if we assume that the continuation is dss .
the spacetime diagram of the generic dss continued solution is given in figure 3 .
figure 3the spacetime diagram of all generic dss continuations of the scalar field critical solution , from .
there is also a unique continuation where the singularity is replaced by a regular centre except at the spacetime point at the base of the ch , which is still a strong curvature singularity .
the lines with arrows are lines of constant adapted coordinate x , with the arrow indicating the direction of / towards larger curvature .
the spacetime diagram of all generic dss continuations of the scalar field critical solution , from .
there is also a unique continuation where the singularity is replaced by a regular centre except at the spacetime point at the base of the ch , which is still a strong curvature singularity .
the lines with arrows are lines of constant adapted coordinate x , with the arrow indicating the direction of / towards larger curvature .
choptuik s results have an obvious bearing on the issue of cosmic censorship ( see for a general review of cosmic censorship ) . roughly speaking
, fine - tuning to the black hole threshold provides a set of data which is codimension one in the space of generic , smooth , asymptotically flat initial data , and whose time evolution contains at least the point singularity of the critical solution .
the cosmic censorship hypothesis must therefore be formulated as generic smooth initial data for reasonable matter do not form naked singularities .
here we look at the relation between fine - tuning and naked singularities in more detail .
christodoulou proves rigorously that naked singularity formation is not generic , but in a rather larger function space , functions of bounded variation , than one would naturally consider . in particular
, the instability of the naked singularity found by christodoulou is not differentiable on the past light cone .
this is unnatural in the context of critical collapse , where the naked singularity can arise from generic ( up to fine - tuning ) smooth initial data , and the intersection of the past light cone of the singularity with the initial data surface is as smooth as the initial data elsewhere .
it is therefore not clear how this theorem relates to the numerical and analytical results strongly indicating that naked singularities are codimension-1 generic within the space of smooth initial data .
the lapse defined by equation ( 20 ) is bounded above and below in the critical solution .
therefore the redshift measured between constant r observers located at any two points on an outgoing radial null geodesic in the critical spacetime to the past of the ch is bounded above and below . within the exact critical solution , a point with arbitrarily high curvature can therefore be observed from a point with arbitrarily low curvature .
next consider a spacetime where the critical solution in a central region is smoothly matched to an asymptotically flat outer region such that the resulting asymptotically flat spacetime contains a part of the critical solution that includes the singularity and a part of the ch . in this spacetime , a point of arbitrarily large curvature can be seen from with finite redshift .
figure 4conformal diagram of the critical solution matched to an asymptotically flat one ( rc : regular centre , s : singularity , ssh : self - similarity horizon ) .
curved lines are lines of constant coordinate , while converging straight lines are lines of constant coordinate x. let the initial data on the cauchy surface cs be those for the exact critical solution out to the 2-sphere r , and let these data be smoothly extended to some data that are asymptotically flat , so that the future null infinity exists . to the past of the matching surface ms
the redshift from point a to point b is finite by self - similarity , and the redshift from b to c is finite by asymptotic flatness .
conformal diagram of the critical solution matched to an asymptotically flat one ( rc : regular centre , s : singularity , ssh : self - similarity horizon ) .
curved lines are lines of constant coordinate , while converging straight lines are lines of constant coordinate x. let the initial data on the cauchy surface cs be those for the exact critical solution out to the 2-sphere r , and let these data be smoothly extended to some data that are asymptotically flat , so that the future null infinity exists . to the past of the matching surface ms
the redshift from point a to point b is finite by self - similarity , and the redshift from b to c is finite by asymptotic flatness .
now consider the evolution of asymptotically flat initial data that have been fine - tuned to the black hole threshold .
the global structure of such spacetimes has been investigated numerically in [ 111 , 77 , 80 , 182 ] .
empirically , these spacetimes can be approximated near the singularity by equation ( 6 ) .
almost all perturbations decay as the singularity is approached and the approximation becomes better , until the one growing perturbation ( which by the assumption of fine - tuning starts out small ) becomes significant . a maximal value of the curvature
is then reached which is still visible from and which scales as 32\documentclass[12pt]{minimal }
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\begin{document}$${r_{\max } } \propto { ( p - { p_\ast})^{2\gamma}}.$$\end{document } finally , consider the limit of perfect fine - tuning .
the growing mode is then absent , and all other modes decay as the naked singularity is approached .
note that equation ( 6 ) suggests this is true regardless of the direction ( future , past , or spacelike , depending on the value of x ) in which the singularity is approached .
this seems to be in conflict with causality : the issue is sensitively connected to the completeness of the modes zi and to the stability of the ch , and requires more investigation .
a complication in the supercritical case has been pointed out in . in the literature on supercritical evolutions ,
what is quoted as the black hole mass is in fact the mass of the first apparent horizon ( ah ) that appears in the time slicing used by the code ( spacelike or null ) .
the black hole mass can and generically will be larger than the ah mass when it is first measured because of matter falling in later , and the region of maximal curvature may well be inside the event horizon , and hidden from observers at ( see figure 5 for an illustration . ) the true black hole mass can only be measured at , where it is defined to be the limit of the bondi mass mb as the bondi time ub . this was implemented in . only one family of initial data was investigated , but in this family it was found that mb converges to 10 of the initial bondi mass in the fine tuning limit .
as the underlying physics is perfectly scale - invariant in the massless scalar field model , the minimum mass must be determined by the family of initial data through the infall of matter into the black hole .
simulations of critical collapse of a perfect fluid in a cosmological context show a similar lower bound due to matter falling back after shock formation , but this may not be true for all initial data .
figure 5the final event horizon of a black hole is only known when the infall of matter has stopped .
radiation at 1 collapses to form a small black hole which settles down , but later more radiation at 2 falls in to give rise to a larger final mass .
fine - tuning of a parameter may result in m , ( p p*) but the final mass m2 would be approximately independent of p. the final event horizon of a black hole is only known when the infall of matter has stopped .
radiation at 1 collapses to form a small black hole which settles down , but later more radiation at 2 falls in to give rise to a larger final mass .
fine - tuning of a parameter may result in m , ( p p*) but the final mass m2 would be approximately independent of p. given the scaling power law for the black hole mass in critical collapse , one would like to know what happens if one takes a generic 1-parameter family of initial data with both electric charge and angular momentum ( for suitable matter ) , and fine - tunes the parameter p to the black hole threshold .
a simple model for charged matter is a complex scalar field coupled to electromagnetism with the substitution a
( note that in geometric units , black hole charge q has dimension of length , but the charge parameter e has dimension 1/length . ) gundlach and martn - garca have studied scalar massless electrodynamics in spherical symmetry perturbatively . clearly , the real scalar field critical solution of choptuik is a solution of this system too .
in fact , it remains a critical solution within massless scalar electrodynamics in the sense that it still has only one growing perturbation mode within the enlarged solution space .
some of its perturbations carry electric charge , but as they are all decaying , electric charge is a subdominant effect .
the charge of the black hole in the critical limit is dominated by the most slowly decaying of the charged modes . from this analysis , a universal power - law scaling of the black hole charge 33\documentclass[12pt]{minimal }
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\begin{document}$$q \sim { ( p - { p_\ast})^\delta}$$\end{document } was predicted . the predicted value 0.88 of the critical exponent ( in scalar electrodynamics ) was subsequently verified in collapse simulations by hod and piran and later again by petryk .
( the mass scales with 0.37 as for the uncharged scalar field . )
no other type of criticality can be found in the phase space of this system as dispersion and black holes are the only possible end states , though black holes with |q| m can be formed .
general considerations similar to those in section 2.6 led gundlach and martn - garca to the general prediction that the two critical exponents are always related , for any matter model , by the inequality 34\documentclass[12pt]{minimal }
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\begin{document}$$\delta \geq 2\gamma$$\end{document } ( with the equality holding if the critical solution is charged ) , so that black hole charge can always be treated perturbatively at the black hole threshold .
an example of the richer phenomenology in the presence of a scale in the field equations is the spherical massive scalar field with a potential m coupled to gravity : in one region of phase space , with characteristic scales smaller than 1/m , the black hole threshold is dominated by the choptuik solution and type ii critical phenomena occur . in another
it is dominated by metastable oscillating boson stars ( whose mass is of order 1/m in geometric units ) and type i critical phenomena occur .
( for the real scalar field , the type i critical solution is an ( unstable ) oscillating boson star while for the complex scalar field it can be a static ( unstable ) boson star . )
when the scalar field with a potential is coupled to electromagnetism , type ii criticality is still controlled by a solution which asymptotically resembles the uncharged choptuik spacetime , but type i criticality is now controlled by charged boson stars .
there are indications that subcritical type i evolutions lead to slow , large amplitude oscillations of stable boson stars [ 141 , 142 , 176 ] and not to dispersion to infinity , as had been conjectured in .
another interesting extension is the study of the dynamics of a real scalar field with a symmetric double - well potential , in which the system displays type i criticality between the two possible vacua .
critical collapse is really relevant for cosmic censorship only if it is not restricted to spherical symmetry .
martn - garca and gundlach have analysed all nonspherical perturbations of the scalar field critical solution by solving a linear eigenvalue problem with an ansatz of regularity at the centre and the ssh .
they find that the only growing mode is the known spherical one , while all other spherical modes and all non - spherical modes decay .
this strongly suggests that the critical solution is an attractor of codimension one not only in the space of spherically symmetric data but ( modulo linearisation stability ) of all data in a finite neighbourhood of spherical symmetry .
more recently , choptuik and collaborators have carried out axisymmetric time evolutions for the massless scalar field using adaptive mesh refinement .
they find that in the limit of fine - tuning generic axisymmetric initial data the spherically symmetric critical solution is approached at first but then deviates from spherical symmetry and eventually develops two centres , each of which approaches the critical solution and bifurcates again in a universal way .
this suggests that the critical solution has non - spherical growing perturbation modes , possibly a single l = 2 even parity mode ( in axisymmetry , only m = 0 is allowed ) .
there appears to be a conflict between the time evolution results and the perturbative results , which needs to be resolved by more work ( see section 5.2 ) .
perturbing the scalar field around spherical symmetry , angular momentum comes in to second order in perturbation theory .
all angular momentum perturbations were found to decay , and a critical exponent , 0.76 for the angular momentum was derived for the massless scalar field in .
the pioneering work of choptuik on the spherical massless scalar field has been followed by a plethora of further investigations .
we have chosen the following rough categories :
systems in which the field equations , when reduced to spherical symmetry , form a single wave - like equation , typically with explicit r - dependence in its coefficients .
this includes yang - mills fields , sigma models , vector and spinor fields , scalar fields in 2 + 1 or in 4 + 1 and more spacetime dimensions , and scalar fields in a semi - classical approximation to quantum gravity.perfect fluid matter , either in an asymptotically flat or a cosmological context .
the linearised euler equations are in fact wave - like , but the full non - linear equations admit shock heating and are therefore not even time - reversal symmetric.collisionless matter described by the vlasov equation is a partial differential equation on particle phase space as well as spacetime . therefore even in spherical symmetry , the matter equation is a partial differential equation in 4 dimensions ( rather than two ) . intuitively speaking ,
there are infinitely more matter degrees of freedom than in the scalar field or in non - spherical vacuum gravity.spherically symmetric nonlinear wave equations on 3 + 1 minkowski spacetime , and other nonlinear partial differential equations which show a transition between singularity formation and dispersal .
some of these examples were constructed because they may have intrinsic physical relevance ( semi - classical gravity , primordial black holes ) , others as toy models for 3 + 1-dimensional gravity , and others mostly out of a purely mathematical interest .
table 1critical collapse in spherical symmetry.mattertypecollapse simulationscritical solutionperturbations of critical solutionperfect fluid , p = kii[72 , 161]css [ 72 , 153 , 161][153 , 139 , 101 , 103]- in 4 + 1 , 5 + 1 , 6 + 1iicss vlasovi?[183 , 168]real scalar field:- massless , minimally coupledii[47 , 48 , 49]dss [ 98 , 154]- massiveioscillating iidss [ 112 , 106][112 , 106]- conformally couplediidss- 4 + 1ii- 5 + 1iimassive complex scalar fieldi , iimassless scalar electrodynamicsiidss massive vector fieldiidss massless diraciicss vacuum brans - dickeistatic 2-d sigma model:- complex scalar ( = 0)iidss - axion - dilaton ( = 1)iicss [ 69 , 110]- scalar - brans - dicke ( > 0)ii[150 , 147]css , dss- general including < 0iicss , dss su(2 ) yang - millsistatic iidss iiicoloured bh [ 14 , 202][193 , 201 , 18]su(2 ) yang - mills - higgs(idem)(idem)su(2 ) skyrme modelistatic iidss so(3 ) mexican hatiidss
systems in which the field equations , when reduced to spherical symmetry , form a single wave - like equation , typically with explicit r - dependence in its coefficients .
this includes yang - mills fields , sigma models , vector and spinor fields , scalar fields in 2 + 1 or in 4 + 1 and more spacetime dimensions , and scalar fields in a semi - classical approximation to quantum gravity
the linearised euler equations are in fact wave - like , but the full non - linear equations admit shock heating and are therefore not even time - reversal symmetric .
collisionless matter described by the vlasov equation is a partial differential equation on particle phase space as well as spacetime .
therefore even in spherical symmetry , the matter equation is a partial differential equation in 4 dimensions ( rather than two ) .
intuitively speaking , there are infinitely more matter degrees of freedom than in the scalar field or in non - spherical vacuum gravity .
spherically symmetric nonlinear wave equations on 3 + 1 minkowski spacetime , and other nonlinear partial differential equations which show a transition between singularity formation and dispersal .
we have already discussed in section 3.6 the effects of adding a potential to the evolution of the scalar field .
an alternative generalization is a modification of the kinetic term in the lagrangian , with the general form 35\documentclass[12pt]{minimal }
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\begin{document}$${1 \over 2}{g^{\mu \nu}}{g_{ij}}({\phi ^k}){\nabla _
\mu}{\phi ^i}{\nabla _ \nu}{\phi ^j}$$\end{document } for an n - dimensional vector field ( x ) with i = 1 , , n , and where gij( ) is a fixed nonlinear function acting as a metric on the so - called target space of the fields . such a system is called a nonlinear model , harmonic map or wave map .
the fields and metric gij are dimensionless , and this allows the introduction of dimensionless parameters in the system , which can not be asymptotically neglected using the arguments of section 2.6 .
( compare with the potential v( ) , which has dimensions ( length ) , and hence requires dimensionful parameters . )
the case n = 1 gives nothing new , and so hirschmann and eardly ( he from now on ) studied the n = 2 case with a target manifold with constant curvature , proportional to a real dimensionless constant . using a single complex coordinate the action of the system can be written as 36\documentclass[12pt]{minimal }
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\begin{document}$$\int { { d^4}x\sqrt g \left [ { { r \over 2 } - { { \vert\nabla \phi { \vert^2 } } \over { { { ( 1 - \kappa \vert\phi { \vert^2})}^2 } } } } \right].}$$\end{document } for 0 this system is equivalent to the problem of a real massless scalar field coupled to brans - dicke ( bd ) gravity ( with the bd coupling constant given by 8bd = 12 + ) .
liebling and choptuik have shown that there is a smooth transition in the bd system from dss criticality for low ( the flat target space case = 0 is equivalent to a self - gravitating complex massless scalar field , whose critical solution is the original dss spacetime found by choptuik ) to css criticality for larger ( the case = 1 is equivalent to the axion - dilaton system , and has been shown to display css criticality in ) .
generalizing their previous results for = 0 [ 123 , 122 ] , he constructed for each a css solution based on the ansatz ( , x ) = e
(x ) for the critical scalar field . studying its perturbations
he concluded that this solution is critical for > 0.0754 , but has three unstable modes for < 0.0754 and even more for > 0.28 .
below 0.0754 a dss solution takes over , as shown in the simulations of liebling and choptuik , and he conjectured that the transition is a hopf bifurcation , such that the dss cycle smoothly shrinks with growing , collapsing onto the css solution at the transition and then disappearing with a finite value of the echoing period . the close relation between the css and dss critical solutions is also manifest in the construction of their global structures . in particular , the results of and for the css = 0 and = 1 solutions respectively show that the cauchy horizon of the singularity is almost but not quite flat , exactly as was the case with the choptuik dss spacetime ( see section 3.3 ) .
this is an n = 3 sigma model , and it also displays a transition between css and dss criticality , but this is a totally different type of transition , in particular showing a divergence in the echoing period . in a reduction to spherical symmetry ,
the effective action is 37\documentclass[12pt]{minimal }
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\begin{document}$$\int { { d^4}x } \sqrt g \left [ { { r \over 2 } - \eta \left({\vert \nabla \phi { \vert^2 } + { { 2{{\sin}^2}\phi } \over { { r^2 } } } } \right ) } \right],$$\end{document } where r is the area radius , and the coupling constant is dimensionless .
it has been shown ( numerically and then analytically ) that for 0 < 1/2 , there is an infinite sequence of css solutions n labelled by a nodal number n , and having n growing modes .
( the case = 0 , in which the sigma field decouples from gravity , will be revisited below . )
the n - th solution is always regular in the past light cone of the singularity , but is regular up to the future light cone only for < n where 0 0.0688 , 1 0.1518 , and n < n+1 < 1/2 . for larger couplings an apparent horizon develops and the solution can not be smoothly continued .
these results suggest that 0 is a stable naked singularity for < 0 , and 1 acts as a critical solution between naked singularity formation and dispersal for < 0 and between black hole formation and dispersal for 0 < < 1 .
the numerical experiments agree with this scenario in the range 0 < 0.14 .
other css solutions of this system are investigated in , and the possibility of chaos in .
aichelburg and collaborators [ 131 , 144 ] have shown that for 0.2 there is clear dss type ii criticality at the black hole threshold .
the period depends on , monotonically decreasing towards an asymptotic value for . interesting new behavior occurs in the intermediate range 0.14 < < 0.2 that lies between clear css and clear dss .
with decreasing the overall dss includes episodes of approximate css , of increasing length ( measured in the log - scale time ) .
as c 0.170 from above the duration of the css epochs , and hence the overall dss period diverges . for 0.14
< < 0.17 time evolutions of initial data near the black hole threshold no longer show overal dss , but they still show css episodes .
it has been conjectured that this transition from css to dss can be interpreted , in the language of the theory of dynamical systems , as the infinite - dimensional analogue of a 3-dimensional shilnikov bifurcation .
high - precision numerics in further supports this picture : for > c a codimension-1 css solution coexists in phase space with a codimension-1 dss attractor such that the ( 1-dimensional ) unstable manifold of the dss solution lies on the stable manifold of the css solution . for
close to c the two solutions are close and the orbits around the dss solution become slower because they spend more time in the neighbourhood of the css attractor .
a linear stability analysis predicts a law \documentclass[12pt]{minimal }
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\begin{document}$\delta \simeq - { 2 \over \lambda}\log ( \eta - { \eta _ c } ) + b$\end{document } for some constant b , where is the lyapunov exponent of the css solution .
choptuik , chmaj and bizo have found both type i and type ii critical collapse in the spherical einstein - yang - mills system with su(2 ) gauge potential , restricting to the purely magnetic case , in which the matter is described by a single real scalar field .
the situation is very similar to that of the massive scalar field , and now the critical solutions are the well - known static n = 1 bartnik - mckinnon solution for type i and a dss solution ( later constructed in ) for type ii . in both cases the black holes produced in the supercritical regime
are schwarzschild black holes with zero yang - mills field strength , but the final states ( and the dynamics leading to them ) can be distinguished by the value of the yang - mills final gauge potential at infinity , which can take two values , corresponding to two distinct vacuum states .
choptuik , hirschmann and marsa have investigated the boundary in phase space between formation of those two types of black holes , using a code that can follow the time evolutions for long after the black hole has formed .
phase transition whose critical solution is an unstable static black hole with yang - mills hair [ 14 , 202 ] , which collapses to a hairless schwarzschild black hole with either vacuum state of the yang - mills field , depending on the sign of its one growing perturbation mode .
coloured black hole is actually a member of a 1-parameter family parameterized by its apparent horizon radius and outside the horizon it approaches the corresponding bm solution .
millward and hirschmann have further coupled a higgs field to the einstein - yang - mills system .
new possible end states appear : regular static solutions , and stable hairy black holes ( different from the coloured black holes referred to above ) .
again there are type i or type ii critical phenomena depending on the initial conditions .
it is known that the spherical critical solutions within the magnetic ansatz become more unstable when other components of the gauge field are taken into account , and so they will not be critical in the general case .
bizo , chmaj and schmidt have found a way of constructing asymptotically flat vacuum spacetimes in 4 + 1 dimensions which are spherically symmetric while containing gravitational waves ( birkhoff s theorem does not hold in more than 3 + 1 dimensions ) .
recall that in ( 3 + 1 dimensional ) bianchi ix cosmology the manifold is m s where the s is equipped with an su(2 ) invariant ( homogeneous but anisotropic ) metric 38\documentclass[12pt]{minimal }
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\begin{document}$$d{s^2 } = l_1 ^ 2\sigma _ 1 ^ 2 + l_2 ^ 2\sigma _ 3 ^ 2 + l_3 ^ 2\sigma _ 3 ^ 2,$$\end{document } where the are the su(2 ) left - invariant 1-forms , and the li are functions of time only .
similarly , the spacetimes of bizo , chmaj and schmidt are of the product form m s where the li now depend only on r and t. this gives rise to nontrivial dynamics , including a threshold between dispersion and black hole formation . with the additional u(1 )
l1 = l2 ( biaxial solutions ) there is only one dynamical degree of freedom . at the black hole threshold ,
type ii critical phenomena are seen with 0.49 and 0.3289 . in evolutions with the general ansatz where all li are different ( triaxial solutions ) , the u(1 ) symmetry is recovered dynamically in the approach to the critical surface .
however , each biaxial solution , and in particular the critical solution , exists in three copies obtained by permutation of the i .
the boundaries , within the critical surface , between their basins of attraction contain in turn codimension - two dss attractors .
it is conjectured that there is in fact a countable family of dss solutions with unstable modes .
szybka and chmaj give numerical evidence that these boundaries within the critical surface are fractal ( in contrast to the critical surface itself , which is smooth as in all other known systems . )
a similar ansatz can be made in other odd spacetime dimensions , and in 8 + 1 dimensions type ii critical behaviour is again observed .
spacetime in 2 + 1 dimensions is flat everywhere where there is no matter , so that gravity is not acting at a distance in the usual way .
there are no gravitational waves , and black holes can only be formed in the presence of a negative cosmological constant ( see for a review ) .
scalar field collapse in circular symmetry was investigated numerically by pretorius and choptuik , and husain and olivier . in a regime where the cosmological constant is small compared to spacetime curvature
they find type ii critical phenomena with a universal css critical solution , and = 1.20 0.05 .
looking for the critical solution in closed form , garfinkle found a countable family of exact spherically symmetric css solutions for a massless scalar field with = 0 , but his results remain inconclusive .
the q = 4 solution appears to match the numerical evolutions inside the past light cone , but its past light cone is also an apparent horizon .
the q = 4 solution has three growing modes although the top one would give = 8/7 1.14 if only the other two could be ruled out .
, it is possible to embed the = 0 solutions into a family of < 0 ones [ 64 , 65 , 43 ] , which can be constructed along the lines of section 2.6 , so that garfinkle s solution could be the leading term in an expansion in e ( ) .
critical collapse of a massless scalar field in spherical symmetry in 5 + 1 spacetime dimensions was investigated in .
results are similar to 3 + 1 dimensions , with a dss critical solution and mass scaling with 0.424 .
birukou et al [ 13 , 133 ] have developed a code for arbitrary spacetime dimension .
they confirm known results in 3 + 1 ( 0.36 ) and 5 + 1 ( 0.44 ) dimensions , and investigate 4 + 1 dimensions . without a cosmological constant they find mass scaling with 0.41 for one family of initial data and 0.52 for another .
they see wiggles in the ln m versus ln(p p * ) plot that indicate a dss critical solution , but have not investigated the critical solution directly . with a negative cosmological constant and the second family , they find = 0.49 .
bland and kunstatter have made a more precise determination : = 0.4131 0.0001 .
this was motivated by an attempt to explain this exponent using an holographic duality between the strong coupling regime of 4 + 1 gravity and the weak coupling regime of 3 + 1 qcd , which had predicted = 0.409552 .
kol relates a solution that is related to the choptuik solution to a variant of the critical solution in the black - string black hole transition , and claims to obtain analytic estimates for and . this has motivated a numerical determination of and for the spherical massless scalar field in noninteger dimension up to 14 [ 190 , 30 ] .
choptuik , hirschmann and liebling have presented perturbative indications that the static solutions found by van putten in the vacuum brans - dicke system are critical solutions .
they have also performed full numerical simulations , but only starting from small deviations with respect to those solutions .
ventrella and choptuik have performed numerical simulations of collapse of a massless dirac field in a special state : an incoherent sum of two independent left - handed zero - spin fields having opposite orbital angular momentum .
this is prepared so that the total distribution of energy - momentum is spherically symmetric . the freedom in the system is then contained in a single complex scalar field obeying a modified linear wave equation in spherical symmetry .
there are clear signs of css criticality in the metric variables , and the critical complex field exhibits a phase of the form e for a definite ( the hirschmann and eardly ansatz for the complex scalar field critical solutions ) , which can be considered as a trivial form of dss .
garfinkle , mann and vuille have found coexistence of types i and ii criticality in the spherical collapse of a massive vector field ( the proca system ) , the scenario being almost identical to that of a massive scalar field . in the self - similar phase the collapse amplifies the longitudinal mode of the proca field with respect to its transverse modes , which become negligible , and
sarbach and lehner find type i critical behaviour in q + 3-dimensional spacetimes with u(1 )
so(q + 1 ) symmetry in einstein - maxwell theory at the threshold between dispersion and formation of a black string .
evans and coleman performed the first simulations of critical collapse with a perfect fluid with eos p = k ( where is the energy density and p the pressure ) for k = 1/3 ( radiation ) , and found a css critical solution with a mass - scaling critical exponent 0.36 .
koike , hara and adachi constructed that critical solution and its linear perturbations from a css ansatz as an eigenvalue problem , computing the critical exponent to high precision . independently , maison constructed the regular css solutions and their linear perturbations for a large number of values of k , showing for the first time that the critical exponents were model - dependent .
as ori and piran before [ 171 , 172 ] , he claimed that there are no regular css solutions for k > 0.89 , but neilsen and choptuik [ 161 , 162 ] have found css critical solutions for all values of k right up to 1 , both in collapse simulations and by making a css ansatz .
the difficulty comes from a change in character of the sonic point , which becomes a nodal point for k > 0.89 , rather than a focal point , making the ode problem associated with the css ansatz much more difficult to solve .
harada has also found that the critical solution becomes unstable to a kink
( discontinuous at the sonic point of the background solution ) mode for k > 0.89 , but because it is not smooth it does not seem to have any influence on the numerical simulations of collapse . on the other hand ,
the limit k 1 leading to the stiff eos p = is singular in that during evolution the fluid 4-velocity can become spacelike and the density negative .
the stiff fluid equations of motion are in fact equivalent to the massless - scalar field , but the critical solutions can differ , dependending on how one deals with the issue of negative density . summarizing , it is possible to construct the evans - coleman css critical ( codimension-1 ) solution for all values 0
this solution can be identified in the general classification of css perfect - fluid solutions as the unique spacetime that is analytic at the center and at the sound cone , is ingoing near the center , and outgoing everywhere else [ 40 , 41 , 42 ] .
there is even a newtonian counterpart of the critical solution : the hunter ( a ) solution .
have calculated using perturbation theory for the spherically symmetric perfect fluid with p = k for k 1/4 in d = 5 , 6 , 7 dimensions .
p = k is the only eos compatible with exact css ( homothetic ) solutions for perfect fluid collapse and therefore we might think that other equations of state would not display critical phenomena , at least of type ii .
neilsen and choptuik have given evidence that for the ideal gas eos \documentclass[12pt]{minimal }
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\begin{document}$p = k{\rho _ 0}\epsilon$\end{document } ( where 0 is the rest mass density and is the internal energy per rest mass unit ) the black hole threshold also contains a css attractor , and that it coincides with the css exact critical solution of the ultrarelativistic case with the same k. this is interpreted a posteriori as a sign that the critical css solution is highly ultrarelativistic , = ( 1 + )0 0 0 , and hence rest mass is irrelevant .
novak has also shown in the case k = 1 , or even with a more general tabulated eos , that type ii critical phenomena can be found by velocity - induced perturbations of static tov solutions .
a thorough and much more precise analysis by noble and choptuik [ 165 , 166 ] of the possible collapse scenarios of the stiff k = 1 ideal gas has confirmed this surprising result , and again the critical solution ( and hence the critical exponent ) is that of the ultrarelativistic limit problem .
parametrizing , as usual , the tov solutions by the central density c , they find that for low - density initial stars it is not possible to form a black hole by velocity - induced collapse ; for intermediate initial values of c , it is possible to induce type ii criticality for large enough velocity perturbations ; for large initial central densities they always get type i criticality , as we might have anticipated .
noble and choptuik have also investigated the evolution of a perfect fluid interacting with a massless scalar field indirectly through gravity . by tuning of the amplitude of the pulse it is possible to drive a fluid star to collapse . for massive stars type
i criticality is found , in which the critical solution oscillates around a member of the unstable tov branch . for less massive stars
a large scalar amplitude is required to induce collapse , and the black hole threshold is always dominated by the scalar field dss critical solution , with the fluid evolving passively .
non - spherically symmetric perturbations around the spherical critical solution for the perfect fluid can be used to study angular momentum perturbatively .
all nonspherical perturbations of the perfect fluid critical solution decay for equations of state p = k with k in the range 1/9 < k < 0.49 , and so the spherically symmetric critical solution is stable under small deviations from spherical symmetry .
infinitesimal angular momentum is carried by the axial parity perturbations with angular dependence l = 1 . from these two facts
one can derive the angular momentum scaling law at the black hole threshold [ 99 , 103 ] , 39\documentclass[12pt]{minimal }
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\begin{document}$$l \sim { ( p - { p_\ast})^\mu},$$\end{document } which should be valid in the range 1/9 < k < 0.49 . the angular momentum exponent (k ) is related to the mass exponent (k ) by 40\documentclass[12pt]{minimal }
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\begin{document}$$\mu ( k ) = ( 2 + { \lambda _ 1}(k))\gamma ( k),$$\end{document } where 41\documentclass[12pt]{minimal }
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\begin{document}$${\lambda _ 1}(k ) = { { 9k - 1 } \over { 3k + 3}}$$\end{document } is the growth or decay rate of the dominant l = 1 axial perturbation mode . in particular for the value k = 1/3 , where 0.3558 , , = ( 5/2) 0.8895 .
ori and piran have pointed out that there exists a css perfect fluid solution for 0 < k < 0.036 generalizing the larson - penston solution of newtonian fluid collapse , and which has a naked singularity for 0 < k < 0.0105 .
harada and maeda [ 113 , 115 ] have shown that this solution has no growing perturbative modes in spherical symmetry and hence a naked singularity becomes a global attractor of the evolution for the latter range of k. this is also true in the limit k = 0 , which can be considered as the newtonian limit [ 116 , 117 ] .
this seems to violate cosmic censorship , as generic spherical initial data would create a naked singularity .
however , the exact result ( 41 ) holds for any regular css spherical perfect fluid solution , and so all such solutions with k < 1/9 have at least one unstable nonspherical perturbation . therefore the naked singularity is unstable to infinitesimal perturbations with angular momentum when one lifts the restriction to spherical symmetry . in the early universe , quantum fluctuations of the
metric and matter can be important , for example providing the seeds of galaxy formation .
large enough fluctuations will collapse to form primordial black holes . as large quantum fluctuations are exponentially more unlikely than small ones , \documentclass[12pt]{minimal }
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\begin{document}$p(\delta ) \sim { e^{- { \delta ^2}}}$\end{document } , where is the density contrast of the fluctuation , one would expect the spectrum of primordial black holes to be sharply peaked at the minimal that leads to black hole formation , giving rise to critical phenomena .
an approximation to primordial black hole formation is a spherically symmetric distribution of a radiation gas ( p = /3 ) with cosmological rather than asymptotically flat boundary conditions . in [ 163 ,
164 ] type ii critical phenomena were found , which would imply that the mass of primordial black holes formed are much smaller than the naively expected value of the mass contained within the hubble horizon at the time of collapse .
the boundary conditions and initial data were refined in [ 119 , 160 ] , and a minimum black hole mass of 10 of the horizon mass was found , due to matter accreting onto the black hole after strong shock formation .
however , when the initial data are constructed more realistically from only the growing cosmological perturbation mode , no minimum mass is found [ 177 , 159 ] .
a cloud of collisionless particles can be described by the vlasov equation , i.e. , the boltzmann equation without collision term .
this matter model differs from field theories by having a much larger number of matter degrees of freedom : the matter content is described by a statistical distribution f(x , p ) on the point particle phase space , instead of a finite number of fields (x ) . when restricted to spherical symmetry , individual particles move tangentially as well as radially , and so individually have angular momentum , but the stress - energy tensor averages out to a spherically symmetric one , with zero total angular momentum .
the distribution f is then a function f(r , t , p , l ) of radius , time , radial momentum and ( conserved ) angular momentum .
several numerical simulations of critical collapse of collisionless matter in spherical symmetry have been published to date , and remarkably no type ii scaling phenomena has been discovered .
indications of type i scaling have been found , but these do not quite fit the standard picture of critical collapse .
find that black hole formation turns on with a mass gap that is a large part of the adm mass of the initial data , and this gap depends on the initial matter condition .
olabarrieta and choptuik find evidence of a metastable static solution at the black hole threshold , with type i scaling of its life time as in equation ( 13 ) .
however , the critical exponent depends weakly on the family of initial data , ranging from 5.0 to 5.9 , with a quoted uncertainty of 0.2 .
furthermore , the matter distribution does not appear to be universal , while the metric seems to be universal up to an overall rescaling , so that there appears to be no universal critical solution .
more precise computations by stevenson and choptuik , using finite volume hrsc methods , have confirmed the existence of static intermediate solutions and non - universal scaling with exponents ranging now from 5.27 to 11.65 .
martn - garca and gundlach have constructed a family of css spherically symmetric solutions for massless particles that is generic by function counting .
there are infinitely many solutions with different matter configurations but the same stress - energy tensor and spacetime metric , due to the existence of an exact symmetry : two massless particles with energy - momentum p in the solution can be replaced by one particle with 2p .
a similar result holds for the perturbations . as the growth exponent of a perturbation mode
can be determined from the metric alone , this means that there are infinitely many perturbation modes with the same . if there is one growing perturbative mode , there are infinitely many .
therefore a candidate critical solution ( either static or css ) can not be isolated or have only one growing mode .
this argument rules out the existence of both type i and type ii critical phenomena ( in their standard form , i.e. , including universality ) for massless particles in the complete system , but some partial form of criticality could still be found by restricting to sections of phase space in which that symmetry is broken , for example by prescribing a fixed form for the dependence of the distribution function f on angular momentum l , as those numerical simulations have done .
a recent investigation of andrasson and rein with massive particles has confirmed again the existence of a mass gap and the existence of metastable static solutions at the black hole threshold , though there is no estimation of the scaling of their life - times .
more interestingly , they show that the subcritical regime can lead to either dispersion or an oscillating steady state depending on the binding energy of the system
. they also conclude , based on perturbative arguments , that there can not be an isolated universal critical solution . more numerical work
is still required , but current evidence suggests that there are no type ii critical phenomena , and that there is a continuum of critical solutions in type i critical phenomena and hence only limited universality .
it is well known that the yang - mills field does not form singularities from smooth initial conditions in 3 + 1 dimensions , but bizo and tabor have shown singularity formation in 4 + 1 ( the critical dimension for this system from the point of view of energy scaling arguments ) and 5 + 1 ( the first supercritical dimension ) . in 5 + 1
there is the countable family wn of css solutions with n unstable modes , such that w1 acts as a critical solution separating singularity ( w0 ) formation from dispersal to infinity . in 4 + 1
there are no self - similar solutions and the formation of singularities seems to proceed through adiabatic shrinking of a static solution .
completely parallel results can be found for wave maps , for which the critical dimension is 2 + 1 .
for the wave map from 3 + 1 minkowski to the 3-sphere , bizo has shown that there is a countable family of regular ( before the ch ) css solutions labeled by a nodal number 0 , such that each solution has n unstable modes .
simulations of collapse in spherical symmetry [ 23 , 151 ] and in 3 dimensions show that n = 0 is a global attractor and the n = 1 solution is the critical solution ( see also [ 67 , 68 ] for computations of the largest perturbation - eigenvalues of w0 and w1 ) . again , for the wave map from 2 + 1 minkowski to the 2-sphere generic singularity formation proceeds through adiabatic shrinking of a static solution .
these results have led to the suggestion in that criticality ( in the sense of the existence of a codimension-1 solution separating evolution towards qualitatively different end states ) could be a generic and robust feature of evolutionary pde systems in supercritical dimensions , and not an effect particular of gravity . garfinkle and isenberg
examine the threshold between the round end state and pinching off in ricci flow for a familiy of spherically symmetric geometries on s. they have found intermediate approach to a special javelin geometry , but have not investigated whether this is universal .
a scaling of the shape of the event horizon at the moment of merger in binary black hole mergers is noted in , but this is really a kinematic effect .
a number of authors have explored the possibility of finding critical phenomena with css ( rather than dss ) massless scalar critical solutions .
the roberts 1-parameter family of 3 + 1 solutions has been analyzed along this line in [ 173 , 32 , 206 , 137 ] .
this family contains black holes whose masses ( with a suitable matching to an asymptotically flat solution ) scale as ( p 1 ) for p 1 , but such a special family of solutions has no direct relevance for collapse from generic data .
a fully analytic construction of all ( spherical and nonspherical ) linear perturbations of the roberts solution by frolov [ 73 , 74 ] has shown that there is a continuum of unstable spherical modes filling a sector of the complex plane with re 1 , so that it can not be a critical solution .
frolov has also suggested that the critical ( p = 1 ) roberts solution , which has an outgoing null singularity , plus its most rapidly growing ( spherical ) perturbation mode would evolve into the choptuik solution , which would inherit the oscillation in with a period 4.44 of that mode .
a similar transition within a single 1-parameter family of solutions has been pointed out in for the wyman solution .
hayward [ 121 , 66 ] and clement and fabbri [ 64 , 65 ] have also proposed critical solutions with a null singularity , and have attempted to construct black hole solutions from their linear perturbations .
this is probably irrelevant to critical collapse , as the critical spacetime does not have an outgoing null singularity .
the future light cone of that point is not a null singularity but a ch with finite curvature .
pullin has suggested describing critical collapse approximately as a perturbation of the schwarzschild spacetime .
price and pullin have approximated the choptuik solution by two flat space solutions of the scalar wave equation that are matched at a transition edge at constant self - similarity coordinate x. the nonlinearity of the gravitational field comes in through the matching procedure , and its details are claimed to provide an estimate of the echoing period . birmingham and sen considered the formation of a black hole from the collision of two point particles of equal mass in 2 + 1 gravity .
peleg and steif have investigated the collapse of a dust ring . in both cases
the mass of the black holes is a known function of the parameters of the initial condition , giving a critical exponent 1/2 , but no underlying self - similar solution is involved .
expand the initial data for einstein clusters in powers of the radius and , assuming that there are no shell crossings , find a mass scaling exponent of 3/2 for two such expansion coefficients .
universality is not demonstrated , and so the connection with a css solution discussed by harada and mahajan is unclear .
frolov , and frolov , larsen and christensen consider a stationary 2 + 1-dimensional nambu - goto membrane held fixed at infinity in a stationary 3 + 1-dimensional black hole background spacetime .
the induced 2 + 1 metric on the membrane can have wormhole , black hole , or minkowski topology .
the mass of the apparent horizon of induced black hole metrics scales with = 2/3 , superimposed with a wiggle of period \documentclass[12pt]{minimal }
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\begin{document}$3\pi/\sqrt 7$\end{document } in ln p.
the mass scaling is universal with respect to different background black hole metrics , as they can be approximated by rindler space in the mass scaling limit .
horowitz and hubeny , and birmingham have attempted to calculate the critical exponent in toy models from the ads - cft correspondence .
lvarez - gaumh et al . have attempted to use the ads - cft correspondence for calculating from the qcd side for the spherical massless scalar field in 5 dimensions , and the spherical perfect fluid with p = k for k = 1/(d 1 ) in d = 5 , 6 , 7 dimensions .
have calculated critical exponents for the formation of an apparent horizon in the collision of two gravitational shock waves .
burko considers the transition between existence and non - existence of a null branch of the singularity inside a spherically symmetric charged black hole with massless scalar field matter thrown in .
wang has constructed homothetic cylindrically symmetric solutions of 3 + 1 einstein - klein - gordon and studied their cylindrically symmetric perturbations .
it is not clear how these are related to a critical surface in phase space .
type ii critical phenomena provide a relatively natural way of producing arbitrarily high curvatures , where quantum gravity effects should become important , from generic initial data . approaching the planck scale from above
, one would expect to be able to write down a critical solution that is the classical critical solution asymptotically at large scales , as an expansion in inverse powers of the planck length ( see section 2.6 ) .
black hole evolution in semiclassical gravity has been investigated in 1 + 1 dimensional models which serve as toy models for spherical symmetry ( see for a review ) .
the black hole threshold in such models has been investigated in [ 45 , 9 , 194 , 137 , 210 , 174 , 31 ] . in some of these models , the critical exponent is 1/2 for kinematical reasons . in a 3 + 1-dimensional but perturbative approach is taken .
if this is larger than the positive lyapunov exponent 0 , it will become the dominant perturbation for sufficiently good fine - tuning , and therefore sufficiently good fine - tuning will reveal a mass gap .
the mass gap is found also in numerical evolutions of a spherical scalar field in 3 + 1 dimensions with the semiclassical equations obtained in the framework of singularity resolution in loop quantum gravity [ 132 , 211 ] .
numerical studies of critical collapse should go beyond spherical symmetry ( and in the first instance to axisymmetry ) for three reasons :
weak gravitational waves in vacuum general relativity can focus and collapse .
the black hole threshold in this process shows what in critical phenomena in gravitational collapse is intrinsic to gravity rather than the matter model.black holes are characterised by charge and angular momentum as well as mass .
angular momentum is the more interesting of the two because it is again independent of matter , but can not be studied in spherical symmetry.angular momentum resists collapse , but angular momentum in the initial data is needed to make a black hole with angular momentum .
therefore it is an interesting question to ask what happens to the dimensionless ratio j / m at the black hole threshold .
in the following
the black hole threshold in this process shows what in critical phenomena in gravitational collapse is intrinsic to gravity rather than the matter model .
angular momentum is the more interesting of the two because it is again independent of matter , but can not be studied in spherical symmetry .
angular momentum resists collapse , but angular momentum in the initial data is needed to make a black hole with angular momentum .
therefore it is an interesting question to ask what happens to the dimensionless ratio j / m at the black hole threshold .
we have already mentioned that when angular momentum is small , a critical exponent for |j| can be derived in perturbation theory .
this has been done for the perfect fluid ( see section 4.2 ) in first - order perturbation theory and for the massless scalar field ( see section 3.7 ) , where second - order perturbation theory in the scalar field is necessary to obtain an angular momentum perturbation in the stress - energy tensor
. however , neither of the predicted angular momentum scaling laws has been verified in numerical evolutions . for a perfect fluid with eos p
= k with 0 < k < 1/9 , precisely one mode that carries angular momentum is unstable , and this mode and the known spherical mode are the only two unstable modes of the spherical critical solution .
( note by comparison that from dimensional analysis one would not expect an uncharged critical solution to have a growing perturbation mode carrying charge . )
the presence of two growing modes of the critical solution is expected to give rise to interesting phenomena . near the critical solution ,
j and m of the final black hole are expected to depend on the distance to the black hole threshold and the angular momentum of the initial data through universal functions of one variable that are similar to
universal scaling functions in statistical mechanics ( see also the end of section 2.7 ) . while they have not yet been computed , these functions can in principle be determined from time evolutions of a single 2-parameter family of initial data , and then determine j and m for all initial data near the black hole threshold and with small angular momentum .
they would extend the simple power - law scalings of j and m into a region of initial data space with larger angular momentum .
they write the metric as 42\documentclass[12pt]{minimal }
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\begin{document}$$d{s^2 } = - { \alpha ^2}d{t^2 } + { \phi ^4}\left [ { { e^{2\eta/3}}{{(dr + { \beta ^r}dt)}^2 } + { r^2}{e^{2\eta/3}}{{(d\theta + { \beta ^\theta}dt)}^2 } + { e^{- 4\eta/3}}{r^2}{{\sin}^2}\theta d{\varphi ^2 } } \right],$$\end{document } where the lapse a , shift components and , and 3-metric coefficients and are functions of r , t , and . axisymmetry limits gravitational waves to one polarisation out of two , so that there are as many physical degrees of freedom as in a single wave equation . on the initial slice , and \documentclass[12pt]{minimal }
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\begin{document}$k_\theta ^r$\end{document } are given as free data , and \documentclass[12pt]{minimal }
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\begin{document}$\phi , k_r^r$\end{document } , and \documentclass[12pt]{minimal }
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\begin{document}$k_\varphi ^\varphi$\end{document } are determined by solving the hamiltonian constraint and the two independent components of the momentum constraint .
afterwards,\documentclass[12pt]{minimal }
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\begin{document}$\eta , k_\theta ^r , k_r^r$\end{document } , and \documentclass[12pt]{minimal }
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\begin{document}$k_\varphi ^\varphi$\end{document } are evolved , and only is obtained by solving the hamiltonian constraint .
is obtained by solving the maximal slicing condition \documentclass[12pt]{minimal }
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\begin{document}$${k_i}^i = 0$$\end{document } for , and and are obtained from the time derivatives of the quasi - isotropic spatial gauge conditions g = rgrr and gr = 0 . in order to keep their numerical grid as small as possible
the two free functions \documentclass[12pt]{minimal }
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\begin{document}$k_\theta ^r$\end{document } in the initial data were chosen to have the same functional form they would have in a linearised gravitational wave with pure l = 2 , m = 0 angular dependence .
this ansatz reduced the freedom in the initial data to one free function of advanced time .
limited numerical resolution allowed abrahams and evans to find black holes with masses only down to 0.2 of the adm mass .
even this far from criticality , they found power - law scaling of the black hole mass , with a critical exponent 0.36 .
the black hole mass was determined from the apparent horizon surface area , and the frequencies of the lowest quasi - normal modes of the black hole .
there was tentative evidence for scale echoing in the time evolution , with 0.6 , with about three echos seen . here
has the echoing property (er , et ) = (r , t ) , and the same echoing property is expected to hold also for , , , and r. in a subsequent paper , some evidence for universality of the critical solution , echoing period and critical exponent was given in the evolution of a second family of initial data , one in which = 0 at the initial time . in this family , black hole masses down to 0.06 of the adm mass were achieved .
it is striking that at 14 years later , these results have not yet been independently verified .
an attempt with a 3-dimensional numerical relativity code ( but axisymmetric initial data ) , using free evolution in the bssn formulation with maximal slicing and zero shift , to repeat the results of abrahams and evans was not successful .
the reason could be a combination of rather lower resolution than that of abrahams and evans , growing constraint violations , and an inappropriate choice of gauge .
( it is now known that bssn with maximal slicing and zero shift is an ill - posed system . ) in 2003 , choptuik , hirschmann , liebling and pretorius reported on numerical evolutions at the black hole threshold of an axisymmetric massless scalar field . in axisymmetry with scalar field matter
the slicing condition is maximal slicing and the spatial gauge , in cylindrical coordinates , is gzz = g , gz = 0 , similar to the gauge used by abrahams and evans .
the hamiltonian constraint is solved at every time step , and the time derivatives of the spatial gauge conditions are substituted into the momentum constraints to obtain second - order elliptic equations for the two shift components . thus the evolution is partially constrained .
the initial data were either time - symmetric or approximately ingoing , with the scalar field either symmetric or antisymmetric in z. in the symmetric case , even strongly non - spherical data were attracted to the known spherical critical solution for the massless scalar field . scaling with the known was observed in the ricci scalar .
however , with sufficiently good fine - tuning to the black hole threshold , the approximately spherical region that approaches the critical solution suffers an l = 2 ( and by ansatz m = 0 ) instability and splits into two new spherical regions which again approach the critical solution .
the spatial separation of the two new centres is related to the smallest length scale that developed prior to the branching .
there is evidence that with increasing fine - tuning each of these centres splits again .
the antisymmetric initial data can not approach a single spherical critical solution , but the solution splits initially into two approximately spherical regions where the critical solution is approached ( up to an overall sign in the scalar field ) .
the separation of these initial two centres is determined by the initial data , but there is evidence that they in turn split .
all this is consistent with the assumption that the spherical critical solution has , besides the known one spherical unstable mode , precisely one further l = 2 unstable mode .
( without the restriction to axisymmetry , if such a mode exists , it would be 5-fold degenerate with m = 2 , , 2 . )
choptuik and co - workers do not state with certainty that the mode they see in numerical evolutions is a continuum mode , although they have no indication that it is a numerical artifact .
the growth rate of the putative mode is measured to be 0.1 0.4 , which should be compared with the growth rate 2.7 of the spherical mode and the relatively small decay rate of 0.02 claimed in for the least damped mode , which is also an l = 2 mode .
we observe that the range of in figures 6 and 7 of is about 10 , and over this range the plot of the amplitude of the l = 2 perturbation against the log - scale coordinate seems equally consistent with linear growth in as with exponential growth .
( in the notation of , denotes proper time and * the accumulation point of echos , so that the log - scale coordinate used in this review corresponds to ln( * ) in the notation of . )
an interesting extension was made in by considering a complex scalar field giving rise to an axisymmetric spacetime with angular momentum .
the stress - energy tensor of a complex scalar field is 43\documentclass[12pt]{minimal }
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\begin{document}$${t_{ab } } = { 1 \over 2}({\psi _ { , a}}{\bar{\psi}_{,b } } + { \bar{\psi}_{,a}}{\psi _ { , b } } - { g_{ab}}{\bar{\psi}_{,c}}{\psi ^{,c}}).$$\end{document } an axisymmetric spacetime with azimuthal killing vector admits a conserved vector field tab , so that the total angular momentum 44\documentclass[12pt]{minimal }
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\begin{document}$$j = \int\nolimits_\sigma { { t_{ab}}{\xi ^a}{n^b}dv}$$\end{document } is independent of the cauchy surface . with the ansatz 45\documentclass[12pt]{minimal }
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\begin{document}$$\psi ( \rho , z , t , \varphi ) = \psi ( \rho , z , t){e^{im\varphi}}$$\end{document } in adapted coordinates where = / and with m being an integer , the stress - energy tensor becomes axisymmetric and hence compatible with the einstein equations for an axisymmetric spacetime . for any tangent to , in particular a hypersurface t = constant , the angular momentum density measured by a normal observer becomes 46\documentclass[12pt]{minimal }
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\begin{document}$$j = { t_{ab}}{\xi ^a}{n^b } = - { { i m } \over 2}\left({\pi \bar \phi - \bar \pi \phi } \right ) = m{a^2}{n^a}{\delta _ { , a}},$$\end{document } where = ae with and a being real and = na,a . by comparison the energy density measured by a normal observer is 47\documentclass[12pt]{minimal }
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\begin{document}$$\rho = { t_{ab}}{n^a}{n^b } = \prod \bar{\prod}+ { d_a}\phi { d^a}\bar{\phi,}$$\end{document } where da is the derivative operator projected into . this means that the ratio of energy density to angular momentum density can be adjusted arbitrarily in the initial data , including zero angular momentum for a that is real ( up to a constant phase ) . on the other hand , even in the absence of angular momentum a purely real obeys a wave equation with an explicit m/ centrifugal term . for the same reason , regular solutions must have on the axis , and there are no spherically symmetric solutions .
intuitively speaking , the centrifugal force resisting collapse appears unrelated to the angular momentum component of the stress - energy tensor in a way that differs from what one would expect in rotating fluid collapse or rotating ( non - axisymmetric ) vacuum collapse . for all initial data in numerical evolutions
, a critical solution is approached that is discretely self - similar with log - scale period 0.42 .
( by ansatz this solution is axisymmetric but spherical symmetry is ruled out and so the critical solution can not be the choptuik solution . ) the same critical solution is approached in particular for initial data with \documentclass[12pt]{minimal }
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\begin{document}$\pi = 0$\end{document } and hence no angular momentum , and initial data where \documentclass[12pt]{minimal }
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\begin{document}$\pi = \bar \phi$\end{document } and hence with large angular momentum .
a scaling exponent of 0.11 is observed in the ricci scalar in subcritical evolutions .
the critical solution is purely real ( up to an initial data - dependent constant phase ) and hence has no angular momentum . only m = 1 was investigated , but it is plausible that a different critical solution exists for each integer m. far from the black hole threshold j m in the final black hole , but nearer the black hole threshold , j m , where j and m are measured on the apparent horizon when it first forms .
j / m 0 is compatible with a non - rotating critical solution . in the absence of angular momentum , the wave equations for the real and imaginary part of decouple .
assuming that the background critical solution is purely real with = 0 and a 1 , and angular momentum is provided by a perturbation with e , one would expect |a| e and j a|| e. integrating over a region of size e when the black hole forms , we find \documentclass[12pt]{minimal }
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\begin{document}$m \sim { e^{- { \tau _ \ast}}}$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$j \sim { e^{(- 2 + \lambda){\tau _ \ast}}}$\end{document}. then j m would imply = 4 .
study a similar system in spherical symmetry , by arranging 2l + 1 scalar fields given by lm = (r , t)ylm( , ) for m = l , l with the same (r , t ) for all values of m so that the total stress - energy tensor and the spacetime are spherically symmetric .
note that not the lm are added but their stress - energies , and each value of l describes a different matter content. then sees a centrifugal barrier in its evolution equation , but there is no angular momentum in the spacetime .
dss critical behaviour is found , and the logarithmic echoing period and mass scaling exponent both decrease approximately exponentially with l. it is observed empirically that the radius r0 of maximum compactness during evolution ( a measure of the scale of the initial data ) and the accumulation time of echos t * ( measured from the initial data ) obey t * r0/(4.35 ) for all l and families of initial data .
lai has studied type i critical phenomena for boson ( massive complex scalar field ) stars in axisymmetry , the first study of type i in axisymmetry .
he finds that the subcritical end state is a boson star with a large amplitude fundamental mode oscillation . a first investigation of type i critical collapse in an astrophysically motivated scenario was carried out in [ 136 , 204 ] .
eos p = ( 1) , where 2 is a constant , p is the pressure , the rest mass density , and the internal energy per rest mass ( so that (1 + ) is the total energy density ) .
the initial data are constructed with p = k for a constant k , which corresponds to the cold ( constant entropy ) limit of the gamma law eos .
( the evolution starts at finite distance , with an initial velocity calculated in the first post - newtonian approximation ) .
the entire solution is axisymmetric with an additional reflection symmetry that maps one star to the other .
the parameter of the initial data that is varied is the mass of the two stars .
supercritical data form a single black hole , while subcritical data form a single star .
the diagnostics given are plots against time of the lapse and the ricci scalar at the symmetry centre of the spacetime , and of outgoing l = 2 gravitational waves .
collapse of the lapse and blowup of the ricci scalar are taken as indications of black hole formation .
the limited numerical evidence is compatible with type i critical phenomena , with the putative critical solution showing oscillations with the same period in the lapse and ricci scalar .
for the critical solution to be exactly time - periodic , it would have to be spherical in order to not lose energy through gravitational waves , and there is some evidence that indeed it does not radiate gravitational waves .
other 1-parameter families of initial data were obtained by fixing the mass of the stars in the first family very near its critical value and then varying one of the following : the initial separation , the initial speed , and . for all approximately the same scaling law for the survival time of the critical solution was found .
however , as all these initial data are very close together , this only confirms the validity of the general perturbation theory explanation of critical phenomena , but does not provide evidence of universality .
a key question that has not been answered is how shock heating affects the standard critical collapse scenario .
a priori the existence of a universal spherical critical solution is unlikely in the presence of shock heating as there is no dynamical mechanism to arrive at a universal spherical temperature profile , in contrast to non - dissipative matter models or vacuum gravity where all equations are wave - like .
interesting numerical evidence for critical phenomena in the grazing collision of two black holes has been found by pretorius .
he evolved initial data for two equal mass nonrotating black holes , with a reflection symmetry through the orbital plane , parameterised by their relative boost at constant impact parameter .
the threshold in initial data space is between data which merge immmediately and those which do not ( although they will merge later for initial data which are bound ) .
the critical solution is a circular orbit that loses 1 1.5% of the total energy per orbit through radiation . on both sides of the threshold , the number of orbits scales as 48\documentclass[12pt]{minimal }
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\begin{document}$$n \simeq - \gamma \ln \vert p - { p_\ast}\vert$$\end{document } for 0.31 0.38 .
the simulations are currently limited by numerical accuracy to n < 5 and e / e < 0.08 , but pretorius conjectures that the total energy loss and hence the number of orbits is limited only by the irreducible mass of the initial data , a much larger number . in particular , he speculates that for highly boosted initial data such that the total energy of the initial data is dominated by kinetic energy , almost all the energy can be converted into gravitational radiation . the standard dynamical systems picture of critical collapse , with the critical solution an attractor in the threshold hypersurface , appears to be consistent with these observation .
pretorius compares his data with the unstable circular geodesics in the spacetime of the hypothetical rotating black hole that would result if merger occurred promptly .
these give orbital periods , and their linear perturbations give a critical exponent , in rough agreement with the numerical values for the full black hole collision .
pretorius does not comment on the nature of the critical solution , but because of the mass loss through gravitational radiation it can not be strictly stationary .
the mass loss would be compatible with self - similarity , with a helical homothetic vector field , but exact self - similarity would be compatible with the presence of black holes only in the infinite boost limit . therefore the critical solution is likely to be more complicated , and can perhaps be written as an expansion with an exactly stationary or homothetic spacetime as the leading term .
pretorius also speculates that these phenomena generalise to generic initial data with unequal masses and black hole spins which are not aligned .
this seems uncertain , given the claim by levin that the threshold of immediate merger is fractal if the spins are not aligned , and that the system is therefore chaotic .
however , levin s analysis is based on a 2nd order post - newtonian approximation to general relativity , in which there is no radiation reaction , while the rapid energy loss observed here may suppress chaos .
nevertheless , the phase space is much bigger when the orbit is not confined to an orbital plane , and so the critical solution observed here may not be an attractor in the full critical surface .
push the numerics towards higher energies emitted and conjecture that the merger of nonspinning black holes can in principle yield a black hole arbitrarily close to extremal kerr . | as first discovered by choptuik , the black hole threshold in the space of initial data for general relativity shows both surprising structure and surprising simplicity .
universality , power - law scaling of the black hole mass , and scale echoing have given rise to the term critical phenomena .
they are explained by the existence of exact solutions which are attractors within the black hole threshold , that is , attractors of codimension one in phase space , and which are typically self - similar .
critical phenomena give a natural route from smooth initial data to arbitrarily large curvatures visible from infinity , and are therefore likely to be relevant for cosmic censorship , quantum gravity , astrophysics , and our general understanding of the dynamics of general relativity . | Introduction
Theory
The Scalar Field
More Spherical Symmetry
Beyond Spherical Symmetry | take generic initial data in general relativity , adjust any one parameter p of the initial data to the threshold of black hole formation , and compare the resulting spacetimes as a function of p. in many situations , the following critical phenomena are then observed :
near the threshold , black holes with arbitrarily small masses can be created , and the black hole mass scales as 1\documentclass[12pt]{minimal }
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\begin{document}$$m \propto { ( p - { p_\ast})^\gamma},$$\end{document } where p parameterises the initial data and black holes form for p > p*.the critical exponent is universal with respect to initial data , that is , independent of the particular 1-parameter family , although it depends on the type of collapsing matter.in the region of large curvature before black hole formation , the spacetime approaches a self - similar solution which is also universal with respect to initial data , the critical solution . in our view , the main physical motivation for this study is that those critical solutions which are self - similar provide a way of achieving arbitrarily large spacetime curvature outside a black hole , and in the limit a naked singularity , by fine - tuning generic initial data for generic matter to the black hole threshold . by perturbing around spherical symmetry , the stability of the choptuik solution in the full phase space can be investigated , and the scaling of black hole angular momentum can be predicted . the power - law scaling ( 10 ) was obeyed from those smallest masses up to black hole masses of , for some families , 0.9 of the adm mass , that is , over six orders of magnitude . roughly speaking
, fine - tuning to the black hole threshold provides a set of data which is codimension one in the space of generic , smooth , asymptotically flat initial data , and whose time evolution contains at least the point singularity of the critical solution . as the underlying physics is perfectly scale - invariant in the massless scalar field model , the minimum mass must be determined by the family of initial data through the infall of matter into the black hole . fine - tuning of a parameter may result in m , ( p p*) but the final mass m2 would be approximately independent of p. given the scaling power law for the black hole mass in critical collapse , one would like to know what happens if one takes a generic 1-parameter family of initial data with both electric charge and angular momentum ( for suitable matter ) , and fine - tunes the parameter p to the black hole threshold . from this analysis , a universal power - law scaling of the black hole charge 33\documentclass[12pt]{minimal }
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\begin{document}$$q \sim { ( p - { p_\ast})^\delta}$$\end{document } was predicted . an example of the richer phenomenology in the presence of a scale in the field equations is the spherical massive scalar field with a potential m coupled to gravity : in one region of phase space , with characteristic scales smaller than 1/m , the black hole threshold is dominated by the choptuik solution and type ii critical phenomena occur . kol relates a solution that is related to the choptuik solution to a variant of the critical solution in the black - string black hole transition , and claims to obtain analytic estimates for and . a recent investigation of andrasson and rein with massive particles has confirmed again the existence of a mass gap and the existence of metastable static solutions at the black hole threshold , though there is no estimation of the scaling of their life - times . even this far from criticality , they found power - law scaling of the black hole mass , with a critical exponent 0.36 . | [
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ly2784544
has been identified as a selective inhibitor of jak2-v617f
and has undergone clinical trials for the treatment of several myeloproliferative
disorders ( scheme 1 ) .
a significant challenge associated with the formation of the functionalized
imidazopyridazine is the introduction of the benzylic morpholine unit .
a first - generation synthesis employed a silver - catalyzed minisci radical
alkylation with n - phthaloylglycine
( scheme 1 ) .
this method suffered from poor
selectivity for the desired c-8 position , as well as forming significant
quantities of a bis - alkylation product . at large
scale , the minisci chemistry proved challenging due to the insolubility
of the products and the thick mixtures that were formed .
additionally ,
from a route design perspective , formation of the morpholine in this
fashion was undesirable due to the use of a protecting group . in order to address these concerns ,
a second - generation synthesis , which employed
a vanadium - catalyzed addition of n - methylmorpholine - n - oxide ( nmo ) was developed that displayed greater scalability ,
improved yield , and product quality .
this method ultimately delivered
> 1 metric tonne of imidiazopyridizine 2 in a highly
reproducible
manner .
while the exact mechanistic pathway for this reaction has
not been elucidated , one possibility proceeds via the intermediacy
of an -amino radical .
other methods for generation of the intermediate
radical were subsequently investigated , including the use of photochemistry .
initial studies found that under irradiation with a hg - lamp , a range
of sensitizers were successful in promoting the addition of n - methylmorpholine ( nmm ) to the pyridazine core , albeit
in low conversions and with relatively high levels of impurities .
the use of nmm would represent a significant advantage as it is available
at greatly reduced cost and the safety and bulk sourcing concerns associated with anhydrous nmo would be
alleviated .
visible light - mediated photocatalysis has developed
into a defined
research field with a recent increase in both the number and diversity
of reported transformations . within this
arena
the generation
of -amino radicals has been demonstrated
from both unfunctionalized amines , and
those containing adjacent activating groups such as co2h or tms .
while most reports demonstrate the addition of -amino radicals
to activated alkenes , macmillan ( scheme 2a ) has shown the efficient arylation
of simple n - aryl amines with either cyano or chloro-
substituted arenes and heteroarenes . furthermore , given the quantity
of established methods for the functionalization of amine containing
compounds , there remain relatively few efforts that have demonstrated
the applicability of photocatalysis to a pharmaceutical or agrochemical
setting , especially with regard to the formation of challenging bond
constructs .
recent elegant studies by nagib and macmillan as well as dirocco and co - workers , among others have identified photocatalysis as an efficient strategy
for the functionalization of biologically relevant heterocycles ( scheme 2b ) .
these reported methods appear ideally suited
for the rapid generation of a collection of compounds during early
stage discovery chemistry .
later stage development demands a different
set of criteria , notably selectivity , efficiency , and scalability .
a limited number of recent reports have demonstrated the use of photocatalysis
en route to known pharmaceutical compounds , such as the efficient 3-step synthesis of pregabalin , albeit in a racemic fashion and with the key
step conducted on < 100 mg scale .
considering the scarcity of industrially relevant examples
and
the unanswered questions of how visible light photocatalysis should
best be conducted at production scale , it currently remains unclear
whether it can be successfully applied in a process development setting . with these questions in mind
, we envisioned the introduction of
the benzylic morpholine in ly2784544 as an ideal opportunity to test
the viability of a photoredox transformation in a complex pharmaceutical
setting ( scheme 2c ) .
we were particularly drawn
to the mild reaction conditions and functional group tolerance typically
associated with visible light - mediated photocatalysis and furthermore
were encouraged by the precedent for the efficient formation of -amino
radicals .
we started with
initial conditions
similar to those reported for the -arylation of n - aryl amines but employed oxygen as
the terminal oxidant by running the reactions open to air .
our light
source consisted of 2 4 w blue led light strips which raised the temperature of the reaction
to 35 c . after 7 h , using 2 mol % of ir(ppy)3 , with 20 equiv of nmm , all of the imidazopyridazine
starting material had been consumed ( as measured by hplc ) .
the desired exo isomer 2 ( the product of addition from
a speculated radical at the exocyclic ch3 position ) was
identified as the major product ( 63% area / area ( a / a ) hplc ) ( scheme 3 ) .
an expected major impurity , the endo regioisomer 3 ( the product of addition from a speculated
radical at the endocyclic ch2 group ) , was present in 11%
( or 5.7:1 exo : endo ) which is greater
than that observed in the second generation route employing nmo and
vo(acac)2 where the endo isomer was formed
in < 5% .
consistent with the previous minisci alkylation the competitive
addition of the -amino radical to the c-7 position of the imidazopyridazine
( 4 ) was observed , albeit in greatly reduced quantity
( 3% vs 25% ) .
further impurities unique to
the photocatalysis protocol were identified
as the reduction of the carbon - chlorine bond ( 5 ) ( 6% ) ,
which is not unexpected given the precedent for photocatalysed aryl
halide bond reduction .
more interestingly
the product of formal methylation of the imidazopyridazine ( 6 ) ( 5% ) and of the addition of dma ( 7 ) ( 12% )
were also present . while the crude 63% a /
a of the desired product
appeared high under these conditions , the reaction was inconsistent
and the isolation of pure 2 via chromatography proved
challenging .
furthermore , the bis - alkylation product 8 , a significant impurity in the n - phthaloylglycine
minisci reaction , could not be reliably resolved using the existing
hlpc method . in order to quantify the reaction products with
more certainty ,
an analytical uplc ( ultra performance liquid chromatography ) method
was developed with both a photodiode array ( pda ) and mass detection
( qda ) analyzer , which proved pivotal for the further optimization
of this reaction .
these analytical systems allowed the analysis of
a reaction mixture to a higher level of sensitivity in greatly reduced
time . at this juncture , we decided to control the reaction temperature
via the use of a jacketed beaker connected to a recirculating chiller .
previously , the temperature of the reaction had been influenced by
the light - source , a feature that can be often overlooked .
running
the reaction at 22 c and using a 1 w led strip , while monitoring
with the updated analytical methods , the reaction was found to be
complete ( < 5% 1 remained ) in 16 h. the desired exo isomer 2 , was again the main component
in the reaction mixture ( 65% a /
a ) with a calculated uplc yield of
57% and 59% respectively , for the two quantitation methods ( uplc - pda
and uplc - qda ) ( table 1 entry 1 ) . under these conditions
the exo : endo ratio was 5.5:1 , while the other quantifiable
impurities were individually controlled to < 5% . through improved chromatography conditions , 2
this agreement between the two analytical
yields and the isolated material provided us with confidence in our
analytical method going forward .
we next sought to utilize the
jacketed beaker system by exploring the reaction at lower temperature .
at 5 c a similar yield and exo : endo ratio
could be obtained in only 8 h , potentially due to higher oxygen
solubility at this temperature ( table 1 , entry
2 ) .
further evidence of the important
role of oxygen was observed when the reaction was run at twice the
scale , resulting in a significant decrease in yield ( entry 3 ) .
this
is attributed to a decrease of relative surface area of solution exposed
to air . further probing the effect of
oxygen we found a deoxygenated reaction gave minimal product ( < 10% )
( entry 4 ) . conducting the reaction under an atmosphere of oxygen gave
both a reduction in the quantity of 2 and in the exo : endo ratio ( entry 5 ) .
furthermore ,
a control reaction utilizing a balloon of air equally gave reduced
yield and exo : endo ratio ( entry
6 ) . in an effort to address the issue of scalability
, air was continuously
sparged through the reaction leading to identical reactions at different
scales , but with significantly decreased exo : endo ratio and with increased reaction time ( entries 7 and
8) .
the poor scalability of this batch reaction was not considered
a critical factor at this juncture .
any scale up would likely be more
efficiently conducted via continuous processing ; therefore , optimization was continued at the current scale .
the
reaction also showed a high degree of sensitivity to the light
source . in the absence of led light , or ir(ppy)3 , less
than <
5% 2 was observed . varying the total led power ,
independently of temperature , showed that increasing led power had
a detrimental effect on selectivity ( figure 1 ) .
most strikingly , after 4 h with 9
w led irradiation < 10% 1 remained ; however , the exo product 2 was only observed in 23% yield .
the remaining material balance ( by uplc ) contained elevated amounts
of previously identified side products ( 5 5% , 4 7% ) but also comprised a large quantity of uncharacterized material
that made quantitation of other known impurities such as 6 and 7 difficult ( figure 1 right ) .
uplc chromatograms
showing reaction profile at 4 h using a 1 w
led ( left ) and a 9 w led ( right ) . while the exo product 2 is
stable
to the reaction conditions under 1 w led irradiation , increasing the
power to 8 w and resubjection to the reaction conditions gave ketone
reduction ( 9 ) as the major product ( 35% ) among a complex
mixture also containing 6 ( 7% ) and dechlorinated 2 ( 7% ) .
the addition of 5 equiv of formic acid provided a
cleaner reaction profile leading to methylation product 6 , isolated in 20% yield ( scheme 4 ) .
this strongly
suggests that 6 is derived from 2 and presumably
is formed via c
no significant decomposition
of 2 was observed in the absence of ir(ppy)3 or nmm , and was greatly reduced in the absence of oxygen ( 72% 2 remained after 8 h ) suggesting this potentially useful transformation is a photocataylsed process . given the limited
solubility of naoac in dma a range of both organic
and inorganic bases were screened .
the
initial choice of 1 equiv of naoac proved optimal with other bases
delivering reduced yield , and surprisingly , reduced exo : endo ratio ( table 2 , entries
16 ) .
the exo : endo ratio
also varied with the amount of nmm . using 60 equiv a 1.2:1 exo : endo ratio was obtained ( entry 7 ) ,
lowering the equivalents below 20 displayed a clear trend for increased exo : endo ratio with decreasing quantities
of nmm , albeit at the expense of 2 ( entries 810 ) .
this is potentially rationalized by competition between radical addition
of the -amino radical and c h abstraction from a molecule
of nmm .
the primary -amino radical could abstract the weaker
secondary c h bond to give the secondary -amino radical
that then may add to the imidazopyridazine core to ultimately generate
the endo isomer 3 .
reactions performed
at a 0.231 mmol
scale , at 5 c with 1.0 mol % ir(ppy)3 , 1.0 equiv
of base , 20 equiv of nmm for 8 h unless otherwise stated . calculated
by uplc ( pda ) .
reaction
performed at a 0.774 mmol
scale , at 35 c with 2.0 mol % ir(ppy)3 , for 12 h. calculated via h nmr
analysis of the crude reaction mixture .
reaction time of 10 h. seeking to lower the cost burden associated with the
use of iridium ,
we screened a wide range of other common photocatalysts .
all ruthenium complexes tested under the standard
reaction conditions returned only starting material ( table 3 , entries 12 ) .
these photocatalysts would
likely operate via reductive quenching of the excited state by nmm
and their consistent failure to promote the reaction may indicate
a general problem when applying reductive quenching to this particular
system .
more promisingly , 5 mol % of the organic photocatalyst eosin
y provided a 22% yield of 2 after 15 h , but with a reduced exo : endo ratio of 3.7:1 ( entry 3 ) .
other
iridium - based photocatalysts were capable of promoting the reaction
but in greatly reduced yield ( 1121% yield ) and with lower exo : endo selectivity ( entries 46 ) .
the unique efficiency of ir(ppy)3 with regard to yield
and exo : endo selectivity is intriguing ,
especially given the similar reduction potentials of ir(fppy)3 ( e1/2 reactions performed at a 0.231 mmol
scale , at 5 c with 1.0 mol % photocatalyst , 1.0 equiv of base ,
20 equiv of nmm for 8 h unless otherwise stated .
reaction time of
10 h. reaction time of 15
h. screening a range of
solvents typically employed in visible light
photocatalysis revealed dmf as comparable to dma at room temperature
( 53% , 5.3:1 exo : endo , 16 h ) .
dmso
provided an increased exo : endo ratio
but significantly lower conversion and yield ( 25% 1 ,
38% 2 , 6.5:1 exo : endo , 16 h ) . increasing the reaction time to 24 h provided 50% yield
of 2 , with 13% 1 remaining , but further
increase in reaction time
mecn did not
efficiently solubilize 1 at 5 c and at 20 c
provided only 12% of the desired product 2 .
concentration
and analysis of the mecn reaction mixture revealed the presence of
nmo , the morpholine source in the corresponding
vo(acac)2 chemistry .
a subsequent control reaction showed
that nmo is not an effective starting material in this process .
interestingly
the use of dmpu gave 45% of the solvent addition products 10 and 11 .
excluding the nmm from the reaction provided
clean conversion to a 1:5 ratio of exo : endo with a combined 53% yield of the inseparable products ( scheme 5 ) .
h
abstraction of the solvent from the ho2 radical , generated following oxygen quenching of the ir(ppy)3 excited state and protonation , and could provide a method
for heterocyclic functionalization in its own right .
given the high exo : endo ratio
observed for the nmo / vo(acac)2 system run in etoh , we next
screened a range of polar protic solvent additives . while the addition of etoh or meoh had limited effect on
both the yield and exo : endo ratio ,
the use of water ( 10:1 dma : h2o ) gave considerably improved exo : endo selectivity ( 12:1 ) at the expense
of reaction time , requiring 24 h to achieve 55% yield with 10% 1 remaining .
the increased reaction time is possibly due to
the lower solubility of ir(ppy)3 in the solvent mixture ,
whereas the cause of increased in exo : endo remains to be elucidated .
further optimization of the dma : h2o conditions by reducing
the reaction temperature to 0 c and using only 0.5 mol % of
ir(ppy)3 resulted in a precipitation of 2 during
the course of the reaction . while this served to both ease isolation
and prevent the product from further reaction ,
premature precipitation
at moderate levels of conversion effectively blocked light penetration
and stalled the reaction .
the solids could be filtered and the filtrate
further progressed ; however , this method proved impractical and gave
material with reduced purity ( 8090% a / a uplc ) and yield ( 4549% ) .
attempts to increase the scale of the reaction while employing these
conditions , again proved problematic .
the scale of the reaction was
increased by a factor of 4 ( 300 mg , 0.92 mmol 1 ) and
the surface area by two , in an effort to mitigate the previously observed
rate decrease upon scale up . at this scale
the premature precipitation
of 2 was a more significant issue , and the reaction time
increased to 72 h. even after this duration only a 37% isolated yield
of 2 was obtained in 80% purity . returning to the standard
scale , and diluting the reaction to 0.09 m retained 2 in solution for an increased duration , allowing greater conversion
( approximately 75% of 2 , 12:1 exo : endo ) .
under the fully optimized process , significant quantities
of 2 precipitated after 32 h. addition of a solution
of 3:1 h2o : etoh followed by filtration , provided 56% isolated
yield with a 90% purity ( a / a uplc ) ( scheme 6 ) .
the purity of this material could be further improved to > 98%
with simple extraction into a solution of 1 n hcl and reisolation
via basification to ph 12 . the final optimized reaction
is superior to the initial first generation
minisci route with regard to selectivity for 2 , step
efficiency , and yield .
furthermore , replacement of agno3 and tfa with 0.5 mol % ir(ppy)3 is an attractive prospect
from a waste management perspective , although the limited scalability
and cost concerns surrounding the use of iridium represent significant
issues to be addressed .
ultimately , any procedure on scale employing
photocatalysis would likely need to be conducted via continuous processing ;
this offers an exciting opportunity to address these current limitations
and research in this direction is underway . despite the
challenges that were encountered in attempts to scale
the methodology , the demonstration of efficiency and selectivity prompted
us to explore the reaction as a tool for discovery chemistry .
both
the previous routes are not ideal for the synthesis of analogues ,
limited by high step count or the availability of the requisite amine - n - oxide . testing a range of simple , commercially available
tertiary amines
we were rapidly able to build a small collection of
imidazopyridizine analogues that would be difficult to prepare by
other means ( scheme 7 ) .
the isolated yields
were moderate ( 2056% ) , yet synthetically useful , and the scope
of the tertiary amine component encompassed aliphatic , benzylic , and
aromatic n - methyl tertiary amines .
in certain cases
( such as 12 ) the reaction appeared to stall and increasing
both the catalyst loading and the equivalents of amine had limited
effect .
radical addition was selective through the n - methyl group when employing n , n - dimethylglycine methyl ester or n , n - dimethybenzylamine , possibly due to the increased nucleophilicity
of a primary radical when compared to the secondary radical . tetramethylethylenediamine ( tmeda ) proved a
particularly efficient
amine in terms of both reaction time and regioisomeric ratio ( r.r ) ( 14 , 7 h , > 10:1 r.r ) .
postulating that this may be due to an efficient intramolecular deprotonation
of the amine radical cation through a 5-membered transition state ,
we tested tetramethylhexanediamine .
this amine proceeded with an equally
high regioisomeric ratio ( r.r > 10:1 ) but with
decreased
yield and increased reaction time ( 15 , 18 h , 24% ) suggesting
the improved selectivity is more likely the kinetic effect of an increased
number of n - methyl groups . in the case of n , n - dimethylethylamine
1.0 mol % ir(ppy)3 was employed to increase the reaction rate to compete productively
with evaporation of the amine ( bp 3638 c ) , a problem
not encountered with the other amines .
the configurational stability
of a stereogenic center alpha to nitrogen was also tested under the
reaction conditions , with minimal erosion of enantiomeric ratio ( e.r ) observed ( 21 ) .
this implies that radical
formation at this position is disfavored or the resultant radical
can not efficiently re - enter the catalytic cycle via h atom abstraction .
n - methylpyrrolidine , n - ethylmorpholine
and triethylamine failed to give any discernible product , while 1-methypiperizine
gave a complex mixture , presumably due to the presence of a secondary
amine .
intriguingly , given their reactivity in related systems employing
electron deficient alkenes or dicyanobenzene , diphenylmethylamine
and n - phenylmorpholine returned only starting material .
attempts were then made to assess the viability of the reaction to
a limited selection of other imidazopyridazines that are potential
intermediates in the synthesis of ly2784544 .
imidazopyridazine 22 ( scheme 8) with an ethyl ester at
c-3 has been extensively studied as an alternative precursor for the
nmo / vo(acac)2 chemistry , although it showed initially poor exo : endo selectivity ( 3:1 ) when compared
to 1 ( not shown ) .
similarly ,
poor exo : endo selectivity was observed
in this visible light - mediated system giving a 1.1:1 exo : endo ratio and only moderate isolated yield ( 36% 22 , 19% 24 , not shown ) .
attempting to improve
the exo : endo ratio via the use of
the 10:1 dma : h2o solvent conditions employed previously
led to no improvement in selectivity ( scheme 8) .
interestingly under these conditions the formation of methylated
imidazopyridazine 25 , analogous to the formal methylation
product 7 , could also be observed ( scheme 8) .
furthermore , while no reaction was observed
on the parent heterocyclic
core 28 without substitution at the c-3 position , the
benzylic imidazopyridiazine 26 gave a low isolated 14%
yield of the exo product 27 .
the next
step in the synthesis of ly2784544 after 2 is the carbonyl
reduction giving 27 , a transformation that required significant
investigation .
the formation of the key
benzylic morpholine unit from 26 would therefore offer
the attractive prospect of eliminating the carbonyl reduction sequence .
additionally the reactivity of 26 , albeit moderate , suggested
the intriguing possibility of extending this reaction to less activated
heterocycles . to this end , a selection of pyridazines that have
previously been
shown to undergo minisci - type radical addition of n - protected amino acid derived radicals were initially tested .
unfortunately , no significant addition products
could be detected other than in the case of 3,6-dichloropyridazine 29 , where a mixture of products consistent with radical addition
were detected but proved difficult to isolate .
other simple heterocyclic scaffolds representative of common
fragments found in pharmaceutical and agrochemical compounds were
subsequently investigated ( scheme 8) .
in all
cases the majority of the starting material was recovered and no significant
formation of radical addition products could be detected .
the nmo / vo(acac)2 chemistry also displays limited substrate scope for the heterocyclic
portion , raising questions over the seemingly unique reactivity of
the substituted imidazopyridazine and the mechanistic pathways of
both these reactions . while the current methodology appears limited
with regards to the generality of the heterocyclic portion , the ease
and simplicity of the reaction set up , combined with the availability
of n - methyl tertiary amine coupling partners renders
this an attractive reaction to test on existing compound libraries .
given the demonstrated sensitivity
of this reaction to a range of factors such as oxygen , light source ,
base , and photocatalyst , it is a strong possibility that multiple
reaction mechanisms may be operational .
one simplistic mechanistic
pathway is depicted in figure 2 whereby the
ir(ppy)3 undergoes initial photoexcitation to a long - lived
excited state that can undergo bimolecular quenching with oxygen to
generate ir(iv ) and the o2 radical .
the resultant ir(iv ) ( e1/2 = + 0.77 vs sce ) may then oxidize nmm to the radical cation and return to the ground state .
the primary
-amino radical 30 is then generated via deprotonation
of the radical cation before addition to imidazopyridazine 1 and rearomatization to yield benzylic morpholine 2 .
volmer analysis , showed that imidazopyridazine 1 is a quencher
of the excited state ir(ppy)3 in the presence of oxygen , and therefore can not exclude mechanisms proceeding
from this initial event .
we probed the
possibility of radical propagation contributing to the mechanistic
complexity by conducting a lights - on - lights - off experiment .
minimal reaction was observed during the periods
of dark suggesting a long - lived radical propagation is not an major
pathway under these conditions .
additionally
the exo : endo selectivity observed
is opposed to that expected if the intermediate -amino radical
was formed via c h abstraction .
a potential mechanism , which we
deem unlikely , is the generation
of an iminium species and friedel crafts type addition of the
imidazopyridazine core .
the use of preformed iminium species 31 in the absence of photocatalyst gave no observable coupling
product .
however , the use of iminium 31 under photocatalytic
conditions in the absence of oxygen provided 2 with high
selectivity , albeit in low yield ( 13% , > 20:1 exo : endo , scheme 9 )
. this may
demonstrate
the possibility of a redox neutral process by which reduction of the
iminium by the excited state of ir(ppy)3 provides the -amino
radical 30 .
alternatively direct reduction of the imidazopyridazine
followed by addition to the iminium provides another mechanistic possibility
that can not be ruled out .
in conclusion , we have successfully developed
a visible light - mediated
photochemical transformation to address a challenging bond construction
en route to the jak2 inhibitor ly2784544 .
the introduction of this
benzylic morpholine unit occurs in moderate yield , with good selectivity
and product purity and is competitive with previously reported routes
in these respects .
importantly , nmm is utilized directly , which has
considerable benefit when compared to the nmm surrogates employed
in the first and second generation syntheses .
furthermore , the methodology
was amenable to a range of simple , commercially available tertiary
amines and delivered a range of analogues in synthetically useful
yields .
while the developed batch conditions are not currently scalable ,
the potential application to continuous processing is an attractive
method to allow the further development of this step , and efforts
in this direction are ongoing . during the course of this investigation
a number of interesting photoredox transformations have been observed ,
such as formal methylation via c n bond cleavage , functionalization
of c
overall , we believe that this initial demonstration
warrants the wider consideration of visible light - mediated photocatalysis
as a tool in process development as well as the potential application
of this specific transformation to the diversification of pharmaceutical
and agrochemical scaffolds .
solvents ( mecn ,
dmf , dmso ) were obtained from a solvent purification system .
reactions
that were monitored by tlc were visualized by a dual short wave / long
wave uv lamp and stained with an ethanolic solution of potassium permanganate .
column flash chromatography was performed using 230400 mesh
silica gel or via automated column chromatography .
jacketed beakers were
obtained from chemglass and connected to a recirculating heater chiller
with sil 180 silicone oil as the coolant .
h , c and f nmr spectra were recorded using an internal
deuterium lock on 400 , 500 , or a 700 mhz spectrometer .
all signals
are reported in ppm with the internal reference of the specified solvent .
data are presented as follows :
integration , multiplicity ( s = singlet , d = doublet , t = triplet ,
q = quartet , m = multiplet , br = broad , app = apparent , dd = doublet
of doublet , dt = doublet of triplet , etc . ) and coupling constant ( j / hz ) .
infrared spectra were recorded on a ft - ir fitted
with an atr accessory .
photocatalyst quenching was conducted on a fluorimeter and the values
represent an average of 3 samples .
uplc analysis was conducted on
a uplc with a beh c18 column ( 1.7 m 2.1 50 mm ) .
chiral
hplc analysis was conducted on a hplc with chiral pak od - h column
( 4.6 250 mm ) at 254 nm at 22 c . to a 25 ml round bottom flask ( rbf ) open
to air
was added 1 ( 125 mg , 0.386 mmol , 1.0 equiv ) , ir(ppy)3 ( 5.00 mg , 0.008
mml , 0.02 equiv ) , sodium acetate ( 32.0 mg , 390 mmol , 1.0 equiv ) , dma
( 2.5 ml ) and n - methylmorpholine ( 0.850 ml , 7.71 mmol ,
20 equiv ) and the yellow heterogeneous solution was irradiated with
2 4 w led strips placed in a circular loop around the flask .
aluminum foil was placed over the lights around the flask to contain
the light ( see figure s1 , supporting information ) .
after 1 h the internal temperature of the flask had stabilized
at 35 c .
max ( atr)/cm 3048 , 2361 , 1603 , 1569 , 1502 ,
1479 ,
1411 , 1318 , 1268 , 1197 , 914 , 887 , 806 , 761 and 611 ; h
nmr ( 500 mhz , cdcl3 ) h 8.26 ( 1h , dd , j 4.5 , 1.7 ) , 7.95 ( 1 h , dd , j 9.1 , 1.7 ) ,
7.63 ( 1h , dd , j 8.2 , 7.5 ) , 7.29 ( 1h , dd , j 8.4 , 2.0 ) , 7.19 ( 1h , dd , j 9.1 , 4.5 ) ,
7.10 ( 1h , dd , j 9.9 , 1.9 ) and 2.68 ( 3 h , s ) ; c nmr ( 126 mhz , cdcl3 ) c 180.3 ,
160.4 ( d , j 255.4 ) , 153.0 , 143.0 , 140.3 , 138.9 ( d , j 10.4 ) , 131.4 ( d , j 3.7 ) , 127.2 ( d , j 14.0 ) , 125.3 ( d , j 3.5 ) , 125.2 , 119.9 ,
116.6 ( d , j 25.5 ) and 16.45 ; f nmr ( 471
mhz , cdcl3 ) f 112.5 ( t , j 8.5 ) ; m / z ( esi+ ) hrms
[ m + h ] c14h10clfn3o found
290.0488 , calcd .
max ( atr)/cm 2927 , 1630 , 1601 , 1554 , 1504 , 1485 , 1396 , 1286 , 1217 , 1074 , 919 ,
822 , and 623 ; h nmr ( 500 mhz , cdcl3 ) h 7.61 ( 1h , t , j 7.9 ) , 7.28 ( 1h , dd , j 8.3 , 1.8 ) , 7.11 ( 1h , dd , j 10.0 , 1.8 ) ,
7.01 ( 1h , d , j 1.0 ) , 2.68 ( 3h , s ) and 2.66 ( 1h , d , j 1.0 ) ; c nmr ( 101 mhz , cdcl3 ) c 180.3 , 160.5 ( d , j 255 ) , 152.1 , 146.7 , 139.7 ,
139.1 ( d , j 10.5 ) , 138.6 , 131.41 ( d , j 3.7 ) , 126.7 ( d , j 13.7 ) , 125.8 , 125.3 ( d , j 3.2 ) , 120.9 , 116.5 ( d , j 25.7 ) , 16.8
and 16.40 ; f nmr ( 471 mhz , cdcl3 ) f 112.4 ( t , j 8.8 ) ; m / z ( esi+ ) hrms [ m + h ] c15h10cl2fn3o found 338.0263 , calcd .
338.0259 .
max ( atr)/cm 2925 , 1649 , 1630 , 1601 , 1554 , 1504 , 1396 , 1286 , 1074 , 919 , 822 and
617 ; h nmr ( 500 mhz , cdcl3 ) h ( as a mixture of rotamers confirmed by vt nmr ) 7.617.58
( 1h , m ) , 7.307.26 ( 1h , m ) , 7.217.09 ( 1h , m ) , 7.01
( 1h , s , major rotamer ) , 6.92 ( 1h , s , minor rotamer ) , 4.95 ( 2h , s ) ,
3.18 ( 3h , s , major rotamer ) , 3.05 ( 3h , s , minor rotamer ) , 2.67 ( 3h ,
s ) , 2.21 ( 3h , s , major rotamer ) and 2.14 ( 3h , s , minor rotamer ) ; c nmr ( 126 mhz , cdcl3 ) c data
for major rotamer 180.0 , 171.4 , 160.4 ( 1d , j 255.4 ) ,
152.2 , 147.0 , 139.1 ( d , j 10.7 ) , 138.2 , 137.2 , 131.3
( 1d , j 3.6 ) , 126.5 ( d , j 13.6 ) ,
125.7 , 125.2 ( d , j 3.4 ) , 119.1 , 116.4 ( d , j 25.6 ) , 46.2 , 37.4 , 21.6 , and 16.2 ; f nmr
( 471 mhz , cdcl3 ) f 112.4 ( t , j 8.6 ) ; m / z ( esi+ ) hrms
[ m + h ] c18h15cl2fn4o2 found 409.0625 , calcd .
was added 2 ( 97.8 mg , 0.231 mmol , 1.0 equiv ) , sodium
acetate ( 19.0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 1.50
mg , 2.31 mol , 0.01 equiv ) , dma ( 1.50 ml ) and n - methylmorpholine ( 510 l , 4.63 mmol , 20 equiv ) to give a heterogeneous
yellow solution .
the flask was placed in a 25 ml jacketed beaker with iproh as the internal coolant connected to a recirculating
heater chiller set to 5 c .
the flask was stirred for 2 min open
to air before switching on 2 4w led strips and stirring for
8 h. uplc analysis ( figure s13 ) showed
27% a / a of starting material 1 , 33% a / a of the reduced
product 9 and also included the c6 carbon - chlorine bond
reduction [ ( 4-chloro-2-fluorophenyl)(2-methyl-8-(morpholinomethyl)imidazo[1,2-b]pyridazin-3-yl)methanone ] 10% a / a and the c n bond
fragmentation product 6 9% a / a . while 9 could
not be efficiently isolated via chromatography , analysis of the h nmr spectrum of the crude reaction mixture following
work
up also confirmed its presence as the major compound among a complex
mixture . to a 10 ml rbf
was added 2 ( 97.8 mg , 0.231
mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231 mmol , 1.0 equiv ) ,
ir(ppy)3 ( 1.50 mg , 2.31 mol , 0.01 equiv ) , dma ( 1.50
ml ) , n - methylmorpholine ( 510 l , 4.63 mmol ,
20 equiv ) and formic acid ( 44.0 l , 1.16 mmol , 5 equiv ) to give
a heterogeneous bright yellow / green solution . the flask was placed
in a 25 ml jacketed beaker with iproh as the internal
coolant connected to a recirculating heater chiller set to 5 c .
the flask was stirred for 2 min open to air before switching on 2
4w led strips and stirring for 22 h. uplc analysis ( figure s14 ) showed 34% a / a of starting material 2 and
the reaction was worked up via the addition of etoac ( 50
ml ) followed by washing with water ( 50 ml ) and 5% licl ( 2 25
ml ) .
the organic phase was then dried ( na2so4 ) and concentrated in vacuo to give a crude product that was purified
via automated column chromatography ( 12 g silica column , 70:10:10
hexanes : etoac : ch2cl2 ) to give 6 ( 16.0 mg , 20% yield ) as a white solid with spectroscopic data in
accordance with that reported .
to four identical
10 ml rbfs was added 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) ,
sodium acetate ( 19.0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol , 0.005 equiv ) , dma ( 1.50 ml ) and n - methylmorpholine ( 510 l , 4.63 mmol , 20 equiv ) to
give a heterogeneous yellow solution .
the flask was placed in a 25
ml jacketed beaker with iproh as the internal coolant
connected to a recirculating heater chiller set to 0 c .
the
flask was stirred for 2 min open to air before switching on a 1 w
led strips .
the reaction was stirred for 32 h before ( 3:1 water : etoh ,
2.0 ml ) was added and the reaction was stirred for a further 30 min .
the solids were filtered , washed with water then dissolved in ch2cl2 and dried with na2so4 .
the result material was dried overnight under a vacuum ( < 1 mbar )
to give 2 ( 5256 mg , 56% average yield ) .
a portion
of this solid ( 50 mg ) was dissolved in etaoc ( 30 ml ) and extracted
twice with 1 n hcl ( 25 ml ) .
the aqueous hcl was basified to ph 12
with naoh and extracted twice with ch2cl2 , dried
( na2so4 ) , filtered and concentrated to give 2 ( 29 mg , 58% recovery , > 98% a / a ) , in accordance
with
the literature.h nmr ( 500 mhz , cdcl3 ) h 7.62 ( 1h , t , j 7.9 ) , 7.38 ( 1h , td , j 0.7 , 0.3 ) , 7.30
( 1h , dd , j 8.3 ) , 7.12 ( 1h , dd , j 10.0 , 1.9 ) , 4.00 ( 2h , s ) , 3.79 ( 4h , br s ) , 2.68 ( 3 h , s ) and 2.61
( 3h , s ) . because of the limited
scalability of the reaction employing nmm , reactions employing other
amines were conducted at 0.231 mmol scale in triplicate and combined
prior to work up and isolation .
to a 10 ml rbf was added 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg ,
0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol ,
0.005 equiv ) , dma ( 1.50 ml ) and amine ( 4.63 mmol , 20 equiv ) to give
a heterogeneous yellow solution .
the flask was placed in a 25 ml jacketed
beaker with iproh as the internal coolant connected
to a recirculating heater chiller set to 5 c .
the flask was
stirred for 2 min open to air before switching on a 1w led strip .
the reaction was monitored via uplc and after a given time worked
up with the addition of etoac ( 50 ml ) followed by washing with water
( 2 50 ml ) and 5% licl ( 2 25 ml ) .
the organic phase was
then dried ( na2so4 ) and concentrated in vacuo
to give a crude product that was purified via automated column chromatography
under the stated conditions . following the general procedure
with the absence of amine
; three identical reactions using 2 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 1.52 mg , 2.31 mol , 0.01
equiv ) , dmpu ( 1.50 ml ) for 41 h at 5 c were combined and worked
up together .
the resulting crude material ( 1:5 exo : endo ) was purified via automated column chromatography
( 12 g silica column , 50:50 etoac : hexanes to etoac followed by a second
column 10:90 acetone : ch2cl2 to 20:80 acetone : ch2cl2 ) to give 10 and 11 ( 164 mg , 53% yield , 1:5 exo : endo ) as white solid .
h nmr ( 700 mhz , cdcl3 ) h 7.62
( 1h , t , j 7.9 ) , 7.30 ( 1h , dd , j 8.2 ,
1.7 ) , 7.11 ( 1h , dd , j 10.1 , 1.6 ) , 6.97 ( 1h , s ) , 5.19
( 1h , d , j 3.9 ) , 3.143.07 ( 2h , m ) , 2.99 ( 3h ,
s ) , 2.98 ( 3h , s ) , 2.66 ( 3h , s ) , 2.572.53 ( 1h , m ) and 2.38
( 1h , dd , j 13.8 , 2.9 ) ; c nmr ( 176 mhz ,
cdcl3 ) c 180.1 160.5 ( d , j 255 ) , 152.1 , 147.0 , 140.0 , 139.2 , ( d , j 10.6 ) ,
138.8 , 131.4 ( d , j 3.6 ) , 126.7 ( d , j 14.1 ) , 125.5 , 125.3 ( d , j 3.2 ) , 120.0 , 116.5 ( d , j 25.6 ) , 65.4 , 64.6 , 55.4 , 54.8 , 27.3 , 23.7 , and 16.3 .
f nmr ( 377 mhz , cdcl3 ) f 112.4
( t , j 8.8 ) ; m / z ( esi+ ) hrms [ m + h ] c20h19cl2fn5o2 found 450.0894 , calcd .
450.0894 . following
the general procedure three identical reactions using 1 ( 75.0 mg ,
0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and n - methylpiperidine ( 562
l , 4.63 mmol , 20 equiv ) for 40 h at 5 c were combined
and worked up together . prior to work up 12 was present
at 45% a / a ( uplc pda ) with > 10:1 exo : endo ( uplc ms ) .
the resulting crude material was purified
via automated column chromatography ( 24 g silica column , 80:20 hexanes : etoac
1% et3n ) to give 12 ( 87 mg , 30% yield ) as
a white solid .
max ( atr)/cm 2938 , 2360 , 2336 , 1636 , 1603 , 1417 , 1277 , 1074 , 918 , 856 and 622 ; h nmr ( 500 mhz , cdcl3 ) h 7.61
( 1h , t , j 7.9 ) , 7.39 ( 1h , s ) , 7.30 ( 1h , dd , j 8.3 , 1.3 ) , 7.12 ( 1h , dd , j 10.0 , 1.7 ) ,
3.94 ( 2h , s ) , 2.68 ( 4h , s ) , 2.53 ( 4h , br ) , 1.65 ( 4h , app quintet , j 5.6 ) and 1.521.50 ( 2h , m ) ; c nmr
( 176 mhz , cdcl3 ) c 180.5 , 160.3 ( d , j 255 ) , 152.0 , 147.3 , 140.0 , 138.9 ( d , j 10.6 ) , 138.5 , 131.2 ( d , j 3.5 ) , 126.6 ( d , j 14.1 ) , 125.5 , 125.1 ( d , j 3.2 ) , 119.1 ,
116.3 ( d , j 25.7 ) , 56.4 , 55.0 , 26.0 , 23.9 , and 16.3 .
f nmr ( 471 mhz , cdcl3 ) f 112.4
( t , j 8.5 ) ; m / z ( esi+ ) hrms [ m + h ] c20h20cl2fn4o found 421.0992 , calcd .
421.0998 . following the general procedure three identical
reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium
acetate ( 19.0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8
mg , 1.16 mol , 0.005 equiv ) , dma ( 1.50 ml ) and n , n - dimethylglycine methylester ( 557 l , 4.63
mmol , 20 equiv ) for 28 h at 5 c were combined and worked up
together . prior to work up 13 was present at 60%
a / a ( uplc pda ) with 10:1 exo : endo ( uplc ms ) .
the resulting crude material was purified via automated
column chromatography ( 24 g silica column , 70:30 hexanes : etoac 1%
et3n ) to give 13 ( 172 mg , 56% yield ) as a
cream solid .
max ( atr)/cm 2948 , 2359 , 2335 , 1741 , 1634 , 1604 , 1505 , 1479 , 1415 , 1275 , 1074 ,
917 , 872 , 817 , 736 , and 622 ; h nmr ( 500 mhz , cdcl3 ) h 7.62 ( 1h , t , j 7.9 ) ,
7.45 ( 1h , t , j 1.1 ) , 7.30 ( 1h , dd , j 8.3 , 1.9 ) , 7.12 ( 1h , dd , j 10.0 , 1.9 ) , 4.19 ( 2h ,
d j 1.1 ) , 3.74 ( 3h , s ) 3.45 ( 2h , s ) 2.67 ( 3h , s )
and 2.51 ( 3h , s ) ; c nmr ( 176 mhz , cdcl3 ) c 180.0 , 171.0 , 160.3 ( d , j 255 ) , 152.1 , 147.3 ,
139.0 ( d , j 10.6 ) , 138.9 , 138.3 , 131.2 ( d , j 3.5 ) , 126.6 ( d , j 14.1 ) , 125.5 , 125.1(d , j 3.2 ) , 119.3 , 116.3 ( d , j 25.7 ) , 58.2 ,
54.5 , 51.7 , 42.7 , and 16.3 ; f nmr ( 471 mhz , cdcl3 ) f 112.4 ( t , j 8.9 ) ; m / z ( esi+ ) hrms [ m + h ]
c19h18cl2fn4o3 found 439.0736 , calcd .
439.0740 . following
the general procedure two identical reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 (
0.8 mg , 1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and tetramethylenediamine ( 349 l , 2.31
mmol , 10 equiv ) for 7 h at 5 c were combined and worked up together .
prior to work up 14 was present at 70% a / a ( uplc
pda ) with >
the resulting crude material was purified via automated column chromatography
( 24 g silica column , 10:90 hexanes : etoac 1% et3n ) to give 14 ( 88 mg , 44% yield ) as a light yellow solid .
max ( atr)/cm 2767 , 2361 , 1622 , 1601 , 1558 ,
1507 , 1414 , 1289 , 1086 , 919 , 879 , 816 , 761 , and 596 ; h
nmr ( 700 mhz , cdcl3 ) h 7.62 ( 1h , t ) ,
7.48 ( 1h , s ) , 7.30 ( 1h , dd , j 8.2 , 1.8 ) , 7.12 ( 1h ,
dd , j 9.9 ) , 4.03 ( 2h , s ) , 2.68 ( 3h , s ) , 2.63 ( 2h ,
t , j 6.8 , ) , 2.47 ( 2h , t , j 6.8 ) ,
2.38 ( 3h , s ) and 2.25 ( 6h , s ) ; c nmr ( 176 mhz , cdcl3 ) c 180.1 , 160.3 ( d , j 255 ) ,
152.1 , 147.4 , 140.1 , 139.0 ( d , j 10.4 ) , 138.5 , 131.2
( d , j 3.5 ) , 126.6 ( d , j 14.2 ) , 125.6 ,
125.1 ( d , j 3.2 ) , 119.4 , 116.4 ( d , j 25.6 ) , 57.6 , 56.0 , 55.7 , 46.0 , 43.2 , and 16.3 ; f nmr
( 471 mhz , cdcl3 ) f 112.4 ( t , j 8.7 ) ; m / z ( esi+ ) hrms
438.1264 . following
the general procedure three identical reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and tetramethyl-1,6-hexanediamine ( 991 l ,
4.63 mmol , 20 equiv ) for 20 h at 5 c were combined and worked
up together . prior to work up 15 was present at 55%
a / a ( uplc pda ) with > 10:1 exo : endo ( uplc ms ) .
excess tetramethyl-1,6-hexanediamine was removed via
kugelrohr distillation at 5060 c and < 1 mbar .
the
resulting crude material was purified via automated column chromatography
( 24 g silica column , etoac 1% et3n ) to give 15 ( 82 mg , 24% yield ) as a light brown solid .
max ( atr)/cm 2923 , 2775 , 1636 , 1610 , 1506 , 1414 ,
1214 , 1074 , 920 and 818 ; h nmr ( 700 mhz , cdcl3 ) h 7.60 ( 1h , t , j 7.9 ) , 7.38
( 1h , s ) , 7.28 ( 1h , dd , j 8.3 , 1.87.11 ( 1h , dd , j 9.9 ) , 3.95 ( 2h , s ) , 2.49 ( 2h , t , j 7.4 )
2.29 ( 3h , s ) , 2.22 ( 2h , t , j 7.4 ) , 2.21 ( 6h , s ) ,
1.53 ( 2h , dt , j 14.7 , 7.3 ) , 1.45 ( 2h , dt , j 14.7 , 7.3 ) and 1.361.29 ( 4h , m ) ; c nmr ( 176 mhz , cdcl3 ) c 180.2 , 160.4
( d , j 255 ) , 152.1 , 147.4 , 140.5 , 138.9 ( d , j 10.3 ) , 138.5 , 131.2 ( d , j 3.5 ) , 126.6
( d , j 14.0 ) , 125.5 , 125.1 ( d , j 3.3 ) ,
119.2 , 116.3 ( d , j 25.4 ) , 59.8 , 58.1 , 55.3 , 45.5 ,
42.8 , 27.7 , 27.4 , 27.4 , 27.3 and 16.3 ; f nmr ( 471 mhz ,
cdcl3 ) f 112.4 ( t , j 8.9 ) ; m / z ( esi+ ) hrms [ m + h ]
c24h31cl2fn5o found 494.1890 , calcd .
494.1884 . following
the general procedure three identical reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 1.52 mg , 2.31 mol , 0.01
equiv ) , dma ( 1.50 ml ) and dimethylethylamine ( 502 l , 4.63 mmol ,
20 equiv ) for 8 h at 5 c were combined and worked up together .
prior to work up 16 was present at 63% a / a ( uplc
pda ) with 67:1 exo : endo ( uplc ms ) .
the resulting crude material was purified via automated
column chromatography ( 24 g silica column , 80:20 hexanes : etoac 1%
et3n ) to give 16 ( 145 mg , 53% yield ) as a
cream solid .
max ( atr)/cm 2804 , 1634 , 1603 , 1507 , 1414 , 1278 , 1213 , 1077 , 1039 , 919 , 859 ,
and 734 ; h nmr ( 500 mhz , cdcl3 ) h 7.62 ( 1h , t , j 7.9 ) , 7.40 ( 1h , t , j 1.3 ) , 7.30 ( 1h , dd , j 8.3 , 1.9 ) , 7.12
( 1h , dd , j 9.9 , 1.9 ) , 3.98 ( 2h , d , j 1.3 ) , 2.68 ( 3h , s ) , 2.59 ( 2h , q , j 7.1 ) , 2.33 ( 3h ,
s ) and 1.14 ( 3h , t , j 7.1 ) ; c nmr ( 176
mhz , cdcl3 ) c 180.2 , 160.3 ( d , j 255 ) , 152.1 , 147.4 , 140.5 , 139.0 ( d , j 10.4 ) , 138.5 , 131.2 ( d , j 3.5 ) , 126.6 ( d , j 14.0 ) , 125.5 , 125.1 ( d , j 3.3 ) , 119.3 ,
116.4 ( d , j 25.6 ) , 54.9 , 52.0 , 42.3 , 16.3 , and 12.6 ; f nmr ( 471 mhz , cdcl3 ) f 112.4
( t , j 8.5 ) ; m / z ( esi+ ) hrms [ m + h ] c18h18cl2fn4o found 395.0840 , calcd .
395.0842 . following
the general procedure three identical reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and 1,4-dimethylpiperizine ( 621 l , 4.63
mmol , 20 equiv ) for 18 h at 5 c were combined and worked up
together . prior to work up 17 was present at 55%
a / a ( uplc pda ) with 56:1 exo : endo ( uplc ms ) .
the resulting crude material was purified
via automated column chromatography ( 12 g silica column , etoac 1%
et3n ) to give 17 ( 109 mg , 36% yield ) as a
light brown solid .
max ( atr)/cm 2797 , 1629 , 1603 , 1498 , 1414 , 1271 , 1224 , 1212 , 1075 , 1013 , 917 ,
826 and 764 ; h nmr ( 700 mhz , cdcl3 ) h 7.63 ( 1h , t , j 7.9 ) , 7.36 ( 1h , s ) , 7.29
( 1h , dd , j 8.3 , 1.7 ) , 7.11 ( 1h , dd , j 9.9 , 1.7 ) , 4.00 ( 2h , d , j 1.0 ) , 2.67 ( 3h , s ) overlapping
2.64 ( 4h , br s ) , 2.51 ( 4h , br s ) and 2.32 ( 3h , s ) ; c
nmr ( 176 mhz , cdcl3 ) c 180.2 , 160.4 ( d , j 255 ) , 152.1 , 147.3 , 139.3 , 139.0 ( d , j 10.3 ) , 138.4 , 131.3 ( d , j 3.5 ) , 126.6 ( d , j 14.1 ) , 125.6 , 125.1 ( d , j 3.3 ) , 119.1 ,
116.4 ( d , j 25.6 ) , 55.5 , 55.0 , 53.4 , 46.0 and 16.3 ; f nmr ( 471 mhz , cdcl3 ) f 112.4
( t , j 8.8 ) ; m / z ( esi+ ) hrms [ m + h ] c20h21cl2fn5o found 436.1102 , calcd .
436.1107 . following the general procedure three identical reactions
using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate
( 19.0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16
mol , 0.005 equiv ) , dma ( 1.50 ml ) and bu-4-methylpiperazine-1-carboxylate
( 695 mg
, 3.47 mmol , 15 equiv ) for 20 h at 5 c were combined
and worked up together . prior to work up 18 was present
at 55% a / a ( uplc pda ) with 34:1 exo : endo ( uplc pda ) .
the resulting crude material was
purified via automated column chromatography ( 12 g silica column ,
90:10 dcm : etoac to 50:50 etoac : dcm ) to give 18-exo ( 99 mg , 27% yield ) as a light cream solid and 18-endo ( 56 mg , 15% yield ) as a cream solid .
max ( atr)/cm 1694 , 1634 , 1604 , 1418 , 1238 ,
1164 , 1128 , 1073 , 1008 , 920 , 850 , and 831 ; h nmr ( 400
mhz , cdcl3 ) h 7.62 ( 1h , t , j 7.9 ) , 7.37 ( 1h , s ) , 7.30 ( 1h , dd , j 8.2 , 1.9 ) ,
7.12 ( 1h , dd , j 10.0 , 1.8 ) , 4.01 ( 2h , d , j 0.9 ) , 3.51 ( 4h , t , j 4.8 ) 2.68 ( 3h , s ) ,
2.55 ( 4h , t , j 4.8 ) and 1.47 ( 9h , s ) ; c nmr ( 176 mhz , cdcl3 ) c 180.2 , 160.5
( d , j 255 ) , 154.8 , 152.3 , 147.4 , 139.3 ( d , j 10.4 ) , 139.0 , 138.5 , 131.4 ( d , j 3.5 ) ,
126.7 ( d , j 14.0 ) , 125.8 , 125.3 ( d , j 3.3 ) , 119.2 , 116.5 ( d , j 25.5 ) , 80.1 , 55.8 , 53.4 ,
20.6 , and 16.4 ; f nmr ( 471 mhz , cdcl3 ) f 112.4 ( t , j 8.6 ) ; m / z ( esi+ ) hrms [ m + h ] c24h26cl2fn5o3 found 522.1481 ,
calcd .
max ( atr)/cm 1696 ,
1636 , 1604 , 1415 , 1270 ,
1147 , 1077 , 920 , 860 , and 728 ; h nmr ( 500 mhz , cdcl3 ) h 7.62 ( 1h , t , j 7.9 ) ,
7.34 ( 1h , s ) , 7.31 ( 1h , dd , j 8.3 , 1.9 ) , 7.13 ( 1h ,
dd , j 9.9 , 1.8 ) , 4.16 ( 2h , s ) , 3.96 ( 1h , dd , j 10.2 , 2.8 ) , 3.05 ( 1h , br s ) , 2.94 ( 1h , d , j 11.4 ) , 2.77 ( 1h , br s ) , 2.68 ( 3h , s ) , 2.39 ( 1h , td , j 11.9 , 3.3 ) , 2.17 ( 3h , s ) , 1.63 ( 1h , br s ) and 1.46 ( 9h , s ) ; c nmr ( 176 mhz , cdcl3 ) c 180.2 ,
160.5 ( d , j 255 ) , 152.7 ( 2 c ) , 147.3 , 140.6 ,
139.2 ( d , j 10.4 ) , 138.0 , 131.4 ( d , j 3.5 ) , 126.7 ( d , j 13.9 ) , 126.0 , 125.3 ( d , j 3.2 ) , 119.1 , 116.6 ( d , j 25.6 ) , 61.2 ,
54.6 , 43.8 , 28.5 , 28.5 , and 16.5 ; f nmr ( 471 mhz , cdcl3 ) f 112.4 ( app m ) ; m / z ( esi+ ) hrms [ m + h ] c24h26cl2fn5o3 found 522.1479 ,
calcd .
522.1469 . following
the general procedure three identical reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg ,
1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and dimethylanaline ( 441 l , 3.47 mmol ,
15 equiv ) for 15 h at 5 c were combined and worked up together .
prior to work up 19 was present at 60% a / a ( uplc
pda ) .
the resulting crude material was purified via automated column
chromatography ( 12 g silica column , 0% etoac to 30% etoac ) to give 19 ( 123 mg , 40% yield ) as a light yellow solid that was contaminated
with 5% of n - methyl - n - phenylformamide .
max ( atr)/cm 2888 , 2360 ,
2330 , 1632 , 1599 , 1505 , 1417 , 1204 , 1075 , 917 , 848 , 817 , and 748 ; h nmr ( 700 mhz , cdcl3 ) h 7.64
( 1h , t , j 7.9 ) , 7.31 ( 1h , dd , j 8.3 ,
1.8 ) , 7.277.24 ( 2h , m ) , 7.13 ( 1h , dd , j 9.9 ,
1.7 ) , 7.00 ( 1h , s ) , 6.80 ( 1h , t , j 7.3 ) , 6.71 ( 2h , d , j 8.2 ) , 4.95 ( 2h , d , j 0.7 ) , 3.15 ( 3h , s ) and 2.72 ( 3h , s ) ; c nmr
( 176 mhz , cdcl3 ) c 180.3 , 160.4 ( d , j 255 ) , 152.3 , 148.7 , 147.4 , 139.5 , 139.1 ( d , j 10.7 ) , 137.7 , 131.3 ( d , j 3.7 ) , 126.5 ( d , j 14.0 ) , 125.9 , 125.2 ( d , j 3.3 ) , 118.1 ,
117.9 , 116.4 ( d , j 25.6 ) , 112.3 , 52.4 , 39.4 , and
16.4 ( one c was not be resolved ) ; f nmr ( 377 mhz , cdcl3 ) f 112.3 ( t , j 8.8 ) ; m / z ( esi+ ) hrms [ m + h ]
c22h18cl2fn4o found 443.0837 , calcd .
443.0836 . following the general procedure three identical
reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium
acetate ( 19.0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8
mg ,
1.16 mol , 0.005 equiv ) , dma ( 1.50 ml ) and dimethylbenzylamine
( 346 l , 2.31 mmol , 10 equiv ) for 15 h at 5 c were combined
and worked up together . prior to work up 20 was present
at 70% a / a ( uplc pda ) and in > 10:1 exo : endo ( uplc ms ) .
the resulting crude material was
purified
via automated column chromatography ( 12 g silica column , 50:50 ch2cl2:hexanes then a gradient of etoac : hexanes ) to
give 20 ( 171 mg , 54% yield ) as a light yellow solid .
max ( atr)/cm 2794 , 2361 , 1634 ,
1604 , 1413 , 1285 , 1076 , 919 , 732 , and 698 ; h nmr ( 500
mhz , cdcl3 ) h 7.61 ( 1h , t , j 7.9 ) , 7.46 ( 1h , dt , j , 1.9 , 1.3 ) , 7.397.37
( 2h , m ) , 7.33 ( 2h , dd , j 7.0 , 1.6 ) , 7.317.24
( 3h , m ) , 7.11 ( 1h , dd , j 9.9 , 1.9 ) , 4.00 ( 2h , s ) ,
3.70 ( 2h , s ) , 2.67 ( 3h , s ) and 2.35 ( 3h , s ) ; c nmr ( 176
mhz , cdcl3 ) c 180.1 160.3 ( d , j 255 ) , 152.1 , 147.4 , 140.4 , 139.0 ( d , j 10.6 ) , 138.5 , 131.2 ( d , j 3.6 ) , 126.6 ( d , j 13.9 ) , 127.5 , 126.7 , 126.3 , 125.6 , 125.1 ( d , j 3.2 ) , 119.2 , 116.3 ( d , j 25.6 ) , 62.6 , 54.5 , 43.2 ,
and 16.4 ; f nmr ( 377 mhz , cdcl3 ) f 112.4 ( t , j 8.8 ) ; m / z ( esi+ ) hrms [ m + h ] c23h19cl2fn4o found 457.0993 , calcd .
following
the general procedure three identical reactions using 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and n - methyl - l - prolinol
( 555 l , 4.63 mmol , 20 equiv ) for 15 h at 5 c were combined
and worked up together . prior to work up 21 was present
at 25% a / a ( uplc pda ) and in > 10:1 exo : endo ( uplc ms ) .
the resulting crude material was
purified
via automated column chromatography ( 12 g silica column , 20:80 etoac : hexanes
to 40:60 etoac : hexanes ) to give 21 ( 60 mg , 20% yield )
as a light brown oil .
+ 19.6 ( c 1.0 , chcl3 ) ; chiral hplc analysis chiralpak od - h ( 5% ipa : hexane , flow rate
1.0 ml min , 254 nm ) tr major ( s ) 13.8 min , tr minor ( r ) 16.5 min 96:4 er . the
racemic standard was prepared in an identical fashion from ( rac)-n - methyl - prolinol to give a light brown solid .
max ( atr)/cm 2946 , 2361 , 1335 , 1634 , 1602 ,
1558 , 1506 , 1418 , 1286 , 213 , 1077 , 919 , 821 , and 735 ; h nmr ( 700 mhz , cdcl3 ) h 7.60 ( 1h , t , j 7.8 ) , 7.29 ( 1h , dd , j 8.2 , 1.2 ) , 7.11
( 1h , dd , j 9.9 , 1.3 ) , 7.10 ( 1h , s ) , 5.32 ( 1h , br
s ) , 4.63 ( 1h , d , j 14.1 ) , 3.82 ( 1h , dd , j 11.7 , 2.7 ) , 3.58 ( 1h , d , j 14.1 ) , 3.52 ( 1h , dd , j 11.7 , 5.5 ) , 2.992.96 ( 1h , m ) , 2.952.92
( 1h , m ) , 2.68 ( 3h , s ) , 2.392.36 ( 1h , m ) , 1.941.89
( 1h , m ) , 1.791.71 ( 2h , m ) , 1.701.64 ( 1h , m ) ; c nmr ( 176 mhz , cdcl3 ) c 180.1
160.5 ( d , j 255 ) , 152.1 , 147.0 , 140.0 , 139.2 , ( d , j 10.6 ) , 138.8 , 131.4 ( d , j 3.6 ) , 126.7
( d , j 14.1 ) , 125.5 , 125.3 ( d , j 3.2 ) ,
120.0 , 116.5 ( d , j 25.6 ) , 65.4 , 64.6 , 55.4 , 54.8 ,
27.3 , 23.7 , and 16.3 ; f nmr ( 377 mhz , cdcl3 ) f 112.2 ( t , j 8.7 ) ; m / z ( esi+ ) hrms [ m + h ] c20h20cl2fn4o2 found
437.0943 , calcd .
437.0942 . following
the general procedure two identical reactions using 1 ( 55.4 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19.0 mg , 0.231
mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg ,
1.16 mol , 0.005
equiv ) , dma ( 1.50 ml ) and n - methylmorpholine ( 510
l , 4.63 mmol , 20 equiv ) for 12 h at 5 c were combined
and worked up together .
the resulting crude material ( 1:1 exo : endo ) was purified via automated column
chromatography ( 12 g silica column , 50:20:30 hexanes : etoac : ch2cl2 ) to give exo-23 ( 57 mg , 36% yield ) as a white solid and endo-24 that was further purified via acid base extraction to give
( 29 mg , 19% ) h nmr ( 500 mhz , cdcl3 ) h 7.40 ( 1h , s ) ,
4.46 ( 2h , q , j 7.1 ) , 3.98 ( 2h , s ) , 3.78 ( 4h , t , j 4.4 ) , 2.72 ( 3h , s ) , 2.60 ( 4h , t , j 4.4 )
and 1.43 ( 3h , t , j 7.1 ) .
max ( atr)/cm 2982 , 2851 , 1696 , 1230 , 1417 ,
1288 , 1225 , 1115 , 989 , 952 , 876 , 758 , and 636 ; h nmr ( 500
mhz , cdcl3 ) h 7.39 ( 1h , s ) , 4.46 ( 2h ,
q , j 7.1 ) , 4.13 ( 1h , d , j 8.2 ) ,
3.99 ( 1h , dd , j 11.2 , 2.9 ) , 3.96 ( 1h , d , j 11.4 ) , 3.74 ( 1h , t j 11.4 ) , 3.29 ( 1h ,
t , j 9.8 ) , 2.88 ( 1h , d , j 11.7 ) ,
2.73 ( 3h , s ) , 2.56 ( 1h , d , j 10.6 ) , 2.17 ( 3h , s )
and 1.43 ( 3h , t , j 7.1 ) ; c nmr ( 176
mhz , cdcl3 ) c 159.5 , 151.6 , 147.9 , 139.3 ,
139.1 , 118.8 , 117.9 , 71.5 , 67.4 , 61.0 , 54.6 , 43.8 , 16.8 , and 14.5
( one c was not resolved ) ; m / z ( esi+ )
hrms [ m + h ] c15h20cln4o3 found 339.1226 , calcd .
339.1218 . following
the general procedure two identical
reactions using 22 ( 55.4 mg , 0.231 mmol , 1.0 equiv ) ,
sodium acetate ( 19,0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol , 0.005 equiv ) , dma ( 1365 l ) , water
( 137 l ) and n - methylmorpholine ( 510 l ,
4.63 mmol , 20 equiv ) for 16 h at 5 c were combined and worked
up together .
the resulting crude material ( 1:1.3 exo : endo ) was purified via automated column chromatography
( 12 g silica column , 50:20:30 hexanes : etoac : ch2cl2 ) to give 25 ( 7 mg , 6% yield ) , 23-exo that was further purified via acid base extraction to
remove ethyl 2-methylimidazo[1,2-b]pyridazine-3-carboxylate
( s3 ) to give ( 26 mg , 17% ) and 24-endo ( 37 mg , 24% yield ) max ( atr)/cm 1679 ,
1528 , 1417 , 1347 , 1295 , 1252 , 1143 , 1130 , 11063 ,
930 , 884 765 and 715 h nmr ( 500 mhz , cdcl3 )
h 7.04 ( 1h , d , j 1.1 ) , 4.46 ( 2h ,
q , j 7.1 ) , 2.73 ( 3h , s ) , 2.66 ( 3h , d , j 1.1 ) and 1.44 ( 3h , t , j 7.1 ) ; c nmr
( 176 mhz , cdcl3 ) c 159.6 , 151.1 , 147.3 ,
140.2 , 138.2 , 120.4 , 117.6 , 60.9 , 16.7 , 16.7 , and 14.5 ; m / z ( esi+ ) hrms [ m + h ] c11h13cln3o2 found 254.0693 , calcd . 254.0691 .
max ( atr)/cm 3113 , 2934 , 1703 , 1425 , 1385 , 1323 , 1266 , 1188 , 1083 ,
1040 , 805 , and 768 h nmr ( 500 mhz , cdcl3 ) h 8.56 ( 1h , dd , j 4.5 , 1.7 ) , 7.97 ( 1 h , dd , j 9.1 , 1.7 ) , 7.22 ( 1h , dd , j 9.1 , 4.5 ) ,
4.49 ( 2h , q , j 7.1 ) , 2.77 ( 3h , s ) and 1.45 ( 3h , t , j 7.1 ) ; c nmr ( 176 mhz , cdcl3 ) c 159.8 , 151.8 , 143.6 , 140.7 , 124.9 , 119.1 , 117.0 , 60.8 , 16.9 ,
and 14.5 m / z ( esi+ ) hrms [ m + h ]
c10h12n3o2 found 206.0926 , calcd . 206.0924 . following the general procedure five identical reactions using 27 ( 71.6 mg , 0.231 mmol , 1.0 equiv ) , sodium acetate ( 19,0
mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 0.8 mg , 1.16 mol ,
0.01 equiv ) , dma ( 1365 l ) , water ( 137 l ) and n - methylmorpholine ( 510 l , 4.63 mmol , 20 equiv ) for
20 h at 5 c were combined and worked up together .
the resulting
crude material ( 1.2:1 exo : endo )
was purified via automated column chromatography ( 12 g silica column ,
50:40:10 hexanes : etoac : ch2cl2 ) to give 27 ( 64 mg , 14% yield in accordance with the literature.h nmr ( 500
mhz , cdcl3 ) h 7.19 ( 1h , t , j 1.3 ) , 7.16 ( 1h , d , j 8.2 ) , 7.06 ( 1h , dd , j 9.7 and 2.1 ) , 7.01 ( 1h , ddd , j 8.2 , 2.1
and 0.7 ) , 4.27 ( 2 h , s ) , 3.96 ( 2h , d , j 1.3 , ) , 3.77
( 4h , t , j 4.6 ) , 2.59 ( 4h , t , j 4.6 ) ,
2.46 ( 3h , d , j 0.5 ) . following the general
procedure 1 ( 75.0 mg , 0.231 mmol , 1.0 equiv ) , ir(ppy)3 ( 2.27 mg , 3.47 mol , 0.015 equiv ) , 31 ( 627
mg , 4.63 mmol , 20 equiv ) , made via the literature report of shustov
and khlebnikov anhydrous dma ( 5 ml ) ,
gave a slurry that was sparged with nitrogen for 15 min and then sealed .
a 1 w led strip was turned on for 24 h at 22 c before the reaction
was worked up and purified to give 2 ( 13 mg , 13% yield ) .
prior to purification h nmr spectroscopic analysis and
uplc pda analysis of the crude reaction mixture showed a exo : endo ratio of > 20:1 . | we
report a detailed investigation into the application of visible
light - mediated photocatalysis to a challenging bond construction in
a complex pharmaceutical target .
the optimized reaction allowed the
direct coupling of n - methylmorpholine with an unfunctionalized
pyridazine in good yield and selectivity , and with high purity of
the product isolated via crystallization .
the reaction also facilitated
the expedient synthesis of a range of analogues via the use of other
commercially available n - methyl substituted tertiary
amines , and therefore it represents an attractive tool for medicinal
chemistry .
furthermore , a number of other interesting photoredox reactions
were discovered during the course of this investigation , such as a
formal methylation reaction via c n bond cleavage , functionalization
of c
h bonds alpha to amides , and a visible light - mediated
iminium ion reduction . | Introduction
Results and Discussion
Conclusion
Experimental Section | in order to address these concerns ,
a second - generation synthesis , which employed
a vanadium - catalyzed addition of n - methylmorpholine - n - oxide ( nmo ) was developed that displayed greater scalability ,
improved yield , and product quality . initial studies found that under irradiation with a hg - lamp , a range
of sensitizers were successful in promoting the addition of n - methylmorpholine ( nmm ) to the pyridazine core , albeit
in low conversions and with relatively high levels of impurities . furthermore , given the quantity
of established methods for the functionalization of amine containing
compounds , there remain relatively few efforts that have demonstrated
the applicability of photocatalysis to a pharmaceutical or agrochemical
setting , especially with regard to the formation of challenging bond
constructs . a limited number of recent reports have demonstrated the use of photocatalysis
en route to known pharmaceutical compounds , such as the efficient 3-step synthesis of pregabalin , albeit in a racemic fashion and with the key
step conducted on < 100 mg scale . with these questions in mind
, we envisioned the introduction of
the benzylic morpholine in ly2784544 as an ideal opportunity to test
the viability of a photoredox transformation in a complex pharmaceutical
setting ( scheme 2c ) . at this juncture , we decided to control the reaction temperature
via the use of a jacketed beaker connected to a recirculating chiller . further optimization of the dma : h2o conditions by reducing
the reaction temperature to 0 c and using only 0.5 mol % of
ir(ppy)3 resulted in a precipitation of 2 during
the course of the reaction . furthermore , replacement of agno3 and tfa with 0.5 mol % ir(ppy)3 is an attractive prospect
from a waste management perspective , although the limited scalability
and cost concerns surrounding the use of iridium represent significant
issues to be addressed . the isolated yields
were moderate ( 2056% ) , yet synthetically useful , and the scope
of the tertiary amine component encompassed aliphatic , benzylic , and
aromatic n - methyl tertiary amines . this amine proceeded with an equally
high regioisomeric ratio ( r.r > 10:1 ) but with
decreased
yield and increased reaction time ( 15 , 18 h , 24% ) suggesting
the improved selectivity is more likely the kinetic effect of an increased
number of n - methyl groups . in the case of n , n - dimethylethylamine
1.0 mol % ir(ppy)3 was employed to increase the reaction rate to compete productively
with evaporation of the amine ( bp 3638 c ) , a problem
not encountered with the other amines . attempts were then made to assess the viability of the reaction to
a limited selection of other imidazopyridazines that are potential
intermediates in the synthesis of ly2784544 . while the current methodology appears limited
with regards to the generality of the heterocyclic portion , the ease
and simplicity of the reaction set up , combined with the availability
of n - methyl tertiary amine coupling partners renders
this an attractive reaction to test on existing compound libraries . given the demonstrated sensitivity
of this reaction to a range of factors such as oxygen , light source ,
base , and photocatalyst , it is a strong possibility that multiple
reaction mechanisms may be operational . in conclusion , we have successfully developed
a visible light - mediated
photochemical transformation to address a challenging bond construction
en route to the jak2 inhibitor ly2784544 . furthermore , the methodology
was amenable to a range of simple , commercially available tertiary
amines and delivered a range of analogues in synthetically useful
yields . during the course of this investigation
a number of interesting photoredox transformations have been observed ,
such as formal methylation via c n bond cleavage , functionalization
of c
overall , we believe that this initial demonstration
warrants the wider consideration of visible light - mediated photocatalysis
as a tool in process development as well as the potential application
of this specific transformation to the diversification of pharmaceutical
and agrochemical scaffolds . to a 25 ml round bottom flask ( rbf ) open
to air
was added 1 ( 125 mg , 0.386 mmol , 1.0 equiv ) , ir(ppy)3 ( 5.00 mg , 0.008
mml , 0.02 equiv ) , sodium acetate ( 32.0 mg , 390 mmol , 1.0 equiv ) , dma
( 2.5 ml ) and n - methylmorpholine ( 0.850 ml , 7.71 mmol ,
20 equiv ) and the yellow heterogeneous solution was irradiated with
2 4 w led strips placed in a circular loop around the flask . | [
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] |
the pharmaceutical field over the past decade has faced continuing challenges in bringing new drug entity to market . in addition
, the cost of developing new drug entity keeps rising and today stands at more than us$ 800 m per new drug entity .
drug delivery research continues to find new therapies for the prevention and treatment of exiting and new diseases .
so , a valuable role is played by drug delivery system by providing optimized products for existing drugs in terms of either enhanced or improved presentation of drug to the systemic circulation [ 1 , 2 ] .
treatment of an acute disease or a chronic illness has been mostly accomplished by delivery of drugs to patients using various pharmaceutical dosage forms .
traditionally , the oral drug delivery has been most widely utilized route of administration among all the routes that have been explored for the systemic delivery of drugs .
conventional oral drug delivery systems are known to provide an immediate release of drug , in which one can not control the release of the drug and can not maintain effective concentration at the target site for longer period of time .
the oral bioavailability of some drug by conventional drug delivery is very low due to presence of food , in stabilization at ph of the gi tract , degradation by enzymes of gi fluid , change in gi motility , and so forth [ 3 , 4 ] . controlled drug delivery systems offer temporal and/or spatial control over the release of drug .
oral controlled drug delivery systems represent the most popular form of controlled drug delivery systems for the obvious advantages of oral route of drug administration .
these dosage forms offer many advantages , such as nearly constant drug level at the site of action , prevention of peak - valley fluctuations , reduction in dose of drug , reduced dosage frequency , avoidance of side effects , and improved patient compliance [ 5 , 6 ] .
the oral controlled release system shows a typical pattern of drug release in which the drug concentration is maintained in between the minimum effective concentration ( mec ) and maximum safe concentration ( msc ) for a prolonged period of time , thereby ensuring sustained therapeutic action ( figure 1 ) .
osmotic devices are most promising strategy - based systems for controlled drug delivery [ 79 ] .
osmosis can be defined as the net movement of water across a selectively permeable membrane driven by a difference in osmotic pressure across the membrane .
it is driven by a difference in solute concentrations across the membrane that allows passage of water , but rejects most solute molecules or ions .
osmotic pressure created by osmogen is used as driving force for these systems to release the drug in controlled manner .
these systems can be used for both route of administration , that is , oral and implantation .
osmotic pump offers many advantages over other controlled drug delivery systems , that is , they are easy to formulate and simple in operation , improved patient compliance with reduced dosing frequency and more consistence , and prolonged therapeutic effect with uniform blood concentration .
moreover they are inexpensive and their production scaleup is easy [ 10 , 11 ] .
osmotic drug - delivery systems suitable for oral administration typically consist of a compressed tablet core that is coated with a semipermeable membrane coating .
this coating has one or more delivery ports through which a solution or suspension of the drug is released over time .
the core consists of a drug formulation that contains an osmotic agent and a water swellable polymer .
the rate at which the core absorbs water depends on the osmotic pressure generated by the core components and the permeability of the membrane coating .
as the core absorbs water , it expands in volume , which pushes the drug solution or suspension out of the tablet through one or more delivery ports [ 12 , 13 ] .
the key distinguishing feature of osmotic drug delivery systems ( compared with other technologies used in controlled - release formulations ) is that they release drug at a rate that is independent of the ph and hydrodynamics of the external dissolution medium .
the result is a robust dosage form for which the in vivo rate of drug release is comparable to the in vitro rate , producing an excellent in vitro / in vivo correlation .
another key advantage of the present osmotic systems is that they are applicable to drugs with a broad range of aqueous solubilities [ 14 , 15 ] .
the historical development of osmotic systems includes seminal contributions such as the rose - nelson pump , the higuchi - leeper pumps , the alzet and osmet systems , the elementary osmotic pump , and the push - pull or gitsr system .
recent advances include the development of the controlled porosity osmotic pump [ 21 , 22 ] , systems based on asymmetric membranes [ 2325 ] , and other approaches .
( 1 ) semipermeable membranesince the membrane in osmotic systems is semipermeable in nature , any polymer that is permeable to water but impermeable to solute can be selected .
acetyl content is described by the degree of substitution ( ds ) , that is , the average number of hydroxyl groups on the anhydroglucose unit of the polymer replaced by substituting group .
some of the polymers that can be used for above purpose include cellulose esters such as cellulose acetate , cellulose diacetate , cellulose triacetate , cellulose propionate , cellulose acetate butyrate , and cellulose ethers like ethyl cellulose .
apart from cellulose derivatives , some other polymers such as agar acetate , amylose triacetate , betaglucan acetate , poly(vinyl methyl ) ether copolymers , poly(orthoesters ) , poly acetals and selectively permeable poly(glycolic acid ) , poly(lactic acid ) derivatives , and eudragits can be used as semipermeable film - forming materials .
since the membrane in osmotic systems is semipermeable in nature , any polymer that is permeable to water but impermeable to solute can be selected .
acetyl content is described by the degree of substitution ( ds ) , that is , the average number of hydroxyl groups on the anhydroglucose unit of the polymer replaced by substituting group .
some of the polymers that can be used for above purpose include cellulose esters such as cellulose acetate , cellulose diacetate , cellulose triacetate , cellulose propionate , cellulose acetate butyrate , and cellulose ethers like ethyl cellulose .
apart from cellulose derivatives , some other polymers such as agar acetate , amylose triacetate , betaglucan acetate , poly(vinyl methyl ) ether copolymers , poly(orthoesters ) , poly acetals and selectively permeable poly(glycolic acid ) , poly(lactic acid ) derivatives , and eudragits can be used as semipermeable film - forming materials .
( 2 ) hydrophilic and hydrophobic polymers these polymers are used in the formulation development of osmotic systems for making drug containing matrix core .
the highly water soluble compounds can be coentrapped in hydrophobic matrices and moderately water soluble compounds can be coentrapped in hydrophilic matrices to obtain more controlled release .
generally , mixtures of both hydrophilic and hydrophobic polymers have been used in the development of osmotic pumps of water - soluble drugs .
the selection is based on the solubility of the drug as well as the amount and rate of drug to be released from the pump .
mostly , swellable polymers are used for the pumps containing moderately water - soluble drugs .
since they increase the hydrostatic pressure inside the pump due to their swelling nature , the nonswellable polymers are used in case of highly water - soluble drugs .
ionic hydrogels such as sodium carboxymethyl cellulose are preferably used because of their osmogenic nature .
more precise controlled release of drugs can be achieved by incorporating these polymers into the formulations .
hydrophilic polymers such as hydroxy ethyl cellulose , carboxy methylcellulose , hydroxy propyl methylcellulose , high - molecular - weight poly(vinyl pyrrolidone ) , and hydrophobic polymers such as ethyl cellulose and wax materials can be used for this purpose .
these polymers are used in the formulation development of osmotic systems for making drug containing matrix core .
the highly water soluble compounds can be coentrapped in hydrophobic matrices and moderately water soluble compounds can be coentrapped in hydrophilic matrices to obtain more controlled release .
generally , mixtures of both hydrophilic and hydrophobic polymers have been used in the development of osmotic pumps of water - soluble drugs .
the selection is based on the solubility of the drug as well as the amount and rate of drug to be released from the pump .
mostly , swellable polymers are used for the pumps containing moderately water - soluble drugs .
since they increase the hydrostatic pressure inside the pump due to their swelling nature , the nonswellable polymers are used in case of highly water - soluble drugs .
ionic hydrogels such as sodium carboxymethyl cellulose are preferably used because of their osmogenic nature .
more precise controlled release of drugs can be achieved by incorporating these polymers into the formulations .
hydrophilic polymers such as hydroxy ethyl cellulose , carboxy methylcellulose , hydroxy propyl methylcellulose , high - molecular - weight poly(vinyl pyrrolidone ) , and hydrophobic polymers such as ethyl cellulose and wax materials can be used for this purpose .
( 3 ) wicking agentsa wicking agent is defined as a material with the ability to draw water into the porous network of a delivery device .
the wicking agents are those agents which help to increase the contact surface area of the drug with the incoming aqueous fluid .
the use of the wicking agent helps to enhance the rate of drug released from the orifice of the drug .
physisorption is a form of absorption in which the solvent molecules can loosely adhere to surfaces of the wicking agent via van der waals interactions between the surface of the wicking agent and the adsorbed molecule .
the function of the wicking agent is to carry water to surfaces inside the core of the tablet , thereby creating channels or a network of increased surface area .
a wicking agent is defined as a material with the ability to draw water into the porous network of a delivery device .
the wicking agents are those agents which help to increase the contact surface area of the drug with the incoming aqueous fluid .
the use of the wicking agent helps to enhance the rate of drug released from the orifice of the drug .
physisorption is a form of absorption in which the solvent molecules can loosely adhere to surfaces of the wicking agent via van der waals interactions between the surface of the wicking agent and the adsorbed molecule .
the function of the wicking agent is to carry water to surfaces inside the core of the tablet , thereby creating channels or a network of increased surface area .
( 4 ) solubilizing agentsfor osmotic drug delivery system , highly water - soluble drugs would demonstrate a high release rate that would be of zero order . thus , many drugs with low intrinsic water solubility are poor candidates for osmotic delivery .
addition of solubilizing agents into the core tablet dramatically increases the drug solubility .
for osmotic drug delivery system
, highly water - soluble drugs would demonstrate a high release rate that would be of zero order .
thus , many drugs with low intrinsic water solubility are poor candidates for osmotic delivery .
nonswellable solubilizing agents are classified into three groups , agents that inhibit crystal formation of the drugs or otherwise act by complexation with the drugs ( e.g. , pvp , poly(ethylene glycol ) ( peg 8000 ) and -cyclodextrin),a micelle - forming surfactant with high hlb value , particularly nonionic surfactants ( e.g. , tween 20 , 60 , and 80 , polyoxyethylene or poly ethylene containing surfactants and other long - chain anionic surfactants such as sls),citrate esters ( e.g. , alkyl esters particularly triethyl citrate ) and their combinations with anionic surfactants .
the combinations of complexing agents such as polyvinyl pyrrolidone ( pvp ) and poly(ethylene glycol ) with anionic surfactants such as sls are mostly preferred .
agents that inhibit crystal formation of the drugs or otherwise act by complexation with the drugs ( e.g. , pvp , poly(ethylene glycol ) ( peg 8000 ) and -cyclodextrin ) , a micelle - forming surfactant with high hlb value , particularly nonionic surfactants ( e.g. , tween 20 , 60 , and 80 , polyoxyethylene or poly ethylene containing surfactants and other long - chain anionic surfactants such as sls ) , citrate esters ( e.g. , alkyl esters particularly triethyl citrate ) and their combinations with anionic surfactants . the combinations of complexing agents such as polyvinyl pyrrolidone ( pvp ) and poly(ethylene glycol ) with anionic surfactants such as sls are mostly preferred .
upon penetration of biological fluid into the osmotic pump through semipermeable membrane , osmogens are dissolved in the biological fluid , which creates osmotic pressure buildup inside the pump and pushes medicament outside the pump through delivery orifice .
generally combinations of osmogens are used to achieve optimum osmotic pressure inside the system ( table 1 ) .
upon penetration of biological fluid into the osmotic pump through semipermeable membrane , osmogens are dissolved in the biological fluid , which creates osmotic pressure buildup inside the pump and pushes medicament outside the pump through delivery orifice .
generally combinations of osmogens are used to achieve optimum osmotic pressure inside the system ( table 1 ) .
they produce an integral composite that is useful for making the wall of the device operative . the surfactants act by regulating the surface energy of materials to improve their blending into the composite and maintain their integrity in the environment of use during the drug release period .
typical surfactants such as poly oxyethylenated glyceryl recinoleate , polyoxyethylenated castor oil having ethylene oxide , glyceryl laurates , and glycerol ( sorbiton oleate , stearate , or laurate ) are incorporated into the formulation .
they produce an integral composite that is useful for making the wall of the device operative .
the surfactants act by regulating the surface energy of materials to improve their blending into the composite and maintain their integrity in the environment of use during the drug release period .
typical surfactants such as poly oxyethylenated glyceryl recinoleate , polyoxyethylenated castor oil having ethylene oxide , glyceryl laurates , and glycerol ( sorbiton oleate , stearate , or laurate ) are incorporated into the formulation .
( 7 ) coating solventssolvents suitable for making polymeric solution that is used for manufacturing the wall of the osmotic device include inert inorganic and organic solvents that do not adversely harm the core and other materials .
the typical solvents include methylene chloride , acetone , methanol , ethanol , isopropyl alcohol , butyl alcohol , ethyl acetate , cyclohexane , carbon tetrachloride , and water .
the mixtures of solvents such as acetone - methanol ( 80 : 20 ) , acetone - ethanol ( 80 : 20 ) , acetone - water ( 90 : 10 ) , methylene chloride - methanol ( 79 : 21 ) , methylene chloride - methanol - water ( 75 : 22 : 3 ) can be used .
solvents suitable for making polymeric solution that is used for manufacturing the wall of the osmotic device include inert inorganic and organic solvents that do not adversely harm the core and other materials .
the typical solvents include methylene chloride , acetone , methanol , ethanol , isopropyl alcohol , butyl alcohol , ethyl acetate , cyclohexane , carbon tetrachloride , and water .
the mixtures of solvents such as acetone - methanol ( 80 : 20 ) , acetone - ethanol ( 80 : 20 ) , acetone - water ( 90 : 10 ) , methylene chloride - methanol ( 79 : 21 ) , methylene chloride - methanol - water ( 75 : 22 : 3 ) can be used .
( 8) plasticizersin pharmaceutical coatings , plasticizers , or low molecular weight diluents are added to modify the physical properties and improve film - forming characteristics of polymers .
plasticizers can turn a hard and brittle polymer into a softer , more pliable material , and possibly make it more resistant to mechanical stress .
plasticizers lower the temperature of the second order - phase transition of the wall or the elastic modules of the wall and also increase the workability , flexibility , and permeability of the coating solvents .
generally from 0.001 to 50 parts of a plasticizer or a mixture of plasticizers are incorporated into 100 parts of costing materials .
peg-600 , peg-200 , triacetin ( ta ) , dibutyl sebacate , ethylene glycol monoacetate , ethylene glycol diacetate , triethyl phosphate , and diethyl tartrate used as plasticizer in formulation of semipermeable membrane [ 38 , 39 ] .
in pharmaceutical coatings , plasticizers , or low molecular weight diluents are added to modify the physical properties and improve film - forming characteristics of polymers .
plasticizers can turn a hard and brittle polymer into a softer , more pliable material , and possibly make it more resistant to mechanical stress .
plasticizers lower the temperature of the second order - phase transition of the wall or the elastic modules of the wall and also increase the workability , flexibility , and permeability of the coating solvents .
generally from 0.001 to 50 parts of a plasticizer or a mixture of plasticizers are incorporated into 100 parts of costing materials .
peg-600 , peg-200 , triacetin ( ta ) , dibutyl sebacate , ethylene glycol monoacetate , ethylene glycol diacetate , triethyl phosphate , and diethyl tartrate used as plasticizer in formulation of semipermeable membrane [ 38 , 39 ] .
( 9 ) pore - forming agentsthese agents are particularly used in the pumps developed for poorly water - soluble drugs and in the development of controlled porosity or multiparticulate osmotic pumps .
the microporous wall may be formed in situ by a pore - former by its leaching during the operation of the system .
the pore - formers can be inorganic or organic and solid or liquid in nature .
for example , alkaline metal salts such as sodium chloride , sodium bromide , potassium chloride , potassium sulphate , potassium phosphate , and so forth , alkaline earth metals such as calcium chloride and calcium nitrate , carbohydrates such as sucrose , glucose , fructose , mannose , lactose , sorbitol , and mannitol , and diols and polyols such as poly hydric alcohols , polyethylene glycols , and polyvinyl pyrrolidone can be used as pore - forming agents .
triethyl citrate ( tec ) and triacetin ( ta ) are also used to create pore in the membrane .
these agents are particularly used in the pumps developed for poorly water - soluble drugs and in the development of controlled porosity or multiparticulate osmotic pumps .
the microporous wall may be formed in situ by a pore - former by its leaching during the operation of the system .
the pore - formers can be inorganic or organic and solid or liquid in nature . for example , alkaline metal salts such as sodium chloride , sodium bromide , potassium chloride , potassium sulphate , potassium phosphate , and so forth , alkaline earth metals such as calcium chloride and calcium nitrate , carbohydrates such as sucrose , glucose , fructose , mannose , lactose , sorbitol , and mannitol , and diols and polyols such as poly hydric alcohols , polyethylene glycols , and polyvinyl pyrrolidone can be used as pore - forming agents .
triethyl citrate ( tec ) and triacetin ( ta ) are also used to create pore in the membrane .
osmotic delivery systems contain at least one delivery orifice in the membrane for drug release .
the size of delivery orifice must be optimized in order to control the drug release from osmotic systems . on the other hand , size of delivery orifice
should not also be too large , otherwise , solute diffusion from the orifice may take place .
if the size of delivery orifice is too small , zero - order delivery will be affected because of development of hydrostatic pressure within the core .
this hydrostatic pressure may not be relieved because of the small orifice size and may lead to deformation of delivery system , thereby resulting in unpredictable drug delivery .
optimum orifice diameter is in the range of 0.0750.274 mm . at orifice size of 0.368 mm and above , control over the delivery rate
delivery orifices in the osmotic systems can be created with the help of a mechanical drill .
laser drilling is one of the most commonly used techniques to create delivery orifice in the osmotic tablet .
laser beam is fired onto the surface of the tablet that absorbs the energy of the beam and gets heated ultimately causing piercing of the wall and , thus forming orifice .
it is possible to control the size of the passageway by varying the laser power , firing duration ( pulse time ) , thickness of the wall , and the dimensions of the beam at the wall . in some of the oral osmotic systems
the system described consists of a incorporation of pore - forming agents into the coating solution .
pore - forming agents are water soluble : upon contact with the aqueous environment , they dissolve in it and leach out from membrane , creatingorifice .
rose and nelson , the australian scientists , were initiators of osmotic drug delivery . in 1955 , they developed an implantable pump for the delivery of drugs to the sheep and cattle gut . the rose - nelson implantable pump shown in figure 2
is composed of three chambers : a drug chamber , a salt chamber holding solid salt , and a water chamber .
the movement of water from the water chamber towards salt chamber is influenced by difference in osmotic pressure across the membrane . conceivably , volume of salt chamber increases due to water flow , which distends the latex diaphragm dividing the salt and drug chambers : eventually , the drug is pumped out of the device .
the kinetics of pumping from rose nelson pump is given by the following equation :
( 1)dmtdt=(dvdt)c ,
where dmt / dt is the drug release rate , dv / dt is the volume flow of water into the salt chamber , and c represents the concentration of drug in the drug chamber .
( 2)dmtdt = acl ,
where , a is the area of semi permeable membrane , is the osmotic pressure gradient , is the permeability of semipermeable membrane , and l is the thickness of semi permeable membrane.these basic equations are applicable to the osmotically driven controlled drug delivery devices .
the saturated salt solution created a high osmotic pressure compared to that pressure required for pumping the suspension of active agent .
therefore , the rate of water entering into the salt chamber remains constant as long as sufficient solid salt is present in die salt chamber to maintain a saturated solution and thereby a constant osmotic pressure driving force is generated .
the major problem associated with rose - nelson pumps was that the osmotic action began whenever water came in contact with the semipermeable membrane .
this needed pumps to be stored empty and water to be loaded prior to use .
higuchi and leeper have proposed a number of variations of the rose - nelson pump and these designs have been described in us patents [ 46 , 47 ] , which represent the first series of simplifications of the rose - nelson pump made by the alza corporation .
the higuchi - leeper pump has no water chamber , and the activation of the device occurs after imbibition of the water from the surrounding environment .
this variation allows the device to be prepared loaded with drug and can be stored for long prior to use .
higuchi - leeper pumps contain a rigid housing and a semi permeable membrane supported on a perforated frame ; a salt chamber containing a fluid solution with an excess of solid salt is usually present in this type of pump . upon administration / implantation , surrounding biological
fluid penetrates into the device through porous and semipermeable membrane and dissolves the mgso4 , creating osmotic pressure inside the device that pushes movable separator toward the drug chamber to remove drug outside the device .
this type of pump is implanted in body of an animal for delivery of antibiotics or growth hormones to animals .
pulsatile delivery could be achieved by using higuchi leeper pump ; such modifications are described and illustrated in figure 4 .
the pulsatile release of drug is achieved by drilling the orifice in elastic material that stretches under the osmotic pressure .
pulse release of drug is obtained after attaining a certain critical pressure , which causes the orifice to open .
the pressure then reduces to cause orifice closing and the cycle repeats to provide drug delivery in a pulsatile fashion .
the orifice should be small enough to be substantially closed when the threshold level of osmotic pressure is not present .
higuchi and theeuwes in early 1970s developed another variant of the rose - nelson pump , even simpler than the higuchi - leeper pump .
this device is illustrated in figure 5 . in this device , the rigid housing consisted of a semipermeable membrane .
this membrane is strong enough to withstand the pumping pressure developed inside the device due to imbibition of water .
the drug is loaded in the device only prior to its application , which extends advantage for storage of the device for longer duration .
the release of the drug from the device is governed by the salt used in the salt chamber and the permeability characteristics of the outer membrane .
small osmotic pumps of this form are available under trade name alzet made by alza corporation in 1976 .
they are used frequently as implantable controlled release delivery systems in experimental studies requiring continuous administration of drugs .
such a implantable alzet pump is shown in figure 6 . rose - nelson pump was further simplified in the form of elementary osmotic pump [ 20 , 52 ] , which made osmotic delivery as a major method of achieving controlled drug release . elementary osmotic pump shown in figure 7
was invented by theeuwes in 1974 and it essentially contains an active agent having a suitable osmotic pressure ; it is fabricated as a tablet coated with semi permeable membrane , usually cellulose acetate [ 32 , 53 ] .
when this coated tablet is exposed to an aqueous environment , the osmotic pressure of the soluble drug inside the tablet draws water through the semi permeable coating and a saturated aqueous solution of drug is formed inside the device .
the membrane is nonextensible and the increase in volume due to imbibition of water raises the hydrostatic pressure inside the tablet , eventually leading to flow of saturated solution of active agent out of the device through a small orifice .
the pump initially releases the drug at a rate given by the following equation ;
( 3)dmtdt=(dvdt)cs ,
where dv / dt depicts the water flow into the tablet and cs is the solubility of the agent inside the tablet .
push - pull osmotic pump is delivered both poorly water soluble and highly water soluble drugs at a constant rate .
one layer ( the upper layer ) contains drug in a formulation of polymeric , osmotic agent , and other tablet excipients .
this polymeric osmotic agent has the ability to form a suspension of drug in situ .
when this tablet later imbibes water , the other layer contains osmotic and colouring agents , polymer and tablet excipients .
these layers are formed and bonded together by tablet compression to form a single bilayer core .
after the coating has been applied , a small hole is drilled through the membrane by a laser or mechanical drill on the drug layer side of the tablet . when the system is placed in aqueous environment ,
the osmotic attraction in the drug layer pulls water into the compartment to form in situ a suspension of drug .
the osmotic agent in the nondrug layer simultaneously attracts water into that compartment , causing it to expand volumetrically , and the expansion of nondrug layer pushes the drug suspension out of the delivery orifice [ 20 , 52 ] .
it is an osmotic tablet wherein the delivery orifices ( holes ) are formed in situ through leaching of water soluble pore - forming agents incorporated in semipermeable membrane ( spm ) ( e.g. , urea , nicotinamide , sorbitol , etc . ) .
drug release rate from cpop depends on various factors like coating thickness , solubility of drug in tablet core , level of leachable pore - forming agent(s ) and the osmotic pressure difference across the membrane [ 54 , 55 ] .
the stomach irritation problems are considerably reduced , as drug is released from the whole of the device surface rather from a single hole .
further , no complicated laser - drilling unit is required because the holes are formed in situ .
various l - oros systems available to provide controlled delivery of liquid drug formulations include l - oros hardcap , l - oros softcap , and a delayed liquid bolus delivery system .
each of these systems includes a liquid drug layer , an osmotic engine or push layer , and a semipermeable membrane coating .
when the system is in contact with the aqueous environment , water permeates across the rate - controlling membrane and activates the osmotic layer ( figure 11 ) [ 9 , 57 ] .
the expansion of the osmotic layer results in the development of hydrostatic pressure inside the system , thereby forcing the liquid formulation to be delivered at the delivery orifice .
whereas l - oros hardcap and l - oros softcap systems are designed to provide continuous drug delivery , the l - oros delayed liquid bolus delivery system is designed to deliver a pulse of liquid drug ( figure 10 ) [ 34 , 58 ] .
the delayed liquid bolus delivery system comprises three layers : a placebo delay layer , a liquid drug layer , and an osmotic engine , all surrounded by a rate - controlling semipermeable membrane ( spm ) .
the delivery orifice is drilled on the placebo layer end of the capsule shaped device . when the osmotic engine expands , the placebo is released first , delaying release of the drug layer ( figure 10 ) .
drug release can be delayed from 1 to 10 hours , depending on permeability of the rate - controlling membrane and the size of placebo .
figure 12 shows that sandwiched osmotic tablet is composed of polymeric push layer sandwiched between two drug layers with two delivery orifices .
when placed in the aqueous environment , the middle push layer containing the swelling agents ' swells and the drug is released from the two orifices situated on opposite sides of the tablet ; thus sandwiched osmotic tablets ( sots ) can be suitable for drugs prone to cause local irritation of the gastric mucosa ( table 2 ) [ 32 , 55 , 60 ] .
osmotic drug delivery systems typically consist of a drug core containing osmogen that is coated with a semipermeable membrane .
this coating has one or more delivery ports through which a solution or suspension of the drug is released over time .
various osmotic systems include rose - nelson pump , the higuchi - leeper pumps , the alzet and osmet systems , the elementary osmotic pump , and the push - pull pump .
recent advances include the development of the controlled porosity osmotic pump , l - oros pump , and sandwiched osmotic tablet . in future
, various attempts are made to produce successful osmotic system like pulsatile delivery based on expandable orifice , lipid osmotic pump , telescopic capsule containing mini osmotic pump for delayed release , osmotic bursting osmotic pump , and so forth . | conventional drug delivery systems are known to provide an immediate release of drug , in which one can not control the release of the drug and can not maintain effective concentration at the target site for longer time .
controlled drug delivery systems offer spatial control over the drug release .
osmotic pumps are most promising systems for controlled drug delivery .
these systems are used for both oral administration and implantation .
osmotic pumps consist of an inner core containing drug and osmogens , coated with a semipermeable membrane .
as the core absorbs water , it expands in volume , which pushes the drug solution out through the delivery ports .
osmotic pumps release drug at a rate that is independent of the ph and hydrodynamics of the dissolution medium .
the historical development of osmotic systems includes development of the rose - nelson pump , the higuchi - leeper pumps , the alzet and osmet systems , the elementary osmotic pump , and the push - pull system .
recent advances include development of the controlled porosity osmotic pump , and systems based on asymmetric membranes .
this paper highlights the principle of osmosis , materials used for fabrication of pumps , types of pumps , advantages , disadvantages , and marketed products of this system . | 1. Introduction
2. Osmotically Controlled Drug Delivery Systems
3. Materials Used in Formulation of Osmotic Pumps
4. Creation of Delivery Orifice
5. Types of Osmotic Pumps
6. Conclusion | conventional oral drug delivery systems are known to provide an immediate release of drug , in which one can not control the release of the drug and can not maintain effective concentration at the target site for longer period of time . controlled drug delivery systems offer temporal and/or spatial control over the release of drug . these dosage forms offer many advantages , such as nearly constant drug level at the site of action , prevention of peak - valley fluctuations , reduction in dose of drug , reduced dosage frequency , avoidance of side effects , and improved patient compliance [ 5 , 6 ] . the oral controlled release system shows a typical pattern of drug release in which the drug concentration is maintained in between the minimum effective concentration ( mec ) and maximum safe concentration ( msc ) for a prolonged period of time , thereby ensuring sustained therapeutic action ( figure 1 ) . osmotic devices are most promising strategy - based systems for controlled drug delivery [ 79 ] . osmotic pump offers many advantages over other controlled drug delivery systems , that is , they are easy to formulate and simple in operation , improved patient compliance with reduced dosing frequency and more consistence , and prolonged therapeutic effect with uniform blood concentration . osmotic drug - delivery systems suitable for oral administration typically consist of a compressed tablet core that is coated with a semipermeable membrane coating . as the core absorbs water , it expands in volume , which pushes the drug solution or suspension out of the tablet through one or more delivery ports [ 12 , 13 ] . the key distinguishing feature of osmotic drug delivery systems ( compared with other technologies used in controlled - release formulations ) is that they release drug at a rate that is independent of the ph and hydrodynamics of the external dissolution medium . the historical development of osmotic systems includes seminal contributions such as the rose - nelson pump , the higuchi - leeper pumps , the alzet and osmet systems , the elementary osmotic pump , and the push - pull or gitsr system . recent advances include the development of the controlled porosity osmotic pump [ 21 , 22 ] , systems based on asymmetric membranes [ 2325 ] , and other approaches . ( 2 ) hydrophilic and hydrophobic polymers these polymers are used in the formulation development of osmotic systems for making drug containing matrix core . the selection is based on the solubility of the drug as well as the amount and rate of drug to be released from the pump . these polymers are used in the formulation development of osmotic systems for making drug containing matrix core . the selection is based on the solubility of the drug as well as the amount and rate of drug to be released from the pump . it is possible to control the size of the passageway by varying the laser power , firing duration ( pulse time ) , thickness of the wall , and the dimensions of the beam at the wall . the kinetics of pumping from rose nelson pump is given by the following equation :
( 1)dmtdt=(dvdt)c ,
where dmt / dt is the drug release rate , dv / dt is the volume flow of water into the salt chamber , and c represents the concentration of drug in the drug chamber . higuchi and leeper have proposed a number of variations of the rose - nelson pump and these designs have been described in us patents [ 46 , 47 ] , which represent the first series of simplifications of the rose - nelson pump made by the alza corporation . the higuchi - leeper pump has no water chamber , and the activation of the device occurs after imbibition of the water from the surrounding environment . higuchi and theeuwes in early 1970s developed another variant of the rose - nelson pump , even simpler than the higuchi - leeper pump . rose - nelson pump was further simplified in the form of elementary osmotic pump [ 20 , 52 ] , which made osmotic delivery as a major method of achieving controlled drug release . the pump initially releases the drug at a rate given by the following equation ;
( 3)dmtdt=(dvdt)cs ,
where dv / dt depicts the water flow into the tablet and cs is the solubility of the agent inside the tablet . the osmotic agent in the nondrug layer simultaneously attracts water into that compartment , causing it to expand volumetrically , and the expansion of nondrug layer pushes the drug suspension out of the delivery orifice [ 20 , 52 ] . each of these systems includes a liquid drug layer , an osmotic engine or push layer , and a semipermeable membrane coating . osmotic drug delivery systems typically consist of a drug core containing osmogen that is coated with a semipermeable membrane . various osmotic systems include rose - nelson pump , the higuchi - leeper pumps , the alzet and osmet systems , the elementary osmotic pump , and the push - pull pump . recent advances include the development of the controlled porosity osmotic pump , l - oros pump , and sandwiched osmotic tablet . | [
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] |
many plants that grow in climates with a cold winter require growth for several months at low temperatures a process called vernalization to promote flowering in spring , when days lengthen and temperatures increase . without this period of cold , plants would grow leaves in the spring , but would fail to flower .
it was noticed that this phenomenon , familiar to every horticulturist , had distinctive features ; something occurred during those cold months that left a mark , which , in effect , released a switch that permitted flowering in spring .
experimental manipulation of temperature showed that this mark was very stable and could persist for at least a year after a vernalization treatment .
although stable during a plant 's life , the marks were erased between generations , as the progeny of vernalized plants themselves required vernalization in order to flower . in recent years , the field has looked beyond the genome and tested whether epigenetic changes ( i.e. , ones that are n't based on dna sequence alteration ) were involved .
one epigenetic system involves dna methylation , and in plants this is often used for genome defense ; for example , to inactivate invading dna such as transposons , viruses , and transgenes .
a second epigenetic system is implemented by two collections of genes known as the polycomb group ( pcg ) and the trithorax group ( trxg ) .
again , methylation is involved , but in this case it is not the dna itself that is modified , it is the histone proteins that package dna in the nucleus ( figure 1a ) . unlike the changes wrought by dna methylation ,
there is ( so far ) little clear - cut evidence that pcg- or trxg - mediated changes are passed between generations in plants .
rather , they are used to provide cells with memories of events that occur during the life of an organism .
typically this is a developmental memory , hence the pcg and trxg components were first identified on account of gross developmental abnormalities resulting from their mutation .
however , they are also used to record transient environmental events that occur during the life of the plant .
this can be used to predict and adapt to future environments and may be especially relevant in plants , which are sessile and so ca n't easily escape their circumstances . here
, we discuss important advances in our understanding of how pcg genes provide a memory of winter .
secondly , we describe the emerging role of pcg and trxg genes in controlling stem cell fate in flowers .
the nucleosome is an octamer containing two molecules each of histone h2a , h2b , h3 , and h4 . for simplicity ,
only one of each of the two h2a and h3 tails are shown in the figure .
( b , c , d ) likely components and functions of arabidopsis pcg and trxg protein complexes equivalent to animal prc2 , prc1 , and trx complexes are shown .
( b ) during vernalization in arabidopsis , the phd - prc2 complex catalyzes h3k27me3 methylation through the swn histone methyltransferase subunit .
vin3 , vel1 , and vrn5 are plant - specific , whereas the other four members are homologs of the animal prc2 core components .
because three of the four core components of animal prc2 have been duplicated in arabidopsis , it is likely that several related complexes exist that differ in components and target gene specificities .
for example , in some complexes , clf may replace swn as the histone methyltransferase unit .
prc1 may catalyze h2ak120ub ubiquitination via its e3 ligase components atring1a , atring1b , atbmi1a , and atbmi1b .
emf1 is likely another ( plant - specific ) component whose precise function is unclear .
( d ) ult1 and atx1 may be components of an arabidopsis trxg complex that catalyses h3k4me3 .
atx1 , arabidopsis homolog of trithorax 1 ; clf , curly leaf ; emf1 , embryonic flower 1 ; fie , fertilization independent endosperm ; lhp1 , like heterochromatin protein 1 ; msi1 , multicopy suppressor of ira 1 ; pcg , polycomb group ; phd - prc2 , plant homeo domain polycomb repressive complex 2 ; prc1 , polycomb repressive complex 1 ; swn , swinger ; trxg , trithorax group ; ult1 , ultrapetala1 ; vel1 , vernalization5/vin3-like 1 ; vin3 , vernalization insensitive 3 ; vrn2 , vernalization2 ; vrn5 , vernalization5 .
the mechanism of the vernalization response has been best studied in the model plant arabidopsis thaliana , where it has been found to depend on the ability of the pcg system to silence the key gene mediating the vernalization response , flowering locus c ( flc ) .
flc is a potent repressor of flowering , most likely because its protein product binds and represses two genes , flowering locus t ( ft ) and suppressor of overexpression of constans 1 ( soc1 ) , which are central players in promoting flowering .
thus in order for plants to become competent to respond to environmental factors promoting flowering ( warm temperatures and long days ) , flc must be switched off . during cold treatments ,
expression of flc is progressively reduced and a vernalization - specific complex of pcg proteins ( termed phd - prc2 , which stands for plant homeo domain polycomb repressive complex 2 ) ( figure 1b ) is recruited to flc .
biochemical purification of this complex indicates that it contains the plant equivalents of the four core components of an animal pcg protein complex known as prc2 ( polycomb repressive complex 2 ) .
prc2 acts as a histone methyltransferase , specifically catalyzing trimethylation of lysine 27 on the histone h3 tail ( h3k27me3 ) .
in addition to the core prc2 components , phd - prc2 contains several plant - specific components that are probably required to boost the histone methyltransferase activity of the complex .
consistent with this , h3k27me3 levels at flc increase during cold treatments and persist afterwards when plants are moved to warm conditions .
h3k27me3 is also necessary for maintenance of flc silencing when plants are removed from the cold . how then does h3k27me3 cause gene silencing ?
although it is necessary for silencing of flc , it is not sufficient : in vernalization1 ( vrn1 ) mutants , flc activity is restored after vernalization , despite the persistence of high h3k27 methylation levels .
h3k27me3 is not thought to directly cause chromatin compaction or inhibit transcription but is probably recognized by proteins that repress transcription of flc . in animals ,
a second complex of pcg proteins , called prc1 , is also required for pcg - mediated silencing .
although the prc1 complex is less well conserved than prc2 , an important recent finding is that several of its members are found in plants and play a similar role in pcg - mediated repression .
one prc1 member , polycomb , binds h3k27me3 , and in arabidopsis the related like heterochromatin 1 ( lhp1 ) protein has a similar function ( figure 1c ) .
it is also likely that prc2 itself binds h3k27me3 , which may help in the stable propagation of this mark through cell division , by methylating newly synthesized ( unmodified ) histones as they are deposited into chromatin , for example .
three other components of prc1 in animals , ring1a , ring1b , and bmi1 , form a ubiquitin ligase complex that monoubiquitinates histone h2a at lysine 119 ( h2ak119ub ) .
although h2ak119ub may help repress transcription directly by blocking rna polymerase ii transcriptional elongation from the promoters of pcg target genes , its importance is challenged by the observation that prc1 can cause chromatin compaction and transcriptional repression independently of its h2a ubiquitination activity .
be this as it may , two groups have now identified arabidopsis homologs of ring1a , ring1b , and bmi1 ( figure 1c ) and the similarity of their knockout phenotypes with those of mutants inactivating prc2 is compelling evidence that they have a similar role to animal prc1 .
in addition , the plant ring proteins can monoubiquitinate h2ak120 ( the residue in plants that most closely corresponds to the animal h2ak119 , although most of the 13 arabidopsis h2a genes do n't encode this residue ) in in - vitro assays and are required for h2a ubiquitination in vivo in arabidopsis .
although so far only a handful of pcg targets have been shown to be misregulated in the arabidopsis atring / atbmi1 mutants , it will obviously be interesting to see if the arabidopsis atring / atbmi genes have any role in the vernalization - induced repression of flc .
because the pcg components are mostly expressed fairly constitutively and seemingly lack specific dna - binding activity , a major problem has been to explain how they are recruited to specific targets such as flc .
numerous recent studies in animals have shown that noncoding rna play a role in recruitment , and two noncoding rnas have now been implicated in vernalization - induced recruitment of phd - prc2 .
one of these , termed coldair , is a long ( about 1 kb ) noncoding sense rna produced from sequences within the large first intron of flc ( figure 2 ) .
expression of coldair is cold - induced and several observations suggest that it is functionally important for flc silencing .
firstly , two earlier studies using transgene reporters showed that sequences within the large first intron of flc are needed for its stable silencing by cold .
in particular , transgenes with deletions that removed part or all of the coldair gene showed fairly normal downregulation during cold treatments but regained activity when plants were returned to warm conditions . secondly , several approaches showed that curly leaf ( clf ) , the catalytic subunit of the phd - prc2 complex , binds coldair rna .
this may help recruit phd - prc2 to flc ; for example , if the coldair transcript remains tethered to its site of transcription at flc ( figure 2 ) or alternatively if the affinity of phd - prc2 for chromatin is altered by its binding coldair .
consistent with this , downregulation of coldair using rna interference strongly reduces the recruitment of clf to flc and also impairs the maintenance of flc silencing following cold treatments . a second noncoding rna , termed coolair , is also produced from flc .
coolair expression is driven by a promoter located downstream of the flc coding region ( figure 2 ) and produces several antisense transcripts that differ according to their splicing or 3 end .
two genes , fpa and fca , promote flowering by downregulating flc expression . although fpa and fca encode proteins that regulate mrna processing , until recently it was problematic that they had no discernable effect on flc mrna processing .
two groups have now resolved this issue by demonstrating that they regulate the processing and relative amounts of the different coolair transcripts .
however , fpa and fca are not required for the vernalization response , and the function of coolair in vernalization is less clear .
notably , flc transgenes that lack sequences downstream of flc that include the coolair promoter retain a normal vernalization response .
although this suggests that coolair may not be necessary , interpretation is complicated because these plants retain an endogenous flc gene that produces coolair transcripts that could act in trans on the flc transgene .
it is also noteworthy that coldair is not sufficient for a vernalization response ; thus , the introduction of flc intragenic sequences that contain coldair into a heterologous reporter did not confer a vernalization response on the reporter .
one possibility , suggested by heo and sung , is that induction of coolair by cold may reduce transcription from the normal promoter 5 end of the flc protein coding region and promote coldair expression from its cryptic promoter within the first flc intron .
the open reading frame of flc is shown in turquoise , with exons depicted as green boxes and introns as black lines .
however , only the coldair transcript is thought to be bound by phd - prc2 and so may be more important in recruiting phd - prc2 to the flc locus .
flc , flowering locus c ; phd - prc2 , plant homeo domain polycomb repressive complex 2 ; rna pol ii , rna polymerase ii .
although many plants show a vernalization response with epigenetic features , the target genes and even the mechanisms may be different than those in arabidopsis . in sugar beet ,
vernalization results in stable repression of a floral repressor , which , although it is unrelated to flc , acts in a similar fashion to repress an ft homolog .
it is not clear yet if this is also pcg mediated . in cereals , vernalization causes the stable upregulation of vrn1 , which promotes flowering . here
, the epigenetic memory is of an active state , and is correlated with h3k4me3 methylation at vrn1 .
it is likely , although not yet proven , that this will involve the trxg genes as these are known to catalyze h3k4me3 methylation and mediate stable gene activation .
once flower induction occurs , the indeterminate vegetative shoot apical meristem at the shoot apex changes its identity to become an inflorescence meristem , which makes floral meristems on its flanks .
the floral meristem produces whorls of floral organ primordia , and so gives rise to the flowers .
like all meristems , the continued production of organ primordia requires the activity of a pool of stem cells in the centre of the meristem .
unlike the other meristems , the floral meristem is determinate so that organ production ceases once the carpels are initiated in the innermost whorl of the flower .
mutations in pcg and trxg genes are well known to cause changes in floral organ identity , reflecting their role in controlling floral homeotic gene activity . however , a second less well emphasized aspect of their mutant phenotype is an increase in floral organ number .
this reflects an emerging role in controlling when the flower stem cell pool is terminated . in arabidopsis shoots ,
the stem cells at the top of the meristem secrete the clavata3 ( clv3 ) signal peptide , which restricts the expression of the transcription factor wuschel ( wus ) in the organizing centre beneath the stem cells . in turn
, wus promotes stem cell fate and as a result also promotes clv3 expression non - cell - autonomously in cells above the organizing centre .
this clv3/wus feedback loop mediates homeostasis of the stem cells via a rapid response to fluctuations in either clv3 or wus expression [ 23 - 25 ] .
however , flowers are determinate structures and as a consequence , a second feedback loop terminates the stem cell pool of floral meristems .
in contrast to the fast and continuously operating clv3/wus system , this feedback loop , built by wus and the mads - box transcription factor agamous ( ag ) , occurs just once .
early in flower development ( stage 3 ) , wus and another transcription factor , leafy , together activate expression of ag in the centre of the floral meristem . in turn ,
ag represses wus expression but there is a delay of about two days before wus expression terminates at stage 6 .
in addition , the first step ( the activation of ag expression by wus ) also exhibits a delay : wus expression occurs during floral stages 2 - 5 . by contrast , ag is not detectable in floral meristem until stage 3 .
the reason for these delays has been unclear but two recent publications reveal the relevance of epigenetic regulation by the pcg and trxg ( figure 3 ) .
serial floral stages with the expression of wus , ag , and knu are shown .
underneath , the genomic regions of these genes are shown schematically with likely binding of transcription factors , expression status , and histone modifications . at the initial stage of flower development ( stage 1 ) ,
wus is activated at stage 2 , but the transcriptional activation of ag by wus is inhibited by h3k27me3 . at stage 3 , the ag locus undergoes activation by progressive ult1/atx1-dependent h3k4me3 and likely demethylation of h3k27 .
the activation of knu by ag is initially inhibited by h3k27me3 at the knu locus between stages 3 to 5 .
the level of h3k27me3 at the knu locus is gradually depleted , perhaps by dilution via successive rounds of cell division , and at stage 6 knu is expressed , causing wus repression .
the punctual termination of wus expression needs additional prc1 activity , which suggests that the wus locus accumulates h3k27me3 and h2ak120ub .
ag , agamous ; atx1 , arabidopsis homolog of trithorax 1 ; knu , knuckels ; ult1 , ultrapetala1 ; wus , wuschel . carles and
fletcher investigated whether the activation of ag is dependent on the arabidopsis sand ( sp100 , aire-1 , nucp41/75 , deaf-1 ) domain protein ultrapetala1 ( ult1 ) and furthermore , whether ult1 is involved in trxg function .
the authors were inspired by the gain - of - function phenotypes of 35s::ult1 transgenic plants , which resemble clf mutants in showing increased ag expression in leaves and inflorescences .
furthermore , ult1 mutants suppress the clf mutant phenotype in ult1 clf double mutants and show reduced ag expression .
this fits with idea that ult1 is a trxg factor and works antagonistically to the pcg to promote ag activity , and may also explain why wus is misexpressed in ult1 mutants .
consistent with this antagonism , 35s::ult1 seedlings , similar to clf mutants , have reduced levels of the repressive mark h3k27me3 at ag , whereas ult1 mutants have increased h3k27me3 levels .
the structure of ult1 makes it unlikely that it is itself an h3k4me3 methyltransferase , rather it may interact with such an enzyme as part of a trxg complex or promote trxg recruitment to targets .
consistent with this , ult1 interacts physically with the trxg protein arabidopsis homolog of trithorax 1 ( atx1 ) , which is responsible for local h3k4me3 deposition ( figure 1d ) .
atx1 can not be the only factor for ult1-dependent h3k4me3 at the ag locus because atx1 mutants , unlike ult1 mutants , do not show indeterminacy of floral meristems and also retain 85% of global h3k4me3 levels .
furthermore , although the ult1-dependent activation of ag by a slow increase of h3k4me3 may provide a time buffer that the transcription factor wus on its own may not provide , it remains unclear whether ult1 and wus work in parallel or in the same pathway .
intriguingly , the delay in the second step of the ag / wus feedback loop the final suppression of wus by ag may also involve epigenetic control .
sun et al . identified knuckels ( knu ) , which encodes a c2h2-type zinc finger transcription factor , as a direct target of ag and a key intermediate in the suppression of wus expression .
genetic analysis and overexpression experiments imply that knu is activated by ag and represses wus expression .
curiously , although ag binds the knu promoter directly , there is a two day lag between the activation of ag and that of knu .
several observations indicate that knu is a pcg target : first , early in floral meristem development the knu locus is strongly decorated with h3k27me3 ; second , in various pcg mutants knu is misexpressed . using an elegant system for synchronizing floral development , sun et al .
show that between floral development stage 3 , when ag is activated , and stage 6 , when knu is activated and wus expression terminated , h3k27me3 is gradually removed from the knu locus . but how can the mads domain transcription factor ag cause histone demethylation ?
one possibility is that ag recruits h3k27me3 demethylases , which have been identified in animals but not yet in plants .
a simple alternative is that ag somehow inhibits the binding of prc1 and prc2 complexes to the knu locus and the h3k27me3 mark is gradually diluted as unmodified histones are incorporated onto replicated dna during cell division .
either way , the h3k27me3 at knu is not sufficient to prevent knu activation by ag but does delay it . similarly , two other recent studies that looked at the activation of another pcg target , in this case by cold , also conclude that h3k27me3 is important for slowing the kinetics of induction .
although the tardy induction of knu in flowers suggests a mechanism for the delayed suppression of wus by ag , it can not be the only factor that terminates wus expression .
thus , knu mutants have much weaker effects on floral determinacy than ag mutants , leaving space for other ag - dependent mechanisms .
as wus is itself a target of h3k27me3 , it is also possible that its repression by knu is just the initial step that facilitates a long - term epigenetic repression by prc1 . supporting this thesis ,
wus is misexpressed in mutants of components of prc1 , which bear extra floral organs .
the mechanism of the vernalization response has been best studied in the model plant arabidopsis thaliana , where it has been found to depend on the ability of the pcg system to silence the key gene mediating the vernalization response , flowering locus c ( flc ) .
flc is a potent repressor of flowering , most likely because its protein product binds and represses two genes , flowering locus t ( ft ) and suppressor of overexpression of constans 1 ( soc1 ) , which are central players in promoting flowering .
thus in order for plants to become competent to respond to environmental factors promoting flowering ( warm temperatures and long days ) , flc must be switched off . during cold treatments ,
expression of flc is progressively reduced and a vernalization - specific complex of pcg proteins ( termed phd - prc2 , which stands for plant homeo domain polycomb repressive complex 2 ) ( figure 1b ) is recruited to flc .
biochemical purification of this complex indicates that it contains the plant equivalents of the four core components of an animal pcg protein complex known as prc2 ( polycomb repressive complex 2 ) .
prc2 acts as a histone methyltransferase , specifically catalyzing trimethylation of lysine 27 on the histone h3 tail ( h3k27me3 ) .
in addition to the core prc2 components , phd - prc2 contains several plant - specific components that are probably required to boost the histone methyltransferase activity of the complex .
consistent with this , h3k27me3 levels at flc increase during cold treatments and persist afterwards when plants are moved to warm conditions .
h3k27me3 is also necessary for maintenance of flc silencing when plants are removed from the cold . how then does h3k27me3 cause gene silencing ?
although it is necessary for silencing of flc , it is not sufficient : in vernalization1 ( vrn1 ) mutants , flc activity is restored after vernalization , despite the persistence of high h3k27 methylation levels .
h3k27me3 is not thought to directly cause chromatin compaction or inhibit transcription but is probably recognized by proteins that repress transcription of flc . in animals ,
a second complex of pcg proteins , called prc1 , is also required for pcg - mediated silencing .
although the prc1 complex is less well conserved than prc2 , an important recent finding is that several of its members are found in plants and play a similar role in pcg - mediated repression .
one prc1 member , polycomb , binds h3k27me3 , and in arabidopsis the related like heterochromatin 1 ( lhp1 ) protein has a similar function ( figure 1c ) .
it is also likely that prc2 itself binds h3k27me3 , which may help in the stable propagation of this mark through cell division , by methylating newly synthesized ( unmodified ) histones as they are deposited into chromatin , for example .
three other components of prc1 in animals , ring1a , ring1b , and bmi1 , form a ubiquitin ligase complex that monoubiquitinates histone h2a at lysine 119 ( h2ak119ub ) .
although h2ak119ub may help repress transcription directly by blocking rna polymerase ii transcriptional elongation from the promoters of pcg target genes , its importance is challenged by the observation that prc1 can cause chromatin compaction and transcriptional repression independently of its h2a ubiquitination activity .
be this as it may , two groups have now identified arabidopsis homologs of ring1a , ring1b , and bmi1 ( figure 1c ) and the similarity of their knockout phenotypes with those of mutants inactivating prc2 is compelling evidence that they have a similar role to animal prc1 .
in addition , the plant ring proteins can monoubiquitinate h2ak120 ( the residue in plants that most closely corresponds to the animal h2ak119 , although most of the 13 arabidopsis h2a genes do n't encode this residue ) in in - vitro assays and are required for h2a ubiquitination in vivo in arabidopsis .
although so far only a handful of pcg targets have been shown to be misregulated in the arabidopsis atring / atbmi1 mutants , it will obviously be interesting to see if the arabidopsis atring / atbmi genes have any role in the vernalization - induced repression of flc .
because the pcg components are mostly expressed fairly constitutively and seemingly lack specific dna - binding activity , a major problem has been to explain how they are recruited to specific targets such as flc .
numerous recent studies in animals have shown that noncoding rna play a role in recruitment , and two noncoding rnas have now been implicated in vernalization - induced recruitment of phd - prc2 .
one of these , termed coldair , is a long ( about 1 kb ) noncoding sense rna produced from sequences within the large first intron of flc ( figure 2 ) .
expression of coldair is cold - induced and several observations suggest that it is functionally important for flc silencing .
firstly , two earlier studies using transgene reporters showed that sequences within the large first intron of flc are needed for its stable silencing by cold .
in particular , transgenes with deletions that removed part or all of the coldair gene showed fairly normal downregulation during cold treatments but regained activity when plants were returned to warm conditions . secondly , several approaches showed that curly leaf ( clf ) , the catalytic subunit of the phd - prc2 complex , binds coldair rna .
this may help recruit phd - prc2 to flc ; for example , if the coldair transcript remains tethered to its site of transcription at flc ( figure 2 ) or alternatively if the affinity of phd - prc2 for chromatin is altered by its binding coldair .
consistent with this , downregulation of coldair using rna interference strongly reduces the recruitment of clf to flc and also impairs the maintenance of flc silencing following cold treatments . a second noncoding rna , termed coolair , is also produced from flc .
coolair expression is driven by a promoter located downstream of the flc coding region ( figure 2 ) and produces several antisense transcripts that differ according to their splicing or 3 end .
two genes , fpa and fca , promote flowering by downregulating flc expression . although fpa and fca encode proteins that regulate mrna processing , until recently it was problematic that they had no discernable effect on flc mrna processing .
two groups have now resolved this issue by demonstrating that they regulate the processing and relative amounts of the different coolair transcripts .
however , fpa and fca are not required for the vernalization response , and the function of coolair in vernalization is less clear .
notably , flc transgenes that lack sequences downstream of flc that include the coolair promoter retain a normal vernalization response .
although this suggests that coolair may not be necessary , interpretation is complicated because these plants retain an endogenous flc gene that produces coolair transcripts that could act in trans on the flc transgene .
it is also noteworthy that coldair is not sufficient for a vernalization response ; thus , the introduction of flc intragenic sequences that contain coldair into a heterologous reporter did not confer a vernalization response on the reporter .
one possibility , suggested by heo and sung , is that induction of coolair by cold may reduce transcription from the normal promoter 5 end of the flc protein coding region and promote coldair expression from its cryptic promoter within the first flc intron .
the open reading frame of flc is shown in turquoise , with exons depicted as green boxes and introns as black lines .
however , only the coldair transcript is thought to be bound by phd - prc2 and so may be more important in recruiting phd - prc2 to the flc locus .
flc , flowering locus c ; phd - prc2 , plant homeo domain polycomb repressive complex 2 ; rna pol ii , rna polymerase ii .
although many plants show a vernalization response with epigenetic features , the target genes and even the mechanisms may be different than those in arabidopsis . in sugar beet ,
vernalization results in stable repression of a floral repressor , which , although it is unrelated to flc , acts in a similar fashion to repress an ft homolog .
it is not clear yet if this is also pcg mediated . in cereals , vernalization causes the stable upregulation of vrn1 , which promotes flowering . here
, the epigenetic memory is of an active state , and is correlated with h3k4me3 methylation at vrn1 .
it is likely , although not yet proven , that this will involve the trxg genes as these are known to catalyze h3k4me3 methylation and mediate stable gene activation .
once flower induction occurs , the indeterminate vegetative shoot apical meristem at the shoot apex changes its identity to become an inflorescence meristem , which makes floral meristems on its flanks .
the floral meristem produces whorls of floral organ primordia , and so gives rise to the flowers .
like all meristems , the continued production of organ primordia requires the activity of a pool of stem cells in the centre of the meristem . unlike the other meristems , the floral meristem is determinate so that organ production ceases once the carpels are initiated in the innermost whorl of the flower .
mutations in pcg and trxg genes are well known to cause changes in floral organ identity , reflecting their role in controlling floral homeotic gene activity . however , a second less well emphasized aspect of their mutant phenotype is an increase in floral organ number .
this reflects an emerging role in controlling when the flower stem cell pool is terminated . in arabidopsis shoots ,
the stem cells at the top of the meristem secrete the clavata3 ( clv3 ) signal peptide , which restricts the expression of the transcription factor wuschel ( wus ) in the organizing centre beneath the stem cells . in turn
, wus promotes stem cell fate and as a result also promotes clv3 expression non - cell - autonomously in cells above the organizing centre . this clv3/wus feedback loop mediates homeostasis of the stem cells via a rapid response to fluctuations in either clv3 or wus expression [ 23 - 25 ] .
however , flowers are determinate structures and as a consequence , a second feedback loop terminates the stem cell pool of floral meristems .
in contrast to the fast and continuously operating clv3/wus system , this feedback loop , built by wus and the mads - box transcription factor agamous ( ag ) , occurs just once .
early in flower development ( stage 3 ) , wus and another transcription factor , leafy , together activate expression of ag in the centre of the floral meristem . in turn ,
ag represses wus expression but there is a delay of about two days before wus expression terminates at stage 6 .
in addition , the first step ( the activation of ag expression by wus ) also exhibits a delay : wus expression occurs during floral stages 2 - 5 . by contrast , ag is not detectable in floral meristem until stage 3 .
the reason for these delays has been unclear but two recent publications reveal the relevance of epigenetic regulation by the pcg and trxg ( figure 3 ) .
serial floral stages with the expression of wus , ag , and knu are shown .
underneath , the genomic regions of these genes are shown schematically with likely binding of transcription factors , expression status , and histone modifications . at the initial stage of flower development ( stage 1 ) ,
wus is activated at stage 2 , but the transcriptional activation of ag by wus is inhibited by h3k27me3 . at stage 3 , the ag locus undergoes activation by progressive ult1/atx1-dependent h3k4me3 and likely demethylation of h3k27 .
the activation of knu by ag is initially inhibited by h3k27me3 at the knu locus between stages 3 to 5 .
the level of h3k27me3 at the knu locus is gradually depleted , perhaps by dilution via successive rounds of cell division , and at stage 6 knu is expressed , causing wus repression .
the punctual termination of wus expression needs additional prc1 activity , which suggests that the wus locus accumulates h3k27me3 and h2ak120ub .
ag , agamous ; atx1 , arabidopsis homolog of trithorax 1 ; knu , knuckels ; ult1 , ultrapetala1 ; wus , wuschel . carles and
fletcher investigated whether the activation of ag is dependent on the arabidopsis sand ( sp100 , aire-1 , nucp41/75 , deaf-1 ) domain protein ultrapetala1 ( ult1 ) and furthermore , whether ult1 is involved in trxg function .
the authors were inspired by the gain - of - function phenotypes of 35s::ult1 transgenic plants , which resemble clf mutants in showing increased ag expression in leaves and inflorescences .
furthermore , ult1 mutants suppress the clf mutant phenotype in ult1 clf double mutants and show reduced ag expression .
this fits with idea that ult1 is a trxg factor and works antagonistically to the pcg to promote ag activity , and may also explain why wus is misexpressed in ult1 mutants .
consistent with this antagonism , 35s::ult1 seedlings , similar to clf mutants , have reduced levels of the repressive mark h3k27me3 at ag , whereas ult1 mutants have increased h3k27me3 levels .
the structure of ult1 makes it unlikely that it is itself an h3k4me3 methyltransferase , rather it may interact with such an enzyme as part of a trxg complex or promote trxg recruitment to targets .
consistent with this , ult1 interacts physically with the trxg protein arabidopsis homolog of trithorax 1 ( atx1 ) , which is responsible for local h3k4me3 deposition ( figure 1d ) .
atx1 can not be the only factor for ult1-dependent h3k4me3 at the ag locus because atx1 mutants , unlike ult1 mutants , do not show indeterminacy of floral meristems and also retain 85% of global h3k4me3 levels .
furthermore , although the ult1-dependent activation of ag by a slow increase of h3k4me3 may provide a time buffer that the transcription factor wus on its own may not provide , it remains unclear whether ult1 and wus work in parallel or in the same pathway .
intriguingly , the delay in the second step of the ag / wus feedback loop the final suppression of wus by ag may also involve epigenetic control .
sun et al . identified knuckels ( knu ) , which encodes a c2h2-type zinc finger transcription factor , as a direct target of ag and a key intermediate in the suppression of wus expression .
genetic analysis and overexpression experiments imply that knu is activated by ag and represses wus expression .
curiously , although ag binds the knu promoter directly , there is a two day lag between the activation of ag and that of knu .
several observations indicate that knu is a pcg target : first , early in floral meristem development the knu locus is strongly decorated with h3k27me3 ; second , in various pcg mutants knu is misexpressed . using an elegant system for synchronizing floral development , sun et al .
show that between floral development stage 3 , when ag is activated , and stage 6 , when knu is activated and wus expression terminated , h3k27me3 is gradually removed from the knu locus . but how can the mads domain transcription factor ag cause histone demethylation ?
one possibility is that ag recruits h3k27me3 demethylases , which have been identified in animals but not yet in plants .
a simple alternative is that ag somehow inhibits the binding of prc1 and prc2 complexes to the knu locus and the h3k27me3 mark is gradually diluted as unmodified histones are incorporated onto replicated dna during cell division .
either way , the h3k27me3 at knu is not sufficient to prevent knu activation by ag but does delay it . similarly , two other recent studies that looked at the activation of another pcg target , in this case by cold , also conclude that h3k27me3 is important for slowing the kinetics of induction .
although the tardy induction of knu in flowers suggests a mechanism for the delayed suppression of wus by ag , it can not be the only factor that terminates wus expression .
thus , knu mutants have much weaker effects on floral determinacy than ag mutants , leaving space for other ag - dependent mechanisms .
as wus is itself a target of h3k27me3 , it is also possible that its repression by knu is just the initial step that facilitates a long - term epigenetic repression by prc1 . supporting this thesis ,
wus is misexpressed in mutants of components of prc1 , which bear extra floral organs .
it is likely that whole genome profiling studies will soon clarify how generally noncoding rnas are involved in pcg recruitment ; for example , by sequencing the rnas that are associated with prc2 components , as has recently been done in animals . it will be important to test whether these rnas can act in trans , that is , to recruit prc2 to loci that are not adjacent to the site of their production .
if so , the mechanism of recruitment is an issue , as it is unlikely to be simply by sequence homology between the rna and the target gene . whether or not prc2 binds specific rna sequences or secondary structures is also an interesting question as , at least in in - vitro assays , components such as clf bind rna and single - strand dna quite generally
. it will be important to know whether the atring1/atbmi1 proteins and their putative mark ( h2ak120ub ) colocalize with prc2 targets , or rather are restricted to a subset of targets , and if the latter , whether these targets show more stable silencing .
lastly , it will be interesting to learn whether the pcg and trxg play a more general role in recording transient environmental events than simply the vernalization response . | many plants respond to winter with epigenetic factors that gradually dampen repression of flowering so that they can flower in spring .
the study of this process was important for the identification of the plant polycomb group ( pcg ) of proteins and their role in the epigenetic control of plant gene expression .
fittingly , these studies continue to illuminate our understanding of pcg function .
we discuss recent advances , particularly the role of noncoding rna in the recruitment of pcg to target genes , and the role of the pcg in regulating the stem cell pool in flowers . | Introduction and context
Recent advances
Vernalization and flowering time
Shaping floral architecture: stemming the stem cell pool
Future directions
Competing Interests | a second epigenetic system is implemented by two collections of genes known as the polycomb group ( pcg ) and the trithorax group ( trxg ) . here
, we discuss important advances in our understanding of how pcg genes provide a memory of winter . secondly , we describe the emerging role of pcg and trxg genes in controlling stem cell fate in flowers . the mechanism of the vernalization response has been best studied in the model plant arabidopsis thaliana , where it has been found to depend on the ability of the pcg system to silence the key gene mediating the vernalization response , flowering locus c ( flc ) . although h2ak119ub may help repress transcription directly by blocking rna polymerase ii transcriptional elongation from the promoters of pcg target genes , its importance is challenged by the observation that prc1 can cause chromatin compaction and transcriptional repression independently of its h2a ubiquitination activity . be this as it may , two groups have now identified arabidopsis homologs of ring1a , ring1b , and bmi1 ( figure 1c ) and the similarity of their knockout phenotypes with those of mutants inactivating prc2 is compelling evidence that they have a similar role to animal prc1 . although so far only a handful of pcg targets have been shown to be misregulated in the arabidopsis atring / atbmi1 mutants , it will obviously be interesting to see if the arabidopsis atring / atbmi genes have any role in the vernalization - induced repression of flc . numerous recent studies in animals have shown that noncoding rna play a role in recruitment , and two noncoding rnas have now been implicated in vernalization - induced recruitment of phd - prc2 . although many plants show a vernalization response with epigenetic features , the target genes and even the mechanisms may be different than those in arabidopsis . unlike the other meristems , the floral meristem is determinate so that organ production ceases once the carpels are initiated in the innermost whorl of the flower . intriguingly , the delay in the second step of the ag / wus feedback loop the final suppression of wus by ag may also involve epigenetic control . the mechanism of the vernalization response has been best studied in the model plant arabidopsis thaliana , where it has been found to depend on the ability of the pcg system to silence the key gene mediating the vernalization response , flowering locus c ( flc ) . although h2ak119ub may help repress transcription directly by blocking rna polymerase ii transcriptional elongation from the promoters of pcg target genes , its importance is challenged by the observation that prc1 can cause chromatin compaction and transcriptional repression independently of its h2a ubiquitination activity . be this as it may , two groups have now identified arabidopsis homologs of ring1a , ring1b , and bmi1 ( figure 1c ) and the similarity of their knockout phenotypes with those of mutants inactivating prc2 is compelling evidence that they have a similar role to animal prc1 . although so far only a handful of pcg targets have been shown to be misregulated in the arabidopsis atring / atbmi1 mutants , it will obviously be interesting to see if the arabidopsis atring / atbmi genes have any role in the vernalization - induced repression of flc . numerous recent studies in animals have shown that noncoding rna play a role in recruitment , and two noncoding rnas have now been implicated in vernalization - induced recruitment of phd - prc2 . however , fpa and fca are not required for the vernalization response , and the function of coolair in vernalization is less clear . although many plants show a vernalization response with epigenetic features , the target genes and even the mechanisms may be different than those in arabidopsis . unlike the other meristems , the floral meristem is determinate so that organ production ceases once the carpels are initiated in the innermost whorl of the flower . however , flowers are determinate structures and as a consequence , a second feedback loop terminates the stem cell pool of floral meristems . this fits with idea that ult1 is a trxg factor and works antagonistically to the pcg to promote ag activity , and may also explain why wus is misexpressed in ult1 mutants . it will be important to know whether the atring1/atbmi1 proteins and their putative mark ( h2ak120ub ) colocalize with prc2 targets , or rather are restricted to a subset of targets , and if the latter , whether these targets show more stable silencing . | [
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] |
post - translational histone modifications
are essential cellular
processes that regulate the accessibility of dna to the cell s
transcriptional apparatus .
one such modification is acetylation of
lysine side - chains that renders these neutral in charge and thereby
alters the electrostatic interactions between , for example , histone
tails and dna or bromodomains . as important as the enzymes that generate
the post - translational modifications are the enzymes that reverse
them .
one class of enzymes that mediate the reversal of histone modifications
is comprised of the histone deacetylases ( hdacs ) , which catalyze deacetylations of acetylated lysine side - chains
in histones and other cellular proteins .
deacetylation of histone tails leads to a condensed
and inaccessible chromatin structure at the site of modification . besides histones ,
( de)acetylations are now known
to regulate a large body of enzymes in the cell , thus
making hdacs key entities in the regulation of eukaryotic cells . in particular , hdacs are often up - regulated in cancers , and the inhibition of hdacs is now believed to
be a promising way to improve the treatment of several cancers .
verinostat ( suberoylanilide hydroxamic acid ;
saha ) is one such hdac inhibitor approved by the fda for the treatment
of t - cell lymphoma .
structural variation of the l1 and l2 loops is highlighted
with a dashed line .
( b ) ribbon representation of hdac8 tyr306phe ( pdb
code 2v5w ) in
gray bound to a cleavable substrate in green licorice .
insert shows
a zoom into the interaction of the binding rail residue asp101 and
the substrate backbone .
the family of human hdacs encompasses at least 11 classical
zinc - dependent
isoforms with various cellular functions and can be categorized into four major classes based on phylogenetic
analysis and sequence similarity .
for example , the classical class
i hdacs include isoforms hdac1 , hdac2 , hdac3 , and hdac8 , which is
the focus of this study .
crystal structures of hdacs and hdac8 in
particular have yielded detailed insight into the structure and conformational
diversity of this protein when bound to inhibitors and substrates , whereas
only one structure of hdac8 without a binding partner is available .
crystal structures of hdac8 complexed with inhibitors
show different conformations of the hdac8 surface , and computational
studies have shown that hdac8 can interconvert between different entrance
topologies on the nanosecond time scale .
thus , the surface around the entrance tunnel to the
active site is malleable , thereby allowing for interaction with different
binding partners ( figure 1a ) . despite
the malleability of the surface that surrounds the entrance
to the active site tunnel , crystal structures of hdac8 exhibit a
structurally
highly conserved binding configuration formed by residues tyr100 and
asp101 , which we will refer to as the
binding rail ( figure 1b ) .
for example , vannini
et al . showed that asp101 forms hydrogen bonds with the backbone of
a substrate thereby positioning and stabilizing the substrate for
the deacetylation reaction .
when a substrate - like
ligand is bound , tyr100 and asp101 stabilize the binding via stacking
of aromatic groups or hydrogen bonds , respectively .
the binding rail
residues show this conformation in every available crystal structure
where electron density is not missing for these residues .
moreover ,
mutating asp101 to ala renders hdac8 inactive , which attests to the
importance of this residue and the binding rail in general for the
catalytic activity .
the binding
rail is located in the l2 loop ( figure 1 ) .
the l2 loop , which consists of residues 83108 ,
has two parts : residues 8392 that are distal to the entrance
tunnel and residues 93108 that are proximal and include the
binding rail , tyr100 and asp101 .
the distal part of the l2 loop is
found in different conformations depending on the inhibitor bound ,
although electron density is often lacking for this part of the loop .
in some crystal structures almost the entire l2 loop is missing electron
density ( pdb codes 3sfh , 1vkg ) probably
because no favorable interactions between the binding rail and the
inhibitors can be formed .
the ligands in these structures are not
substrate - like and can not form hydrogen bonds with asp101 nor can
they -stack with tyr100 .
the hdac8 structure reported without
a ligand bound shows no electron density for residues 85103
( pdb code 3f07 chain c ) .
thus , previous studies suggest that the l2 loop is highly
dynamic , the weaker the interaction between the ligand and the binding
rail the more dynamic the loop . moreover , in some crystal structures
the l2 loop is involved in symmetry related crystal contacts , which
affects the b - factors in this region , further confounding a concise
interpretation of the loop flexibility from crystal structures alone . the l1 loop , which spans residues 3135 ( figure 1a ) , is also located in the vicinity of the entrance
to the catalytic site .
the l1 loop is directly above an internal cavity ,
which is believed to play a role in the catalytic mechanism by functioning as an exit tunnel for the acetate
product after catalysis . as for the l2 loop described above
, the l1
loop is found in very different conformations in different crystal
structures ( figure 1a ) .
specifically , the c position of lys33 in this loop deviates by more than
0.5 nm when bound to different inhibitors ( pdb codes 1t69 and 1t64 ) , demonstrating
its flexibility and ability to undergo large - scale motions .
both ,
the l1 and l2 loops are regions of the highest sequence variation
among the class i hdacs .
correlation between hdac8 activity
and loop flexibility has been
suggested previously by somoza et al . and dowling et al .
in
particular , it was shown that a zinc ion can bind to the distal part
of the l2 loop and thereby stabilize its conformation .
this stabilization down - regulates the activity ,
which demonstrates that flexibility is important for function .
moreover , a recent crystal structure and study
of hdac3 shows strong evidence that flexibility
of both the l1 and l2 loops is essential for catalytic function of
hdac3 .
hdac3 and hdac8 are both class i histone deacetylases , and
they have high sequence and structural similarity ( 41% sequence identity
and structure similarity z - score of 55.8 for crystal structures 1t64 and 4a69 ) , which indicate
that flexibility of the l1 and l2 loops is also important for hdac8
and possibly for class i hdacs in general . a mechanism describing
how the loop dynamics and different conformations
tune the activity of hdacs and the involvement of dynamics in the
catalytic cycle is , to the best of our knowledge , not present in the
literature .
to explore these questions and the possible function of
the malleable surface we first carried out two 1.1 s fully
atomistic molecular dynamics ( md ) simulations of hdac8 ( with and without
inhibitor ) in explicit solvent followed by a 13.2 s simulation
of hdac8 on the anton supercomputer . as these simulations
showed interesting conformational transitions we designed mutants
to interfere with such transitions and
we employed a fluorogenic assay as well as
a novel real - time progression nmr assay we have developed , where we
use part of the acetylated n - terminal tail of p53 as a substrate .
overall , our data indicate that interconversions between different
l1 and l2 loop conformations may be key for the enzymatic activity .
as expected from the previously solved structures
and experimental results , the simulation of free hdac8 shows that
the surface in the vicinity of the entrance to the catalytic site
is very flexible and in particular the l1 and l2 loops of hdac8 interconvert
between different states ( figure 2 ) , some of
which are closely related to different crystal structures ( figure s1 ) .
for example , in one of the l1 loop
conformations present during the simulation an extra cavity and a
large groove become accessible , as has been observed in crystal structures
with different ligands and in other computational studies .
for the l2 loop , the binding rail has two distinct conformations ,
which we will refer to as in and
out
( colored light- and dark - green , respectively , in figure 2a ) . in the
in conformation tyr100 and asp101
build a rail toward the catalytic site as observed in crystal structures ( figures 1b and s1d and
video s1 ) , which has been attributed to substrate binding and
positioning ( figure 1b ) . on the contrary , in the
out conformation ,
the binding rail residues 100 and 101 are oriented away from the entrance
to the catalytic site .
in
and out conformations takes place at tyr100 , as shown
in the analysis of the backbone angle ( figure 2b ) .
in conformation , as illustrated in figure s1d , where , for example , the binding rail is mainly
in the in conformation between 800 and 1000 ns .
in ( pale colors ) and out ( dark
colors ) conformation of the binding rail ( green residues ) .
( b ) microkinetic
processes and states over the simulation time : ( i ) binding rail flips ,
measured via of tyr100 ( ii ) l1:l2 salt - bridge formation between
lys33 and asp8789 measured using a lys n-asp o distance cutoff of 0.35 nm ( iii ) presence
of an -helix at residues 9397 as calculated by stride .
since , as outlined above , the
binding rail is very important for
function , we examined the simulations with the aim of revealing possible
mechanisms that interfere with the binding rail conformation .
we observed
three features of the l1 and l2 dynamics that can be represented by
three microkinetic processes , that is , ( 1 ) the binding rail flips
between in and
out conformations ,
( 2 ) l1:l2 interaction , dominated by salt - bridge formation between
lys33 of l1 and the triple asp repeat of l2 ( residues 8789 ) ,
and ( 3 ) formation of a helix in the l2 loop .
these processes seem
to be correlated ( figure 2b ) , and hence we
divide the trajectory into three consecutive states : f1 , f2 and f3 ,
which each show a characteristic pattern regarding the microkinetic
processes .
in
conformation
and flips to the out conformation after a couple of
nanoseconds .
after the initial flipping out , the binding rail is only
found in its
this behavior of the binding rail appears to be cooperative with conformational
changes along the l2 loop from tyr100 toward the triple asp repeat
and the formation of salt bridges between the triple asp repeat of
the l2 loop and the lys33 of the l1 loop , characteristic for the l1:l2
interaction .
out
state between 160 and 420 ns where the l1 and l2 loop interact ( video s1 ) .
these electrostatic interactions are
not present in the subsequent f3 state , which allows the l2 loop to
adopt conformations where the binding rail becomes loose and can flip
between the in and out conformations .
thus , after 420 ns of simulation time , when reaching the f3 state ,
we see several
it seems that the binding rail behavior can be
steered by the presence
or absence of the l1:l2 interactions .
we observe a number of transitions
between the microkinetic states , which suggests that the energy landscape
between the states is shallow .
hence , a small perturbation to the
energy landscape , such as point mutations or binding of small regulators
and inhibitors could stabilize one of the states and thereby shift
the populations , as has been shown for low - lying thermally excited
states in other proteins . to further investigate a possible
correlation between the binding rail and the l1:l2 interaction , we
perturbed the energy landscape of hdac8 by simulating the response
to a small and flexible hdac inhibitor , saha , followed by the experimental
investigation of the l1:l2 interactions using point mutations assessed
by in vitro biochemical assays .
overall , we seek
to characterize the response of the interactions , dynamics , and structure
to perturbations and relate this to the catalytic function of hdac8 .
flexible behavior of the ligand and the loops at the entrance
surface in the hdac8:saha complex is expected , since crystallographic
b - factors are large in these regions , while high r values are also observed for this ligand ( video
s2 )
. states of the l1 and l2 loop conformations when interacting with
saha ( chemical structure shown in the insert ) .
( a ) : hdac8:saha complex
snapshot of the simulation with saha ( licorice ) and hdac8 ( gray cartoon )
where the binding rail is in its
positions of lys33 , asp8789 , tyr100 , and asp101 are illustrated
with colored spheres .
( b ) : microkinetic processes over the simulation
time as defined in figure 2 : ( i ) angle
of tyr100 , indicating binding - rail conformation ; ( ii ) l1:l2 salt bridge
presence between lys33 and asp8789 ; ( iii ) presence of an -helix
at residues 9397 .
again we decomposed the trajectory into the three microkinetic
processes ( figure 3b ) , clearly revealing two
distinct states , which we name i1 and i2 . in the i1 state , after the
initial interaction of the binding rail with saha is lost , the aromatic
ring of saha adopts a number of binding modes different from the crystal
structure , although the hydroxamate group always remains bound to
the catalytic zinc ( video s2 ) .
overall ,
in the i1 state saha can adopt a conformation where its aromatic cap
group is buried in a pocket that becomes accessible in the vicinity
of the entrance tunnel and the l1 loop .
in this conformation , where
saha is buried in the pocket at the l1 loop , lys33 exhibits frequent
electrostatic contacts with the triple asp repeat of the distal part
of the l2 loop , while the binding rail is in the out
conformation , preventing any contacts with the inhibitor .
thus , the
frequent interactions of l1 and l2 loops prevent helix formation and
keep the binding rail trapped in its out conformation .
this is the same mechanism that we observed for the free form in the
f2 state , where the distal part of the l2 loop interacts with the
l1 loop . in the i2 state , which is reached after 500 ns , the
l1:l2 interactions are less frequent , allowing the formation of a
helix in the l2 loop ( residues 9397 ) . during the i2 to i1
transition at 780 ns the l1:l2 interactions again
the l1:l2 interactions in the
hdac8:saha complex are much more
pronounced than in free hdac8 , trapping the binding rail in its
nevertheless , it is clear
that the states sampled by the perturbed form ( hdac8:saha ) and the
free form of hdac8 show many similarities , in particular , the f2 and
the i1 state have many common features , such as the conformation of
the l1 loop and the formation of salt bridges between lys33 and the
triple asp repeat in the l2 loop .
hence , we conclude that the mechanism
of l1:l2 interaction steering the binding rail behavior we observe
is robust and can be triggered by small perturbations such as the
binding of a small molecule .
in the 1.1 s duration simulations ,
we have only observed
a few transitions between the states . to enhance the sampling of the
transitions we used metadynamics simulations .
however , these proved
unsuccessful as using collective variables such as the l1l2
loop distance and backbone angle of tyr100 did not enhance sampling
of the transitions .
we infer that there are unknown processes transverse
to the chosen collective variables that limit the sampling rate and
lead to strong hysteresis . as an alternative route to improve
the sampling of the state transitions
,
we used the anton supercomputer to carry out a much longer , unbiased ,
md simulation . while simulating free hdac8 for 13.2 s
did not
yield proportionally more sampling of the state transitions , the longer
simulation confirms our previous observation that l1:l2 interactions
steer the binding rail backbone conformation ( figure s2 ) , and we can see several states where there are
l1:l2 interactions present or absent . since these microkinetic
processes steer a functionally important
part of the protein , we hypothesize that the observed processes play
a role in the catalytic cycle of hdac8 .
it is unfortunately not feasible
to simulate the whole catalytic mechanism including binding , cleaving
of substrate and product egression .
even obtaining statistically robust
measurements of the loop state transition is barely achievable , as
we have shown using anton . given these limitations , only experimental
evidence can substantiate this hypothesis .
hence , we expressed hdac8
mutants in which the l1:l2 interactions are altered and measured their
activity .
salt - bridge formation
between the l1 and l2 loop is characteristic of the l1:l2 interaction ,
which led us to design three mutants that change the charges of the
loops in a systematic manner and thereby modulate the l1:l2 interactions .
the three mutants are : lys33glu ; asp87arg / asp88arg / asp89arg , which
we abbreviate as asp8789arg ; and a charge - swap mutant lys33glu / asp8789arg .
we first assessed the enzymatic activity of the wild - type hdac8 and
these mutants with a fluorogenic assay using boc - lys(ac)-7-amino-4-methylcoumarin
( mal ) as a substrate .
the wild - type hdac8
has a kcat / km of 38 4 m s for the
mal substrate while , as shown in table 1 , mutating
lys33 to glu leaves the enzyme with only 8% activity and mutation
of the three asp residues 8789 to arg results in 54% residual
activity .
now , if the l1:l2 interaction were irrelevant for enzymatic
activity , then one would expect that the charge - swap mutant lys33glu / asp8789arg
should have a relative activity given by the product of the two individual
relative activities of the mutants , lys33glu and asp8789arg .
interestingly , the charge - swap mutant shows 15% of wild - type activity ,
which is significantly larger than what would be expected in a model
where the enzyme activity were independent of the lys33:asp8789
interaction .
although the three mutations
alter the l1:l2 interaction , they
could also affect substrate binding and cause other changes due to
the change of charge and steric effects .
specifically , lys33 is not
far from the binding rail itself , and mutation of this residue could
directly interfere with substrate binding .
moreover , the mal substrate
is by no means a perfect mimic of a natural substrate since the methylcoumarin
fluorophore is attached directly to the c - terminus of the acetylated
lysine , which like the substrate in the crystal structure 2v5w probably points
directly toward lys33 .
also , the amino acids introduced by mutation
( up to four ) are of different sizes to those in the wild - type enzyme ;
it is therefore very likely that we observe a combination of an alteration
of the l1:l2 interaction together with other secondary effects . to address these issues and to confirm that the l1:l2 interaction
generally tunes the enzymatic activity of hdac8 , we established a
real - time nmr assay with a more natural - like substrate . in this assay
the activity of the hdac8 mutants was measured against an acetylated
lysine embedded in a 21 amino acid residue peptide based on a part
of the n - terminal tail of the tumor suppressor p53 gshlkskkgqstsrhk - k(ac)-lmfk ,
where the acetylated lys ( k(ac ) ) corresponds to the 382 position in
p53 .
monitoring the amount of substrate and product over time followed
by fitting to the michaelis
menten differential equations allows
us to determine kcat / km values for hdac8 with this natural - like substrate ( see supporting information ) . for wild - type
hdac8 , kcat / km = 289 6 m s in the
real - time p53 based assay , which is approximately one - third
of that reported for the fluor - de - lys assay ( biomol ) previously .
as shown in table 1 ,
the activity of the lys33glu mutant is minimal , with only 2%
of the wild - type activity .
the asp8789arg mutant and the charge - swap
mutant lys33glu / asp8789arg show very similar kcat / km values , at 10%
of the wild - type activity .
hence , for this more natural - like substrate
the rescue effect resulting from reintroducing attracting charges
on the l1 and l2 loops is larger than in the fluorogenic assay and
yields an activity which exceeds that of the asp8789arg mutation
alone .
again , the importance of the lys33:asp8789 interaction
for activity is further emphasized as the product of the relative
activity of the two individual mutants , 0.018 0.101
0.002 , is significantly lower than the relative activity of the charge - swap
lys33glu / asp8789arg mutant , 0.115 . the influence on
activity and the rescue effect that we observe
above for the l1:l2 interaction is in the same range as that of other
mechanisms that have been shown to down - regulate hdac8 , such as zn binding to the l2 loop and phosphorylation of ser39 . the latter has been linked to cellular regulation of hdac8 , where
the activity of the phosphorylated ser39p and the mimicking mutant
ser39glu is 26-fold less than that of the wild - type .
the above results show that there are
substrate - specific contributions
to the effects of the mutations and that the charge - altering mutations
in the l1 and l2 loops are not independent .
hence , the l1:l2 interaction
is an important component within the catalytic cycle of hdac8 .
the
explicit influence on the kon , koff , and kcat rates
is hidden in the ( kcat / km ) ratio , but we consistently see a rescue effect by swapping
the charges on both loops , supporting our hypothesis that this mechanism
has a direct influence on the catalytic cycle .
note that the residues
mutated are not conserved , while a direct interaction between the
l1 and l2 loop has not been observed before . a detailed analysis
of the md and interactions present in the three
mutants
unfortunately ,
the sampling issues in such md simulations would be worse for the
mutations as compared to the wild - type , since certain states are predicted
to be depopulated .
we therefore developed an alternative approach
to further bridge the experimental and theoretical results , wherein
the mutation is performed in silico . specifically ,
as shown in supporting information , mutating
lys33 to glu leads to a depopulation of the f2 state due to its higher
estimated free energy ( figure s3 ) .
hence ,
our experimental data supports the md simulations , thereby substantiating
our hypothesis .
the catalytic cycle of hdac8 involves at least three steps , including
binding of substrate , cleaving of the acetylated lysine , and release
of the products .
characterization of the interconversions between
these states during catalysis is central to both understanding the
underlying mechanism of hdac8 and facilitating development of novel
inhibitors of hdacs .
the structure of an inactive mutant of
hdac8 bound to a cleavable substrate ( pdb code 2v5w ) has been previously
determined , and we will assume that this structure largely represents
a substrate binding state .
in conformation ,
forming hydrogen bonds with the substrate , there being no obvious
contacts between the l1 loop and the triple asp repeat of the l2 loop .
moreover , the l2 loop is structured in the binding state with electron
density for both backbone and side - chains ( figure 1b ) .
our simulations suggest that the l1 loop conformations
and dynamic
interactions are able to steer the structure of the l2 loop and thereby
influence the conformational sampling of the binding rail . only in
the free form
is there an effective sampling of the binding conformation
of the l2 loop ( figure 2b ) , where specifically
in the f3 state the binding rail frequently samples the
when the distal part of l2 interacts with l1 , it traps
the binding rail in the out state .
conversely , when
there is no interaction with the l1 loop , the binding rail can dynamically
sample the in conformation , i.e. , a binding state .
we therefore hypothesize that the observed microkinetic processes ,
in particular the l1:l2 interaction , are able to tune the binding
rail availability , hence tuning enzymatic activity .
based on our md simulations we propose a model in which dynamic
interactions of the l1 and l2 loops steer the behavior of the binding
rail of hdac8 and should thus be regarded as part of the catalytic
process of the enzyme . informed by these simulations we designed mutations
that interfere with this interaction and were able to show , using
two different assays , that this interaction has an effect on the activity
of the enzyme .
moreover , it has been shown that zinc binding
to a second site
at the distal l2 loop down regulates the enzyme . in the structure
where this site is occupied by zinc
it is clear that the asp8789
residues are not available to form salt - bridges with the l1 loop .
interestingly , the regions that we have identified to
be important for catalytic activity of hdac8 were also recently shown
to be important in hdac3 , where two binding partners are needed for
activation . in the recently published
crystal structure of hdac3 ,
the activating binding partners clamp
the interface adjacent to the l1 and l2 loop .
this clamping yields
a structure of hdac3 similar to that of hdac8 , possibly allowing for
a similar l1:l2 interaction and allosteric mechanism . a similar feature
can also be observed in hdac2 , where a recent crystal structure revealed
a more helical l2 loop , but a longer l1 loop , again allowing for interloop
interactions .
our theoretical work
on the atomistic scale has yielded a hypothesis
we have been able to substantiate using biochemical experiments on
the macroscopic scale .
therefore , the mechanism we propose furnishes
a conceptual platform by means of which one can rationalize the influence
of l1 and l2 dynamic interactions on enzymatic activity of hdac8 and
possibly other hdacs .
the first set of unbiased md
simulations were performed using gromacs 4.5.3 , the amber99sb - ildn force field , and tip3p water .
0.9 nm cutoffs were used for electrostatic and van der waals
interactions and the particle mesh ewald ( pme ) method was applied
for long - range electrostatics .
we used
an extensive equilibration protocol , where each system was simulated
for 5 ns in an nvt ensemble followed by 5 ns in an npt ensemble during
which we applied positional restraints of 1000 kj mol nm on heavy atoms of the protein and increased
the temperature .
all production runs were performed in an npt ensemble
using a nos hover thermostat at 300 k , an isotropic
parrinello rahman pressure coupling and periodic boundary conditions .
the neighbor list was updated every 5 steps using 2 fs integration
time steps while keeping bonds involving hydrogens and heavy atoms
constrained using the p - lincs algorithm .
the topology for saha was generated using ambertools 1.5 with the
resp methodology after geometry optimization at the b3lyp 6 - 31 g * level
using orca 2.8.0 .
protonation states
of histidines were calculated using the h++ server and the protonation states of histidines 142 and 143 close
to the catalytic site were taken from ( ref ( 16 ) ) as protonated on the nitrogen .
data was analyzed using gromacs , vmd 1.9 , and matlab ( mathworks ) .
sequence similarities and structure similarity z - score
of crystal structures were obtained from the dali server .
the anton production simulation was run
in the npt ensemble with the same force field and water model as the
gromacs simulations .
1.1 nm cutoffs were used for short - range interactions ,
and a 64 64 64 pme mesh was used for long - range electrostatic
interactions .
a berendsen thermostat and barostat were applied to maintain 300 k and 1 bar as the simulation
temperature and pressure , respectively .
hdac8 was expressed as described
elsewhere . in brief , escherichia
coli codon - optimized coding sequence of hdac8 wild - type was
obtained from genscript ( piscataway , usa ) in a pet-29b+ vector containing
an n - terminal his - nusa - tag separated
from the hdac8 coding sequence by a linker containing a specific tev
cleavage site .
wild - type and mutant constructs were
expressed using bl21(de3 ) cells in 1 or 2 l of lb medium at 21 c
overnight .
cells were harvested , resuspended in lysis buffer [ 50 mm
tris ph 8.0 , 3 mm mgcl2 , 500 mm kcl , 5 mm -mercaptoethanol ,
5 mm imidazole , 5% glycerol , 0.25% igepal , 1 tablet of complete protease
inhibitor ( roche ) per 50 ml , traces of dnase i ( roche ) , and lysozyme
( sigma ) ] , before lysis through sonication and subsequent centrifugation
of the lysate at 25 000 g for 45 min .
purification
over a first ni - nta column ( ge healthcare ) using an imidazole gradient
( 5200 mm ) was followed by dialysis into cleavage buffer ( 50
mm tris ph 8.0 , 150 mm kcl , 5 mm -mercaptoethanol , 5% glycerol )
and cleavage by his - tagged tev - protease . cleaved hdac8 was separated
from the his - nusa - tag , the tev - protease and nonspecific contaminants
by passage through a second ni - nta column .
the flow - through was pooled ,
concentrated , and subjected to a gel filtration column ( s75 , ge healthcare )
in gel - filtration buffer ( 50 mm tris ph 8.0 , 150 mm kcl , 1 mm tcep ,
5% glycerol ) .
trichostatin a ( tsa )
was purchased from enzo life sciences and porcine pancreatic trypsin
( type ix - s ) from sigma .
the in vitro hdac assay used is based on a homogeneous
fluorogenic hdac assay .
aliquots were
prepared for hdac8 wild - type and each mutant to yield a 0.4 , 1 , and
2 m final concentration in the total reaction volume of 60
l in assay buffer ( 50 mm tris ph 8.0 , 137 mm nacl , 2.7 mm kcl ,
1 mm mgcl2 , 1 mg / ml bsa ) .
mal substrate solution of 50
mm in dmso was diluted in assay buffer and added to the enzyme solution
to yield a final concentration of 250 m in the reaction volume .
the hdac8:mal solution was incubated at 25 c for 30 or 60
min , after which 50 l of the reaction solution was pipetted
on a 96-well white nbs microplate , where the wells had been preloaded
with 50 l developer solution ( 10 mg / ml trypsin and 4 m
tsa in assay buffer ) .
the microplate was left for 30 min at ambient
temperature before the fluorescence was measured on a bmg fluostar
optima plate reader with excitation at 380 nm and emission at 460
nm .
activities for different reaction times and concentrations were
compared to the wild - type enzyme and averaged as summarized in table 1 .
errors were estimated as the root - mean - square
deviation of the activity measured in the six assays for each mutant
( two time points and three concentrations ) . the kcat / km for the wild - type enzyme
was determined using substrate concentrations
of 25 , 50 , 100 , 200 , 400 , and 600 m and an enzyme concentration
of 400 nm .
reactions were incubated for 15 , 30 , and 45 min ( figure s4 ) .
the 21 amino acid peptide used
as substrate in the assay was purchased from pepceuticals ( leicestershire ,
uk ) with the sequence based on the n - terminal tail of p53 : gshlkskkgqstsrhk - k(ac)-lmfk ,
where k(ac ) denotes acetylated lysine , which corresponds to the lys382
position in p53 .
the substrate was dissolved in nmr assay buffer ( 25
mm tris ph 8.0 , 137 mm nacl , 2.7 mm kcl , and 1 mm mgcl2 ) and 10% ( v / v ) d2o was added .
enzyme was
added to start the reaction with a final enzyme concentration of 500
nm in a total volume of 550 l for each nmr sample .
1d h nmr spectra were recorded ( each 231 s ) over a time course
of typically 1624 h. substrate and product peaks were integrated
for each spectrum to yield the progression of substrate to product
conversion .
menten differential equations were
solved with
known starting conditions for a grid of kcat / km values yielding a first estimate
of kcat / km by fitting a first - order polynomial through the minimum of the surface .
the fit was refined by calculating on a line perpendicular to first determined kcat / km .
errors reported
represent the 1 confidence interval assuming a 5% uncertainty
in measuring the nmr signal , based on the signal - to - noise ratio of
the spectra .
nmr spectra were recorded on a bruker avance 500
( karlsruhe , germany ) .
| the human histone deacetylase 8 ( hdac8 )
is a key hydrolase in gene
regulation and has been identified as a drug target for the treatment
of several cancers .
previously the hdac8 enzyme has been extensively
studied using biochemical techniques , x - ray crystallography , and computational
methods .
those investigations have yielded detailed information about
the active site and have demonstrated that the substrate entrance
surface is highly dynamic .
yet it has remained unclear how the dynamics
of the entrance surface tune and influence the catalytic activity
of hdac8 . using long time scale all atom molecular dynamics simulations
we have found a mechanism whereby the interactions and dynamics of
two loops tune the configuration of functionally important residues
of hdac8 and could therefore influence the activity of the enzyme .
we subsequently investigated this hypothesis using a well - established
fluorescence activity assay and a noninvasive real - time progression
assay , where deacetylation of a p53 based peptide was observed by
nuclear magnetic resonance spectroscopy .
our work delivers detailed
insight into the dynamic loop network of hdac8 and provides an explanation
for a number of experimental observations . | Introduction
Results
and Discussion
Conclusions
Methods | in particular , hdacs are often up - regulated in cancers , and the inhibition of hdacs is now believed to
be a promising way to improve the treatment of several cancers . verinostat ( suberoylanilide hydroxamic acid ;
saha ) is one such hdac inhibitor approved by the fda for the treatment
of t - cell lymphoma . crystal structures of hdacs and hdac8 in
particular have yielded detailed insight into the structure and conformational
diversity of this protein when bound to inhibitors and substrates , whereas
only one structure of hdac8 without a binding partner is available . crystal structures of hdac8 complexed with inhibitors
show different conformations of the hdac8 surface , and computational
studies have shown that hdac8 can interconvert between different entrance
topologies on the nanosecond time scale . despite
the malleability of the surface that surrounds the entrance
to the active site tunnel , crystal structures of hdac8 exhibit a
structurally
highly conserved binding configuration formed by residues tyr100 and
asp101 , which we will refer to as the
binding rail ( figure 1b ) . a mechanism describing
how the loop dynamics and different conformations
tune the activity of hdacs and the involvement of dynamics in the
catalytic cycle is , to the best of our knowledge , not present in the
literature . as these simulations
showed interesting conformational transitions we designed mutants
to interfere with such transitions and
we employed a fluorogenic assay as well as
a novel real - time progression nmr assay we have developed , where we
use part of the acetylated n - terminal tail of p53 as a substrate . as expected from the previously solved structures
and experimental results , the simulation of free hdac8 shows that
the surface in the vicinity of the entrance to the catalytic site
is very flexible and in particular the l1 and l2 loops of hdac8 interconvert
between different states ( figure 2 ) , some of
which are closely related to different crystal structures ( figure s1 ) . overall , we seek
to characterize the response of the interactions , dynamics , and structure
to perturbations and relate this to the catalytic function of hdac8 . in the i1 state , after the
initial interaction of the binding rail with saha is lost , the aromatic
ring of saha adopts a number of binding modes different from the crystal
structure , although the hydroxamate group always remains bound to
the catalytic zinc ( video s2 ) . since these microkinetic
processes steer a functionally important
part of the protein , we hypothesize that the observed processes play
a role in the catalytic cycle of hdac8 . to address these issues and to confirm that the l1:l2 interaction
generally tunes the enzymatic activity of hdac8 , we established a
real - time nmr assay with a more natural - like substrate . in this assay
the activity of the hdac8 mutants was measured against an acetylated
lysine embedded in a 21 amino acid residue peptide based on a part
of the n - terminal tail of the tumor suppressor p53 gshlkskkgqstsrhk - k(ac)-lmfk ,
where the acetylated lys ( k(ac ) ) corresponds to the 382 position in
p53 . for wild - type
hdac8 , kcat / km = 289 6 m s in the
real - time p53 based assay , which is approximately one - third
of that reported for the fluor - de - lys assay ( biomol ) previously . the latter has been linked to cellular regulation of hdac8 , where
the activity of the phosphorylated ser39p and the mimicking mutant
ser39glu is 26-fold less than that of the wild - type . based on our md simulations we propose a model in which dynamic
interactions of the l1 and l2 loops steer the behavior of the binding
rail of hdac8 and should thus be regarded as part of the catalytic
process of the enzyme . informed by these simulations we designed mutations
that interfere with this interaction and were able to show , using
two different assays , that this interaction has an effect on the activity
of the enzyme . interestingly , the regions that we have identified to
be important for catalytic activity of hdac8 were also recently shown
to be important in hdac3 , where two binding partners are needed for
activation . | [
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] |
cardiac manifestations are highly prevalent in patients with systemic lupus erythematosus ( sle).1 , 2 while electrical abnormalities can occur , isolated atrioventricular ( av ) conduction disease , ranging from first degree to complete av block ( avb ) , is rarely seen in older children and adults with sle such that pacemakers are infrequently required.3 , 4 , 5 , 6 , 7 , 8 a few reports have described abnormal atrial electrical activity in the form of atrial standstill , which was speculated to be secondary to recurrent flares of sle , pericarditis , myocarditis , and/or myocardial arteritis.9 in contrast , complete avb and ventricular cardiomyopathy are wellestablished immunologic complications in neonates of mothers with antiro / ssa antibodies , such that pacemakers are frequently indicated . however , atrial conduction disorders , including atrial inexcitability or standstill , have not been described in this setting .
we , therefore , sought to further characterize the electrophysiological manifestations of maternal sle on neonatal atria .
we identified all patients diagnosed with congenital complete avb in the absence of structural heart disease between june 1971 and december 2012 at sainte justine hospital , montreal , canada . within this cohort of patients
, we further identified those in whom avb was associated with maternal antissa and/or antissb antibodies .
data abstracted from medical records included demographic information , presence or absence of maternal autoimmune antibodies , details regarding pacemaker implantation and all reinterventions , pacemaker interrogations , ecgs , echocardiograms , and histopathological reports when available .
continuous variables are expressed as median and interquartile range ( 25th , 75th percentile ) .
the authors had full access to and take full responsibility for the integrity of the data .
a total of 31 patients were diagnosed with isolated congenital complete avb ( figure 1 ) , 13 ( 42% ) of whom had positive maternal antibodies . of the 18 patients without maternal antissa / ssb antibodies ,
all underwent uneventful epicardial pacemaker implantation , with no reported difficulties associated with atrial sensing or pacing .
all maintained normal atrial pacing and sensing thresholds on followup and none developed cardiomyopathy . among the 13 patients with maternal antibodymediated congenital complete avb
a 1.5yearold asymptomatic patient with complete avb and a junctional escape rate > 50 bpm did not yet receive a pacemaker .
five ( 42% ) of the 12 patients with pacemakers were found to have notable atrial electrical abnormalities in the setting of normal serum electrolytes .
characteristics of these 5 patients are summarized in table 2 and their case presentations are further described below .
* two patients with no known la electrical abnormalities had a singlechamber ventricular pacemaker . since la pacing and sensing thresholds were not assessed ,
characteristics of the 12 patients with isolated immunologic complete avb and pacemaker implantation avb indicates atrioventricular block .
characteristics of the 5 patients with immunologic complete avb and left atrial electrical abnormalities asd indicates atrial septal defect ; avb , atrioventricular block ; avvr , atrioventricular valve regurgitation ; crt , cardiac resynchronization therapy ; la , left atrium ; laa , left atrial appendage ; lv , left ventricle / ventricular ; minus sign denotes no ; pht , pulmonary hypertension ; plus sign denotes yes ; ra , right atrium ; raa , right atrial appendage .
a baby girl , born at 36 weeks , had fetal evidence of bradycardia at 21 weeks associated with firstdegree avb . cardiac anatomy and function were normal , with no evidence of hydrops .
her mother was positive for lupus autoantibodies . despite maternal treatment with dexamethasone , avb progressed to complete block within a month .
a small pericardial effusion with moderate biventricular av valve regurgitation prompted a premature csection . at birth , she had a junctional escape rhythm at 75 bpm .
postnatal echocardiography revealed resolution of the pericardial effusion and av valve regurgitation . a dualchamber epicardial pacemaker
was implanted at 1 month of age in the context of recurrence of the pericardial effusion , a junctional escape rate in the 60s , persistent pulmonary hypertension ( 50% of systemic systolic pressure ) despite phosphodiesterase inhibitors , and failure to thrive .
atrial standstill was readily observed . the left atrial appendage ( laa ) and left atrium ( la ) appeared abnormally white ( figure 2 ) .
thus , the atrial lead was placed on the right atrium ( ra ) , and the ventricular lead on the left ventricle with good sensing and capture thresholds .
although the immediate postoperative period was uneventful , the patient 's condition suddenly deteriorated on the night of day 3 . following a feed , the patient became tachypneic and clamped , and died despite immediate and prolonged attempts at resuscitation .
pacemaker interrogation a few hours later showed no evidence of lead dysfunction . on cardiac autopsy ,
microscopic examination demonstrated the absence of a clear av node , which was replaced by fibrous tissue .
nonspecific changes were noted of the la and laa consisting of a mild fibrous pericarditis associated with degenerative phenomena described as dark microdebris similar to calcic salts arranged in a fine layer close to the la epicardium . surgical view for patient 1 , through a left anterolateral thoracotomy and after reclining the pericardium ( head of the patient to the right ) .
the mother , who had a diagnosis of sle with positive antibodies , was started on dexamethasone at 26 weeks .
the patient was born at 37 weeks by csection in predominantly 2:1 avb with a mean junctional escape rate of 65 bpm .
the patient remained asymptomatic despite progression to complete avb , with a gradually decreasing junctional escape rate to 44 bpm and progressive left ventricular dilation ( z score of 4.9 for the left ventricular enddiastolic diameter ) , yet with a normal shortening fraction for age .
an epicardial dualchamber pacemaker was implanted at 59 months of age through a left anterolateral thoracotomy .
sensing was appropriate , but capture could not be achieved anywhere on the laa or la despite outputs up to 8 v at 0.52 ms .
the atrial lead was , therefore , fixed to the right atrial appendage without difficulty .
multiple biopsies of the laa were performed , which showed no evidence of atrial fibrosis or loss of atrial myocytes . left ventricular function remained normal on followup .
the third patient , a 3yearold girl , was born to a woman with positive antissa antibodies .
she was diagnosed with firstdegree avb at 20 weeks of gestation , with no associated cardiac abnormalities or signs of hydrops .
although the mother was started on corticosteroids and treated until the 32nd week , avb progressed to complete block .
labor was induced at 37 weeks , with an uncomplicated vaginal delivery . at birth , she had a mean junctional escape rate of 70 bpm .
progressive signs of fatigue and decreased functional capacity prompted implantation of a dualchamber epicardial pacemaker at 16 months of age via a left anterolateral thoracotomy .
although sensing was adequate over the la , no capture could be achieved despite highoutput pacing .
the atrial lead was fixed to the right atrial appendage , where sensing and pacing thresholds were normal , and the second lead was placed on the left ventricle .
the immediate postoperative course was uneventful , although left ventricular dysfunction developed insidiously . at the age of 2 years , she was hospitalized with fever , vomiting , and signs of heart failure , with a left ventricular ejection fraction of 20% and interventricular dyssynchrony .
two weeks after admission , she underwent pacemaker upgrade to a biventricular epicardial system by adding a right ventricular lead .
findings on ventricular myocardial biopsy were uninformative . despite biventricular pacing and maximal medical therapy ,
she underwent implantation of a left ventricular assist device at 2 years and 9 months , followed by uneventful cardiac transplantation 7 months later .
a biopsy prior to transplantation was compatible with dilated cardiomyopathy , with no evidence of slerelated myocarditis and no perivascular antibodies by immunofluorescence .
the fourth patient , who is now 6 years old , was incidentally found to be in complete avb block at 2.8 years of age .
her mean junctional escape rate by holter monitoring was 39 bpm , with a maximum pause of 3.4 s. she was asymptomatic and had biventricular dilation ( z score of 3.5 ) with normal biventricular systolic function .
an epicardial pacemaker was implanted at 3 years of age , with leads positioned on the laa and left ventricle without complication .
pacemaker interrogation revealed marked delay between the onset of the surface p wave and the local laa signal , suggestive of atrial conduction delay .
left ventricular function progressively decreased on followup , with a shortening fraction of 12% and ejection fraction of 25% 6 months later .
one year after pacemaker implantation , the system was upgraded to epicardial cardiac resynchronization therapy , and the laa lead was replaced by a right atrial lead ( figure 3 ) . at last followup , her left ventricular systolic function had improved , with an ejection fraction of 45% and shortening fraction of 22% .
as shown in figure 3c , a narrow paced qrscomplex was obtained with a pr interval of 120 ms .
a , standard 12lead ecg after dualchamber pacemaker implantation with the atrial lead implanted on the left atrial appendage . sensed av delay is programmed at 50 ms but the pr interval is 220 ms .
b , pacemaker interrogation with programmer strip showing bipolar signals ( surface ecg lead i ) , marker channel , and a egms . due to atrial conduction delay , a long interval ( > 200 ms ) is noted between surface p waves ( asterisks ) and sensed atrial signals ( as on marker channel ; local atrial signals on a egm channel ) .
c , standard 12lead ecg after upgrade to biventricular pacing system and transfer of the atrial lead to the high right atrium .
the pr interval is 128 ms and the qrs complex is narrow ( 98 ms ) .
the fifth patient was diagnosed with complete avb at 25 weeks of gestation with escape rates around 70 bpm and normal cardiac anatomy and function .
given ventricular escape rates progressing down to the low 60s , an epicardial dualchamber pacemaker was implanted at 2 weeks of age , with the atrial lead placed on the la . sensing was adequate and the pacing threshold was 2 v at 0.5 ms .
the second lead was placed onto the left ventricle with adequate sensing and pacing thresholds .
two weeks later , dysfunction of the atrial lead was noted with undersensing and an increased pacing threshold .
progressive left ventricular dilation with mild systolic dysfunction was observed on followup , prompting initiation of an angiotensinconvertingenzyme inhibitor .
when the generator required replacement at 5 years of age , a transvenous dualchamber pacemaker system was implanted without complication .
a baby girl , born at 36 weeks , had fetal evidence of bradycardia at 21 weeks associated with firstdegree avb . cardiac anatomy and function were normal , with no evidence of hydrops .
her mother was positive for lupus autoantibodies . despite maternal treatment with dexamethasone , avb progressed to complete block within a month .
a small pericardial effusion with moderate biventricular av valve regurgitation prompted a premature csection . at birth , she had a junctional escape rhythm at 75 bpm .
a dualchamber epicardial pacemaker was implanted at 1 month of age in the context of recurrence of the pericardial effusion , a junctional escape rate in the 60s , persistent pulmonary hypertension ( 50% of systemic systolic pressure ) despite phosphodiesterase inhibitors , and failure to thrive .
atrial standstill was readily observed . the left atrial appendage ( laa ) and left atrium ( la ) appeared abnormally white ( figure 2 ) .
thus , the atrial lead was placed on the right atrium ( ra ) , and the ventricular lead on the left ventricle with good sensing and capture thresholds . although the immediate postoperative period was uneventful , the patient 's condition suddenly deteriorated on the night of day 3 . following a feed , the patient became tachypneic and clamped , and died despite immediate and prolonged attempts at resuscitation .
pacemaker interrogation a few hours later showed no evidence of lead dysfunction . on cardiac autopsy ,
microscopic examination demonstrated the absence of a clear av node , which was replaced by fibrous tissue .
nonspecific changes were noted of the la and laa consisting of a mild fibrous pericarditis associated with degenerative phenomena described as dark microdebris similar to calcic salts arranged in a fine layer close to the la epicardium . surgical view for patient 1 , through a left anterolateral thoracotomy and after reclining the pericardium ( head of the patient to the right ) .
the mother , who had a diagnosis of sle with positive antibodies , was started on dexamethasone at 26 weeks .
the patient was born at 37 weeks by csection in predominantly 2:1 avb with a mean junctional escape rate of 65 bpm .
the patient remained asymptomatic despite progression to complete avb , with a gradually decreasing junctional escape rate to 44 bpm and progressive left ventricular dilation ( z score of 4.9 for the left ventricular enddiastolic diameter ) , yet with a normal shortening fraction for age .
an epicardial dualchamber pacemaker was implanted at 59 months of age through a left anterolateral thoracotomy .
sensing was appropriate , but capture could not be achieved anywhere on the laa or la despite outputs up to 8 v at 0.52 ms .
the atrial lead was , therefore , fixed to the right atrial appendage without difficulty .
multiple biopsies of the laa were performed , which showed no evidence of atrial fibrosis or loss of atrial myocytes . left ventricular function remained normal on followup .
the third patient , a 3yearold girl , was born to a woman with positive antissa antibodies .
she was diagnosed with firstdegree avb at 20 weeks of gestation , with no associated cardiac abnormalities or signs of hydrops .
although the mother was started on corticosteroids and treated until the 32nd week , avb progressed to complete block .
labor was induced at 37 weeks , with an uncomplicated vaginal delivery . at birth
progressive signs of fatigue and decreased functional capacity prompted implantation of a dualchamber epicardial pacemaker at 16 months of age via a left anterolateral thoracotomy .
although sensing was adequate over the la , no capture could be achieved despite highoutput pacing .
the atrial lead was fixed to the right atrial appendage , where sensing and pacing thresholds were normal , and the second lead was placed on the left ventricle .
the immediate postoperative course was uneventful , although left ventricular dysfunction developed insidiously . at the age of 2 years
, she was hospitalized with fever , vomiting , and signs of heart failure , with a left ventricular ejection fraction of 20% and interventricular dyssynchrony .
two weeks after admission , she underwent pacemaker upgrade to a biventricular epicardial system by adding a right ventricular lead .
findings on ventricular myocardial biopsy were uninformative . despite biventricular pacing and maximal medical therapy ,
she underwent implantation of a left ventricular assist device at 2 years and 9 months , followed by uneventful cardiac transplantation 7 months later .
a biopsy prior to transplantation was compatible with dilated cardiomyopathy , with no evidence of slerelated myocarditis and no perivascular antibodies by immunofluorescence .
the fourth patient , who is now 6 years old , was incidentally found to be in complete avb block at 2.8 years of age .
her mean junctional escape rate by holter monitoring was 39 bpm , with a maximum pause of 3.4 s. she was asymptomatic and had biventricular dilation ( z score of 3.5 ) with normal biventricular systolic function .
an epicardial pacemaker was implanted at 3 years of age , with leads positioned on the laa and left ventricle without complication .
pacemaker interrogation revealed marked delay between the onset of the surface p wave and the local laa signal , suggestive of atrial conduction delay .
left ventricular function progressively decreased on followup , with a shortening fraction of 12% and ejection fraction of 25% 6 months later .
one year after pacemaker implantation , the system was upgraded to epicardial cardiac resynchronization therapy , and the laa lead was replaced by a right atrial lead ( figure 3 ) . at last followup ,
her left ventricular systolic function had improved , with an ejection fraction of 45% and shortening fraction of 22% .
as shown in figure 3c , a narrow paced qrscomplex was obtained with a pr interval of 120 ms .
a , standard 12lead ecg after dualchamber pacemaker implantation with the atrial lead implanted on the left atrial appendage . sensed av delay is programmed at 50 ms but the pr interval is 220 ms .
b , pacemaker interrogation with programmer strip showing bipolar signals ( surface ecg lead i ) , marker channel , and a egms . due to atrial conduction delay , a long interval ( > 200 ms ) is noted between surface p waves ( asterisks ) and sensed atrial signals ( as on marker channel ; local atrial signals on a egm channel ) .
c , standard 12lead ecg after upgrade to biventricular pacing system and transfer of the atrial lead to the high right atrium .
the pr interval is 128 ms and the qrs complex is narrow ( 98 ms ) .
the fifth patient was diagnosed with complete avb at 25 weeks of gestation with escape rates around 70 bpm and normal cardiac anatomy and function .
given ventricular escape rates progressing down to the low 60s , an epicardial dualchamber pacemaker was implanted at 2 weeks of age , with the atrial lead placed on the la .
sensing was adequate and the pacing threshold was 2 v at 0.5 ms .
the second lead was placed onto the left ventricle with adequate sensing and pacing thresholds .
two weeks later , dysfunction of the atrial lead was noted with undersensing and an increased pacing threshold .
progressive left ventricular dilation with mild systolic dysfunction was observed on followup , prompting initiation of an angiotensinconvertingenzyme inhibitor . when the generator required replacement at 5 years of age , a transvenous dualchamber pacemaker system was implanted without complication .
herein , we report previously undescribed atrial electrical abnormalities in patients with isolated complete avb in the setting of maternal sle antibodies
. left atrial inexcitability and/or atrial conduction delay was highly prevalent ( 42% ) in our series of patients with maternal antissa and/or antissb antibodies , in contrast to no patient with nonimmunemediated avb .
moreover , these atrial electrical anomalies were of clinical consequence and led to repositioning of the epicardial lead from the laa / la to the right atrial appendage / ra either at the initial intervention or during subsequent surgery .
persistent atrial standstill is characterized by a slow , often junctional , escape rhythm , with absent p waves by surface and endocavitary ecgs in combination with a lack of atrial excitability by direct electrical stimulation.10 the atrial inexcitability observed in our patients does not meet this definition since ra was not appreciably altered and normal surface p waves were observed .
the concept of partial atrial standstill was previously reported in an adult without sle who had absent p waves , with electrical and mechanical atrial activity confined to the laa.11
atrial standstill in the fetus or infant is exceedingly rare.12 to our knowledge , it has not previously been described in the context of immunologic or nonimmunemediated avb .
patients with chronic atrial stretch due to asynchronous pacing13 or atrial septal defects14 , 15 may demonstrate features of sinus node remodeling and atrial standstill .
this raises the possibility that the atrial inexcitability observed in our patients was secondary to avb and av dyssynchrony as opposed to a primary or immunemediated pathophysiological mechanism .
nevertheless , the absence of atrial inexcitability observed in our series of patients without immunemediated avb argues against an atrial remodeling hypothesis.16
interatrial conduction block occurs in 12% of adults with avb.17 it should be evaluated at the time of epicardial pacemaker implantation in order to optimize lead placement and av delay programming.18
the pale macroscopic appearance of the la in 2 patients raised suspicion for endocardial fibroelastosis.19 however , histopathology was limited to nonspecific changes .
it remains possible that disease of a patchy nature might have been missed despite multiple biopsies directed at the grossly abnormal la tissue .
alternatively , it may be hypothesized that functional abnormalities rather than anatomical defects underlie the observed electrical changes . in a recent series of 18 cardiac autopsies with neonatal lupus , no histological evidence of damage to the av node or surrounding tissue
was found in 2 cases with advanced avb.20 there is evidence to suggest that other electrical structures , such as the sinus node , can be affected in a functional way in patients with immunologic complete avb.20 for example , in a murine model of congenital avb , maternal sera containing antibodies against ssa / rossb / la ribonucleoproteins inhibit ltype calcium channel currents in isolated cardiac myocytes and induce sinus bradycardia , without anatomical changes.21 although the pathophysiology remains to be elucidated , a similar mechanism may potentially be implicated in our patients .
mutations in the gene encoding the cardiac sodium channel alpha subunit , scn5a , have been described in cases of familial atrial standstill22 and in a case with atrial standstill associated with progressive sinus node dysfunction and hispurkinje system disease.23 genetic testing targeting sodium channel genes was not performed in our patients such that a genetic polymorphism leading to ion channel malfunctioning and atrial inexcitability can not be excluded .
conduction defects and cardiomyopathy are wellcharacterized immunologic complications of transplacental passage of maternal autoantibodies directed against fetal ssa / ro or ssb / la ribonucleoproteins .
these autoantibodies initiate a complex cascade of maternal antibodytriggered inflammation , ultimately leading to fibrosis and scarring.24 , 25 , 26 the incidence of avb is estimated to be 2% for firstborns , with a recurrence risk of 16% to 18%.27 , 28 , 29 at least 50% to 66% of afflicted children will require permanent pacing , often during the first year of life.27 , 30 , 31 the lack of previously reported la inexcitability may reflect , in part , the fact that singlechamber ventricular pacemakers are often implanted . in our own series , 2 of 7 patients with immunologic av block but considered not to have atrial electrical abnormalities underwent ventricular pacing only , such that the true incidence of la inexcitability may be underestimated .
prenatal diagnosis of maternal lupusinduced avb was made in 4 of 5 of our patients with la electrical abnormalities . in 2 such cases ,
corticosteroids were initiated once firstdegree avb was diagnosed but were ineffective . despite some supportive evidence,30
there is a lack of clear efficacy of steroids on arresting progression toward complete avb .
although several studies have reported that antissa / ro levels in maternal sera are associated with a high risk of cardiac complications,28 , 32 exact values were unavailable in our study , precluding correlations with atrial electrical disease . in fetal survivors with antibodymediated avb and normal cardiac function at birth , reported rates of
lateronset dilated cardiomyopathy range from 5% to 10%.29 , 33 in our series , 5 of 12 patients ( 42% ) with immunologic avb and pacemaker implantation developed dilated cardiomyopathy .
larger studies are required to confirm the higher incidence of heart failure in the presence of atrial electrical disease .
our institutional pacemaker programming strategy changed over the course of the study to reflect the literature , albeit limited , suggesting a link between faster ventricular pacing rates and cardiomyopathy in the setting of congenital avb.34 two of 3 patients with ddd pacemakers initially programmed to track up to 180 bpm later received cardiac resynchronization therapy systems .
in the 2 patients with the most recent device implantations , upper tracking rates were initially limited to 90 and 100 bpm , with subsequent 10bpm increments on a monthly basis , if left ventricular function remained normal .
preoperative echocardiograms performed in our patients did not systematically target la or laa mechanical contraction , such as with mitral doppler inflow and mitral annulus tissue doppler ( awave and awave , respectively ) .
a previous report has described persistent la mechanical standstill assessed by doppler and tissue doppler imaging in a 64yearold patient with normal pwaves by ecg after a biatrial maze procedure for atrial fibrillation.35 this case underscores our observation that la inexcitability is not necessarily associated with the absence of p waves by ecg .
it remains to be determined whether echocardiographic evaluation of la and laa function in patients with immunologic avb could guide the surgeon in selecting a different approach to the classic left anterolateral thoracotomy , in order to place the atrial lead on the ra rather than la .
persistent atrial standstill is characterized by a slow , often junctional , escape rhythm , with absent p waves by surface and endocavitary ecgs in combination with a lack of atrial excitability by direct electrical stimulation.10 the atrial inexcitability observed in our patients does not meet this definition since ra was not appreciably altered and normal surface p waves were observed .
the concept of partial atrial standstill was previously reported in an adult without sle who had absent p waves , with electrical and mechanical atrial activity confined to the laa.11
atrial standstill in the fetus or infant is exceedingly rare.12 to our knowledge , it has not previously been described in the context of immunologic or nonimmunemediated avb .
patients with chronic atrial stretch due to asynchronous pacing13 or atrial septal defects14 , 15 may demonstrate features of sinus node remodeling and atrial standstill .
this raises the possibility that the atrial inexcitability observed in our patients was secondary to avb and av dyssynchrony as opposed to a primary or immunemediated pathophysiological mechanism .
nevertheless , the absence of atrial inexcitability observed in our series of patients without immunemediated avb argues against an atrial remodeling hypothesis.16
interatrial conduction block occurs in 12% of adults with avb.17 it should be evaluated at the time of epicardial pacemaker implantation in order to optimize lead placement and av delay programming.18
the pale macroscopic appearance of the la in 2 patients raised suspicion for endocardial fibroelastosis.19 however , histopathology was limited to nonspecific changes .
it remains possible that disease of a patchy nature might have been missed despite multiple biopsies directed at the grossly abnormal la tissue .
alternatively , it may be hypothesized that functional abnormalities rather than anatomical defects underlie the observed electrical changes . in a recent series of 18 cardiac autopsies with neonatal lupus , no histological evidence of damage to the av node or surrounding tissue
was found in 2 cases with advanced avb.20 there is evidence to suggest that other electrical structures , such as the sinus node , can be affected in a functional way in patients with immunologic complete avb.20 for example , in a murine model of congenital avb , maternal sera containing antibodies against ssa / rossb / la ribonucleoproteins inhibit ltype calcium channel currents in isolated cardiac myocytes and induce sinus bradycardia , without anatomical changes.21 although the pathophysiology remains to be elucidated , a similar mechanism may potentially be implicated in our patients .
mutations in the gene encoding the cardiac sodium channel alpha subunit , scn5a , have been described in cases of familial atrial standstill22 and in a case with atrial standstill associated with progressive sinus node dysfunction and hispurkinje system disease.23 genetic testing targeting sodium channel genes was not performed in our patients such that a genetic polymorphism leading to ion channel malfunctioning and atrial inexcitability can not be excluded .
conduction defects and cardiomyopathy are wellcharacterized immunologic complications of transplacental passage of maternal autoantibodies directed against fetal ssa / ro or ssb / la ribonucleoproteins .
these autoantibodies initiate a complex cascade of maternal antibodytriggered inflammation , ultimately leading to fibrosis and scarring.24 , 25 , 26 the incidence of avb is estimated to be 2% for firstborns , with a recurrence risk of 16% to 18%.27 , 28 , 29 at least 50% to 66% of afflicted children will require permanent pacing , often during the first year of life.27 , 30 , 31 the lack of previously reported la inexcitability may reflect , in part , the fact that singlechamber ventricular pacemakers are often implanted . in our own series , 2 of 7 patients with immunologic av block but considered not to have atrial electrical abnormalities underwent ventricular pacing only , such that the true incidence of la inexcitability may be underestimated .
prenatal diagnosis of maternal lupusinduced avb was made in 4 of 5 of our patients with la electrical abnormalities . in 2 such cases ,
corticosteroids were initiated once firstdegree avb was diagnosed but were ineffective . despite some supportive evidence,30
there is a lack of clear efficacy of steroids on arresting progression toward complete avb .
although several studies have reported that antissa / ro levels in maternal sera are associated with a high risk of cardiac complications,28 , 32 exact values were unavailable in our study , precluding correlations with atrial electrical disease . in fetal survivors with antibodymediated avb and normal cardiac function at birth , reported rates of
lateronset dilated cardiomyopathy range from 5% to 10%.29 , 33 in our series , 5 of 12 patients ( 42% ) with immunologic avb and pacemaker implantation developed dilated cardiomyopathy .
larger studies are required to confirm the higher incidence of heart failure in the presence of atrial electrical disease .
our institutional pacemaker programming strategy changed over the course of the study to reflect the literature , albeit limited , suggesting a link between faster ventricular pacing rates and cardiomyopathy in the setting of congenital avb.34 two of 3 patients with ddd pacemakers initially programmed to track up to 180 bpm later received cardiac resynchronization therapy systems . in the 2 patients with the most recent device implantations , upper tracking rates were initially limited to 90 and 100 bpm , with subsequent 10bpm increments on a monthly basis , if left ventricular function remained normal .
preoperative echocardiograms performed in our patients did not systematically target la or laa mechanical contraction , such as with mitral doppler inflow and mitral annulus tissue doppler ( awave and awave , respectively ) .
a previous report has described persistent la mechanical standstill assessed by doppler and tissue doppler imaging in a 64yearold patient with normal pwaves by ecg after a biatrial maze procedure for atrial fibrillation.35 this case underscores our observation that la inexcitability is not necessarily associated with the absence of p waves by ecg .
it remains to be determined whether echocardiographic evaluation of la and laa function in patients with immunologic avb could guide the surgeon in selecting a different approach to the classic left anterolateral thoracotomy , in order to place the atrial lead on the ra rather than la .
electrical abnormalities of the la and laa appear to be common in patients with congenital lupusinduced avb and include la inexcitability and atrial conduction delay .
the ra is seemingly spared from clinically recognizable electrical abnormalities and the surface p wave is not conspicuously altered .
these la electrical abnormalities bear clinical relevance , particularly with regard to influencing the selection of an epicardial atrial pacing site . to date
, there is no histological evidence of extensive atrial fibrosis such that immunemediated functional impairment of electrical activity is suspected .
further research is required to elucidate the underlying pathophysiological mechanism of this newly recognized entity , determine whether it may be diagnosed noninvasively such as by echocardiography , explore whether it is a marker of poorer outcome ( eg , left ventricular systolic dysfunction ) , and assess whether directed therapies ( eg , corticosteroids ) may alter the disease course .
dr khairy is supported by a canada research chair in electrophysiology and congenital heart disease . | backgroundcongenital atrioventricular block is a wellestablished immunologic complication of maternal systemic lupus erythematosus .
we sought to further characterize the electrophysiological manifestations of maternal systemic lupus erythematosus on neonatal atria.methods and resultscases of isolated congenital atrioventricular block treated at our center over the past 41 years were identified .
data were extracted from clinical charts , pacemaker interrogations , ecgs , echocardiograms , and histopathological reports , when available . of 31 patients with isolated congenital atrioventricular block ,
18 were negative for maternal antibodies and had normal epicardial atrial sensing and pacing thresholds .
in contrast , 12 of 13 patients with positive maternal antibodies had epicardial pacemakers , 5 ( 42% ) of whom had left atrial ( la ) inexcitability and/or atrial conduction delay . in 3 patients , the la
could not be captured despite highoutput pacing .
the fourth patient had acutely successful la appendage and left ventricular lead placement . at early followup , an increased delay between the surface pwave and intracardiac atrial depolarization was observed , indicative of atrial conduction delay .
the fifth patient exhibited la lead dysfunction , with atrial undersensing and an increased capture threshold , 2 weeks after implantation .
biopsies of la appendages performed in 2 patients showed no evidence of atrial fibrosis or loss of atrial myocytes.conclusionsherein , we report previously undescribed yet prevalent electrophysiological ramifications of maternal systemic lupus erythematosus , which extend beyond congenital atrioventricular block to encompass alterations in la conduction , including la inexcitability .
these manifestations can complicate epicardial pacemaker implantation in newborns . in the absence of histological evidence of extensive atrial fibrosis , immunemediated functional impairment of electrical activity
is suspected . | Introduction
Methods
Results
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Discussion
Atrial Standstill and IntraAtrial Conduction Delay
Electrical Abnormalities Without Major Histopathological Anomalies
Electrical Abnormalities in the Offspring of Mothers With SLE
Preoperative Echocardiography
Conclusions
Sources of Funding
Disclosures | cardiac manifestations are highly prevalent in patients with systemic lupus erythematosus ( sle).1 , 2 while electrical abnormalities can occur , isolated atrioventricular ( av ) conduction disease , ranging from first degree to complete av block ( avb ) , is rarely seen in older children and adults with sle such that pacemakers are infrequently required.3 , 4 , 5 , 6 , 7 , 8 a few reports have described abnormal atrial electrical activity in the form of atrial standstill , which was speculated to be secondary to recurrent flares of sle , pericarditis , myocarditis , and/or myocardial arteritis.9 in contrast , complete avb and ventricular cardiomyopathy are wellestablished immunologic complications in neonates of mothers with antiro / ssa antibodies , such that pacemakers are frequently indicated . we , therefore , sought to further characterize the electrophysiological manifestations of maternal sle on neonatal atria . data abstracted from medical records included demographic information , presence or absence of maternal autoimmune antibodies , details regarding pacemaker implantation and all reinterventions , pacemaker interrogations , ecgs , echocardiograms , and histopathological reports when available . a total of 31 patients were diagnosed with isolated congenital complete avb ( figure 1 ) , 13 ( 42% ) of whom had positive maternal antibodies . of the 18 patients without maternal antissa / ssb antibodies ,
all underwent uneventful epicardial pacemaker implantation , with no reported difficulties associated with atrial sensing or pacing . five ( 42% ) of the 12 patients with pacemakers were found to have notable atrial electrical abnormalities in the setting of normal serum electrolytes . characteristics of the 5 patients with immunologic complete avb and left atrial electrical abnormalities asd indicates atrial septal defect ; avb , atrioventricular block ; avvr , atrioventricular valve regurgitation ; crt , cardiac resynchronization therapy ; la , left atrium ; laa , left atrial appendage ; lv , left ventricle / ventricular ; minus sign denotes no ; pht , pulmonary hypertension ; plus sign denotes yes ; ra , right atrium ; raa , right atrial appendage . multiple biopsies of the laa were performed , which showed no evidence of atrial fibrosis or loss of atrial myocytes . pacemaker interrogation revealed marked delay between the onset of the surface p wave and the local laa signal , suggestive of atrial conduction delay . thus , the atrial lead was placed on the right atrium ( ra ) , and the ventricular lead on the left ventricle with good sensing and capture thresholds . multiple biopsies of the laa were performed , which showed no evidence of atrial fibrosis or loss of atrial myocytes . pacemaker interrogation revealed marked delay between the onset of the surface p wave and the local laa signal , suggestive of atrial conduction delay . herein , we report previously undescribed atrial electrical abnormalities in patients with isolated complete avb in the setting of maternal sle antibodies
. left atrial inexcitability and/or atrial conduction delay was highly prevalent ( 42% ) in our series of patients with maternal antissa and/or antissb antibodies , in contrast to no patient with nonimmunemediated avb . nevertheless , the absence of atrial inexcitability observed in our series of patients without immunemediated avb argues against an atrial remodeling hypothesis.16
interatrial conduction block occurs in 12% of adults with avb.17 it should be evaluated at the time of epicardial pacemaker implantation in order to optimize lead placement and av delay programming.18
the pale macroscopic appearance of the la in 2 patients raised suspicion for endocardial fibroelastosis.19 however , histopathology was limited to nonspecific changes . in the 2 patients with the most recent device implantations , upper tracking rates were initially limited to 90 and 100 bpm , with subsequent 10bpm increments on a monthly basis , if left ventricular function remained normal . nevertheless , the absence of atrial inexcitability observed in our series of patients without immunemediated avb argues against an atrial remodeling hypothesis.16
interatrial conduction block occurs in 12% of adults with avb.17 it should be evaluated at the time of epicardial pacemaker implantation in order to optimize lead placement and av delay programming.18
the pale macroscopic appearance of the la in 2 patients raised suspicion for endocardial fibroelastosis.19 however , histopathology was limited to nonspecific changes . in the 2 patients with the most recent device implantations , upper tracking rates were initially limited to 90 and 100 bpm , with subsequent 10bpm increments on a monthly basis , if left ventricular function remained normal . electrical abnormalities of the la and laa appear to be common in patients with congenital lupusinduced avb and include la inexcitability and atrial conduction delay . to date
, there is no histological evidence of extensive atrial fibrosis such that immunemediated functional impairment of electrical activity is suspected . | [
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] |
electrostatic
energy constitutes one of the most important components
of the total intermolecular interaction energy and may substantially
influence the overall interaction strength of the molecular species .
this is especially true in the case of polar systems where electrostatic
interactions are often the driving force for specific complex formation
and stabilization .
on the other hand , it is difficult
to achieve ab initio or dft level results for macromolecules as such
computations are extremely expensive and time consuming , if indeed
feasible . in this view
, it is not surprising that accurate and fast
evaluation of electrostatic interactions in molecular systems is one
of the most challenging tasks in the rapidly developing field of macromolecular
chemistry , including molecular recognition , protein modeling , and
drug design . in the field of quantum chemistry
, the intermolecular
electrostatic
energy represents the coulombic interaction between two isolated molecular
charge densities , tota(r ) and totb(r ) , not disturbed by the neighboring molecule(s )
( molecular complex counterparts , solvent molecules , etc.).1the effect of polarization
of the molecular
charge density by the electric field of the neighboring unperturbed
charge densities is quantified in the form of induction energy .
these
two ( electrostatic and induction ) , together with dispersion and exchange
repulsion , constitute the most important components of the total interaction
energy .
for a small molecular system , they can be directly computed
within the framework of perturbation theories , such as dft - sapt ( symmetry - adapted
perturbation theory based on the density functional theory ) , for example . up to now , the most common approach to deal with
the interactions
in macromolecular systems
is based on force fields . in classical force
fields , there are only two nonbonded components of energy : electrostatics
and van der waals .
the van der waals component is usually computed
with a lennard jones potential and the electrostatic component
with coulomb s law . to compute the electrostatic energy , the
molecular charge distribution is commonly approximated by an appropriate
set of atom - centered point charges . in more advanced force fields ,
higher levels of multipole expansion ( dipoles , quadrupoles , etc . )
are implemented or extra point charges ( often representing lone pairs )
are added to specific atoms . in classic additive force fields ,
the
charges are fixed and often are not affected by the local electrostatic
environment .
electronic polarization is accounted for in an average
fashion for a specific environment ( commonly water ) , and a cancellation
of errors between the electrostatic and van der waals contributions
is commonly present .
considerable effort is devoted nowadays to develop
next - generation polarizable force fields that incorporate in a direct
way specific models for polarizability .
the force field method is aimed at relatively good estimation
of
the total interaction energy and is not optimized to properly describe
the individual components of energy . indeed , in the case of the electrostatic
component , for example , the point multipole expansion does not account
for charge overlap effects and hence excludes the penetration energy .
[ commonly , electrostatic interactions between molecules are described
by point charge or point
these models are computationally inexpensive and give accurate
descriptions of interaction when the molecules are not very much close
together .
when molecules ( atoms ) are in close contact due to electron
distribution overlap , these models gives an error called the penetration
effect .
this is the difference between the exact electrostatic interaction
energy computed by integral over the continuous distributions of charge
and electrostatic interaction energy computed from multipolar expansion
approximation .
note that such defined penetration energy depends not
only on the level of charge density overlap but also on the level
at which multipole expansion is truncated .
] penetration is not taken
directly into account in the classical force field approach , but it
is compensated by a less repulsive van der waals potential . as a consequence ,
electrostatic energies computed with coulomb s law from point
charges come close to exact values only at large interatomic distances .
in general ,
all interaction energy components in the force field exhibit
rather large errors at typical vdw distances , which nevertheless tend
to approximately cancel out in a systematic way .
the performance of
the force fields for various energy components has been recently tested
against the dft - sapt results .
there is
ongoing research to develop more accurate , but still fast , methods
for estimation of electrostatic properties that take directly into
account both density asphericity and penetration . among them
there is the pseudoatom database approach that
has its
roots in crystallography . up to now , there are three well - established
databanks : generalized experimental library of multipolar atom model
( elmam / elmam2 ) , theoretical invariom database , and
university of buffalo pseudoatom databank ( ubdb ) . the
aforementioned databanks can be employed , besides being a source of
aspherical scattering factors in an x - ray diffraction data refinement ,
to reconstruct the electron density distributions of macromolecules
and , in turn , to derive the electrostatic properties of such complex
systems .
,
the ubdb approach has been tested against quantum
mechanical results computed for a small set of amino acids and similar
molecules and for a more extensive set of nucleic
acid bases .
the test suggests that the
ubdb approach leads to results of quality not much worse than from
quantum mechanics , but still it is much faster .
does the
quality of the electrostatic properties derived from the ubdb method
more closely resemble the quantum mechanical or force field results ?
does ubdb give more physical electrostatic properties than popular
force fields , which for many systems are the only feasible source
of information regarding electrostatic properties but were not designed
to properly describe electrostatics .
hence , in the following paper ,
we present a thorough comparison of the recently extended ubdb with
the amber and charmm force fields approach to electrostatics .
a similar
analysis has already been done in the case of the elmam2 database , in which the interactions in amino acid - based
systems were compared to amber results . here , we have chosen a larger
and more diverse set of compounds that are widely used as a benchmark
in many computational studies , that is , the s66 and jsch-2005 biologically
oriented training sets .
such a strategy will serve as a comprehensive
overview of the ubdb method performance and limitations and will indicate
the reliability of its application .
the university
of buffalo databank ( ubdb ) has been used together
with the lsdb program to reconstruct electron
density distributions of biological and organic molecules .
the ubdb
offers the possibility of structural refinement of x - ray diffraction
data with the use of aspherical scattering factors computed from the
transferable aspherical atom model ( taam ) .
apart from x - ray data refinement ,
the ubdb can be applied for evaluation of electrostatic properties
of molecular complexes using a reconstructed electron density distribution .
the databank is based on isolated molecule charge densities computed
in vacuum and therefore does not take into account intermolecular
polarization effects .
in addition to the previous databank version , the current version of the ubdb contains 253
atom definitions consisting of 21 hydrogen , 129 carbon , 33 nitrogen ,
39 oxygen , 10 sulfur , 9 phosphorus , 2 chlorine , 4 fluorine , 2 bromine ,
and 3 boron atom types . for the purposes of this work , the
database
has been updated with seven new atom types commonly present in small
organic molecules shown in figure 1 .
the symbol d represents a dummy
atom required for definition of the coordinate system .
the ubdb has been extended following the procedure
applied in the
previous version of the databank .
good
quality experimental molecular geometries of model molecules were
obtained from the cambridge structural database ( csd ) .
hydrogen atom positions were corrected by extending
x h distances to their standard neutron diffraction values .
theoretical structure factors were obtained
from single - point wave functions computed at the b3lyp/6 - 31 g * * level to which the hansen and coppens pseudoatom multipole model was fitted . in the pseudoatom model , individual
atomic densities are described in terms of spherical core and valence
densities with an expansion of atom - centered real spherical harmonic
functions2where core and val are spherical
and valence densities , respectively .
the
third term contains the sum of angular function ylm(, ) to take into
account aspherical deformations .
the coefficients pval and plm are population
for the spherical and multipole density , respectively . the
and are scaling parameters that determine the expansion / contraction
of spherical and multipolar valence densities , respectively .
the derived
multipole parameters were averaged for chemically similar atoms and
together with atom type definitions were added to the ubdb . for
this analysis , the all atom additive general amber force field ( gaff ) and charmm general force field ( cgenff ) were selected . to parametrize molecular complexes
with the gaff atom types ,
in addition , the program can calculate partial charges
with different models and then generate an amber force field model
for an organic input molecule followed by atom type , bond , angle ,
and dihedrals . to obtain partial charges ,
two popular methods were
employed : am1-bcc and restrained electrostatic
potential fitting ( resp ) .
the am1-bcc
methodology begins with the semi - empirical calculation of mulliken
( am1 ) charges used to describe features of an electron distribution
such as formal charge and delocalization , and in the final step , it
generates bond charge corrections ( bccs ) parametrized to reproduce
ab initio ( hf/6 - 31 g * ) electrostatic potentials .
the resp charges were
computed from the molecular electrostatic potential using the antechamber
package . in our study ,
the first set was obtained from the molecular
electrostatic potential computed at the hf/6 - 31 g * level in vacuum
( here abbreviated simply as resp ) .
the second set comes from a molecular
electrostatic potential computed at the same level but with a self - consistent
reaction field ( scrf ) method ( hereafter ,
the set is abbreviated as resp - scrf ) , which was intended to mimic
the solvated environment .
the polarizable continuum model ( pcm ) with an external dielectric constant of 78.39
was used . the integral equation formalism method for pcm
was used
with the united atom topological model ( ua0 ) for the cavity , using
the default parameters of gaussian 03 .
it is known that the standard resp methodology produces charges
that are systematically overestimated by approximately as
much as the dipole is enhanced for a water molecule in the tip3p or spc models of
water over its gas - phase value .
the resp - scrf method was used to understand to what extent
highly polarized partial charges improve the quality of the molecular
mechanics approximation , a phenomenon observed by some researchers .
conversely , the cgenff program assigns
atomic point charges by
analogy with a set of model compounds , the charges
on which were explicitly optimized for use in the charmm force field .
it provides a wide range of atom types present in organic molecules ,
including heterocyclics , followed by generation
of topology from charmm ver .
the water partial charges were taken from the tip3p water model and used in all point charge methods
tested . to compute the electrostatic interaction energy ( ees ) , a variety of calculations were performed .
in the
ubdb approach , the molecular electron density was reproduced from
the ubdb .
the lsdb program was used to
transfer the multipole populations from the ubdb to all structures .
the xdprop module of the xd2006 package was used to calculate interaction energies from the derived
charge density using the exact potential multipole method ( epmm ) .
the epmm evaluates the exact coulomb integral in the inner region
( 4.5 ) and combines it with a buckingham - type multipole
approximation for long - range interatomic interactions . in the case of the point
charge methods ,
energies of nonbonded interactions were calculated between all pairs
of atoms within a specified interatomic cutoff distance .
the ees was computed using a standard molecular mechanics force
fields term3which corresponds to the coulomb electrostatic
interaction in vacuum between a pair of atom - centered partial charges qi and qj , separated by a distance rij .
the ees term was computed separately for dimer ( ab ) and for each monomer
( a and b ) , and then the sum of ees for
monomers was subtracted from the ees for
the dimer . such a procedure is equivalent to direct computation of
the intermolecular ees by application
of the summation in eq 3 . such calculated energies were compared with
the corresponding reference
values ( abbreviated as ref ) obtained directly from wave functions
computed for monomers in vacuum at the b3lyp / aug - cc - pvtz level using gaussian03 .
the spdfg program was used to calculate
the exact ees from the wave function .
the numerical rys quadrature is implemented in the spdfg code for
the computation of one- and two - electron coulomb integrals . for details , refer to the supporting information .
the ees values obtained from spdfg
were taken as reference points as they have excellent agreement with epol(1 ) obtained from the symmetry - adapted perturbation
theory based on density function theory ( dft - sapt ) .
the
dft - sapt method gives directly all physical components of interaction
energy : electrostatic epol(1 ) , induction ,
and dispersion ( taking into account of overlap effects ) and their
exchange counterparts .
[ induction interaction ( also known as polarization )
is the interaction between a permanent multipole on one molecule with
an induced multipole on another .
when a molecule is placed in the
field of other molecules , moments will be induced on that molecule
due to its polarizability . whereas , dispersion interaction ( also called
london interactions ) is an attractive component of the intermolecular
interaction energy and acts between all type of molecules , polar or
not .
it arises due to the formation of a time variable instantaneous
dipole of one system and the induced multipole of the second system . ]
the first - order sapt energy ( electrostatic+exchange ) was computed
with monomer densities and density matrices from the kohn sham
dft .
the second - order sapt energy ( induction , dispersion+exchange )
from the ( time - dependent ) coupled perturbed theory utilized kohn sham
response functions . for the s66 data set ,
the correlation between epol(1 ) from dft - sapt and ees obtained from the spdfg program
was 0.99 and the rmse was 0.5 kcal mol ( see the supporting information for details ) . to quantify the
molecular electrostatic potential ,
we have calculated
quantitative descriptors of the electrostatic potential mapped on
the molecular van der waals surface as proposed by politzer and co - workers .
the electrostatic potential ( ri ) of a single molecule was computed over a grid with 0.1
step size around the molecule with a 3.00 margin using
the vmopro module of the mopro package .
statistical
quantitative descriptors for electrostatic potential were evaluated
at the van der waals surface of thickness 0.3 . the reference
electrostatic potential grids were calculated using gaussian03 at
the b3lyp / aug - cc - pvtz level .
all quantitative notation used here is
the same as in politzer s original papers .
the descriptors
were computed for each monomer and further averaged with the same
molecules .
this was done because some molecules ( e.g. , uracil ) had
slightly different geometries in different complexes .
the mopro package was used ,
applying the buckingham approximation as described in the formula
given by spackman and coppens at the molecular center of mass .
molecular dipole
moments evaluated from atomic point charges were calculated in the
mopro package as well .
the reference dipole moment magnitudes were
directly obtained from the b3lyp / aug - cc - pvtz wave functions computed
in gaussian03 . as with the electrostatic potential ,
dipoles were computed
for each monomer and further averaged with the same molecules . in this study
s66 is a benchmark
data set characterized by well - balanced interaction energies for noncovalent
interactions relevant to biological chemistry .
it includes small organic
molecules with a variety of interaction motifs and represents most
typical noncovalent interactions .
a total of 66 molecular complexes
from the s66 data set can be divided in three subgroups : 23 complexes
representing all possible types of of hydrogen bonding , 23 complexes
representing dispersion - dominated interactions ( ,
aliphatic
aliphatic , and aliphatic ) , and in
the remaining 20 complexes , the interactions consist of a combination
of dispersion and electrostatic contributions .
the jsch-2005 data
set is more focused on biological systems and contains almost exclusively
nucleic acid bases , amino acids , and their derivatives .
three subgroups contain nucleic acid
bases divided according to the type of predominant noncovalent interaction :
hydrogen - bonded base pairs ( 38 structures ) , interstrand base pairs
( 32 structures ) , and stacked base pairs ( 54 structures ) .
the remaining
fourth subgroup contains 19 amino acid complexes ( see supporting information for details ) .
one of the
hydrogen - bonded base pairs ( cytosine ... protonated cytosine ) was excluded
from this study because it was previously reported to have two resonance
structures , where a different nitrogen
atom bears a + 1 formal charge .
several force fields assign different
charges when given either of two resonance structures of protonated
cytosine , resulting in a high discrepancy in interaction energy .
equilibrium
geometries of both data sets are taken from hobza and co - workers .
in addition to equilibrium geometries , we have also analyzed
the s66 8 data set representing
eight points along the dissociation curve for each complex from s66 ,
altogether 528 dimers .
it has already been reported that electrostatic energies obtained
from the combined method of ubdb and epmm reproduce quite well the
electrostatic energies obtained from quantum chemical calculations .
the rmse between ubdb+epmm and adf / b3lyp / tzp for 11 molecular dimers
of -glycine , n - acetylglycine , and l-(a)-lactic acid was 1.9
kcal mol . in another
study of 10 amino acid dimers of glycine , serine , and leucine ,
the
rmse was 5 kcal mol between ubdb+epmm and adf / b3lyp / tzp
as compared to amber99 , charmm27 , mm3 , and mmff values ranging from
7 to 8 kcal mol . in
a recent paper ,
the electrostatic energy was analyzed for nucleic
acid base dimers , and the rmse was 3.7 kcal mol between ubdb+epmm and gaussian03/b3lyp/6 - 31g**. a good linear correlation
between the ubdb+epmm and the quantum chemical reference results was
observed , with the slope close to unity ( table 1 , figure 2 ) .
the am1-bcc , resp , and
cgenff results differ much more from the reference , with rmses equal
to 4 kcal mol .
the strength of electrostatic
interactions is about 50% less favorable on average for all three
point charge methods .
comparison of electrostatic interaction energies computed
from
tested charge models with reference to theoretical results ( b3lyp / aug - cc - pvtz )
for the s66 data set .
the quality of energies obtained from the am1-bcc charges
is nearly
as good as that of the results that were obtained using resp charges ,
despite the much simpler methodology applied in the latter case .
the resp charges perform only slightly better than am1-bcc ,
probably due to the fact that the empirical corrections used in am1-bcc
were parametrized to reproduce the resp charges derived from hf/6 - 31g*.
the smaller computational cost of computing the am1-bcc charges , as
compared to those obtained from dft , make the am1-bcc method more
attractive
. one can wonder whether the resp charges would perform
better if
a higher level of ab inito theory was used .
the reason why it is recommended
to derive charges at the hf/6 - 31 g * level is that the always attractive three - body correlations could be accounted
for in an average way . to achieve a more pronounced effect without
relying on the deficiencies of the 6 - 31 g * basis set
only , a self - consistent
reaction field ( scrf ) method can be used to mimic the polarizable
environment , which is an approach sometimes used in drug design .
this
particular choice of method hyperpolarizes the vacuum charges in a
way that could be considered appropriate for condensed - phase simulation .
we noticed a significant improvement in the quality of estimated energies
calculated from point charges , which were obtained from the hf/6 - 31 g *
resp combined with scrf , with rmse equal to 1.9 kcal mol .
the results based on cgenff charges are almost as good as
those
obtained from the am1-bcc method .
the am1-bcc method relies on calculations
performed directly for particular molecules , and the cgenff uses empirical
( extended bond charge increment ) rules to assign charges by analogy
to a set of model compounds optimized to accurately interact with
water .
this charge assignment does not involve electronic structure
calculations of any kind and is therefore much faster than the other
methods .
taking into account the above
facts , it maybe concluded that cgenff performs quite well .
considering
the type of dominating interactions , the largest absolute
errors in the ees estimation were observed
for the hydrogen - bonded complexes ( table 2 ) .
the values of ees from ubdb+epmm were
the most similar to the ref among all tested methods ( rmse = 1.4 kcal
mol ) .
the am1-bcc and cgenff charges gave the
largest discrepancies ( rmse = 6.3 and 6.8 kcal mol , respectively ) , and the resp - scrf charges were second close to the
ref .
the ees for hydrogen bonding seems
to be the most sensitive , on an absolute scale , to any simplifications
in the electron density description . on the other hand ,
ees in dispersion - dominated complexes seems to be least
sensitive , on an absolute scale , to the method used for estimation
( rmse 2.5 kcal mol for all point charge
methods ) with the exception for the ubdb+epmm method , leading to energies
evidently closer to the ref ( rmse = 0.9 kcal mol ) .
however , when the error of estimation is related to the
values
of estimated energies ( % error ) , it appears that these are dispersion - dominated
complexes for which electrostatic energy is estimated as the worst
on a relative scale .
the am1-bcc , resp , resp - scrf , and cgenff methods
give rise to ees in dispersion - dominated
complexes that are too weak by about 100% and the ubdd+epmm by 16% .
relatively , the results for hydrogen - bonded complexes are estimated
as the best .
the relative error was only 2% for ubdb+epmm , 11% for
resp+scrf , and around 33% for the remaining methods .
the reason for
the above behavior is undoubtedly related to fact that electrostatic
contribution to interaction energies in dispersion - dominated systems
are very small ( here , 2.7 kcal mol on
average ) at the limit of the tested method s accuracy . on the
other hand ,
hydrogen - bonding interactions are much stronger ( here ,
13.2 kcal mol on average ) and dominated by electrostatics
that are very sensitive to the accurate description of anisotropy
of interacting charge densities .
it is clear that the ubdb+epmm method
is in very good agreement with the ref for estimating ees in all types of interactions ( hydrogen bonding , dispersion
dominated , and mixed ) .
r -
correlation coefficient of linear regression fit inbetween reference
and aproximated values , slope - the slope of the linear regression
line , rmse - root mean square error ( rmse = ( t = 1n(fi
yi))/n ) , mae - mean
absolute error ( mae = ( 1/n)t = 1n| fi
yi| ) , me - mean signed error
( me = t = 1n(fi
yi)/n ) , % error - the error as a percent
of the exact value ( ( ( fi yi)/fi ) 100% ) , where fi and yi are the reference and approximated values ,
respectively . the accurate
description of the entire potential energy surface is of great importance
for any method that is applied to calculations for nonequilibrium
geometries or molecular dynamics simulations .
the ees is one of the most important contributions to the intermolecular
interaction energy , especially at long interatomic distances , where
density overlap does not occur and the remaining contributions to
energy ( dispersion , induction , etc . )
usually , at long
distances , the point multipole approximation is good enough to compute
accurate energies of electrostatic interactions .
proper estimation
of electrostatics at short distances becomes an issue because here
the electron density overlap appears and the multipole approximation
becomes insufficient .
the difference between the proper ees and the ees estimated from
the point multipole approximation is referred to as the penetration
electrostatic energy . to study the performance of tested methods
at both long- and short - range distances ,
we have analyzed s66 molecular
complexes at eight scan points along the dissociation curve ( seven
different intermolecular distances from the equilibrium point and
at equilibrium ) .
plots depicting the behavior of the estimated ees depend on the distance for dimers , which
represent examples of different subgroups , are given in figure 3 .
electrostatic interaction energy in kcal mol computed by ubdb+epmm and various point charge methods compared
with the ref ( b3lyp / aug - cc - pvtz ) energy .
the values of ees estimated by the
ubdb+epmm method quite nicely follow the reference ones .
the error
of ees estimation in the case of ubdb+epmm
increases with decreasing distance ; it starts from 0.4 kcal mol and reaches a maximum of 1.9 kcal mol at the shortest distances .
the ees obtained
from point charges agrees well with the ref only at larger distances .
the rmse was usually below 1.0 kcal mol for separations
equal to 1.25 times the equilibrium distance or larger ( figure 4 and
table ii of the supporting information ) .
interestingly , the resp - scrf method is the only one that overestimates
the strength of the electrostatic interactions at longer distances ,
a phenomenon also reported by other authors .
when molecules approach each other , the strength of electrostatic
interactions become more and more severely underestimated , with rmse
up to 8 kcal mol at 0.9 times the equilibrium
distance . indeed , the underestimation is already significant at equilibrium
distances , as observed in the s66 data set .
however , it should be
noted that the vdw radii in force fields are chosen such that the
interaction distances of hydrogen - bonded complexes are underestimated
by about 0.2 with respect to the vacuum , as explained
in the introduction . in this light ,
the discrepancies
in interaction energies will be compensated by the van der waals term .
the effect of such compensation would be comparable to about a 10%
horizontal shift in figure 3 .
applying such
a shift would bring the am1-bcc , resp , and cgenff results in much
better ( albeit still not perfect ) agreement with the reference and
would lead to severe overestimation for resp - scrf .
therefore , the
latter method may not be suitable for full force field calculations
in which the interaction geometry is allowed to relax .
again , ees estimated from polarized resp charges ( resp - scrf )
were much closer to the reference values than those from vacuum resp
charges .
this is especially true for ions and polar molecules , which
is in agreement with the finding of other researchers .
visualization
of the differences in the rmse ( kcal mol ) in ees for s66 8 at different
intermolecular distances .
the ees scan nicely illustrates
how
neglecting the charge density overlap influences the values of ees , while the interaction distance shortens .
the scans also help to understand why the electrostatic energies in
dispersion - dominated complexes are so badly estimated by the tested
point charge models .
in several dispersion - dominated dimers , the ees computed from point charges become more and
more positive with intermolecular distance shortening , as illustrated
in figure 3 for the benzene
this is probably due to an insufficient level of multipole
expansion . with atomic point charges only , unfavorable orientations
of local dipole moments may lead to repulsive energies .
the jsch-2005
data set is composed of larger molecules , and the range of ees is much larger than in the case of s66 .
the range of ees energies for hydrogen - bonded
base pair complexes was from 7.9 to 49.5 kcal mol .
in stacked complexes , the interaction energies were
usually larger than 22.8 kcal mol and
reached 3.7 kcal mol .
the interaction energies of charged
amino acid pairs were very negativ , and in the case of e49-k6(1bq9 ) ,
the ees value reached 96.5 kcal
mol .
the ees energy
obtained from the ubdb+epmm method was in good agreement with the
ref with a correlation coefficient 0.98 ; the point charge methods
deviated more from the ref ( table 4 and figure 5 ) .
the overall statistics from electrostatic
energies computed for
the jsch-2005 data set show a similar behavior to the s66 data set .
all tested methods gave rise to the electrostatic interactions that
were too weak on average .
the second closest is the resp - scrf method , and am1-bcc ,
resp , and cgenff gave the largest discrepancies but were similar to
each other . for each tested method ,
the absolute errors are larger
than for the s66 data set , but the relative errors remain similar . comparison
of electrostatic interaction energies with the ref values
for the jsch-2005 data set .
the jsch-2005 data set was not as well characterized as the
s66
set in terms of type of interactions dominated in molecular dimers
and the size of molecules .
nonetheless , some trends observed in the
case of the s66 subgroups are also visible here . in the planar subgroup ,
in which hydrogen bonding dominates , the resp - scrf is almost as close
to the ref as ubdb+epmm . in the stacked subgroup , in which dispersion
interactions dominate ,
all point charge methods give similar absolute
errors , although here the relative error is much smaller than it was
for the similar subset of s66 .
the rmse for ubdb+empmm ranged from
1.2 to 5.8 kcal mol , representing a significant
improvement over point charge methods , which range from 1.0 to 13.4
kcal mol ( table 5 ) .
electrostatic
potentials mapped on the van der waals surfaces ( mepss ) in all examined
models show positive or negative potential values characteristic for
particular types of functional groups ( figures 6 , 7 , and 8) .
politzer
and co - workers have shown that quantitative information can be derived
from the meps and used to predict several molecular properties .
we used some of the meps descriptors proposed by them to do quantitative
evaluation of electrostatic potentials computed from all tested methods .
the charged molecules of the jsch2005 data set were excluded for this
study because of the undefined origin of formal charges .
we focused
our analysis on the average value of positive potential on the surface v and its variance , the
negative potential v and its variance
, and on the average value of the total
potential on the surface v. it appears
that all the tested methods ( ubdb , am1-bcc , resp , and cgenff ) approximate
average values of positive and negative potentials and their variance
with similar errors .
the statistics tends to be slightly more favorable
for resp and slightly poorer for cgenff .
interestingly , v is much better approximated than v in all tested methods : rmses 0.003 e / a0 and 0.006 e / a0 for v and v , respectively . in the
case of v , some clear discrepancies
between methods are visible .
although all tested methods lead to values
of the overall potential on the van der waals surface shifted toward
negative values , compared to the reference ( figure 9 ) , v values from ubdb are much
closer to the reference values ( rmsd = 0.002 e / a0 ) and remain slightly positive for almost all
molecules ( tables 6 and 7 ) .
isolated molecule electrostatic potential ( esp ) of uracil mapped
on the van der waals surface for ( a ) ref , ( b ) ubdb , ( c ) am1-bcc , ( d )
resp , and ( e ) cgenff models .
the maximum negative and positive values
of esp correspond to the values 0.11 and 0.11 e / bohr , respectively .
isolated molecule electrostatic potential ( esp ) of guanine mapped
on the van der waals surface for ( a ) ref , ( b ) ubdb , ( c ) am1-bcc , ( d )
resp , and ( e ) cgenff models . the maximum negative and positive values
of esp correspond to the values 0.11 and 0.11 e / bohr , respectively .
isolated molecule electrostatic potential ( esp ) of pyridine mapped
on the van der waals surface for ( a ) ref , ( b ) ubdb , ( c ) am1-bcc , ( d )
resp , and ( e ) cgenff models .
the maximum negative and positive values
of esp correspond to the values 0.11 and 0.11 e / bohr , respectively .
visualization of the differences in the properties of isolated
molecule electrostatic potential mapped on the van der waals surface
( e / bohr ) between ref and the given model .
resp
was designed to accurately fit the electrostatic potential resulting
from a quantum calculation , meaning that it should reproduce the dipole
moment of the quantum mechanical charge distribution well , and this
was the case ( table 8 , figure 10 ) .
the r for the resp dipole
for both s66 and jsch-2005 data sets together was 0.99 , and the lowest
rmse is observed for resp . slightly worse correlations are observed
for ubdb and am1-bcc , and the worst is cgenff . nonetheless , the magnitude
of the rms errors were in a similar range for all tested methods ,
with the lowest one being 0.46 d for resp and the largest being 0.92
d for cgenff .
the performance of the cgenff partial charges in the
molecular dipole moment estimation is therefore satisfactory .
it appears
that the ubdb method is not better than point charge methods for molecular
dipole moment magnitude approximation .
isolated molecule dipole
moments computed from tested methods are
compared with those computed directly from the electronic wave function
from b3lyp / aug - cc - pvtz calculations ( ref ) . in the case of resp
dipole moment magnitudes , there is a
small
trend visible ; with an increasing value of dipole moment magnitudes ,
resp dipoles become more and more overestimated .
this effect is much
more pronounced for the resp - scrf dipole moments ( see supporting information for details ) .
the above
trends illustrate the consequences of overestimated atomic charges
derived from prepolarized charge densities .
application of a continuous
solvent model with an external dielectric constant of 78.39 ( resp - scrf
charges ) gives rise to quite a significant overestimation of molecular
dipole moments ( more than 30% in some cases ) and brings values of ees closer to the referential ones in a way that
imitates some penetration effects .
it is worth stressing here that
the reference ees does not contain any
contribution from polarization effects and fully accounts for penetration
effects .
in fact , more proper reference values for ees estimated from polarized charges would be a sum of
electrostatic and induction energies .
the
performance of the ubdb for electrostatic interaction energy
( ees ) estimation was analyzed with the
s66 and jsch-2005 data sets .
the results were compared with ees estimated from various widely used molecular
mechanics force fields and from quantum mechanical computation . for
a total of 208 different molecular complexes ,
electrostatic energies
were calculated at their equilibrium geometries as well as at shorter
and longer distances .
the energy obtained from ubdb+epmm is in good
agreement with reference energies obtained at the spdfg / b3lyp / aug - cc - pvtz
level of theory ( rmse = 1.1 and 3.2 kcal mol for
s66 and jsch-2005 , respectively ) .
the results clearly show that the
ubdb+epmm method much more closely resembles the reference quantum
mechanical results in comparison to the point charge - based force field
methods ( am1-bcc , resp , and cgenff ; rmse 4.0 and 7.0
kcal mol for s66 and jsch-2005 , respectively ) .
the discrepancies between energy estimated for point charge methods
and ref were somewhat satisfactory at longer distances but increase
when passing to shorter distances due to severe underestimation of
electrostatic interaction strengths in that region .
this is because
the point charge approximation does not incorporate penetration effects
arising at short distances and also because the point charges were
parametrized for bulk - phase interaction distances , which are shorter
than the vacuum - phase distances found in the s66 and jsch-2005 data
sets .
it is assumed that these errors are compensated by stronger
vdw interactions in the force fields being studied . along with
electrostatic interaction energies , the following properties
related to electron density distributions have been compared extensively :
electrostatic potential and dipole moments . here , the benefit of using
ubdb - derived densities is only visible for the case of the average
value of total electrostatic potential mapped on the molecular van
der waals surface .
other properties , including molecular dipole moment
magnitudes , were quite well approximated by all tested methods including
ubdb ; those differences that were apparent suggest that resp may be
marginally the best one and cgenff the worst .
all the results
indicate that cgenff point charges were almost
as good as am1-bcc .
bearing in mind that the latter were computed
on demand for a particular molecule and the former assigns charges
by analogy without involving electronic structure calculations , the
cgenff charges performed very well .
our results emphasize that the
point charge model poorly represents the charge density distribution
at equilibrium distances , which is a major drawback in electrostatic
interaction energy estimation by force field methods .
however , it
should be emphasized that the force field charges are designed to
mimic a condensed - phase environment in a mean field way , and often
their deficiencies are compensated by cancellation of errors between
electrostatic and vdw interaction such that a larger discrepancy with
respect to the exact gas - phase target data is somewhat expected .
the
smaller difference between reference and ubdb+epmm results strongly
suggests that this is a better method for electrostatic energy estimation
than the others .
the strength of the ubdb+epmm method most probably
lies in the fact that for short distances exact evaluation of ees is possible having access to ubdb - derived
charge densities . | accurate and fast evaluation of electrostatic
interactions in molecular
systems is one of the most challenging tasks in the rapidly advancing
field of macromolecular chemistry and drug design .
electrostatic interactions
are of crucial importance in biological systems .
they are well represented
by quantum mechanical methods ; however , such calculations are computationally
expensive . in this study
, we have evaluated the university of buffalo
pseudoatom databank ( ubdb)1,2 approach for approximation
of electrostatic properties of macromolecules and their complexes .
we selected the s663 and jsch-20054 data sets ( 208 molecular complexes in total )
for this study .
these complexes represent a wide range of chemical
and biological systems for which hydrogen bonding , electrostatic ,
and van der waals interactions play important roles .
reference electrostatic
energies were obtained directly from wave functions at the b3lyp / aug - cc - pvtz
level of theory using the sapt ( symmetry - adapted perturbation theory )
scheme for calculation of electrostatic contributions to total intermolecular
interaction energies .
electrostatic energies calculated on the basis
of the ubdb were compared with corresponding reference results .
results
were also compared with energies computed using a point charge model
from popular force fields ( am1-bcc and resp used in amber and cgenff
from charmm family ) .
the energy trends are quite consistent ( r2 0.98 ) for the ubdb method as compared
to the amber5 and charmm force field methods6(r2 0.93
on average )
. the rsmes do not exceed 3.2 kcal mol1 for the ubdb and are in the range of 3.77.6 kcal mol1 for the point charge models .
we also investigated
the discrepancies in electrostatic potentials and magnitudes of dipole
moments among the tested methods .
this study shows that estimation
of electrostatic interaction energies using the ubdb databank is accurate
and reasonably fast when compared to other known methods , which opens
potential new applications to macromolecules . | Introduction
Theory
and Computation Methods
Results and Discussion
Conclusions | in this view
, it is not surprising that accurate and fast
evaluation of electrostatic interactions in molecular systems is one
of the most challenging tasks in the rapidly developing field of macromolecular
chemistry , including molecular recognition , protein modeling , and
drug design . for a small molecular system , they can be directly computed
within the framework of perturbation theories , such as dft - sapt ( symmetry - adapted
perturbation theory based on the density functional theory ) , for example . hence , in the following paper ,
we present a thorough comparison of the recently extended ubdb with
the amber and charmm force fields approach to electrostatics . apart from x - ray data refinement ,
the ubdb can be applied for evaluation of electrostatic properties
of molecular complexes using a reconstructed electron density distribution . such calculated energies were compared with
the corresponding reference
values ( abbreviated as ref ) obtained directly from wave functions
computed for monomers in vacuum at the b3lyp / aug - cc - pvtz level using gaussian03 . the reference
electrostatic potential grids were calculated using gaussian03 at
the b3lyp / aug - cc - pvtz level . the reference dipole moment magnitudes were
directly obtained from the b3lyp / aug - cc - pvtz wave functions computed
in gaussian03 . a total of 66 molecular complexes
from the s66 data set can be divided in three subgroups : 23 complexes
representing all possible types of of hydrogen bonding , 23 complexes
representing dispersion - dominated interactions ( ,
aliphatic
aliphatic , and aliphatic ) , and in
the remaining 20 complexes , the interactions consist of a combination
of dispersion and electrostatic contributions . comparison of electrostatic interaction energies computed
from
tested charge models with reference to theoretical results ( b3lyp / aug - cc - pvtz )
for the s66 data set . the reason for
the above behavior is undoubtedly related to fact that electrostatic
contribution to interaction energies in dispersion - dominated systems
are very small ( here , 2.7 kcal mol on
average ) at the limit of the tested method s accuracy . the ees is one of the most important contributions to the intermolecular
interaction energy , especially at long interatomic distances , where
density overlap does not occur and the remaining contributions to
energy ( dispersion , induction , etc . ) electrostatic interaction energy in kcal mol computed by ubdb+epmm and various point charge methods compared
with the ref ( b3lyp / aug - cc - pvtz ) energy . we focused
our analysis on the average value of positive potential on the surface v and its variance , the
negative potential v and its variance
, and on the average value of the total
potential on the surface v. it appears
that all the tested methods ( ubdb , am1-bcc , resp , and cgenff ) approximate
average values of positive and negative potentials and their variance
with similar errors . although all tested methods lead to values
of the overall potential on the van der waals surface shifted toward
negative values , compared to the reference ( figure 9 ) , v values from ubdb are much
closer to the reference values ( rmsd = 0.002 e / a0 ) and remain slightly positive for almost all
molecules ( tables 6 and 7 ) . visualization of the differences in the properties of isolated
molecule electrostatic potential mapped on the van der waals surface
( e / bohr ) between ref and the given model . isolated molecule dipole
moments computed from tested methods are
compared with those computed directly from the electronic wave function
from b3lyp / aug - cc - pvtz calculations ( ref ) . the energy obtained from ubdb+epmm is in good
agreement with reference energies obtained at the spdfg / b3lyp / aug - cc - pvtz
level of theory ( rmse = 1.1 and 3.2 kcal mol for
s66 and jsch-2005 , respectively ) . the results clearly show that the
ubdb+epmm method much more closely resembles the reference quantum
mechanical results in comparison to the point charge - based force field
methods ( am1-bcc , resp , and cgenff ; rmse 4.0 and 7.0
kcal mol for s66 and jsch-2005 , respectively ) . our results emphasize that the
point charge model poorly represents the charge density distribution
at equilibrium distances , which is a major drawback in electrostatic
interaction energy estimation by force field methods . | [
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] |
neonates represent an ideal population for the investigation of the biological dispositions that guide humans to acquire their native language .
neonate studies reveal how we begin processing speech , and inform us about the cognitive faculties that we possess before the accumulation of significant linguistic experience .
newborns stay awake only for short periods of time ; and when they are awake they eat , interact with caretakers , cry , or are in a quiet state of drowsiness .
in addition , most of the current neuroimaging techniques are impractical for testing healthy young participants because these techniques a ) use machines that produce high levels of acoustic noise , b ) require the application of liquids or gel in the newborn 's head , or c ) have very low tolerance to facial and body movement .
functional near - infrared spectroscopy ( fnirs ) is an imaging technique that has been employed for clinical purposes ( wolf et al . , 2007 ) and more recently in cognitive research as well .
it is regarded as one of the most appropriate to study language faculties and cognitive capacities in the newborn 's brain ( see aslin and mehler , 2005 ; minagawa - kawai et al . , 2008 ; lloyd - fox et al . , 2010
; obrig et al . , 2010 , for reviews on fnirs developmental studies , but see also dehaene - lambertz and pea , 2001 ; kushnerenko et al .
, 2001 ; cheour et al . , 2002 , 2004 ; imada et al . , 2006 , for examples of language studies in newborns using magnetoencephalography and electroencephalography ) .
fnirs is non - invasive , and there is no need to use any substance , not even to keep the device in place on the infant 's head .
it is ideal to study how neonates process auditory stimuli because the device makes hardly any noise .
moreover , we have observed that the number of participants providing useful fnirs data is very high compared to many behavioral methods used to investigate neonatal cognition : in typical behavioral paradigms used to study auditory capacities , rejection rate may reach beyond 50% . in our fnirs studies , the number of infants that are excluded from the analysis ranges between 20 and 25% .
one reason for this is that , in fnirs studies regarding auditory perception , participants need not be excluded because of behaviors like falling asleep or refusing the pacifier , very common in neonates .
however , infants must be relatively quiet because head movements can easily displace the fnirs probes .
in what follows , we describe some studies carried out with newborns in our laboratory using fnirs , with particular focus on the technical and methodological aspects .
functional near - infrared spectroscopy works by measuring changes in cerebral blood flow volume and oxygen saturation using optical means .
it is based on the emission of near - infrared laser light on the subject 's scalp .
( 2011 ) provide excellent reviews about the workings of fnirs and its applications to infant research .
our laboratory is located in the hospital azienda ospedaliera santa maria della misericordia in udine , in the same building as the obstetrics and neonatology department .
we use the etg-4000 machine ( hitachi medical corporation , tokyo , japan ) , which emits continuous near - infrared light at two wavelengths-695 nm and 830 nm - through optical fibers .
the sampling rate is 10 hz , and the total laser power output per fiber we use is 0.75 mw .
a silicon holder usually called probe keeps the optical fibers at a fixed distance from one another . in each probe , five fibers act as emitters and four as detectors , allowing simultaneous recording from 12 points per probe .
two probes are used , one for each of the cerebral hemispheres ( figure 1 ) , providing a total of 24 recording sites .
probes containing the emitters ( red marks ) and detectors ( blue marks ) of near - infrared light , and their positioning on the neonate 's head .
because we are interested in normal development and language acquisition , we focus our studies on healthy hearing newborns .
infants are considered eligible to participate in our studies if their gestational age is between 38 and 42 weeks , their apgar score is at least eight in the first minute of life , no problems were observed in the hearing test , and do not present hematomes .
moreover , to maximize the likelihood of monitoring the same areas of the brain across different infants , we recruit only those whose head diameter ranges between 33.5 and 36.0 cm .
each infant is tested individually inside a dimly lit sound - attenuated booth ( figure 2 ) .
newborns come to the test session when they are in a quiet state of rest , either awake or sleeping .
they remain in their own nursery crib and are tested lying in it , without receiving any reinforcement . a nurse or a pediatrician assists the neonates .
the neonate rests in his o her crib , assisted by a nurse or pediatrician .
sounds are presented by two loudspeakers located about 70 cm in front of the neonate 's head .
one of the parents may be present during the experimental session , sitting on an armchair .
because of our focus on language - related processes , we have mostly worked with auditory stimuli
. these are presented at an appropriate intensity via two loudspeakers inside the experimental booth .
the fnirs machine and the control computer are placed outside the booth , to avoid undesired noise and heating in the testing room .
they choose whether to be inside the testing booth ( provided that they will remain still and silent throughout the session ) or outside , observing the infant 's behavior through the online video recording .
parents are informed of how fnirs functions , and they sign a consent form after they have understood how the experiment works and all their questions have been answered .
a long tradition in developmental research has used behavioral methods such as the high - amplitude sucking paradigm for tackling many infant speech perception questions .
thus , during our first experiences with fnirs , we aimed to determine whether previous results obtained with these behavioral methods could be reproduced . while replicating previous findings , the fnirs studies aimed to provide a link between behavioral observations and their underlying brain mechanisms .
our very first study assessed the specialization of the brain to process speech stimuli at birth . in healthy adult participants ,
the left - hemisphere dominance for speech is a well - known fact supported by a wealth of clinical , behavioral , and brain imaging studies .
the question that arose among developmental cognitive scientists was whether the left and right hemispheres are equal in function at birth and then specialized through experience , or whether at birth both hemispheres are already predisposed to process distinct types of stimuli .
subsequent research with young infants suggested that the second alternative was the most probable ( segalowitz and chapman , 1980 ; best , 1988 ; bertoncini et al .
many researchers used indirect behavioral measures to assess a diversity of questions : for instance , bertoncini et al .
( 1989 ) tested 2-weeks - old infants with a dichotic listening technique during a high - amplitude sucking experimental session .
two stimuli , a syllable and a musical stimulus , were presented simultaneously to the infant , one in each ear .
when infants listened to repeated syllables with their right ear , they displayed a change in behavior when a different syllable was presented , which was interpreted as a discrimination response .
instead , no discrimination occurred if infants listened to the linguistic material with their left ear .
( 2003 ) used fnirs to investigate the patterns of brain activation in response to auditory stimulation in full - term neonates .
their study thus provided a direct measure to assess hemispheric specialization while participants listened to normal speech , backward speech , or silence .
( 2003 ) found that the newborn left - hemisphere shows greater hemodynamic activity in response to normal speech than to backward speech or silence .
in addition , no area of the right hemisphere showed differential activation when contrasting forward and backward speech .
first , their study supported the notion of an early left - hemisphere specialization for speech , a fact intrinsically related to the emergence of the language faculty .
second , it validated fnirs as a technique capable of both replicating previous behavioral results and enriching them by providing direct measurements of the activity in left and right perisylvian areas when processing speech . being a relatively young technique ( see wolf et al . , 2007 , for a history of near - infrared spectroscopy techniques ) , fnirs must be evaluated also in terms of the reliability of the provided measures . in this respect ,
for instance , plichta et al . ( 2006 ) reported a high reproducibility at the group level , but not when re - testing single participants .
( 2003 ) have been successfully reproduced in our laboratory in several occasions after the original work .
below we present in detail the two main testing protocols that we use in our research with neonates . we started our exploration of cognitive core language acquisition mechanisms by borrowing block designs from the fmri tradition .
newborns are presented with sets of stimuli ( blocks ) corresponding to the different experimental conditions .
the duration of the blocks and their content vary according to the purposes of each study .
for example , pea et al . ( 2003 ) presented neonates with continuous stimuli : backward or forward infant - directed utterances in blocks of 15 s of duration . instead , in the gervain et al .
( 2008 ) studies , blocks were composed of 10 discrete items separated by pauses of varying length ( 0.51.5 s ) , yielding blocks of about 18 s. there were two kinds of blocks , which were presented in an interleaved fashion , avoiding the presentation of more than two consecutive blocks of the same condition ( figure 3a ) . from the first published study , our group used and continued using a variable separation of blocks ( 2535 s ) to avoid the effects of spontaneous oscillations frequently detected in fnirs recordings ( diehl et al . ,
for the statistical analyses we usually consider oxyhemoglobin or total hemoglobin concentration changes during the time window spanning from 10 to 20 s after the onset of stimulation , which roughly coincides with the plateau of the hemodynamic response . in each experimental block a given channel is rejected either because of movement artifact that is , if the hemodynamic signal shows a variation per unit of time above a given threshold level or saturation of the optical channel due to displacement of the probes .
only infants with a minimum amount of accepted block - channel pairs are further considered .
each rectangle represents a block , consisting in either continuous auditory stimulation or a series of discrete sounds separated by short pauses of about 1 s. consecutive blocks are separated by silent intervals of varying duration .
( a ) the block design consists in the intermixed presentation of n blocks belonging to each of the conditions x and y. ( b ) the familiarization - recognition design consists in a familiarization phase ( f ) in which all neonates listen to n blocks of stimulation . after a silent retention interval
here , half of the group of infants listens to m stimuli blocks of the same kind ( s ) of the familiarization , whereas the other half listens to a different kind of stimuli ( d ) .
block designs are useful to contrast brain responses to two or more experimental conditions ( e.g. , backward speech , forward speech , silence ) .
one advantage is that one can compare conditions without the need of additional ad hoc control groups .
however , a trade - off between number of contrasts and length of the experimental session should be considered , because the longer the session , the higher is the probability of the infant becoming fussy , and therefore of rejection .
it is important to highlight that experiments using this design have proven sensible not only for the study of prosodic or acoustic properties , but also for assessing learnability of specific structures in the first days of life : gervain et al .
( 2008 ) examined newborns ability to learn and detect repetition patterns ( e.g. , words like mubaba , penana ) , showing that these sequences evoke greater activation in specific areas of the newborn brain as compared to non - repeating sequences ( e.g. , words like mubage ,
behavioral methods have delivered very useful data that increased our understanding of cognitive development and language acquisition .
the habituation - dishabituation method was the most frequently used to test neonates , yielding important findings that advanced our knowledge of how infants acquire language . among
many other colleagues that applied this paradigm , we highlight the work by eimas et al .
( 1971 ) , the cardinal study who first discovered that 1- and 4-month - olds distinguish phonemes that differ by one feature , for example [ b ] from [ p ] or [ b ] from [ d ] .
furthermore , some years later kuhl ( 1983 ) explored how infants categorize syllables despite the variability of the speech stimuli , and mehler et al .
( 1978 ) discovered that newborns recognize their own mother 's voice ( see also jusczyk , 1997 , for an extended review on infant 's speech - processing studies based on behavioral methods ) . notwithstanding the important findings obtained with the habituation - dishabituation paradigm , the studies using it suffered from the aforementioned problems of behavioral methods such as high rejection rate .
moreover , using only behavioral paradigms restricted investigations to discrimination capacities mostly , leaving practically unattended other cognitive functions equally important for language acquisition .
for instance , very little is known about the memory capacities of neonates , in spite of the great progress that cognitive scientists and neuroscientists have made in identifying brain regions underlying memory processes in adults ( e.g. , nyberg and cabeza , 2000 ; baddeley et al . , 2009 ) .
one of the most important challenges of fnirs is to address questions that are difficult to assess behaviorally .
benavides - varela , gmez , bion , macagno , peretz and mehler ( in preparation ) adapted a habituation - deshabituation paradigm for testing newborn 's memory for speech sounds with fnirs , which could also provide indications of neural activity associated with encoding and recognition at birth .
benavides - varela and colleagues tested neonates between 2 and 5 days old in a recognition memory task .
newborns listened to a single cvcv word repeated for 10 blocks ( each block contained six identical words ) for a total of 6 min of exposure ( see figure 3b ) .
a 2-min - long silent pause separated the familiarization and test phases , providing a means of tapping infants memory .
after the pause , the same word of the familiarization phase was presented to half of the infants , whereas the other half listened to a novel word .
differential responses in hemoglobin concentration between the two groups of newborns would indicate that the familiar stimulus is remembered .
results were auspicious : participants hearing a novel word showed higher relative concentrations of oxyhemoglobin in the first block of the test phase , as compared to neonates who heard again the familiar word .
fnirs revealed these differences in a bilaterally distributed network , involving temporo - parietal and anterior areas .
these results open a field of possibilities for studying and better understanding the development of early memory capacities , and represent a promising step forward with respect to behavioral approaches . the use of fnirs has the advantage that the experimental session can be shortened or lengthened to study important variables such as amount of exposure or long - lasting retention .
this represented a problem in behavioral studies , because infants often fall asleep during long silent pauses , or because the behavioral responses of the control groups may become noisy ( see jusczyk et al . ,
furthermore , the findings of benavides - varela and colleagues suggest that 6 min of familiarization(see footnote 4 ) are enough for newborns to form a lasting representation of the presented word . this exposure time is considerably shorter than the ones used in comparable behavioral studies .
the usual implementation of the habituation - dishabituation procedure requires the adoption of a criterion for shifting from habituation phase to test phase , depending on each infant 's behavior .
we point out that the fnirs version of this method allowed us to equate the amount of exposure each infant received in the familiarization phase .
this is particularly important in memory studies , in which the amount of exposure partly determines the robustness of the memory trace , and therefore of the recognition response .
finally , we stress the fact that the fnirs technique not only provides a yes / no answer to the experimental question at hand , but also informs us of the activation of several cortical areas .
we believe that this type of design will prove useful for studies aiming beyond infants discrimination abilities and preferences , representing an opportunity to track the time course of learning , and the encoding and recognition processes in newborns and infants at different stages of development .
we started our exploration of cognitive core language acquisition mechanisms by borrowing block designs from the fmri tradition .
newborns are presented with sets of stimuli ( blocks ) corresponding to the different experimental conditions .
the duration of the blocks and their content vary according to the purposes of each study .
for example , pea et al . ( 2003 ) presented neonates with continuous stimuli : backward or forward infant - directed utterances in blocks of 15 s of duration . instead , in the gervain et al .
( 2008 ) studies , blocks were composed of 10 discrete items separated by pauses of varying length ( 0.51.5 s ) , yielding blocks of about 18 s. there were two kinds of blocks , which were presented in an interleaved fashion , avoiding the presentation of more than two consecutive blocks of the same condition ( figure 3a ) . from the first published study , our group used and continued using a variable separation of blocks ( 2535 s ) to avoid the effects of spontaneous oscillations frequently detected in fnirs recordings ( diehl et al . , 1998 ) .
for the statistical analyses we usually consider oxyhemoglobin or total hemoglobin concentration changes during the time window spanning from 10 to 20 s after the onset of stimulation , which roughly coincides with the plateau of the hemodynamic response . in each experimental block a given channel is rejected either because of movement artifact that is , if the hemodynamic signal shows a variation per unit of time above a given threshold level or saturation of the optical channel due to displacement of the probes .
only infants with a minimum amount of accepted block - channel pairs are further considered .
each rectangle represents a block , consisting in either continuous auditory stimulation or a series of discrete sounds separated by short pauses of about 1 s. consecutive blocks are separated by silent intervals of varying duration .
( a ) the block design consists in the intermixed presentation of n blocks belonging to each of the conditions x and y. ( b ) the familiarization - recognition design consists in a familiarization phase ( f ) in which all neonates listen to n blocks of stimulation . after a silent retention interval
here , half of the group of infants listens to m stimuli blocks of the same kind ( s ) of the familiarization , whereas the other half listens to a different kind of stimuli ( d ) .
block designs are useful to contrast brain responses to two or more experimental conditions ( e.g. , backward speech , forward speech , silence ) .
one advantage is that one can compare conditions without the need of additional ad hoc control groups .
however , a trade - off between number of contrasts and length of the experimental session should be considered , because the longer the session , the higher is the probability of the infant becoming fussy , and therefore of rejection .
it is important to highlight that experiments using this design have proven sensible not only for the study of prosodic or acoustic properties , but also for assessing learnability of specific structures in the first days of life : gervain et al .
( 2008 ) examined newborns ability to learn and detect repetition patterns ( e.g. , words like mubaba , penana ) , showing that these sequences evoke greater activation in specific areas of the newborn brain as compared to non - repeating sequences ( e.g. , words like mubage , penaku ) .
behavioral methods have delivered very useful data that increased our understanding of cognitive development and language acquisition .
the habituation - dishabituation method was the most frequently used to test neonates , yielding important findings that advanced our knowledge of how infants acquire language . among
many other colleagues that applied this paradigm , we highlight the work by eimas et al .
( 1971 ) , the cardinal study who first discovered that 1- and 4-month - olds distinguish phonemes that differ by one feature , for example [ b ] from [ p ] or [ b ] from [ d ] . furthermore , some years later kuhl ( 1983 ) explored how infants categorize syllables despite the variability of the speech stimuli , and mehler et al .
( 1978 ) discovered that newborns recognize their own mother 's voice ( see also jusczyk , 1997 , for an extended review on infant 's speech - processing studies based on behavioral methods ) . notwithstanding the important findings obtained with the habituation - dishabituation paradigm , the studies using it suffered from the aforementioned problems of behavioral methods such as high rejection rate .
moreover , using only behavioral paradigms restricted investigations to discrimination capacities mostly , leaving practically unattended other cognitive functions equally important for language acquisition .
for instance , very little is known about the memory capacities of neonates , in spite of the great progress that cognitive scientists and neuroscientists have made in identifying brain regions underlying memory processes in adults ( e.g. , nyberg and cabeza , 2000 ; baddeley et al . , 2009 ) .
one of the most important challenges of fnirs is to address questions that are difficult to assess behaviorally . benavides - varela , gmez , bion , macagno , peretz and mehler ( in preparation ) adapted a habituation - deshabituation paradigm for testing newborn 's memory for speech sounds with fnirs , which could also provide indications of neural activity associated with encoding and recognition at birth . benavides - varela and colleagues tested neonates between 2 and 5 days old in a recognition memory task .
newborns listened to a single cvcv word repeated for 10 blocks ( each block contained six identical words ) for a total of 6 min of exposure ( see figure 3b ) .
a 2-min - long silent pause separated the familiarization and test phases , providing a means of tapping infants memory .
after the pause , the same word of the familiarization phase was presented to half of the infants , whereas the other half listened to a novel word .
differential responses in hemoglobin concentration between the two groups of newborns would indicate that the familiar stimulus is remembered .
results were auspicious : participants hearing a novel word showed higher relative concentrations of oxyhemoglobin in the first block of the test phase , as compared to neonates who heard again the familiar word .
fnirs revealed these differences in a bilaterally distributed network , involving temporo - parietal and anterior areas .
these results open a field of possibilities for studying and better understanding the development of early memory capacities , and represent a promising step forward with respect to behavioral approaches .
the use of fnirs has the advantage that the experimental session can be shortened or lengthened to study important variables such as amount of exposure or long - lasting retention .
this represented a problem in behavioral studies , because infants often fall asleep during long silent pauses , or because the behavioral responses of the control groups may become noisy ( see jusczyk et al . , 1995 ) .
furthermore , the findings of benavides - varela and colleagues suggest that 6 min of familiarization(see footnote 4 ) are enough for newborns to form a lasting representation of the presented word . this exposure time is considerably shorter than the ones used in comparable behavioral studies .
the usual implementation of the habituation - dishabituation procedure requires the adoption of a criterion for shifting from habituation phase to test phase , depending on each infant 's behavior .
we point out that the fnirs version of this method allowed us to equate the amount of exposure each infant received in the familiarization phase .
this is particularly important in memory studies , in which the amount of exposure partly determines the robustness of the memory trace , and therefore of the recognition response .
finally , we stress the fact that the fnirs technique not only provides a yes / no answer to the experimental question at hand , but also informs us of the activation of several cortical areas .
we believe that this type of design will prove useful for studies aiming beyond infants discrimination abilities and preferences , representing an opportunity to track the time course of learning , and the encoding and recognition processes in newborns and infants at different stages of development .
developmental scientists face the challenge of devising reliable methodologies to assess cognitive capacities in neonates .
the incorporation of the fnirs imaging technique promotes our understanding of early language acquisition and memory capacities .
we hope that the successful experiences with fnirs in several laboratories ( e.g. , pea et al . , 2003 ; bortfeld et al . , 2007 ;
, 2009 ; lloyd - fox et al . , 2010 ; obrig et al . ,
2010 ) will encourage further exploitation of this valuable tool for studying early human development .
an important line of methodological progress is focusing on designs that combine different paradigms ( behavioral and neuroimaging ) or techniques ( e.g. , fnirs and electroencephalography ) , either across different experimental sessions or in simultaneous recordings .
the use of two or more techniques has the potential to provide complementary information about the functioning of the newborn 's brain . a pioneer study by telkemeyer et al .
( 2009 ) has already used nirs and eeg concurrently to test auditory processing in newborns .
we are confident that such adaptations will play a central role in improving our knowledge about human innate cognitive capacities .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | the measurement of newborns brain hemodynamic activity has improved our understanding of early cognitive processes , in particular of language acquisition . in this paper , we describe two experimental protocols adapted to study neonates speech - processing capacities using functional near - infrared spectroscopy ( fnirs ) : the block design and the familiarization - recognition design .
we review some of their benefits and disadvantages , and refer to research issues that can be explored by means of these protocols .
we also illustrate the use of the two experimental designs through representative fnirs studies that reveal specific patterns of activation of the newborn brain during speech perception , learning of repetition structures , and word recognition . | Introduction
Equipment and Experimental Setup
Reliability of the Method
Experimental Protocols
Block design
Familiarization-recognition design
Concluding Remarks
Conflict of Interest Statement | in addition , most of the current neuroimaging techniques are impractical for testing healthy young participants because these techniques a ) use machines that produce high levels of acoustic noise , b ) require the application of liquids or gel in the newborn 's head , or c ) have very low tolerance to facial and body movement . functional near - infrared spectroscopy ( fnirs ) is an imaging technique that has been employed for clinical purposes ( wolf et al . it is regarded as one of the most appropriate to study language faculties and cognitive capacities in the newborn 's brain ( see aslin and mehler , 2005 ; minagawa - kawai et al . moreover , we have observed that the number of participants providing useful fnirs data is very high compared to many behavioral methods used to investigate neonatal cognition : in typical behavioral paradigms used to study auditory capacities , rejection rate may reach beyond 50% . one reason for this is that , in fnirs studies regarding auditory perception , participants need not be excluded because of behaviors like falling asleep or refusing the pacifier , very common in neonates . functional near - infrared spectroscopy works by measuring changes in cerebral blood flow volume and oxygen saturation using optical means . probes containing the emitters ( red marks ) and detectors ( blue marks ) of near - infrared light , and their positioning on the neonate 's head . because of our focus on language - related processes , we have mostly worked with auditory stimuli
. , 2007 , for a history of near - infrared spectroscopy techniques ) , fnirs must be evaluated also in terms of the reliability of the provided measures . ( a ) the block design consists in the intermixed presentation of n blocks belonging to each of the conditions x and y. ( b ) the familiarization - recognition design consists in a familiarization phase ( f ) in which all neonates listen to n blocks of stimulation . , words like mubaba , penana ) , showing that these sequences evoke greater activation in specific areas of the newborn brain as compared to non - repeating sequences ( e.g. , words like mubage ,
behavioral methods have delivered very useful data that increased our understanding of cognitive development and language acquisition . ( 1978 ) discovered that newborns recognize their own mother 's voice ( see also jusczyk , 1997 , for an extended review on infant 's speech - processing studies based on behavioral methods ) . a 2-min - long silent pause separated the familiarization and test phases , providing a means of tapping infants memory . after the pause , the same word of the familiarization phase was presented to half of the infants , whereas the other half listened to a novel word . differential responses in hemoglobin concentration between the two groups of newborns would indicate that the familiar stimulus is remembered . these results open a field of possibilities for studying and better understanding the development of early memory capacities , and represent a promising step forward with respect to behavioral approaches . the use of fnirs has the advantage that the experimental session can be shortened or lengthened to study important variables such as amount of exposure or long - lasting retention . this is particularly important in memory studies , in which the amount of exposure partly determines the robustness of the memory trace , and therefore of the recognition response . finally , we stress the fact that the fnirs technique not only provides a yes / no answer to the experimental question at hand , but also informs us of the activation of several cortical areas . ( a ) the block design consists in the intermixed presentation of n blocks belonging to each of the conditions x and y. ( b ) the familiarization - recognition design consists in a familiarization phase ( f ) in which all neonates listen to n blocks of stimulation . however , a trade - off between number of contrasts and length of the experimental session should be considered , because the longer the session , the higher is the probability of the infant becoming fussy , and therefore of rejection . , words like mubaba , penana ) , showing that these sequences evoke greater activation in specific areas of the newborn brain as compared to non - repeating sequences ( e.g. behavioral methods have delivered very useful data that increased our understanding of cognitive development and language acquisition . a 2-min - long silent pause separated the familiarization and test phases , providing a means of tapping infants memory . the use of fnirs has the advantage that the experimental session can be shortened or lengthened to study important variables such as amount of exposure or long - lasting retention . this is particularly important in memory studies , in which the amount of exposure partly determines the robustness of the memory trace , and therefore of the recognition response . finally , we stress the fact that the fnirs technique not only provides a yes / no answer to the experimental question at hand , but also informs us of the activation of several cortical areas . the incorporation of the fnirs imaging technique promotes our understanding of early language acquisition and memory capacities . the use of two or more techniques has the potential to provide complementary information about the functioning of the newborn 's brain . | [
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] |
empowerment appears to mainly affect the use of prenatal care services and with the promotion of empowerment during pregnancy , we can improve mothers reproductive health ( 1 ) .
it is necessary for all women to make proactive decisions about their health and pregnancy in order to demonstrate their many capabilities and control their fate ( 2 ) .
empowerment during pregnancy helps women attain the necessary skills to correctly approach problems that may emerge .
these skills would also be useful in future situations , particularly during the maternal period ( 3 ) .
the international conference on population and development ( icpd ) in 1994 emphasized the maternal empowerment as an essential component for reaching gestational and population health ( 4 ) .
one of the most critical responsibilities of governments is removing women s empowerment barriers , and improving gestational health by any approach possible . also , the icpd in beijing ( 1995 ) considered the empowerment of women as a necessary component for reaching sustainable development in all aspects of life , and improving gestational health ( 4 , 5 ) .
some research demonstrates that mothers with higher skills and competency pass their pregnancy period with fewer problems and display a higher performance when taking care of and raising their children after labor ( 68 ) . such greater empowerment , consequently results in the establishment of a stronger family ( 9 ) .
not many studies regarding empowerment during pregnancy are conducted in iran . according to a study
, there was relationship between female empowerment and gestational behavior , and researchers emphasized that increasing women s potential and capabilities through advancing their knowledge level , improving their training levels , and emphasizing their empowerment are at the forefront of national issues ( 6 ) . in another study that examined the effects of an empowerment - based educational package on prenatal empowerment , results showed that educational programs can increase pregnant mothers empowerment ( 7 ) .
in addition , researchers in two studies on iranian womens empowerment concluded that social factors and training are necessary tools for empowerment , and paying attention to women s empowerment is important ( 10 , 11 ) .
it can be accepted that these studies were not sufficient and the predictor of prenatal empowerment in iran was unknown .
but in some african countries , research has been conducted on prenatal empowerment ( 1 , 12 , 13 ) . the cultural context of these countries differs from iran , and empowerment depends on the context of the respective countries ( 14 ) . maternal mortality ratio ( mmr ) in certain developing countries can be reduced via the promotion of women s empowerment during pregnancy ( 9 , 15 ) .
lack of women s empowerment is the main cause of maternal mortality and without empowerment , the goal of reducing the rate of maternal mortality can not be reached ( 16 ) .
the annual reduction rate of mmr in the islamic republic of iran in 19902000 was 8.9% in comparison with 3% between 20052015 , showed that the reduction in mmr remained near steady in the following years ( 17 ) .
it has been shown that there is a relationship between socioeconomic inequalities and mmr in iran ( 18 ) . particularly in certain ethnic groups , such as the turkman in golestan province ,
the mmr is 2.2 times higher than fars ethnicity group ( 19 ) . in this situation ,
when a husband s knowledge is inferior , and the mother s capabilities of making decisions for their health care are hindered , it causes an increase in maternal mortality ( 20 ) . in order to solve this problem ,
we need to evaluate the predictors of prenatal empowerment to empower pregnant women by health care providers and encourage them to make their own decisions regarding their health during pregnancy ( 15 ) .
this cross - sectional study was conducted to evaluate pregnancy empowerment predictors while considering the importance of maternal empowerment in the context of iran .
this cross - sectional study was conducted in golestan province , northwestern iran , an area which has divers ethnicity .
first an exact number of urban medical centers and clinics were listed ; then golestan were divided into five different regions ( including central , eastern , western , southern and northern regions ) and two health centers from each region ( totally 10 centers ) were selected randomly . to determine the sample size ,
this data were entered into the spss software , and then using pass software , and correlation assessment between empowerment dimensions in kameda pregnancy empowerment and the spritzer psychological empowerment in the first sample , and considering =0.05 , =0.2 and 0.80 power , finally a minimum sample size of 141 samples was estimated . with this information , 180nulliparous pregnant mothers , or mothers who had delivered during the last two months were selected .
after the exclusion of 19 participants , either because of lack of interest to participation , illiteracy or being foreign nationals , 161 mothers personally completed questionnaires during three months in spring 2014 .
questionnaires were then checked by a midwife , and returned to the mothers if any questions remained unanswered , until completion .
sense of spiritual support was quantified , based on four questions on the subject of spirituality : trust in god in all things , acceptance of the judgment of god , appealing to imams to fulfill wishes , and belief in god through hardship .
ownership score was measured by the quantity of the mothers property and belongings , for example house , car and home were each rated as n=0 and y=1 .
an iranian questionnaire , was used in this survey , for measurement of socio - political , educational , and mental - financial predictors of empowerment in pregnant mothers . this 32-item questionnaire s validity and reliability , were investigated by authors of this study in 2015 .
the score of this scale was between 32 and 128 , and the average content validity index ( s - cvi / ave ) of this scale was estimated as 91.34% .
the internal consistency of the scale was determined as 93.3% using the -cronbach coefficient , which was higher than 70% for all sub - scales ( 21 ) . to study empowerment predictors in pregnancy ,
the following requirements are considered : the mean error value must be zero and the errors should have a constant variance .
the errors must be independent and durbin - watson ( dw ) statistics must be in the range of 1.52.5 , and the errors must have normal distribution .
independent variables are linearly non - correlated , so that collinearity tests must be performed on them in a way that vif<10 and tolerance>0.1 ( 22 ) . to investigate three groups of empowerment predictors , first , each group of predictors should be separately computed and then the overall empowerment predictors are to be analyzed .
the permission for this study was issued after the study was approved by research committee and regional committee of ethics on 25.01.2014 .
the study protocol meets the ethical guidelines of the 1975 declaration of helsinki and all ethical considerations such as voluntarily participation with complete awareness , and consenting for the study were practiced .
mothers were reassured about the privacy of their data and they were informed that only the researcher had access to collected data , and personal information ( name and surname ) , were entered as code numbers into software , and all data were securely protected .
this cross - sectional study was conducted in golestan province , northwestern iran , an area which has divers ethnicity .
first an exact number of urban medical centers and clinics were listed ; then golestan were divided into five different regions ( including central , eastern , western , southern and northern regions ) and two health centers from each region ( totally 10 centers ) were selected randomly . to determine the sample size ,
this data were entered into the spss software , and then using pass software , and correlation assessment between empowerment dimensions in kameda pregnancy empowerment and the spritzer psychological empowerment in the first sample , and considering =0.05 , =0.2 and 0.80 power , finally a minimum sample size of 141 samples was estimated . with this information , 180nulliparous pregnant mothers , or mothers who had delivered during the last two months were selected .
after the exclusion of 19 participants , either because of lack of interest to participation , illiteracy or being foreign nationals , 161 mothers personally completed questionnaires during three months in spring 2014 .
questionnaires were then checked by a midwife , and returned to the mothers if any questions remained unanswered , until completion .
sense of spiritual support was quantified , based on four questions on the subject of spirituality : trust in god in all things , acceptance of the judgment of god , appealing to imams to fulfill wishes , and belief in god through hardship .
ownership score was measured by the quantity of the mothers property and belongings , for example house , car and home were each rated as n=0 and y=1 .
an iranian questionnaire , was used in this survey , for measurement of socio - political , educational , and mental - financial predictors of empowerment in pregnant mothers . this 32-item questionnaire s validity and reliability , were investigated by authors of this study in 2015 .
the score of this scale was between 32 and 128 , and the average content validity index ( s - cvi / ave ) of this scale was estimated as 91.34% .
the internal consistency of the scale was determined as 93.3% using the -cronbach coefficient , which was higher than 70% for all sub - scales ( 21 ) .
to study empowerment predictors in pregnancy , the linear regression method was applied . to perform a linear regression analysis ,
the following requirements are considered : the mean error value must be zero and the errors should have a constant variance .
the errors must be independent and durbin - watson ( dw ) statistics must be in the range of 1.52.5 , and the errors must have normal distribution .
independent variables are linearly non - correlated , so that collinearity tests must be performed on them in a way that vif<10 and tolerance>0.1 ( 22 ) . to investigate three groups of empowerment predictors , first , each group of predictors should be separately computed and then the overall empowerment predictors are to be analyzed .
the permission for this study was issued after the study was approved by research committee and regional committee of ethics on 25.01.2014 .
the study protocol meets the ethical guidelines of the 1975 declaration of helsinki and all ethical considerations such as voluntarily participation with complete awareness , and consenting for the study were practiced .
mothers were reassured about the privacy of their data and they were informed that only the researcher had access to collected data , and personal information ( name and surname ) , were entered as code numbers into software , and all data were securely protected .
of the pregnant mothers , 92.5% were gravid 1 and most of families ( 87% ) lived independently with their husbands ( table 1 ) .
the average empowerment for three dimensions is presented in table 2 . before importing variables in the regression model ,
the requirements of linear regression was considered ( tables 3 , 4 ) , and the correlation of all variables should be assessed , so only variables that correlate with dependent variable are imported to the regression model . thus , the correlation matrix of the dependent and independent variables is studied initially . in this paper ,
the durbin - watson statistics and mean error value were calculated , where these values were suitable . subsequently , the linear regression model was performed .
initially , variables including mother s age , marital age , employment of mothers , participation in prenatal education classes , marital satisfaction score , and sense of spiritual support score , as correlated factors in educational empowerment , are imported to the linear regression model .
the results show that the participation in pregnancy classes , spiritual support and mother s age , are predictors of educational empowerment indexes ( table 3 ) .
mothers with higher age , who participated in prenatal education and received better emotional support , indicate higher educational empowerment .
in other words , the educational empowerment formula of the pregnant mothers with constant of 0.998 is as follows : educational dimension of empowerment=0.998 + 0.479 ( participation in prenatal education classes ) + 0.347 ( sense of spiritual support ) + 0.023 ( mother s age ) .
variables including mother s age , marital age , marital life length , fars ethnicity , mother and father literacy level , living status , ownership score , participation in prenatal educational classes , employment of mother and ownership of a property by credit arrangement , are imported to the linear regression model as correlated factors in autonomy dimension of empowerment .
the results show that marital life length , employment of mothers , and living in her own home are as a predictors of mental - financial independent empowerment indexes ( table 3 ) .
mothers of senior age and longer marital period , and those who are living in their own private home indicate higher empowerment in independency and autonomy . in other words ,
the autonomy dimension of empowerment formula in pregnant mothers with constant of 1.426 is as follows : autonomy dimension of empowerment=1.426 + 0.305(employment of mother ) + 0.178(living in own home ) + 0.067(marital life length ) + 0.040(marriage age ) .
the linear regression model was used to evaluate socio - political variables including : mother s age , marriage age , employment of mother , mother s level of literacy , and sense of spiritual support .
and employment of mother are predictors in socio - political empowerment ( table 3 ) .
the study indicates that mothers with higher marriage age and high sense of spiritual support who are employed , have higher socio - political empowerment . in other words ,
the socio - political dimension of empowerment formula in pregnant mothers with constant of 2.122 is as follows : socio - political dimension of empowerment=1.554 + 0.151(sense of spiritual support ) + 0.147(employment of mother ) + 0.016(mother marriage age ) .
maternal age , the age of marriage , the length of marital life , employment of mother , mother and father s literacy level , spiritual support and participation in prenatal educational classes were variables that correlated with the total empowerment score . to evaluate the total empowerment in pregnant women the data was imported to the linear regression model .
the results show that the participation in pregnancy classes , spiritual support
, marriage age and employment of mother are important predictors of total empowerment indexes ; and mothers with higher marriage age and those who were employed and participated in prenatal educational classes and have high spiritual support , indicate higher total empowerment ( table 3 ) . in other words ,
the total empowerment formula in pregnant mothers with constant of 15.253 is as follows : total empowerment=15.253 + 2.537(employment of mother ) + 2.515(participation in prenatal education classes ) + 2.307(sense of spiritual support ) + 0.248(marriage age ) .
variables including the level of mother s education , autonomy and socio - political empowerment were imported to the linear regression model .
the results show that educational empowerment has the greatest impact on total empowerment ( table 3 ) ; therefore , total empowerment formula in pregnant mothers with constant of 0.409 is as follows : total empowerment=0.409 + 4.886(educational dimension ) + 3.022(autonomy empowerment ) + 2.987(socio - political empowerment ) .
of the pregnant mothers , 92.5% were gravid 1 and most of families ( 87% ) lived independently with their husbands ( table 1 ) .
the average empowerment for three dimensions is presented in table 2 . before importing variables in the regression model ,
the requirements of linear regression was considered ( tables 3 , 4 ) , and the correlation of all variables should be assessed , so only variables that correlate with dependent variable are imported to the regression model . thus , the correlation matrix of the dependent and independent variables is studied initially . in this paper ,
the durbin - watson statistics and mean error value were calculated , where these values were suitable . subsequently , the linear regression model was performed .
initially , variables including mother s age , marital age , employment of mothers , participation in prenatal education classes , marital satisfaction score , and sense of spiritual support score , as correlated factors in educational empowerment , are imported to the linear regression model .
the results show that the participation in pregnancy classes , spiritual support and mother s age , are predictors of educational empowerment indexes ( table 3 ) .
mothers with higher age , who participated in prenatal education and received better emotional support , indicate higher educational empowerment .
in other words , the educational empowerment formula of the pregnant mothers with constant of 0.998 is as follows : educational dimension of empowerment=0.998 + 0.479 ( participation in prenatal education classes ) + 0.347 ( sense of spiritual support ) + 0.023 ( mother s age ) .
variables including mother s age , marital age , marital life length , fars ethnicity , mother and father literacy level , living status , ownership score , participation in prenatal educational classes , employment of mother and ownership of a property by credit arrangement , are imported to the linear regression model as correlated factors in autonomy dimension of empowerment .
, employment of mothers , and living in her own home are as a predictors of mental - financial independent empowerment indexes ( table 3 ) . mothers of senior age and longer marital period , and those who are living in their own private home indicate higher empowerment in independency and autonomy . in other words ,
the autonomy dimension of empowerment formula in pregnant mothers with constant of 1.426 is as follows : autonomy dimension of empowerment=1.426 + 0.305(employment of mother ) + 0.178(living in own home ) + 0.067(marital life length ) + 0.040(marriage age ) .
the linear regression model was used to evaluate socio - political variables including : mother s age , marriage age , employment of mother , mother s level of literacy , and sense of spiritual support .
and employment of mother are predictors in socio - political empowerment ( table 3 ) .
the study indicates that mothers with higher marriage age and high sense of spiritual support who are employed , have higher socio - political empowerment . in other words ,
the socio - political dimension of empowerment formula in pregnant mothers with constant of 2.122 is as follows : socio - political dimension of empowerment=1.554 + 0.151(sense of spiritual support ) + 0.147(employment of mother ) + 0.016(mother marriage age ) .
maternal age , the age of marriage , the length of marital life , employment of mother , mother and father s literacy level , spiritual support and participation in prenatal educational classes were variables that correlated with the total empowerment score . to evaluate the total empowerment in pregnant women the data was imported to the linear regression model .
the results show that the participation in pregnancy classes , spiritual support , marriage age and employment of mother are important predictors of total empowerment indexes ; and mothers with higher marriage age and those who were employed and participated in prenatal educational classes and have high spiritual support , indicate higher total empowerment ( table 3 ) . in other words ,
the total empowerment formula in pregnant mothers with constant of 15.253 is as follows : total empowerment=15.253 + 2.537(employment of mother ) + 2.515(participation in prenatal education classes ) + 2.307(sense of spiritual support ) + 0.248(marriage age ) .
variables including the level of mother s education , autonomy and socio - political empowerment were imported to the linear regression model .
the results show that educational empowerment has the greatest impact on total empowerment ( table 3 ) ; therefore , total empowerment formula in pregnant mothers with constant of 0.409 is as follows : total empowerment=0.409 + 4.886(educational dimension ) + 3.022(autonomy empowerment ) + 2.987(socio - political empowerment ) .
as the result of this study shows , the level of empowerment in women during pregnancy will be predictable , based on three main factors : a ) the individual characteristic of the pregnant woman , such as , mother s age , marriage age , the length of marriage ; b ) the socio - economic status of the mother , for example , mother s employment , living in her own home ; and c ) the educational factors , including , participation in prenatal education classes and having spiritual support .
the employment , learning professional skills and financial stability are the common elements that most researchers agreed on ( 10 ) . in fact ,
studies showed that having higher education helps women to reach a better financial and legal stability that effect on their level of health , and a direct effect in their empowerment ( 23 ) .
renkert ( 2001 ) believed that to progressively increase the level of education would cause a higher independency in decision making and individual empowerment ( 8) . in this study
, there is a high correlation between education and employment , and these two factors together should not be inserted in regression model with high correlation ( 22 ) .
so , due to the fact that higher education increases the chance of employment of women , we include employment in our linear regression model .
the finding showed that employment plays the predicator role in all empowerment dimensions except education .
this study shows that with the increase in mother employment , the marriage age , socio - political , autonomy and total empowerment increase too .
surely , employment and learning professional skills have more significant effect on economical dimensions in the empowerment of women .
in fact , women s employment helps their social , political , and psychological dimensions of their empowerment ( 11 ) . financial resources such as ,
having a job and monthly income are the most important elements of women s empowerment ( 10 , 11 ) . as table 3
shows , living in their own home , and ownership , predict the autonomy factor of empowerment .
if we consider home ownership as a predictor for a better financial status , women who live in their own home have higher empowerment .
ahmad ( 2010 ) believed that in developing countries , to improve mothers health , the economic , social and educational situation of mothers must improve ( 15 ) .
there were relations between the educational - economical situation and decision making power for seeking health care ( 15 , 24 ) .
another study performed in iran , considered the socio - economical factors as the determinants of empowerment ( 6 ) .
this study considered mother employment as the main factor in mother and child health , and as the predicator of mother s mental - financial autonomy empowerment ( 13 ) .
ahmad ( 2010 ) believes that the women with more empowerment have more autonomy and they are healthier ( 15 ) .
decision making power and control over her own life are the elements of autonomic empowerment , which are affected directly from mothers employment ( 15 , 25 ) . in many studies , employment , economical status and wealth and ownership of properties
mothers age and wealth have been the main elements that effect on the presence of their spouse at prenatal care visits .
ahmad found out in his studies that there was a positive and strong relation between key elements of social economical situation and health of mothers in 31 developing countries , which includes 20% of world population ( 15 ) . in this study
we observed that age of marriage predicts socio - political , autonomy and total empowerment . a mothers age predicts educational empowerment and the length of marital life predicts autonomy empowerment .
there was relation between economical situation , level of education and mother empowerment which was controlled by mother s age ( 15 ) . in a study conducted in iran , mother s age , marriage age , age at first pregnancy , and the educational level ,
in fact it seems that age has a fundamental effect on mother s empowerment ( 15 ) .
other elements such employment , economical situation , spiritual and educational status are the following effective factors in women s empowerment . in addition
, education is the most important empowerment tool . as it improves women s knowledge , skills , and self - confidence , which consequently result in female participation in the developmental , process ( 4 ) .
in this study , mothers participation in prenatal educational classes has been considered as a predictor of educational and total empowerment . since educational empowerment
has the highest coefficient in regression model and on total empowerment , its plausible to promote educational and total empowerment with training and educational programs that emphasize on empowering mothers .
the effect of training programs on mother empowerment has been observed in jahdi s study , which showed that training is motivational and the actuating force in empowering women during pregnancy .
and it is the first major guide line in planning and engaging programs to empower women .
pregnancy training empowers women to understand and adapt to physical and behavioral changes during pregnancy . also , group training under supervision of health care provider , offers mothers the opportunity to listen and learn from others experiences and knowledge during class discussions .
group training also promotes bonding and friendship among expectant mothers , and it increase social and self - care empowerment among them ( 7 ) . in a study on relation between empowerment and pregnant behavior , it suggested that the training is a determinant element of empowerment . and it considered that countries need to pay a special attention to increase women s knowledge and training , in order to empower women and to increase their capability and power over their pregnancy ( 6 ) .
a study on195 women that evaluated the effect of monitoring women empowerment by health care providers , emphasized on the importance of education during pregnancy and the role of health care providers in educating women during pregnancy ( 29 ) .
public training is confirmed as an effective element in empowerment in other studies too ( 1 , 10 ) .
as it mentioned before , the finding of this study confirmed the effect of spiritual support in women empowerment during pregnancy .
kidwai ( 2013 ) believed that there is a relation between spirituality of a person and the level of distress she may suffer ; and the course of this relation ( positive or negative ) related to a person s cultural and personal background ( 30 ) .
in fact spiritual belief and belief in god gives people a positive or negative perspective toward dealing with distress .
people who have positive attitudes and have believed in god have more mental and emotional comfort ( 3133 ) . believing in as high a power as god , empowers the believers and increases their coping skills ( 34 , 35 ) . in iran ,
due to islamic religious believes people who find themselves under the grace of god , have more comfort and have less tension and stress .
in this study , three predictors of mother s empowerment , including socio - economic , autonomy and educational status have been surveyed by a scale which has been designed particularly for nulliparous iranian pregnant women . but to plan a comprehensive educational program that covers all aspects of empowerment during pregnancy for all groups of pregnant women , it is important to survey multiparous pregnant women , as well as high risk pregnant women using a larger sample size .
it can be concluded from this study that employment and the age of marriage are the main predicators of empowerment .
since employed women tend to marry in more mature ages and with a better socio - economic situation , they have more empowerment during pregnancy . according to the findings of this study
so , it is necessary to have an accurate program which emphasize in training during pregnancy . in this program
we should be able to recognize mothers in need , and provide a special training program for them to increase their health and empowerment during pregnancies , which guide them to a safer pregnancy and motherhood .
our study showed that mothers who find themselves under god s protection and feel spiritual support , have more empowerment .
thus , having spiritual support and participating in pregnancy classes are the two important predictors of educational empowerment and empowerment in total . | introductionconsidering that empowering expectant mothers is an important issue to maintain a healthy pregnancy , this study was conducted to evaluate the predictors of empowerment among iranian pregnant women.methodsthis cross sectional study was conducted in golestan , north of iran in 2015 .
a total number of 161 pregnant women were selected through random cluster sampling from urban health centers , using pass software .
the socio - political , educational , and mental - financial predictors of empowerment were measured using a self - structured questionnaire during pregnancy and was analyzed by a linear regression model using spss version 16.resultsthe findings of linear regression showed that educational dimension of empowerment had the highest coefficient in the regression model , on total empowerment ( eta standardized coefficient [ ]=0.696 with dw=1.830 and means error=0 ) .
the total empowerment score of pregnant women was controlled by individual factors such as the age of marriage ( -0.228 ) , employment ( -0.210 ) , and educational factors such as participation in prenatal education classes ( -0.246 ) , and moral issues such as sense of spiritual support ( -0.217).conclusionby recognizing and observing predictors of empowerment during pregnancy , health care providers can increase women s power over their pregnancy .
educational predictors of empowerment were the most important factors to empower women during pregnancy .
the objective of childbirth education classes , therefore , should shift from simply giving information to women , towards giving them appropriate knowledge in order to provide them with empowerment during pregnancy . | 1. Introduction
2. Material and Methods
2.1. Research design, setting and data collection
2.2. Empowerment Scale
2.3. Statistical analysis
2.4. Ethical considerations
3. Results
3.1. Demographic characteristic of participant
3.2. Educational Dimension of Empowerment
3.3. Autonomy (Mental-financial in dependency) Dimension of Empowerment
3.4. Socio-Political predictors of Empowerment
3.5. Total Empowerment score
3.6. Total Empowerment
4. Discussion
5. Limitations of the study
6. Conclusions | in order to solve this problem ,
we need to evaluate the predictors of prenatal empowerment to empower pregnant women by health care providers and encourage them to make their own decisions regarding their health during pregnancy ( 15 ) . this cross - sectional study was conducted to evaluate pregnancy empowerment predictors while considering the importance of maternal empowerment in the context of iran . an iranian questionnaire , was used in this survey , for measurement of socio - political , educational , and mental - financial predictors of empowerment in pregnant mothers . an iranian questionnaire , was used in this survey , for measurement of socio - political , educational , and mental - financial predictors of empowerment in pregnant mothers . before importing variables in the regression model ,
the requirements of linear regression was considered ( tables 3 , 4 ) , and the correlation of all variables should be assessed , so only variables that correlate with dependent variable are imported to the regression model . initially , variables including mother s age , marital age , employment of mothers , participation in prenatal education classes , marital satisfaction score , and sense of spiritual support score , as correlated factors in educational empowerment , are imported to the linear regression model . in other words , the educational empowerment formula of the pregnant mothers with constant of 0.998 is as follows : educational dimension of empowerment=0.998 + 0.479 ( participation in prenatal education classes ) + 0.347 ( sense of spiritual support ) + 0.023 ( mother s age ) . variables including mother s age , marital age , marital life length , fars ethnicity , mother and father literacy level , living status , ownership score , participation in prenatal educational classes , employment of mother and ownership of a property by credit arrangement , are imported to the linear regression model as correlated factors in autonomy dimension of empowerment . the linear regression model was used to evaluate socio - political variables including : mother s age , marriage age , employment of mother , mother s level of literacy , and sense of spiritual support . in other words ,
the socio - political dimension of empowerment formula in pregnant mothers with constant of 2.122 is as follows : socio - political dimension of empowerment=1.554 + 0.151(sense of spiritual support ) + 0.147(employment of mother ) + 0.016(mother marriage age ) . maternal age , the age of marriage , the length of marital life , employment of mother , mother and father s literacy level , spiritual support and participation in prenatal educational classes were variables that correlated with the total empowerment score . before importing variables in the regression model ,
the requirements of linear regression was considered ( tables 3 , 4 ) , and the correlation of all variables should be assessed , so only variables that correlate with dependent variable are imported to the regression model . initially , variables including mother s age , marital age , employment of mothers , participation in prenatal education classes , marital satisfaction score , and sense of spiritual support score , as correlated factors in educational empowerment , are imported to the linear regression model . in other words , the educational empowerment formula of the pregnant mothers with constant of 0.998 is as follows : educational dimension of empowerment=0.998 + 0.479 ( participation in prenatal education classes ) + 0.347 ( sense of spiritual support ) + 0.023 ( mother s age ) . variables including mother s age , marital age , marital life length , fars ethnicity , mother and father literacy level , living status , ownership score , participation in prenatal educational classes , employment of mother and ownership of a property by credit arrangement , are imported to the linear regression model as correlated factors in autonomy dimension of empowerment . the linear regression model was used to evaluate socio - political variables including : mother s age , marriage age , employment of mother , mother s level of literacy , and sense of spiritual support . maternal age , the age of marriage , the length of marital life , employment of mother , mother and father s literacy level , spiritual support and participation in prenatal educational classes were variables that correlated with the total empowerment score . to evaluate the total empowerment in pregnant women the data was imported to the linear regression model . as the result of this study shows , the level of empowerment in women during pregnancy will be predictable , based on three main factors : a ) the individual characteristic of the pregnant woman , such as , mother s age , marriage age , the length of marriage ; b ) the socio - economic status of the mother , for example , mother s employment , living in her own home ; and c ) the educational factors , including , participation in prenatal education classes and having spiritual support . and it considered that countries need to pay a special attention to increase women s knowledge and training , in order to empower women and to increase their capability and power over their pregnancy ( 6 ) . in this study , three predictors of mother s empowerment , including socio - economic , autonomy and educational status have been surveyed by a scale which has been designed particularly for nulliparous iranian pregnant women . but to plan a comprehensive educational program that covers all aspects of empowerment during pregnancy for all groups of pregnant women , it is important to survey multiparous pregnant women , as well as high risk pregnant women using a larger sample size . | [
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] |
the alternaria sp . isolate
was prepared for scale - up
chemical analysis by culturing it for 4 weeks on a solid - state medium
composed of cheerios breakfast cereal supplemented with a 0.3% sucrose
solution .
the fungal biomass was extracted with etoac , yielding
a crude extract that was subsequently processed over several types
of sorbents ( including silica gel , sephadex lh20 , and c18 ) to provide purified metabolites 117 .
compound 1 was obtained
as a pale yellow , amorphous
powder , and its molecular formula was established as c34h30o14s based on hresims data ( [ m + na ] ion at m / z 717.1253 , calcd
717.1248 ) indicating 20 degrees of unsaturation . the h
and c nmr spectra of 1 provided evidence
for approximately half of the expected proton and carbon resonances ,
which were assigned as belonging to 11 nonprotonated carbons , three
methines , three methyls , and three exchangeable protons .
the initial
analysis of the data set led us to suspect that 1 might
be a dimeric compound .
further examination of the h and c nmr data ( table 1 ) indicated that
the resonances observed for 1 were similar to those for
the known metabolite sulochrin ( 4 ) , which we also purified from the same fungal extract .
the
major differences between 1 and 4 as revealed
by nmr were the loss of signals for a methine unit ( h 6.91 ; c 107.6 ) and the gain of a new nonprotonated
sp carbon resonance ( c 109.1 ) .
the necessity
of including a sulfur atom in 1 as required by the molecular
formula led us to propose that the sulfur served as a thioether bridge
linking the c-2 and c-2 carbons .
thus , the structure of the
new sulochrin dimer was proposed as illustrated ( 1 ) ,
and the metabolite was given the trivial name polluxochrin .
metabolite 1 shares structural similarities to other sulochrin dimers
including , disulochrin , as well as guignasulfide , which is a demethoxy analogue of 1 .
hresims provided a pseudomolecular ion at m / z 685.0981 [ m + na ] ( calcd 685.0986 )
that supported the molecular formula c33h26o13s .
inspection
of the h and c nmr data for 2 revealed that the spectra superficially appeared as the superimposition
of two sets of signals ( table 2 ) with one set
virtually identical to those generated for compound 1 and the other set bearing substantial similarity to methyl-(1,6-dihydroxy-3-methylxanthone)-8-carboxylate
( 5 ) , which was also purified
from the fungal extract .
the additional unit of unsaturation afforded
via the incorporation of 5 in this metabolite readily
accounted for the overall difference of one unsaturation unit between
metabolites 1 and 2 .
the change from a single
c-9 diaryl ketone signal in 1 ( c 197.8 )
( table 1 ) to pyrone ( c 179.2 )
and diaryl ( c 197.7 ) ketone signals in 2 ( table 2 ) further supported the asymmetric
dimeric nature of the new metabolite .
this assertion was subsequently
confirmed by hsqc and hmbc experiments ( figures
s9 and s10 ) , as well as data generated from a single - crystal
x - ray diffraction experiment ( figure 1 ) .
ortep structure generated
from the x - ray diffraction data for a
single crystal of 2 .
compounds 1 and 2 were determined to
be susceptible to transformation under aqueous conditions . upon incubation
for 48 h in various aqueous solvent mixtures , compound 1 was observed by lc - pda - esims to transform into 2 , which
subsequently converted to another compound ( 3 ) ( figure s1 ) .
the transformation process was determined
to be temperature dependent , and even mild heating for brief periods
led to the rapid loss of 1 and generation of 2 and 3 . to obtain
material for structure characterization ,
5.0 mg of 1 in aqueous meoh was held at 40 c for
6 h , generating 3.5 mg of 3 .
the purified product was
examined by hresims , which yielded a pseudomolecular ion at m / z 629.0757 [ m h ] ( calcd 629.0754 ) corresponding to the molecular formula c32h22o12s .
an analysis of the h and c nmr data for the product ( table s1 ) indicated that 3 was structurally similar to compounds 1 and 2 .
immediately apparent was that the c nmr diaryl ketone signal in 1 ( c-9/9 ,
c 197.8 ) had been replaced by a pyrone ketone signal
in 3 ( c-9/9 , c 179.1 ) ( tables 1 and s1 ) .
subsequent
2d nmr experiments ( figures s14 and s15 ) confirmed that these changes resulted in the formation of a new
compound that consisted of a thioether - linked dimer of 5 connected to the sulfur atom through c-2 and c-2. in contrast
to metabolite 1 , compound 3 exhibited two
nearly superimposable sets of nmr signals .
the doubling of the signals
is proposed to result from the hindered rotation of the s
a similar cyclization phenomenon
has been observed for the transformation of metabolite 4 to 5 , but in this case , the reaction rate was much
slower , taking upward of a week to occur .
these results imply that
the sulfur atom might serve to accelerate the intramolecular cyclization
of 1 into 2 and 3 .
the
molecular formula for compound 7 was determined
by hresims to be c13h16o5 .
an analysis
of the 1d and 2d nmr ( in acetone - d6 and
dmso - d6 ) for 7 indicated
that this metabolite had the same planar structure as pyrenochaetic
acid d , but the published account of
this metabolite did not address the compound s absolute configuration .
to address this problem , we prepared crystals of 7 for
x - ray diffraction analysis ( figure 3 ) .
the metabolite s absolute configuration was determined
to be 8s by the hooft method .
this was further supported by comparing the ecd experimental
data for 7 with a computationally derived theoretical
spectrum ( figure 4a , refer to the supporting information for a discussion of how
the theoretical ecd data for 7 were prepared ) .
ortep structure generated from the x - ray diffraction data
for a
single crystal of 7 . calculated and experimental ecd data for compounds 7 ( a ) and 10 ( b ) .
compound 8 was determined by hresims to have
the same
molecular formula as 7 .
analysis of the h
and c nmr data for 8 ( tables 3 and 4 ) revealed that it possessed
the same carbon skeleton as the co - occurring metabolite pyrenochaetic
acid c ( 6 ) .
however , the
carbon and proton signals attributable to an oxidized methylene ( ch2 - 10 , h 3.42 ; c 60.4 ) were
replaced by signals for an aliphatic methyl . taking into account the
hresims data ,
it was proposed that compound 8 contained
a new c-10 hydroxyl group relative to 6 . combining the
proton and carbon chemical shift patterns for 8 with
the h
h cosy correlations among h-8
and h-9 and h-10 , as well as the hmbc correlations between h-8 and
h-9 with c-7 ( figure 2 ) , the position of the
new hydroxyl group was unambiguously assigned .
pyrenochaetic acid f ( 9 ) was
purified as a colorless ,
amorphous solid , and its molecular formula was established to be c15h18o6 based on hresims data ( [ m
h ] ion at m / z 293.1034 , calcd 293.1031 ) .
the compound
exhibited nmr resonances similar to 8 with the addition
of proton and carbon signals corresponding to an acetoxy group ( tables 3 and 4 ) .
the inclusion of
the acetoxy group in the structure was confirmed on the basis of an
hmbc correlation from h-10 ( h 4.03 ) to c-14 ( c 170.8 ) ( figure 2 ) .
pyrenochaetic
acid g ( 10 ) was obtained as a colorless ,
amorphous solid .
an [ m h ] molecular ion
at m / z 251.0932 ( calcd 251.0925 )
in the hresims spectrum was in agreement with the molecular formula
c13h16o5 .
the hmbc correlation data ( h-7 to c-3 , c-4 , c-5 ,
and c-8 , as well as h-9 and h-10 to c-8 ) indicated that the oxidation
pattern of c-7 and c-8 in 7 was reversed in 10 ( i.e. , c-7 bore the hydroxyl group and c-8 was a ketone ) ( tables 3 and 4 , figure 2 ) .
the metabolite s absolute configuration was determined
to be 7r by comparing experimental ecd data with
a theoretical spectrum of the compound ( figure 4b , refer to the supporting information for a discussion of how the theoretical ecd data for 10 were prepared ) .
thus , the structure of the new metabolite was confirmed
as illustrated for 10 and was named pyrenochaetic acid
g. the molecular formula of 11 was determined to
be c13h14o5 based on hresims , which
provided
an [ m h ] ion at m / z 249.0776 ( calcd 249.0768 ) . a comparison of the 1d nmr
data for 11 with 10 ( tables 3 and 4 ) revealed that the carbon and
proton signals attributable to the c-7 methine were missing and had
been replaced by a downfield ketone resonance ( c 196.3 ) .
compound 11 was named pyrenochaetic acid h. dimethylamide
asterrate ( 13 ) possessed the molecular
formula c19h21no7 as determined by
hresims , indicating that the metabolite possessed 10 degrees of unsaturation .
the compound exhibited h and c nmr resonances
( table 5 ) that were similar to the known , co - occurring
metabolite asterric acid ( 12 ) , with the major exceptions being the addition of two new methyl groups
( c-9 : c 34.3 , h 2.91 ;
c-10 : c 37.5 , h 2.85 ) .
considering the chemical shift and hmbc correlation data for 13 ( h 2.91 and 2.85 to c 166.2 ) , the new methyl groups were rationalized to be part of a
tertiary amide that had replaced the carboxylic acid in 12 .
compound 14 was obtained as a pale yellow , amorphous
solid . on the basis of the hresims data ( [ m + h ] ion at m / z 639.1708 , calcd 639.1714 )
analysis of h
nmr , c nmr , and hsqc data ( table 6 ) revealed that this metabolite possessed 17 nonprotonated carbons ,
eight methines , three methylenes , and four methyl groups . further
analysis of the 2d nmr data ( figure 5 ) revealed
that 14 shared the same planar structure as blennolide
g. when the nmr solvent was changed from
dmso - d6 to cdcl3 ( as reported
in the literature ) , compound 14 provided h and c nmr spectra that were identical to blennolide
g ; however , their specific rotation values were in essence the opposite
of one another { 14 : [ ]d 68.8 ( c 0.125 , chcl3 ) ; blennolide
g : [ ]d + 81.1 ( c 0.29 ,
chcl3)}. further analysis of the ecd data for 14 ( figure s80 ) revealed a cotton effect
pattern that was the inverse of that generated for blennolide g. thus ,
metabolite 14 was determined to be the antipode of blennolide
g. selected h
metabolites 15 , 16 , and 17 shared the same molecular formula
( c32h30o14 ) , as well as many other
similarities among their h and c nmr spectral
data ( table 6 ) . in spite of this ,
compound 15 stood out as displaying half of the expected proton and
carbon resonances , which indicated that it was a symmetrical dimer .
hmbc correlation data ( figure 5 ) were essential
for establishing the c-6c-6 linkage and overall planar
structure of 15 .
it was subsequently determined that
metabolite 16 shared the same planar structure as 15 ( figure 5 ) . however , the presence
of two distinct sets of resonances representing the two monomeric
portions of 16 denoted that this metabolite was an asymmetric
diastereomer of 15 .
in contrast to 15 and 16 , hmbc correlations from h-7 to c-8 and h-7 to c-6
( figure 5 ) indicated that metabolite 17 was covalently bonded via a c-8c-6 linkage .
since compounds 1517 were not active
in our panel of assays and were prone to consistent degradation during
both freezer storage and room - temperature handling , our investigation
of their relative configurations was discontinued .
all the purified
compounds were screened for cancer cell cytotoxicity , antibacterial and
antifungal activities , and inhibition
of fungal biofilm formation . only compounds 13 exhibited anti - mrsa activity , with
mic values of 4.1 , 4.9 , and 3.2 m ( 2.9 , 3.2 , and 2.0 g / ml ) ,
respectively , whereas the mic for chloramphenicol was 5 m ( 1.6
g / ml ) .
metabolites 13 also
showed weak mammalian cell cytotoxicity effects against pancreatic
cancer cells ( mia paca-2 ) with ic50 values of 50.8 , 30.3 ,
and 29.3 m , respectively .
optical rotation data
were determined on a rudolph research autopol iii automatic polarimeter .
accurate
mass data were collected on an agilent 6538 hresi qtof ms coupled
to an agilent 1290 hplc .
lc - ms data were obtained on a shimadzu lc - ms
2020 system ( esi quadrupole ) coupled to a photodiode array detector ,
with a phenomenex kinetex column ( 2.6 m c18 column ,
100 , 75 3.0 mm ) .
the hplc system utilized scl-10a vp
pumps and a system controller with luna 5 m c18 columns
( 110 , 250 21.2 mm , 10 ml / min and 110 , 250
10 mm , 4 ml / min ) .
x - ray data were collected using a diffractometer
with a bruker apex ccd area detector and graphite - monochromated mo
k radiation ( = 0.710 73 ) .
the
fungal isolate ( internal strain designation wailua pda-3 ) was obtained
from a soil sample collected in the vicinity of wailua falls , hawaii .
the isolate was identified as similar to alternaria longissima based on its sequence analysis ( 99% sequence homology ) .
km088044 ) was
compared by blast analysis to sequences publicly available through
the ncbi database .
the fungal isolate was cultured under solid - state
conditions on a medium composed of cheerios breakfast cereal supplemented
with a 0.3% sucrose solution with 0.005% chloramphenicol .
the fungus
was grown for 4 weeks at room temperature ( 25 c ) .
the scale - up solid - phase
cultures were pooled and extracted twice overnight with etoac , and
the organic solvent was partitioned against water .
the resultant organic
layers were concentrated under vacuum . the resulting extract ( 71 g )
ch2cl2 ) was subjected to c18 vlc ( h2o meoh ) to give four subfractions .
subfraction 1 ( eluted
with 4:6 h2o meoh ) was further purified by semipreparative
hplc ( mobile phase 1:1 h2o meoh with 0.1% formic
acid in water ) to afford compounds 6 ( 2.0 mg ) , 7 ( 15.0 mg ) , 8 ( 2.5 mg ) , 9 ( 2.8
mg ) , 10 ( 3.0 mg ) , 11 ( 2.3 mg ) , and 12 ( 6.0 mg ) .
subfraction 3 ( eluted with 8:2 h2o meoh )
was applied to a sephadex lh20 column and eluted with 1:1 ch2cl2meoh .
h2o with 0.1%
formic acid in water ) yielded 1 ( 8.0 mg ) , 2 ( 7.5 mg ) , 4 ( 25 mg ) , 5 ( 14 mg ) , 13 ( 6.5 mg ) , 14 ( 4.0 mg ) , 15 ( 5.1
mg ) , 16 ( 4.7 mg ) , and 17 ( 5.0 mg ) .
pale yellow , amorphous
solid ; uv ( meoh ) max ( log ) 210 ( 4.27 ) , 286
( 3.81 ) , 346 ( 3.54 ) nm ; ir ( film ) max 3600 , 2953 ,
1712 , 1641 , 1514 , 1454 , 1344 , 1211 , 1091 , 1022 , 831 cm ; h and c nmr see table 1 ; hresims [ m + na]m / z 717.1253 ( calcd for c34h30o14sna , 717.1248 ) .
pale
yellow , block - like
crystals ; uv ( meoh ) max ( log ) 208 ( 4.37 ) ,
238 ( 4.34 ) , 364 ( 4.05 ) nm ; ir ( film ) max 3600 , 2930 ,
1712 , 1645 , 1585 , 1514 , 1365 , 1209 , 1089 , 1020 , 831 cm ; h and c nmr see table 2 ; hresims [ m + na]m / z 685.0981 ( calcd for c33h26o13sna , 685.0986 ) .
pale
yellow powder ; uv
( meoh ) max ( log ) 210 ( 4.39 ) , 246 ( 4.12 ) ,
298 ( 3.56 ) , 364 ( 3.90 ) nm ; ir ( film ) max 3610 , 2935 ,
1739 , 1699 , 1651 , 1514 , 1365 , 1269 , 1211 , 1008 , 827 cm ; h and c nmr see table
s1 ; hresims [ m h]m / z 629.0757 ( calcd for c32h21o12s , 629.0754 ) .
colorless cubic
crystals ; [ ]d 5.0 ( c 0.18 , etoh ) ; uv ( meoh ) max ( log )
212 ( 4.14 ) , 252 ( 3.68 ) , 302 ( 3.32 ) nm ; cd ( meoh ) max ( ) 254 ( 6.82 ) 317 ( 2.50 ) ; ir ( film ) max 2972 , 1699 , 1541 , 1456 , 1413 , 1315 , 1226 , 1097
, 974 cm ; h and c nmr see tables 3 and 4 ; hresims [ m
h]m / z 251.0931
( calcd for c13h15o5 , 251.0925 ) .
colorless , amorphous
solid ; uv ( meoh ) max ( log ) 212 ( 4.12 ) , 246
( 3.61 ) , 298 ( 3.23 ) nm ; ir ( film ) max 2941 , 1697 ,
1541 , 1456 , 1413 , 1315 , 1234 , 1095 cm ;
h and c nmr see tables 3 and 4 ; hresims [ m h]m / z 251.0931 ( calcd for c13h15o5 , 251.0925 ) .
colorless , amorphous
solid ; uv ( meoh ) max ( log ) 210 ( 4.19 ) , 298
( 3.34 ) nm ; ir ( film ) max 2966 , 1737 , 1687 , 1546 ,
1454 , 1413 , 1238 , 1097 , 1039 cm ; h
and c nmr see tables 3 and 4 ; hresims [ m
colorless ,
amorphous solid ; [ ]d 11.4 ( c 0.18 , meoh ) ; uv ( meoh ) max ( log )
216 ( 4.20 ) , 248 ( 3.79 ) , 296 ( 3.37 ) nm ; cd ( meoh ) max ( ) 244 ( 6.48 ) , 275 ( 1.85 ) ; ir ( film ) max 2965 , 1699 , 1577 , 1456 , 1413 , 1313 , 1226 , 1091 cm ; h and c nmr see tables 3 and 4 ; hresims [ m h]m / z 251.0932 ( calcd
for c13h15o5 , 251.0925 ) .
colorless ,
amorphous solid ; uv ( meoh ) max ( log ) 212
( 4.19 ) , 270 ( 3.88 ) , 326 ( 3.46 ) nm ; ir ( film ) max 2981 , 1685 , 1548 , 1460 , 1311 , 1255 , 1096 cm ; h and c nmr see tables 3 and 4 ; hresims [ m
pale yellow ,
amorphous solid ; uv ( meoh ) max ( log ) 212
( 4.33 ) , 284 ( 3.32 ) , 310 ( 3.34 ) nm ; ir ( film ) max 3439 , 1625 , 1469 , 1357 , 1249 , 1205 , 1149 , 1060 cm ; h and c nmr see table 5 ; hresims [ m + h]m / z 376.1392 ( calcd for c19h22no7 ,
376.1391 ) . pale yellow ,
amorphous solid ; [ ]d 68.8 ( c 0.125 , chcl3 ) ; uv ( meoh ) max ( log ) : 206 ( 4.26 ) , 260 ( 3.97 ) , 340 ( 3.85 ) nm ; cd ( meoh )
max ( ) 211 ( 8.37 ) , 223 ( 14.2 ) , 328
( 3.35 ) ; ir ( film ) max 2967 , 1788 , 1739 ,
1612 , 1435 , 1361 , 1213 , 1047 cm ; h
and c nmr see table 6 ; hresims
[ m + h]m / z 639.1708
( calcd for c32h31o14 , 639.1714 ) .
pale yellow , amorphous
solid ; [ ]d 75.3 ( c 0.473 , chcl3 ) ; uv ( meoh ) max ( log )
208 ( 4.36 ) , 258 ( 4.30 ) , 364 ( 3.84 ) nm ; ir ( film ) max 3439 , 2965 , 1788 , 1645 , 1433 , 1359 , 1273 , 1207 , 1056 cm ; h and c nmr see table 6 ; hresims [ m + na]m / z 661.1530 ( calcd for c32h30o14na , 661.1528 ) .
pale yellow , amorphous
solid ; [ ]d 35.0 ( c 0.035 , chcl3 ) ; uv ( meoh ) max ( log )
206 ( 4.32 ) , 256 ( 4.25 ) , 360 ( 3.79 ) nm ; ir ( film ) max 3458 , 2962 , 1791 , 1647 , 1433 , 1359 , 1286 , 1207 , 1012 cm ; h and c nmr see table 6 ; hresims [ m + na]m / z 661.1531 ( calcd for c32h30o14na , 661.1528 ) .
pale yellow , amorphous
solid ; [ ]d 28.8 ( c 0.125 , chcl3 ) ; uv ( meoh ) max ( log )
210 ( 4.50 ) , 256 ( 4.43 ) , 360 ( 3.93 ) nm ; ir ( film ) max 3441 , 1788 , 1745 , 1647 , 1469 , 1354 , 1280 , 1172 , 1055 cm ; h and c nmr see table 6 ; hresims [ m + na]m / z 661.1527 ( calcd for c32h30o14na , 661.1528 ) .
a yellow plate - shaped
crystal of dimensions 0.46
0.26 0.08 mm of 2 and a colorless prism - shaped
crystal of dimensions 0.44 0.32 0.18 mm of 7 were selected for structural analysis .
intensity data for these
two compounds were collected using a diffractometer with a bruker
apex ccd area detector and graphite - monochromated mo k radiation ( 0.710 73
) .
details of the x - ray crystal structure analysis for 2 and 7 are available in the supporting information .
the x - ray crystallographic data for 2 and 7 have been deposited with the cambridge
crystallographic data center under accession numbers ccdc 993668 and 993669 , respectively .
these data can be accessed free of charge at http://www.ccdc.cam.ac.uk/. conformational analyses were carried out using spartan10 .
geometry , frequency , and ecd calculations were
applied at the dft level with gaussian 09 .
specdis 1.60 was used to average single ecd or uv vis spectra
after boltzmann statistical weighting .
mammalian cell cytotoxicity assays
were performed on pancreatic cancer ( mia paca-2 ) cells by adding 5000
cells per well into 96-well plates .
the cells were allowed to adhere
overnight at 37 c in a humidified incubator ( 5% co2 atmosphere ) .
test compounds were diluted in dmso and added to the
wells so that the final concentration of dmso per well did not exceed
1% by volume .
the plates containing treated and control cells were
incubated for 48 h , and cell viability was determined by mtt assay .
compounds were
tested against methicillin - resistant staphylococcus aureus ( mrsa ) strain atcc 700787 , which also
exhibits reduced susceptibility to vancomycin .
a stock culture was
diluted in brain heart infusion broth and delivered into presterilized
96-well plates .
stocks prepared in dmso were added to each well of
the 96-well plate ( 1% final dmso concentration ) .
optical density measurements
( od600 ) of the cultures were determined on a tecan infinite m200 plate
reader .
the plates were incubated for 17 h in a humidified incubator
at 37 c .
plates were removed and orbitally shaken , and a final
od600 value was recorded .
the final od600 reading was subtracted from
the initial od600 reading to obtain the change in od600 ( antimicrobial
activity was determined based on the change in optical density ) . | polluxochrin ( 1 ) and
dioschrin ( 2 ) , two
new dimers of sulochrin linked by thioether bonds , were purified from
an alternaria sp .
isolate obtained from a hawaiian
soil sample .
the structures of the two metabolites were established
by nmr , mass spectrometry data , and x - ray analysis .
metabolite 1 was determined to be susceptible to intramolecular cyclization
under aqueous conditions , resulting in the generation of 2 as well as another dimeric compound , castochrin ( 3 ) .
an additional nine new metabolites were also obtained , including four
new pyrenochaetic acid derivatives ( 811 ) , one new asterric acid analogue ( 13 ) , and four new
secalonic acid analogues ( 1417 ) .
bioassay analysis of these compounds revealed 13 displayed antimicrobial and weak cytotoxic activities . | Results
and Discussion
Experimental
Section | thus , the structure of the
new sulochrin dimer was proposed as illustrated ( 1 ) ,
and the metabolite was given the trivial name polluxochrin . inspection
of the h and c nmr data for 2 revealed that the spectra superficially appeared as the superimposition
of two sets of signals ( table 2 ) with one set
virtually identical to those generated for compound 1 and the other set bearing substantial similarity to methyl-(1,6-dihydroxy-3-methylxanthone)-8-carboxylate
( 5 ) , which was also purified
from the fungal extract . the change from a single
c-9 diaryl ketone signal in 1 ( c 197.8 )
( table 1 ) to pyrone ( c 179.2 )
and diaryl ( c 197.7 ) ketone signals in 2 ( table 2 ) further supported the asymmetric
dimeric nature of the new metabolite . this assertion was subsequently
confirmed by hsqc and hmbc experiments ( figures
s9 and s10 ) , as well as data generated from a single - crystal
x - ray diffraction experiment ( figure 1 ) . compounds 1 and 2 were determined to
be susceptible to transformation under aqueous conditions . the transformation process was determined
to be temperature dependent , and even mild heating for brief periods
led to the rapid loss of 1 and generation of 2 and 3 . combining the
proton and carbon chemical shift patterns for 8 with
the h
h cosy correlations among h-8
and h-9 and h-10 , as well as the hmbc correlations between h-8 and
h-9 with c-7 ( figure 2 ) , the position of the
new hydroxyl group was unambiguously assigned . thus , the structure of the new metabolite was confirmed
as illustrated for 10 and was named pyrenochaetic acid
g. the molecular formula of 11 was determined to
be c13h14o5 based on hresims , which
provided
an [ m h ] ion at m / z 249.0776 ( calcd 249.0768 ) . on the basis of the hresims data ( [ m + h ] ion at m / z 639.1708 , calcd 639.1714 )
analysis of h
nmr , c nmr , and hsqc data ( table 6 ) revealed that this metabolite possessed 17 nonprotonated carbons ,
eight methines , three methylenes , and four methyl groups . further
analysis of the 2d nmr data ( figure 5 ) revealed
that 14 shared the same planar structure as blennolide
g. when the nmr solvent was changed from
dmso - d6 to cdcl3 ( as reported
in the literature ) , compound 14 provided h and c nmr spectra that were identical to blennolide
g ; however , their specific rotation values were in essence the opposite
of one another { 14 : [ ]d 68.8 ( c 0.125 , chcl3 ) ; blennolide
g : [ ]d + 81.1 ( c 0.29 ,
chcl3)}. further analysis of the ecd data for 14 ( figure s80 ) revealed a cotton effect
pattern that was the inverse of that generated for blennolide g. thus ,
metabolite 14 was determined to be the antipode of blennolide
g. selected h
metabolites 15 , 16 , and 17 shared the same molecular formula
( c32h30o14 ) , as well as many other
similarities among their h and c nmr spectral
data ( table 6 ) . only compounds 13 exhibited anti - mrsa activity , with
mic values of 4.1 , 4.9 , and 3.2 m ( 2.9 , 3.2 , and 2.0 g / ml ) ,
respectively , whereas the mic for chloramphenicol was 5 m ( 1.6
g / ml ) . subfraction 1 ( eluted
with 4:6 h2o meoh ) was further purified by semipreparative
hplc ( mobile phase 1:1 h2o meoh with 0.1% formic
acid in water ) to afford compounds 6 ( 2.0 mg ) , 7 ( 15.0 mg ) , 8 ( 2.5 mg ) , 9 ( 2.8
mg ) , 10 ( 3.0 mg ) , 11 ( 2.3 mg ) , and 12 ( 6.0 mg ) . details of the x - ray crystal structure analysis for 2 and 7 are available in the supporting information . | [
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the self - etching approach has been proposed in an effort to simplify the dentin / enamel bonding systems .
the elimination of separate etching and rinsing steps simplified the bonding technique and has been responsible for the increased popularity of these systems in daily practice33 .
self - etching adhesive systems differ from etch - and - rinse adhesives in several aspects , such as the initial ph , type of acidic monomer , number of bottles and steps , concentration of water and solvents , and the hydrophilicity of the bonding layer .
they are classified into two- or one - step systems depending on the number of procedures required for bonding , as well as in mild , moderate and acidic systems depending on their initial ph31 .
the clinical application of these systems would be even further promising if their performance were at least similar to that of three - step etch - and - rinse adhesive systems , which are considered clinically favorable on bonding to enamel and dentin23 because both substrates need to be hybridized in most clinical situations . as the ph of self - etching systems is not similar to that of phosphoric acid used in the etch - and - rinse approach , increasing concern about their performance on intact
is slightly ground during a bevel or cut during cavity preparation , which provides a more receptive substrate for bonding13 .
however , there are cases such as bonding of orthodontic brackets or conservative and preventive restorative procedures , where bonding should be made on intact enamel . therefore , it is clinically important to determine the performance of such systems on ground vs. unground enamel substrates .
some studies have demonstrated that self - etching adhesives can provide resin - enamel bond strength values as high as those obtained by phosphoric acid treatment , as long as the bonding is performed on ground enamel13 .
however , other authors have not observed any difference on the performance of self etching systems when applied on ground and unground enamel10 .
one of the differences that might have accounted for the conflicting results of these two studies was the way enamel was prepared . while in the former study13 enamel
was prepared with a high - speed diamond bur , silicon carbide paper was used the latter study10 .
it is likely that the use of high - speed diamond bur may expose deeper enamel surface than the use of silicon carbide paper .
based on that , we hypothesize that the kind of enamel preparation might affect the bond strength of self - etching adhesive systems to enamel .
therefore , the purpose of this study was to evaluate the bond strength and etching pattern of enamel after application of two - step self - etching and etch - and - rinse adhesive systems to unground , bur - prepared and silicon carbide paper - treated enamel .
the null hypothesis tested was that the bonding performance of the self - etching adhesives does not vary for the studied parameters .
this study was approved by the institutional review board of the dental school ( faculty of dentistry , university of so paulo , fousp ) under protocol number 208/03 .
fifty - four extracted third molars were obtained and immersed in 0.5% chloramine at 4c during 7 days5 before the beginning of the laboratorial setting .
the teeth were then sectioned in a mesiodistal direction in order to obtain tooth halves that were randomly distributed to each experimental condition .
the obtained buccal and lingual surfaces were cleaned with a pumice and water and examined under a stereomicroscope to ensure that they were free of surface cracks , decalcification or any sign of previous grinding .
the occlusal third of the buccal and lingual surfaces were usually outside the bonding area due to their inclination .
the tooth halves were then randomly assigned to 3 groups according to the type of enamel surface preparation : group 1 : pumice prophylaxis ( no grinding was done ) ; group 2 : after prophylaxis , a wheel medium - grit diamond bur ( # 4142 , particle size ca 100 mm , kg sorensen , barueri , sp , brazil ) was applied on enamel surface using a high - speed handpiece with water coolant . this procedure created 0.5-mm - deep grooves on the surface , which was then flattened with a tapered round - end fine - grit diamond bur ( # 4138 , particlesize ca 46 mm , kg sorensen ) ; group 3 : after prophylaxis , the enamel was ground with a 60-grit silicon carbide paper14,30 under water cooling for 60 s. each group was further divided into six subgroups according to the adhesive system used .
four two - step self - etching adhesive systems were used : a mild system ( ph = 2.0 ) ( clearfil se bond - cse ; kuraray medical inc , tokyo , japan ) , two intermediate strong systems ( ph = 1.5 ) ( optibond solo plus self - etch primer - op ; kerr co , orange , ca , usa and adhese - ad ; ivoclar vivadent , schaan , liechtenstein ) and an acidic system ( ph<1 ) ( tyrian self priming etchant - ty ; bisco inc , schaumburg , il , usa ) . the ph of each system is provided by their manufacturers and have also been measured in a previous study14 .
two and three - step etch - and - rinse adhesive systems - adper scotchbond multi - purpose plus - sbmp and adper single bond - sb ( 3m / espe , st paul , mn , usa ) , respectively - were used as controls .
all adhesive systems were applied under controlled environmental conditions ( 24c/60% relative humidity ) by a single operator , following the bonding protocols summarized in figure 1 .
special care was taken to ensure that the enamel surfaces were adequately coated by monomer after solvent evaporation . in the event matte enamel
was observed , an additional coat of adhesive was applied to produce shiny surfaces prior to light - curing with a vip unit ( 600 mw / cm , bisco inc , schaumburg , il , usa ) .
next , a 3-mm - high composite resin ( filtek z250 , 3m / espe ) block was incrementally built up on the treated enamel surface and each 1-mm thick increment was light cured individually .
after storage in distilled water at 37c for 24 h , the specimens were sectioned longitudinally in both " x " and " y " directions across the bonded interface with a diamond saw in a labcut 1010 machine ( extec ; enfield , ct , usa ) to obtain approximately 5 - 7 bonded sticks per tooth with a cross - sectional area of about 0.8 mm .
the number of prematurely debonded sticks ( pd ) per tooth during specimen preparation was recorded .
each stick was examined with a stereomicroscope ( x10 ) in order to check the inclination of the bonding interfaces in the four sides of each stick .
the cross - sectional area of each stick was measured with a digital caliper accurate to the nearest 0.01 mm ( absolute , mitutoyo , tokyo , japan ) and recorded for calculation of the bond strength .
each stick was then individually fixed to a custom - made testing jig and tested in microtensile strength to failure in a universal testing machine ( emic , so jos dos pinhais , pr , brazil ) running at a crosshead speed of 0.5 mm / min .
the bond failure modes were evaluated at x400 with light stereomicroscope ( hmv- 2 , shimadzu , tokyo , japan ) and classified as cohesive ( failure exclusively within enamel or composite resin ) and adhesive / mixed ( failure at resin / enamel interface or mixed with cohesive failure of the neighboring substrates ) . a bond strength index ( bsi )
the bs index is a weighted mean assuming the relative contribution of the possible mode of failures , according to the following equation ( values in mpa ) :
ba / m mean bond strength of sticks with adhesive / mixed fracture pattern ;
% a / m percentage of sticks with adhesive / mixed fracture pattern ;
cd cohesive strength of enamel ; % d percentage of sticks that failed cohesively in enamel ;
cr cohesive strength of resin ;
% r percentage of sticks that failed cohesively in resin ;
bds bond strength attributed to sticks that spontaneously debonded during preparation ; % ds percentage of sticks debonded during preparation .
the cohesive strength of the composite resin and the cohesive strength of enamel are considered as the mean value of all the specimens ( from a single tooth ) that failed in that manner .
the mean value attributed to specimens that failed prematurely during preparation is arbitrary , and corresponded to approximately half of the minimum bond strength value that could be measured in this study .
the microtensile bsis were subjected to a two - way repeated measures analysis of variance and tukey 's post - hoc test ( =0.05 ) for pair - wise comparisons .
the effect of conditioning with 35% phosphoric acid and the self - etching primers on ground and unground buccal or lingual enamel surfaces was analyzed .
enamel surfaces that were not treated with the conditioners and bur - cut and silicon carbide paper - treated surfaces were also observed for comparison purposes .
teeth ( n=2/condition ) were bisected longitudinally in a mesiodistal direction to provide two halves .
a deep lingual guiding slit was prepared with a diamond bur to facilitate subsequent fracture of the etched surfaces .
free enamel surfaces were cleaned with pumice / water slurry - prophylaxis ( group 1 ) . after cleaning , the enamel surfaces from group 2 were bur - cut and those from group 3 were ground with wet 60-grit silicon carbide paper for 60 s , as described for the microtensile bond strength testing .
enamel surfaces from groups 1 , 2 and 3 were treated with 35% phosphoric acid and self - etching primers as described in figure 1 .
phosphoric acid - etched enamel was rinsed with water spray for 15 s , while enamel etched with self - etching primers was rinsed with alternate baths of ethanol and acetone ( 20 s each ) in order to remove the monomers6 .
thereafter , the same specimens were gently split with a hammer and scalpel blade along the pre - formed slits to provide a sagittal view of the etched enamel .
specimens were stored in a desiccator containing silica gel for 12 h. following that , they were mounted on aluminum stubs with colloidal silver and gold sputter - coated ( balzers scd 050 sputter coater , bal - tec , germany ) to be observed under a scanning electron microscope ( philips xl30 ; eindhoven , the netherlands ) at 15 kv of accelerating voltage .
both the buccal and lingual etched surfaces as well as the sagittally fractured surfaces of the same tooth were examined .
fifty - four extracted third molars were obtained and immersed in 0.5% chloramine at 4c during 7 days5 before the beginning of the laboratorial setting .
the teeth were then sectioned in a mesiodistal direction in order to obtain tooth halves that were randomly distributed to each experimental condition .
the obtained buccal and lingual surfaces were cleaned with a pumice and water and examined under a stereomicroscope to ensure that they were free of surface cracks , decalcification or any sign of previous grinding .
the occlusal third of the buccal and lingual surfaces were usually outside the bonding area due to their inclination .
the tooth halves were then randomly assigned to 3 groups according to the type of enamel surface preparation : group 1 : pumice prophylaxis ( no grinding was done ) ; group 2 : after prophylaxis , a wheel medium - grit diamond bur ( # 4142 , particle size ca 100 mm , kg sorensen , barueri , sp , brazil ) was applied on enamel surface using a high - speed handpiece with water coolant .
this procedure created 0.5-mm - deep grooves on the surface , which was then flattened with a tapered round - end fine - grit diamond bur ( # 4138 , particlesize ca 46 mm , kg sorensen ) ; group 3 : after prophylaxis , the enamel was ground with a 60-grit silicon carbide paper14,30 under water cooling for 60 s. each group was further divided into six subgroups according to the adhesive system used .
four two - step self - etching adhesive systems were used : a mild system ( ph = 2.0 ) ( clearfil se bond - cse ; kuraray medical inc , tokyo , japan ) , two intermediate strong systems ( ph = 1.5 ) ( optibond solo plus self - etch primer - op ; kerr co , orange , ca , usa and adhese - ad ; ivoclar vivadent , schaan , liechtenstein ) and an acidic system ( ph<1 ) ( tyrian self priming etchant - ty ; bisco inc , schaumburg , il , usa ) . the ph of each system is provided by their manufacturers and have also been measured in a previous study14 .
two and three - step etch - and - rinse adhesive systems - adper scotchbond multi - purpose plus - sbmp and adper single bond - sb ( 3m / espe , st paul , mn , usa ) , respectively - were used as controls .
all adhesive systems were applied under controlled environmental conditions ( 24c/60% relative humidity ) by a single operator , following the bonding protocols summarized in figure 1 .
special care was taken to ensure that the enamel surfaces were adequately coated by monomer after solvent evaporation . in the event matte enamel
was observed , an additional coat of adhesive was applied to produce shiny surfaces prior to light - curing with a vip unit ( 600 mw / cm , bisco inc , schaumburg , il , usa ) .
next , a 3-mm - high composite resin ( filtek z250 , 3m / espe ) block was incrementally built up on the treated enamel surface and each 1-mm thick increment was light cured individually .
after storage in distilled water at 37c for 24 h , the specimens were sectioned longitudinally in both " x " and " y " directions across the bonded interface with a diamond saw in a labcut 1010 machine ( extec ; enfield , ct , usa ) to obtain approximately 5 - 7 bonded sticks per tooth with a cross - sectional area of about 0.8 mm .
the number of prematurely debonded sticks ( pd ) per tooth during specimen preparation was recorded .
each stick was examined with a stereomicroscope ( x10 ) in order to check the inclination of the bonding interfaces in the four sides of each stick .
the cross - sectional area of each stick was measured with a digital caliper accurate to the nearest 0.01 mm ( absolute , mitutoyo , tokyo , japan ) and recorded for calculation of the bond strength .
each stick was then individually fixed to a custom - made testing jig and tested in microtensile strength to failure in a universal testing machine ( emic , so jos dos pinhais , pr , brazil ) running at a crosshead speed of 0.5 mm / min .
the bond failure modes were evaluated at x400 with light stereomicroscope ( hmv- 2 , shimadzu , tokyo , japan ) and classified as cohesive ( failure exclusively within enamel or composite resin ) and adhesive / mixed ( failure at resin / enamel interface or mixed with cohesive failure of the neighboring substrates ) . a bond strength index ( bsi )
the bs index is a weighted mean assuming the relative contribution of the possible mode of failures , according to the following equation ( values in mpa ) :
ba / m mean bond strength of sticks with adhesive / mixed fracture pattern ;
% a / m percentage of sticks with adhesive / mixed fracture pattern ;
cd cohesive strength of enamel ; % d percentage of sticks that failed cohesively in enamel ;
cr cohesive strength of resin ;
% r percentage of sticks that failed cohesively in resin ;
bds bond strength attributed to sticks that spontaneously debonded during preparation ; % ds percentage of sticks debonded during preparation .
the cohesive strength of the composite resin and the cohesive strength of enamel are considered as the mean value of all the specimens ( from a single tooth ) that failed in that manner . the mean value attributed to specimens that failed prematurely during preparation is arbitrary , and corresponded to approximately half of the minimum bond strength value that could be measured in this study .
the microtensile bsis were subjected to a two - way repeated measures analysis of variance and tukey 's post - hoc test ( =0.05 ) for pair - wise comparisons .
the effect of conditioning with 35% phosphoric acid and the self - etching primers on ground and unground buccal or lingual enamel surfaces was analyzed .
enamel surfaces that were not treated with the conditioners and bur - cut and silicon carbide paper - treated surfaces were also observed for comparison purposes . teeth ( n=2/condition ) were bisected longitudinally in a mesiodistal direction to provide two halves .
a deep lingual guiding slit was prepared with a diamond bur to facilitate subsequent fracture of the etched surfaces .
free enamel surfaces were cleaned with pumice / water slurry - prophylaxis ( group 1 ) . after cleaning , the enamel surfaces from group 2 were bur - cut and those from group 3 were ground with wet 60-grit silicon carbide paper for 60 s , as described for the microtensile bond strength testing .
enamel surfaces from groups 1 , 2 and 3 were treated with 35% phosphoric acid and self - etching primers as described in figure 1 .
phosphoric acid - etched enamel was rinsed with water spray for 15 s , while enamel etched with self - etching primers was rinsed with alternate baths of ethanol and acetone ( 20 s each ) in order to remove the monomers6 .
thereafter , the same specimens were gently split with a hammer and scalpel blade along the pre - formed slits to provide a sagittal view of the etched enamel .
specimens were stored in a desiccator containing silica gel for 12 h. following that , they were mounted on aluminum stubs with colloidal silver and gold sputter - coated ( balzers scd 050 sputter coater , bal - tec , germany ) to be observed under a scanning electron microscope ( philips xl30 ; eindhoven , the netherlands ) at 15 kv of accelerating voltage .
both the buccal and lingual etched surfaces as well as the sagittally fractured surfaces of the same tooth were examined .
the mean cross - sectional area ranged from 0.73 0.2 mm and no difference was detected among the treatment groups ( p>0.05 ) .
the mean bsis ( mpa ) and standard deviations , as well as the number of tested sticks and the number of prematurely debonded specimens are shown in table 1 .
neither the main factor surface treatment ( p=0.48 ) nor the interaction adhesive x surface treatment were statistically significant ( p=0.06 ) .
the highest resin - enamel mean bond strength value was observed for the two etch - and - rinse adhesive systems ( sbmp and sb ) .
op , ad and ty showed the lowest resin - enamel mean bond strength values , which were statistically different from all others adhesives .
cse showed the highest mean bond strength value among the self - etching adhesive systems ( table 2 ) .
the bond failures modes were predominantly of the mixed type . scanning electron microscopy ( sem ) micrographs of ground and unground enamel surfaces treated with phosphoric acid and the self - etching primers are shown in figures 3 to 7 . the effect of prophylaxis , diamond bur or sic paper treatments was also analyzed ( figure 2 ) .
enamel surface after prophylaxis was very smooth , with small and shallow grooves and few exposed enamel rods .
an apparently rougher surface than that created by prophylaxis with more grooves could be seen after enamel grinding with 60-grit sic paper .
diamond bur - prepared enamel showed larger and deeper grooves than those created by the other methods of preparation as well as an apparently rougher smear layer .
regardless of the surface treatment , only the diamond bur removed the prismless enamel ( figure 2 ) .
sem micrographs of the enamel surfaces etched with 35% phosphoric acid revealed little difference among the methods of preparation ( figure 3 ) . in all specimens ,
the smear layer was totally removed by phosphoric acid and enamel rods were exposed and etched .
the selective etching of prism cores ( type 1 pattern ) and prism peripheries ( type 2 pattern ) along with areas without selective demineralization could be observed in the same specimen .
the enamel surfaces following treatment with cse showed a predominant smooth surface with shallow depressions along the enamel surface ( figure 4 ) . when mild self - etching primer was used on prepared surfaces
the etching pattern created by the cse primer was more evident on the intact enamel surface .
the etching appearance of ad and op presented similar morphological features ( figures 5 and 6 respectively ) .
the ph of these moderate self - etching primers allowed them to remove the smear layer created by the sic paper and by the diamond bur in a more effective way than the mild cse primer .
areas of un - etched and smooth surfaces were intercalated with some deep and large grooves on enamel .
the demineralization pattern of these systems was more pronounced when they were applied on the diamond bur - prepared surface , i.e. , prismatic enamel surface . unlike the enamel microporosities produced by 35% phosphoric acid , the porosities produced by the most acidic self - etching primer , ty , were less numerous , deeper and larger ( figure 7 ) .
it was also evident the presence of fine surface roughening on the enamel surface with an uneven conditioning pattern .
regardless of the preparation method and the ph of the primer , none of the evaluated self - etching primers was able to expose the enamel rods as did the 35% phosphoric acid .
the mean cross - sectional area ranged from 0.73 0.2 mm and no difference was detected among the treatment groups ( p>0.05 ) .
the mean bsis ( mpa ) and standard deviations , as well as the number of tested sticks and the number of prematurely debonded specimens are shown in table 1 .
neither the main factor surface treatment ( p=0.48 ) nor the interaction adhesive x surface treatment were statistically significant ( p=0.06 ) .
the highest resin - enamel mean bond strength value was observed for the two etch - and - rinse adhesive systems ( sbmp and sb ) .
op , ad and ty showed the lowest resin - enamel mean bond strength values , which were statistically different from all others adhesives .
cse showed the highest mean bond strength value among the self - etching adhesive systems ( table 2 ) .
scanning electron microscopy ( sem ) micrographs of ground and unground enamel surfaces treated with phosphoric acid and the self - etching primers are shown in figures 3 to 7 .
the effect of prophylaxis , diamond bur or sic paper treatments was also analyzed ( figure 2 ) .
enamel surface after prophylaxis was very smooth , with small and shallow grooves and few exposed enamel rods .
an apparently rougher surface than that created by prophylaxis with more grooves could be seen after enamel grinding with 60-grit sic paper .
diamond bur - prepared enamel showed larger and deeper grooves than those created by the other methods of preparation as well as an apparently rougher smear layer .
regardless of the surface treatment , only the diamond bur removed the prismless enamel ( figure 2 ) .
sem micrographs of the enamel surfaces etched with 35% phosphoric acid revealed little difference among the methods of preparation ( figure 3 ) . in all specimens , the smear layer was totally removed by phosphoric acid and enamel rods were exposed and etched .
the selective etching of prism cores ( type 1 pattern ) and prism peripheries ( type 2 pattern ) along with areas without selective demineralization could be observed in the same specimen .
the enamel surfaces following treatment with cse showed a predominant smooth surface with shallow depressions along the enamel surface ( figure 4 ) . when mild self - etching primer was used on prepared surfaces
the etching pattern created by the cse primer was more evident on the intact enamel surface .
the etching appearance of ad and op presented similar morphological features ( figures 5 and 6 respectively ) .
the ph of these moderate self - etching primers allowed them to remove the smear layer created by the sic paper and by the diamond bur in a more effective way than the mild cse primer .
areas of un - etched and smooth surfaces were intercalated with some deep and large grooves on enamel .
the demineralization pattern of these systems was more pronounced when they were applied on the diamond bur - prepared surface , i.e. , prismatic enamel surface . unlike the enamel microporosities produced by 35% phosphoric acid , the porosities produced by the most acidic self - etching primer , ty , were less numerous , deeper and larger ( figure 7 ) .
it was also evident the presence of fine surface roughening on the enamel surface with an uneven conditioning pattern .
regardless of the preparation method and the ph of the primer , none of the evaluated self - etching primers was able to expose the enamel rods as did the 35% phosphoric acid .
the null hypothesis was not totally accepted because the bond strength and the morphology of surface enamel varied depending on the adhesive system .
the morphological structure of the intact peripheral enamel surface is different from that of the middle and inner enamel layer .
it has been widely reported that the superficial layer of enamel is less reactive to acids than the middle layer . as the acidity of the self - etching adhesive systems
are relatively lower than that of phosphoric acid treatment , the presence of this superficial enamel layer has been partially blamed for the lower performance of the self - etching systems in this substrate3,11,13,15,16 .
this study demonstrated that the 0.5-mm - deep grinding produced by the diamond bur was the only treatment able to homogeneously remove the prismless enamel .
pumice prophylaxis and sic paper abrasion did not remove this layer . despite these morphological results ,
it was impossible to distinguish under sem differences in the surface micromorphology of ground ( sic paper and diamond bur ) and unground enamel after conditioning with phosphoric acid , since all treatments depicted the presence of enamel rods
. this may be one of the reasons why differences between enamel surface preparations were not found in the phosphoric acid group .
this finding is in accordance with other studies that evaluated the performance of etch - and- rinse adhesives on ground and unground enamel10,13,22,27 . when the morphological findings of the same adhesive system were compared on ground and unground enamel some differences in the etching pattern
the two moderate ( op and ad ) and the most acidic ( ty ) self - etching systems demineralized the ground enamel surface more effectively than the intact enamel . despite these variations ,
some studies have demonstrated that enamel abrasion with diamond burs can improve the bond strength of self - etching systems to enamel13,27 .
other studies , in agreement with the present findings , have observed that the bond strength to unground or ground enamel does not differ7,10 or it is dependent on the adhesive system evaluated22 .
this apparent controversy means that the lower performance of self - etching adhesive systems on enamel3,11,13,15,16 can not be solely attributed to the presence of the prismless enamel .
variation in adhesive viscosity , surface tension , acidity of the self - etch system , chemical interaction of acidic monomers with enamel34 , water concentration9 , cohesive strength of the adhesives24,28 are important features to be considered .
this is somewhat true that although a more retentive etching pattern was observed for the moderate ( op and ad ) and particularly for the most acidic ( ty ) adhesives in comparison to the mild cse , higher bond strength values were not observed for ty .
the ty adhesive applied on diamond bur - prepared enamel produced an etching pattern that resembled that of 35% phosphoric acid etching , although the delineation of enamel rods was not as evident as that created by phosphoric acid .
contrary to these findings , other studies have demonstrated that more acidic self - etching systems were able to produce an extremely defined pattern of enamel etching , similar to phosphoric acid conditioning1,8 even when applied on intact enamel surface21 .
it is likely that differences in the composition of adhesive systems employed in these studies could have played a role on this apparent controversy .
in addition to the concentration and the type of acidic monomers that directly alter the acidity of self - etching adhesive systems18 , differences in the water concentration among in the systems can also contribute to partial or total dissociation of the acid functionalities18 .
it has been demonstrated that increasing water concentration from 10 to 20 vol% resulted in an increase in the degree of ionization of an acidic monomer , lowering the ph of the self - etching primer solution and further increasing their depth of demineralization9 .
sem examination of the surface morphology of enamel has shown that cse produced a very mild etching effect8 , with the bulk of the surface remaining unetched .
this means that among the adhesive systems tested in the present study , cse has only superficial interaction with enamel and a reduced potential for micromechanical interlocking . despite these morphological findings , cse achieved the highest bond strength values among the self - etching adhesive systems .
indeed , this finding is in accordance with previous studies , which demonstrated that mild self - etching adhesive systems perform well when compared to more acidic systems either in intact enamel or dentin4,11,12,15 .
it is clear from the micromorphological findings of this study that the use of a stronger acid resulted in a more dramatic dissolution and a more defined etching pattern , as previously reported21 .
low bond strength values are usually reported for more acidic self - etching adhesive systems when they are compared under microtensile bond strength approach4,11,31 .
the absence of relationship between the depth of demineralization and the strength of bonds produced by the more aggressive self - etching and etch - and - rinse adhesive systems on enamel shown in the present investigation is consistent with previous works10,21,29 .
as already reported , this means that other factors , apart from the etching pattern , may have a more important role on the bond strength values .
for instance , cse is a self - etching primer that contains 10- methacryloxydecyl dihydrogen phosphate ( 10-mdp ) as functional monomer dissolved in water to result in a ph of around 2 .
the excellent performance of this system in vitro
4,12,15 and in vivo investigations32 may be partially attributed to the additional chemical interaction of hydroxyapatite with the functional monomer 10-mdp34 .
this can theoretically contribute to the actual adhesive potential to enamel that consists of nearly only mineral substance , with which 10-mdp can chemically react .
20 , using a modeling approach , demonstrated that the theoretical strength of the resin - dentin bond strength should be proportional to the strength of the adhesive used to infiltrate demineralized dentin .
this was also confirmed in some laboratorial studies24,28 . when bonding to dentin , a strong relationship between the resin- dentin bond strengths and the mechanical properties of cured resin was observed for the self - etching adhesive systems24 .
the high initial ph of more acidic systems appear to dramatically weaken the bonding performance , either via chemical interacting with the adhesive layer placed next26 or via the presence of solvents within the polymer , which render the adhesive layer thinner and may weaken the polymer formed2 , thus compromising their bond strength to enamel .
as the acidity of self - etching adhesives is increased with the incorporation of higher concentration of hydrophilic and acidic monomers , the problems that are associated with acid - base incompatibility and water permeability become even more accentuated .
the acidic components of these adhesives may also adversely interact with the composite 's photoinitiator and so weaken the bonding complex2,6 .
another point to be considered is the reported low hydrolytic stability of methacrylates in acidic solutions17,19 . according to some authors17,19 , the ester portion of functional methacrylate , such as hema , used for a self - etching primer can become hydrolyzed in aqueous solutions when the ph values were below 2 .
they also suggested that the hydrolysis rate of the ester portion of methacrylates is also dependent on the storage temperature , which means that the self - etching primer should be kept refrigerated when not in use .
although phosphorus - containing monomers , such as mdp , are said to be more stable in acid environment , they should also be kept refrigerated when not in use .
in summary , it may be concluded that preparation of enamel surface ( prophylaxis , diamond bur or sic - paper ) did not influence the bonding performance of the self - etching adhesive systems evaluated in this study .
the highest bond strength values were observed for the etch - and - rinse adhesive systems . | objectives : to assess the bond strength and the morphology of enamel after application of self - etching adhesive systems with different acidities .
the tested hypothesis was that the performance of the self - etching adhesive systems does not vary for the studied parameters.material and methods : composite resin ( filtek z250 ) buildups were bonded to untreated ( prophylaxis ) and treated ( burcut or sic - paper ) enamel surfaces of third molars after application of four self - etching and two etch - and - rinse adhesive systems ( n=6/condition ) : clearfil se bond ( cse ) ; optibond solo plus self - etch ( op ) ; adhese ( ad ) ; tyrian self priming etching ( ty ) , adper scotchbond multi - purpose plus ( sbmp ) and adper single bond ( sb ) .
after storage in water ( 24 h/37c ) , the bonded specimens were sectioned into sticks with 0.8 mm2 cross - sectional area and the microtensile bond strength was tested at a crosshead speed of 0.5 mm / min .
the mean bond strength values ( mpa ) were subjected to two - way anova and tukey 's test ( =0.05 ) .
the etching patterns of the adhesive systems were also observed with a scanning electron microscope.results:the main factor adhesive system was statistically significant ( p<0.05 ) .
the mean bond strength values ( mpa ) and standard deviations were : cse ( 20.53.5 ) , op ( 11.32.3 ) , ad ( 11.22.8 ) , ty ( 11.13.0 ) , sbmp ( 21.94.0 ) and sb ( 24.93.0 ) .
different etching patterns were observed for the self - etching primers depending on the enamel treatment and the ph of the adhesive system.conclusion:although there is a tendency towards using adhesive systems with simplified application procedures , this may compromise the bonding performance of some systems to enamel , even when the prismless enamel is removed . | INTRODUCTION
MATERIAL AND METHODS
Microtensile Bond Strength
Enamel Etching Pattern
RESULTS
Microtensile Bond Strength
Enamel Etching Pattern
DISCUSSION
CONCLUSION | self - etching adhesive systems differ from etch - and - rinse adhesives in several aspects , such as the initial ph , type of acidic monomer , number of bottles and steps , concentration of water and solvents , and the hydrophilicity of the bonding layer . based on that , we hypothesize that the kind of enamel preparation might affect the bond strength of self - etching adhesive systems to enamel . therefore , the purpose of this study was to evaluate the bond strength and etching pattern of enamel after application of two - step self - etching and etch - and - rinse adhesive systems to unground , bur - prepared and silicon carbide paper - treated enamel . the null hypothesis tested was that the bonding performance of the self - etching adhesives does not vary for the studied parameters . four two - step self - etching adhesive systems were used : a mild system ( ph = 2.0 ) ( clearfil se bond - cse ; kuraray medical inc , tokyo , japan ) , two intermediate strong systems ( ph = 1.5 ) ( optibond solo plus self - etch primer - op ; kerr co , orange , ca , usa and adhese - ad ; ivoclar vivadent , schaan , liechtenstein ) and an acidic system ( ph<1 ) ( tyrian self priming etchant - ty ; bisco inc , schaumburg , il , usa ) . two and three - step etch - and - rinse adhesive systems - adper scotchbond multi - purpose plus - sbmp and adper single bond - sb ( 3m / espe , st paul , mn , usa ) , respectively - were used as controls . after storage in distilled water at 37c for 24 h , the specimens were sectioned longitudinally in both " x " and " y " directions across the bonded interface with a diamond saw in a labcut 1010 machine ( extec ; enfield , ct , usa ) to obtain approximately 5 - 7 bonded sticks per tooth with a cross - sectional area of about 0.8 mm . the microtensile bsis were subjected to a two - way repeated measures analysis of variance and tukey 's post - hoc test ( =0.05 ) for pair - wise comparisons . four two - step self - etching adhesive systems were used : a mild system ( ph = 2.0 ) ( clearfil se bond - cse ; kuraray medical inc , tokyo , japan ) , two intermediate strong systems ( ph = 1.5 ) ( optibond solo plus self - etch primer - op ; kerr co , orange , ca , usa and adhese - ad ; ivoclar vivadent , schaan , liechtenstein ) and an acidic system ( ph<1 ) ( tyrian self priming etchant - ty ; bisco inc , schaumburg , il , usa ) . two and three - step etch - and - rinse adhesive systems - adper scotchbond multi - purpose plus - sbmp and adper single bond - sb ( 3m / espe , st paul , mn , usa ) , respectively - were used as controls . after storage in distilled water at 37c for 24 h , the specimens were sectioned longitudinally in both " x " and " y " directions across the bonded interface with a diamond saw in a labcut 1010 machine ( extec ; enfield , ct , usa ) to obtain approximately 5 - 7 bonded sticks per tooth with a cross - sectional area of about 0.8 mm . the microtensile bsis were subjected to a two - way repeated measures analysis of variance and tukey 's post - hoc test ( =0.05 ) for pair - wise comparisons . the highest resin - enamel mean bond strength value was observed for the two etch - and - rinse adhesive systems ( sbmp and sb ) . the highest resin - enamel mean bond strength value was observed for the two etch - and - rinse adhesive systems ( sbmp and sb ) . the null hypothesis was not totally accepted because the bond strength and the morphology of surface enamel varied depending on the adhesive system . when the morphological findings of the same adhesive system were compared on ground and unground enamel some differences in the etching pattern
the two moderate ( op and ad ) and the most acidic ( ty ) self - etching systems demineralized the ground enamel surface more effectively than the intact enamel . in summary , it may be concluded that preparation of enamel surface ( prophylaxis , diamond bur or sic - paper ) did not influence the bonding performance of the self - etching adhesive systems evaluated in this study . the highest bond strength values were observed for the etch - and - rinse adhesive systems . | [
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among the cancers affecting women , cervical cancer has the second highest occurrence worldwide . in developing countries where 80% of the deaths caused by this disease occur ,
the most important risk factor associated with the development of cervical cancer is the infection of cervical epithelial cells by high - risk human papilloma virus genotype ( hr - hpv ) .
the tumor development can take 1025 years , during this time the cervical epithelium develops a non - invasive neoplastic lesion called cervical intraepithelial neoplasia ( cin ) .
the world health organization distinguishes three cin grades ( cin1 , cin2 and cin3 ) according to the degree of epithelial abnormality
. this can easily give the faulty impression of a solidified sculpture , as if the three cin grades are static events , whereas in reality a cin lesion is a dynamic process that can persist and progress but also regress .
when cin2 - 3 is diagnosed in a histological punch biopsy , definitive therapy by cone excision is usually promptly performed .
the reason for this aggressive invasive therapy is that 530% of cin2 - 3 patients will eventually develop invasive cancer when left untreated .
however , at the same time many ( 1040% ) cin2 - 3 s will regress spontaneously .
recently , it has been shown that functional biomarkers such as ki67 , prb , p53 and cytokeratine 13/14 are helpful in the prediction of regression or not , and the aggregate information provided by these biomarkers exceeds the value of the classic grading system .
consequently , many more cin - lesions that can either regress or progress could be more accurately treated .
although these methods are very promising , the fixation process of fresh tissues , required for conventional microscopical and many immunohistochemical stains leads to loss of water - soluble proteins that could be of interest .
in addition it affects the remaining proteins by introducing different crosslinks that can make it difficult to obtain them for identification after the fixation process [ 811 ] . some of the protein modifications introduced by the fixation process may also lead to misidentifications .
cytokines and chemokines are examples of such proteins whose characterization in cin - lesions could give valuable information about the patient s immune reaction against the hpv - infection [ 1214 ] .
as the hpv - infection induces a local immune response in the cervix that is barely detectable at plasma / serum level it is important to establish a standardized method for collection of a sample that truly represents the cervix microenvironment . in order to achieve this
, several studies have been performed using different sample types like cervicovaginal washings [ 14 , 16 ] , cervical mucus and cells supernatant from cytobrush collection , employing different analysis technologies .
one of these technologies is surface - enhanced laser desorption - ionization time of flight mass spectrometry ( seldi - tof ms ) which is a powerful tool for identifying differentially expressed proteins .
these proteins can be potentially diagnostic and prognostic biomarkers in neoplasia [ 19 , 20 ] .
the aim of this study was to develop a protein - saving biopsy processing method with similarities to the method published by celis et al . , and to utilize seldi - tof ms to analyze the proteins from the biopsies , while at the same time preserving the tissue for conventional microscopic and immunohistochemical studies . in this pilot study
, we will investigate if it is possible to separate normal cervical tissue from cin2 - 3 lesions .
the current study is a sub - project from a larger prospective study , approved by the regional medical ethics committee of helse vest , norway , the norwegian data inspection , and the health directorate of norway , # 33.06 , # 17185 and # 07/330 .
the criterion for selection in this large prospective study is a cytological abnormal smear followed by cervical biopsy in women with previously normal smears . of these 256 patients with cervical punch biopsy samples ,
45 diagnosed as cin2 - 3 ( n = 45 ) were selected . from women in the same age range , biopsies that were histologically and immunohistochemically normal (
biopsies of normal and cervical neoplastic epithelium were collected and immediately placed in polystyrene tubes ( sarsted , nurmbrecht , germany ) containing 5 ml of rpmi1640 culture medium ( gibco , carslbad , usa ) and stored for 24 h at 4c .
afterwards , the supernatants were collected , split into aliquots of 500 l and stored at 80c until analysis . after 24 h incubation in the rpmi1640 medium ,
the biopsies were routinely fixed in buffered 4% formaldehyde , embedded in paraffin , cut at 4 m , and stained with hematoxylin , eosin and safran ( he s ) for routine microscopic examination .
p16 and mib-1 immunostainings were used to confirm the diagnosis and human papilloma virus genotype determinations were done in all cases .
all biopsies were reviewed by two pathologists , who were blinded to the original diagnosis and follow - up ; in case of discrepancies the cases were re - reviewed with a double - head microscope by two pathologists ( eg , jb ) who also used the immunohistochemical stains and a consensus diagnosis was always obtained .
the cases were diagnosed as follows : normal ( n = 10 ) and cin2 - 3 ( n = 45 ) . the protocol for immunohistochemistry , type / manufacturer and dilution of the antibodies p16 and ki67 has been described before . for the epithelial biomarkers
the following antibodies and dilutions were used : ck-13 ( clone ks-1a3 , novocastra , newcastle upon tyne , uk ) 1/200 ; ck-14 ( clone ll002 , novocastra , newcastle upon tyne , uk ) 1/40 ; prb ( clone 13a10 , novocastra , newcastle upon tyne , uk ) 1/25 ; p53 ( clone do-7 , dako , glostrup , denmark , s3002 ) 1/200 ; phosphohistone = pph3 ( polyclonal cell signaling solutions , upstate # 06 - 570 ; lake placid , ny , usa ) 1/1500 . the section adjacent to the sections used for immunostainings was cut and stained with h&e to ensure the presence of the same cin lesion in all test sections ( sandwich - technique ) .
protein concentrations were assessed using a spectrophotometer ( thermo scientific nanodrop spectrophotometer ) measuring the absorbance at 280 nm .
samples were subjected to seldi - tof ms profiling according to the manufacturer s instructions ( ciphergen biosystems , fremont , ca , usa ) .
the biopsy supernatants were diluted 1:5 with 50 mm sodium acetate ( ph 4.3 ) and then bound to a cm10 proteinchip array .
they were incubated for two hours at room temperature on a platform shaker , then washed twice with 50 mm sodium acetate buffer , followed by two times 1 l of energy absorbing molecule ( = eam ) solution ( consisting of 50% saturated synaptic acid dissolved in 50% acetonitrile and 0.5% trifluoroacetic acid ) .
two replicates were prepared on different cm10 proteinchips by two different analysts on two different days .
the time - of - flight spectra were generated on the protein biological system ii mass spectrometer reader ( ciphergen biosystems , fremont , ca , usa ) , using a laser intensity of 170 , and a detector sensitivity of seven .
the seldi - tof ms data analysis was performed in three steps : 1 ) peak detection , 2 ) selection of peaks with the highest discriminatory power and 3 ) building a multivariate model based on the selection in step 2 .
the peak detection was done using the ciphergen seldi software version 3.2 after internal and external mass calibration followed by normalization ( tic intensity ) of all spectra as one group .
the mass range from 2000 to 20000 da contained the majority of the peptides / proteins in the samples , and was selected .
masses less than 2000 da was excluded as these are known to contain adducts and artifacts from the eam - solution and other chemical contaminants .
the peak detection includes baseline subtraction , calibration of mass accuracy and automatic peak detection .
each spectrum was then assigned to one of two groups , normal or cin2 - 3 . to select peaks with the highest discriminatory power , the biomarker wizard ( ciphergen ) was used for peak detection and clustering of all the spectra .
this was done using a signal - to - noise ( s / n ) ratio of 5 and 15% of all spectra for the first pass detection and clustering , and an s / n ratio of 2 for the second pass .
the cluster results were then imported into spss version 15 ( spss norway as , oslo , norway ) , cart ( salford , san diego , ca , usa ) and medcalc ( medcalc software , mariakerke , belgium ) for binary logistic regression analysis and classification and regression tree analysis .
a t - test was done , a correlation test using all 40 peaks followed by the development of a binary logistic regression model using the peaks that were found not to be correlated .
the current study is a sub - project from a larger prospective study , approved by the regional medical ethics committee of helse vest , norway , the norwegian data inspection , and the health directorate of norway , # 33.06 , # 17185 and # 07/330 .
the criterion for selection in this large prospective study is a cytological abnormal smear followed by cervical biopsy in women with previously normal smears . of these 256 patients with cervical punch biopsy samples ,
45 diagnosed as cin2 - 3 ( n = 45 ) were selected . from women in the same age range , biopsies that were histologically and immunohistochemically normal (
for soluble protein recovery , biopsies of normal and cervical neoplastic epithelium were collected and immediately placed in polystyrene tubes ( sarsted , nurmbrecht , germany ) containing 5 ml of rpmi1640 culture medium ( gibco , carslbad , usa ) and stored for 24 h at 4c .
afterwards , the supernatants were collected , split into aliquots of 500 l and stored at 80c until analysis .
after 24 h incubation in the rpmi1640 medium , the biopsies were routinely fixed in buffered 4% formaldehyde , embedded in paraffin , cut at 4 m , and stained with hematoxylin , eosin and safran ( he s ) for routine microscopic examination .
p16 and mib-1 immunostainings were used to confirm the diagnosis and human papilloma virus genotype determinations were done in all cases .
all biopsies were reviewed by two pathologists , who were blinded to the original diagnosis and follow - up ; in case of discrepancies the cases were re - reviewed with a double - head microscope by two pathologists ( eg , jb ) who also used the immunohistochemical stains and a consensus diagnosis was always obtained .
the cases were diagnosed as follows : normal ( n = 10 ) and cin2 - 3 ( n = 45 ) .
the protocol for immunohistochemistry , type / manufacturer and dilution of the antibodies p16 and ki67 has been described before . for the epithelial biomarkers
the following antibodies and dilutions were used : ck-13 ( clone ks-1a3 , novocastra , newcastle upon tyne , uk ) 1/200 ; ck-14 ( clone ll002 , novocastra , newcastle upon tyne , uk ) 1/40 ; prb ( clone 13a10 , novocastra , newcastle upon tyne , uk ) 1/25 ; p53 ( clone do-7 , dako , glostrup , denmark , s3002 ) 1/200 ; phosphohistone = pph3 ( polyclonal cell signaling solutions , upstate # 06 - 570 ; lake placid , ny , usa ) 1/1500 . the section adjacent to the sections used for immunostainings was cut and stained with h&e to ensure the presence of the same cin lesion in all test sections ( sandwich - technique ) .
protein concentrations were assessed using a spectrophotometer ( thermo scientific nanodrop spectrophotometer ) measuring the absorbance at 280 nm .
samples were subjected to seldi - tof ms profiling according to the manufacturer s instructions ( ciphergen biosystems , fremont , ca , usa ) .
the biopsy supernatants were diluted 1:5 with 50 mm sodium acetate ( ph 4.3 ) and then bound to a cm10 proteinchip array .
they were incubated for two hours at room temperature on a platform shaker , then washed twice with 50 mm sodium acetate buffer , followed by two times 1 l of energy absorbing molecule ( = eam ) solution ( consisting of 50% saturated synaptic acid dissolved in 50% acetonitrile and 0.5% trifluoroacetic acid ) .
two replicates were prepared on different cm10 proteinchips by two different analysts on two different days .
the time - of - flight spectra were generated on the protein biological system ii mass spectrometer reader ( ciphergen biosystems , fremont , ca , usa ) , using a laser intensity of 170 , and a detector sensitivity of seven .
the seldi - tof ms data analysis was performed in three steps : 1 ) peak detection , 2 ) selection of peaks with the highest discriminatory power and 3 ) building a multivariate model based on the selection in step 2 .
the peak detection was done using the ciphergen seldi software version 3.2 after internal and external mass calibration followed by normalization ( tic intensity ) of all spectra as one group . the mass range from 2000 to 20000 da contained the majority of the peptides / proteins in the samples , and
masses less than 2000 da was excluded as these are known to contain adducts and artifacts from the eam - solution and other chemical contaminants .
the peak detection includes baseline subtraction , calibration of mass accuracy and automatic peak detection .
each spectrum was then assigned to one of two groups , normal or cin2 - 3 . to select peaks with the highest discriminatory power , the biomarker wizard ( ciphergen ) was used for peak detection and clustering of all the spectra .
this was done using a signal - to - noise ( s / n ) ratio of 5 and 15% of all spectra for the first pass detection and clustering , and an s / n ratio of 2 for the second pass .
the cluster results were then imported into spss version 15 ( spss norway as , oslo , norway ) , cart ( salford , san diego , ca , usa ) and medcalc ( medcalc software , mariakerke , belgium ) for binary logistic regression analysis and classification and regression tree analysis . a t - test was done , a correlation test using all 40 peaks followed by the development of a binary logistic regression model using the peaks that were found not to be correlated .
the median age of the patients at the time that the biopsies were obtained was 30.8 years ( range 2640 ) for the cin2 - 3 group and for the normal biopsies group , 33.1 years ( range 3234 ) ( these differences were not significant ) .
the mean protein concentrations of the normal ( 1.00 mg / ml ) and the cin2 - 3 ( 1.09 mg / ml ) samples were also not significantly different .
we did evaluate the effect of different incubation times on the histology and immunohistochemistry on cervical biopsies .
figure 1 gives typical examples , showing that after 24 h incubation , there are no visible differences compared with 0 h incubation .
note that there are no visible differences after 24 h incubation typical examples of cervical biopsies after 0 and 24 h incubation in rpmi .
note that there are no visible differences after 24 h incubation the peak detection and clustering process using biomarker wizard resulted in a total of 40 protein peaks .
the development of a binary logistic model resulted in three peaks found to have independent value in discriminating the two groups .
figures 2 and 3 show zoom views of the differential peaks and examples of full spectra ( m / z 200010000 ) for the 2 groups analyzed . as fig .
2 shows , the peak with m / z 3430 is down - regulated in cin2 - 3 samples compared to normal samples , while the peaks with m / z 5077 and 7794 up - regulated .
the reproducibility between the technical replicates and between samples can be seen in fig . 3 where the replicates for three different cin2 - 3 samples are visualized as gel traces ( gel view ) .
2zoom views of seldi - tof ms spectra show different expressions in protein profiles between normal ( three bottom graphs ) and cin2 - 3 ( three top graphs ) for the three peaksfig .
3one normal trace for sample 1 combined with gel traces ( gel view ) showing the protein profiles for both replicates of three representative cin2 - 3 samples ( labelled 13 ) covering the m / z range from 2000 to 10000 zoom views of seldi - tof ms spectra show different expressions in protein profiles between normal ( three bottom graphs ) and cin2 - 3 ( three top graphs ) for the three peaks one normal trace for sample 1 combined with gel traces ( gel view ) showing the protein profiles for both replicates of three representative cin2 - 3 samples ( labelled 13 ) covering the m / z range from 2000 to 10000 the reproducibility of the analysis was tested by comparing all 40 peaks between replicate 1 and 2 .
only three of the 40 peaks were significant different between replicate 1 and replicate 2 , i.e. m / z 3318 ( p = 0.01 ) , m / z 4023(p = 0.0001 ) and m / z 10885 ( p = 0.03 ) . all other peaks had p values higher than 0.10 and the three discriminatory peaks were not different at all ( p > 0.80 ) .
when the multivariate analyses were repeated leaving out the peaks that were significantly different between the two replicates , the discriminatory results between the normal and cin2 - 3 samples were the same as before .
the average intensities , confidence intervals and the probabilities of no difference ( t - test ) between the normal and cin2 - 3 groups assuming independency are shown .
figures 4 , 5 and 6 shows the discrimination of the two groups using the three seldi - tof ms peaks as variables . by using the developed three - step binary logistic model , an overall correct classification of 88%
14 of the 20 normal samples and 84 of 90 of the cin2 - 3 samples were correctly classified .
table 1mean intensities and confidence intervals ( in parenthesis ) for proteins differentially expressed between normal cervical tissue and cin2 - 3 samples .
the probability of no differences ( t - test ) between normal and cin2 - 3 samples is presented as p - valuesprotein m / znormal ( n = 10)cin2 - 3 ( n = 45)p - value ( normal vs cin2 - 3)mean intensitymean intensity343019.0 ( 13.5424.52)7.3 ( 6.438.16)<0.0001507710.7 ( 5.6715.82)26.9 ( 22.7431.07)0.000677941.7 ( 1.152.24)7.3 ( 6.228.44)<0.0001fig .
4scatterplot showing the discrimination of the two groups using m / z 3430 and 7794 as variables .
5scatterplot showing the discrimination of the two groups using m / z 5077 and 7794 as variables .
6a three - dimensional scatterplot showing the discrimination of the two groups using all three peaks .
red triangles : cin2 - 3 samples mean intensities and confidence intervals ( in parenthesis ) for proteins differentially expressed between normal cervical tissue and cin2 - 3 samples . the probability of no differences ( t - test ) between normal and cin2 - 3 samples is presented as p - values scatterplot showing the discrimination of the two groups using m / z 3430 and 7794 as variables .
red triangles : cin2 - 3 samples scatterplot showing the discrimination of the two groups using m / z 5077 and 7794 as variables .
red triangles : cin2 - 3 samples a three - dimensional scatterplot showing the discrimination of the two groups using all three peaks .
this study presents the results obtained from analyzing protein extracts obtained by a novel method by seldi - tof ms .
we have shown that water - soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and cin2 - 3 .
we found that 24 h immersion of cervical punch biopsies in rpmi at 4c can be used to harvest 1 mg / ml water soluble proteins without compromising the routine immunohistochemical analysis of the tissue .
moreover , the water soluble proteins from the biopsies give good discrimination between samples with normal and cin2 - 3 epithelia .
seldi - tof ms technology has been applied for several years to address the need for new markers for early cancer detection [ 19 , 2224 ] or for different lymphomas .
most studies have compared diseased and healthy persons and have been useful for a better understanding of the development of several types of cancer .
however , it is more difficult to develop biomarkers which can discriminate between the different development stages of a neoplasia as the differences are much smaller than between healthy and diseased persons . yet
, the model presented here shows that this still is possible with the technology used .
cin represents the first step in the development of cervical cancer through the three different cin phases . in a future study , it is important to analyze whether seldi - tof ms or other mass spectrometric techniques can discriminate between cins that will regress spontaneously from cins that will persist or progress to invasive cancer .
furthermore , the protein collection method that was developed is of great interest for studying not only cervical neoplasia but it could also be applied to other organ tracts .
the method of placing a biopsy in a cell culture medium for 24 h at 4c is considerably longer than 1 h in pbs at 37c used by celis et al . , and enables the collection of proteins from a biopsy without interfering with the essential diagnostic information .
the three peaks found to separate normal tissue and cin2 - 3 lesions did not have the same m / z - values as those reported found from seldi - tof ms analysis of cell lysates of normal and invasive cervical cancer or from plasma samples of cin patients .
the protein concentrations in the samples were comparable to those found in samples from mucosal collection using sponges .
in addition , the rpmi immersion method has additional value to conventional ffpe analysis as it enables the collection and analysis of water soluble proteins which are not available from formalin - fixed paraffin - embedded tissue protein extracts .
proteins like cytokines and chemokines are collected in a volume enabling the analysis by different techniques like seldi - tof ms , lc - ms / ms or by ultrasensitive elisa techniques like electrochemiluminescence .
an essential aspect of a new laboratory test with possible diagnostic and therapeutic implications is the variation sources in all steps of the analysis .
pre - analytical ( a. each time getting the same samples , by using the same biopsy device ; b. taking two samples from the same patient and put them in two different rpmi tubes ; c. standardize the incubation conditions and time ) ; 2 .
analytical part ( a. binding and cleanup on the seldi - chip ; b. reproducibility of the seldi - tof measurement of the same sample ; c. take two samples from the same rpmi sample and test the reproducibility ) ; 3 . post - analytical ( a. peak processing and clustering ; b. data analysis ) . as to sampling device
two neighbouring biopsy samples in the cervix from the same patient also gave comparable results .
we standardized the incubation conditions ( rpmi , 4c ) and time ( 24 h ) .
as to the analytical part , all the technical aspects of the seldi chip were standardized as described .
as to peak processing , clustering and data analysis , we used strictly protocolized standard operation procedures .
the results are promising but should be confirmed in a larger set of independent samples . in an ongoing study
not published yet , a subset of supernatants has been characterized using lc - ms / ms .
further work to identify the candidate biomarkers found using this model will be done using lc - ms / ms ( one- or multidimensional [ 32 , 33 ] ) of tryptic digests after fractionation by chromatography or two - dimensional gel electrophoresis .
an identification of the possible biomarker candidates found using this model will possibly give new insight to the mechanisms related to the human papilloma virus infection .
immersion of cervical punch biopsies for 24 h in rpmi tissue culture medium allows the analysis of water soluble proteins that can assist in the diagnosis of cervical intraepithelial neoplasia without interfering with the diagnosis .
we conclude that the water soluble proteins from the biopsies provide good discrimination between normal and cin2 - 3 samples .
this is a promising strategy to study the dynamic behavior of cins and hopefully will allow identification of regressive and non - regressive cin2 - 3 lesions and a better indication of patient - tailored treatment . | backgroundcervical intraepithelial neoplasia ( cin ) , a frequently encountered disease caused by human papilloma virus ( hpv ) is often diagnosed in formaldehyde - fixed paraffin embedded ( ffpe ) punch biopsies . since it is known that this procedure strongly affects the water - soluble proteins contained in the cervical tissue we decided to investigate whether a water - soluble protein - saving biopsy processing method can be used to support the diagnosis of normal and cin.methodscervical punch biopsies from 55 women were incubated for 24 h at 4c in rpmi1640 medium for protein analysis prior to usual ffpe processing and p16 and ki67-supported histologic consensus diagnosis was assessed .
the biopsy supernatants were subjected to surface - enhanced laser desorption - ionization time of flight mass spectrometry ( seldi - tof ms ) for identifying differentially expressed proteins . binary logistic regression and classification and regression trees ( cart ) were used to develop a classification model.resultsthe age of the patients ranged from 26 to 40 years ( median 29.7 ) .
the consensus diagnoses were normal cervical tissue ( n = 10 ) and cin2 - 3 ( n = 45 ) .
the mean protein concentration was 1.00 and 1.09 mg / ml in the normal and cin2 - 3 group , respectively .
the peak detection and clustering process resulted in 40 protein peaks .
many of these peaks differed between the two groups , but only three had independent discriminating power .
the overall classification results were 88%.conclusionswater - soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and cin2 - 3 . | Background
Methods
Study population
Sample collection
Pathology
Immunohistochemistry
ProteinChip SELDI-TOF MS analysis
Data analysis
Results
Discussion
Conclusions | one of these technologies is surface - enhanced laser desorption - ionization time of flight mass spectrometry ( seldi - tof ms ) which is a powerful tool for identifying differentially expressed proteins . of these 256 patients with cervical punch biopsy samples ,
45 diagnosed as cin2 - 3 ( n = 45 ) were selected . from women in the same age range , biopsies that were histologically and immunohistochemically normal (
biopsies of normal and cervical neoplastic epithelium were collected and immediately placed in polystyrene tubes ( sarsted , nurmbrecht , germany ) containing 5 ml of rpmi1640 culture medium ( gibco , carslbad , usa ) and stored for 24 h at 4c . the cases were diagnosed as follows : normal ( n = 10 ) and cin2 - 3 ( n = 45 ) . the cluster results were then imported into spss version 15 ( spss norway as , oslo , norway ) , cart ( salford , san diego , ca , usa ) and medcalc ( medcalc software , mariakerke , belgium ) for binary logistic regression analysis and classification and regression tree analysis . of these 256 patients with cervical punch biopsy samples ,
45 diagnosed as cin2 - 3 ( n = 45 ) were selected . from women in the same age range , biopsies that were histologically and immunohistochemically normal (
for soluble protein recovery , biopsies of normal and cervical neoplastic epithelium were collected and immediately placed in polystyrene tubes ( sarsted , nurmbrecht , germany ) containing 5 ml of rpmi1640 culture medium ( gibco , carslbad , usa ) and stored for 24 h at 4c . the cases were diagnosed as follows : normal ( n = 10 ) and cin2 - 3 ( n = 45 ) . the cluster results were then imported into spss version 15 ( spss norway as , oslo , norway ) , cart ( salford , san diego , ca , usa ) and medcalc ( medcalc software , mariakerke , belgium ) for binary logistic regression analysis and classification and regression tree analysis . the median age of the patients at the time that the biopsies were obtained was 30.8 years ( range 2640 ) for the cin2 - 3 group and for the normal biopsies group , 33.1 years ( range 3234 ) ( these differences were not significant ) . the mean protein concentrations of the normal ( 1.00 mg / ml ) and the cin2 - 3 ( 1.09 mg / ml ) samples were also not significantly different . note that there are no visible differences after 24 h incubation the peak detection and clustering process using biomarker wizard resulted in a total of 40 protein peaks . 2zoom views of seldi - tof ms spectra show different expressions in protein profiles between normal ( three bottom graphs ) and cin2 - 3 ( three top graphs ) for the three peaksfig . 3one normal trace for sample 1 combined with gel traces ( gel view ) showing the protein profiles for both replicates of three representative cin2 - 3 samples ( labelled 13 ) covering the m / z range from 2000 to 10000 zoom views of seldi - tof ms spectra show different expressions in protein profiles between normal ( three bottom graphs ) and cin2 - 3 ( three top graphs ) for the three peaks one normal trace for sample 1 combined with gel traces ( gel view ) showing the protein profiles for both replicates of three representative cin2 - 3 samples ( labelled 13 ) covering the m / z range from 2000 to 10000 the reproducibility of the analysis was tested by comparing all 40 peaks between replicate 1 and 2 . the probability of no differences ( t - test ) between normal and cin2 - 3 samples is presented as p - values scatterplot showing the discrimination of the two groups using m / z 3430 and 7794 as variables . we have shown that water - soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and cin2 - 3 . we found that 24 h immersion of cervical punch biopsies in rpmi at 4c can be used to harvest 1 mg / ml water soluble proteins without compromising the routine immunohistochemical analysis of the tissue . the three peaks found to separate normal tissue and cin2 - 3 lesions did not have the same m / z - values as those reported found from seldi - tof ms analysis of cell lysates of normal and invasive cervical cancer or from plasma samples of cin patients . immersion of cervical punch biopsies for 24 h in rpmi tissue culture medium allows the analysis of water soluble proteins that can assist in the diagnosis of cervical intraepithelial neoplasia without interfering with the diagnosis . | [
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the common thymine oxidation product in dna , 5,6-dihydroxy-5,6dihydro-2-thymine , known as thymine glycol ( tg ) , is formed by exposure to ionizing radiation as well as a variety of chemical oxidizing agents .
tg is also formed by oxidation of 5-methylcytosine followed by hydrolytic deamination of the unstable 5-methylcytosine glycol .
once formed , the c5 and c6 atoms in tg are chiral , and thus , tg exists in dna as two diastereomeric pairs of epimers , the 5r cis , trans pair ( 5r,6s;5r,6r ) and the 5s cis , trans pair ( 5s,6r;5s,6s ) ( scheme 1 ) . for the 5r pair of epimers , the rate of epimerization at the nucleoside level is 5.8 10 min . the 5r pair is more abundant and more stable . for both the 5r and 5s pairs , the cis epimers predominate at the nucleoside level .
tg inhibits dna synthesis by many prokaryotic and eukaryotic dna polymerases one nucleotide before and opposite the lesion site , although several dna polymerases lacking 3 5 exonuclease activity can bypass it , albeit more slowly than a control .
the bypass of tg by y - family dna polymerases is stereospecific , with pol bypassing the 5r epimers more efficiently and pol bypassing the 5s epimers more efficiently .
the human hnth1 glycosylase , which repairs pyrimidine lesions arising from oxidative damage , shows a 13:1 preference for excising the 5r epimers vs the 5s epimers , whereas the hneil1 glycosylase shows a 1.5:1 preference for excising the 5r epimers vs the 5s epimers .
have shown that the repair of tg by dna n - glycosylases / ap lyases is modulated by the cistrans epimerization of these two sets of diastereomers .
the base excision repair of these lesions also depends upon the identity of the opposing base .
the hnth glycosylase repairs the cis-5r,6s tg more efficiently when it is placed opposite adenine than when placed opposite guanine .
the more abundant 5r tg lesion was structurally examined in the 5-atga-3 sequence , paired opposite da .
these studies concluded that tg induced a localized structural perturbation , and that tg was extrahelical .
it had also been reported that the structure of the 5r tg lesion in the 5-gtgc-3 sequence was disordered .
these nmr studies did not address the potential structural consequences of cis - trans epimerization of tg in duplex dna .
more recently , a binary primer - template complex , containing a site - specifically 5r - tg - modified template , was crystallized with the replicative rb69 dna polymerase . the resulting structure , representing the situation immediately following incorporation of datp opposite tg , revealed the presence of the cis 5r,6s tg epimer at the active site . the cis 5r,6s tg epimer was intrahelical and formed a watsoncrick base pair with the da at the primer 3-terminus .
this confirmed modeling studies in which the cis 5r,6s tg epimer was predicted to pair with da .
moreover , in the crystal structure with the rb69 polymerase , the tg methyl group was in the axial conformation , hindering stacking of the adjacent 5-template guanine . these structural results provided a possible rationale for earlier observations that extension past the 5r tg lesion by the klenow fragment of escherichia coli dna polymerase i or t4 dna polymerase was prohibited .
presently , the 5r - tg adduct has been incorporated site - specifically into 5-d(gtgcgtggtttgt)-3 and annealed with either 5-d(acaaacacgcac)-3 , forming a duplex containing the tga base pair , corresponding to the oxidative damage of thymine in dna , or 5-d(acaaacgcgcac)-3 , forming a duplex containing the mismatched tg : g base pair , corresponding to the formation of tg following oxidative damage and deamination of 5-methylcytosine in dna . for the duplex containing the tga pair ,
the cis-5r,6s and trans-5r,6r epimers exist in a cis : trans ratio of 7:3 at 30 c .
in contrast , for the duplex containing the tg : g pair , the cis : trans equilibrium strongly favors the cis-5r,6s epimer ; the level of the trans-5r,6r epimer remains below the level of detection by nmr .
the anomer of the 5r tg lesion remains the predominant deoxyribose c1 epimer in these double stranded oligodeoxynucleotides .
these data suggest that the potentially significant levels of the trans-5r,6r tg epimer in duplex dna should not be ignored with respect to the biological processing of the 5r tg lesion , corroborating observations that the repair of tg by dna n - glycosylases / ap lyases is modulated by the cis - trans epimerization .
mass spectrometric analysis yielded the anticipated molecular ion peak with mass 3732 ( m / z ) .
enzymatic hydrolysis of the oligodeoxynucleotide to nucleosides , followed by c-18 hplc chromatography , also yielded a single peak ( figure s1 in the supporting information ) .
additional peaks corresponding to the da , dc , dg , and dt mononucleosides were observed in the anticipated intensity ratios .
nmr data for each of the duplexes containing the tgg or tga base pairs was collected immediately upon sample preparation and subsequently repeated after 4 wks ; no changes in the spectra were observed , suggesting that the samples had achieved equilibrium .
figure 1 shows an expansion of the noesy spectrum including the noes between purine h8 and pyrimidine h6 protons and the corresponding deoxyribose h1 protons for the 5- d(gtgcgtggtttgt)-35-d(acaaacacgcac)-3 duplex , containing the tga base pair .
there was no break in the sequential noe connectivity for the modified strand ( figure 1a ) .
the g h1tg h6 noe was observed , as was the tg h6tg h1 noe .
there was also no break in connectivity for the complementary strand ( figure 1 , panel b ) .
the 5- d(gtgcgtggtttgt)-35-d(acaaacgcgcac)-3 duplex containing the tgg pair also showed no break in connectivity ( figure 1 , panel c ) .
again , the g h1tg h6 noe was observed , as was the tg h6tg h1 noe
. there was also no break in connectivity for the complementary strand of the duplex containing the tgg pair ( figure 1 , panel d ) .
sequential noesy assignments of purine h8 and pyrimidine h6 protons to deoxyribose h1 protons , for the duplexes containing either the tga or tgg base pairs .
the deoxyribose protons for both duplexes were assigned from a combination of cosy and noesy data . with the exception of several of the h4 protons , and the stereotopic assignments of the h5 and h5 sugar protons ,
noe intensities were used to assign the deoxyribose h2 and h2 resonances based on the fact that h2h3 cross peak was anticipated to have a greater intensity than the h2h3 cross peak .
the assignments of the nonexchangeable protons for the two 5r tg - modified duplexes , and the corresponding unmodified duplex , are provided in tables s1 , s2 , and s3 in the supporting information .
the assignments of the remaining watsoncrick hydrogen - bonded imino and amino protons of the two modified oligodeoxynucleotides were made using standard methods .
the spectra were similar for both of the 5r - tg - modified duplexes ( figure 2 ) . in both instances ,
the g n1h imino resonance was broad at 5 c and disappeared when the temperature was increased to 15 c , indicating that the presence of tg influenced watsoncrick hydrogen bonding at the 5 neighbor gc base pair ( figure 3 ) . in contrast , for the unmodified sample , the g n1h imino resonance was sharp and was observed at temperatures as high as 40 c . for both modified duplexes , there was no cross peak between the broad g n1h resonance and g n1h , located at base pair cg ( figure 2b and d ) .
the imino resonances for base pairs ta , gc , cg , gc , ta , ta , ta , and gc were observed ( figure 2a and c ) .
the imino resonances for the terminal base pairs gc and ta were not observed , attributed to exchange broadening with water .
p>(a ) expanded plot showing noes from the imino protons to amino protons for the duplex containing the tga base pair .
the cross peaks are assigned as a , gnh2g n1h ; b , a h2g n1h ; c , cnh2g n1h1 ; d , gnh2g n1h ; e , cnh2g n1h ; f , c h5g n1h ; g , gnh2g n1h ; h , a h2g n1h ; i , cnh2g n1h ; j , cnh2g n1h ; k , c h5g n1h ; l , gnh2g n1h ; m , cnh2g n1h ; n , anh2t n3h ; o , a h2t n3h ; p , anh1t n3h ; q , anh2t n3h ; r , anh2t n3h ; s , a h2t n3h ; t , a h2t n3h ; u ,
anh1t n3h ; v , a h2t n3h ; w , anh2t n3h ; , anh2t n3h ; y , a h2t n3h ; z. anh1t n3h ; a , a h2t n3h .
( c ) expanded plot showing noes from the imino protons to amino protons for the duplex containing the tgg base pair .
the cross peaks are assigned as a , gnh2g n1h ; b , cnh2g n1h ; c , c h5g n1h ; d , gnh2g n1h ; e , a h2g n1h ; f , cnh2g n1h ; g , cnh2g n1h ; h , c h5g n1h ; i , gnh2g n1h ; j , a h2g n1h ; k , cnh2g n1h ; l , c h5g n1h ; m , gnh2g n1h ; n , cnh2g n1h ; o , a h2t n3h ; p , anh2t n3h ; q , anh2t n3h ; r , anh2t n3h ; s , a h2t n3h ; t , a h2t n3h ; u , a h2t n3h ; v , cnh2t n3h ; w , anh2t n3h ; , a h2t n3h ; y , a h2t n3h ; z , anh1t n3h ; a , a h2t n3h .
( d ) expanded plot showing the sequential noe connectivity for the imino protons of the duplex containing the tgg base pair .
the data was collected at 800 mhz at 250 ms mixing time and a temperature of 7 c .
temperature - dependent analysis of imino protons of the duplexes containing ( a ) the ta base pair , ( b ) the tga base pair , and ( c ) the tgg base pairs , as monitored by h nmr . for the duplex containing
the tga pair analysis of noesy data showed the presence of two cross - peaks arising from dipolar couplings between tg ch3 and tg h6 ( figure 4 ) .
these were attributed to exchange between two chemical species , involving both the tg ch3 and tg h6 protons ( figure 4 , panels a and b ) .
the integrated volumes of the two exchange cross peaks were consistent at multiple noe mixing times .
for the tga duplex integration of the two tg ch3 resonances indicated that the two species were present at an equilibrium ratio of 7:3 ( figure 5 ) .
for the major species , the tg ch3 protons exhibited a chemical shift of 0.49 ppm , while the tg h6 proton resonated at 4.58 ppm .
these chemical shifts were consistent with values reported for the tg ch3 and h6 protons in earlier nmr studies in dna .
a total of 23 noe cross peaks were assigned between tg ch3 and h6 in the major species and dna ( 7 for tg h6 and 16 for tg ch3 ) in the tga duplex .
the resonances for the tg ch3 and h6 protons of the minor species were significantly downfield relative to those from the major species , located at 1.24 ppm and 4.91 ppm , respectively .
the tg ch3 resonance for the minor species was overlapped with the t ch3 resonance ( figure 5 ) . for the minor species
, there was only one noe cross peak observed to a dna proton , observed between tg h6 and tg h2. a single set of resonances was observed for the g and g dna protons , indicating that the two species observed for tg did not extend to the neighboring nucleotides .
( a ) noesy data collected for the duplex containing the tga base pair at a noe mixing time of 250 ms .
( b ) noesy data collected for the duplex containing the tga base pair at a noe mixing time of 150 ms .
( c ) noesy data collected for the duplex containing the tgg base pair at a noe mixing time of 250 ms .
( d ) noesy data collected for the duplex containing the tgg base pair at a noe mixing time of 150 ms .
( a ) for the duplex containing thetga base pair the resonances arising from the trans-5r,6r configuration of tg ch3 and t ch3 overlapped .
( b ) single resonance assigned to the cis-5r,6s configuration of tg ch3 was observed for the duplex containing the tgg base pair ( 20 mm sodium phosphate , ph 7.0 , 100 mm nacl , 30 c ) .
for the duplex containing the tgg pair analysis of noesy data obtained at multiple mixing times did not exhibit chemical exchange cross peaks for either the tg ch3 and tg h6 protons ( figure 4c and d ) .
the tg ch3 protons exhibited a chemical shift of 0.91 ppm , while the tg h6 proton resonated at 4.70 ppm ( figure 5 ) .
a total of 20 noe cross peaks were assigned between tg ch3 and h6 in major species and dna ( nine for tg h6 and eleven for tg ch3 ) .
the two tg species observed for the duplex containing the tga base pair were assigned as arising from slow exchange ( nmr time scale ) between the cis-5r,6s and trans-5r,6r epimers .
seven noesy cross peaks were observed between the tg h6 resonance of the major species and surrounding protons ( table 1 ) .
their intensities at mixing times of 80 and 250 ms were compared with the corresponding distances predicted for each epimer on the basis of molecular modeling .
the spectral overlap of tg h3 and tg h6 resonances in the major species hindered the assessment of the tg h3tg h1 and tg h6tg h1 cross peaks .
for the cis-5r,6s configuration , tg h6 and tg ch3 are spatially proximate , yielding a strong tg h6tg ch3 noe even at the short mixing time of 150 ms ( figure 4a and b ) .
likewise , the g h1tg h6 and g h8tg h6 noes were diagnostic of the cis-5r,6s configuration . on this basis , the major species , present at 70% population , was assigned as the cis epimer .
the configuration of the single species present in the duplex containing the tgg base pair was determined to be cis by the same approach ( figure 4 , panel c and d ) . for the duplex containing the tgg base pair the tg h3 and tg h6 resonances were separated ( 4.53 and 4.70 ppm , respectively ) .
noesy data were collected for the duplex containing for the tga base pair , at mixing times of 80 , 150 , 200 , and 250 ms . for the major species present , the cis-5r,6s lesion , the relative intensities of the noe cross - peaks corresponding to the deoxyribose h1 , h2 , h2 , and h3 protons were evaluated .
these data revealed that the intensity of the tg h1tg h2 noe was greater than the tg h1tg h2 noe , which placed h1 in the configuration ( figure 6 ) .
deoxyribose h1 , h2 , h2 , and h3 noe intensities assigned to the cis-5r,6s configuration , at a mixing time of 150 ms , for the duplex containing the tga base pair .
for either the cis-5r,6s or trans-5r,6r configurations , the tg ch3 may be in either axial or equatorial conformations .
calculations using gaussian 03 ( table 2 ) predicted that the cis-5r,6s configuration was at lower energy than the trans-5r,6r configuration , which was consistent with the present experimental observations , as well as previous experimental observations .
the calculations also predicted that for the cis-5r,6s configuration , the tg ch3 group favored the axial conformation , whereas for the trans-5r,6r configuration , the tg ch3 group favored the equatorial conformation .
of note was the observation that the calculations predicted that the trans-5r,6r configuration with the methyl group in the equatorial conformation was the next most energetically favorable configuration .
the 5-d(gtgcgtggtttgt)-35-d(acaaacacgcac)-3 and 5-d(gtgcgtggtttgt)-35-d(acaaacgcgcac)-3 duplexes were analyzed by uv melting and compared to the unmodified 5-d(gtgcgtgtttgt)-35-d(acaaacacgcac)-3 duplex .
all of the duplexes displayed hyperchromic shifts in uv absorption as temperature was increased from 5 to 80 c .
the tm values calculated for the concentration of 1.0 10 m are reported in table 3 .
both the duplexes containing either the tga or the tgg base pairs exhibited a 13 c reduction in tm relative to the unmodified duplex , ta .
the thermodynamic parameters were extracted from individual melting curves and vant hoff plots ( figure 7 ) .
these were comparable , indicating that both tg - modified duplexes melted via one step transitions .
in addition , there were no concentration - dependent melting effects observed within this concentration range ( table 3 ) .
the free energy values ( with respect to duplex formation ) were calculated at 25 and 37 c .
the values at both temperatures were negative , consistent with spontaneous formation of the duplexes at temperatures below the tm of the samples .
both 5r tg - modified samples showed increased ( less negative ) free energies of approximately 3 kcal / mol when compared to the unmodified sample at both 25 and 37 c .
each of the duplexes exhibited negative values for enthalpy and entropy ( with respect to duplex formation ) .
this indicated that in all cases , duplex formation was enthalpically favored and entropically disfavored .
however , the melting of the duplex containing the tgg pair was enthalpically favored as compared to the duplex containing the tga base pair .
( a ) hybridization plots ( alpha curves ) for the duplexes containing the unmodified ta base pair ( ) , the tga base pair ( ) , and the tgg base pair ( ) .
( b ) vant hoff plots of tm vs ln(ct/4 ) for the duplex containing the unmodified ta base pair ( ) , the tga base pair ( ) , and the tgg base pair ( ) . calculated for 1.0 10 m oligodeoxynucleotide duplex concentration .
reported errors are standard deviations . determined from individual melting curves . determined from ( tm )
tg is a substrate for base excision repair , both in escherichia coli and in mammalian cells . in e. coli ,
repair of tg is initiated by endonuclease iii ( nth ) and endonuclease viii ( nei ) .
both randomly introduced tg lesions and abasic sites were substrates for the uvrabc repair enzymes of e. coli .
subsequently it was determined that tg was excised from dna in vitro by human ner enzymes . however , dna containing dihydrothymine , a lesion with a similar structure to thymine glycol , but which can not undergo epimerization between cis and trans epimers , was not incised .
the present data reveal that in the duplex oligodeoxynucleotide containing the tga base pair , the 5r tg adduct exists as an 7:3 equilibrium mixture of cis 5r,6s and trans 5r,6r epimers , which equilibrate in slow exchange on the nmr time scale .
this ratio is comparable to the 87% cis to 13% trans ratio of epimers reported at the nucleoside level . on the other hand , the duplex containing the mismatched tgg base pair exists in solution predominantly as the cis 5r,6s epimer .
overall , we conclude that depending upon the identity of the complementary nucleotide , significant levels of the trans 5r,6r epimer may be present in duplex dna , and that the 5r tg lesion should be considered to exist in duplex dna as an equilibrium mixture of the two epimers .
the nmr data argue that for both duplexes , the purine placed opposite the 5r tg lesion stacks into the duplexes .
there is no disruption of sequential noe connectivity for the complementary strand either with the duplex containing the tga pair or the duplex containing the tgg pair ( figure 1 ) .
this suggests that for both duplexes , there is minimal structural perturbation to the complementary strand .
also , for both duplexes , tg does not interrupt the sequential noe connectivity in the modified strand ( figure 1 ) .
thus , it seems possible that irrespective of its placement opposite either a or g , tg remains either stacked or partially stacked within the duplex .
the present data also corroborate the conclusion that tg disrupts hydrogen bonding interactions , either when placed opposite to a or g. the tg n3 imino proton is not observed in the h nmr spectrum , which is attributed to its rapid exchange with solvent .
significantly , for the duplexes containing the tgg or tga base pairs , the g n3h imino or anh proton resonances , respectively , were not observed , suggesting that they also undergo rapid exchange with solvent .
the thermodynamic measurements indicate that both the duplexes containing the tga or tgg base pairs have similar 13 c decreases in thermal melting temperatures , and both exhibit similar 3 kcal / mol g values , which result from enthalpy - driven reductions in duplex stabilities ( table 3 ) .
notably , when the 5r tg lesion was examined in the 5-atga-3 sequence , paired opposite da , it was concluded that it induced a significant , localized structural change with the tg being largely extrahelical . in that instance , an extrahelical orientation of tg was concluded from the observation that it exhibited an increased solvent accessible surface area relative to thymidine .
it had also been reported that the structure of the 5r tg lesion in the 5-gtgc-3 sequence was disordered .
it seems plausible that the orientation of tg may be dependent upon dna sequence , as well as the identity of the complementary nucleotide . the structural refinement of tg in the present sequence contexts , when placed opposite either da or dg , will now be of considerable interest .
the evidently weak hydrogen bonding interactions between the tga or tgg base pairs in these oligodeoxynucleotide duplexes suggests that differences in stacking interactions and/or steric interactions involving the opposing purine and the flanking base pairs play an important role in mediating the position of the equilibrium between the cis 5r,6s and trans 5r,6r epimers .
clark et al . concluded that the cis 5r,6s epimer favors the axial conformation of the tg ch3 group .
in addition , the present dft calculations predict that the trans 5r,6r epimer with the tg ch3 group in the equatorial conformation is the second most energetically favorable configuration ( table 2 ) .
this may be important in determining the equilibrium between the cis 5r,6s and trans 5r,6r epimers in duplex dna .
for the duplex containing the tga base pair , tg provides a reasonable steric match for the normal ta pair with a. epimerization to the trans 5r,6r epimer might allow the tg ch3 group to shift to the equatorial conformation , which might reduce its steric clash with the 5-neighbor nucleotide g ( figure 8) .
in contrast , when placed opposite dg in the tgg pair , it seems possible that tg is oriented similarly to a wobble gt pair ; detailed nmr studies to examine this hypothesis are in progress
. this would shift tg toward the major groove , as compared to its orientation when placed opposite a , and would enable the tg ch3 group to be maintained in the energetically more favorable axial conformation , consistent with the observation that in the tgg pair the cis 5r,6s epimer is favored ( figure 8) .
this hypothesis also draws support from the observation that tg is weakly mutagenic , causing < 0.5% tc transitions , which also could be explained by the formation of gtg wobble pairs during error - prone translesion dna replication .
( a ) insertion of the cis-5r,6s tg lesion into the duplex opposite da , in a watsoncrick orientation , with the axial conformation of tg ch3 , induces steric clash with the 5 neighbor , g. ( b ) epimerization of the cis-5r,6s tg lesion to the trans-5r,6r lesion allows the tg ch3 to assume an equatorial conformation that causes less steric interaction with tg ch3 and the g nucleotide .
( c ) formation of a wobble tgg base pair allows tg to be displaced toward the major groove , resulting in less steric clash between the axial conformation of tg ch3 in the cis-5r,6s tg configuration and the 5-neighbor , g. the presence of the 5r tg lesion in the duplexes containing the tga or tgg base pairs perturbs the 5-neighbor base pair gc .
this observation is consistent with the predominance of the cis-5r,6s epimer in both instances , and the conclusion that the cis-5r,6s epimer favors the axial conformation of the tg ch3 group .
this is corroborated by the observation that the imino resonance attributed to base pair gc broadens due to solvent exchange and disappears from the h nmr spectrum 35 c lower in the 5r - tg modified dna as compared to the corresponding unmodified oligodeoxynucleotide duplex ( figure 3 ) .
for the duplex containing the tga pair , nmr analysis of both the exchangeable tg amine and an amine resonances suggest that these protons undergo increased exchange with solvent . taken together ,
similarly , for the oligodeoxynucleotide containing the tgg mismatch , the tm was reduced by 13 c .
although it has been recognized that tg exists in dna as two diastereomeric pairs of epimers , solution structural studies of the 5r tg lesion ( no structural studies of the 5s tg lesion have been conducted ) have not commented upon this equilibrium .
consequently , it will be of interest to re - evaluate the solution structures of these lesions , and the structural refinement of the two duplexes discussed herein , is in progress .
the structure of a binary primer - template complex with the replicative rb69 dna polymerase , representing the situation immediately following incorporation of datp opposite tg , revealed the presence of the cis-5r,6s tg epimer at the active site .
the cis-5r,6s tg epimer was intrahelical and was positioned to form a watsoncrick base pair with the da at the primer 3-terminus .
this confirmed modeling studies in which the cis-5r,6s tg epimer was predicted to pair with da .
the tg methyl group was in the axial conformation , hindering stacking of the adjacent 5-template guanine .
tg blocks dna replication by both replicative and repair polymerases . with either the klenow fragment of e. coli dna polymerase i or t4 dna polymerase , extension past the 5r tg lesion is prohibited , as opposed to dntp insertion
however , pyrimidines 5 to template tg allow residual polymerase read - through more often than do purines . if not repaired , the 5r tg lesion is lethal to cells .
the interconversion of the 5r tg lesion between the cis-5r,6s and trans-5r,6r epimers , and the observation that the position of this equilibrium depends upon the identity of the nucleotide placed opposite to the 5r tg lesion is likely to influence the recognition and repair of these lesions in duplex dna .
it is tempting to speculate that hneil1s ability to release tg much more efficiently in a tgg pair compared to a tga pair may be related to its exclusive cis stereochemistry in the former pair .
by contrast , the rate of release of tg from tga is greater by hnth1 compared to tgg , when [ s ] [ e ] but not when [ e ] [ s ] , which could be due to product inhibition .
it has been proposed that hnth1 and hneil1 only release the 5r ( or 5s ) tg lesions as either the cis or trans epimers , and that under single turnover conditions , the interconversion of the two epimers represents the rate - limiting step for complete excision of tg .
this notion would imply that under single turnover conditions , the efficiency of excision of tg should depend upon the position of the equilibrium between tg epimers at specific sites in dna , and that the conditions that modulate this equilibrium in duplex dna should be further characterized .
for the 5r tg lesion , the equilibrium between the cis-5r,6s - and trans-5r,6r- epimers depends upon the identity of the purine in the complementary strand . for the duplex containing the tga pair , the equilibrium ratio of cis-5r,6s : trans-5r,6r epimers was 7:3 at 30 c .
in contrast , for a duplex containing the tgg pair , the cis-5r,6s : trans-5r,6r equilibrium strongly favored the cis-5r,6s epimer ; the level of the trans-5r,6r epimer remained below the level of detection by nmr .
the observations that the cis-5r,6s - thymine glycol lesion exists in equilibrium with its trans-5r,6r epimer in duplex dna and that the position of this equilibrium is affected by the complementary base extends upon observations that this equilibrium modulates the biological processing of tg .
the oligodeoxynucleotides 5-d(gtgcgtgtttgt)-3 , 5-d(acaaacgcgcac)-3 and 5-d(acaaacacgcac)-3 , purified by anion exchange chromatography , were purchased from the midland certified reagent co. ( midland , tx ) .
the tg - dodecamer 5-d(gtgcgtggtttgt)-3 , tg = 5r tg , was synthesized and purified as reported .
complementary oligodeoxynucleotides were annealed in 20 mm sodium phosphate buffer , containing 100 mm nacl , 10 mm nan3 , and 50 m na2edta ( ph 7.0 ) , heated to 70 c for 10 min and subsequently cooled to ambient temperature .
samples were suspended in a matrix consisting of 0.5 m 3-hydroxypicolinic acid in 1:1 ch3cn : h2o and spotted onto sample plates .
the accelerating voltage was 20 kv , with a grid voltage of 85.00% , guide wire voltage of 0.050% , and a delay of 100 ns .
dna purification by hplc was conducted using a phenomenex gemini c-18 column ( 250 mm 10 mm ) .
elution was performed using a linear gradient from 6 to 30% ch3cn over 25 min .
the gels and buffers were prepared using beckman coulter ssdna 100-r kits ( beckman - coulter , inc . ,
fullerton , ca ) . an injection voltage of 10.0 kv was used for 8 s , followed by a separation voltage of 14.1 kv for 35 min , using a 33 cm capillary .
elution times were referenced to an internal standard ( beckman orange g product number 241524 ) .
uv - absorption thermal denaturation experiments were conducted on a cary 4e uvvis spectrophotometer ( varian , inc . ) .
samples were suspended in 10 mm phosphate buffer , containing 500 mm nacl , and 10 mm na2edta ( ph 7.0 ) .
four concentrations of approximately 0.1 , 0.5 , 0.8 , and 1.0 m in 1 cm capped cuvettes were placed in the spectrometer s multicell temperature regulation block along with a blank . during the serial analysis of the samples ,
temperature was increased or decreased , depending on experiment , at a rate of 0.3 c /min and absorbance was measured at 260 nm . temperature was allowed to equilibrate for 5 min at 5 and 80 c prior to each sweep .
thermodynamic parameters were generated by analysis of absorbance vs temperature profiles within in the meltwin ( v3.5 mcdowell ) and thermal ( v 2.0 cary ) applications ( varian instruments , palo alto , ca ) .
the thermodynamic values ( h and s ) were obtained either from individual melting curves , or from vant hoff analysis ( sigmaplot v9.0 ) gibbs free energies ( g ) were determined at 25 and 37 c .
oligodeoxynucleotide ( 0.20.5 a260 unit ) was dissolved in 30 l 100 mm tris - hcl buffer containing 10 mm mgcl2 ( ph 7.0 ) and incubated with dnase i ( 8 u , promega ) , snake venom phosphodiesterase i ( 0.02 u , sigma aldrich ) , and e. coli alkaline phosphatase ( 1.7 u ; sigma aldrich ) at 37 c for 90 min . the product mixture was analyzed by hplc using a waters ymc ods - aq column ( 250 mm 4.6 mm i.d . ) .
elution was performed using a 15 min linear gradient from 1% to 10% ch3cn , followed by a 5 min linear gradient from 10 to 20% ch3cn .
adducted nucleosides were identified by comparison with authentic samples based on retention times and uv spectra .
sample concentrations were 1.5 mm , in 20 mm sodium phosphate , 100 mm nacl ( ph 7.0 ) . to examine nonexchangeable protons ,
samples were suspended in 99.996% d2o . for observation of exchangeable protons , samples were suspended in 9:1 h2o : d2o .
the nonexchangeable proton experiments were performed at 30 0.5 c , exchangeable proton experiments were performed at 5 0.5 c .
noesy spectra for the nonexchangeable protons were recorded at mixing times of 80 , 150 , 200 , and 250 ms .
typical acquisition parameters for nmr experiments were as follows : 512 real data points in the d1 dimension with 32 scans per fid , 2k real data points in the d2 dimension , sweep width of 10 ppm , and a relaxation delay of 2.0 s. the residual water resonance was suppressed using presaturation .
noesy spectra of exchangeable protons were obtained using watergate h2o suppression and a sweep width of 20 ppm .
the binomial water suppression delay of 188 s at 800 mhz ( 158 s at 500 mhz ) was selected to avoid suppression of amino resonances .
the programs xwinnmr ( v 3.5 patch level 6 , bruker inc . , billerica , ma ) and nmrpipe were used for data processing .
geometry optimization and frequency calculations were performed using the b3lyp density functional method with the 631 g * , 631 g * * , 631+g * , and 6311++g * * basis sets .
potential points were written out with a density of 6 points per unit area for an electrostatic potential ( esp ) fit .
coordinates were chosen for cis tg ( ch3 axial ) , cis tg ( ch3 equatorial ) , trans tg ( ch3 axial ) , trans tg ( ch3 equatorial ) , and the aldehyde intermediate . in all cases , | thymine glycol ( tg ) , 5,6-dihydroxy-5,6-dihydrothymine , is formed in dna by the reaction of thymine with reactive oxygen species .
the 5r tg lesion was incorporated site - specifically into 5-d(g1t2g3c4g5tg6g7t8t9t10g11t12)-3 ; tg = 5r tg .
the tg - modified oligodeoxynucleotide was annealed with either 5-d(a13c14a15a16a17c18a19c20g21c22a23c24)-3 , forming the tg6a19 base pair , corresponding to the oxidative damage of thymine in dna , or 5-d(a13c14a15a16a17c18g19c20g21c22a23c24)-3 , forming the mismatched tg6g19 base pair , corresponding to the formation of tg following oxidative damage and deamination of 5-methylcytosine in dna . at 30 c , the equilibrium ratio of cis-5r,6s : trans-5r,6r epimers was 7:3 for the duplex containing the tg6a19 base pair .
in contrast , for the duplex containing the tg6g19 base pair , the cis-5r,6s : trans-5r,6r equilibrium favored the cis-5r,6s epimer ; the level of the trans-5r,6r epimer remained below the level of detection by nmr .
the data suggested that tg disrupted hydrogen bonding interactions , either when placed opposite to a19 or g19 .
thermodynamic measurements indicated a 13 c reduction of tm regardless of whether tg was placed opposite dg or da in the complementary strand . although both pairings increased the free energy of melting by 3 kcal / mol , the melting of the tgg pair was more enthalpically favored than was the melting of the tga pair .
the observation that the position of the equilibrium between the cis-5r,6s and trans-5r,6r thymine glycol epimers in duplex dna was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of tg [ ocampo - hafallam .
t. ; altamiranoa . ; basua .
k. ; chanm .
k. ; ocampoj .
e. ; cummingsa.jr . ; boorsteinr .
j. ; cunninghamr .
p. ; teeborg .
w.dna repair ( amst)2006 , 5 , 444454 ] . | Introduction
Results
Discussion
Summary
Experimental Section | presently , the 5r - tg adduct has been incorporated site - specifically into 5-d(gtgcgtggtttgt)-3 and annealed with either 5-d(acaaacacgcac)-3 , forming a duplex containing the tga base pair , corresponding to the oxidative damage of thymine in dna , or 5-d(acaaacgcgcac)-3 , forming a duplex containing the mismatched tg : g base pair , corresponding to the formation of tg following oxidative damage and deamination of 5-methylcytosine in dna . for the duplex containing the tga pair ,
the cis-5r,6s and trans-5r,6r epimers exist in a cis : trans ratio of 7:3 at 30 c . in contrast , for the duplex containing the tg : g pair , the cis : trans equilibrium strongly favors the cis-5r,6s epimer ; the level of the trans-5r,6r epimer remains below the level of detection by nmr . these data suggest that the potentially significant levels of the trans-5r,6r tg epimer in duplex dna should not be ignored with respect to the biological processing of the 5r tg lesion , corroborating observations that the repair of tg by dna n - glycosylases / ap lyases is modulated by the cis - trans epimerization . the two tg species observed for the duplex containing the tga base pair were assigned as arising from slow exchange ( nmr time scale ) between the cis-5r,6s and trans-5r,6r epimers . however , the melting of the duplex containing the tgg pair was enthalpically favored as compared to the duplex containing the tga base pair . ( b ) vant hoff plots of tm vs ln(ct/4 ) for the duplex containing the unmodified ta base pair ( ) , the tga base pair ( ) , and the tgg base pair ( ) . overall , we conclude that depending upon the identity of the complementary nucleotide , significant levels of the trans 5r,6r epimer may be present in duplex dna , and that the 5r tg lesion should be considered to exist in duplex dna as an equilibrium mixture of the two epimers . the present data also corroborate the conclusion that tg disrupts hydrogen bonding interactions , either when placed opposite to a or g. the tg n3 imino proton is not observed in the h nmr spectrum , which is attributed to its rapid exchange with solvent . in contrast , when placed opposite dg in the tgg pair , it seems possible that tg is oriented similarly to a wobble gt pair ; detailed nmr studies to examine this hypothesis are in progress
. ( c ) formation of a wobble tgg base pair allows tg to be displaced toward the major groove , resulting in less steric clash between the axial conformation of tg ch3 in the cis-5r,6s tg configuration and the 5-neighbor , g. the presence of the 5r tg lesion in the duplexes containing the tga or tgg base pairs perturbs the 5-neighbor base pair gc . the interconversion of the 5r tg lesion between the cis-5r,6s and trans-5r,6r epimers , and the observation that the position of this equilibrium depends upon the identity of the nucleotide placed opposite to the 5r tg lesion is likely to influence the recognition and repair of these lesions in duplex dna . this notion would imply that under single turnover conditions , the efficiency of excision of tg should depend upon the position of the equilibrium between tg epimers at specific sites in dna , and that the conditions that modulate this equilibrium in duplex dna should be further characterized . for the 5r tg lesion , the equilibrium between the cis-5r,6s - and trans-5r,6r- epimers depends upon the identity of the purine in the complementary strand . for the duplex containing the tga pair , the equilibrium ratio of cis-5r,6s : trans-5r,6r epimers was 7:3 at 30 c . in contrast , for a duplex containing the tgg pair , the cis-5r,6s : trans-5r,6r equilibrium strongly favored the cis-5r,6s epimer ; the level of the trans-5r,6r epimer remained below the level of detection by nmr . the observations that the cis-5r,6s - thymine glycol lesion exists in equilibrium with its trans-5r,6r epimer in duplex dna and that the position of this equilibrium is affected by the complementary base extends upon observations that this equilibrium modulates the biological processing of tg . | [
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] |
it is well established that maternal smoking during pregnancy is associated with lower birth size in offspring .
the evidence relating to the associations between maternal smoking during pregnancy and offspring post - natal growth is , however , less consistent .
some studies have found that height deficits in children born to mothers who smoke during pregnancy persist , whereas others have shown that the gap narrows soon after birth .
more recently , there has also been interest in the potential effects of maternal smoking during pregnancy on offspring obesity . a systematic review and meta - analysis ( n = 84 563 ) showed that smoking during pregnancy is associated with increased odds of being overweight in the offspring [ pooled odds ratio ( or ) 1.50 , 95% confidence interval ( 95% ci ) 1.361.65 ] , with results not attenuated by adjustment for confounders . however , a recent study comparing siblings for whom maternal smoking during pregnancy was discordant ( an approach which can partially control for unmeasured confounders ) concluded that observed associations between smoking during pregnancy and offspring obesity are likely to be confounded by shared familial characteristics rather than causally related to intrauterine mechanisms .
previous publications from the avon longitudinal study of parents and children ( alspac ) have demonstrated associations between maternal smoking during pregnancy and reduced height at the age of 7.5 years , and increased adiposity at the ages of 7 and 9.9 years using single measures of height and adiposity .
we build on this work to explore associations between maternal smoking during pregnancy and individual trajectories of height and adiposity between birth and the age of 10 years .
this will enable us to explore the extent to which differences in height and adiposity at birth between the offspring of smokers and non - smokers are overcome with age , and identify the ages at which any such changes occur . in order to investigate the extent to which any observed associations are due to confounding
first , we control for a wide set of socio - economic and familial variables .
secondly , we compare the associations of maternal smoking during pregnancy with those of her partner 's smoking during pregnancy in order to explore the existence of any unmeasured familial confounders including socio - economic , behavioural and genetic factors .
if the associations of maternal and her partner 's smoking with offspring growth trajectories are similar , this is suggestive of confounding by familial factors rather than any causal intrauterine effect of maternal smoking . as proof of principle of the validity of this approach
, we have previously shown that the association of maternal smoking in pregnancy with offspring birthweight ( an association accepted as causal via intrauterine factors ) is considerably stronger than the association of partner smoking with offspring birthweight in alspac .
finally , we compare the growth trajectories of offspring of women who smoke during pregnancy with those of women who do not smoke during pregnancy but ( re-)start soon after delivery , since if the growth patterns in these two groups are similar then confounding is more likely .
pregnant women resident in one of the three bristol - based health districts with an expected delivery date between april 1 , 1991 and december 31 , 1992 were invited to participate . of these women ,
14 541 were recruited ; there were 14 062 live - born children , 13 988 of whom were alive at 1 year .
follow - up has included parent- and child - completed questionnaires , links to routine data and clinic attendance .
ethical approval was obtained from the alspac law and ethics committee and the local research ethics committees .
dichotomous indicators of any / no smoking during pregnancy were based on self - report data , the details of which are found in the supplementary data , available as supplementary data at ije online .
in addition to the dichotomous variables , a three - category measure of smoking dose was created for both mothers and their partners ; individuals were classified as non - smokers , light smokers ( 10 cigarettes / day ) and heavy smokers ( > 10 cigarettes / day ) .
a three - category indicator of maternal post - natal smoking status was created : did not smoke during pregnancy and had not ( re-)started smoking by 8 weeks post delivery , did not smoke during pregnancy but had ( re-)started smoking by 8 weeks post delivery , or smoked during pregnancy .
height and weight data were available for alspac participants from several sources ; measurement methods and number of measures per child are detailed in the supplementary data , available as supplementary data at ije online .
within alspac , the only measures of adiposity repeated across the whole of childhood were those based on height and weight . body mass index ( bmi , kg / m ) is the most common way of adjusting weight for height .
patterns of bmi change in early childhood are extremely complicated . given this , we decided not to model bmi from birth .
rather , ponderal index ( pi , kg / m ) was used as the measure of adiposity from birth to the age of 2 years .
individual trajectories of height between the ages 0 and 10 years , pi between the ages 0 and 2 years and bmi between the ages 2 and 10 years were estimated using random - effects linear spline models ( two levels : measurement occasion and individual ) .
these models allow for the change in scale and variance of growth measures over time and use all available data from all eligible children under a missing at random assumption .
they allow for individual variation in trajectories , since random effects allow each individual to have different intercepts and slopes ( rates of growth in each linear spline period ) .
trajectories were modelled separately for boys and girls , and not beyond the age of 10 years since puberty would necessitate individual spline points due to variation in age at puberty onset .
full statistical methodology is in the supplementary data , available as supplementary data at ije online .
variables considered as potential confounders were maternal education , household occupational social class , parity , maternal age , maternal height , maternal bmi , gestational age at birth and breastfeeding .
measurements are detailed in the supplementary data , available as supplementary data at ije online .
associations between maternal / partner smoking during pregnancy and confounders were assessed by tabulations and logistic regressions .
analyses were restricted to children alive at the age of 1 year , with at least one measure of height / adiposity between the ages 0 and 10 years , data on maternal and her partner 's smoking during pregnancy and all confounders .
where relevant , analyses were further restricted to those with dose / maternal post - natal smoking data .
associations between growth trajectories and ( i ) maternal / partner smoking during pregnancy , ( ii ) maternal / partner smoking dose and ( iii ) maternal post - natal smoking were modelled by including interaction term(s ) in the random - effects models between the smoking variables and the intercept ( birth length , pi at birth , or bmi at the age of 24 months ) and each growth coefficient ( rate of height growth , pi change or bmi change in each linear spline period ) . an example model is shown in the supplementary data , available as supplementary data at ije online .
analyses were carried out using the statistical packages stata11 , mlwin v2.24 and the stata command
the eligible sample for the main analysis was 9424 offspring for height models , 9321 for pi models and 8887 for bmi models ( 6467% of the cohort members alive at the age of 1 year ) .
of the mothers included in our analysis , 20.5% of them and 36.1% of their partners smoked during the pregnancy .
overall , 3471 ( 39% ) of households were discordant for parental smoking ( further details in supplementary data , available as supplementary data at ije online ) .
sample sizes and prevalences for dose response and post - natal smoking analysis are detailed in supplementary data and supplementary tables s1 and s2 , available as supplementary data at ije online .
although the participants included in our analyses were of higher socio - economic position than those excluded ( supplementary table s3 , available as supplementary data at ije online ) , the association between maternal smoking during pregnancy and birth length did not differ between participants included in our analyses and those excluded due to missing data on confounders ( supplementary table s4 , available as supplementary data at ije online ) .
women who smoked during pregnancy tended to be of lower socio - economic position , higher parity , younger age and shorter height than those who did not ( supplementary table s5 , available as supplementary data at ije online ) ; similar associations with these characteristics were observed for partner smoking ( supplementary table s6 , available as supplementary data at ije online ) .
there was some evidence that associations with maternal education , age and height and breastfeeding were stronger for partner smoking than maternal smoking ; the reverse was true for household social class and no differences were observed for parity , gestational age or maternal bmi ( supplementary table s7 , available as supplementary data at ije online ) .
the multi - level models identified ( supplementary figures s1s3 , available as supplementary data at ije online ) :
four periods of length / height growth ( boys : birth to 3 , 310 , 1029 , 29120 months ; girls : birth to 2 , 211 , 1132 , 32120 months);two periods of pi change in boys ( birth to 2 and 224 months);three periods of pi change in girls ( birth to 1 , 14 and 424 months ) ; andsix periods of bmi change ( boys : 2456 , 5667 , 6773 , 7379 , 79105 and 105120 months ; girls : 2460 , 6065 , 6575 , 7581 , 81103 and 103120 months ) .
four periods of length / height growth ( boys : birth to 3 , 310 , 1029 , 29120 months ; girls : birth to 2 , 211 , 1132 , 32120 months ) ; two periods of pi change in boys ( birth to 2 and 224 months ) ; three periods of pi change in girls ( birth to 1 , 14 and 424 months ) ; and six periods of bmi change ( boys : 2456 , 5667 , 6773 , 7379 , 79105 and 105120 months ; girls : 2460 , 6065 , 6575 , 7581 , 81103 and 103120 months ) . maternal smoking during pregnancy
is associated with lower birth length ( tables 1 and 2 ) and this association remains after adjustment for confounders and after mutual adjustment for partner 's smoking .
in fully adjusted models , maternal smoking is associated with a 0.67 cm ( se = 0.09 ) lower birth length in girls and 0.63 cm ( se = 0.08 ) in boys . in unadjusted models ,
partner 's smoking is also associated with lower birth length , but this is attenuated by adjustment for confounders and maternal smoking ( table 3 ) .
there is strong evidence for a statistical difference between the coefficients of maternal and partner 's smoking for birth length ( p < 0.001 ) ( tables 1 and 2 ) . for girls
, there is evidence of a dose response in the relationship between maternal smoking and offspring birth length , with the lower birth length among smokers compared with non - smokers being twice the magnitude for heavy smokers ( 1.02 cm ) compared with light smokers ( 0.53 cm ) ; in boys there was no dose
response , with differences in comparison with non - smokers being the same for heavy and light smokers ( both 0.64 cm ) ( table 4 ) .
there is no reduction in birth length for the offspring of women who do not smoke during pregnancy but ( re-)start within 8 weeks of delivery in either girls or boys ( table 5 ) .
table 1the associations between maternal and partner smoking during pregnancy and trajectories of height , pi and bmi in female offspringmaternal smoking during pregnancypartner smoking during pregnancyp valuemean ( sd ) growth rate per monthcrude coefficient ( se)adjusted coefficient ( se)adjusted coefficient with further adjustment for paternal smoking ( se)crude coefficient ( se)adjusted coefficient ( se)adjusted coefficient with further adjustment for maternal smoking ( se)maternal partner hetero- geneityheight , n = 4592 birth length , cm49.8 ( 1.1)0.7644 ( 0.0910)0.6555 ( 0.0837)0.6680 ( 0.0888)0.2375 ( 0.0759)0.1440 ( 0.0693)0.0367 ( 0.0731)<0.001 growth 1 , cm / month3.9 ( 0.2)0.0403 ( 0.0473)0.0375 ( 0.0470)0.0281 ( 0.0502)0.0308 ( 0.0392)0.0301 ( 0.0389)0.0220 ( 0.0416)0.93 growth 2 , cm / month1.8 ( 0.2)0.0450 ( 0.0136)0.0467 ( 0.0136)0.0430 ( 0.0145)0.0214 ( 0.0113)0.0212 ( 0.0113)0.0083 ( 0.0121)0.07 growth 3 , cm / month0.9 ( 0.09)0.0124 ( 0.0063)0.0125 ( 0.0063)0.0117 ( 0.0068)0.0058 ( 0.0052)0.0055 ( 0.0052)0.0018 ( 0.0056)0.26 growth 4 , cm / month0.5 ( 0.04)0.0066 ( 0.0024)0.0068 ( 0.0024)0.0073 ( 0.0026)0.0009 ( 0.0020)0.0010 ( 0.0020)0.0012 ( 0.0021)0.01pi , n = 4544 pi at birth , kg / m26.7 ( 1.4)0.0680 ( 0.1094)0.0723 ( 0.1102)0.0888 ( 0.0966)0.0007 ( 0.0904)0.0001 ( 0.0907)0.0339 ( 0.0966)0.37 pi change 1 , kg / m / month0.4 ( 1.7)0.3285 ( 0.1592)0.3338 ( 0.1591)0.3813 ( 0.1712)0.0077 ( 0.1320)0.0085 ( 0.1320)0.1079 ( 0.1419)0.03 pi change 2 , kg / m / month0.2 ( 0.6)0.0252 ( 0.0589)0.0243 ( 0.0589)0.0264 ( 0.0634)0.0023 ( 0.0486)0.0024 ( 0.0486)0.0048 ( 0.0523)0.70 pi change 3 , kg / m / month0.4 ( 0.07)0.0052 ( 0.0088)0.0053 ( 0.0088)0.0071 ( 0.0094)0.0022 ( 0.0073)0.0022 ( 0.0073)0.0043 ( 0.0078)0.35bmi , n = 4353 ( kg / m / month ) bmi change 10.01 ( 0.02)0.0026 ( 0.0040)0.0024 ( 0.0040)0.0041 ( 0.0043)0.0019 ( 0.0033)0.0022 ( 0.0033)0.0034 ( 0.0036)0.18 bmi change 20.06 ( 0.1)0.0236 ( 0.0380)0.0240 ( 0.0381)0.0032 ( 0.0408)0.0555 ( 0.0300)0.0570 ( 0.0300)0.0583 ( 0.0323)0.24 bmi change 30.02 ( 0.08)0.0375 ( 0.0319)0.0372 ( 0.0319)0.0142 ( 0.0340)0.0540 ( 0.0247)0.0543 ( 0.0247)0.0503 ( 0.0264)0.40 bmi change 40.01 ( 0.1)0.0108 ( 0.0352)0.0107 ( 0.0353)0.0273 ( 0.0375)0.0291 ( 0.0272)0.0293 ( 0.0272)0.0367 ( 0.0290)0.18 bmi change 50.07 ( 0.03)0.0008 ( 0.0036)0.0006 ( 0.0036)0.0027 ( 0.0038)0.0042 ( 0.0028)0.0043 ( 0.0028)0.0049 ( 0.0030)0.12 bmi change 60.04 ( 0.03)0.0095 ( 0.0043)0.0096 ( 0.0043)0.0078 ( 0.0046)0.0064 ( 0.0034)0.0064 ( 0.0034)0.0042 ( 0.0036)0.54coefficients are for interactions between smoking during pregnancy and the intercept ( birth length , pi at birth or bmi at the age of 2 years ) or slope ( growth rate in each linear spline period ) , that is they represent the additive effect of maternal or partner smoking during pregnancy on the intercept or growth rate
. slopes are centimetre per month for height growth , kilograms per cubic metre per month for pi and kilograms per square metre per month for bmi.adjusted for maternal education , household social class , parity , maternal age , maternal height , maternal bmi , gestational age at birth and breastfeeding.p - values are for heterogeneity tests of the difference of maternal partner coefficients , with mutual adjustment for maternal / partner smoking.height growth period 1 : birth to 2 months ; height growth period 2 : 211 months ; height growth period 3 : 1132 months ; height growth period 4 : 32120 months ; pi change period 1 : birth to 1 month ; pi change period 2 : 14 months ; pi change period 3 : 424 months ; bmi change period 1 : 2456 months ; bmi change period 2 : 5667 months ; bmi change period 3 : 6773 months ; bmi change period 4 : 7379 months ; bmi change period 5 : 79105 months ; bmi change period 6 : 105120 months .
table 2the associations between maternal and partner smoking during pregnancy and trajectories of height , pi and bmi in male offspringmaternal smoking during pregnancypartner smoking during pregnancyp - valuemean ( sd ) growth rate per monthcrude coefficient ( se)adjusted coefficient ( se)adjusted coefficient with further adjustment for paternal smoking ( se)crude coefficient ( se)adjusted coefficient ( se)adjusted coefficient with further adjustment for maternal smoking ( se)maternal partner hetero- geneityheight , n = 4832 birth length , cm50.7 ( 1.2)0.6849 ( 0.0891)0.5865 ( 0.0785)0.6275 ( 0.0830)0.0873 ( 0.0765)0.0590 ( 0.0664)0.1095 ( 0.0698)<0.001 growth 1 , cm / month3.8 ( 0.2)0.0155 ( 0.0401)0.0198 ( 0.0396)0.0262 ( 0.0424)0.0098 ( 0.0339)0.0078 ( 0.0335)0.0152 ( 0.0358)0.46 growth 2 , cm / month1.7 ( 0.2)0.0497 ( 0.0183)0.0504 ( 0.0183)0.0476 ( 0.0196)0.0199 ( 0.0155)0.0210 ( 0.0155)0.0063 ( 0.0166)0.11 growth 3 , cm / month1.0 ( 0.1)0.0054 ( 0.0065)0.0055 ( 0.0065)0.0033 ( 0.0069)0.0053 ( 0.0055)0.0063 ( 0.0055)0.0052 ( 0.0059)0.83 growth 4 , cm / month0.5 ( 0.04)0.0027 ( 0.0022)0.0025 ( 0.0023)0.0020 ( 0.0024)0.0019 ( 0.0018)0.0018 ( 0.0019)0.0012 ( 0.0020)0.80pi , n = 4777 pi at birth , kg / m26.2 ( 1.2)0.2557 ( 0.1013)0.2296 ( 0.1022)0.2059 ( 0.1087)0.1298 ( 0.0865)0.1233 ( 0.0867)0.0569 ( 0.0921)0.30 pi change 1 , kg / m / month0.5 ( 0.8)0.2482 ( 0.0678)0.2494 ( 0.0678)0.2311 ( 0.0726)0.1125 ( 0.0580)0.1145 ( 0.0580)0.0436 ( 0.0621)0.05 pi change 2 , kg / m / month0.4 ( 0.08)0.0055 ( 0.0076)0.0053 ( 0.0076)0.0048 ( 0.0081)0.0026 ( 0.0065)0.0027 ( 0.0065)0.0011 ( 0.0069)0.73bmi , n = 4534 ( kg / m / month ) bmi change 10.01 ( 0.05)0.0118 ( 0.0156)0.0114 ( 0.0156)0.0117 ( 0.0169)0.0035 ( 0.0120)0.0031 ( 0.0119)0.0003 ( 0.0130)0.57 bmi change 20.03 ( 0.02)0.0081 ( 0.0092)0.0091 ( 0.0092)0.0079 ( 0.0101)0.0054 ( 0.0072)0.0054 ( 0.0072)0.0027 ( 0.0078)0.68 bmi change 30.03 ( 0.04)0.0108 ( 0.0153)0.0087 ( 0.0153)0.0154 ( 0.0164)0.0108 ( 0.0120)0.0103 ( 0.0120)0.0144 ( 0.0129)0.15 bmi change 40.02 ( 0.04)0.0171 ( 0.0174)0.0163 ( 0.0174)0.0095 ( 0.0186)0.0179 ( 0.0132)0.0180 ( 0.0132)0.0153 ( 0.0141)0.80 bmi change 50.02 ( 0.03)0.0012 ( 0.0043)0.0017 ( 0.0043)0.0006 ( 0.0045)0.0049 ( 0.0033)0.0049 ( 0.0033)0.0050 ( 0.0035)0.33 bmi change 60.05 ( 0.03)0.0054 ( 0.0024)0.0057 ( 0.0024)0.0043 ( 0.0026)0.0044 ( 0.0020)0.0045 ( 0.0020)0.0033 ( 0.0021)0.77coefficients are for interactions between smoking during pregnancy and the intercept ( birth length , pi at birth or bmi at the age of 2 years ) or slope ( growth rate in each linear spline period ) , i.e. they represent the additive effect of maternal or partner smoking during pregnancy on the intercept or growth rate .
slopes are centimetre per month for height growth , kilograms per cubic metre per month for pi , and kilograms per square metre per month for bmi.adjusted for maternal education , household social class , parity , maternal age , maternal height , maternal bmi , gestational age at birth and breastfeeding.p - values are for heterogeneity tests of the difference of maternal partner coefficients , with mutual adjustment for maternal / partner smoking.height growth period 1 : birth to 3 months ; height growth period 2 : 310 months ; height growth period 3 : 1029 months ; height growth period 4 : 29120 months ; pi change period 1 : birth to 2 months ; pi change period 2 : 224 months ; bmi change period 1 : 2460 months ; bmi change period 2 : 6065 months ; bmi change period 3 : 6575 months ; bmi change period 4 : 7581 months ; bmi change period 5 : 81103 months ; bmi change period 6 : 103120 months .
table 3predicted differences in height and adiposity between the offspring of smokers and non - smokerspredicted change ( se ) comparing offspring of smokers with non - smokerspredicted measurement for offspring of non - smokers ( se)maternal smokingpartner smokinggirls height ( cm ) birth50.75 ( 0.11)0.67 ( 0.09)+0.04 ( 0.07 ) age 2 years86.52 ( 0.12)0.37 ( 0.12)+0.13 ( 0.10 ) age 10 years140.67 ( 0.16)1.11 ( 0.27)+0.22 ( 0.22)adiposity pi at birth ( kg / m)26.37 ( 0.12)0.09 ( 0.10)+0.03 ( 0.10 ) bmi at age 2 years ) ( kg / m)16.55 ( 0.10)+0.14 ( 0.10)+0.05 ( 0.08 ) bmi at age 10 years17.79 ( 0.11)+0.39 ( 0.14)+0.35 ( 0.11)boys height ( cm ) birth51.55 ( 0.10)0.63 ( 0.08)+0.11 ( 0.07 ) age 2 years88.33 ( 0.11)0.26 ( 0.12)+0.04 ( 0.10 ) age 10 years141.53 ( 0.15)0.46 ( 0.26)0.10 ( 0.22)adiposity pi at birth ( kg / m)25.86 ( 0.10)0.21 ( 0.11)0.06 ( 0.09 ) bmi at age 2 years16.85 ( 0.19)0.14 ( 0.48)+0.07 ( 0.37 ) bmi at age 10 years16.22 ( 0.08)+0.24 ( 0.08)+0.10 ( 0.07)values are predicted from the multi - level models , based on models adjusted for confounders and mutually adjusted for both maternal and partner 's smoking .
height / lengths are in centimetres , pi is in kilograms cubic per metre , bmi is in kilograms per square metre .
table 4dose response in predicted differences in height and adiposity between the offspring of smokers and non - smokerspredicted change ( se ) comparing offspring of smokers with non - smokerspredicted measurement for offspring of non - smokers ( se)light maternal smoking : 10 cigarettes / dayheavy maternal smoking : > 10 cigarettes / daylight partner smoking : 10 cigarettes / dayheavy partner smoking : > 10 cigarettes / daygirls height ( cm ) n = 3351 birth50.39 ( 0.14)0.53 ( 0.12)1.02 ( 0.15)+0.04 ( 0.11)+0.11 ( 0.10 ) age 2 years86.23 ( 0.15)0.21 ( 0.17)0.31 ( 0.22)0.08 ( 0.16)0.01 ( 0.15 ) age 10 years140.55 ( 0.20)0.74 ( 0.38)1.70 ( 0.51)0.31 ( 0.34)0.19 ( 0.33)adiposity n = 3189 pi at birth ( kg / m)26.25 ( 0.15)+0.01 ( 0.17)0.53 ( 0.21)0.06 ( 0.15)+0.08 ( 0.14 ) bmi at age 2 years ( kg / m)16.49 ( 0.12)+0.29 ( 0.14)0.15 ( 0.17)+0.10 ( 0.13)+0.05 ( 0.11 ) bmi at age 10 years17.73 ( 0.13)+0.22 ( 0.19)+0.47 ( 0.25)+0.29 ( 0.17)+0.43 ( 0.17)boys height ( cm ) n = 3544 birth51.11 ( 0.15)0.64 ( 0.12)0.64 ( 0.16)+0.18 ( 0.11)+0.03 ( 0.11 ) age 2 years87.94 ( 0.15)0.06 ( 0.17)0.27 ( 0.23)+0.09 ( 0.16)0.26 ( 0.15 ) age 10 years141.26 ( 0.20)0.24 ( 0.36)0.73 ( 0.50)0.04 ( 0.33)0.33 ( 0.32)adiposity n = 3288 pi at birth ( kg / m)25.83 ( 0.14)0.07 ( 0.15)0.37 ( 0.20)+0.04 ( 0.14)0.09 ( 0.14 ) bmi at age 2 years16.89 ( 0.25)+0.40 ( 0.67)2.10 ( 1.02)+0.45 ( 0.58)+0.24 ( 0.52 ) bmi at age 10 years16.23 ( 0.11)+0.09 ( 0.11)+0.14 ( 0.16)0.08 ( 0.10)+0.21 ( 0.10)values are predicted from the multi - level models , based on models adjusted for confounders and mutually adjusted for both maternal and partner s smoking .
height / lengths are in centimetres , pi is in kilograms per cubic metre , bmi is in kilograms per square metre .
maternal smoking dose calculated from the highest smoking level reported from three antenatal questionnaires . partner smoking dose calculated from reported smoking behaviour in a questionnaire at 18 weeks ' gestation .
table 5differences in predicted differences in height and adiposity between the offspring of women who do not smoke during or after pregnancy , those who stop smoking during pregnancy but restart soon afterwards , and those who smoke during pregnancypredicted change ( se ) comparing offspring with those whose mothers are non - smokerspredicted measurement for offspring of non - smokers ( se)restarters : no smoking during pregnancy but resumed within 8 weeks of deliverypregnancy smokersgirls height ( cm ) n = 4302 birth50.55 ( 0.13)+0.20 ( 0.20)0.66 ( 0.08 ) age 2 years86.31 ( 0.13)+0.17 ( 0.30)0.27 ( 0.12 ) age 10 years141.04 ( 0.17)0.08 ( 0.69)0.96 ( 0.27)adiposity n = 4095 pi at birth ( kg / m)26.25 ( 0.13)0.10 ( 0.28)0.05 ( 0.12 ) bmi at age 2 years ( kg / m)16.53 ( 0.11)0.25 ( 0.24)+0.15 ( 0.10 ) bmi at age 10 years17.83 ( 0.12)0.11 ( 0.35)+0.54 ( 0.14)boys height ( cm ) n = 4503 birth51.21 ( 0.13)+0.33 ( 0.20)0.55 ( 0.08 ) age 2 years87.96 ( 0.13)+0.08 ( 0.29)0.26 ( 0.12 ) age 10 years141.20 ( 0.16)0.23 ( 0.62)0.60 ( 0.25)adiposity n = 4245 pi at birth ( kg / m)25.92 ( 0.12)+0.25 ( 0.27)0.17 ( 0.11 ) bmi at age 2 years16.84 ( 0.20)+0.19 ( 1.05)0.26 ( 0.47 ) bmi at age 10 years16.22 ( 0.10)+0.26 ( 0.20)+0.24 ( 0.08)values are predicted from the multi - level models , based on models adjusted for confounders and mutually adjusted for both maternal and partner s smoking .
height / lengths are in centimetres , pi is in kilograms per cubic metre , bmi is in kilograms square per .
non - smokers are those who reported no smoking in any of the three antenatal questionnaires .
restarters are those who reported no smoking in any of the three antenatal questionnaires but who reported that they had ( re-)started smoking since the birth in a questionnaire at 8 weeks after delivery .
pregnancy smokers are those who reported smoking at any time during pregnancy in the three antenatal questionnaires .
the associations between maternal and partner smoking during pregnancy and trajectories of height , pi and bmi in female offspring coefficients are for interactions between smoking during pregnancy and the intercept ( birth length , pi at birth or bmi at the age of 2 years ) or slope ( growth rate in each linear spline period ) , that is they represent the additive effect of maternal or partner smoking during pregnancy on the intercept or growth rate .
slopes are centimetre per month for height growth , kilograms per cubic metre per month for pi and kilograms per square metre per month for bmi . adjusted for maternal education , household social class , parity , maternal age , maternal height , maternal bmi , gestational age at birth and breastfeeding .
p - values are for heterogeneity tests of the difference of maternal partner coefficients , with mutual adjustment for maternal / partner smoking .
height growth period 1 : birth to 2 months ; height growth period 2 : 211 months ; height growth period 3 : 1132 months ; height growth period 4 : 32120 months ; pi change period 1 : birth to 1 month ; pi change period 2 : 14 months ; pi change period 3 : 424 months ; bmi change period 1 : 2456 months ; bmi change period 2 : 5667 months ; bmi change period 3 : 6773 months ; bmi change period 4 : 7379 months ; bmi change period 5 : 79105 months ; bmi change period 6 : 105120 months .
the associations between maternal and partner smoking during pregnancy and trajectories of height , pi and bmi in male offspring coefficients are for interactions between smoking during pregnancy and the intercept ( birth length , pi at birth or bmi at the age of 2 years ) or slope ( growth rate in each linear spline period ) , i.e. they represent the additive effect of maternal or partner smoking during pregnancy on the intercept or growth rate .
slopes are centimetre per month for height growth , kilograms per cubic metre per month for pi , and kilograms per square metre per month for bmi . adjusted for maternal education , household social class , parity , maternal age , maternal height , maternal bmi , gestational age at birth and breastfeeding .
p - values are for heterogeneity tests of the difference of maternal partner coefficients , with mutual adjustment for maternal / partner smoking .
height growth period 1 : birth to 3 months ; height growth period 2 : 310 months ; height growth period 3 : 1029 months ; height growth period 4 : 29120 months ; pi change period 1 : birth to 2 months ; pi change period 2 : 224 months ; bmi change period 1 : 2460 months ; bmi change period 2 : 6065 months ; bmi change period 3 : 6575 months ; bmi change period 4 : 7581 months ; bmi change period 5 : 81103 months ; bmi change period 6 : 103120 months . predicted differences in height and adiposity between the offspring of smokers and non - smokers values are predicted from the multi - level models , based on models adjusted for confounders and mutually adjusted for both maternal and partner 's smoking .
height / lengths are in centimetres , pi is in kilograms cubic per metre , bmi is in kilograms per square metre .
response in predicted differences in height and adiposity between the offspring of smokers and non - smokers values are predicted from the multi - level models , based on models adjusted for confounders and mutually adjusted for both maternal and partner s smoking .
height / lengths are in centimetres , pi is in kilograms per cubic metre , bmi is in kilograms per square metre . maternal smoking dose calculated from the highest smoking level reported from three antenatal questionnaires .
partner smoking dose calculated from reported smoking behaviour in a questionnaire at 18 weeks ' gestation .
differences in predicted differences in height and adiposity between the offspring of women who do not smoke during or after pregnancy , those who stop smoking during pregnancy but restart soon afterwards , and those who smoke during pregnancy values are predicted from the multi - level models , based on models adjusted for confounders and mutually adjusted for both maternal and partner s smoking .
height / lengths are in centimetres , pi is in kilograms per cubic metre , bmi is in kilograms square per .
non - smokers are those who reported no smoking in any of the three antenatal questionnaires .
restarters are those who reported no smoking in any of the three antenatal questionnaires but who reported that they had ( re-)started smoking since the birth in a questionnaire at 8 weeks after delivery .
pregnancy smokers are those who reported smoking at any time during pregnancy in the three antenatal questionnaires . in the first year of life , the offspring of women who smoke during pregnancy grow faster than the offspring of non - smokers ( growth periods 1 and 2 in tables 1 and 2 ) . in girls ,
the association between partner 's smoking and offspring growth in the first few months of life ( growth period 1 in tables 1 and 2 ) is similar to the association with maternal smoking after mutual adjustment ( p - value for heterogeneity is 0.93 ) ; in boys , the coefficients are in opposite directions with maternal smoking associated with faster growth and partner 's smoking associated with slower growth , but there is no statistical evidence of heterogeneity ( p = 0.46 ) and the coefficients have large standard errors in both girls and boys .
the positive association between maternal smoking in pregnancy and growth in later infancy ( growth period 2 in tables 1 and 2 ) is not attenuated by adjustment for measured confounders or mutual adjustment for partner 's smoking .
a positive association for growth in this period is also seen for partner 's smoking , but the magnitude is 4-fold weaker than for maternal smoking , with some evidence of statistical heterogeneity between the maternal and partner coefficients ( p = 0.07 for girls , 0.11 for boys ) . later in childhood , between the ages 1 and 10 years , the offspring of women who smoke grow more slowly in height than those of non - smokers .
this association is weak but not attenuated by measured confounders . in boys , the associations with growth between the ages 1 and 10 years are approximately similar for maternal and partner 's smoking ; in girls , there is some indication of maternal smoking being more strongly associated with slower growth than partner smoking in the final growth period only ( growth period 4 in table 1 ) ; p - value for heterogeneity is 0.01 . by the age of 10 years , the daughter of a woman who smoked during pregnancy is on average 1.11 cm ( se = 0.27 ) shorter than the daughter of a non - smoker once measured confounders and partner 's smoking have been adjusted for ; the difference is 0.22 cm ( se = 0.22 ) for partner 's smoking ( table 3 ) .
the equivalent differences in boys are 0.46 cm ( se = 0.26 ) for maternal smoking and 0.10 cm ( se = 0.22 ) for partner smoking .
the offspring of women who ( re-)started smoking within 8 weeks of delivery show a much smaller height deficit than those whose mothers smoked during pregnancy ( table 5 ) .
the offspring of women who smoke during pregnancy have a pi at birth 0.09 kg / m ( girls , se = 0.10 ) or 0.21 kg / m ( boys , se = 0.11 ) lower than offspring of women who do not smoke in pregnancy ( tables 1 and 2 ) in adjusted models . for both boys and girls
, this association is not attenuated by adjustment for measured confounders . in mutually adjusted models ,
the negative association of maternal smoking with pi at birth is stronger for maternal than partner smoking .
however , the standard errors of coefficients for both maternal and partner smoking and pi at birth are large , and there is little statistical evidence of a difference between the associations of maternal and partner 's smoking with pi at birth ( p - values for heterogeneity of maternal and partner coefficients 0.30 for boys , 0.37 for girls )
. there is , however , an indication of a dose response , with the reduction in pi at birth being much greater in the offspring of women who smoke heavily during pregnancy compared with the reduction association with light smoking ( table 4 ) .
the associations with post - natal smoking and pi at birth are qualitatively different for boys and girls ( formal interaction tests were not performed ) ; in girls both maternal smoking during pregnancy and post - natal smoking are associated with a reduced pi at birth , whereas in boys the reduction in pi at birth is confined to maternal smoking during pregnancy ( table 5 ) .
male offspring of women who smoke have faster rates of pi increase in the first 2 months of life , and female offspring have slower rates of pi decrease in the first 1 month of life and faster rates of pi increase between the ages 1 and 4 months . among girls ,
the associations with pi changes are in opposite directions for maternal and partner 's smoking , although there is only statistical evidence of a difference between the coefficients for the first 1 month of life ( p = 0.03 ) . among boys , the associations with pi changes in the first 2 months are 5-fold stronger for maternal smoking than for partner 's smoking ( p - value for heterogeneity of coefficients is 0.05 ) .
all of these associations , however , are of small magnitude when compared with the mean and standard deviation of average pi change rates , and have large standard errors .
there is little evidence of differences in pi changes later in infancy and bmi changes between the ages 2 and 10 years between the offspring of smokers and non - smokers .
some of the coefficients are of greater magnitude for maternal smoking and others are stronger for partner 's smoking , but all effect sizes are very small with large standard errors and there is no statistical evidence of maternal partner differences in any of the coefficients . by the age of 2 years ,
the male offspring of women who smoked during pregnancy remain slightly less adipose than the offspring of non - smokers ; their bmi is on average 0.14 kg / m ( se = 0.48 ) lower than the offspring of smokers . in girls , however , maternal smoking during pregnancy is associated with higher bmi by the age of 2 years of 0.14 kg / m ( se = 0.10 ) ( table 3 ) . when the cumulative effect of all adiposity changes up to the age of 10 years is calculated , maternal smoking during pregnancy has a similar association with offspring adiposity compared with partner 's smoking for girls ; but for boys , there is a stronger association for maternal smoking . after adjustment for confounders , maternal smoking in pregnancy is associated with higher bmi at the age of 10 years by an average 0.39 kg / m ( se = 0.14 ) in girls and 0.24 kg / m ( se = 0.08 ) in boys .
the observed increases in bmi by the age of 10 years for partner 's smoking during pregnancy are 0.35 kg / m ( se = 0.11 ) for girls and 0.10 kg / m ( se = 0.07 ) for boys ( table 3 ) . the difference in bmi at the age of 10 years compared with the baseline group of non - smokers is greater for the offspring of women who smoked heavily during pregnancy than for the offspring of women who smoked lightly ( table 4 ) .
however , a similar difference between heavy and light smokers is observed for partner smoking . in girls ,
the difference in bmi at the age of 10 years compared with the baseline group of non - smokers is greater for the offspring of women who smoked during pregnancy than for women who ( re-)started smoking within 8 weeks of delivery , but in boys these groups have a similar elevation in bmi at the age of 10 years ( table 5 ) .
our data are consistent with maternal smoking reducing offspring birth length through a causal intrauterine effect ; this association was strong , resistant to adjustment for observed confounding factors , not observed for partner 's smoking during pregnancy and not observed in the offspring of women who ( re-)started smoking within 8 weeks of delivery .
this is consistent with the large body of literature suggesting a causal effect of maternal smoking during pregnancy on birth size .
several potential mechanisms for the causal effect of smoking on birth size have been postulated , including the vasoconstrictive action of nicotine and fetal hypoxia , and it is possible that these mechanisms could result in lasting changes to the infant that would affect post - natal growth . in our study , there was some indication of different post - natal growth patterns in the offspring of maternal smokers compared with non - smokers , with children of smokers growing more rapidly in infancy but more slowly later in childhood .
however , differences in post - natal height growth rates between the offspring of smokers and non - smokers are relatively small , and could be explained by chance .
thus the height differential , which persists across childhood as previously observed in this cohort , appears to be largely due to smaller birth length rather than to different post - natal height growth patterns .
animal studies have suggested an association between maternal nicotine exposure and changes in adipose tissue and glucose metabolism , which would be consistent with maternal smoking during pregnancy having a causal effect on greater offspring adiposity .
although many observational studies in human populations have also demonstrated an association between maternal smoking during pregnancy and increased offspring adiposity , including one showing stronger maternal than paternal effects and another carried out on a cohort born between 1959 and 1966 , when smoking during pregnancy was more common and less socio - economically confounded , and average bmi levels were lower , the degree to which this association is causal via intrauterine mechanisms is uncertain , with confounding by familial factors potentially very important . in our data ,
the offspring of women who smoke during pregnancy have a slightly lower pi at birth , and faster rates of adiposity change in the first few months of life . by the age of 2 years
, male children born to women who smoked during pregnancy remain of slightly lower bmi than those born to non - smokers or to women whose partners smoked , but female offspring of smokers have slightly higher bmi . by the age of 10 years ,
associations between maternal smoking during pregnancy and adiposity changes were not attenuated by adjustment for measured confounders .
for pi at birth , we did not demonstrate statistical heterogeneity between the coefficients for maternal and partner 's smoking , indicating that this observed maternal
partner difference may be due to chance , although a dose response was observed such that the reduction in pi at birth was largely restricted to the offspring of women who smoked heavily ( > 10 cigarettes / day ) .
for pi changes in the first months of life , there is some evidence that the coefficients for maternal and partner 's smoking are different , indicating a possible effect of maternal smoking during pregnancy on adiposity changes in the early months of life .
however , the coefficient sizes were small with large standard errors , thus the associations could be due to chance and require replication in other studies . for pi changes in later infancy and bmi changes between the ages 2 and 10 years , however ,
the associations with maternal and partner 's smoking are equivalent , suggesting that any associations between smoking during pregnancy and adiposity changes after early infancy may be due to either confounding by unmeasured familial factors .
these findings contrast with results from a recent publication , which reported that in brazilian children born either in 1993 or 2004 , maternal smoking in pregnancy was associated with greater offspring bmi at ages 12 and 48 months , with no association with partner 's smoking and statistical evidence for differences between maternal and paternal smoking .
the population differences between the two studies and the inability of the brazilian study to examine associations into later childhood might explain these differences . although we found a dose response such that the offspring of women who smoke heavily ( > 10 cigarettes / day ) have a higher bmi at the age of 10 years than the offspring of light smokers , a similar dose response is observed for partner 's smoking . at least in boys , the increase in bmi at the age of 10 years is also similar for the offspring of women who ( re-)started smoking within 8 weeks of delivery .
together , these observations suggest that the associations we observe in later childhood for bmi are due to confounding rather than a causal effect of maternal smoking in pregnancy . the main strengths of our analyses are the large sample size , the use of repeat measures of height and adiposity , the multiple approaches to control for both measured and unmeasured confounding factors and the use of dose information and data on post - natal smoking . one limitation of our study is that we have used self - reported data on smoking during pregnancy , although a meta - analysis of studies comparing self - reported smoking with biochemical measures provides reassurance for using self - reported smoking behaviour , since it was found to have good sensitivity and specificity .
this meta - analysis did not , however , include studies during pregnancy , when the bias may be greater due to the stigma attached to pregnancy smoking
. however , the fact that we do not see a reduction in birth length for the offspring of women who report not having smoked during pregnancy , but ( re-)started within 8 weeks of delivery , provides support for the validity of the self - reported smoking data .
we have reported results separately for boys and girls because the growth trajectories were modelled separately by gender ; thus we can not formally test for interactions by gender .
any gender differences may be due to chance , and differences observed between girls and boys should be interpreted with caution .
thus , overall , these analyses are consistent with an intrauterine effect of maternal smoking on birth length and possibly also on adiposity at birth and on height and adiposity changes in infancy .
we do not find any strong evidence of an intrauterine effect on changes in height or adiposity after infancy .
apart from the associations with birth length , most other coefficient sizes were small with large standard errors , and therefore these associations require replication in other studies and utilization of other methodologies such as mendelian randomization .
uk economic and social research council ( res-060 - 23 - 0011 funded salary for l.d.h . at the start of this work ) ; uk medical research council population health scientist fellowship ( to l.d.h . ) ; sir henry wellcome postdoctoral fellowship from the wellcome trust ( to m .-
; the uk medical research council ( mrc ) , the wellcome trust and the university of bristol provide core funding support for alspac ; the uk mrc ( g0600705 ) and the university of bristol provide core funding for the mrc centre of causal analyses in translational epidemiology . | background maternal smoking during pregnancy is associated with reduced offspring birth length and has been postulated as a risk factor for obesity .
causality for obesity is not established .
causality is well - supported for birth length , but evidence on persistence of height deficits is inconsistent.methods we examined the association between maternal smoking during pregnancy and trajectories of offspring height ( 010 years , n = 9424 ) , ponderal index ( pi ) ( 02 years , n = 9321 ) and body mass index ( bmi ) ( 210 years , n = 8887 ) in the avon longitudinal study of parents and children . to strengthen inference ,
measured confounders were controlled for , maternal and partner smoking associations were compared , dose response and associations with post - natal smoking were examined.results maternal smoking during pregnancy was associated with shorter birth length , faster height growth in infancy and slower growth in later childhood . by 10 years , daughters of women who smoke during pregnancy are on average 1.11 cm ( se = 0.27 ) shorter after adjustment for confounders and partner smoking ; the difference is 0.22 cm ( se = 0.22 ) for partner 's smoking .
maternal smoking was associated with lower pi at birth , faster pi increase in infancy , but not with bmi changes 210 years .
associations were stronger for maternal than partner smoking for pi at birth and pi changes in infancy , but not for bmi changes after 2 years . a similar dose
response in both maternal and partner smoking was seen for bmi change 210 years.conclusion maternal smoking during pregnancy has an intrauterine effect on birth length , and possibly on adiposity at birth and changes in height and adiposity in infancy .
we do not find evidence of a specific intrauterine effect on height or adiposity changes after the age of 2 years . | Introduction
Subjects and Methods
Results
Discussion
Supplementary Data
Funding
Supplementary Material | table 2the associations between maternal and partner smoking during pregnancy and trajectories of height , pi and bmi in male offspringmaternal smoking during pregnancypartner smoking during pregnancyp - valuemean ( sd ) growth rate per monthcrude coefficient ( se)adjusted coefficient ( se)adjusted coefficient with further adjustment for paternal smoking ( se)crude coefficient ( se)adjusted coefficient ( se)adjusted coefficient with further adjustment for maternal smoking ( se)maternal partner hetero- geneityheight , n = 4832 birth length , cm50.7 ( 1.2)0.6849 ( 0.0891)0.5865 ( 0.0785)0.6275 ( 0.0830)0.0873 ( 0.0765)0.0590 ( 0.0664)0.1095 ( 0.0698)<0.001 growth 1 , cm / month3.8 ( 0.2)0.0155 ( 0.0401)0.0198 ( 0.0396)0.0262 ( 0.0424)0.0098 ( 0.0339)0.0078 ( 0.0335)0.0152 ( 0.0358)0.46 growth 2 , cm / month1.7 ( 0.2)0.0497 ( 0.0183)0.0504 ( 0.0183)0.0476 ( 0.0196)0.0199 ( 0.0155)0.0210 ( 0.0155)0.0063 ( 0.0166)0.11 growth 3 , cm / month1.0 ( 0.1)0.0054 ( 0.0065)0.0055 ( 0.0065)0.0033 ( 0.0069)0.0053 ( 0.0055)0.0063 ( 0.0055)0.0052 ( 0.0059)0.83 growth 4 , cm / month0.5 ( 0.04)0.0027 ( 0.0022)0.0025 ( 0.0023)0.0020 ( 0.0024)0.0019 ( 0.0018)0.0018 ( 0.0019)0.0012 ( 0.0020)0.80pi , n = 4777 pi at birth , kg / m26.2 ( 1.2)0.2557 ( 0.1013)0.2296 ( 0.1022)0.2059 ( 0.1087)0.1298 ( 0.0865)0.1233 ( 0.0867)0.0569 ( 0.0921)0.30 pi change 1 , kg / m / month0.5 ( 0.8)0.2482 ( 0.0678)0.2494 ( 0.0678)0.2311 ( 0.0726)0.1125 ( 0.0580)0.1145 ( 0.0580)0.0436 ( 0.0621)0.05 pi change 2 , kg / m / month0.4 ( 0.08)0.0055 ( 0.0076)0.0053 ( 0.0076)0.0048 ( 0.0081)0.0026 ( 0.0065)0.0027 ( 0.0065)0.0011 ( 0.0069)0.73bmi , n = 4534 ( kg / m / month ) bmi change 10.01 ( 0.05)0.0118 ( 0.0156)0.0114 ( 0.0156)0.0117 ( 0.0169)0.0035 ( 0.0120)0.0031 ( 0.0119)0.0003 ( 0.0130)0.57 bmi change 20.03 ( 0.02)0.0081 ( 0.0092)0.0091 ( 0.0092)0.0079 ( 0.0101)0.0054 ( 0.0072)0.0054 ( 0.0072)0.0027 ( 0.0078)0.68 bmi change 30.03 ( 0.04)0.0108 ( 0.0153)0.0087 ( 0.0153)0.0154 ( 0.0164)0.0108 ( 0.0120)0.0103 ( 0.0120)0.0144 ( 0.0129)0.15 bmi change 40.02 ( 0.04)0.0171 ( 0.0174)0.0163 ( 0.0174)0.0095 ( 0.0186)0.0179 ( 0.0132)0.0180 ( 0.0132)0.0153 ( 0.0141)0.80 bmi change 50.02 ( 0.03)0.0012 ( 0.0043)0.0017 ( 0.0043)0.0006 ( 0.0045)0.0049 ( 0.0033)0.0049 ( 0.0033)0.0050 ( 0.0035)0.33 bmi change 60.05 ( 0.03)0.0054 ( 0.0024)0.0057 ( 0.0024)0.0043 ( 0.0026)0.0044 ( 0.0020)0.0045 ( 0.0020)0.0033 ( 0.0021)0.77coefficients are for interactions between smoking during pregnancy and the intercept ( birth length , pi at birth or bmi at the age of 2 years ) or slope ( growth rate in each linear spline period ) , i.e. by the age of 10 years , the daughter of a woman who smoked during pregnancy is on average 1.11 cm ( se = 0.27 ) shorter than the daughter of a non - smoker once measured confounders and partner 's smoking have been adjusted for ; the difference is 0.22 cm ( se = 0.22 ) for partner 's smoking ( table 3 ) . | [
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synapses transfer information from sensory cells or neurons to other neurons or distinct target cell types , such as muscle cells .
a plethora of presynaptic proteins orchestrate neurotransmitter release at the presynaptic active zone ( az ) .
these proteins are organized into three main compartments , which are ultrastructurally defined and classically referred to as ( 1 ) the cytomatrix at the active zone ( caz ) with ( 2 ) presynaptic electron dense projections that are clustering ( 3 ) synaptic vesicles ( zhai and bellen 2004 ) .
the presynaptic dense projections appear highly variable in size and shape , which have been hypothesized to follow the function of a given synapse type .
they seem to be present at all neuronal azs but differ greatly in terms of order , density and morphology as well as molecular composition ( zhai and bellen 2004 ) .
for example , rather small structures of less than 100 nm height are found at mammalian conventional central nervous system ( cns ) synapses where they form a presynaptic grid , also termed a
remarkably regularly arranged structures can be observed at neuromuscular junctions of the frog ( harlow et al .
2012 ) . moreover , presynaptic dense projections are not an evolutionary invention of vertebrates , as insects such as the fruitfly drosophila melanogaster also harbor elaborated dense projections termed
t - bars , which are found at almost every synapse type ( for review , see wichmann and sigrist 2010 ) . the anatomical hallmark of tonically releasing sensory mammalian photoreceptor synapses , a huge plate - like dense projection that tethers hundreds of synaptic vesicles ( schmitz et al .
2000 ) , was discovered in the 1950s ( de robertis and franchi 1956 ) , when transmission electron microscopy started to become a commonly used technique .
electron microscopy allowed researchers to visualize the ultrastructure of cells in detail for the first time ( de robertis and bennett 1955 ) , bringing exciting new knowledge about morphology , organization and communication of cells in general and synapses in particular ( see , for example : de robertis and bennett 1955 ; de robertis and franchi 1956 ) . at this time
, synaptic vesicles were discovered at guinea pig retinal synapses , where they were called
synaptic vesicle was coined by de robertis and bennett ( 1955 ) , who were inspecting bullfrog and earthworm synapses . in parallel ,
the work of de robertis and franchi ( 1956 ) on photoreceptors of light- or dark - exposed rabbits provided the first experimental evidence correlating synaptic vesicle numbers and presynaptic activity .
a few years later , the large presynaptic dense structures of these synapses were named
ribbons , when their characteristic shape with extended longitudinal axis was recognized in serial 3d reconstructions of guinea pig retinas ( sjostrand 1958 ) .
subsequently , synaptic ribbons were also found to decorate cochlear afferent hair cell synapses ( smith and sjostrand 1961 ) .
golgi or horseradish peroxidase labeling in combination with transmission electron microscopy were also and still are , widely used to visualize neurons ( meller et al .
1968 ; levay 1973 ; white and rock 1980 ; defelipe et al . 1986 ) and to understand the anatomy of the inner ear .
for example , the afferent spiral ganglion neurons ( sgns ) of the cochlear nerve , which carry the information about an acoustical signal from the inner ear to the brainstem , were studied intensely in various mammals such as guinea pig , mouse or cat ( spoendlin 1972 , 1975 , 1979 ; paradiesgarten and spoendlin 1976 ; bodian 1978 ; kiang et al .
1982 ; liberman 1982a ; ginzberg and morest 1984 ; ryugo and rouiller 1988 ; liberman et al .
these studies revealed that inner and outer hair cells are innervated by different sgn types ( kiang et al .
1982 ) , outer hair cells ( ohcs ) by unmyelinated ( 5 % ) and inner hair cells ( ihcs ) by myelinated ( 95 % ) afferent fibers ( spoendlin 1969 , 1975 ) .
each of the myelinated , bipolar type i sgns sends a peripheral unmyelinated and unbranched neurite to form a synapse with a single ihc ribbon synapse ( liberman 1980 ; liberman et al .
; reviewed in meyer and moser 2010 ) . therefore , recordings from sgns enable the investigation of the function of individual azs within an ihc .
type i sgns show different intensity thresholds and dynamic ranges in cat ( liberman and kiang 1978 ) .
paired recordings from hair cells and postsynaptic neurons have provided insight into synaptic sound encoding and its presynaptic determinants ( palmer and russell 1986 ) .
finally , observations of postsynaptic excitatory potentials by recordings from near the synapse revealed the first information on the presynaptic release mechanism ( furukawa et al .
each ihc contains 530 azs , dependent on species and tonotopic position along the cochlea , generally peaking at the region with the greatest sound sensitivity for the particular species ( francis et al .
liberman and co - workers were among the pioneers coupling structural investigations of the mammalian auditory system to its function . in his seminal study , liberman s ( 1982b ) functional characterization of cat single auditory nerve fibers
was followed by horseradish peroxidase labeling to individually back - trace the innervation location at the respective ihc azs .
this approach allowed the author to relate functional parameters such as spontaneous firing rates and firing thresholds to morphology of type i sgns , described , for example , by the dimension and location of their unmyelinated terminals on the ihcs .
these studies together led to the hypothesis that ribbon synapses within a given ihc are structurally and functionally heterogeneous ( which will be discussed later in this review ) and pointed to the further need for detailed structure function analyses .
horseradish peroxidase labeling combined with electron microscopy also provided insights into presynaptic vesicle cycling in hair cells ( siegel and brownell 1986 ) .
more recently , hair cell synapses have increasingly attracted research activity and novel as well as classical methods have been employed for assessing their structure and function in combination with genetic or pharmacological manipulation of the synapses or noise exposure . quantitative electron microscopy analysis employing electron tomography of different functional states as well as freeze - fracture and subsequent electron microscopy
have been introduced by roberts and others for studies of hair cell synapses ( roberts et al .
molecular manipulations involving germline mutagenesis as well as virus - mediated gene transfer were established .
further , patch - clamp recordings have characterized ca currents ( e.g. , lewis and hudspeth 1983 ; fuchs et al .
2000 ; brandt et al . 2003 ) and membrane turnover ( e.g. , parsons et al . 1994 ; moser and beutner 2000 ; schnee et al .
technically very challenging postsynaptic patch - clamp recordings have provided insight into the excitatory postsynaptic currents ( glowatzki and fuchs 2002 ) and , combined with presynaptic recordings , have elucidated hair cell synaptic mechanisms with superb resolution ( e.g. , keen and hudspeth 2006 ; goutman and glowatzki 2007 ; li et al . 2009 ) .
immunohistochemistry combined with high - resolution microscopy as well as transcriptomic and proteomic analyses have informed on the molecular composition of hair cell synapses ( khimich et al .
finally , fluorescence imaging has been implemented for studies of hair cell synapse function ( tucker and fettiplace 1995 ; issa and hudspeth 1996 ; zenisek et al .
2003 ; griesinger et al . 2005 ; frank et al . 2009 ; revelo et al . 2014 ) .
ribbon - type azs cope with a demanding task : synaptic vesicles need to be released indefatigably and rapidly recycled at individual synapses in order to maintain high firing rates of sgns that fire at hundreds of hz even during continued stimulation ( reviewed in matthews and fuchs 2010 ; pangri et al .
2012 ) . sustained exocytosis amounts to up to 70 hz from each release site , of which about a dozen comprise the readily - releasable vesicle pool ( rrp ) .
this was demonstrated in mouse ihcs ( pangri et al . 2010 ) and is to our knowledge one of the highest release rates per site described to date ( pangri et al .
this process requires very efficient means of clearing previously exocytosed membrane and proteins from the site followed by immobilization and priming of new vesicles for the next round of release .
moreover , the release of the neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less ( kiang et al .
1967 ; palmer and russell 1986 ; kppl 1997 ; goutman 2012 ; li et al . 2014 ) .
how the molecular machinery of ihc azs meets these requirements is just starting to emerge .
it is becoming clear that ultrastructural assessment of functional synapse states is required in addition to the powerful combination of molecular manipulation and physiological characterization .
in this review , we will emphasize recent approaches coupling functional and structural investigations of release at the level of ihcs and their ribbon synapses , as well as recent findings regarding vesicular recycling after transmitter release .
how does the subcellular organization of sensory ihcs enable mechanotransduction and transmitter release at high rates ?
ihcs are epithelial cells by origin and exhibit several characteristics that distinguish them from neurons .
most notably , they show a strong polarization with respect to both long and short cell axes . the polarization along the apicobasal axis follows a clear compartmentalization , e.g. , apparent by the hair bundle harboring the mechanotransduction apparatus of the apical membrane . graded receptor potentials
are formed by mechanoelectrical ( apical ) and voltage - gated ( basal ) conductances ( corey and hudspeth 1979 ; roberts et al .
actin - filled stereocilia protrude into the endolymph in a highly organized manner and their sophisticated supramolecular mechanotransduction apparatus enables ultrasensitive detection of sound - born vibrations of the cochlear partition ( reviewed in kazmierczak and mller 2012 ) .
while the molecular identity of the mechanotransducer channel still awaits definitive demonstration , recent work indicates the transmembrane channel - like proteins ( tmc)-1 and -2 as promising candidates ( pan et al . 2013 ) .
opening of the apical mechanotransducer channels depolarizes the ihc , subsequently activating cav1.3 ca channels ( platzer et al .
2004 ) at the presynaptic az in the basolateral membrane , where the incoming ca triggers neurotransmitter release .
the density of ribbon synapses shows a strong basoapical gradient , with the supranuclear portion of the hair cell being devoid of azs ( francis et al .
, the cuticular plate likely serves as an anchor for the stereociliar actin bundles , containing a rich protein network with cytoskeletal proteins such as actin , -actinin and tropomyosin ( slepecky and chamberlain 1985 ; zine and romand 1993 ) .
moreover , the striated organelle , located underneath the cuticular plate , likely modulates the stereociliar actin bundles ( vranceanu et al .
microtubules are primarily found beneath the cuticular plate ( slepecky and chamberlain 1985 ; steyger et al .
1990 ) but appear connected to cytoskeletal proteins in the cuticular plate , for example via acf7a ( actin crosslinking family protein 7a ) , as suggested for zebrafish neuromast hair cells ( antonellis et al . 2014 ) .
1990 ) , providing the mechanical strength of hair cells ( szarama et al . 2012 ) and tracks for efficient cargo protein trafficking along the apicobasal axis ( furness et al . 1990 ) .
in addition to the cellular apicobasal polarity , hair cells also show planar cell polarity , which is reflected in the orderly orientation of their hair bundles ( reviewed in ezan and montcouquiol 2013 ; sienknecht et al .
2014 ) . whether the basolateral organization of the hair cells is similarly instructed by planar cell polarity
remains to be tested . in the next sections , we will focus on the organization of the basal portion of ihcs and discuss structure and function of hair cell ribbon synapses
. emphasis will be on the molecular machinery of the synapse , synapse development , synaptic heterogeneity and synaptic vesicle recycling .
phylogenetically , ribbons in sensory cells are old structures that occur not only in mammals but also in fishes , amphibians and birds . in the mammalian organ of corti , they were first described by smith and sjstrand ( 1961 ) and are found in both sensory cell types , i.e. , ihcs and ohcs ( sobkowicz et al .
1982 ) . the discovery of the protein ribeye , initially purified from bovine retina , ( schmitz et al .
2000 ) , as the main and structure - yielding component of ribbons in rat photoreceptors ( schmitz et al .
( zenisek et al . 2003 ) , zebrafish photoreceptors and bipolar cells ( wan et al . 2005 ) and mouse cochlear hair cells ( khimich et al . 2005 ; see also immunogold labeling in fig .
1a ) highlights the conservation of the ribbon in vertebrate evolution ( schmitz 2009 ) .
nonetheless , ribbons still vary greatly in size and shape ( lenzi and von gersdorff 2001 ; moser et al . 2006 ; matthews and fuchs 2010 ) , likely reflecting structural adaptation to the specific needs of the respective synaptic connection for sensory coding.fig .
a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement ( contrast enhanced image in a ) , see also schematic representation ( a ) .
b a p14 mature ribbon with the typical multi - lamellar pattern ( contrast enhanced image in b ) , see also scheme in b
c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools ( yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density ) . c the membrane - proximal vesicles ( orange ) are arranged around the presynaptic density ( magenta ) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in ( d ) , scale bar ( d ) 100 nm ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; d 2-color sted image of immunolabeled bassoon ( magenta ) and cav1.3 channel clusters ( green ) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed .
scale image:700 700 nm ; e , e mathematic model showing the total mean steady state [ ca ] profile at the az membrane ( e ) ; e effective number of cav1.3 channels contributing to total mean steady state [ ca ] as shown in ( e ) .
( c , c , d , e , e modified from wong et al .
2014 , embo j ; reprinted with permission 2014 wong et al . ) .
f schematic summary of the protein arrangement at mature ihc ribbon synapses spatial distribution of ihc az proteins .
a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement ( contrast enhanced image in a ) , see also schematic representation ( a ) .
b a p14 mature ribbon with the typical multi - lamellar pattern ( contrast enhanced image in b ) , see also scheme in b . scale bars ( a , a , b ) 100 nm .
c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools ( yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density ) . c the membrane - proximal vesicles ( orange ) are arranged around the presynaptic density ( magenta ) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in ( d ) , scale bar ( d ) 100 nm ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; d 2-color sted image of immunolabeled bassoon ( magenta ) and cav1.3 channel clusters ( green ) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed .
scale image:700 700 nm ; e , e mathematic model showing the total mean steady state [ ca ] profile at the az membrane ( e ) ; e effective number of cav1.3 channels contributing to total mean steady state [ ca ] as shown in ( e ) .
( c , c , d , e , e modified from wong et al .
2014 , embo j ; reprinted with permission 2014 wong et al . ) .
f schematic summary of the protein arrangement at mature ihc ribbon synapses ribeye is composed of two major domains : while the a domain organizes the assembly of the synaptic ribbon and is unique in structure , the b domain is structurally nearly identical to the transcription repressor ctbp2 , which is encoded by the same gene but uses a different transcription initiation site ( schmitz et al .
the b domain is also assumed to be involved in tethering of synaptic vesicles to the ribbon ( schmitz et al .
2000 ; schmitz 2009 ) , though the proteins that form tethers remain to be identified .
ribeye appears to organize ribbon shape directly based on its domain structure ( schmitz 2009 ) and its aggregation properties ( magupalli et al .
a trifold lamellar pattern has been described for photoreceptor ribbons and assigned to the polarized arrangement of ribeye ( schmitz 2009 ) . also at mature ihc ribbons , a lamellar substructure is observed that harbors multiple lamellar foldings ( sobkowicz et al .
this effect can be attributed to the differences in the ribbon shape in ihcs and photoreceptors . in immature ihc ribbons ,
the lamellar pattern is not prominent but instead a dotted pattern can be observed ( fig .
recently , at zebrafish hair cell ribbon synapses , it was found that the ribeye a and b domain segregate along the vertical axis of the ribbons , with the b domain more located towards the basal end ( sheets et al .
ribeye , in contrast to other az proteins , is not found in invertebrates such as the fruitfly drosophila melanogaster ; however , bruchpilot ( brp ) , the homolog of the vertebrate az protein cast ( caz - associated structural protein)/erc2 , functions as the main building block of t - bars ( kittel et al .
in fact , only the n - terminus is conserved and shows sequence homologies to cast , whereas the c - terminus is only found in dipteran insects and rather resembles cytoskeletal elements such as plectin due to its numerous coiled - coil domains ( wagh et al .
2006 ) . moreover , the c - terminus mediates vesicle tethering to the t - bar ( hallermann et al .
the structurally related proteins bassoon and piccolo as well as cast , elks , rab3-interacting molecule ( rim ) and munc13 are present ( betz et al . 1998 ; fenster et al . 2000 ; dresbach et al . 2001 ; deguchi - tawarada et al .
additionally , ctbp2 and ctbp1 have also been found at conventional azs ( tom dieck et al . 2005 ) . except for piccolo , which at ribbon synapses is solely expressed as a shorter splice variant nicknamed as piccolino ( regus - leidig et al .
2013 ) , these proteins also largely form the caz at photoreceptor ribbon synapses ( wang et al .
the components of hair cell azs , on the other hand , are still largely unexplored , except for bassoon ( khimich et al . 2005 ) and piccolo / piccolino ( khimich et al . 2005 ; regus - leidig et al .
in fact , a recent study indicates that ihc synapses operate without munc13-like priming factors ( vogl et al .
bassoon , together with ribeye , is responsible for the ribbon shape and , hence , might contribute to its function .
studies of ihcs from bassoon mutant mice indicated an anchoring function of bassoon ( khimich et al . 2005 ; frank et al .
2013 ) , in line with findings at photoreceptor ribbon synapses ( dick et al . 2003 ; tom dieck et al .
the fraction of ribbon - occupied synapses remaining in bassoon - deficient ihcs seems to depend on age and residual levels of full - length bassoon ( khimich et al .
the fraction of ribbonless synapses increased from 50 % at postnatal day 11 ( p11 ) up to 88 % at p70 . a lower and relatively constant fraction of 56 % ribbonless synapses
was found in a gene - trap bassoon mutant ( bsn ) likely due to a weak residual synaptic expression of bassoon ( jing et al .
. however , the anchorage of the remaining ribbons seems impaired ( frank et al .
maintained az ultrastructure , bsn animals exhibited a larger number of ca channels at ihc synapses compared to bsn mice and also displayed an intermediate phenotype regarding sustained ihc exocytosis ( jing et al .
in contrast , the size of the readily releasable vesicle pool ( rrp ) was strikingly reduced in both mutants . moreover , single unit recordings of the sgns show comparably severe defects in bsn and bsn mice , as both genotypes had impaired sound onset coding and lower evoked and spontaneous spike rates . taken together , these results indicate that the remaining , loosely anchored ribbons might function inadequately ( jing et al .
this further suggests that the mere presence of the ribbon , even with tethered vesicles , is not sufficient to maintain normal transmitter release and sustain the rrp at ihc ribbon synapses .
moreover , it seems that bassoon contributes to organizing the ihc az beyond anchoring the ribbon .
this has been concluded from impaired clustering of ca channels at bsn ribbon - occupied synapses ( frank et al .
2010 ) and indicates a potential direct contribution of bassoon in organizing the az ( frank et al . 2010 ; hallermann and silver 2013 ) .
in contrast to conventional synapses , where often only the combined knockdown of bassoon and its homolog piccolo causes synaptic defects ( altrock et al .
2008 ; mukherjee et al . 2010 ; waites et al . 2011 , 2013 ) , these proteins seem not to act redundantly at ribbon synapses . as mentioned above , at hair cell and photoreceptor ribbons , only the short isoform of piccolo , piccolino , is expressed , which lacks a large c - terminal part ( regus - leidig et al .
binding sites for the proteins abp1 , pra1 , git1 and profilin ( wang et al . 1999 ; fenster et al .
2003 ) are still present , whereas binding sites for , e.g. , bassoon or rim are lacking ( regus - leidig et al .
accordingly , piccolino exhibits a different spatial distribution at ribbon synapses of photoreceptors , where it is found directly on the ribbon , as indicated by pre - embedding immunogold - labelings , using an antibody recognizing the n - terminus of the protein ( limbach et al .
( 2014 ) revealed a striking impairment in the ribbon structure upon piccolino rnai - based knockdown .
instead , a high proportion of attached spherical ribbons was found that resemble ribbon precursors of photoreceptor ribbons suggesting a role of piccolino in structural ribbon maturation ( regus - leidig et al .
ribeye and presumably piccolino as well as bassoon present the main structural components of the ribbon and/or the anchorage of the ribbon to the az . in order to resolve the function of ribbons ,
the ribbon was suggested to ( 1 ) promote a large readily releasable pool of vesicles via establishing / stabilizing many ca channels and vesicular release sites ( khimich et al .
2010 ) , ( 2 ) facilitate vesicle replenishment at the az ( conveyor belt model , e.g. , bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001 ; snellman et al .
2011 ) , ( 3 ) facilitate multivesicular release ( edmonds 2004 ; fuchs 2005 ) , or ( 4 ) serve as a diffusion barrier to enable high local ca concentrations ( graydon et al .
functional interpretations have also been provided for the morphologically distinct populations of synaptic vesicles at ribbon synapses but in each case remain to be validated . ribbon - associated vesicles structurally attached through filamentous protein tethers
the ones at the base of the ribbon face the presynaptic plasma membrane ( membrane - proximal vesicles ; fig . 1c , c ) and are often tethered to the membrane and/or to the presynaptic density ( tethered vesicles ) .
lateral to this subpopulation of ribbon - associated vesicles , there are few additional membrane - proximal and even membrane - tethered vesicles that are not in close vicinity to the ribbon .
while further testing is required , current evidence suggests that the membrane - proximal vesicles comprise the rrp ( e.g. , in retinal bipolar cells : von gersdorff et al . 1996 ;
zenisek et al . 2000 ; frog saccular hair cells : lenzi et al . 1999 , 2002 ; rutherford and roberts 2006 ; and mouse inner hair cells : khimich et al .
support for this hypothesis comes from the approximate matching between the morphologically estimated number of membrane - proximal vesicles and the functionally defined size of the rrp , i.e. , the fast component of exocytosis upon depolarization - evoked ca influx ( von gersdorff et al .
1996 ; pangri et al . 2010 ; frank et al . 2010 ) , as well as from the observation that these vesicles are most heavily depleted upon stimulation ( lenzi et al . 2002 ; pangri et al .
furthermore , the tethering of vesicles to the az membrane might reflect a structural correlate of fusion competence ( siksou et al .
new high - resolution imaging approaches using rapid freezing methods and/or electron tomography have revealed that synaptic vesicles in proximity to the membrane exhibit several morphologically distinct stages .
tethers of different numbers and lengths connecting synaptic vesicles to the az membrane could be observed at conventional synapses and synaptosome preparations ( siksou et al .
2007 , 2009 ; fernndez - busnadiego et al . 2010 , 2013 ) but also at ihc ribbon synapses ( frank et al . 2010 ) .
moreover , cryo - electron tomography , a method allowing visualization of hydrated and unstained tissue , revealed that tethering of synaptic vesicles in synaptosomes prepared from hippocampal tissue precedes the full contact of a synaptic vesicle with the membrane ( fernndez - busnadiego et al .
single long tethers initially seem to be formed and synaptic vesicles likely enter the rrp via the formation of several short tethers ( < 5 nm ) . in line with this hypothesis , this fraction of vesicles could be depleted by application of hypertonic sucrose , that is thought to trigger rrp release ( rosenmund and stevens 1996 ) .
moreover , the formation of short tethers could be inhibited using tetanus toxin , pointing towards the fact that neuronal soluble nsf attachment protein receptors ( snare ) proteins are involved in this process ( fernndez - busnadiego et al .
2013 ) play a crucial role in tethering vesicles to the membrane at conventional cns synapses .
interestingly , munc13 and caps priming factors seem not to operate at the ihc ribbon synapse ( vogl et al .
some authors even argue that the entire ribbon - associated vesicle population is fusion - competent and , therefore , can be released within a few milliseconds or less ( heidelberger et al .
1994 ; edmonds 2004 ) . evidence for a priming function of the ribbon has recently been presented ( snellman et al .
in contrast to caz proteins , which are , at least in part , conserved at ihc azs , the molecular machinery involved in the regulation of synaptic vesicle fusion seems to deviate strongly from that of
as mentioned above , neurotransmitter release at conventional synapses is mediated by neuronal snares , namely snap-25 , syntaxin 1 and synaptobrevin 1 or 2 ( reviewed in jahn and fasshauer 2012 ) .
snare activity can be blocked by neurotoxin - mediated cleavage or genetic manipulations ( schiavo et al .
ribbon synapses , the snare protein machinery appears to be present and functional ( brandsttter et al .
1996 ; morgans et al . 1996 ; morgans 2000 ; von kriegstein and schmitz 2003 ; uthaiah and hudspeth 2010 ; cooper et al .
in contrast , ihc exocytosis seems to be insensitive to neurotoxins and genetic ablation of neuronal snares ( nouvian et al .
2011 ) and , hence , a functional role of syntaxin 1 , synaptobrevin 1 and 2 as well as snap-25 in ihc exocytosis is questionable . while some studies detected snare mrnas and proteins in the sensory epithelium and hair cells ( safieddine and wenthold 1999 ; uthaiah and hudspeth 2010 ; nouvian et al . 2011 ) , neither snap-25 , synaptobrevin 13 nor syntaxin 1 could be detected by immunofluorescence imaging in mouse ihcs ( nouvian et al .
2010 ; reisinger et al . 2011 ) and complexins ( strenzke et al . 2009
in contrast , the multi - c2 domain protein otoferlin plays a central role for hair cell exocytosis ( roux et al .
the absence or mutation of otoferlin causes deafness or temperature - sensitive hearing impairment in humans ( yasunaga et al . 1999 ; varga et al .
2008 ) and rodents ( roux et al . 2006 ; longo - guess et al . 2007 ;
ultrastructurally , otoferlin is also found at ribbon synapses , mostly but not exclusively at synaptic vesicles and the az membrane ( roux et al .
sufficient amounts of otoferlin appear to be required for correct vesicular fusion and replenishment ( roux et al .
otoferlin is suggested to act as the ca sensor in ihcs ( roux et al .
2006 ; johnson and chapman 2010 ) , due to its ca - binding capabilities ( roux et al . 2006 ; ramakrishnan et al .
2010 ) and facilitates snare - mediated liposome fusion ( johnson and chapman 2010 ) . in its absence , no depolarization - evoked rrp exocytosis is observed in ihcs ( roux et al .
2010 ) . transgenic expression of synaptotagmin 1 , the major ca sensor of neuronal synaptic vesicle exocytosis , failed to restore ihc exocytosis and hearing in otoferlin ko mice , which may not be too surprising given the overall low conservation of the molecular composition between conventional and ihc synapses ( reisinger et al .
next to proteinaceous exocytosis machineries , the actual mechanisms of vesicle fusion as well as the transport of vesicles to the ihc release site are still largely unknown .
the large and , in terms of amplitude and shape , heterogeneous excitatory postsynaptic currents ( epscs ) measured at postsynaptic afferent boutons of sgns have been interpreted to result from multivesicular ( multiquantal ) release at ihc azs ( glowatzki and fuchs 2002 ) .
large epscs ensure rapid and temporal precise spike generation of sgns ( rutherford et al .
2012 ) and the relevance of such large epscs for achieving a high synchronization index of postsynaptic firing ( i.e. , better phase locking precision ) has recently been shown in the frog papilla ( li et al .
several multiquantal release scenarios at ihc ribbons have been discussed : ( 1 ) synchronized vesicle fusion of several single vesicles as well as ( 2 ) compound fusion , following homotypic vesicle - to - vesicle fusion and ( 3 ) sequential fusion involving homotypic vesicle - to - vesicle fusion while release occurs ( glowatzki and fuchs 2002 ; edmonds 2004 ; neef et al .
( 2014 ) indicated that univesicular ( uniquantal ) release can explain the large size of sgn epscs and that the control of release by a dynamic vesicular fusion pore can account for the observed epsc heterogeneity . at this point
, none of the above discussed mechanisms can definitively be ruled out or confirmed and future work , including detailed morphological analysis using electron microscopy of defined functional states , will be required to advance our understanding of exocytosis mechanism at ihc ribbon synapses . in case vesicles
do not homotypically fuse with other vesicles at the ribbon while releasing , a transport mechanism of the vesicles to the membrane has to exist .
the conveyor belt model , transporting the vesicle actively along the ribbon to the membrane , was one of the first models to be introduced ( bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001 ) .
accordingly , a kinesin polypeptide , kif3a , was identified on photoreceptor ribbons that could serve as a motor for vesicle transport involving the filamentous tethers observed at the ribbon ( muresan et al .
1999 ) , which were also proposed to function as stepping stones for synaptic vesicles ( usukura and yamada 1987 ; parsons and sterling 2003 ) .
recently , the tethers at the ribbon were suggested to be directly involved in coordinating vesicle transport towards the membrane via crowd surfing , based on passive diffusion following the gradient established by exocytic vesicle consumption at the base of the ribbon ( graydon et al .
the tethers simply need to bind the vesicles and prevent them from detaching until they reach the az membrane , where release maintains the diffusion gradient ( graydon et al .
2014 ) . however , future experiments involving mutant analyses will be necessary to identify the proteins mediating vesicular tethering to the ribbon and estimate their affinity to the vesicles and the functional relevance of the tethers for synaptic transmission
. moreover , it will be interesting to investigate whether and how the tethering can be influenced by factors such as activity or even developmental stage .
for example , maturation from pre - hearing to hearing significantly determines structure and function of the ribbon synapses and the spatial arrangement of az proteins such as the ca channels or bassoon , as will be emphasized in the next section . during maturation of the organ of corti ,
. how do morphological alterations during the transition from a pre - hearing to a hearing state correlate to functional maturation of ribbon synapses ? generally , synaptic contacts are ultrastructurally defined as pre- and postsynaptic electron - dense membranes that are closely aligned . the postsynaptic density ( psd )
is clearly visible as an electron - dense structure beneath the postsynaptic membranes directly juxtaposed to the presynaptic az .
the innervation pattern of sgn fibers at hair cells within the immature rodent cochlea is significantly different from the mature configuration and massive rearrangements of the fibers that occur before the onset of hearing .
hereby , type i sgn fibers retract from the ohcs , whereas type ii sgn fibers disappear from the ihcs ( perkins and morest 1975 ; echteler 1992 ; simmons 2002 ) .
these developmental processes of fiber innervation take place in the first postnatal week in three distinct phases : ( 1 ) in e18-p0 animals , fibers of both afferent types extend towards all hair cells ; ( 2 ) between p0 and p3 a refinement occurs , where the outer spiral bundle forms that innervate the ohcs ; and ( 3 ) the type i fibers retract from the ohcs around p3p6 , accompanied by synaptic pruning , while they keep their projections on the ihcs ( huang et al .
ampa - typed glutamate receptors and scaffold proteins like bassoon and shank1 disappear during the maturation process from ohcs .
glua2/3 subunits remain stable throughout development and into adulthood , while glua4 subunit expression significantly increase in adult type i fibers ( huang et al .
recently , the molecular arrangement of afferent synapses in relation to functional changes at the ihcs has been addressed in more detail using a combination of confocal , stimulated emission depletion ( sted ) and electron microscopy , as well as ihc presynaptic physiology and computational modeling ( wong et al .
it is known that , in the early pre - hearing stages between p6 and p9 , several small apposing pre- and postsynaptic densities mark nascent synapses .
some of the presynaptic densities are occupied by synaptic ribbons , which are small and round in shape and attached via two triangular - shaped proteinaceous anchors ( sobkowicz et al .
however , floating ribbons were also frequently observed in close proximity to az areas at these developmental stages ( wong et al .
serial 3d electron microscopic reconstructions corroborated the notion of several discontinuous pre- and postsynaptic specializations .
such synaptic sites are organized as loose suprastructures on the bouton surface and are likely functional , as immunohistochemistry indicates the presence of presynaptic ca channels and postsynaptic ampa receptors ( wong et al .
2000 ; hell 2007 ) , revealed that cav1.3 channels are arranged in small round spots ( wong et al .
2014 ) rather than the stripes previously described for mature azs ( frank et al . 2010
in addition , a huge number of extrasynaptic cav1.3 channels can be observed in immature ihcs ( zampini et al .
2014 ) , which enable the cells to fire ca action potentials ( kros et al . 1998 ; brandt et al .
these action potentials evoke exocytosis in the pre - hearing stage ( beutner and moser 2001 ; glowatzki and fuchs 2002 ; johnson et al .
2005 ) but show lower ca efficiency ( beutner and moser 2001 ; brandt et al .
2003 ; johnson et al . 2005 ) and a supra - linear ca dependence ( johnson et al . 2005 ) .
the pre - sensory ihc activity appears to drive bursting activity in the developing auditory system ( glowatzki and fuchs 2002 ; tritsch et al .
2007 , 2010 ; wong et al . 2013 ; clause et al . 2014 ) . in this context , the regulation of presynaptic firing by paracrine and/or efferent synaptic control is being subject to intense research ( glowatzki and fuchs 2000 ; tritsch et al .
efferent innervation , moreover , seems to play an important role in the maturation process of ihcs ( glowatzki and fuchs 2000 ; marcotti 2004 ; goutman et al .
efferent fibers originate from the superior olivary complex and , before onset of hearing , form transient axosomatic contacts with ihcs ( simmons et al .
later , they largely retract from ihcs and rather form axodendritic contacts to the afferent terminals ( pujol et al .
the transient efferent inhibition is thought to counteract the ihc depolarization resulting from the resting mechanotransducer current ( gloc and holt 2003 ; waguespack et al .
2007 ; lelli et al . 2009 ) . upon genetically induced impairment of the efferent input , the linearization of ca dependent exocytosis
is affected ( johnson et al . 2007 ) and the maturation of ihc afferent synapses is also disturbed ( johnson et al .
2013b ) . around the onset of hearing ( at around p11 ; mikaelian and ruben 1965 ) ,
when graded receptor potentials start governing transmitter release , extrasynaptic cav1.3 channels get pruned and spatial coupling of ca channels and vesicular release sites is tightened .
ca efficiency of exocytosis and a near - linear ca dependence of rrp exocytosis when probed with changes in the number of open ca channels ( wong et al .
2014 ) . therefore , while the intrinsic ca dependence of exocytosis apparently does not change upon the onset of hearing , experimental data and biophysical modeling of exocytosis at mature and immature az topographies support the notion of a developmental switch from the more ca microdomain - like control of exocytosis by several ca channels per vesicle to a more ca nanodomain - like control of exocytosis ( wong et al . 2014 ; fig . 1e , e ) .
interestingly , in adult gerbils , the open probability of ca channels in ihcs increased due to a preference of the ca channel for the bursting mode ( zampini et al .
structurally , alongside ca channels , other presynaptic az components become reorganized such as the bassoon containing presynaptic density ( fig .
these alterations are accompanied by changes of the postsynaptic glutamate receptor fields that also develop to one continuous ring - like cluster ( wong et al .
moreover , ribbons increase in size and undergo striking changes of shape . at the ultrastructural level , their cross - sectional shape changes from predominantly round ( fig .
1a ) to a rather oval- , droplet- or wedge - like shape between p14 and p20 ( wong et al . 2014
the two rootlets seem to merge to a continuous presynaptic density that contains the scaffolding protein bassoon , as revealed by immunogold labeling ( wong et al .
shortly after onset of hearing at p14 , a large proportion of ribbons with two rootlets can still be found , whereas about a week later the morphological maturation appears to be completed ( wong et al .
factors that participate in the maturation of ihc synapses , next to the efferent olivocochlear transmission ( see above ; johnson et al .
2013a ) , are thyroid hormone ( sendin et al . 2007 ) and myosin 6 ( heidrych et al . 2009 ; roux et al . 2009 ) . for both , a higher proportion of morphological immature ribbons have been observed in genetic deletion models . in conclusion , during development from pre - hearing to hearing , ihc ribbon synapses undergo major morphological and functional refinements , resulting in tighter spatial coupling between ca influx and exocytosis ( wong et al .
the number of ca channels , vesicular release sites and ribbon - associated vesicles seems to scale with the size and number of ribbons at the az ( martinez - dunst et al .
; graydon et al . 2011 ; kantardzhieva et al . 2013 ; wong et al . 2013 , 2014 ) .
strengthening of presynaptic transmitter release might therefore be accomplished by increasing ribbon or az size and/or ribbon numbers per az . moreover ,
finally , lateral olivocochlear efferent fibers might modulate postsynaptic excitability and thereby affect afferent synaptic strength . to establish which of these mechanisms contribute to determining and regulating synaptic strength of hair cell synapses
interestingly , the size and shape of ribbons appear to be highly variable and dynamic .
in fact , in photoreceptors , these parameters strongly correlate with activity in light ( silent ) or dark ( active ) conditions ( spiwoks - becker et al .
similarly , in ihcs , a diverse spectrum of ribbons has also been observed ( bodian 1978 ; sobkowicz et al .
the specific ultrastructural properties seem to depend on several factors : ( 1 ) the maturation / age ( see section above ) , ( 2 ) position within the inner hair cell and maybe also ( 3 ) dynamic adaptation to activity .
a pioneering study in cats was one of the first to identify the correlation between structural heterogeneity of ribbon synapses and functional characteristics of auditory nerve fibers ( merchan - perez and liberman 1996 ) .
surprisingly , large azs with big and/or several ribbons , supposedly reflecting large presynaptic strength , seem to drive sgns with low spontaneous rate and high thresholds ( see also scheme in fig .
whereas this conundrum remains unsolved , the mechanisms of functional presynaptic heterogeneity are now beginning to be understood .
evidence for such heterogeneity within individual ihcs was obtained using confocal imaging of presynaptic ca influx ( frank et al .
this study showed that presynaptic ca signals varied substantially in amplitude and voltage - dependence among the azs within individual ihcs .
the amplitude of the ca signal scaled with ribbon size as approximated by simultaneous imaging of a fluorescently tagged ribeye - binding peptide ( frank et al .
2009 ) and seemed to be greater at the neural side of the ihcs ( meyer et al .
linking such estimates to the functional and morphological properties of the postsynaptic neurons will be an important task for future studies .
so far , correlative arguments based on coincidental changes in maximal strength of presynaptic ca influx and postsynaptic spiking during development and upon genetic disruption as well as modeling have been brought forward to argue that strong synapses drive sgns that have high spontaneous rates and low thresholds ( wong et al .
interestingly , an inverse correlation of pre- and postsynaptic parameters of synaptic strength has recently been reported for mouse ihcs : liberman et al .
the authors favored the interpretation that the sgns inserting at the neural ( modiolar ) face of ihcs exhibit low spontaneous rates and high thresholds despite their corresponding large ihc azs , because they have a smaller complement of ampa receptors than those at the neural ( pillar ) side .
this would agree with the conclusion of the classical study , which showed a neural
abneural gradient of az size using electron microscopy for cat ihcs whereby large azs faced sgns with low spontaneous rates and high thresholds ( merchan - perez and liberman 1996 ) . in a laborious approach , the authors traced 11 functionally - characterized fibers to the ihcs using serial 3d reconstructions of ultrathin sections . in this way , it was possible to directly correlate morphological parameters such as ribbon length , fiber contact area , synaptic plaque area and synaptic vesicle numbers to the functional parameters determined prior to fiber labeling using single unit recordings .
recently , such a gradient was also suggested for mouse ihcs and reported to be influenced by the lateral olivocochlear innervation ( yin et al .
the segregation of nerve fibers on neural and abneural sides was further observed in a study investigating the abundance of mitochondria in postsynaptic terminals . here , postsynaptic boutons facing the abneural side seem to harbor more mitochondria ( francis et al .
2004 ) . monitoring epscs from single afferent boutons , which is a suitable method to address synaptic function on the level of individual release sites ( glowatzki and fuchs 2002 ) , further showed differences among synapses . in these experiments ,
varying fractions of multiphasic epscs were observed and proposed to underlie the diverse firing properties of sgns ( grant et al .
a schematic of an organ of corti showing afferent and efferent innervations at ihcs . modified from meyer and moser 2010 , curr opin otolaryngol head neck surg , reprinted with permission from 2010 wolters kluwer health .
b mean and sd of f ( gray ) as a function of depolarizing potential ( v
m ) , obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus .
f ( mean gray ) and i
ca ( mean black ) show a similar voltage dependence ( thin lines corresponding sds ) .
b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs .
pronounced variability in the voltage dependence of activation , even within the same cell ( dashed traces individual data curves from 3 ca microdomains in an ihc ) . modified from frank et al .
c , c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate ( sr ) fibers .
sections through a high - sr ( c ) and a low - sr ( c ) synapse containing the synaptic ribbon are shown with cisternal ( maroon ) and vesicular ( green ) profiles .
d mean density ( se ) of docked vesicles , i.e. , within 20 nm of the presynaptic density along the presynaptic membrane .
d mean number ( se ) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses .
2013 , j comp neurol , reprinted with permission from 2013 wiley periodicals ) principle of functional heterogeneity in ihcs .
a schematic of an organ of corti showing afferent and efferent innervations at ihcs . modified from meyer and moser 2010 , curr opin otolaryngol head neck surg , reprinted with permission from 2010 wolters kluwer health .
b mean and sd of f ( gray ) as a function of depolarizing potential ( v
m ) , obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus .
f ( mean gray ) and i
ca ( mean black ) show a similar voltage dependence ( thin lines corresponding sds ) .
b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs .
pronounced variability in the voltage dependence of activation , even within the same cell ( dashed traces individual data curves from 3 ca microdomains in an ihc ) . modified from frank et al .
c , c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate ( sr ) fibers .
sections through a high - sr ( c ) and a low - sr ( c ) synapse containing the synaptic ribbon are shown with cisternal ( maroon ) and vesicular ( green ) profiles .
d mean density ( se ) of docked vesicles , i.e. , within 20 nm of the presynaptic density along the presynaptic membrane .
d mean number ( se ) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses .
2013 , j comp neurol , reprinted with permission from 2013 wiley periodicals ) further insights into the morphological heterogeneity require modern 3d reconstructions of larger volumes such as serial block face scanning electron microscopy ( denk and horstmann 2004 ) or focused ion beam scanning electron microscopy ( see , e.g. , knott et al . 2008 ; kreshuk et al .
using these methods , a detailed 3d view of single ihcs including afferent and efferent innervations would be possible , finally allowing correlation of parameters , such as presynaptic ribbon size and postsynaptic bouton diameter , as a function of position on the ihc for a large number of synapses , together with the functional assessment of pre- and postsynaptic properties in cochlear explants by electrophysiology ( goutman and glowatzki 2007 ) and/or imaging of ihc ca and exocytosis ( e.g. , frank et al .
tight coupling between exo- and endocytosis is a prerequisite for maintaining the enormous vesicle turnover rates at ribbon synapses
clathrin - coated structures but also large cisterns without clathrin - coats , are observed close to synaptic ribbons ( siegel and brownell 1986 ; sendin et al .
2007 ; frank et al . 2010 ; kantardzhieva et al . 2013 ; neef et al . 2014 ; revelo et al . 2014 ) .
2013 ) set out to determine whether such cisterns participate in vesicle reformation and what differences can be observed in correlation to the functional properties of high and low spontaneous rate fibers ( fig .
an extensive quantitative analysis of ribbons , vesicles and cisterns from serial sections of cat ihc ribbon synapses suggested a
fewer cisterns and more synaptic vesicles are found , which indeed points towards a contribution of cisterns to locally restricted vesicle formation .
other studies used membrane capacitance measurements to provide an initial functional assessment of endocytic membrane retrieval at ihc azs ( moser and beutner 2000 ; beutner and moser 2001 ; neef et al .
2014 ) . moreover , ph - sensitive gfp ( phluorin ; miesenbck et al . 1998 ) targeted to the intraluminal face of vesicle membranes by attachment to vesicular glutamate transporters ( zhu et al .
2009 ) has become an important tool in studying exo- and endocytosis , not only from neurons but also ihcs ( neef et al .
additionally , a novel membrane tracer specifically tailored to use in the organ of corti has been devised and applied to investigate endocytosis ( revelo et al .
2014 ) , as the commonly used styryl dye fm1 - 43 penetrates stereociliar mechanotransduction channels and hence is of limited use to study endocytosis , in ihcs ( gale et al .
, expression analysis and immunohistochemistry have revealed the presence of several important molecular players of endocytosis such as dynamins , amphiphysin , clathrin ( neef et al . 2014 ) and adaptor protein 2 ( ap-2 ) ( duncker et al .
a very recent dna microarray study investigating ihc and ohc transcriptomes might even give more insight into proteins involved in vesicle recycling ( liu et al .
currently , in ihcs , three distinct mechanisms are considered to mediate endocytosis : slow cme , fast bulk endocytosis and potentially kiss - and - run or
cme is the main pathway of membrane retrieval for mild stimulation and proceeds at a constant rate ; it represents the linear component of endocytosis following exocytosis of the rrp ( fig .
this mechanism is not only inhibited by the clathrin - inhibitor pitstop-2 but also by disruption of dynamin 1 via pharmacological and genetic means ( neef et al .
in contrast , a different study reported inhibition of sustained exocytosis by the presumptive dynamin inhibitor dynasore but did not investigate endocytic membrane retrieval ( duncker et al .
finally , when exocytosis exceeds three to four rrp equivalents , ihcs additionally recruit a faster mode of membrane retrieval , which proceeds with an exponential time course within a few seconds .
3a ) and , indeed , there is plenty of evidence for the invagination and fission of large stretches of plasma membrane in the vicinity of hair cell azs ( lenzi et al .
2002 ; frank et al . 2010 ; pangri et al . 2010 ; kamin et al . 2014 ; neef et al . 2014 ; revelo et al .
both mechanisms seem to engage in different phases of release : cme supports vesicle cycling during mild stimulation but bulk endocytosis finally occurs after prolonged stimulation , providing a mechanism that assures the balance between exo- and endocytosis in ihcs and thus , assures high release rates ( neef et al .
3endocytosis in inner hair cells . a , a representative recordings in response to 20 ms ( a ) or 200 ms ( a ) depolarizations . after the c
m increase upon 20 ms depolarization
, the slope - corrected c
m traces ( middle ) typically showed a linear decay ( a ) .
the 200-ms - long depolarization resulted in a combination of exponential and linear decay ( a ) .
b 3d reconstructions of resting ( b ) , stimulated ( b ) and recovered ihcs ( b , b ) .
most organelles , including the tubular ones , are replaced by small vesicles during the recovery periods .
insets magnified regions from the four different cell regions ( cuticular plate , top , nuclear and basal regions ) . note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery .
c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin ( first row ) , for vglut3 and syntaxin 6 ( sx 6 , second row ) , for otoferlin and syntaxin 16 ( sx 16 , third row ) and finally for syntaxin 6 and syntaxin 16 ( fourth row ) .
the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope . dashed white lines the plasma membrane of the ihcs .
d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 ( or syntaxin 16 ) correlate in the mcling - labeled organelles at the top and nuclear levels .
organelles with a different molecular composition recycle membrane in different regions , taking up mcling .
apical endocytosis takes up the membrane into round organelles , a sizeable proportion of which is similar to late endosomes ( light blue ) .
endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers , syntaxin 16 and syntaxin 6 .
this suggests that these organelles participate in constitutive pathways , probably by maintaining membrane traffic between the plasma membrane and the trans - golgi . at the base of the cell
, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3 , rab3 and also otoferlin .
endocytosis in inner hair cells . a , a representative recordings in response to 20 ms ( a ) or 200 ms ( a ) depolarizations . after the c
m increase upon 20 ms depolarization
, the slope - corrected c
m traces ( middle ) typically showed a linear decay ( a ) .
the 200-ms - long depolarization resulted in a combination of exponential and linear decay ( a ) .
b 3d reconstructions of resting ( b ) , stimulated ( b ) and recovered ihcs ( b , b ) .
most organelles , including the tubular ones , are replaced by small vesicles during the recovery periods .
insets magnified regions from the four different cell regions ( cuticular plate , top , nuclear and basal regions ) . note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery .
c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin ( first row ) , for vglut3 and syntaxin 6 ( sx 6 , second row ) , for otoferlin and syntaxin 16 ( sx 16 , third row ) and finally for syntaxin 6 and syntaxin 16 ( fourth row ) .
the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope . dashed white lines the plasma membrane of the ihcs .
d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 ( or syntaxin 16 ) correlate in the mcling - labeled organelles at the top and nuclear levels .
organelles with a different molecular composition recycle membrane in different regions , taking up mcling .
apical endocytosis takes up the membrane into round organelles , a sizeable proportion of which is similar to late endosomes ( light blue ) .
endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers , syntaxin 16 and syntaxin 6 .
this suggests that these organelles participate in constitutive pathways , probably by maintaining membrane traffic between the plasma membrane and the trans - golgi . at the base of the cell
, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3 , rab3 and also otoferlin .
2014 , reprinted with permission from 2014 revelo et al . ) but where does the retrieval of synaptic vesicle membrane take place and where and how are synaptic vesicles regenerated following membrane retrieval in hair cells ? does synaptic endocytosis comply with the apicobasal compartmentalization of the hair cell ? at photoreceptor ribbons , a periactive zone , marked by the presence of endocytic proteins , was identified directly adjacent to the az area ( in a range of 120250 nm from the ribbon ) using high - resolution and electron microscopy ( wahl et al .
does this also apply to ihc ribbons ? at the resolution of confocal microscopy , a similar perisynaptic accumulation of endocytic proteins has , so far , not been found ( neef et al .
clearly , electron micrographs indicate that both bulk and cme endocytosis take place near the az ( siegel and brownell 1986 ; lenzi et al .
2002 ; frank et al . 2010 ; kamin et al . 2014 ; neef et al .
a radically different model of ihc endocytosis has been sketched based on life imaging of fm1 - 43 uptake into ihcs , whereby exocytosed membrane was postulated to move towards the ihc apex for endocytosis and recycling via the golgi apparatus ( griesinger et al .
recent elaborative studies using various styryl dyes and more suitable fluorescent membrane markers lead us to reconsider this hypothesis .
first , it was corroborated that many styryl dyes including fm1 - 43 permeate into the cytosol of ihcs via the mechanotransducer channels ( kamin et al .
therefore , on the ultrastructural level , the photo - oxidation technique revealed a fuzzy dark diaminobenzidin ( dab ) smear inside the cytosol in addition to the precipitate in membrane - bound organelles likely resulting from internalized fm1 - 43 .
the stimulation - induced endocytic uptake of fm1 - 43 could still be followed by observing an electron - dense precipitate within vesicular structures , which allows the determination of the presence or absence as well as the localization of stained structures under resting ( fig .
3b ) and recovery ( fig . 3b , b ) conditions in serial 3d reconstructed ihcs ( kamin et al . 2014 ) .
at rest , endosome - like organelles were detected in the apex of the ihcs , whereas larger tubulo - cisternal organelles dominated at the nuclear region . at the basal region , only a few labeled structures were present .
stimulation massively increased the amount of basolateral membrane trafficking , reflected by the appearance of labeled small vesicles and endosome - like vacuoles ; however , no changes in the apical and nuclear regions could be observed ( kamin et al .
strikingly , the basolateral cisterns were replaced in the basal region by small , synaptic - like vesicles during a few minutes of recovery from stimulation , suggesting a highly efficient mechanism of vesicle regeneration from cisternal membranes internalized by bulk endocytosis .
the combination of fm1 - 43 uptake and photo - oxidation therefore suggests that synaptic vesicle recycling takes place at the basal part , close to ribbons at least during synaptic activity ( kamin et al .
recently , a novel membrane tracer , named membrane - binding fluorophore - cysteine - lysine - palmitoyl group ( mcling ) , which does not permeate the mechanotransducer channel , tightly binds biological membranes and can be fixed , has been developed ( revelo et al .
2014 ) . in combination with super - resolution light microscopy ( i.e. , sted ) , the spatial organization and pathways of endocytosis in ihcs could be further investigated . in order to improve the spatial resolution of sted microscopy in the axial direction ,
thin sections of ihcs were imaged after embedding the organs of corti in melamine , which maintains the fluorescence ( revelo et al .
the apical , nuclear and basal regions under conditions of rest , stimulation and recovery from stimulation , were investigated and the uptake of mcling monitored . in order to reveal the molecular identity of mcling - labeled structures and thereby identify the respective endocytic pathways , samples were co - stained with different protein markers for the endoplasmic reticulum ( er ) and golgi as well as synaptic vesicle endo- and exocytosis . in these experiments , a strong correlation for basolateral mcling localization with vglut3 , rab3 and otoferlin immunofluorescence was found .
otoferlin as well as syntaxin 16 , a late endosomal marker , colocalized with apical and nuclear mcling ( fig .
moreover , the lysosomal - associated membrane protein 1 ( lamp1 ) colocalized with mcling in the apical region of the ihc .
stimulation led to a selective uptake of mcling at the base of the ihc , corroborating the notion of local recycling of synaptic vesicles that was postulated based on electron microscopy and photo - oxidation ( kamin et al .
the local recycling hypothesis was further supported by the finding that exogenous vglut1-phluorin fluorescence not only transiently appeared at ribbon - type azs but remained there for tens of seconds after stimulation ( neef et al .
finally , the mcling experiments revealed large membranous organelles near synapses , which were replaced by small organelles a few minutes after stimulation , thereby providing direct evidence of bulk endocytosis and vesicle regeneration from the internalized plasma membrane .
the association of otoferlin with all three putative membrane recycling pathways suggests a more general role of this protein in endocytosis .
otoferlin has recently been assigned a role in vesicle endocytosis due to its interaction with ap-2 . using a high - resolution liquid chromatography coupled with a mass spectrometry approach ,
multiple subunits of ap-2 were identified as interaction partners of otoferlin in the mammalian cochlea and the proposed interactions were biochemically confirmed by co - immunoprecipitation ( duncker et al . 2013 ) .
ap-2 plays a role in clathrin - mediated endocytosis via binding to clathrin - coated vesicles budding from the plasma membrane ( keyel et al .
2010 ) and has been shown to be expressed in ihcs ( duncker et al .
future work is required to clarify the role of ap-2 in hair cell endocytosis and the relevance of its interaction with otoferlin .
recently , major progress has been made towards dissecting the molecular anatomy and physiology of hair cell ribbon synapses .
this includes powerful single synapse techniques such as ( 1 ) patch - clamp of postsynaptic afferent terminals of sgns , ( 2 ) high resolution -functional imaging of presynaptic ihc ca dynamics and membrane turnover , as well as ( 3 ) super - resolution light microscopy and electron tomography following high - pressure freezing .
however , in order to investigate the release mechanisms of ihcs and firmly correlate structure and function , the development of new functional and morphological approaches is required . functional and morphological analysis of single synapses will be necessary and some questions require reading out both pre- and postsynaptic properties at the same time .
the commonly used k stimulation of cochlear tissue likely mimics strong physiological steady - state stimulation .
but this stimulation does not provide the temporal resolution to allow the observation of the release kinetics at ihc ribbon synapses .
especially , knowledge about short - term plastic changes is lacking , since it is not possible to apply very short stimuli ( i.e. , millisecond range ) and investigate the cells during and at defined times after stimulation .
therefore , approaches are needed that meet two requirements : ( 1 ) a precise stimulation protocol combined with ( 2 ) rapid immobilization of the sample , e.g. , by using high - pressure freezing .
one emerging tool that promises to fulfill these requirements is the combination of optogenetic stimulation with high - pressure freezing .
this could involve the expression of a light - sensitive ion channel such as channelrhodopsin-2 ( chr-2 ) from the green algae chlamydomonas reinhardii ( nagel et al . 2003 ) in hair cells and stimulation would ideally be performed within a chamber that should be mounted in a freezing machine in order to minimize the time delay before freezing .
recently , synaptic recovery of motoneurons from c. elegans was analyzed using optogenetic stimulation in combination with high - pressure freezing ( kittelmann et al .
moreover , after a single light stimulus , docked vesicles fused along a broad az on c. elegans motoneurons expressing chr-2 .
these vesicles were replenished with a time constant of about 2 s. further , endocytosis occurred within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions ( watanabe et al .
moreover , a study on optically stimulated cultured hippocampal neurons revealed an ultrafast endocytosis mechanism at central synapses ( watanabe et al .
these initial experiments indicate that optogenetics , in combination with high - pressure freezing ( flash and freeze ; watanabe et al .
2013b ) and subsequent electron tomography , might provide sufficient resolution to study the ultrastructure of spatiotemporally defined functional states and thus provide a completely new view on the release mechanism of ihc ribbon synapses .
how does the subcellular organization of sensory ihcs enable mechanotransduction and transmitter release at high rates ?
ihcs are epithelial cells by origin and exhibit several characteristics that distinguish them from neurons .
most notably , they show a strong polarization with respect to both long and short cell axes . the polarization along the apicobasal axis follows a clear compartmentalization , e.g. , apparent by the hair bundle harboring the mechanotransduction apparatus of the apical membrane . graded receptor potentials
are formed by mechanoelectrical ( apical ) and voltage - gated ( basal ) conductances ( corey and hudspeth 1979 ; roberts et al . 1990 ) .
actin - filled stereocilia protrude into the endolymph in a highly organized manner and their sophisticated supramolecular mechanotransduction apparatus enables ultrasensitive detection of sound - born vibrations of the cochlear partition ( reviewed in kazmierczak and mller 2012 ) .
while the molecular identity of the mechanotransducer channel still awaits definitive demonstration , recent work indicates the transmembrane channel - like proteins ( tmc)-1 and -2 as promising candidates ( pan et al .
opening of the apical mechanotransducer channels depolarizes the ihc , subsequently activating cav1.3 ca channels ( platzer et al .
; dou et al . 2004 ) at the presynaptic az in the basolateral membrane , where the incoming ca triggers neurotransmitter release .
the density of ribbon synapses shows a strong basoapical gradient , with the supranuclear portion of the hair cell being devoid of azs ( francis et al .
, the cuticular plate likely serves as an anchor for the stereociliar actin bundles , containing a rich protein network with cytoskeletal proteins such as actin , -actinin and tropomyosin ( slepecky and chamberlain 1985 ; zine and romand 1993 ) .
moreover , the striated organelle , located underneath the cuticular plate , likely modulates the stereociliar actin bundles ( vranceanu et al .
microtubules are primarily found beneath the cuticular plate ( slepecky and chamberlain 1985 ; steyger et al .
1990 ) but appear connected to cytoskeletal proteins in the cuticular plate , for example via acf7a ( actin crosslinking family protein 7a ) , as suggested for zebrafish neuromast hair cells ( antonellis et al . 2014 ) .
1990 ) , providing the mechanical strength of hair cells ( szarama et al . 2012 ) and tracks for efficient cargo protein trafficking along the apicobasal axis ( furness et al . 1990 ) .
in addition to the cellular apicobasal polarity , hair cells also show planar cell polarity , which is reflected in the orderly orientation of their hair bundles ( reviewed in ezan and montcouquiol 2013 ; sienknecht et al .
2014 ) . whether the basolateral organization of the hair cells is similarly instructed by planar cell polarity
, we will focus on the organization of the basal portion of ihcs and discuss structure and function of hair cell ribbon synapses .
emphasis will be on the molecular machinery of the synapse , synapse development , synaptic heterogeneity and synaptic vesicle recycling .
phylogenetically , ribbons in sensory cells are old structures that occur not only in mammals but also in fishes , amphibians and birds . in the mammalian organ of corti , they were first described by smith and sjstrand ( 1961 ) and are found in both sensory cell types , i.e. , ihcs and ohcs ( sobkowicz et al .
1982 ) . the discovery of the protein ribeye , initially purified from bovine retina , ( schmitz et al .
2000 ) , as the main and structure - yielding component of ribbons in rat photoreceptors ( schmitz et al .
2003 ) , zebrafish photoreceptors and bipolar cells ( wan et al . 2005 ) and mouse cochlear hair cells ( khimich et al . 2005 ; see also immunogold labeling in fig .
1a ) highlights the conservation of the ribbon in vertebrate evolution ( schmitz 2009 ) .
nonetheless , ribbons still vary greatly in size and shape ( lenzi and von gersdorff 2001 ; moser et al . 2006 ; matthews and fuchs 2010 ) , likely reflecting structural adaptation to the specific needs of the respective synaptic connection for sensory coding.fig .
a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement ( contrast enhanced image in a ) , see also schematic representation ( a ) .
b a p14 mature ribbon with the typical multi - lamellar pattern ( contrast enhanced image in b ) , see also scheme in b . scale bars ( a , a , b ) 100 nm .
c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools ( yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density ) . c the membrane - proximal vesicles ( orange ) are arranged around the presynaptic density ( magenta ) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in ( d ) , scale bar ( d ) 100 nm ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; d 2-color sted image of immunolabeled bassoon ( magenta ) and cav1.3 channel clusters ( green ) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed .
scale image:700 700 nm ; e , e mathematic model showing the total mean steady state [ ca ] profile at the az membrane ( e ) ; e effective number of cav1.3 channels contributing to total mean steady state [ ca ] as shown in ( e ) .
( c , c , d , e , e modified from wong et al .
2014 , embo j ; reprinted with permission 2014 wong et al . ) .
f schematic summary of the protein arrangement at mature ihc ribbon synapses spatial distribution of ihc az proteins .
a ribeye is the main component of the ribbon as shown by pre - embedding immunogold labeling of a p14 ihc synaptic ribbon using an anti - ctbp2 antibody ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; a representative image of an electron micrograph of a round - shaped p9 immature ribbon exhibiting a dotted pattern possibly caused by ribeye arrangement ( contrast enhanced image in a ) , see also schematic representation ( a ) .
b a p14 mature ribbon with the typical multi - lamellar pattern ( contrast enhanced image in b ) , see also scheme in b . scale bars ( a , a , b ) 100 nm .
c a serial 3d reconstruction of a mature ribbon with two distinct morphological vesicle pools ( yellow : ribbon - associated vesicles ; orange : membrane - proximal vesicles ; red : ribbon ; blue : az membrane ; magenta : presynaptic density ) . c the membrane - proximal vesicles ( orange ) are arranged around the presynaptic density ( magenta ) that is containing the scaffolding protein bassoon as shown by the pre - embedding immunogold labeling in ( d ) , scale bar ( d ) 100 nm ( courtesy of susann michanski , innerearlab , university medical center , gttingen , germany ) ; d 2-color sted image of immunolabeled bassoon ( magenta ) and cav1.3 channel clusters ( green ) in mature ihcs : stripelike morphology and closely aligned immunofluorescence of bassoon and cav1.3 can be observed . scale image:700 700 nm ; e , e mathematic model showing the total mean steady state [ ca ] profile at the az membrane ( e ) ; e effective number of cav1.3 channels contributing to total mean steady state [ ca ] as shown in ( e ) .
( c , c , d , e , e modified from wong et al .
2014 , embo j ; reprinted with permission 2014 wong et al . ) .
f schematic summary of the protein arrangement at mature ihc ribbon synapses ribeye is composed of two major domains : while the a domain organizes the assembly of the synaptic ribbon and is unique in structure , the b domain is structurally nearly identical to the transcription repressor ctbp2 , which is encoded by the same gene but uses a different transcription initiation site ( schmitz et al . 2000 ) and exhibits enzymatic activity ( schwarz et al .
the b domain is also assumed to be involved in tethering of synaptic vesicles to the ribbon ( schmitz et al .
2000 ; schmitz 2009 ) , though the proteins that form tethers remain to be identified .
ribeye appears to organize ribbon shape directly based on its domain structure ( schmitz 2009 ) and its aggregation properties ( magupalli et al .
a trifold lamellar pattern has been described for photoreceptor ribbons and assigned to the polarized arrangement of ribeye ( schmitz 2009 ) . also at mature ihc ribbons , a lamellar substructure is observed that harbors multiple lamellar foldings ( sobkowicz et al .
this effect can be attributed to the differences in the ribbon shape in ihcs and photoreceptors . in immature ihc ribbons ,
the lamellar pattern is not prominent but instead a dotted pattern can be observed ( fig .
recently , at zebrafish hair cell ribbon synapses , it was found that the ribeye a and b domain segregate along the vertical axis of the ribbons , with the b domain more located towards the basal end ( sheets et al .
ribeye , in contrast to other az proteins , is not found in invertebrates such as the fruitfly drosophila melanogaster ; however , bruchpilot ( brp ) , the homolog of the vertebrate az protein cast ( caz - associated structural protein)/erc2 , functions as the main building block of t - bars ( kittel et al . 2006 ; wagh et al .
in fact , only the n - terminus is conserved and shows sequence homologies to cast , whereas the c - terminus is only found in dipteran insects and rather resembles cytoskeletal elements such as plectin due to its numerous coiled - coil domains ( wagh et al .
moreover , the c - terminus mediates vesicle tethering to the t - bar ( hallermann et al .
the structurally related proteins bassoon and piccolo as well as cast , elks , rab3-interacting molecule ( rim ) and munc13 are present ( betz et al . 1998 ; fenster et al . 2000 ; dresbach et al . 2001 ; deguchi - tawarada et al . 2006 ; wang et al . 2009 ; sdhof 2012 ) .
additionally , ctbp2 and ctbp1 have also been found at conventional azs ( tom dieck et al . 2005 ) . except for piccolo , which at ribbon synapses is solely expressed as a shorter splice variant nicknamed as piccolino ( regus - leidig et al .
2013 ) , these proteins also largely form the caz at photoreceptor ribbon synapses ( wang et al .
the components of hair cell azs , on the other hand , are still largely unexplored , except for bassoon ( khimich et al . 2005 ) and piccolo / piccolino ( khimich et al . 2005 ; regus - leidig et al .
in fact , a recent study indicates that ihc synapses operate without munc13-like priming factors ( vogl et al .
bassoon , together with ribeye , is responsible for the ribbon shape and , hence , might contribute to its function .
studies of ihcs from bassoon mutant mice indicated an anchoring function of bassoon ( khimich et al . 2005 ; frank et al . 2010
; jing et al . 2013 ) , in line with findings at photoreceptor ribbon synapses ( dick et al . 2003 ; tom dieck et al .
the fraction of ribbon - occupied synapses remaining in bassoon - deficient ihcs seems to depend on age and residual levels of full - length bassoon ( khimich et al .
the fraction of ribbonless synapses increased from 50 % at postnatal day 11 ( p11 ) up to 88 % at p70 .
a lower and relatively constant fraction of 56 % ribbonless synapses was found in a gene - trap bassoon mutant ( bsn ) likely due to a weak residual synaptic expression of bassoon ( jing et al .
maintained az ultrastructure , bsn animals exhibited a larger number of ca channels at ihc synapses compared to bsn mice and also displayed an intermediate phenotype regarding sustained ihc exocytosis ( jing et al .
in contrast , the size of the readily releasable vesicle pool ( rrp ) was strikingly reduced in both mutants . moreover , single unit recordings of the sgns show comparably severe defects in bsn and bsn mice , as both genotypes had impaired sound onset coding and lower evoked and spontaneous spike rates .
taken together , these results indicate that the remaining , loosely anchored ribbons might function inadequately ( jing et al .
this further suggests that the mere presence of the ribbon , even with tethered vesicles , is not sufficient to maintain normal transmitter release and sustain the rrp at ihc ribbon synapses .
moreover , it seems that bassoon contributes to organizing the ihc az beyond anchoring the ribbon .
this has been concluded from impaired clustering of ca channels at bsn ribbon - occupied synapses ( frank et al .
2010 ) and indicates a potential direct contribution of bassoon in organizing the az ( frank et al . 2010 ; hallermann and silver 2013 ) .
in contrast to conventional synapses , where often only the combined knockdown of bassoon and its homolog piccolo causes synaptic defects ( altrock et al .
2003 ; leal - ortiz et al . 2008 ; mukherjee et al . 2010 ; waites et al . 2011 , 2013 ) , these proteins seem not to act redundantly at ribbon synapses .
as mentioned above , at hair cell and photoreceptor ribbons , only the short isoform of piccolo , piccolino , is expressed , which lacks a large c - terminal part ( regus - leidig et al .
binding sites for the proteins abp1 , pra1 , git1 and profilin ( wang et al .
2003 ) are still present , whereas binding sites for , e.g. , bassoon or rim are lacking ( regus - leidig et al .
accordingly , piccolino exhibits a different spatial distribution at ribbon synapses of photoreceptors , where it is found directly on the ribbon , as indicated by pre - embedding immunogold - labelings , using an antibody recognizing the n - terminus of the protein ( limbach et al .
( 2014 ) revealed a striking impairment in the ribbon structure upon piccolino rnai - based knockdown .
instead , a high proportion of attached spherical ribbons was found that resemble ribbon precursors of photoreceptor ribbons suggesting a role of piccolino in structural ribbon maturation ( regus - leidig et al .
ribeye and presumably piccolino as well as bassoon present the main structural components of the ribbon and/or the anchorage of the ribbon to the az . in order to resolve the function of ribbons ,
the ribbon was suggested to ( 1 ) promote a large readily releasable pool of vesicles via establishing / stabilizing many ca channels and vesicular release sites ( khimich et al .
2010 ) , ( 2 ) facilitate vesicle replenishment at the az ( conveyor belt model , e.g. , bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001 ; snellman et al .
2011 ) , ( 3 ) facilitate multivesicular release ( edmonds 2004 ; fuchs 2005 ) , or ( 4 ) serve as a diffusion barrier to enable high local ca concentrations ( graydon et al .
functional interpretations have also been provided for the morphologically distinct populations of synaptic vesicles at ribbon synapses but in each case remain to be validated . ribbon - associated vesicles structurally attached through filamentous protein tethers
the ones at the base of the ribbon face the presynaptic plasma membrane ( membrane - proximal vesicles ; fig . 1c , c ) and are often tethered to the membrane and/or to the presynaptic density ( tethered vesicles ) .
lateral to this subpopulation of ribbon - associated vesicles , there are few additional membrane - proximal and even membrane - tethered vesicles that are not in close vicinity to the ribbon . while further testing is required , current evidence suggests that the membrane - proximal vesicles comprise the rrp ( e.g. , in retinal bipolar cells : von gersdorff et al .
1996 ; zenisek et al . 2000 ; frog saccular hair cells : lenzi et al . 1999 , 2002 ;
rutherford and roberts 2006 ; and mouse inner hair cells : khimich et al . 2005 ; frank et al .
support for this hypothesis comes from the approximate matching between the morphologically estimated number of membrane - proximal vesicles and the functionally defined size of the rrp , i.e. , the fast component of exocytosis upon depolarization - evoked ca influx ( von gersdorff et al . 1996 ; pangri et al . 2010 ; frank et al . 2010 ) , as well as from the observation that these vesicles are most heavily depleted upon stimulation ( lenzi et al . 2002 ; pangri et al .
furthermore , the tethering of vesicles to the az membrane might reflect a structural correlate of fusion competence ( siksou et al .
new high - resolution imaging approaches using rapid freezing methods and/or electron tomography have revealed that synaptic vesicles in proximity to the membrane exhibit several morphologically distinct stages .
tethers of different numbers and lengths connecting synaptic vesicles to the az membrane could be observed at conventional synapses and synaptosome preparations ( siksou et al .
2007 , 2009 ; fernndez - busnadiego et al . 2010 , 2013 ) but also at ihc ribbon synapses ( frank et al . 2010 ) .
moreover , cryo - electron tomography , a method allowing visualization of hydrated and unstained tissue , revealed that tethering of synaptic vesicles in synaptosomes prepared from hippocampal tissue precedes the full contact of a synaptic vesicle with the membrane ( fernndez - busnadiego et al .
single long tethers initially seem to be formed and synaptic vesicles likely enter the rrp via the formation of several short tethers ( < 5 nm ) . in line with this hypothesis
, this fraction of vesicles could be depleted by application of hypertonic sucrose , that is thought to trigger rrp release ( rosenmund and stevens 1996 ) . moreover
, the formation of short tethers could be inhibited using tetanus toxin , pointing towards the fact that neuronal soluble nsf attachment protein receptors ( snare ) proteins are involved in this process ( fernndez - busnadiego et al .
2013 ) play a crucial role in tethering vesicles to the membrane at conventional cns synapses .
interestingly , munc13 and caps priming factors seem not to operate at the ihc ribbon synapse ( vogl et al . 2015 ) .
some authors even argue that the entire ribbon - associated vesicle population is fusion - competent and , therefore , can be released within a few milliseconds or less ( heidelberger et al .
1994 ; edmonds 2004 ) . evidence for a priming function of the ribbon has recently been presented ( snellman et al .
in contrast to caz proteins , which are , at least in part , conserved at ihc azs , the molecular machinery involved in the regulation of synaptic vesicle fusion seems to deviate strongly from that of
as mentioned above , neurotransmitter release at conventional synapses is mediated by neuronal snares , namely snap-25 , syntaxin 1 and synaptobrevin 1 or 2 ( reviewed in jahn and fasshauer 2012 ) .
snare activity can be blocked by neurotoxin - mediated cleavage or genetic manipulations ( schiavo et al .
ribbon synapses , the snare protein machinery appears to be present and functional ( brandsttter et al .
1996 ; morgans et al . 1996 ; morgans 2000 ; von kriegstein and schmitz 2003 ; uthaiah and hudspeth 2010 ; cooper et al .
in contrast , ihc exocytosis seems to be insensitive to neurotoxins and genetic ablation of neuronal snares ( nouvian et al . 2011 ) and , hence , a functional role of syntaxin 1 , synaptobrevin 1 and 2 as well as snap-25 in ihc exocytosis is questionable . while some studies detected snare mrnas and proteins in the sensory epithelium and hair cells ( safieddine and wenthold 1999 ; uthaiah and hudspeth 2010 ; nouvian et al .
2011 ) , neither snap-25 , synaptobrevin 13 nor syntaxin 1 could be detected by immunofluorescence imaging in mouse ihcs ( nouvian et al .
moreover , snare regulators such as synaptotagmins 13 ( beurg et al . 2010 ; reisinger et al . 2011 ) and complexins ( strenzke et al .
in contrast , the multi - c2 domain protein otoferlin plays a central role for hair cell exocytosis ( roux et al .
the absence or mutation of otoferlin causes deafness or temperature - sensitive hearing impairment in humans ( yasunaga et al . 1999 ; varga et al .
2008 ) and rodents ( roux et al . 2006 ; longo - guess et al . 2007 ;
ultrastructurally , otoferlin is also found at ribbon synapses , mostly but not exclusively at synaptic vesicles and the az membrane ( roux et al .
sufficient amounts of otoferlin appear to be required for correct vesicular fusion and replenishment ( roux et al .
otoferlin is suggested to act as the ca sensor in ihcs ( roux et al .
2006 ; johnson and chapman 2010 ) , due to its ca - binding capabilities ( roux et al . 2006 ; ramakrishnan et al . 2009 , 2014 ; johnson and chapman 2010 ; pangri et al .
2010 ) and facilitates snare - mediated liposome fusion ( johnson and chapman 2010 ) . in its absence , no depolarization - evoked rrp exocytosis is observed in ihcs ( roux et al .
2010 ) . transgenic expression of synaptotagmin 1 , the major ca sensor of neuronal synaptic vesicle exocytosis , failed to restore ihc exocytosis and hearing in otoferlin ko mice , which may not be too surprising given the overall low conservation of the molecular composition between conventional and ihc synapses ( reisinger et al .
next to proteinaceous exocytosis machineries , the actual mechanisms of vesicle fusion as well as the transport of vesicles to the ihc release site are still largely unknown .
the large and , in terms of amplitude and shape , heterogeneous excitatory postsynaptic currents ( epscs ) measured at postsynaptic afferent boutons of sgns have been interpreted to result from multivesicular ( multiquantal ) release at ihc azs ( glowatzki and fuchs 2002 ) .
large epscs ensure rapid and temporal precise spike generation of sgns ( rutherford et al . 2012 ) and the relevance of such large epscs for achieving a high synchronization index of postsynaptic firing ( i.e. , better phase locking precision ) has recently been shown in the frog papilla ( li et al .
several multiquantal release scenarios at ihc ribbons have been discussed : ( 1 ) synchronized vesicle fusion of several single vesicles as well as ( 2 ) compound fusion , following homotypic vesicle - to - vesicle fusion and ( 3 ) sequential fusion involving homotypic vesicle - to - vesicle fusion while release occurs ( glowatzki and fuchs 2002 ; edmonds 2004 ; neef et al .
recent findings suggest an alternative candidate mechanism for ihc exocytosis . combining experimental approaches and mathematical modeling chapochnikov et al .
( 2014 ) indicated that univesicular ( uniquantal ) release can explain the large size of sgn epscs and that the control of release by a dynamic vesicular fusion pore can account for the observed epsc heterogeneity . at this point
, none of the above discussed mechanisms can definitively be ruled out or confirmed and future work , including detailed morphological analysis using electron microscopy of defined functional states , will be required to advance our understanding of exocytosis mechanism at ihc ribbon synapses . in case vesicles
do not homotypically fuse with other vesicles at the ribbon while releasing , a transport mechanism of the vesicles to the membrane has to exist .
the conveyor belt model , transporting the vesicle actively along the ribbon to the membrane , was one of the first models to be introduced ( bunt 1971 ; gray and pease 1971 ; vollrath and spiwoks - becker 1996 ; lenzi and von gersdorff 2001 ) .
accordingly , a kinesin polypeptide , kif3a , was identified on photoreceptor ribbons that could serve as a motor for vesicle transport involving the filamentous tethers observed at the ribbon ( muresan et al .
1999 ) , which were also proposed to function as stepping stones for synaptic vesicles ( usukura and yamada 1987 ; parsons and sterling 2003 ) .
recently , the tethers at the ribbon were suggested to be directly involved in coordinating vesicle transport towards the membrane via
crowd surfing , based on passive diffusion following the gradient established by exocytic vesicle consumption at the base of the ribbon ( graydon et al .
the tethers simply need to bind the vesicles and prevent them from detaching until they reach the az membrane , where release maintains the diffusion gradient ( graydon et al .
2014 ) . however , future experiments involving mutant analyses will be necessary to identify the proteins mediating vesicular tethering to the ribbon and estimate their affinity to the vesicles and the functional relevance of the tethers for synaptic transmission
. moreover , it will be interesting to investigate whether and how the tethering can be influenced by factors such as activity or even developmental stage .
for example , maturation from pre - hearing to hearing significantly determines structure and function of the ribbon synapses and the spatial arrangement of az proteins such as the ca channels or bassoon , as will be emphasized in the next section .
during maturation of the organ of corti , ribbon synapses and sgn fibers undergo drastic morphological changes
. how do morphological alterations during the transition from a pre - hearing to a hearing state correlate to functional maturation of ribbon synapses ? generally , synaptic contacts are ultrastructurally defined as pre- and postsynaptic electron - dense membranes that are closely aligned . the postsynaptic density ( psd )
is clearly visible as an electron - dense structure beneath the postsynaptic membranes directly juxtaposed to the presynaptic az .
the innervation pattern of sgn fibers at hair cells within the immature rodent cochlea is significantly different from the mature configuration and massive rearrangements of the fibers that occur before the onset of hearing .
hereby , type i sgn fibers retract from the ohcs , whereas type ii sgn fibers disappear from the ihcs ( perkins and morest 1975 ; echteler 1992 ; simmons 2002 ) .
these developmental processes of fiber innervation take place in the first postnatal week in three distinct phases : ( 1 ) in e18-p0 animals , fibers of both afferent types extend towards all hair cells ; ( 2 ) between p0 and p3 a refinement occurs , where the outer spiral bundle forms that innervate the ohcs ; and ( 3 ) the type i fibers retract from the ohcs around p3p6 , accompanied by synaptic pruning , while they keep their projections on the ihcs ( huang et al .
ampa - typed glutamate receptors and scaffold proteins like bassoon and shank1 disappear during the maturation process from ohcs .
glua2/3 subunits remain stable throughout development and into adulthood , while glua4 subunit expression significantly increase in adult type i fibers ( huang et al .
recently , the molecular arrangement of afferent synapses in relation to functional changes at the ihcs has been addressed in more detail using a combination of confocal , stimulated emission depletion ( sted ) and electron microscopy , as well as ihc presynaptic physiology and computational modeling ( wong et al . 2014 ) .
it is known that , in the early pre - hearing stages between p6 and p9 , several small apposing pre- and postsynaptic densities mark nascent synapses .
some of the presynaptic densities are occupied by synaptic ribbons , which are small and round in shape and attached via two triangular - shaped proteinaceous anchors ( sobkowicz et al .
however , floating ribbons were also frequently observed in close proximity to az areas at these developmental stages ( wong et al .
serial 3d electron microscopic reconstructions corroborated the notion of several discontinuous pre- and postsynaptic specializations .
such synaptic sites are organized as loose suprastructures on the bouton surface and are likely functional , as immunohistochemistry indicates the presence of presynaptic ca channels and postsynaptic ampa receptors ( wong et al .
2000 ; hell 2007 ) , revealed that cav1.3 channels are arranged in small round spots ( wong et al .
2014 ) rather than the stripes previously described for mature azs ( frank et al .
in addition , a huge number of extrasynaptic cav1.3 channels can be observed in immature ihcs ( zampini et al .
2014 ) , which enable the cells to fire ca action potentials ( kros et al . 1998 ; brandt et al .
these action potentials evoke exocytosis in the pre - hearing stage ( beutner and moser 2001 ; glowatzki and fuchs 2002 ; johnson et al .
2005 ) but show lower ca efficiency ( beutner and moser 2001 ; brandt et al .
2003 ; johnson et al . 2005 ) and a supra - linear ca dependence ( johnson et al . 2005 ) .
the pre - sensory ihc activity appears to drive bursting activity in the developing auditory system ( glowatzki and fuchs 2002 ; tritsch et al .
2007 , 2010 ; wong et al . 2013 ; clause et al . 2014 ) . in this context , the regulation of presynaptic firing by paracrine and/or efferent synaptic control is being subject to intense research ( glowatzki and fuchs 2000 ; tritsch et al .
efferent innervation , moreover , seems to play an important role in the maturation process of ihcs ( glowatzki and fuchs 2000 ; marcotti 2004 ; goutman et al .
efferent fibers originate from the superior olivary complex and , before onset of hearing , form transient axosomatic contacts with ihcs ( simmons et al .
later , they largely retract from ihcs and rather form axodendritic contacts to the afferent terminals ( pujol et al .
the transient efferent inhibition is thought to counteract the ihc depolarization resulting from the resting mechanotransducer current ( gloc and holt 2003 ; waguespack et al .
2009 ) . upon genetically induced impairment of the efferent input , the linearization of ca dependent exocytosis is affected ( johnson et al . 2007 ) and the maturation of ihc afferent synapses is also disturbed ( johnson et al .
2013b ) . around the onset of hearing ( at around p11 ; mikaelian and ruben 1965 ) ,
when graded receptor potentials start governing transmitter release , extrasynaptic cav1.3 channels get pruned and spatial coupling of ca channels and vesicular release sites is tightened .
ca efficiency of exocytosis and a near - linear ca dependence of rrp exocytosis when probed with changes in the number of open ca channels ( wong et al .
2014 ) . therefore , while the intrinsic ca dependence of exocytosis apparently does not change upon the onset of hearing , experimental data and biophysical modeling of exocytosis at mature and immature az topographies support the notion of a developmental switch from the more ca microdomain - like control of exocytosis by several ca channels per vesicle to a more ca nanodomain - like control of exocytosis ( wong et al . 2014 ; fig . 1e , e ) .
interestingly , in adult gerbils , the open probability of ca channels in ihcs increased due to a preference of the ca channel for the bursting mode ( zampini et al .
structurally , alongside ca channels , other presynaptic az components become reorganized such as the bassoon containing presynaptic density ( fig .
these alterations are accompanied by changes of the postsynaptic glutamate receptor fields that also develop to one continuous ring - like cluster ( wong et al .
moreover , ribbons increase in size and undergo striking changes of shape . at the ultrastructural level , their cross - sectional shape changes from predominantly round ( fig .
1a ) to a rather oval- , droplet- or wedge - like shape between p14 and p20 ( wong et al . 2014
the two rootlets seem to merge to a continuous presynaptic density that contains the scaffolding protein bassoon , as revealed by immunogold labeling ( wong et al .
shortly after onset of hearing at p14 , a large proportion of ribbons with two rootlets can still be found , whereas about a week later the morphological maturation appears to be completed ( wong et al .
. factors that participate in the maturation of ihc synapses , next to the efferent olivocochlear transmission ( see above ; johnson et al .
2013a ) , are thyroid hormone ( sendin et al . 2007 ) and myosin 6 ( heidrych et al . 2009 ; roux et al . 2009 ) . for both , a higher proportion of morphological immature ribbons have been observed in genetic deletion models . in conclusion , during development from pre - hearing to hearing , ihc ribbon synapses undergo major morphological and functional refinements , resulting in tighter spatial coupling between ca influx and exocytosis ( wong et al .
the number of ca channels , vesicular release sites and ribbon - associated vesicles seems to scale with the size and number of ribbons at the az ( martinez - dunst et al .
; graydon et al . 2011 ; kantardzhieva et al . 2013 ; wong et al . 2013 , 2014 ) .
strengthening of presynaptic transmitter release might therefore be accomplished by increasing ribbon or az size and/or ribbon numbers per az . moreover ,
finally , lateral olivocochlear efferent fibers might modulate postsynaptic excitability and thereby affect afferent synaptic strength . to establish which of these mechanisms contribute to determining and regulating synaptic strength of hair cell synapses
interestingly , the size and shape of ribbons appear to be highly variable and dynamic .
in fact , in photoreceptors , these parameters strongly correlate with activity in light ( silent ) or dark ( active ) conditions ( spiwoks - becker et al .
a diverse spectrum of ribbons has also been observed ( bodian 1978 ; sobkowicz et al .
the specific ultrastructural properties seem to depend on several factors : ( 1 ) the maturation / age ( see section above ) , ( 2 ) position within the inner hair cell and maybe also ( 3 ) dynamic adaptation to activity .
a pioneering study in cats was one of the first to identify the correlation between structural heterogeneity of ribbon synapses and functional characteristics of auditory nerve fibers ( merchan - perez and liberman 1996 ) .
surprisingly , large azs with big and/or several ribbons , supposedly reflecting large presynaptic strength , seem to drive sgns with low spontaneous rate and high thresholds ( see also scheme in fig .
whereas this conundrum remains unsolved , the mechanisms of functional presynaptic heterogeneity are now beginning to be understood .
evidence for such heterogeneity within individual ihcs was obtained using confocal imaging of presynaptic ca influx ( frank et al .
this study showed that presynaptic ca signals varied substantially in amplitude and voltage - dependence among the azs within individual ihcs .
the amplitude of the ca signal scaled with ribbon size as approximated by simultaneous imaging of a fluorescently tagged ribeye - binding peptide ( frank et al .
2009 ) and seemed to be greater at the neural side of the ihcs ( meyer et al .
linking such estimates to the functional and morphological properties of the postsynaptic neurons will be an important task for future studies .
so far , correlative arguments based on coincidental changes in maximal strength of presynaptic ca influx and postsynaptic spiking during development and upon genetic disruption as well as modeling have been brought forward to argue that strong synapses drive sgns that have high spontaneous rates and low thresholds ( wong et al . 2013 ) .
interestingly , an inverse correlation of pre- and postsynaptic parameters of synaptic strength has recently been reported for mouse ihcs : liberman et al .
the authors favored the interpretation that the sgns inserting at the neural ( modiolar ) face of ihcs exhibit low spontaneous rates and high thresholds despite their corresponding large ihc azs , because they have a smaller complement of ampa receptors than those at the neural ( pillar ) side .
this would agree with the conclusion of the classical study , which showed a neural
abneural gradient of az size using electron microscopy for cat ihcs whereby large azs faced sgns with low spontaneous rates and high thresholds ( merchan - perez and liberman 1996 ) . in a laborious approach
, the authors traced 11 functionally - characterized fibers to the ihcs using serial 3d reconstructions of ultrathin sections . in this way
, it was possible to directly correlate morphological parameters such as ribbon length , fiber contact area , synaptic plaque area and synaptic vesicle numbers to the functional parameters determined prior to fiber labeling using single unit recordings .
recently , such a gradient was also suggested for mouse ihcs and reported to be influenced by the lateral olivocochlear innervation ( yin et al .
the segregation of nerve fibers on neural and abneural sides was further observed in a study investigating the abundance of mitochondria in postsynaptic terminals . here
, postsynaptic boutons facing the abneural side seem to harbor more mitochondria ( francis et al .
monitoring epscs from single afferent boutons , which is a suitable method to address synaptic function on the level of individual release sites ( glowatzki and fuchs 2002 ) , further showed differences among synapses . in these experiments ,
varying fractions of multiphasic epscs were observed and proposed to underlie the diverse firing properties of sgns ( grant et al .
a schematic of an organ of corti showing afferent and efferent innervations at ihcs . modified from meyer and moser 2010 , curr opin otolaryngol head neck surg , reprinted with permission from 2010 wolters kluwer health .
b mean and sd of f ( gray ) as a function of depolarizing potential ( v
m ) , obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus .
f ( mean gray ) and i
ca ( mean black ) show a similar voltage dependence ( thin lines corresponding sds ) . b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs .
pronounced variability in the voltage dependence of activation , even within the same cell ( dashed traces individual data curves from 3 ca microdomains in an ihc ) . modified from frank et al .
c , c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate ( sr ) fibers .
sections through a high - sr ( c ) and a low - sr ( c ) synapse containing the synaptic ribbon are shown with cisternal ( maroon ) and vesicular ( green ) profiles .
d mean density ( se ) of docked vesicles , i.e. , within 20 nm of the presynaptic density along the presynaptic membrane .
d mean number ( se ) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses .
rectangle the area of significant differences between low- and high - sr synapses . se standard error ; sr spontaneous rate .
2013 , j comp neurol , reprinted with permission from 2013 wiley periodicals ) principle of functional heterogeneity in ihcs .
a schematic of an organ of corti showing afferent and efferent innervations at ihcs . modified from meyer and moser 2010 , curr opin otolaryngol head neck surg , reprinted with permission from 2010 wolters kluwer health .
b mean and sd of f ( gray ) as a function of depolarizing potential ( v
m ) , obtained from spot - detection experiments at the center of the ca microdomain ; f was averaged over the last 15 ms of a 20-ms stimulus .
f ( mean gray ) and i
ca ( mean black ) show a similar voltage dependence ( thin lines corresponding sds ) .
b heterogeneous voltage dependence and ca channel number of synaptic ca channel clusters in ihcs .
pronounced variability in the voltage dependence of activation , even within the same cell ( dashed traces individual data curves from 3 ca microdomains in an ihc ) . modified from frank et al .
c , c colorized spatial distribution of vesicles and cisterns around the ribbon in low- and high - spontaneous rate ( sr ) fibers .
sections through a high - sr ( c ) and a low - sr ( c ) synapse containing the synaptic ribbon are shown with cisternal ( maroon ) and vesicular ( green ) profiles .
d mean density ( se ) of docked vesicles , i.e. , within 20 nm of the presynaptic density along the presynaptic membrane .
d mean number ( se ) of cisterns within 20 nm of the presynaptic density versus distance along the presynaptic membrane is shown for all synapses .
2013 , j comp neurol , reprinted with permission from 2013 wiley periodicals ) further insights into the morphological heterogeneity require modern 3d reconstructions of larger volumes such as serial block face scanning electron microscopy ( denk and horstmann 2004 ) or focused ion beam scanning electron microscopy ( see , e.g. , knott et al . 2008
using these methods , a detailed 3d view of single ihcs including afferent and efferent innervations would be possible , finally allowing correlation of parameters , such as presynaptic ribbon size and postsynaptic bouton diameter , as a function of position on the ihc for a large number of synapses , together with the functional assessment of pre- and postsynaptic properties in cochlear explants by electrophysiology ( goutman and glowatzki 2007 ) and/or imaging of ihc ca and exocytosis ( e.g. , frank et al .
tight coupling between exo- and endocytosis is a prerequisite for maintaining the enormous vesicle turnover rates at ribbon synapses
clathrin - coated structures but also large cisterns without clathrin - coats , are observed close to synaptic ribbons ( siegel and brownell 1986 ; sendin et al .
2007 ; frank et al . 2010 ; kantardzhieva et al . 2013 ; neef et al . 2014 ; revelo et al . 2014 ) .
2013 ) set out to determine whether such cisterns participate in vesicle reformation and what differences can be observed in correlation to the functional properties of high and low spontaneous rate fibers ( fig .
an extensive quantitative analysis of ribbons , vesicles and cisterns from serial sections of cat ihc ribbon synapses suggested a
fewer cisterns and more synaptic vesicles are found , which indeed points towards a contribution of cisterns to locally restricted vesicle formation .
other studies used membrane capacitance measurements to provide an initial functional assessment of endocytic membrane retrieval at ihc azs ( moser and beutner 2000 ; beutner and moser 2001 ; neef et al .
moreover , ph - sensitive gfp ( phluorin ; miesenbck et al . 1998 ) targeted to the intraluminal face of vesicle membranes by attachment to vesicular glutamate transporters ( zhu et al .
2009 ) has become an important tool in studying exo- and endocytosis , not only from neurons but also ihcs ( neef et al .
additionally , a novel membrane tracer specifically tailored to use in the organ of corti has been devised and applied to investigate endocytosis ( revelo et al .
2014 ) , as the commonly used styryl dye fm1 - 43 penetrates stereociliar mechanotransduction channels and hence is of limited use to study endocytosis , in ihcs ( gale et al .
, expression analysis and immunohistochemistry have revealed the presence of several important molecular players of endocytosis such as dynamins , amphiphysin , clathrin ( neef et al .
2014 ) and adaptor protein 2 ( ap-2 ) ( duncker et al . 2013 ) in ihcs . a very recent dna microarray study investigating ihc and ohc transcriptomes might even give more insight into proteins involved in vesicle recycling ( liu et al .
currently , in ihcs , three distinct mechanisms are considered to mediate endocytosis : slow cme , fast bulk endocytosis and potentially kiss - and - run or
cme is the main pathway of membrane retrieval for mild stimulation and proceeds at a constant rate ; it represents the linear component of endocytosis following exocytosis of the rrp ( fig .
this mechanism is not only inhibited by the clathrin - inhibitor pitstop-2 but also by disruption of dynamin 1 via pharmacological and genetic means ( neef et al . 2014 ) .
in contrast , a different study reported inhibition of sustained exocytosis by the presumptive dynamin inhibitor dynasore but did not investigate endocytic membrane retrieval ( duncker et al .
finally , when exocytosis exceeds three to four rrp equivalents , ihcs additionally recruit a faster mode of membrane retrieval , which proceeds with an exponential time course within a few seconds .
3a ) and , indeed , there is plenty of evidence for the invagination and fission of large stretches of plasma membrane in the vicinity of hair cell azs ( lenzi et al .
both mechanisms seem to engage in different phases of release : cme supports vesicle cycling during mild stimulation but bulk endocytosis finally occurs after prolonged stimulation , providing a mechanism that assures the balance between exo- and endocytosis in ihcs and thus , assures high release rates ( neef et al .
3endocytosis in inner hair cells . a , a representative recordings in response to 20 ms ( a ) or 200 ms ( a ) depolarizations .
after the c
m increase upon 20 ms depolarization , the slope - corrected c
m traces ( middle ) typically showed a linear decay ( a ) .
the 200-ms - long depolarization resulted in a combination of exponential and linear decay ( a ) .
b 3d reconstructions of resting ( b ) , stimulated ( b ) and recovered ihcs ( b , b ) .
most organelles , including the tubular ones , are replaced by small vesicles during the recovery periods .
insets magnified regions from the four different cell regions ( cuticular plate , top , nuclear and basal regions ) . note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery .
c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin ( first row ) , for vglut3 and syntaxin 6 ( sx 6 , second row ) , for otoferlin and syntaxin 16 ( sx 16 , third row ) and finally for syntaxin 6 and syntaxin 16 ( fourth row ) .
the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope . dashed white lines the plasma membrane of the ihcs .
d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 ( or syntaxin 16 ) correlate in the mcling - labeled organelles at the top and nuclear levels .
organelles with a different molecular composition recycle membrane in different regions , taking up mcling .
apical endocytosis takes up the membrane into round organelles , a sizeable proportion of which is similar to late endosomes ( light blue ) .
endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers , syntaxin 16 and syntaxin 6 .
this suggests that these organelles participate in constitutive pathways , probably by maintaining membrane traffic between the plasma membrane and the trans - golgi . at the base of the cell
, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3 , rab3 and also otoferlin .
a , a representative recordings in response to 20 ms ( a ) or 200 ms ( a ) depolarizations . after the c
m increase upon 20 ms depolarization , the slope - corrected c
m traces ( middle ) typically showed a linear decay ( a ) .
the 200-ms - long depolarization resulted in a combination of exponential and linear decay ( a ) .
b 3d reconstructions of resting ( b ) , stimulated ( b ) and recovered ihcs ( b , b ) .
most organelles , including the tubular ones , are replaced by small vesicles during the recovery periods .
insets magnified regions from the four different cell regions ( cuticular plate , top , nuclear and basal regions ) . note the increased number of endosome - like organelles at the base of the cell after stimulation and during recovery .
c mcling - labeled organs of corti were immunostained for vglut3 and otoferlin ( first row ) , for vglut3 and syntaxin 6 ( sx 6 , second row ) , for otoferlin and syntaxin 16 ( sx 16 , third row ) and finally for syntaxin 6 and syntaxin 16 ( fourth row ) .
the samples were cut into 20-nm sections and were imaged using an epifluorescence microscope . dashed white lines the plasma membrane of the ihcs .
d graphic representation of pearson s correlation coefficients : otoferlin and syntaxin 6 ( or syntaxin 16 ) correlate in the mcling - labeled organelles at the top and nuclear levels .
organelles with a different molecular composition recycle membrane in different regions , taking up mcling .
apical endocytosis takes up the membrane into round organelles , a sizeable proportion of which is similar to late endosomes ( light blue ) .
endocytosis in the top and nuclear regions reaches tubular organelles containing otoferlin and two endosome markers , syntaxin 16 and syntaxin 6 .
this suggests that these organelles participate in constitutive pathways , probably by maintaining membrane traffic between the plasma membrane and the trans - golgi . at the base of the cell
, stimulation induces the formation of membrane infoldings and cisterns that are characterized by the presence of vglut3 , rab3 and also otoferlin .
( c e modified from revelo et al . 2014 , reprinted with permission from 2014 revelo et al . ) but where does the retrieval of synaptic vesicle membrane take place and where and how are synaptic vesicles regenerated following membrane retrieval in hair cells ? does synaptic endocytosis comply with the apicobasal compartmentalization of the hair cell ? at photoreceptor ribbons , a periactive zone , marked by the presence of endocytic proteins , was identified directly adjacent to the az area ( in a range of 120250 nm from the ribbon ) using high - resolution and electron microscopy ( wahl et al .
does this also apply to ihc ribbons ? at the resolution of confocal microscopy , a similar perisynaptic accumulation of endocytic proteins has , so far , not been found ( neef et al .
clearly , electron micrographs indicate that both bulk and cme endocytosis take place near the az ( siegel and brownell 1986 ; lenzi et al .
a radically different model of ihc endocytosis has been sketched based on life imaging of fm1 - 43 uptake into ihcs , whereby exocytosed membrane was postulated to move towards the ihc apex for endocytosis and recycling via the golgi apparatus ( griesinger et al .
recent elaborative studies using various styryl dyes and more suitable fluorescent membrane markers lead us to reconsider this hypothesis .
first , it was corroborated that many styryl dyes including fm1 - 43 permeate into the cytosol of ihcs via the mechanotransducer channels ( kamin et al .
therefore , on the ultrastructural level , the photo - oxidation technique revealed a fuzzy dark diaminobenzidin ( dab ) smear inside the cytosol in addition to the precipitate in membrane - bound organelles likely resulting from internalized fm1 - 43 .
the stimulation - induced endocytic uptake of fm1 - 43 could still be followed by observing an electron - dense precipitate within vesicular structures , which allows the determination of the presence or absence as well as the localization of stained structures under resting ( fig .
b ) conditions in serial 3d reconstructed ihcs ( kamin et al . 2014 ) . at rest , endosome - like organelles were detected in the apex of the ihcs , whereas larger tubulo - cisternal organelles dominated at the nuclear region . at the basal region , only a few labeled structures were present .
stimulation massively increased the amount of basolateral membrane trafficking , reflected by the appearance of labeled small vesicles and endosome - like vacuoles ; however , no changes in the apical and nuclear regions could be observed ( kamin et al .
strikingly , the basolateral cisterns were replaced in the basal region by small , synaptic - like vesicles during a few minutes of recovery from stimulation , suggesting a highly efficient mechanism of vesicle regeneration from cisternal membranes internalized by bulk endocytosis .
the combination of fm1 - 43 uptake and photo - oxidation therefore suggests that synaptic vesicle recycling takes place at the basal part , close to ribbons at least during synaptic activity ( kamin et al .
recently , a novel membrane tracer , named membrane - binding fluorophore - cysteine - lysine - palmitoyl group ( mcling ) , which does not permeate the mechanotransducer channel , tightly binds biological membranes and can be fixed , has been developed ( revelo et al .
2014 ) . in combination with super - resolution light microscopy ( i.e. , sted ) , the spatial organization and pathways of endocytosis in ihcs could be further investigated . in order to improve the spatial resolution of sted microscopy in the axial direction ,
thin sections of ihcs were imaged after embedding the organs of corti in melamine , which maintains the fluorescence ( revelo et al .
the apical , nuclear and basal regions under conditions of rest , stimulation and recovery from stimulation , were investigated and the uptake of mcling monitored . in order to reveal the molecular identity of mcling - labeled structures and thereby identify the respective endocytic pathways , samples were co - stained with different protein markers for the endoplasmic reticulum ( er ) and golgi as well as synaptic vesicle endo- and exocytosis . in these experiments , a strong correlation for basolateral mcling localization with vglut3 , rab3 and otoferlin immunofluorescence was found .
otoferlin as well as syntaxin 16 , a late endosomal marker , colocalized with apical and nuclear mcling ( fig .
moreover , the lysosomal - associated membrane protein 1 ( lamp1 ) colocalized with mcling in the apical region of the ihc .
stimulation led to a selective uptake of mcling at the base of the ihc , corroborating the notion of local recycling of synaptic vesicles that was postulated based on electron microscopy and photo - oxidation ( kamin et al .
the local recycling hypothesis was further supported by the finding that exogenous vglut1-phluorin fluorescence not only transiently appeared at ribbon - type azs but remained there for tens of seconds after stimulation ( neef et al .
finally , the mcling experiments revealed large membranous organelles near synapses , which were replaced by small organelles a few minutes after stimulation , thereby providing direct evidence of bulk endocytosis and vesicle regeneration from the internalized plasma membrane .
the association of otoferlin with all three putative membrane recycling pathways suggests a more general role of this protein in endocytosis .
otoferlin has recently been assigned a role in vesicle endocytosis due to its interaction with ap-2 . using a high - resolution liquid chromatography coupled with a mass spectrometry approach ,
multiple subunits of ap-2 were identified as interaction partners of otoferlin in the mammalian cochlea and the proposed interactions were biochemically confirmed by co - immunoprecipitation ( duncker et al . 2013 ) .
ap-2 plays a role in clathrin - mediated endocytosis via binding to clathrin - coated vesicles budding from the plasma membrane ( keyel et al .
2010 ) and has been shown to be expressed in ihcs ( duncker et al .
future work is required to clarify the role of ap-2 in hair cell endocytosis and the relevance of its interaction with otoferlin .
recently , major progress has been made towards dissecting the molecular anatomy and physiology of hair cell ribbon synapses .
this includes powerful single synapse techniques such as ( 1 ) patch - clamp of postsynaptic afferent terminals of sgns , ( 2 ) high resolution -functional imaging of presynaptic ihc ca dynamics and membrane turnover , as well as ( 3 ) super - resolution light microscopy and electron tomography following high - pressure freezing .
however , in order to investigate the release mechanisms of ihcs and firmly correlate structure and function , the development of new functional and morphological approaches is required . functional and morphological analysis of single synapses will be necessary and some questions require reading out both pre- and postsynaptic properties at the same time .
the commonly used k stimulation of cochlear tissue likely mimics strong physiological steady - state stimulation .
but this stimulation does not provide the temporal resolution to allow the observation of the release kinetics at ihc ribbon synapses .
especially , knowledge about short - term plastic changes is lacking , since it is not possible to apply very short stimuli ( i.e. , millisecond range ) and investigate the cells during and at defined times after stimulation .
therefore , approaches are needed that meet two requirements : ( 1 ) a precise stimulation protocol combined with ( 2 ) rapid immobilization of the sample , e.g. , by using high - pressure freezing .
one emerging tool that promises to fulfill these requirements is the combination of optogenetic stimulation with high - pressure freezing .
this could involve the expression of a light - sensitive ion channel such as channelrhodopsin-2 ( chr-2 ) from the green algae chlamydomonas reinhardii ( nagel et al . 2003 ) in hair cells and stimulation would ideally be performed within a chamber that should be mounted in a freezing machine in order to minimize the time delay before freezing .
recently , synaptic recovery of motoneurons from c. elegans was analyzed using optogenetic stimulation in combination with high - pressure freezing ( kittelmann et al .
moreover , after a single light stimulus , docked vesicles fused along a broad az on c. elegans motoneurons expressing chr-2 .
these vesicles were replenished with a time constant of about 2 s. further , endocytosis occurred within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions ( watanabe et al .
moreover , a study on optically stimulated cultured hippocampal neurons revealed an ultrafast endocytosis mechanism at central synapses ( watanabe et al .
these initial experiments indicate that optogenetics , in combination with high - pressure freezing ( flash and freeze ; watanabe et al .
2013b ) and subsequent electron tomography , might provide sufficient resolution to study the ultrastructure of spatiotemporally defined functional states and thus provide a completely new view on the release mechanism of ihc ribbon synapses . | in the mammalian cochlea , sound is encoded at synapses between inner hair cells ( ihcs ) and type i spiral ganglion neurons ( sgns ) .
each sgn receives input from a single ihc ribbon - type active zone ( az ) and yet sgns indefatigably spike up to hundreds of hz to encode acoustic stimuli with submillisecond precision .
accumulating evidence indicates a highly specialized molecular composition and structure of the presynapse , adapted to suit these high functional demands .
however , we are only beginning to understand key features such as stimulus secretion coupling , exocytosis mechanisms , exo endocytosis coupling , modes of endocytosis and vesicle reformation , as well as replenishment of the readily releasable pool . relating structure and function
has become an important avenue in addressing these points and has been applied to normal and genetically manipulated hair cell synapses . here , we review some of the exciting new insights gained from recent studies of the molecular anatomy and physiology of ihc ribbon synapses . | Introduction
The structure of the inner hair cell is set up for efficient signaling
Molecular anatomy and physiology of hair cell ribbon synapses
Structural and functional maturation of inner hair cell ribbon synapses
Dynamics and heterogeneity of hair cell ribbon synapses
Synaptic vesicle recycling in inner hair cells
Outlook | a plethora of presynaptic proteins orchestrate neurotransmitter release at the presynaptic active zone ( az ) . for example , the afferent spiral ganglion neurons ( sgns ) of the cochlear nerve , which carry the information about an acoustical signal from the inner ear to the brainstem , were studied intensely in various mammals such as guinea pig , mouse or cat ( spoendlin 1972 , 1975 , 1979 ; paradiesgarten and spoendlin 1976 ; bodian 1978 ; kiang et al . 1982 ) , outer hair cells ( ohcs ) by unmyelinated ( 5 % ) and inner hair cells ( ihcs ) by myelinated ( 95 % ) afferent fibers ( spoendlin 1969 , 1975 ) . each of the myelinated , bipolar type i sgns sends a peripheral unmyelinated and unbranched neurite to form a synapse with a single ihc ribbon synapse ( liberman 1980 ; liberman et al . more recently , hair cell synapses have increasingly attracted research activity and novel as well as classical methods have been employed for assessing their structure and function in combination with genetic or pharmacological manipulation of the synapses or noise exposure . quantitative electron microscopy analysis employing electron tomography of different functional states as well as freeze - fracture and subsequent electron microscopy
have been introduced by roberts and others for studies of hair cell synapses ( roberts et al . immunohistochemistry combined with high - resolution microscopy as well as transcriptomic and proteomic analyses have informed on the molecular composition of hair cell synapses ( khimich et al . in this review , we will emphasize recent approaches coupling functional and structural investigations of release at the level of ihcs and their ribbon synapses , as well as recent findings regarding vesicular recycling after transmitter release . in the next sections , we will focus on the organization of the basal portion of ihcs and discuss structure and function of hair cell ribbon synapses
. f schematic summary of the protein arrangement at mature ihc ribbon synapses ribeye is composed of two major domains : while the a domain organizes the assembly of the synaptic ribbon and is unique in structure , the b domain is structurally nearly identical to the transcription repressor ctbp2 , which is encoded by the same gene but uses a different transcription initiation site ( schmitz et al . for example , maturation from pre - hearing to hearing significantly determines structure and function of the ribbon synapses and the spatial arrangement of az proteins such as the ca channels or bassoon , as will be emphasized in the next section . recently , the molecular arrangement of afferent synapses in relation to functional changes at the ihcs has been addressed in more detail using a combination of confocal , stimulated emission depletion ( sted ) and electron microscopy , as well as ihc presynaptic physiology and computational modeling ( wong et al . recently , major progress has been made towards dissecting the molecular anatomy and physiology of hair cell ribbon synapses . 2013b ) and subsequent electron tomography , might provide sufficient resolution to study the ultrastructure of spatiotemporally defined functional states and thus provide a completely new view on the release mechanism of ihc ribbon synapses . whether the basolateral organization of the hair cells is similarly instructed by planar cell polarity
, we will focus on the organization of the basal portion of ihcs and discuss structure and function of hair cell ribbon synapses . f schematic summary of the protein arrangement at mature ihc ribbon synapses spatial distribution of ihc az proteins . f schematic summary of the protein arrangement at mature ihc ribbon synapses ribeye is composed of two major domains : while the a domain organizes the assembly of the synaptic ribbon and is unique in structure , the b domain is structurally nearly identical to the transcription repressor ctbp2 , which is encoded by the same gene but uses a different transcription initiation site ( schmitz et al . for example , maturation from pre - hearing to hearing significantly determines structure and function of the ribbon synapses and the spatial arrangement of az proteins such as the ca channels or bassoon , as will be emphasized in the next section . recently , the molecular arrangement of afferent synapses in relation to functional changes at the ihcs has been addressed in more detail using a combination of confocal , stimulated emission depletion ( sted ) and electron microscopy , as well as ihc presynaptic physiology and computational modeling ( wong et al . recently , major progress has been made towards dissecting the molecular anatomy and physiology of hair cell ribbon synapses . this includes powerful single synapse techniques such as ( 1 ) patch - clamp of postsynaptic afferent terminals of sgns , ( 2 ) high resolution -functional imaging of presynaptic ihc ca dynamics and membrane turnover , as well as ( 3 ) super - resolution light microscopy and electron tomography following high - pressure freezing . | [
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] |
the development of
effective strategies for modulating protein protein
interactions ( ppis ) has the potential to vastly expand the range of
druggable proteins . targeting the typically large
, flat surfaces involved
with molecules of high affinity and specificity can be readily achieved
by mimicking the natural binding partner protein s interacting
surface in the form of constrained peptides or peptidomimetics ( reviewed
in refs ( 15 ) ) .
the constraint serves the dual purpose of preorganizing the structure
and thereby increasing the binding affinity as well as enhancing the
pharmacokinetic properties such as in vivo stability and cell penetration . toward this goal , a class of molecules called
have been developed , which are characterized
by a bioactive , -helical conformation that is induced by chemically
cross - linking two side chains .
constraining non - helical , extended , or irregularly structured peptide
motifs presents a different challenge , as it is harder to rationally
design a linker so as to stabilize a binding - competent conformation
( e.g. , ref ( 19 ) ) .
given
that these types of motifs are found in up to 50% of all ppis , there is a pressing need to address this challenge . here
, we use a two - component double - click chemistry approach to
macrocyclize peptides in an extended , non - helical conformation .
tankyrase 1 and tankyrase 2 ( subsequently
referred to as tnks ) are poly(adp - ribose ) polymerases ( parps ) involved
in a number of cellular processes .
these include control of the mitotic
checkpoint , regulation of telomere length by targeting telomeric
repeat binding factor 1 ( trf1 ) for degradation , and regulation of wnt signaling by targeting axin
for degradation .
altered tnks expression or activity
is implicated in various disease states , and increased expression
of tnks has been observed in many different cancers including breast
cancers , fibrosarcoma , ovarian cancer , glioblastoma , pancreatic adenocarcinoma ,
and gastric cancer . as cancer therapeutics ,
tnks
inhibitors could potentially exploit tumor - specific wnt dependency
( e.g. , in colorectal cancers with apc and kras mutations , which are
resistant to epidermal growth factor receptor inhibitors ) or target
telomere dysfunction or an enhanced mitotic rate .
it has also been
shown that silencing of tnks by rnai has a synthetic lethality effect
in tumor cells with brca1/brca2 gene defects but has minimal effects
in wild - type cells .
thus , tnks inhibitors ,
like parp inhibitors , may be useful for the treatment of breast cancers
caused by mutations in the brca genes . furthermore , tnks inhibitors
may additionally have broader clinical applications .
for example ,
the wnt pathway has been found to be a valid target for treating neurodegenerative
diseases ( reviewed in refs ( 38 and 39 ) ) , such as multiple sclerosis and amyotrophic lateral
sclerosis .
more recently , tnks have been
found to play a role in glucose homeostasis in type ii diabetes .
the first small - molecule tnks inhibitor ( discovered in a wnt pathway inhibitor screen )
and those developed
subsequently are all directed against the catalytic parp domain ; however , there
are problems of cellular toxicity due to off - target effects arising
from the nad+/adp ribose - like characteristic of these active - site
inhibitors ( reviewed in refs ( 35 and 4951 ) ) .
the tnks proteins have a domain structure that
is distinct from other parp family members , as they contain an ankyrin
domain comprising a series of ankyrin - repeat clusters ( arcs ) that
are involved in targeting specific proteins for parylation ( figure 1a ) .
our aim herein
was therefore to develop a new class of highly specific tnks inhibitors
by targeting the substrate - recognition domain .
moreover , guettler
et al . have recently shown that tnks can induce wnt signaling independently
of its catalytic parp activity , mediated instead via an arc - domain
scaffolding function and thus suggesting additional advantages of
therapeutic targeting of this domain .
( a ) domain architecture , comprising
a homopolymeric run of histidine , proline and serine ( hps ) residues ,
the ankyrin repeat cluster ( arc ) , sterile alpha motifs ( sam ) , and
catalytic parp domains .
( b ) structure of human tnks2 arc4 ( gray cartoon )
in complex with substrate peptide lphlqrsppdgqsfrs ( purple ;
pdb i d : 3twr ) ; for clarity , only the central part
of the peptide ( in bold ) is labeled .
( c ) structure of mouse tnks1
arc23 ( gray cartoon ) in complex with mouse axin1_180 ;
only residues 1830 ( green ) and 6078 ( yellow ) of maxin1
are visible in the crystal structure ( pdb i d : 3utm ) . to this end , we have designed
a series of macrocyclized peptides
using a modular two - component strategy that employs click chemistry
to connect a linker and a peptide , thus allowing each to be varied
independently before assembling them in a combinatorial fashion .
our designs were guided by the previously determined crystal structures
of substrate - derived peptides in complex with the fourth arc domain
of tnks2 ( tnks2 arc4 ) ( figure 1b ) and subsequently optimized
with the help of molecular dynamics ( md ) simulations using an iterative
process of modifying the length and rigidity of the linker as well
as its position along the peptide sequence .
we determined the crystal
structures of two of the macrocyclized peptides in complex with tnks ,
leading to further optimization of the design .
we show that these
peptides are able to disrupt tankyrase - substrate complexes in vitro
and to inhibit wnt transcription in a dose - dependent manner .
thus ,
our results provide a promising starting point for a new class of
substrate - competitive inhibitors of tankyrase
. moreover , the methodology
has the potential to be a general approach for inhibiting ppis that
involve non - helical , extended , or irregularly structured molecular
recognition motifs .
we focused the design and
optimization strategy on an 8-residue consensus sequence , reagdgee ,
derived previously from a mutational analysis of tnks substrates . in that study ,
a 10-residue peptide was found
to be sufficient for high - affinity tnks binding ; we demonstrate here
that further truncation to the 8-core binding residues does not weaken
the affinity of the peptide .
computational alanine scanning was first
carried out on structures extracted from three independent 50 ns md
simulations of the complex between tnks2 arc4 and the 8-residue consensus
sequence ( for which there are no available experimental structures )
to assess which positions would be amenable to replacement by an unnatural
amino acid ( uaa ) having azido functionality for conjugation to the
linker .
the analysis indicated arg1 to be an important hot - spot residue ,
whereas ala3 ( mutated to gly in the computational analysis ) , asp5 ,
and glu8 make only modest contributions to the interaction ( figure s1 ) .
the modeling also suggests that positions
4 and 6 are unfavorable for cross - linking due to a high likelihood
of steric clashes of their side chains with the protein surface .
mutations
of glu2 and glu7 were found to be stabilizing , indicating that the
wild - type residues are detrimental to binding and consequently amenable
to replacement by uaas .
thus , a first panel of macrocyclized peptides
was synthesized with azido - functionalized uaas at positions 2 or 3
and 7 or 8 ( figure 2a ) , and they were acetylated at the n - terminus to neutralize the
terminal charge .
the uaa side chains are denoted n1n4 to indicate
the number of methylene ( ch2 ) units ( figure 2a ) .
( a , left ) sequences
of the synthesized
peptides with the uaas denoted as x ; ( right ) chemical
structures of the different uaa side chains , where n2 , n3 , and so
forth indicate the number of ch2 units in the side chain .
( b ) chemical structures of the linkers , where m3m5 indicate
the number of carbon / nitrogen atoms between the two alkynyl groups .
( c ) schematic of the click reaction between the peptides in ( a ) and
linkers in ( b ) ; see figure s2 for full
chemical structures of representative peptides .
we explored a number of different linker designs , varying
both
the chemical properties and rigidity ( figure 2b ) .
all linkers contained an alkyne at each
terminus to cross - link the two uaas in the peptide .
linkers with linear
aliphatic units between the two alkynyl groups are denoted m3m5
to indicate the number of methylene ( ch2 ) units .
a longer linker containing hydrocarbon
units and a urea derivative , which we denote as m7n , was synthesized
from 1,1-carbonyldiimidazole and 1-amino-3-butyne ( protocols
modified from ref ( 57 ) ) .
the linkers with greater rigidity , m3c and m5c , contain aromatic
groups and were synthesized using palladium - catalyzed coupling chemistry .
we previously used our double - click chemistry strategy
to staple -helical peptides derived from the tumor suppressor
p53 for inhibition of the p53-mdm2 interaction . in that case , as for other helix stapling methods , the
stapling positions were identified to be the i , i+4 or i , i+7 residue
pairs so that the intrinsic helical propensity of the peptide brings
the two stapling positions close together in space , thereby helping
to drive the reaction .
the tnks - bound peptides adopt an extended conformation ,
and therefore , we first tested the efficiency of the click reaction
between linker and peptide in this context .
after optimization of
the reaction conditions , we were able to show that the click reaction
between peptide cp4n4 and linker m5 proceeded to completion after
stirring overnight under nitrogen in degassed solvent ( figure 2c ) .
monitoring by liquid chromatography
mass spectrometry ( lcms ) showed that no starting material remained
and that there was a single product with the expected mass .
the purified
and lyophilized product was further analyzed by infrared spectrometry
( ir ) and high - resolution mass spectrometry to verify that the two
azido groups in the peptide reacted with the same linker because other
potential byproducts share the same mass from the 1,3-dipolar cycloaddition .
the disappearance of the typical azido stretch at 2100 cm in the ir spectrum after the reaction suggested that both azido
groups had reacted ( figure s4 ) .
the isotopic
patterns from mass spectrometry showed a mass spacing between two
[ m + 2h ] species of 0.5 amu , consistent with the cross - linking
of a single linker onto one peptide ( figure s5 ) .
these results demonstrate unequivocally that we can extend the
scope of our two - component click macrocyclization strategy to non - helical
peptides .
the affinities of a first panel of macrocyclized peptides
for binding to tnks2 arc4 were determined using a competitive fluorescence
polarization ( fp ) assay with a tamra - labeled linear peptide t - pep1
( tamra - ahx - reagdgee , where ahx is a spacer that comprises 6-aminohexanoic
acid ; tamra - labeled peptides are indicated with
t-
at the beginning of the name ) as the competing ligand ( figure 3 and table s1 ) .
the dissociation constant of the labeled t - pep1 was 0.8
0.1 m from the direct fp measurement , and that of unlabeled
pep1 was 1.2 0.1 m from the competitive fp measurement
in agreement with the values reported in the literature .
two uncross - linked precursor peptides , cp4n4
and cp5n4 , were also tested and found to have affinities that were
similar to or only slightly weaker than pep1 , indicating that incorporation
of the uaas did not significantly perturb the interaction with tnks
arc4 .
all of the macrocyclized peptides in this first panel bound
more weakly than the linear pep1 to tnks arc4 .
we tested several combinations
of uaas and linkers based on cross - linking positions 2 and 7 ( rxagdgxe ;
cp2 ) , including varying the uaa side chain lengths ( n ) between 2 and 4 , and two different linker lengths ( m ) of 5 and 7 ; however , all combinations yielded peptides that exhibited
very weak binding affinities .
macrocyclization of cp3 , containing
uaas in positions 2 and 8 , also yielded weak binding affinities .
macrocyclization
of cp4 and cp5 ( uaas in positions 3 and 7/8 ) , however , yielded higher
binding affinities , and peptide rexgdgxe ( cp4 ) was therefore chosen
for further optimization . determining the optimal peptide - linker combination .
dissociation
constants were determined by competitive fp assay using labeled t - pep1
bound to arc4 as the tracer for the first panel of macrocyclized peptides
in which we varied the positions of the two uaas ( denoted x ) in the
peptide sequence , the lengths of the side chains , and the length of
the linker .
n is the number of methylene
( ch2 ) groups in the side chain of the uaas ;
m is the number of carbon / nitrogen atoms between
the two triazole groups in the linker .
special linkers are m5c , a
rigid linker based on napthalene , and m7n , a linear linker made from
a urea derivative described earlier .
the dissociation
constant of the unlabeled linear peptide , pep1 , is shown for comparison .
uncross - linked cp4n4 and cp5n4 were measured as comparisons to their
cross - linked counterpart .
to aid the design process , we determined the structure
of cp4n4m5 ( figure s2 ) in complex with
tnks2 arc4 ( figure 4 and figures s6 and s7 ) .
this structure
shows that the binding mode of the macrocyclized peptide is very similar
to that of the natural peptides .
specifically , arg1 of cp4n4m5 forms
two salt bridges with glu598 and asp589 of tnks2 arc4 ( figure s6a ) and glu2 with tnks2 lys557 ( figure s6b ) .
the side chains of the two uaas ,
uaa3 and uaa7 , point away from the protein surface , which is crucial
for their ability to form a macrocycle that does not interfere with
protein binding ( figure 4b ) . between uaa3 and uaa7 , residues gly4 , asp5 , and gly6 of the peptide
fit inside a groove on the protein surface ( figure 4a and figure s6c ) with hydrogen bonding between asp5 and tnks2 ser527 ( figure s6d ) and the phenol rings of tyr569 and
tyr536 stacking in parallel on either side of gly6 ( figure s6c ) .
the c - terminal residue of cp4n4m5 , glu8 , interacts
with tnks2 lys604 via a salt bridge ( figure s6e ) .
we note that the linker is positioned away from the binding surface
and does not perturb the interactions between cp4n4m5 and tnks2 arc4
( figure 4b ) , and it
should therefore be possible to install additional functionalities
on the linker without compromising the binding interface . despite
the high 1.35 resolution of the crystal structure , parts of
the uaa side chains and the triazole - containing linkage are poorly
defined in the election density ( figure 4b ) .
this suggests that shortening the uaa
side chains and the linker should lead to tighter binding by further
reducing conformational flexibility .
( a ) crystal structure of tnks2 arc4 ( gray
surface ) in complex with
the macrocyclized peptide cp4n4m5 ( orange carbons ; linker in red ) .
( b ) 2fobs fcalc electron density ( blue mesh ) of the linker and of the
central residues of the peptide ( orange ; linker in red ) contoured
at 1 , rotated approximately 90 along the horizontal axis
compared to ( a ) .
consequently , we next
synthesized uaas with shorter side chains
and incorporated them into the cp4 sequence .
all of the peptides were
synthesized with a tamra label at the n - terminus ( tamra - ahx - rexgdgxe )
for use in the direct fp assay format .
the number ( n ) of ch2 groups in the side chain of each uaa was varied
between 1 and 3 , and the number of carbon / nitrogen atoms ( m ) between the two triazole groups in the linker was varied
between 3 and 5 ( figure 5a and b and table s2 ) .
we found that the
binding affinity was always weakened when non - identical uaas were
used in the same peptide , and it was greatly weakened with uaa side
chain length of n = 1 . upon shortening of the linker ,
the binding affinity progressively increased , and rigidifying the
linker by incorporating an aromatic moiety ( linker m3c ) resulted in
a further increase of the affinity .
the macrocyclized peptide with
the highest binding affinity had n = 2 for both uaa
side chains and the rigid linker m3c ( labeled t - cp4n2m3c , figures s2 and s3 ) ; its affinity is approximately
2-fold higher than that of the linear peptide t - pep1 .
( a ) direct fp measurements
for a subset of the second panel of
tamra - labeled peptides based on the cp4 sequence ( tamra - ahx - rexdgdxe )
with ( b ) their binding affinities listed in a table ( see table s2 for the full list of peptides and their
binding affinities ) .
( c ) crystal structure of tnks2 arc4 ( gray
surface ) in complex with macrocyclized peptide cp4n2m3 ( cyan ; linker
in green ) .
superposition of cp4n2m3 ( cyan / green ) and cp4n4m5 ( orange / red )
in the two structures of the complexed peptides ; tnks2 arc4 is shown
in gray .
( d ) 2fobs fcalc electron density , contoured at 1 , of the linker
and of the central residues of peptide cp4n2m3 .
( e ) overlay of the
three structures : the complex with the natural substrate peptide lphlqrsppdgqsfrs
is shown ( pdb i d : 3twr ) ( protein : pink ; peptide : purple ) .
ankyrin repeats 4 and 5 of tnks2 arc4-cp4n2m3 ( cyan ) move by 23
compared to those in the complex with cp4n4m5 ( orange ) and
the linear peptide ( pink ) .
( f ) cd spectra of linear peptides pep1
( green ) and cp4n4 ( red ) and of cross - linked peptides cp4n4m5 ( orange ) ,
cp4n2m3 ( cyan ) , and the non - binding cross - linked peptide cp4n1m3 ( purple ) .
we then synthesized the unlabeled
version of the highest affinity
peptides from this second round of design and determined the structure
of tnks2 arc4 in complex with one of them , cp4n2m3 ( figure 5 , and figures s2 , s3 , and s7a ) .
when comparing this structure with that of
tnks2 arc4-cp4n4m5 and those of previously published natural peptides , we can see that the linkers of both cp4n4m5
and cp4n2m3 are long enough to retain the binding mode of the natural
peptide ( figure s7b ) .
additionally , the
tnks2 protein itself appears to have some flexibility for accommodating
the slightly different conformations of the peptides .
both peptides
are more flexible at their respective termini ( see figures s7 and s8 ) , whereas the central regions
make close contacts with tnks2 arc4 and adopt virtually identical
conformations in the two macrocyclized peptides ( figure 5c ) .
the central residues gly4-asp5-gly6
are deeply inserted into a binding cleft of the protein , forming several
hydrogen bonding interactions as well as remarkable -stacking
interaction , wherein gly6 is sandwiched between tyr536 and tyr569
( figures 4b and 5d and figure s7c ) .
shortening
of the uaa side chains and the hydrocarbon linker rigidifies the macrocycle ,
which is corroborated by the better - defined electron density of cp4n2m3
( figure 5d and figure s7d ) .
the shorter linker of cp4n2m3 pulls
the side chains of peptide residues 3 and 7 slightly inward when compared
with that of the wild - type peptide ( pdb i d : 3twr ) and cp4n4m5 ( figure 5c and figure s7b ) .
this difference
appears to have an effect on the overall protein
curvature , whereby ankyrin repeats 4 and 5 move by 23
( figure 5e ) .
although
we can not exclude a crystallization artifact caused by changes in
the packing of the complexes in the two different crystal forms ( table s3 ) , we could not observe a change of this
type in any of the previously published linear peptide complexes ( pdb
ids : 3twr , 3tws , 3twt , 3twu , 3twv , 3tww , and 3twx ) , and the resolution of 1.33 should be sufficient
to reliably observe these differences .
this alternative conformation
was very similar to the crystal structure of the apo protein ( pdb
i d : 3twq ; figure s9a ) with the only discernible difference
found in ankyrin repeat 5 .
accordingly , this change in protein conformation
mainly affects the c - terminal end of the bound peptide : whereas glu8
of cp4n4m5 forms a salt bridge with lys604 ( figure s6e ) , in cp4n2m3 , it instead forms an interaction with lys602
( figure s6f ) . observed intermittently in
md simulations of the tnks2
arc4-pep1 complex ( figure s9b ) , this apo - like bound conformation
allows for the formation of a novel water network that connects glu8
and lys602 with the triazole moiety of uaa3 in the tnks2 arc4-cp4n2m3
complex . an md simulation of the isolated tnks2 arc4 sampled mainly
the apo - like bound conformation ( figure s9c ) .
conformations that are somewhat similar to those observed in the
linear peptide and cp4n4m5 complexes , except for slight differences
in ankyrin repeats 3 and 5 , were also sampled ( figure s9c ) .
the fact that the unbound protein is able to
adopt the apo - like bound conformation prior to binding suggests that
conformational selection plays a dominant role in the binding of cp4n2m3 ,
whereas the requirement for modest conformational changes in two of
the ankyrin repeats suggests that induced fit mechanism is likely
to be prominent in the binding of the linear peptides and cp4n4m5 . to better understand the effect of the different macrocycles on
the conformations of the peptides and how any differences might translate
into the order of binding abilities observed , we performed circular
dichroism ( cd ) on the unlabeled peptides ( figure 5f ) .
we find that linear pep1 and uncross - linked
cp4n4 exhibit a random - coil conformation , as does the macrocyclic
cp4n4m5 .
for the higher - affinity binder cp4n2m3 , although its cd spectrum
resembles that of a random coil with a minimum at 196 nm , it also
has a small positive maximum at 217218 nm similar to that
of a collagen ; this distinctive secondary
structure is presumably due to the tighter constraint restricting
the flexibility of the peptide backbone .
it is consistent with our
goal of designing the macrocycles to restrict the conformations of
the peptides to reduce the entropy cost of binding as further evidenced
in the isothermal titration calorimetry data shown below .
when we
further reduce the length of the macrocycle in cp4n1m3 , a macrocyclic
peptide with nondetectable kd for tnks2
arc4 ( figure 5a and
b and table s2 ) , the cd spectrum indicates
that an -helical structure is induced by the tighter constraint .
analysis of the crystal structures also shows clearly that shorter
uaas ( n = 1 ) would constrain the macrocycle too much
for the peptide to form ideal interactions with the protein , leading
to the observed weaker binding for n = 1 peptides
( table s2 ) . for the effects
of macrocyclization on peptide stability
to be
tested , representatives of both the linear and the macrocyclized peptides
were subjected to proteolytic degradation using the aspn protease ,
which cleaves the n - terminal to aspartates , in this case , asp5 , which
is located within the macrocycle .
protection of the central residues ,
gly4-asp5-gly6 , is crucial for the integrity of the peptide for fitting
inside the binding groove of the protein .
tamra - labeled peptides were
used for monitoring the degradation based on the tamra signal at 550
nm wavelength .
the majority of the linear peptide , t - pep1 , was degraded
within 1 h at room temperature , whereas nearly 100% of the macrocyclized
peptide , t - cp4n2m3 , remained intact after 4.5 h of peptidase treatment
( figure s10a ) .
no mass corresponding to
the possible degraded products could be found in the sample of the
treated t - cp4n2m3 after 4.5 h ( figure s10b ) .
isothermal titration calorimetry ( itc ) was used
to characterize the
thermodynamics of the tnks peptide interactions ( figure 6 , figure s11 , and tables s4 and s5 ) .
the smaller ts terms obtained for the macrocyclized peptides compared
with linear peptide pep1 are consistent with the goal of macrocyclization ,
which is to lock the peptide in an active conformation and thereby
minimize the entropic cost of binding ( figure 6 ) .
furthermore , a smaller ts value was obtained for peptide cp4n2m3c with the more
rigid linker compared with that of cp4n2m3 , consistent with the success
of this design feature .
thermodynamic parameters obtained for the binding
of the peptides
to tnks2 arc4 and to tnks1 arc23 , as measured by itc .
error
bars are those obtained from standard derivation of the mean from
duplicate measurements .
tnks proteins contain five arcs , of which arc2 , 4 , and 5
have sites
for recognition by tnks binding motifs .
we therefore also tested the
binding of the peptides to tnks1 arc23 , a construct containing
arc2 and ( non - binding ) arc3 , whose structure had previously been solved .
we obtained similar results to tnks2 arc4 in
terms of the relative s values of pep1 and
the two macrocyclized peptides .
we note that all of the peptides tested
bound more tightly to tnks1 arc23 than to tnks2 arc4 ( tables s4 and s5 ) , although the two arc domains
share almost identical residues at the binding interfaces .
we
next compared the thermodynamic parameters of the peptides with
those of the tnks substrate axin .
axin is a rate - limiting component
of the -catenin destruction complex in the wnt sigaling pathway ,
and parylation of axin by tnks leads to axin degradation by the proteasome .
the crystal structure of mouse tnks1 arc23 had previously
suggested that dimerization of arc23 occurs upon interaction
with a fragment ( residues 180 ) of mouse axin1 because this fragment contains not one but two tnks binding
motifs , both of which are similar in sequence to the consensus sequence
deduced by guettler et al .
( figure 1c ) . the two motifs
of axin ( residues 1830 and 6079 ) each interact with
one arc2 subunit in the arc23 dimer .
we performed itc using
fragments of human axin1 , which share high homology to the mouse counterparts .
our itc data are consistent with the dimerization of tnks1 arc23
seen in the crystal structure in that we also observe a stoichiometry
of two tnks1 arc23 molecules binding to one axin1_180
molecule ( table s5 ) ; a stoichiometry of
1 was obtained for the interaction of tnks2 arc4 with axin1_180 .
furthermore , the tnks2 arc4 binding affinity of axin1_143
( containing only the n - terminal tnks - binding motif ) was similar to
that of axin1_180 ( containing both motifs ) ( table s4 ) , indicating that the n - terminal motif is the main
contributor to the tnks2 arc4 interaction , as was found to be the
case for the tnks1 arc23 interaction .
lastly , the itc data show that both cp4n2m3 and cp4n2m3c are better
binders than axin1 to tnks1 arc23 as well as to tnks2 arc4 ,
a result that was also confirmed by fluorescence polarization ( tables s4 and s5 ) .
axin
interaction using an in vitro pull - down assay with tnks2 arc4 and
gst - tagged human axin1_180 immobilized on glutathione beads
( figure 7 ) .
the immobilized
tnks2 arc4-axin1_180 complex was incubated with increasing
concentrations of linear peptide pep1 or macrocyclized peptide cp4n2m3
for 0.5 h. after washing the resin , the proteins remaining bound to
the resin were analyzed by sds - page .
the results show that pull - down
of tnks2 arc4 was disrupted by the peptides in a dose - dependent manner .
macrocyclized peptide cp4n2m3 was a slightly better inhibitor than
pep1 ( ic50 of 20 and 40 m , respectively ) ,
reflecting its slightly higher tnks binding affinity .
competition of peptides
with gst - tagged human axin1_180
for binding to tnks2 arc4 .
the tnks2 arc4-axin1_180 complex ,
immobilized on glutathione beads , was incubated with increasing concentrations
of peptide .
after washing , the protein remaining bound to the resin
was run on sds - page .
the tnks2 arc4 band was quantified using densitometry
and normalized against the gst - axin1_180 band .
the data are
plotted relative to the sample without peptide incubation ( which was
set at a value of 1 ) .
we attached one of four different cell - penetrating
peptide ( cpp ) sequences onto cp4n2m3c via the linker by synthesizing
the cpp sequences with the m3c linker conjugated to their n - termini
( see schematic in table s6 ) .
the two - component
double - click reaction was then performed between the cpp - conjugated
m3c linker and the peptide t - cp4n2 ( tamra - ahx - rexgdgxe , where x is
n2 ) . in the case of the penetratin peptide ( a 16-residue cell - penetrating
sequence from the antennapedia homeodomain ) , we found that we needed two ahx spacers between the cpp and the
m3c linker for the click reaction to reach completion , as this relatively
long cell - penetrating sequence appeared to otherwise inhibit the click
reaction presumably for steric reasons .
next , we checked the tnks2
arc4 binding affinities of the tamra - labeled cpp - conjugated peptides
using direct fp ( table s7 ) . for the arg9 cell - penetrating sequence , when we used the two ahx spacers
we observed a degree of non - specific binding to tnks2 arc4 , likely
due to the arginine residues attached via the long flexible spacers
competing with the binding peptide for a hotspot interaction on the
protein , and therefore
we did
not observe any non - specific binding for the other cpp - conjugated
peptides shown in table s7 .
all four exhibited
tnks binding affinities that are similar to that of the unconjugated
counterpart t - cp4n2m3c .
we next tested the cell - penetrating capabilities
of the tamra - labeled peptides using u2os and hek 293 t cells .
three
of the cpp - conjugated macrocyclized peptides , t - cp4n2m3c - r6 , t - cp4n2m3c - r9 , and t - cp4n2m3c - antp , showed significant
fluorescence spreading across the whole cell even at the lowest concentration
of 10 m .
in contrast , t - cp4n2m3c - lclr ( lclr is a cell - penetrating
tetrapeptide derived from the x - protein of the hepatitis b virus ) and the non - conjugated peptides t - cp4n2m3 and t - cp4n2m3c exhibited
only punctate fluorescence indicative of endosomal entrapment , and
the linear peptide t - pep1 showed very weak fluorescence or no fluorescence
at all ( figure s12 ) . a
dual - luciferase reporter ( dlr ) assay in wnt - activated hek 293 t cells
was used to determine the ability of the peptides to modulate -catenin
levels and suppress wnt transcription .
unlabeled cpp - conjugates , rather
than the tamra - labeled versions , were used in the experiments because
rhodamine - based fluorescent labels have been found to cause cytotoxicity
once they are transported intracellularly with the aid of cpp peptides .
treatment of hek 293 t cells with an increasing
concentration of unlabeled peptide cp4n2m3c - antp ( containing the penetratin
sequence on the linker ) led to a dose - dependent decrease in wnt pathway
reporter activity with an ic50 around 50 m ( figure 8) .
there was no observed
change in the level of the control renilla luciferase activity , indicating
that peptide treatment had no effect on transcription in general .
moreover , treatment with non - cell - penetrating peptides pep1 or cp4n2m3c
had no effect on wnt reporter activity , nor did treatment with cp4n1m3c - antp
( a cell - penetrating version of the non - binding peptide cp4n1m3c ) ( table s2 ) , thereby satisfying these specificity
controls .
figure s13 shows that none of
the unlabeled peptides were cytotoxic at the concentrations used in
the wnt signaling assays .
effect on wnt - activated hek 293 t cells of treatment
with representative
peptides .
here , we employed molecular dynamics simulations ,
two - component
double - click macrocyclization , and combinatorial use of different
non - natural amino acids and cross - linkers to constrain peptides that
have extended bioactive conformations .
we demonstrate the application
of this approach to design inhibitors of the substrate - recognition
domain of the tnks proteins whose activity is deregulated in numerous
cancers .
one advantage of the two - component strategy is that the peptide
and the linker can be engineered separately and then used in a combinatorial
fashion to efficiently generate an array of molecules . with the consensus - optimized
substrate sequence as a starting point
, we used an iterative process
of macrocyclized peptide design in which we varied the length and
rigidity of the linker as well as its position along the peptide .
the design was further guided by determining the crystal structures
of two of the macrocyclized peptides in complex with tnks . in this
way , we identified peptides with submicromolar tnks binding affinities
that are proteolytically stable and capable of disrupting the interaction
between tnks and the axin substrate in a dose - dependent manner .
we
exploited the two - component nature of the macrocyclization method
to introduce cell - penetrating capability via the linker , and we showed
that these peptides are indeed able to enter cells and modulate -catenin
levels .
thus , these macrocylized peptides represent a promising new
class of substrate - competitive inhibitors of tnks with potential for
suppressing wnt signaling in cancer .
moreover , we have demonstrated
that double - click macrocyclization can be applied to non - helical ,
extended , or irregularly structured peptide conformations ; we have
also recently used it to develop constrained peptide inhibitors of
the hepatocyte nuclear factor 1 transcription factor ( hnf1)importin
interaction in ovarian clear cell carcinoma .
thus , the double - click macrocyclization strategy has a
broad scope , especially given the frequency with which non - helical
motifs are found in mediating ppis .
the structure of human
tnks2 arc4 bound to a 3bp2-derived peptide ( pdb i d : 3twr(53 ) ) was used to model the complex of tnks2 arc4 and the consensus
tankyrase - binding peptide .
the 3bp2-derived peptide was truncated
to the core binding 8-residue motif ( rsppdgqs ) and then modified
into the consensus peptide ( reagdgee ) by using the
tleap module of amber 11 to change the side chains of the mutated
residues .
protonation states of the complex were determined by pdb2pqr . the leap program in amber 11
was then used to solvate the
complex with tip3p water molecules in
a truncated octahedral box , such that its walls were at least 9
away from any atom of the protein or peptide , followed by neutralization
of the system with seven sodium ions .
three independent explicit - solvent
md simulations of the modeled tankyrase complex were carried out using
different initial atomic velocities .
energy minimizations and
md simulations were performed using the ff99sb - ildn force field with the sander and pmemd modules of amber
11 , respectively .
shake was applied to constrain all bonds involving hydrogen atoms ,
allowing for a time step of 2 fs . non - bonded interactions were truncated
at 9 , whereas the particle mesh ewald method was used to account for long - range electrostatic interactions
under periodic boundary conditions .
weak harmonic positional restraints
with a force constant of 2.0 kcal mol were placed on the complex s non - hydrogen
atoms during the minimization and initial equilibration steps .
energy
minimization was carried out using the steepest descent algorithm
for 500 steps , followed by the conjugate gradient algorithm for another
500 steps .
the system was then heated at constant volume to 300 k
over 50 ps , followed by equilibration at a constant pressure of 1
atm for another 50 ps .
subsequent unrestrained equilibration ( 2 ns )
and production ( 50 ns ) runs were carried out at a constant temperature
of 300 k using a langevin thermostat with
a collision frequency of 2 ps and a constant pressure
of 1 atm using a berendsen barostat with
a pressure relaxation time of 2 ps .
the protein structures generated
by the simulations were clustered
using the art-2 algorithm based on the
root - mean - square deviation ( rmsd ) of the c atoms . cut - off radii
of 1.4 and 1.3 were used to generate the clusters for the unbound
and complex simulations , respectively .
the cluster members with the
lowest rmsd from their respective centroids were selected as representative
structures for structural alignment .
computational alanine
scanning was carried out on the peptide from 200 equally spaced complex
structures extracted from the last 30 ns of the md simulations deemed
stably equilibrated .
the differences in the binding free energies
( g ) of the wild - type and alanine mutants
( or glycine mutant for ala3 ) were calculated using the molecular mechanics / generalized
born surface area ( mm / gbsa ) method .
molecular
mechanical energies were calculated with the sander module of amber
11 . the polar contribution to the solvation free energy
was calculated
by the pbsa program using the modified gb model described by onufriev
et al . , and the nonpolar contribution
was estimated from the solvent accessible surface area using the linear
combinations of pairwise overlaps method with set to 0.0072 kcal / and set
to zero .
the entropy term was not considered
due to the high computational cost and the assumption that the entropy
of the mutant does not differ considerably from that of the wild type .
the fmoc - protected
azido - functionalized amino acids were synthesized as described previously .
all tnks binding peptides were synthesized manually
on rink amide mbha resin ( 0.65 mmol / g , 100200 mesh , novabiochem )
using a vac - man laboratory vacuum manifold ( promega ) according to
the standard fmoc - based solid - phase peptide synthesis ( spps ) method .
the n - terminus of the peptide was either capped by acetylation or
coupled with a spacer aminohexanoic acid followed by 5-carboxytetramethylrhodamine
( 5-tamra ) .
cell - penetrating peptides were synthesized manually or
on an automated microwave peptide synthesizer ( liberty blue , cem )
on rink amide mbha ll resin ( 0.38 mmol / g , 100200 mesh , novabiochem ) ,
and linker m3c was attached to the n - terminal end of the peptides
by manual spps .
all peptides were cleaved from the resin in a tfa
cocktail containing 92.5% ( v / v ) tfa , 2.5% ( v / v ) water , 2.5% ( v / v )
triisopropylsilane , and 2.5% ( v / v ) dichloromethane .
the eluate was
dried , and the cleaved peptide was precipitated with diethyl ether ,
redissolved in 1:1 ( v / v ) water / acetonitrile , filtered , and lyophilized .
the crude peptide was purified on a semi - preparative hplc agilent
1260 infinity using a supelcosil abz+plus column ( alkyl - amide phase ,
250 mm 21.2 mm , 5 m ) , eluting with a linear gradient
system ( solvent a : 0.1% ( v / v ) tfa in water ; solvent b : 0.05% ( v / v )
tfa in acetonitrile ) .
the purity of the peptide was checked on an
analytical hplc ( agilent 1260 infinity , supelcosil abz+plus column
( 150 mm 4.6 mm , 3 m ) ) , eluting with a linear gradient
system ( solvent a : 0.05% ( v / v ) tfa in water ; solvent b : 0.05% ( v / v )
tfa in acetonitrile ) .
the double - click reaction was carried out to cross - link
the azido - functionalized peptides with dialkynyl functionalized linkers .
the aliphatic linkers were purchased from sigma - aldrich ,
and all others were synthesized according to protocols previously
described .
all solvents for the reaction were degassed with
nitrogen for 1 h before use .
the dialkynyl linker ( 1.1 equiv ) was
added to the diazido - peptide in 1:1 ( v / v ) water and tert - butanol under nitrogen before the addition of a solution of copper(ii )
sulfate pentahydrate ( 1 equiv ) , tris(3-hydroxypropyltriazolylmethyl)amine
( thpta , 1 equiv ) , and sodium ascorbate ( 3 equiv ) in water .
the reaction
mixture was stirred for 16 h at room temperature and monitored by
lcms .
a second aliquot of linker and catalyst was added for those
reactions that had not run to completion .
the crude product was purified
on the semi - preparative hplc as described above .
the purified macrocyclized
peptide was lyophilized and analyzed by infrared spectrometry ( ir )
and high - resolution mass spectrometry .
genes for human tnks1_179966
and tnks2_488649 were purchased from epoch life science , and
that for human axin1_180 was purchased from life technologies .
tnks1_315662 , tnks2_488649 , axin1_143 , axin1_180
were cloned into expression vector prseta ( ge healthcare ) or pgex
using standard molecular biology methods .
the plasmid containing each construct was transformed to e. coli c41(de3 ) cells , and the colonies were grown in 2ty
media at 37 c until od 600 reached 0.61.0 and were then
induced at 2025 c with 0.20.5 mm iptg for 16
h. cell pellets were collected and resuspended in 50 mm trishcl
buffer ph 8.0 , 500 mm nacl , 2 mm dtt , protease inhibitor cocktail
( sigma - aldrich ) , 10 mm edta for gst - tagged tnks2_488649 , axin1_143 ,
and axin1_180 . for his6-tagged tnks1_315662 ,
10 mm edta was replaced with 10 mm imidazole .
the cells were lysed
using an emulsiflex c5 homogenizer , and the lysate was added to either
ni - nta agarose ( qiagen ) or glutathione sepharose 4b beads ( ge healthcare ) .
the bound his6- or gst - tagged protein was washed with the
same resuspension buffer but without the protease inhibitor , and the
tag was cleaved with thrombin ( 25 u per liter of culture ) overnight
at 25 c .
the desired protein was purified to > 95% homogeneity
on a size - exclusion gel - filtration column ( hiload 16/60 superdex g75 )
equilibrated with loading buffer of 50 mm hepes buffer ph 7.5 , 300
mm nacl , 2 mm dtt .
for protein crystallization , tnks2_488649
was expressed and purified using the same protocol , except that the
loading buffer was 20 mm hepes buffer ph 7.5 , 150 mm nacl , 0.5 mm
tcep .
for the cocrystal structure of
tnks2 488649 in complex with cp4n4m5 , 0.3 l of 1 mm
tnks2_488649 and 5 mm cp4n4m5 in 20 mm hepes ph 7.5 , 150 mm
nacl , 0.5 mm tcep , 5% ( v / v ) dmso were mixed with 0.3 l of precipitant
solution containing 1.60 m trisodium citrate in a sitting drop vapor
diffusion experiment at 19 c .
for the crystal structure of tnks2 488649
in complex with sp4n2m3 , a mixture of 0.3 l of 1 mm tnks2_488649
and 2.9 mm cp4n2m3 in 10 mm hepes ph 7.5 , 75 mm nacl , 0.25 mm tcep ,
5% ( v / v ) dmso was mixed with 0.3 l of precipitant solution
containing 3.20 m ammonium sulfate and 0.1 m trisodium citrate ph
5.0 in a sitting drop vapor diffusion experiment at 19 c .
needle - like
crystals appeared after 4 days and grew to the final length of 420
m after 12 days .
data were collected at diamond light source
beamlines i24 and i04 - 1 and processed with autoproc / xds to 1.35 and 1.33 ,
respectively ( table s3 ) .
phases were obtained
through molecular replacement using phaser with pdb 3twq(53 ) as the search model .
the
structures have been deposited in the protein data bank with codes 5bxu ( tnks2 arc4cp4n4m5
complex ) and 5bxo ( tnks2 arc4cp4n2m3 complex ) . for the comparative analysis
,
the c-atoms of the tnks2 residues were aligned within a 7
radius around the peptide / macrocycle binding site to avoid changes
in curvature having a significant impact on the analysis .
fluorescence polarization
( fp ) assays were performed in 96-well half area black microplates
( corning ) on a clariostar microplate reader ( bmg labtech ) using excitation
filter 54020 nm , dichroic mirror lp 566 nm , and emission filter
59020 nm . all peptides were dissolved in water as stock solutions .
for direct fp assays , the stock concentrations of the tamra - labeled
peptides were determined based on the 5-tamra absorbance at 556 nm
( extinction coefficient = 89,000 m cm ) measured on a nanodrop 2000 ( thermo scientific )
and verified by amino acid analysis ( department of biochemistry , university
of cambridge ) .
tnks2 arc4 ( 488649 ) or tnks1 arc23
( 315662 ) was diluted 2-fold serially in the assay buffer ( 50
mm hepes ph 7.5 , 300 mm nacl , 2 mm dtt , 0.05% ( v / v ) tween 20 ) for
a 12-point titration curve in triplicate .
the diluted tamra - labeled
peptide ( 20 nm , 50 l ) was mixed with the serially diluted tnks2
arc4 or tnks1 arc23 ( 400 m , 50 l ) in
each well and incubated for 30 min at 25 c before the measurement .
data were analyzed on graphpad prism 5.0 , and the dissociation constant , kd , was determined using the following equation
assuming the ratio between the concentration of the bound and that
of the total tamra - labeled peptide is proportional to the fluorescence
polarizationwhere fp is the fluorescence
polarization , b is the minimum fp , t is the maximum fp , l0 is the total concentration of tamra - labeled
peptide , p0 is the total concentration
of protein , and kd is the dissociation
constant . for the competition fp assays ,
the stock concentrations
of the unlabeled macrocyclized peptides or the axin1 fragments were
determined by amino acid analysis .
tamra - labeled pep1 ( 20 nm ) and
tnks2 arc4 ( 3 m ) or tnks1 arc23 ( 1.2 m ) were
incubated in pbs buffer containing 0.05% ( v / v ) tween 20 .
the unlabeled
peptide or axin1 fragment was diluted 2-fold serially in pbs buffer
containing 0.05 ( v / v ) tween 20 for a 12-point titration curve in triplicate .
the diluted peptide or axin1 fragment ( 50 l ) was mixed with
the tamra - pep1/tnks solution ( 50 l ) in each well and incubated
for 30 min at 25 c before the measurement was taken .
cd spectra were
recorded on a chirascan cd spectrometer ( applied photophysics ) , fitted
with a water bath and using a 1 mm path length cell .
unlabeled peptides
( 0.1 mm ) were prepared in water , and the spectra were recorded at
25 c .
four separate measurements were made , and the mean molar
ellipticity , , was then plotted against the wavelength .
all itc experiments
were performed on a microcal itc200 ( ge healthcare ) at 25 c .
tnks2 arc4 ( 488649 ) , tnks1 arc23 ( 315662 ) ,
and axin1 fragments were dialyzed overnight in pbs and 0.5 mm tcep .
the peptide was diluted
from the stock solution using the same dialysis buffer , and the effect
of buffer dilution was accounted for when preparing tnks samples .
( 160280 m ) or tnks1 arc23 ( 60150
m ) was titrated into the sample cell containing the peptide
( 515 m ) or axin1 fragments ( 812 m ) with
an initial injection of 0.2 l over 0.4 s followed by 19 injections
of 2 l over 4 s with a spacing of 60 s , except for tnks2 arc4
and axin1 fragments for which the spacing was 70 s. control experiments
were performed using the same settings as above except that the cell
was filled with dialysis buffer .
data were fitted with nonlinear regression
using a one - site binding model from origin 7.0 ( microcal , inc . ) .
gst - tagged human
axin1_180
was incubated with glutathione sepharose 4b beads ( ge healthcare )
for 1 h at 4 c .
the beads were washed 3 times with wash buffer
( 50 mm trishcl ph 8.0 , 500 mm nacl , 2 mm dtt ) .
approximately
80 g of gst - tagged axin1_180 ( 10 l ) on beads
was incubated with tnks2 arc4 ( 40 l , 89 m ) or his6-tagged tnk1 arc23 ( 50 l , 100 m ) for
0.5 h at room temperature .
the unbound tnks protein was washed away
with wash buffer ( 150 l 2 ) , and the beads were incubated
at room temperature for 0.5 h with the serially diluted unlabeled
peptide ( 50 l , 0200 m ) .
the beads were subsequently
washed twice ( 150 l 2 ) , boiled with sds , and loaded
on the sds - page protein gels . after the run , the gels were stained
with coomassie brilliant blue and imaged .
the intensities of the bands
were analyzed using imagej , and the standard deviations were calculated
from two independent experiments .
endoproteinase aspn ( 50 ng ,
new england biolabs ) was added to a mixture of tamra - labeled peptide
( 400 m ) , tamra ( 100 m ) , and endoproteinase aspn enzyme
buffer ( new england biolabs ) , and the volume was made up to 50 l
with sterile water .
the reaction was incubated with shaking at 550
rpm at 25 c . at each time point , a 5 l aliquot was taken
and centrifuged at 10,000 rpm at 4 c , and the supernatant was
diluted 4 times with water before being loaded onto an analytical
hplc ( agilent 1260 infinity , supelcosil abz+plus column ( 150 mm
4.6 mm , 3 m ) ) and eluted with a linear gradient system ( solvent
a : 0.05% ( v / v ) tfa in water ; solvent b : 0.05% ( v / v ) tfa in acetonitrile ) .
tamra - labeled t - pep1 was run with a linear gradient of 595%
solvent b over 5 min , and tamra - labeled cp4n2m3 was run with 540%
solvent b over 20 min .
the cleavage of the tamra - labeled peptide was
monitored at 550 nm , and the integral of the peptide peak was measured
against that of tamra as the internal standard .
after 270 min , the
remaining sample containing tamra - labeled t - cp4n2m3 was submitted
to lcms , and no mass corresponding to the hydrolyzed macrocyclized
peptide could be observed .
the plot ( figure s10a ) is representative of one of the two independent experiments .
u2os or hek 293 t cells were grown
to 100% confluency before being used in all assays .
cells ( 10 ) were seeded in glass - bottom dishes ( 35 mm diameter , mattek
corporation ) and grown for 16 h at 37 c and 5% co2 in 1 ml of dmem ( 1 ) + glutamax-1 ( life technologies ) .
cells
were then incubated with tamra - labeled peptides ( 1040 m )
for 4.5 h , followed by a further 0.5 h incubation with hoechst 33342
nucleic acid stain ( 5 g / ml ) at 37 c and 5% co2 before being washed with pbs ( 2 1 ml ) and replenished with
1 ml of hbss ( 1 ) ( life technologies ) .
confocal images were taken
at 37 c on a leica tcs sp5 confocal microscope with sequential
excitation at 405 and 543 nm , respectively .
u2os cells ( 2 10 ) or hek 293 t cells ( 2
10 ) were seeded in a 96-well
cell culture plate ( corning costar ) and grown for 16 h at 37 c
and 5% co2 in 100 l of dmem ( 1 ) + glutamax-1
( life technologies ) containing 10% ( v / v ) fetal bovine serum and penicillin - streptomycin .
cells were then incubated with the unlabeled peptides ( 50 and 100
m ) for 5 h at 37 c and 5% co2 .
a maximum ldh
release control was prepared by adding 10 lysis solution ( 10
l , promega ) to the cells and incubated at 37 c for 45
min before the measurement .
the supernatant ( 50 l ) from each
well was transferred to a clear flat - bottom 96-well microplate , and
the assay was performed using a cytotox 96 nonradioactive cytotoxicity
assay ( promega ) according to the manufacturer s protocol .
the
ldh release indicated by the absorbance at 490 nm was measured on
a clariostar microplate reader ( bmg labtech ) .
data were obtained from
triplicate samples , and the standard deviations were calculated from
two independent experiments . hek 293 t were grown in dulbecco s
modified eagles medium ( dmem ) supplemented with 10% fetal calf serum .
wnt pathway activity was induced by treating cells with 20 mm licl
( or kcl as control ) or conditioned media obtained from l - cells expressing
wnt3a for 6 h. cells were transfected with the lipofectamine 2000
transfection reagent according to the manufacturer s protocol
( invitrogen ) . for topflash reporter assays , 100 ng of topflash
plasmid and 10 ng of cmv - renilla plasmid ( as internal control ) were
used to transfect cells in 24-well plates .
transfected cells were
allowed to recover for 8 h , and concurrently treated with unlabeled
macrocyclized peptides and wnt3a conditioned media / licl for a further
16 h. topflash assays were performed using the dual - luciferase reporter
assay system ( promega ) as previously described .
relative luciferase values were obtained from triplicate
samples ( from two independent experiments ) by dividing the firefly
luciferase values ( from topflash ) by the renilla luciferase values
( from cmv - renilla ) , and standard deviations were calculated . | we report a double - click
macrocyclization approach for the design
of constrained peptide inhibitors having non - helical or extended conformations .
our targets are the tankyrase proteins ( tnks ) , poly(adp - ribose ) polymerases
( parp ) that regulate wnt signaling by targeting axin for degradation .
tnks are deregulated in many different cancer types , and inhibition
of tnks therefore represents an attractive therapeutic strategy .
however ,
clinical development of tnks - specific parp catalytic inhibitors is
challenging due to off - target effects and cellular toxicity .
we instead
targeted the substrate - recognition domain of tnks , as it is unique
among parp family members .
we employed a two - component strategy , allowing
peptide and linker to be separately engineered and then assembled
in a combinatorial fashion via click chemistry . using the consensus
substrate - peptide sequence as a starting point , we optimized the length
and rigidity of the linker and its position along the peptide .
optimization
was further guided by high - resolution crystal structures of two of
the macrocyclized peptides in complex with tnks .
this approach led
to macrocyclized peptides with submicromolar affinities for tnks and
high proteolytic stability that are able to disrupt the interaction
between tnks and axin substrate and to inhibit wnt signaling in a
dose - dependent manner .
the peptides therefore represent a promising
starting point for a new class of substrate - competitive inhibitors
of tnks with potential for suppressing wnt signaling in cancer .
moreover ,
by demonstrating the application of the double - click macrocyclization
approach to non - helical , extended , or irregularly structured peptides ,
we greatly extend its potential and scope , especially given the frequency
with which such motifs mediate protein protein interactions . | Introduction
Results
Summary
Experimental
Section | constraining non - helical , extended , or irregularly structured peptide
motifs presents a different challenge , as it is harder to rationally
design a linker so as to stabilize a binding - competent conformation
( e.g. here
, we use a two - component double - click chemistry approach to
macrocyclize peptides in an extended , non - helical conformation . these include control of the mitotic
checkpoint , regulation of telomere length by targeting telomeric
repeat binding factor 1 ( trf1 ) for degradation , and regulation of wnt signaling by targeting axin
for degradation . the first small - molecule tnks inhibitor ( discovered in a wnt pathway inhibitor screen )
and those developed
subsequently are all directed against the catalytic parp domain ; however , there
are problems of cellular toxicity due to off - target effects arising
from the nad+/adp ribose - like characteristic of these active - site
inhibitors ( reviewed in refs ( 35 and 4951 ) ) . to this end , we have designed
a series of macrocyclized peptides
using a modular two - component strategy that employs click chemistry
to connect a linker and a peptide , thus allowing each to be varied
independently before assembling them in a combinatorial fashion . our designs were guided by the previously determined crystal structures
of substrate - derived peptides in complex with the fourth arc domain
of tnks2 ( tnks2 arc4 ) ( figure 1b ) and subsequently optimized
with the help of molecular dynamics ( md ) simulations using an iterative
process of modifying the length and rigidity of the linker as well
as its position along the peptide sequence . we determined the crystal
structures of two of the macrocyclized peptides in complex with tnks ,
leading to further optimization of the design . we show that these
peptides are able to disrupt tankyrase - substrate complexes in vitro
and to inhibit wnt transcription in a dose - dependent manner . thus ,
our results provide a promising starting point for a new class of
substrate - competitive inhibitors of tankyrase
. moreover , the methodology
has the potential to be a general approach for inhibiting ppis that
involve non - helical , extended , or irregularly structured molecular
recognition motifs . dissociation
constants were determined by competitive fp assay using labeled t - pep1
bound to arc4 as the tracer for the first panel of macrocyclized peptides
in which we varied the positions of the two uaas ( denoted x ) in the
peptide sequence , the lengths of the side chains , and the length of
the linker . here , we employed molecular dynamics simulations ,
two - component
double - click macrocyclization , and combinatorial use of different
non - natural amino acids and cross - linkers to constrain peptides that
have extended bioactive conformations . we demonstrate the application
of this approach to design inhibitors of the substrate - recognition
domain of the tnks proteins whose activity is deregulated in numerous
cancers . one advantage of the two - component strategy is that the peptide
and the linker can be engineered separately and then used in a combinatorial
fashion to efficiently generate an array of molecules . with the consensus - optimized
substrate sequence as a starting point
, we used an iterative process
of macrocyclized peptide design in which we varied the length and
rigidity of the linker as well as its position along the peptide . the design was further guided by determining the crystal structures
of two of the macrocyclized peptides in complex with tnks . in this
way , we identified peptides with submicromolar tnks binding affinities
that are proteolytically stable and capable of disrupting the interaction
between tnks and the axin substrate in a dose - dependent manner . thus , these macrocylized peptides represent a promising new
class of substrate - competitive inhibitors of tnks with potential for
suppressing wnt signaling in cancer . moreover , we have demonstrated
that double - click macrocyclization can be applied to non - helical ,
extended , or irregularly structured peptide conformations ; we have
also recently used it to develop constrained peptide inhibitors of
the hepatocyte nuclear factor 1 transcription factor ( hnf1)importin
interaction in ovarian clear cell carcinoma . thus , the double - click macrocyclization strategy has a
broad scope , especially given the frequency with which non - helical
motifs are found in mediating ppis . | [
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] |
hif is a heterodimer consisting of one of three alpha ( ) subunits and a beta ( ) subunit .
hif-1 is the most well - established member of the hif family ; the other two members of the basic helix loop helix - pas ( bhlh - pas ) superfamily are hif-2 ( endothelial pas domain protein 1 or hif1-like - factor ) , which also stabilizes hypoxia and binds to the aryl hydrocarbon receptor nuclear translocator ( arnt ) [ 1 , 2 ] and hif-3 .
hif-1 is a transcriptional activator and a master regulator for the expression of genes involved in the response to hypoxia in most mammalian cells .
hif-1 is prolyl hydroxylated at p402 and p564 in its oxygen - dependent degradation domain ( odd ) under normoxic conditions .
this leads to binding of the hif- unit to the e3 ubiquitin ligase von hippel - lindau protein ( pvhl ) at l574 for degradation [ 5 , 6 ] .
low oxygen tension stabilizes the -subunit and leads to nuclear translocation , formation of a dimer with hif-1 , and recruitment of transcriptional coactivators .
this complex binds to an enhancer domain of the hypoxia responsive element ( hre ) located either at the 5 or 3 region of target genes , including heme oxygenase-1 , vascular endothelial growth factor ( vegf ) , glucose transporter ( glut)-1 , and glut-4 .
interestingly , hif-1 also upregulates glycolytic enzymes and glucose transporters , allowing cells to depend more heavily on glycolysis for energy , suggesting that hif-1 also modulates aerobic metabolism . the vast majority of hif target genes are regulated by hif-1 , whereas exclusively hif-2-dependent genes are scarce and cell type - dependent [ 1215 ] .
most reports show that hif-1 and hif-2 are master regulators of the transcriptional response to hypoxia , but the role of hif-3 under hypoxic conditions is far less clear .
hif-3 is the newest member of the family and may be more restricted than the other hif subunits .
hif-3 sequence has relatively low identity with hif-1/2 . hif-1 and hif-2 have two terminal transactivation domain ( tads ) [ 1 , 10 ] , whereas hif-3 has only one tad .
hif-3 has a unique leucine zipper domain and an lxxll motif , and these unique structural features are evolutionarily conserved .
hif-3 has multiple splice variants , and the inhibitory pas domain protein is the most studied , which is a truncated protein and a dominant - negative inhibitor of hif-1 . hif-1 and hif-2 have 48% amino acid sequence identity and similar protein structures , but are nonredundant and have distinct target genes and mechanisms of regulation .
interestingly , hif-1 appears the most active isoform during short periods ( 224 h ) of intense hypoxia or anoxia ( < 0.1% o2 ) in some cell lines , whereas hif-2 is active under mild or physiological hypoxia ( < 5% o2 ) , and continues to be active even after 4872 h of hypoxia .
thus , in some contexts , hif-1 plays key role in initial response to hypoxia whereas hif-2 drives the hypoxic response during chronic hypoxic exposure [ 19 , 20 ] .
this hif switch results between hif-1 and hif-2 suggests physiological and pathological adaptation required to adapt for cell survival .
interestingly , these isoform plays opposite but balancing roles during the hypoxic response under both physiological and pathophysiological conditions in some cells .
prolyl hydroxylases ( phds ) [ 1 , 2 and 3 ] are evolutionary conserved oxygen sensors in metazoans .
these dioxygenases were discovered after confirming that oxygen - dependent enzymatic activity covalently modifies an hif-1 domain known as the odd domain [ 2123 ] .
phds require 2-oxoglutarate as a cosubstrate , molecular oxygen , and iron liganded by two histidine and one aspartic acid residues to function as hydroxylases .
phds play a key role in the hif - mediated hypoxia signaling pathway , which facilitates cell survival and adaptation in response to capricious environmental oxygen levels .
phds lose their activity under hypoxic conditions , leading to accumulation and nuclear translocation of hif- and activation of hif target genes by binding to hres .
the first identified function of phds was to hydroxylate human hif-1 h - subunits at pro402 and pro564 under normoxic conditions , resulting in their recognition , pvhl ubiquitylation , and degradation by 26s proteasomes .
the phd catalytic domain recognizes a specific lxxlap motif in the odd of the hif- subunits .
phd13 have near ubiquitous tissue expression ; phd-2 is generally the most abundant isoform , with the exception of the testis , where phd-1 is the most highly expressed isoform , and the heart , where phd-3 expression predominates .
loss of phd-1 lowers skeletal muscle and liver oxygen consumption by reprogramming glucose metabolism from primarily oxidative to more anaerobic atp production , suggesting selective loss of phd-1-induced hypoxia tolerance [ 29 , 30 ] .
knockdown of the phd-2 gene , but not those of phd-1 and phd-3 , results in embryonic death due to placental and heart defects , suggesting that phd-2 is essential during mouse embryogenesis .
interestingly , broad spectrum conditional phd-2 knockout in adult mice leads to hyperactive angiogenesis , angiectasia , and congestive heart failure , suggesting that phd-2 is a major negative regulator of vascular growth [ 32 , 33 ] .
furthermore , phd-3 is required for proper anatomical and physiological integrity of the system , as loss of phd-3 results in abnormal sympathoadrenal development and systemic hypotension .
targeted deletion of the phd-3 gene increases angiogenesis and preserves cardiac function by stabilizing hif-1 after infarction , suggesting a potential target for pharmacological management of ischemic myocardial disease .
oxygen scarcity leads mitochondria to produce reactive oxygen species ( ros ) thereby giving alert to cells to the shortage .
ros describe a range of molecules and free radicals ( chemical species with one unpaired electron ) derived from molecular oxygen .
the mitochondrial electron transport chain contains several redox centers that may leak electrons to oxygen , constituting the primary source of superoxide ( precursor of most ros ) in most tissues .
enzymatic sources include nadph oxidases [ 36 , 37 ] as well as cytochrome p450-dependent oxygenases .
proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both superoxide and h2o2 ( and therefore constitutes a source of oh ) and has been proposed to mediate deleterious processes in vivo .
nonenzymatic production of superoxide occurs when a single electron is directly transferred to oxygen by reduced coenzymes or prosthetic groups or by xenobiotics previously reduced by enzymes .
according to the equation(1)dsuperoxidedt = ko2r,where r is an electron donor , the rate of superoxide formation increases with oxygen concentration under normobaric and hyperbaric conditions .
as predicted by ( 1 ) , mitochondrial production of superoxide anion should increase with oxygen concentration , but the proportion of superoxide anion is less than predicted in tissue exposed to atmospheric oxygen [ 41 , 42 ] .
ros activate the hif pathway during hypoxia leading to stabilization of the hif(s ) -isoform .
the generation of ros should decrease with hypoxia according to ( 1 ) , yet many reports show increased oxidative stress under moderately hypoxic conditions [ 4345 ] but not under severe hypoxic conditions .
moreover , substantial evidence indicates a role for the functional respiratory chain in the generation of ros under hypoxic conditions .
a mutation in the respiratory chain [ 45 , 46 ] or complex 1 inhibitors prevent stabilization of hif under hypoxic conditions .
more research is needed to clarify the paradoxical ros formation in the response of tissues to hypoxia .
mitochondria are the largest consumers of cellular o2 and are likely candidates for the location of a cellular oxygen sensor .
mitochondria are both targets and important sources of free radicals . most vital intracellular processes , including blood vessel maintenance , heart contractibility , lung functioning , and neurotransmitter and hormonal support require mitochondria .
mitochondria are the source of atp and maintain oxygen homeostasis at both the systemic and cellular levels .
initial evidence supporting mitochondria as an oxygen sensor came with the discovery that mitochondrial - depleted hep3b cells fail to respire and activate mrnas of erythropoietin , glycolytic enzymes , or vegf during hypoxia .
these cells also fail to increase the generation of ros during hypoxia , suggesting that mitochondrial ( mt ) ros trigger hypoxia - induced transcription .
mitochondria are implicated in multiple hif - dependent and independent pathways through production of mtros .
interestingly , decreasing mtros levels with mitochondrial inhibitors or ros scavengers prevents stabilization of hif-1 under hypoxic conditions , suggesting that ros are important for this effect [ 4547 ] .
interestingly , murine embryonic cells lacking cytochrome c , and therefore mitochondrial activity , have impaired cellular oxygen sensing , which prevents stabilization of hif-1 and hif-2 under hypoxic conditions , suggesting that mtros act upstream of phd to regulate hif-1 and hif-2 .
mitochondrial complex iii is required for hypoxia - induced ros production and cellular oxygen sensing [ 51 , 52 ] .
similarly , embryonic stem cells lacking cytochrome c fail to stabilize hif-1 during hypoxia , as loss of cytochrome c leads to complete reduction of cytochrome c1 and the rieske iron - sulfur protein , which inhibit transfer of electrons from ubiquinol at the qo site .
the complex iii qo site is necessary to increase cytosolic ros under hypoxic conditions , which inhibits phds from degrading the hif-1 protein .
additionally , exogenous h2o2 or mutations leading to h2o2 accumulation stabilize hif-1 during normoxia [ 52 , 54 ] . in accordance
, antioxidants abolish the hypoxic hif response , suggesting that generation of mtros is responsible for propagating the hypoxic signal .
the role of the hif pathway has been demonstrated under hypoxic conditions but it also plays an important role in normoxia .
hif1/ knockout mice have cardiac and vascular malformations and embryos die around mid - gestation , whereas hif-2 knockout mice die presumably of bradycardia due to an inadequate supply of catecholamines during embryonic development .
hif-1 is present in mice under normoxic conditions , increases within distinct cell types in response to systemic hypoxia , and plays an important role in oxygen homeostasis .
hif-2 plays an important role in cardiovascular development and angiogenesis [ 56 , 58 ] .
murine embryonic stem cells lacking the hif-2 gene revealed an association with the response to hypoglycemia rather than hypoxia , suggesting that hif-2 may be more important in the survival response than oxygen level .
hypoxia affects expression of the per1 and clock circadian genes , and hif-1 interacts with per1 under normoxic conditions .
the mouse hif1a gene is expressed from two distinct promoter / first exon combinations , resulting in tissue - specific ( mhif-1alphai.1 ) and ubiquitous ( mhif-1alphai.2 ) forms .
the mhif-1alphai.1 protein is located in the mid - piece of the spermatozoal flagellum and expression is oxygen independent .
the mhif-1alphai.1 isoform is also upregulated in activated t cells under normoxic conditions , suggesting a physiological role for the mhif-1alphai.1 isoform in activated lymphocytes .
interestingly , mice exposed to an elevated temperature strongly upregulate hif-1 in the liver , kidney , and spleen , suggesting a novel mechanism to stabilize hif-1 under normoxic conditions .
furthermore , a recent study demonstrated that treating normal human cells with low - dose radiation induces a hif-1-mediated adaptive and protective metabolic response and increased radiation resistance .
hif-1 is induced and activated at physiological oxygen tensions in a mitogen activated protein kinase - dependent manner and determines the increased cell proliferation rate that is common under these conditions .
hif-1 plays an important role in the adaptation of myocardium to mechanical stress via stress - activated channels .
as discussed previously , hif-1 is required for mesenchymal cell survival , and hif knockout mice have malformed cardiovascular systems and neural tube defects and die during mid - gestation [ 55 , 67 , 68 ] .
hypoxia potentiates interleukin ( il)-1 expression and attenuates selective targeting of il-1 to autophagic degradation in activated macrophages , suggesting that a novel proinflammatory mechanism may participate in atherogenesis .
these observations support the notion that the hif pathway plays important roles in physiological adaptation and is required for normal growth and development .
interestingly , mice with only one hif-1 mutant allele develop normally but have impaired physiological responses to chronic hypoxia , such as reduced polycythemia , right ventricular hypertrophy , pulmonary hypertension , pulmonary vascular remodeling , and electrophysiological responses [ 70 , 71 ] .
the carotid body ( cb ) monitors arterial blood o2 levels and stimulates breathing in response to hypoxemia to ensure an adequate o2 supply . both hif-1 and
hif-2 are expressed in the cb [ 56 , 72 ] , and cb responses to hypoxia are impaired in hif1+/ mice , whereas they are exaggerated in hif2+/ mice .
balanced hif-1 and hif-2 activity is critical for oxygen sensing by the cb and adrenal medulla and for their control of cardiorespiratory function .
similarly , cbs isolated from hif-1 heterozygous mice have a dramatic effect on neural activity and ventilatory adaptation after exposure to hypoxia , suggesting a role for hif-1 at the systemic level .
partial hif-2 deficiency leads to increased levels of hif-1 and nadph oxidase 2 , resulting in an oxidized intracellular redox state , exaggerated hypoxic sensitivity , and cardiorespiratory abnormalities , which are reversed by treatment with a hif-1 inhibitor or a superoxide anion scavenger .
in contrast , partial hif-1 deficiency increases levels of hif-2 and superoxide dismutase 2 , leading to a reduced intracellular redox state , blunted oxygen sensing , and impaired cb and ventilatory responses to chronic hypoxia , which are corrected by a hif-2 inhibitor .
these observations demonstrate that the redox balance , which is determined by mutual antagonism between hif- isoforms , establishes the hypoxic sensing set - point by the cb and adrenal medulla and is required for maintenance of cardiorespiratory homeostasis .
hif-2 plays an important role during the postvasculogenesis stages and is required to remodel the primary vascular network into a mature hierarchical pattern .
hif-2 sense hypoxia during mid - gestational development and translate this signal into an altered gene expression pattern , leading to increased circulating catecholamine levels and proper cardiac function . despite of normoxic environment several tissues are inherently hypoxic suggesting importance of hif pathway in normal development .
interestingly , tissue - specific targeting to delete hif-1 in the cartilaginous growth plate of developing bone showed gross skeletal malformations and die perinatally , probably due to tracheal abnormalities suggesting the role of hypoxia in growth plate development .
oxygen tension determines whether cytotrophoblasts , specialized placental cells proliferate or invade , thereby regulating placental growth and cellular architecture .
moreover , arnt knockout mice placentas shows greatly reduced labyrinthine and spongiotrophoblast layers , and increased numbers of giant cells supporting that hif-1 is essential for mammalian placentation .
additionally , hifs play important roles in modulating the developmental plasticity of stem cells by integrating physiological , transcriptional and epigenetic inputs in placenta . transforming growth factor ( tgf ) -3 , an inhibitor of extravillous trophoblast differentiation ,
in addition , hif-1alpha - deficient mouse embryonic fibroblasts showed impaired migratory capabilities and demonstrated that tgf--3 manifests hypoxia and hif-1-dependent regulation . furthermore , hypoxia signaling plays a central role in cartilage , bone , and hematopoiesis .
hif-1 plays a bimodal role in cartilage homeostasis by enhancing anaerobic glycolysis and inhibiting apoptosis suggesting the potential role of this pathway in treatment of osteoarthritis [ 83 , 84 ] .
hif-2 , was found to be essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice and its function are independent of oxygen - dependent hydroxylation .
elevated levels of hif-1 promotes cartilage formation and maintenance whereas elevated levels of hif-2 favors cartilage destruction and endochondral ossification [ 85 , 86 ] .
taken together this suggests that both hif-1 and hif-2 plays an important role for normal growth and development of skeletal vasculature .
interestingly , hif-1 stimulates glycolysis and actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase-1 ( pdk-1 ) activity .
that study also reported that pdk-1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial tca cycle , which decreases mitochondrial oxygen consumption resulting in a relative increase in intracellular oxygen tension .
another known mechanism to increase respiration efficiency in hypoxic cells is by regulating of cytochrome c oxidase ( cox ) activity .
cox is located in the inner mitochondrial membrane and is a dimer composed of monomers with 13 subunits .
subunits i , ii , and iii are encoded by the mitochondrial genome , constitute the catalytic core of the enzyme , and are highly conserved in eukaryotes .
the crystal structure of bovine cox reveals that subunit iv ( cox4 ) interacts with both cox1 and cox2 .
the first step of cox assembly in mammalian cells is the association of cox1 with cox4 .
cox4 binds atp , within the complex , leading to allosteric inhibition of cox activity at high atp / adp ratios and demonstrating a regulatory role for cox4 .
under hypoxic conditions , hif-1 mutually regulates cox4 subunit expression by stimulating transcription of the genes encoding cox4 - 2 and lon , a mitochondrial protease required for cox4 - 1 degradation .
the effects of manipulating cox-4 subunit expression on cox activity , atp production , o2 consumption , and ros generation indicate that the cox4 subunit switch is a homeostatic response that optimizes respiration efficiency at different o2 concentrations .
simply , pdk-1 inhibits conversion of pyruvate to acetyl - coa , thereby preventing pyruvate entry into the tca cycle and the cox-4 subunits govern mitochondrial respiration efficiency in response to varied oxygen tensions [ 11 , 87 ] .
autophagy may be the fourth adaptive metabolic response required to prevent increased ros levels and cell death in hypoxic cells .
mitochondria are replaced every 24 weeks in rat brain , heart , liver , and kidney .
the destruction of mitochondria is believed to occur via autophagy [ 93 , 94 ] .
autophagy can be induced by environmental stress such as nutrient deprivation and provides a mechanism to dispose of damaged mitochondria [ 95 , 96 ] .
autophagy is induced in the heart subject to hypoxic or ischemic conditions and has been proposed to play either a protective or pathogenic role in heart disease [ 9698 ] .
bnip3 is an accepted hif-1 target gene [ 99 , 100 ] and is associated with autophagy .
bnip3 may disrupt interactions between beclin-1 , a highly conserved protein required to initiate autophagy , and bcl2 or bcl - xl .
in contrast to hif , c - myc is a proto - oncogene that codes for a transcription factor , regulates the expression of 1015% of all genes in the genome [ 102 , 103 ] , and promotes mitochondrial respiration by increasing biogenesis . under physiological conditions
hif-1 inhibits c - myc activity by directly interacting and stimulating a proteasome - dependent pathway [ 104 , 105 ] .
c - myc paradoxically collaborates with hif-1 under stress conditions to induce pdk-1 and hexokinase 2 expression followed by aerobic glycolysis [ 105 , 106 ] and angiogenesis .
the role of hif pathway in cell death is controversial ; hif-1 can induce apoptosis , prevent cell death , or even stimulate cell proliferation .
the oxygen concentration determines whether cell will go apoptosis or not ; oxygen level in the range 00.5% could induce apoptosis whereas cells with oxygen levels in the range of 13% do not undergo apoptosis .
atp is another key determinant of apoptosis ; as long as cells have an enough supply of glycolytic atp during oxygen deprivation , apoptosis can be executed .
moreover , lack of oxygen inhibits the electron transport chain at the inner membrane of the mitochondria thereby causes a reduction in the membrane potential .
this reduction of mitochondrial derived atp causes activation of bax or bak , leading to cytochrome c release into the cytosol .
cytochrome c is released and caspase-9 is activated in oxygen - deprived cells undergoing apoptosis [ 110 , 111 ] . furthermore , p53 protein , an important regulator of apoptosis can induce the bax and bak proteins thereby initiating the cascade leading to apoptosis through cytochrome c .
interestingly , fibroblasts from mice lacking both bax and bak genes are resistant to oxygen deprivation induced apoptosis .
similarly , cell over - expressing bcl-2 or bcl - xl , the anti - apoptotic proteins prevents oxygen deprivation induced apoptosis by inhibiting the release of cytochrome c from the mitochondria [ 110 , 113 , 114 ] .
in addition to energy deprivation , ros generation contributes to hypoxia induced apoptosis . in this case
, the initiator caspase 9 is cleaved directly to the active form by caspases 3 and 12 , without the involvement of cytochrome c in response to hypoxia .
activation of c - jun nh2-terminal kinase ( jnk ) is another mechanism by which hypoxia can induce apoptosis in melanoma cells .
hif pathway is also involved in cell proliferation and promoting metastases [ 117 , 118 ] , vegf is particularly noteworthy target gene of hif pathway involved in cell proliferation and upregulated in most cancers [ 119 , 120 ] .
the hifs can alter cell - cycle progression through putative transcriptional targets such as cyclin d1 and indirect modulation of p21 and p27 .
furthermore , hypoxic induction of hif-1 suppresses cell proliferation and acute hif-1 stabilization at moderate hypoxia ( 1% o2 ) results in cell cycle arrest by inhibiting c - myc transcriptional activity .
in contrast , hif-2 induction promotes cell cycle progression by enhancing c - myc function .
the correlation between hypoxia and intracellular ph ( phi ) was extensively researched in the 198090s .
rapid build - up of intracellular lactate and h as well as an extensive decrease in phosphocreatine concentration is the first sign of hypoxia .
furthermore , metabolic damage is considerably greater if glucose is absent during the insult , suggesting that either anaerobic atp production or low ph may exert some protective effect against acute cell damage .
the regulation of na / h exchange ( nhe ) and phi is vital for maintaining cell viability .
phi modulates a number of important cell functions , including signal transduction pathways involved in the regulation of cell size and proliferation [ 124126 ] .
alterations in phi are also associated with hypoxic pulmonary vasoconstriction [ 127 , 128 ] .
pulmonary arterial smooth muscle cells from chronic hypoxic mice have an elevated basal phi accompanied by an increase in nhe activity , secondary to increased nhe1 isoform expression .
hif-1 plays an important role governing increased nhe , nhe1 expression , and alkalinization of pulmonary arterial smooth muscle cells in response to hypoxia .
tumor cells have a lower extracellular ph ( phe ) and a higher phi than those of normal cells .
low phe promotes invasiveness , whereas high phi provides a competitive advantage to growth of tumor cells compared to normal cells for [ 132 , 133 ] .
hypoxia induces coordinated upregulation of glycolysis , a potential step that may promote tumor cell growth and activate the capacity of tumor - associated carbonic anhydrase ( ca ) ix to acidify phe thereby leading to tumor progression .
hypoxia - inducible caix and caxii proteins promote cell survival and growth by maintaining phi .
moreover , caix and caxii constitute a robust phi - regulating system that confers a tumor growth and survival advantage to cells exposed to a hypoxic and acidic microenvironment .
muscle training induces negative regulators of hif ( phd , fih , and sirtuin-6 ) but lowers pdk-1 expression in elite athletes , thereby contributing to skeletal muscle adaptation to exercise .
endurance exercise improves muscle oxidative capacity , whereas resistance exercise training leads to increased muscle size and strength .
one study investigated the effects of 8 weeks of resistance exercise training performed under hypoxic ( hrt ) or normoxic conditions ( nrt ) on skeletal muscle .
as results , significant increases in muscle endurance , plasma vegf concentration , and capillary - to - fiber ratio were observed following training in the hrt group compared those in the nrt group , suggesting that hrt may also lead to increased muscular endurance and promote angiogenesis in skeletal muscle .
chuvash polycythemia ( cp ) is an autosomal recessive disorder in which regulatory degradation of hif is impaired , resulting in elevated levels of hif under normal oxygen tensions [ 140 , 141 ] .
patients with cp show early and marked phosphocreatine depletion , higher blood lactate accumulation , acidosis in skeletal muscles , and reduced exercise capacity .
interestingly , gene therapy with intramuscular administration of ad2/hif-1/vp16 was not effective for patients with intermittent claudication .
pyruvate dehydrogenase ( pdh ) plays an important role controlling the flux of pyruvate to acetyl - coa .
pdh is inactivated during acute hypoxia thereby promoting conversion of pyruvate to lactate , suggesting an influence of pdh activity on the fate of pyruvate .
the transition from acute to chronic hypoxia desensitizes the hif-1 pathway , leading to a re - establishment of pdh activity and reduced lactate production by exercising muscles .
exercise with intermittent hypoxic training for 3 weeks causes a significant decrease in skeletal muscle hif-1 mrna , suggesting that transcriptional and posttranscriptional regulation of the hif-1 differ in muscle and other cells .
in contrast , the hif-3 subunit plays a negative role in adaptation to hypoxia because inhibiting hif-3 expression leads to increased physical endurance .
in present review we have discussed the physiological adaptations and importance of the hif pathway .
are also upregulated under inflammatory conditions , suggesting their role in maintaining homeostatic conditions and protecting against cellular inflammation .
for example , hif inhibitors have been developed to treat cancer and ischemia , whereas hif activators could be utilized for stroke and spinal cord injuries .
significant developments have been made towards understanding the roles of the hifs under both physiological and pathophysiological conditions .
the interaction of hif-1 with hif-2 and hif-3 during physiological adaptation will provide a great deal of understanding of hif(s ) .
moreover how these transcription factors interact with other known proteins and pathways will help in designing the future therapeutics with minimal side effect . | oxygen homeostasis reflects the constant body requirement to generate energy .
hypoxia ( 0.11% o2 ) , physioxia or physoxia ( 113% ) , and normoxia ( 20% ) are terms used to define oxygen concentration in the cellular environment . a decrease in oxygen ( hypoxia ) or excess oxygen ( hyperoxia ) could be deleterious for cellular adaptation and survival .
hypoxia can occur under both physiological ( e.g. , exercise , embryonic development , underwater diving , or high altitude ) and pathological conditions ( e.g. , inflammation , solid tumor formation , lung disease , or myocardial infarction ) .
hypoxia plays a key role in the pathophysiology of heart disease , cancers , stroke , and other causes of mortality .
hypoxia inducible factor(s ) ( hifs ) are key oxygen sensors that mediate the ability of the cell to cope with decreased oxygen tension .
these transcription factors regulate cellular adaptation to hypoxia and protect cells by responding acutely and inducing production of endogenous metabolites and proteins to promptly regulate metabolic pathways . here , we review the role of the hif pathway as a metabolic adaptation pathway and how this pathway plays a role in cell survival .
we emphasize the roles of the hif pathway in physiological adaptation , cell death , ph regulation , and adaptation during exercise . | 1. Introduction
2. Prolyl Hydroxylases: Oxygen Sensors
3. Hypoxia and Reactive Oxygen Species
4. Hypoxia and Mitochondrial Respiration
5. Physiological Adaptation by HIF(s)
6. Conclusion | hif-1 is the most well - established member of the hif family ; the other two members of the basic helix loop helix - pas ( bhlh - pas ) superfamily are hif-2 ( endothelial pas domain protein 1 or hif1-like - factor ) , which also stabilizes hypoxia and binds to the aryl hydrocarbon receptor nuclear translocator ( arnt ) [ 1 , 2 ] and hif-3 . this complex binds to an enhancer domain of the hypoxia responsive element ( hre ) located either at the 5 or 3 region of target genes , including heme oxygenase-1 , vascular endothelial growth factor ( vegf ) , glucose transporter ( glut)-1 , and glut-4 . most reports show that hif-1 and hif-2 are master regulators of the transcriptional response to hypoxia , but the role of hif-3 under hypoxic conditions is far less clear . interestingly , hif-1 appears the most active isoform during short periods ( 224 h ) of intense hypoxia or anoxia ( < 0.1% o2 ) in some cell lines , whereas hif-2 is active under mild or physiological hypoxia ( < 5% o2 ) , and continues to be active even after 4872 h of hypoxia . thus , in some contexts , hif-1 plays key role in initial response to hypoxia whereas hif-2 drives the hypoxic response during chronic hypoxic exposure [ 19 , 20 ] . phds play a key role in the hif - mediated hypoxia signaling pathway , which facilitates cell survival and adaptation in response to capricious environmental oxygen levels . as predicted by ( 1 ) , mitochondrial production of superoxide anion should increase with oxygen concentration , but the proportion of superoxide anion is less than predicted in tissue exposed to atmospheric oxygen [ 41 , 42 ] . ros activate the hif pathway during hypoxia leading to stabilization of the hif(s ) -isoform . the role of the hif pathway has been demonstrated under hypoxic conditions but it also plays an important role in normoxia . hif-1 is present in mice under normoxic conditions , increases within distinct cell types in response to systemic hypoxia , and plays an important role in oxygen homeostasis . these observations support the notion that the hif pathway plays important roles in physiological adaptation and is required for normal growth and development . both hif-1 and
hif-2 are expressed in the cb [ 56 , 72 ] , and cb responses to hypoxia are impaired in hif1+/ mice , whereas they are exaggerated in hif2+/ mice . interestingly , tissue - specific targeting to delete hif-1 in the cartilaginous growth plate of developing bone showed gross skeletal malformations and die perinatally , probably due to tracheal abnormalities suggesting the role of hypoxia in growth plate development . furthermore , hypoxia signaling plays a central role in cartilage , bone , and hematopoiesis . hif-1 plays a bimodal role in cartilage homeostasis by enhancing anaerobic glycolysis and inhibiting apoptosis suggesting the potential role of this pathway in treatment of osteoarthritis [ 83 , 84 ] . autophagy is induced in the heart subject to hypoxic or ischemic conditions and has been proposed to play either a protective or pathogenic role in heart disease [ 9698 ] . the role of hif pathway in cell death is controversial ; hif-1 can induce apoptosis , prevent cell death , or even stimulate cell proliferation . the oxygen concentration determines whether cell will go apoptosis or not ; oxygen level in the range 00.5% could induce apoptosis whereas cells with oxygen levels in the range of 13% do not undergo apoptosis . furthermore , hypoxic induction of hif-1 suppresses cell proliferation and acute hif-1 stabilization at moderate hypoxia ( 1% o2 ) results in cell cycle arrest by inhibiting c - myc transcriptional activity . as results , significant increases in muscle endurance , plasma vegf concentration , and capillary - to - fiber ratio were observed following training in the hrt group compared those in the nrt group , suggesting that hrt may also lead to increased muscular endurance and promote angiogenesis in skeletal muscle . exercise with intermittent hypoxic training for 3 weeks causes a significant decrease in skeletal muscle hif-1 mrna , suggesting that transcriptional and posttranscriptional regulation of the hif-1 differ in muscle and other cells . in contrast , the hif-3 subunit plays a negative role in adaptation to hypoxia because inhibiting hif-3 expression leads to increased physical endurance . in present review we have discussed the physiological adaptations and importance of the hif pathway . significant developments have been made towards understanding the roles of the hifs under both physiological and pathophysiological conditions . | [
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] |
a 55-year - old female patient with a height of 151 cm and a weight of 55 kg was referred from the hematooncology clinic for left buttock pain caused by right infiltrative ductal breast cancer with left pelvic bone metastasis .
the degree of pain on the 1 - 10 numeric rating scale ( nrs ) was 8 , and it was characterized as a spontaneous tingling and ripping pain . she had received breast conserving surgery for right breast cancer 2 years before , but metastasis to the left pelvic bone had been discovered in an mri one year before .
chemotherapy with adriamycin , cyclophosphamide , and taxol had been performed 6 times , and radiation therapy had been performed 8 times , but there had been no reduction in tumor size .
hence , palliative chemotherapy of gemzar and navelbine had been performed 6 times , and radiation therapy had been performed 6 times . at the time of referral ,
the patient was having difficulty walking due to the pain in the pelvic area . in the mri and bone scan of the pelvic area performed before referral , bone metastasis
was observed in the left ilium , left ischium , and right acetabulum , in addition to a pathologic fracture in the left iliac wing ( fig .
the patient was administered with fentanyl patch 100 g / hr , oxycodone ir 10 mg bid , gabapentin 200 mg tid , and carbamazepine 200 mg bid for pain control ; however , she was continuously complaining of severe pain ( nrs 8/10 ) that worsened when walking . during hospitalization ,
fluoroscopically guided caudal epidural injection of 0.125% chirocaine ( levobupivacaine hydrochloride , abbott korea , korea ) 10 ml and triam ( triamcinolone acetonide , shin poong pharm , korea ) 20 mg was performed twice with a one - week interval .
however , the pain alleviation effect did not continue for more than one day after the procedure .
hence , scrambler therapy ( mc5-a calmare ) was planned . because the patient 's pain was located at the s1 - 2 dermatome of the left buttocks , the scrambler electrodes were attached at 4 normal sensory areas above and below the painful area of the s1 - 2 dermatome for treatment . during the scrambler treatment ,
the nrs score was maintained at 3 in the ward on the day of treatment .
the patient was discharged after 10 sessions of scrambler therapy . during the 10 sessions ,
the dermatome location of pain did not change , but the area of pain slowly contracted ; hence , the painful area of the patient was consulted daily before attaching the electrodes .
pain medications were continually administered without a change in dosage during the scrambler therapy . the pain alleviation effect through scrambler therapy continued for 2 months ; on the third month
the patient was re - hospitalized , and a second round of scrambler therapy was started . during treatment ,
the nrs score decreased from 7/10 to 0/10 on the first day , and this was maintained for 3 - 4 hours after treatment .
subsequently , the nrs score remained at 3.5/10 for 2 months , and pain medication was consistently given without a change in dosage during this period .
a 49-year - old female patient with a height of 152 cm and a weight of 45 kg was referred by her obstetrics and gynaecology for bilateral sacral area pain caused by uteuterine sarcoma and bilateral sacral bone metastasis .
the patient complained of a spontaneous , throbbing pain with an nrs score of 8/10 , and it worsened with changes in position or movement .
total laparoscopic hysterectomy had been performed on the patient for uterine sarcoma one year before . because discomfort in the lower abdomen and symptoms of tenesmus had continued after surgery
, a ct scan of the abdomen had been performed one month after surgery . in the scan , a heterogeneously enhancing mass lesion of 14.6 7.7 cm in the pelvic cavity and
numerous lymph node enlargements in the abdominal cavity had been observed , as well as a soft mass lesion of 3.7 2.9 cm in the left pelvic bone and a lesion of 2.5 cm in the right pelvic bone ( fig .
doxorubicin and cisplatin chemotherapy had been started and further radiation therapy scheduled . at the time of referral ,
the patient was taking oxycodone pr 20 mg bid , as well as oxycodone ir 5 mg when pain increased , as prescribed by the referring department .
however , severe opioid - induced constipation developed , and the patient was refusing an increase in opioids despite the increase in pain level .
thus , scrambler therapy was planned for the patient . as the patient 's area of pain
was located on the bilateral s3 - 5dermatome , the scrambler electrodes were attached to 6 normal sensory areas to the left and right of the s3 - 5dermatome pain area for treatment .
scrambler therapy was performed 10 times for 40 minutes once every day , and oral pain medication was continually administered without a change in dosage .
from the first day of treatment , the nrs score of the affected area decreased from nrs 8/10 to 0/10 , and the nrs score at home after the procedure decreased to 2.5/10 ; this was maintained for approximately 2 weeks after treatment . during the 10 sessions of scrambler therapy , the dermatome location of pain did not change , but the painful area slowly contracted ; thus , the patient 's area of pain was consulted daily before attaching the electrodes .
the pain medications were continually administered without a change in dosage during the scrambler therapy .
one week after completion of scrambler therapy , the patient developed symptoms of diarrhea as she started radiation therapy .
the patient was re - hospitalized , as she wanted a second round of scrambler therapy .
there was no pain during the subsequent scrambler therapy , and the chronic diarrhea improved .
afterwards , the patient was transferred to another hospital near her hometown . a 56-year - old male patient with a height of 166.6 cm and a weight of 59.9 kg
was referred from the gastroenterology department for right chest pain caused by hepatocellular carcinoma and right chest fifth rib metastasis .
the degree of pain was nrs 6/10 , and it was characterized as a spontaneous splitting pain .
transarterial chemoembolization ( tace ) chemotherapy had been performed on the patient for liver cancer for one year .
five months before visiting the cancer pain unit at our hospital , metastasis to the right chest fifth rib had been discovered , and the patient had received radiation therapy . in the subsequent mri and bone scan of the thoracic vertebrae , right fifth rib metastasis with extraosseous mass formation had been observed .
the patient had the underlying diseases of hepatitis b and liver cirrhosis and , at the time of referral , had been using ultracet tab ( acetaminophen 325 mg tramadol hydrochloride 37.5 mg , janssen korea ltd , korea ) 2 tab tid for pain control .
hence , a thoracic epidural injection had been performed during hospitalization , but 3 days after the procedure , the pain had worsened , with the nrs score increasing again to 6/10 from 2/10 ; thus scrambler therapy ( mc5-a calmare ) was planned for the patient .
the patient complained of chest pain in the right fifth rib area , so the scrambler electrodes were attached to 2 normal sensory areas to the left and right of the right fifth rib pain area for treatment .
as the 10 sessions of treatment progressed , it was clear that the pain area was gradually contracting , and the location of the electrodes was adjusted accordingly for treatment .
after 2 sessions of scrambler therapy , the nrs score decreased to 2/10 , and the treatment effect continued until the next morning .
after 10 sessions of scrambler therapy , the nrs score has been maintained at 2/10 , and the pain area maintained at about 80% reduced state , for 2 months .
a 55-year - old female patient with a height of 151 cm and a weight of 55 kg was referred from the hematooncology clinic for left buttock pain caused by right infiltrative ductal breast cancer with left pelvic bone metastasis .
the degree of pain on the 1 - 10 numeric rating scale ( nrs ) was 8 , and it was characterized as a spontaneous tingling and ripping pain . she had received breast conserving surgery for right breast cancer 2 years before , but metastasis to the left pelvic bone had been discovered in an mri one year before .
chemotherapy with adriamycin , cyclophosphamide , and taxol had been performed 6 times , and radiation therapy had been performed 8 times , but there had been no reduction in tumor size .
hence , palliative chemotherapy of gemzar and navelbine had been performed 6 times , and radiation therapy had been performed 6 times . at the time of referral ,
the patient was having difficulty walking due to the pain in the pelvic area . in the mri and bone scan of the pelvic area performed before referral , bone metastasis
was observed in the left ilium , left ischium , and right acetabulum , in addition to a pathologic fracture in the left iliac wing ( fig .
the patient was administered with fentanyl patch 100 g / hr , oxycodone ir 10 mg bid , gabapentin 200 mg tid , and carbamazepine 200 mg bid for pain control ; however , she was continuously complaining of severe pain ( nrs 8/10 ) that worsened when walking . during hospitalization ,
fluoroscopically guided caudal epidural injection of 0.125% chirocaine ( levobupivacaine hydrochloride , abbott korea , korea ) 10 ml and triam ( triamcinolone acetonide , shin poong pharm , korea ) 20 mg was performed twice with a one - week interval .
however , the pain alleviation effect did not continue for more than one day after the procedure .
hence , scrambler therapy ( mc5-a calmare ) was planned . because the patient 's pain was located at the s1 - 2 dermatome of the left buttocks , the scrambler electrodes were attached at 4 normal sensory areas above and below the painful area of the s1 - 2 dermatome for treatment . during the scrambler treatment ,
the nrs score was maintained at 3 in the ward on the day of treatment .
the patient was discharged after 10 sessions of scrambler therapy . during the 10 sessions ,
the dermatome location of pain did not change , but the area of pain slowly contracted ; hence , the painful area of the patient was consulted daily before attaching the electrodes .
pain medications were continually administered without a change in dosage during the scrambler therapy . the pain alleviation effect through scrambler therapy continued for 2 months ; on the third month
the patient was re - hospitalized , and a second round of scrambler therapy was started . during treatment ,
the nrs score decreased from 7/10 to 0/10 on the first day , and this was maintained for 3 - 4 hours after treatment .
subsequently , the nrs score remained at 3.5/10 for 2 months , and pain medication was consistently given without a change in dosage during this period .
a 49-year - old female patient with a height of 152 cm and a weight of 45 kg was referred by her obstetrics and gynaecology for bilateral sacral area pain caused by uteuterine sarcoma and bilateral sacral bone metastasis .
the patient complained of a spontaneous , throbbing pain with an nrs score of 8/10 , and it worsened with changes in position or movement .
total laparoscopic hysterectomy had been performed on the patient for uterine sarcoma one year before . because discomfort in the lower abdomen and symptoms of tenesmus had continued after surgery
, a ct scan of the abdomen had been performed one month after surgery . in the scan , a heterogeneously enhancing mass lesion of 14.6 7.7 cm in the pelvic cavity and
numerous lymph node enlargements in the abdominal cavity had been observed , as well as a soft mass lesion of 3.7 2.9 cm in the left pelvic bone and a lesion of 2.5 cm in the right pelvic bone ( fig .
2 ) . doxorubicin and cisplatin chemotherapy had been started and further radiation therapy scheduled . at the time of referral ,
the patient was taking oxycodone pr 20 mg bid , as well as oxycodone ir 5 mg when pain increased , as prescribed by the referring department .
however , severe opioid - induced constipation developed , and the patient was refusing an increase in opioids despite the increase in pain level .
as the patient 's area of pain was located on the bilateral s3 - 5dermatome , the scrambler electrodes were attached to 6 normal sensory areas to the left and right of the s3 - 5dermatome pain area for treatment .
scrambler therapy was performed 10 times for 40 minutes once every day , and oral pain medication was continually administered without a change in dosage . from the first day of treatment , the nrs score of the affected area decreased from nrs 8/10 to 0/10 , and the nrs score at home after the procedure decreased to 2.5/10 ; this was maintained for approximately 2 weeks after treatment . during the 10 sessions of scrambler therapy , the dermatome location of pain did not change , but the painful area slowly contracted ; thus , the patient 's area of pain was consulted daily before attaching the electrodes .
the pain medications were continually administered without a change in dosage during the scrambler therapy .
one week after completion of scrambler therapy , the patient developed symptoms of diarrhea as she started radiation therapy .
the patient was re - hospitalized , as she wanted a second round of scrambler therapy .
there was no pain during the subsequent scrambler therapy , and the chronic diarrhea improved .
a 56-year - old male patient with a height of 166.6 cm and a weight of 59.9 kg was referred from the gastroenterology department for right chest pain caused by hepatocellular carcinoma and right chest fifth rib metastasis .
the degree of pain was nrs 6/10 , and it was characterized as a spontaneous splitting pain .
transarterial chemoembolization ( tace ) chemotherapy had been performed on the patient for liver cancer for one year .
five months before visiting the cancer pain unit at our hospital , metastasis to the right chest fifth rib had been discovered , and the patient had received radiation therapy . in the subsequent mri and bone scan of the thoracic vertebrae , right fifth rib metastasis with extraosseous mass formation had been observed .
the patient had the underlying diseases of hepatitis b and liver cirrhosis and , at the time of referral , had been using ultracet tab ( acetaminophen 325 mg tramadol hydrochloride 37.5 mg , janssen korea ltd , korea ) 2 tab tid for pain control .
hence , a thoracic epidural injection had been performed during hospitalization , but 3 days after the procedure , the pain had worsened , with the nrs score increasing again to 6/10 from 2/10 ; thus scrambler therapy ( mc5-a calmare ) was planned for the patient .
the patient complained of chest pain in the right fifth rib area , so the scrambler electrodes were attached to 2 normal sensory areas to the left and right of the right fifth rib pain area for treatment . as the 10 sessions of treatment progressed , it was clear that the pain area was gradually contracting , and the location of the electrodes was adjusted accordingly for treatment .
after 2 sessions of scrambler therapy , the nrs score decreased to 2/10 , and the treatment effect continued until the next morning .
after 10 sessions of scrambler therapy , the nrs score has been maintained at 2/10 , and the pain area maintained at about 80% reduced state , for 2 months .
in the above cases , scrambler therapy demonstrated effective treatment in patients complaining of severe cancer pain but unable to experience relief from nerve blocks or medication therapy .
the patient in case 1 had received fluoroscopically guided caudal block for pain in the left buttock caused by right breast cancer and metastasis in the left pelvic area , but the pain alleviation effect was minimal .
after scrambler therapy , however , the nrs score was reduced and maintained at 3.5/10 from 7/10 .
the patient in case 2 was suffering from bilateral sacral pain from uterine carcinoma and bilateral pelvic area metastasis , and she could not increase medication due to side effects from opioids . however , after scrambler therapy , the nrs score was reduced and maintained at 3/10 from 8/10 .
the patient in case 3 had received a thoracic epidural injection for right chest pain caused by hepatocellular carcinoma and metastasis to the right chest fifth rib , but the pain alleviation effect did not continue for more than 3 days . hence , the patient underwent scrambler therapy , and the nrs score was reduced and maintained at 2/10 from 6/10 .
the mechanism of scrambler therapy has not been clearly revealed , similar to other electrical stimulation therapies such as tens or spinal cord stimulation ( scs ) .
marineo , the developer of scrambler therapy , suggested the following mechanism : scrambler therapy provides " no - pain " information to the periphery sensory nerve receptors through attached electrode patches , and this is conveyed to the central nervous system and remembered by the system to relieve patients ' pain .
this electrical stimulus is conducted through c - fiber and a fiber , which usually convey pain , but it is not a method of simply stimulating the periphery pain nerves that cause pain .
it is also different from dulling the senses of the patient , so that he or she can still feel normal stimulation on the treated area after scrambler therapy .
the effect of scrambler therapy appears within 10 seconds of starting treatment , and pain alleviation is maintained continuously for several days or several months after completion of treatment .
the mechanism signifies that remodulation occurs in the periphery and central nervous system or the calcium channels of the synapses , which become the main target for treating neuropathic pain .
finally , the patient feels the information conveyed by scrambler electrodes by the entire dermatome where the electrodes are attached rather than only the area where the electrodes are attached ; thus , it is evident that " no - pain " information is conveyed through the dermatome . however , no research results have yet supported these findings in the present literature . procedures for scrambler therapy start from first clearly defining the pain area .
next , electrodes are attached to the areas proximal and distal to the pain area . here
, it is recommended that the electrodes are attached along the dermatome of the pain area , and they should be positioned in areas where there is no pain .
subsequently , electrical stimulus is applied and the intensity is increased gradually ; the intensity of the electrodes is set to the maximum value at which the patient does not feel discomfort . in this stage , the electrical stimulus conveys 16 types of signals similar to the action potential conveyed through the nervous system . in the initial treatment , the 16 types of action potential ,
frequency from 43 to 52 hz , duration of electrical stimulus from 0.7 to 10 seconds , and amplitude are dynamically adjusted to find the optimal " no - pain " information appropriate for the patient . through these processes ,
the patient feels his or her pain disappearing within 10 seconds of starting therapy . if there is no pain relief , the procedures
in addition , if there are areas where pain remains , more electrodes are attached to alleviate the remaining pain .
scrambler therapy is performed for 40 minutes per session , and the treatment should be performed every day if possible . to the present , there have been 4 papers published regarding the treatment effect of scrambler therapy .
the first study was conducted in 11 patients with pancreatic cancer ; as a result of scrambler therapy , 9 patients were able to discontinue drug treatment .
the second study was conducted in 226 patients complaining of non - cancerous neuropathic pain where medication had no effect ; with scrambler therapy , 80% of patients were improved ( improvement of 50% or more ) , 10% showed partial improvement ( improvement of 25 - 49% ) , and 10% had no improvement ( improvement of less than 24% or visual analogue scale ( vas ) score > 3 ) .
the third study reported the effect of scrambler therapy in 16 patients complaining of peripheral neuralgia caused by chemotherapy .
the pain completely disappeared in 4 out of the 16 patients , and 64% of the pain improved in most patients .
the authors of this study concluded that scrambler therapy is effective in pain alleviation without side effects in patients with peripheral neuralgia caused by chemotherapy .
in contrast to the above three , the most recently published paper was a randomized controlled trial with a control group .
this study compared treatment results between a drug therapy group and a scrambler therapy group in patients with chronic neuropathic pain ; one month after treatment , the nrs score in the control group decreased 28% , from 8.1/10 to 5.8/10 , while the scrambler therapy group decreased 91% , from 8/10 to 0.7/10 ( p < 0.0001 ) .
the pain scores of the control group 2 and 3 months after starting treatment were 5.7/10 and 5.9/10 , respectively , while the scrambler treatment group had scores of 1.4/10 and 2/10 ( p < 0.0001 ) .
the patients in our case study also showed a pain alleviation effect of 70% or more , which is similar to existing papers .
the patient in case 3 showed a reduction in the degree and area of pain of up to 80% .
however , the patients in cases 1 and 2 showed a recurrence of pain with the passage of time , and this is considered to be the result of continuous tissue destruction of cancerous tissue and subsequent pain stimulus .
precedent reports suggested that dosage of analgesics can be significantly reduced when there is improvement from scrambler therapy ; in our cases , however , the patients had mainly complained of local pain caused by bone metastasis of cancerous pain , mostly accompanied by intermittent pain in the cancer area .
we also considered the fact that excessive reduction of analgesics is not recommended in terminal cancer patients .
therefore , we did not promote excessive reduction of analgesics during and after scrambler treatment .
many hypotheses have been put forward to explain the treatment mechanism of scs and tens , such as supraspinal processes , modulation of descending inhibitor pathways , peripheral release of calcitonin , increased gate control for pain threshold , reduction of windup phenomenon , and reduction in impulses from damaged nerves .
additionally , scs reduces the stimulation of damaged nerves and is known to reduce psychological maladaptation for pain .
however , similar to scrambler therapy , the precise treatment mechanism has not been revealed for either of these methods .
scrambler therapy is quite similar to existing tens in that it treats pain by applying electrical stimulus from electrodes attached to the skin .
however , scrambler therapy shows differences with tens in the method of electrical stimulus and treatment effect .
first , tens positions the electrodes on the area of pain , while scrambler therapy positions the electrodes on normal sensory areas surrounding the area of pain .
second , tens has shown a limited effect in patients with postherpetic neuralgia ( phn ) , and the treatment effect also disappears within a few hours .
in contrast , scrambler therapy has shown great results in the pain treatment of phn patients .
third , tens conveys unchanging on - off biphasic electric waves , whereas scrambler therapy provides 16 nonlinear waveforms that change continuously .
in addition , when the treatment effects of scs are examined , patients with complex regional pain syndrome type i showed a vas score reduction from 10/10 to 2/10 when scs was performed , while phn patients showed a vas median value reduction from 9/10 ( interquartiles 7.75 - 10 ) to 1/10 ( interquartiles 1.0 - 2.75 ) .
in addition , when compared with existing drug therapy , scs reduces pain by 50% or more in failed back surgery syndrome patients , and the effect continued for 24 months . in this way
, scs is an effective method for treating chronic pain , but it is invasive and expensive .
there is also a high risk of complications , including one report of a 38% occurrence of hardware - related complications .
scrambler therapy has also proven to be an effective treatment method for patients complaining of various types of chronic pain and peripheral neuralgia caused by chemotherapy .
this case study has also demonstrated a good treatment effect for pain caused by bone metastasis of cancer .
however , the severity and incidence of complications can not be compared with scs , as the only complication from scrambler therapy is skin irritation in the area of electrode attachment .
ricci et al . reported the effects of scrambler therapy on 40 patients with peripheral neuralgia caused by chemotherapy and 33 non- cancerous pain patients .
two weeks after 10 sessions of scrambler therapy , a response rate of 71% was observed in the peripheral neuralgia patients ( 64% complete response where pain was reduced 50% or more , 7% partial response where pain was reduced 25 - 49% ) , and an 85% response rate was seen in non - cancerous patients ( 70% complete response , 15% partial response ) .
there were no statistical differences between the improved group and the non - improved group in factors that may decide response rate to scrambler therapy , such as nociceptive / neuropathic characteristics of pain and degree of improvement , duration of pain ( 1 - 3 months or 3 months or more ) , and characteristics of pain ( continuous pain / continuous plus breakthrough pain / intermittent pain ) .
it is necessary to research the factors that influence response to scrambler therapy through large - scale research in the future .
the authors were able to obtain a satisfactory effect by administering scrambler therapy in patients with cancerous pain caused by bone metastasis of cancer cells and in whom pain control through palliative treatment methods had proven difficult .
scrambler therapy is non - invasive , has no complications , causes minimal discomfort during treatment , and is similar or superior to other existing treatments in effect and duration .
however , there is insufficient comparative research regarding the effect of scrambler therapy for various types of pain . | more than 80% of cancer patients experience cancer pain . among them , more than 50% experience moderate to severe pain . to control cancer pain ,
a variety of methods have been used , including medications and nerve blocks . in some patients ,
however , it is impossible to perform nerve blocks due to caner metastasis into the epidural space , while in other patients , opioid dose escalation is impossible due to opioid side effects ; thus , cancer pain management is difficult .
scrambler therapy is a novel approach for pain control that uses ekg - like pads , which are applied above and below the site of pain .
scrambler therapy synthesizes 16 different types of nerve action potentials that provide " non - pain " information via cutaneous nerves .
the advantages of this treatment are that it is non - invasive and safe and has no significant side effects . in this case series ,
we report the treatment results of using scrambler therapy in three cancer patients with intractable pain . | CASE REPORT
1. Case 1
2. Case 2
3. Case 3
DISCUSSION | the patient was administered with fentanyl patch 100 g / hr , oxycodone ir 10 mg bid , gabapentin 200 mg tid , and carbamazepine 200 mg bid for pain control ; however , she was continuously complaining of severe pain ( nrs 8/10 ) that worsened when walking . because the patient 's pain was located at the s1 - 2 dermatome of the left buttocks , the scrambler electrodes were attached at 4 normal sensory areas above and below the painful area of the s1 - 2 dermatome for treatment . during the 10 sessions of scrambler therapy , the dermatome location of pain did not change , but the painful area slowly contracted ; thus , the patient 's area of pain was consulted daily before attaching the electrodes . hence , a thoracic epidural injection had been performed during hospitalization , but 3 days after the procedure , the pain had worsened , with the nrs score increasing again to 6/10 from 2/10 ; thus scrambler therapy ( mc5-a calmare ) was planned for the patient . the patient was administered with fentanyl patch 100 g / hr , oxycodone ir 10 mg bid , gabapentin 200 mg tid , and carbamazepine 200 mg bid for pain control ; however , she was continuously complaining of severe pain ( nrs 8/10 ) that worsened when walking . because the patient 's pain was located at the s1 - 2 dermatome of the left buttocks , the scrambler electrodes were attached at 4 normal sensory areas above and below the painful area of the s1 - 2 dermatome for treatment . during the 10 sessions of scrambler therapy , the dermatome location of pain did not change , but the painful area slowly contracted ; thus , the patient 's area of pain was consulted daily before attaching the electrodes . hence , a thoracic epidural injection had been performed during hospitalization , but 3 days after the procedure , the pain had worsened , with the nrs score increasing again to 6/10 from 2/10 ; thus scrambler therapy ( mc5-a calmare ) was planned for the patient . after scrambler therapy , however , the nrs score was reduced and maintained at 3.5/10 from 7/10 . marineo , the developer of scrambler therapy , suggested the following mechanism : scrambler therapy provides " no - pain " information to the periphery sensory nerve receptors through attached electrode patches , and this is conveyed to the central nervous system and remembered by the system to relieve patients ' pain . finally , the patient feels the information conveyed by scrambler electrodes by the entire dermatome where the electrodes are attached rather than only the area where the electrodes are attached ; thus , it is evident that " no - pain " information is conveyed through the dermatome . in the initial treatment , the 16 types of action potential ,
frequency from 43 to 52 hz , duration of electrical stimulus from 0.7 to 10 seconds , and amplitude are dynamically adjusted to find the optimal " no - pain " information appropriate for the patient . to the present , there have been 4 papers published regarding the treatment effect of scrambler therapy . the authors of this study concluded that scrambler therapy is effective in pain alleviation without side effects in patients with peripheral neuralgia caused by chemotherapy . this study compared treatment results between a drug therapy group and a scrambler therapy group in patients with chronic neuropathic pain ; one month after treatment , the nrs score in the control group decreased 28% , from 8.1/10 to 5.8/10 , while the scrambler therapy group decreased 91% , from 8/10 to 0.7/10 ( p < 0.0001 ) . precedent reports suggested that dosage of analgesics can be significantly reduced when there is improvement from scrambler therapy ; in our cases , however , the patients had mainly complained of local pain caused by bone metastasis of cancerous pain , mostly accompanied by intermittent pain in the cancer area . first , tens positions the electrodes on the area of pain , while scrambler therapy positions the electrodes on normal sensory areas surrounding the area of pain . in addition , when the treatment effects of scs are examined , patients with complex regional pain syndrome type i showed a vas score reduction from 10/10 to 2/10 when scs was performed , while phn patients showed a vas median value reduction from 9/10 ( interquartiles 7.75 - 10 ) to 1/10 ( interquartiles 1.0 - 2.75 ) . in this way
, scs is an effective method for treating chronic pain , but it is invasive and expensive . the authors were able to obtain a satisfactory effect by administering scrambler therapy in patients with cancerous pain caused by bone metastasis of cancer cells and in whom pain control through palliative treatment methods had proven difficult . scrambler therapy is non - invasive , has no complications , causes minimal discomfort during treatment , and is similar or superior to other existing treatments in effect and duration . however , there is insufficient comparative research regarding the effect of scrambler therapy for various types of pain . | [
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] |
candidiasis is a threat to public health as an opportunistic infection that affects patients with endocrine disorders , malignant disease , hiv / aids , and nosocomial infections .
robin , saccharomycetaceae family ) is the culprit in the majority of cases . c. albicans has a staggering morphological feature that enables it to colonize in a variety of forms ranging from unicellular budding yeast to true hyphae .
a frail alteration to the host system physiology transmutes this harmless commensal yeast into a treacherous pathogen , resulting in malaise .
therefore , it is imperative for the host defense system to alleviate the condition by returning the yeast back to its propitious commensal state .
candida strains are presently gaining resistance to antifungal agents due to their contemporaneous usage , which complicates the annihilation of the yeast .
the revertible nature of candida infestation increases the intricacy of treatment administration , and detracts from the urgency of the development of new antifungal agents .
a great number of people are accustomed to the use of these plants as they are affordable and accessible .
a quarter of all prescriptions in industrialized countries are predicted to possess one or more components derived from plants .
c. spectabilis numbers among the 600 or so species belonging to the genus cassia ; it is widely grown as an ornamental plant in tropical and subtropical areas .
the species is extensively recognized for its diverse biological and pharmacological properties , and has been commonly used by traditional healers for many years5 , 6 ; brazilian healers , for instance , have long incorporated this plant into treatments for flu and colds , and as a laxative and purgative .
pharmacological reports have also identified antifungal , antibacterial , and antioxidant activities . a recent study by torey and sasidharan enunciated the standardization of c. spectabilis leaf methanol extract with respect to authenticity , assay methods , and chemical constituent analysis .
torey et al also tested the antibiofilm activity of c. spectabilis leaf methanol extract on a c. albicans biofilm and found that it was a good antibiofilm agent .
again , sangetha et al tested and reported on the toxicity of c. spectabilis leaf methanol extract against mice .
the researchers affirmed that c. spectabilis is nontoxic and recommended it as a safe natural product for commercial utilization .
however , the mechanism that vitiates c. albicans is yet to be established even though the extract is effective against the pathogenic yeast .
this paper reports on the mechanism of action of standardized c. spectabilis leaf extract against a candidiasis - causing yeast , with the aim of providing a breakthrough for prediction of the medicinal effects of the extract so that it can be optimized for the eradication of candidiasis .
antimicrobial enhancement for clinical purposes has been focused on targeting five essential components of cellular activity : the cell wall , protein synthesis , rna synthesis , dna synthesis , and intermediary metabolism .
fresh c. spectabilis leaves were collected from the university sains malaysia ( usm ) campus at penang , malaysia in april 2009 .
a single specimen was deposited in the herbarium of the school of biological sciences , usm , pulau pinang , malaysia ( voucher number 11033 ) and was authenticated by mr shunmugam vellosamy .
the leaves were then separated and washed with water to remove dirt prior to the drying process ( 40 c for 3 days ) .
the leaf extract was prepared by macerating the dry powdered plant material in methanol ( sigma -aldrich , germany ) for 3 days .
powdered leaves with an approximation of 200 g were macerated with methanol under agitation conditions for 72 hours .
the extracted materials were then filtered with no . 1 whatman filter paper ( whatman , maidstone , uk ) and further vaporized to dryness using the rotary evaporator ( buchi rotary evaporator r110 , buchi , switzerland ) .
the cytotoxicity of the extract on vero cells ( atcc : manassas , va , usa ) was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ( sigma , usa ) ) assay .
the cell lines were grown as a monolayer culture in roswell park memorial institute ( rpmi-1640 ) medium ( gibco , invitrogen , uk ) , supplemented with 10% fetal bovine serum ( fbs , gibco , invitrogen , uk ) , 100 units / ml penicillin ( serva co , germany ) , 100 g / ml streptomycin ( merck co , germany ) , and 1 g nahco3 . the cultures were maintained at 37 c in humidified 5% co2 conditions .
the leaf extract of c. spectabilis was tested for in vitro cytotoxicity using vero cells by the mtt assay as described by mosmann with some modifications .
briefly , 100 l of media ( rpmi-1640 ) was added to the control wells of the 96-well microtiter plate .
then , 100 l of the c. spectabilis methanol extract ( 200 , 100 , 50 , 25 , 12.5 , 6.25 , 3.13 , 1.56 , and 0.78 g / ml ) was added into the respective treatment wells .
the cultured vero cells were harvested by trypsinization and pooled in a 50 ml vial .
then , 100 l of the cell extract was plated into each well ( 1 10 cells per well ) and the microtiter plate was incubated at 37 c in a humidified 5% co2 incubator for 24 hours . after the incubation period
, mtt ( 20 l of 5 mg / ml ) was added into each well and the cells were incubated for 4 hours at 37 c until a purple precipitate was clearly visible under a microscope .
then , the medium together with the mtt ( 190 l ) was aspirated from the wells , dimethyl sulfoxide ( dmso ; 100 l ) was added , and the plates were shaken for 5 minutes .
the absorbance for each well was measured at 540 nm using a microtiter plate reader ( sunrise tecan ) . the percentage cell viability ( cv )
was calculated using the following formula : cv ( % ) = average absorbance of drug wells / average absorbance of control wells 100 a dose response curve was plotted and the half maximal inhibitory concentration ( ic50 ) value was obtained with probit analysis using spss version 16.0 software ( spss inc . , chicago , il , usa ) .
an 18-hour culture of c. albicans was diluted with a sterile physiologic saline solution [ ps ; 0.85% ( w / v ) sodium chloride ] with reference to the 0.5 mcfarland standard to achieve inoculums of approximately 10 colony forming units ( cfu)/ml .
a serial dilution was carried out to give final concentrations between 1.563 mg / ml and 200.00 mg / ml of crude extract of c. spectabilis .
the tubes were inoculated with 500 l yeast suspension per ml sabouraud dextrose broth ( sdb ) ( oxoid , basingstoke , uk ) , homogenized , and incubated at 37 c . after incubation , 20 l was withdrawn from each tube , inoculated on sda agar plates , and incubated at 37 c for 24 hours .
the minimal inhibitory concentration ( mic ) value was determined as the lowest concentration of the crude extract in the broth medium that inhibited a visible growth of the test microorganism .
ion leakage kinetics was performed according to a previously reported method . briefly , c. albicans was suspended in yeast nitrogen base ( ynb ) to achieve a final concentration of 5 10 cells / ml . the mic concentration ( 6.25 mg / ml ) of the c. spectabilis leaf extract was added to the test tubes .
amphotericin b ( ampb ; 10 g / ml ) was the positive control , and methanol was used as the negative control .
samples of 10 ml were retrieved for ion concentration determination immediately after adding the extract and ampb at 15-minute intervals for a period of 60 minutes .
after centrifugation , the supernatant was used for ion determination with a perkin elmer 403 atomic absorbance spectrophotometer .
the concentration of potassium ions was determined after calibration with kcl at 383 nm .
fresh c. albicans isolates were first cultured in yeast peptone dextrose ( ypd ) broth for 14 hours at 37 c .
the culture was divided into two upon which one sample was treated with 6.25 mg / ml of the c. spectabilis leaf extract for 10 hours at 37 c while methanol was added to the other sample and used as an untreated negative control .
the isolation of cell walls from c. albicans was performed following the procedures by pitarch et al .
the cell pellet was washed in ice - cold sterile distilled water and resuspended in ice - cold lysis buffer ( 10 mm tris
. then , 0.5 mm of chilled , acid - washed glass beads ( sigma , st .
after centrifugation , the pellet was successively washed with cold water and gradual concentrations of nacl ( 5% , 2% , and 1% ) in 1 mm pmsf .
sodium dodecyl sulfate / dithiothreitol ( sds / dtt)-extractable proteins were extracted by boiling the purified cell walls in sds extraction buffer ( 50 mm tris
hcl , ph 8.0 , 0.1 m edta , 2% sds , 10 mm dtt ) at 100 c for 10 minutes .
the sample was centrifuged for 10 minutes at 1000 g and the supernatant was precipitated using trichloroacetic acid ( tca ) .
the pellet obtained from the above was used for the subsequent extraction using mild alkali conditions .
the cell walls were resuspended in ice - cold wash buffer ( 0.1 m sodium acetate , ph 5.5 , 1 mm pmsf ) , centrifuged for 10 minutes at 1000 g , and the supernatant was discarded .
the cell walls were then resuspended in ice - cold extraction solution ( 30 mm naoh , 1 mm pmsf ) and the cell wall suspension was incubated at 4 c for 17 hours .
the suspension was centrifuged for 10 minutes at 1000 g and the supernatant was collected and precipitated using tca .
tca ( 20% final concentration ) was added to the supernatant obtained from the procedures above and the samples were incubated for 30 minutes on ice . the suspension was then centrifuged at 10,000 g for 15 minutes and the supernatant was discarded .
the protein pellet was washed twice with cold acetone to remove residual tca and the pellet was air - dried and stored at 80 c until further use .
samples were taken up in sds loading solution and 20 l was loaded onto 1020% biorad criterion gel .
the gel was run for 160 minutes at a voltage of 140 v. the gel was stained using the colloidal coomassie brilliant blue g-250 .
all sds - polyacrylamide gel electrophoresis ( sds - page ) dyes , reagents , and equipment were from bio - rad ( hercules , ca , usa ) .
the -glucosidase activity was measured using p - nitrophenyl--d - glucopyranoside ( sigma , germany ) as a substrate based on a previously described method with some modifications .
intact organisms of 4 10 cells in 1.1 ml pbs were incubated with 6.25 mg / ml of the c. spectabilis extract and 5 mm substrate in 1.0 ml of 0.1 m phosphate - citrate buffer ( ph 6.0 ) for 2 hours at 37 c .
a negative control containing methanol was used for comparison . the reaction was terminated by the addition of 2 ml of ice - cold 0.1 m na2co3 .
the p - nitrophenol that was released in the supernatant was then read at an optical density ( od ) of 405 nm .
the preparation of the standard curve was done by preparing p - nitrophenol with concentrations of 140 , 70 , 35 , 17.5 , 8.75 , and 0 m in test tubes .
a standard curve was plotted using the p - nitrophenol standard and the amount of -glucosidase released was calculated using the following formula:-glucosidaseactivity in the sample(mu / ml)=1slopeod405totalvolumetimesamplevolume the culture conditions and treatment with plant extract were carried out according to a previously reported method .
yyg medium ( 15 ml ) ( 1.17% yeast carbon base , 0.01% yeast extract , 0.27% glucose ) containing 6.25
mg / ml of the c. spectabilis leaf extract was inoculated with 106 cfu of c. albicans and incubated at 37 c for 48 hours while shaking at 60 rpm .
the proteinase assay was undertaken using a modification of the previous method by adding 0.2 ml of the supernatant to 2 ml of 0.1 m citrate buffer ( ph 3.2 ) containing 0.2% bovine serum albumin ( bsa ) and incubating at 37 c for 2 hours .
the mixture was centrifuged at 5000 rpm for 30 minutes and the supernatant was read at an absorbance of 750 nm on a spectrophotometer .
the c. albicans cells were treated with 6.25 mg / ml ( the mic obtained from our previous study ) of the c. spectabilis leaf extract for 36 hours .
after treatment with c. spectabilis extract , scanning electron microscopy ( sem ) observation was carried out on c. albicans biofilm .
the plate containing 25 ml potato dextrose agar medium was seeded with 1 ml of the c. albicans suspension containing 10 cells per ml from a 24-hour - old culture .
the extract ( 1 ml ) , at the concentration of 6.25 mg / ml , was then dropped onto the inoculated agar and was further incubated for another 36 hours at 37 c .
segments ( 510 mm ) were cut from cultures growing on potato dextrose plates at 36 hours for sem examination .
subsequently , the planchette was placed on a petri plate . in a fume hood , a vial cap containing 2% osmium tetroxide in water was placed in an unoccupied quadrant of the plate . after being covered , the plate was sealed with parafilm , and vapor fixation of the sample proceeded for 1 hour .
once the sample was vapor - fixed , the planchette was plunged into slush nitrogen ( 210 c ) and transferred on to the peltier - cooled stage of the freeze dryer ( emitech k750 ) , and freeze drying of the specimen proceeded for 10 hours . finally , the freeze - dried specimen was sputter - coated with 510 nm of gold before viewing in the sem ( fesem leo supra 50 vp ; carl zeiss , germany ) operating at 5 kv at 5.00 k of magnification .
1a the cells appear as a monolayer of elongated cells with nuclei positioned in the middle and attached to the surface of the microtiter plate .
1b shows c. spectabilis - treated ( 50 g / ml ) vero cells this concentration correlates with the ic50 concentration acquired earlier , hence elucidating the damage in 50% of the vero cells , which is depicted with shriveled and detached characteristics on the microtiter plate surface .
1c shows vero cells treated with 200 g / ml of the c. spectabilis leaf extract ; no viable cells are observed here .
the broth dilution method recorded the mic value of 6.25 mg / ml against the c. albicans test strain .
potassium ( k ) ion leakage was estimated using atomic absorption spectroscopy ( aas ) and the resulting patterns are presented in fig .
it was found that the methanol extract of c. spectabilis was effective in leakage of k ions by releasing 1039 ppm whereas ampb ( positive control ) released comparatively higher ions than the extract ( 1115 ppm ) .
the k ions were instantly detected within the culture supernatant upon extract treatment initiation , to which progressive increments were observed peaking at 30 minutes of exposure , after which they declined . by contrast , c. albicans cells treated with ampb managed to achieve their peak after 15 minutes .
untreated c. albicans cells served as a negative control whereby the cells had small traces of k ions in the culture supernatant .
this result illustrates that k ions are leaked from the cells within 60 minutes of interaction between the organism and the extract .
there were two extraction methods : sds / dtt and naoh extractions . on extraction with sds / dtt , the c. albicans cell walls expressed two new proteins ( red arrows ) .
one of these proteins was measured to be in the range of 3750 kda in molecular weight whereas the other protein was between 20 kda and 25 kda in molecular weight . as for the extraction using naoh
, three major proteins were discovered to be absent in the cell walls of treated c. albicans compared to the untreated ones ( white arrows ) . when compared to the untreated sample ,
two of these absent proteins were identified to be within the range 3750 kda in molecular weight while the final one was estimated to be in the range 5075 kda in molecular weight .
a thinner and fainter protein band in the treated sample ( black arrow ) indicated lower protein expression this was observed with both sds / ddt and naoh extraction methods .
the low - expressed proteins were noted to be about 10 kda in the sds / dtt extraction sample and around 37 kda in the naoh extraction sample .
4 depicts the -glucosidase activity of the c. albicans cells treated with 6.25 mg / ml of the leaf extract .
it was found that the cells treated with the extract possessed a significantly higher -glucosidase activity compared to the untreated control .
the activity of the treated cells was 0.432 0.003 mu / ml , while the activity of the untreated cells was 0.048 0.001 mu / ml ( p < 0.05 ) .
it was observed that the cells treated with the c. spectabilis leaf extract ( 6.25 mg / ml ) exhibited a significantly higher proteinase activity compared to the control .
the absorbance value ( at 750 nm ) of the supernatant for the treated cells was 0.033 0.001 , while the untreated cells gave an absorbance value of 0.025 0.001 ( p < 0.05 ) .
the sem photomicrographs of the untreated and c. spectabilis - treated cells of c. albicans at 36 hours are displayed in fig .
6a depicts the untreated c. albicans cells that were used as a negative control ; the presence of biofilm that connects the cells together can be seen along with budding cells . by contrast , the treated cells appear to be loosened from each other indicating a deteriorated biofilm ( fig .
6b ) ; the cells seemed to develop small ruptures on the cell surface actuating in cell convulsion ; the treated cells were more elongated compared to the untreated cells .
in our study , a few characteristics were disclosed ascertaining the possible patterns by which c. spectabilis leaf extract may act upon c. albicans . fig .
the activity of the extract suggests a promising mechanism for altering cell wall and cell membrane components , inhibiting dna , and affecting protein synthesis and metabolism of c. albicans .
an extract possessing distinctive active compounds is associated with multiple mechanisms of action , each ascribable to a specialized compound or group of compounds .
the rigid cell wall of c. albicans is composed mainly of glucans and chitins offering the notion that some compounds might be responsible for the initial step of cell wall penetration , before allowing the rest of the crude extract compounds to access further into the cell causing more damage . the complete procedure for discovering and developing a new therapeutic agent is highly expensive and tedious , often stretching over a 12-year period . identifying
the ic50 value obtained when vero cell lines were treated with c. spectabilis extract was 59.10 g / ml ; this value was considered nontoxic because it was more than 20 g / ml . a previous study conducted by sangetha et al on the same leaf extract using mice oral acute toxicity tests further verifies the nature of c. spectabilis leaf extract as nontoxic .
toxic effects of an antimicrobial agent should be pondered deliberately especially when concerning host cells .
nevertheless , it was surprising that the toxic effect of c. spectabilis leaf extract was exhibited on c. albicans rather than on vero cells , suggesting that the extract displayed selective toxicity towards yeast cells .
although there are many commercial drugs readily available to treat candidiasis , c. spectabilis fortuitously reveals its competence in inhibiting c. albicans growth ( fig .
it can be corroborated that c. spectabilis leaf extract is a potent in vitro anticandidal agent .
cell culture can be used to screen for toxicity both by estimation of the basal functions of the cell ( i.e. , those processes common to all types of cells ) or by tests on specialized cell functions .
general toxicity tests , aimed mainly at detection of the biological activity of test substances , can be carried out on many cell types ( e.g. , fibroblasts , hela cells , and hepatoma cells ) .
a number of parameters including vital staining , cytosolic enzyme release , cell growth , and cloning efficiency are used as end - points to measure toxicity .
hence , in this study vero cells from a monkey were used for the cytotoxicity assay .
the osmotic balance of a cell can be easily affected by the changes in potassium ions ( k ) , and ion leakage from a cell may result in cell lysis .
these ions play crucial roles in activating essential enzymes that function as organic catalysts mediating prominent biochemical reactions .
c. albicans cells treated with c. spectabilis were evaluated for k leakage and the efficacy
ampb binds to ergosterol causing the formation of aqueous channels that increase membrane permeability to univalent cations resulting in cell death . a gradual leakage of k ions by the treated c. albicans cells was shown here ( fig .
2 ) corresponding to the time of treatment exposure ; this result was as good as the effect of the commercial antifungal drug ampb on k leakage in c. albicans .
the leaf extract pattern coined a perception that the extract had altered the membrane of the c. albicans intensifying its permeability to release k ions .
the fungal plasma membrane is constituted predominantly by phospholipids , sphingolipids , and sterols that together serve as a permeability barrier concurrent to the transport of small molecules . among other things
, these components also transmit signals and serve as a matrix for proteins with various functions.29 , 30 therefore , when this membrane is compromised , it will cause an efflux of intracellular potassium .
cox et al studied the influence of tea tree oil on c. albicans and drew the conclusion that leakage of k ions was an indication of cell membrane structure damage .
similarly , wei et al performed an analysis of k leakage to investigate plasma membrane injury in c. albicans and asserted that the outflow of k ions was associated with yeast cell membrane permeability .
this result can also be related to other antifungal mechanisms such as binding of the drug to sterols to increase permeability , inhibition of ergosterol synthesis , or even dysfunctional membranal enzymes .
other methods can also be used to assess the membrane damage caused by antifungal agents , for example , assessment of atp leakage by fungal cells was employed by kulakovskaya et al . according to nagarsekar et al , cytoplasmic membrane damage can be evidenced by leakage of cellular k ions .
hence , it is concluded that the c. spectabilis leaf extract caused membrane damage in the c. albicans cells .
proteomic analysis of c. albicans cell wall proteins was performed to substantiate the association of k ion leakage with cell membrane damage .
the c. albicans cell wall is very significant in terms of biology and pathogenicity .
the mechanical and osmotic balance of the yeast is achieved by its cell wall , which is constructed mainly of carbohydrates with a basic network complex of -glucans , chitin , and mannan.37 , 38 , 39 , 40 the yeast cell wall forms a definite boundary in providing mechanical stability and cell tugor , in addition to its role in the construction of the outer fungal structural surface .
the cell wall , being the first point of contact when subjected to the c. spectabilis leaf extract , pointedly provokes interruption in the organization or functions of the cell wall components .
these alterations within the cell wall organization are inspected at the molecular level through its cell wall protein expression .
considering the fact that proteins are the ultimate gene products and representatives of various physiological and pathological processes of the living cell , the field of proteomics has made allowances for the influence of different conditions on the expression of proteins.41 , 42 for this reason , the cell wall protein expression was investigated to ratify the alteration involving the cell wall constituents caused by the c. spectabilis leaf extract .
different biochemical approaches have been used to identify proteins that are randomly scattered along the cell wall .
the noncovalently attached proteins are extracted using sds / ddt while covalently bound proteins are extracted by naoh treatment ( mild alkali treatment ) .
the presence of new proteins can be proposed as the action of c. spectabilis leaf extract in disintegrating cell wall moieties into smaller peptides .
low protein expression levels may result from the possible disintegration of proteins into smaller peptides making them imperceptible between the bounds of the standard molecular mass weights .
mahmoud and aly reported the differences in cell wall protein expression using sds - page ; they uncovered an increase in phosphopeptidomannan of molecular weights 45 kda , 25 kda , and 15 kda when c. albicans was treated with a polymer .
other conditions are also capable of modifying the composition of the cell wall . for example , an elevated level of candida pir2-related protein was found to be a reason for vulnerability of c. albicans .
then again , the featured changes in cell wall proteins solidly attest the changes in protein expression .
c. albicans is well advanced in secreting an assortment of hydrolytic enzymes such as -glucosidase and proteinase .
it causes destruction to the host cells when it degrades healthy tissues to supply its nutrient needs .
the enzymes -glucosidase and proteinase are culpable in the virulence of c. albicans and were found to be significantly higher in treated c. albicans cells in contrast with untreated control cells .
the enzyme activity was compared between a fluconazole - resistant c. albicans strain and a fluconazole - sensitive strain and they found that -glucosidase plays an insignificant role in the virulence of c. albicans .
the proteinase activity of c. albicans cells is divulged generally as a repercussion of virulence .
some time ago , it was reported that a c. albicans culture filtrate manifested many types of hydrolytic enzymes including proteinases , phospholipases , acid phosphatases , chitinases , esterases , and glucoamylases .
given that c. spectabilis had earlier demonstrated antibiofilm activities towards c. albicans , the feeble yeast cells of cutler s study can be used to rationalize the high proteinase activities .
cassone et al discovered the presence of proteinase in pathogenic strains isolated from cases of candidal vaginitis and those of carriers .
schreiber et al did not find a correlation between the volume of proteinase generated by various clinical and normal c. albicans strains with the invasion level of the strains .
more to the point , another study found similar production of proteinase in a low - virulence mutant of c. albicans and virulent parent strains .
the increase in proteinase activity alone is not sufficient to be considered a virulence factor as dubois et al revealed that a systemic infection - causing mutant c. albicans in mice transformed from being virulent to avirulent when proteinase was overexpressed .
the authors believe that the overexpressed proteinase might have resulted from compromised yeast cell walls , otherwise the mice host immune system would not been able to eliminate these c. albicans unless they were susceptible . in general terms
, the high -glucosidase and proteolytic activity of c. albicans is an indication of altered metabolism caused by the c. spectabilis leaf extract . c. albicans cells were exposed to the c. spectabilis leaf extract for 36 hours , and the morphological alterations were viewed through sem along with other untreated cells .
the cells also appeared to be detached from each other elucidating certain disturbance in the biofilm formation .
one of the major contributors to candidiasis occurring at an alarming rate is the propensity of c. albicans for biofilm formation .
the biofilm forms when single cells attach to a surface and grow into microcolonies , followed by blended production of a complex three - dimensional ( 3d ) structure held by hyphae and exopolymer matrix .
the biofilm acts as a barrier , preventing penetration by immune system components or even antifungal agents.51 , 52 , 53 critically , sem revealed the after - effect of the c. spectabilis leaf extract on c. albicans biofilm construction .
the destruction of the biofilm that occurs in treated cells facilitates the host immune system to eliminate the pathogen , thereby eradicating candidiasis .
taken together , our findings demonstrated that c. spectabilis leaf extract is nontoxic to vero cells and is effective against c. albicans biofilms .
future studies will be necessary to identify the protein changes that occur within the cell wall during treatment , as well as the isolation and the characterization of active compound(s ) with antibiofilm activity . in conclusion , c. spectabilis leaf extract has strong anticandidal activity with various mechanisms of action . | candida albicans has become resistant to the commercially available , toxic , and expensive anti - candida agents that are on the market .
these factors force the search for new antifungal agents from natural resources .
cassia spectabilis had been traditionally employed by healers for many generations .
the possible mechanisms of the c. spectabilis leaf extract were determined by potassium leakage study and the effect of the extract on the constituents of the cell wall and enzymes as well as the morphological changes on c. albicans cells were studied along with cytotoxicity assays .
the cytotoxicity result indicated that the extract is nontoxic as was clearly substantiated by a half maximal inhibitory concentration ( ic50 ) value of 59.10 g / ml .
the treated cells ( c. spectabilis extract ) demonstrated potassium leakage of 1039 parts per million ( ppm ) compared to amphotericin b ( ampb)-treated cells with a released potassium value of 1115 ppm .
the effects of the extract on the cell wall proteins illustrated that there were three major types of variations in the expression of treated cell wall proteins : the presence of new proteins , the absence of proteins , and the amount of expressed protein .
the activities of two enzymes , -glucosidase and proteinase , were determined to be significantly high , thereby not fully coinciding with the properties of the antifungal reaction triggered by c. spectabilis .
the morphology of c. albicans cells treated with the c. spectabilis extract showed that the cells had abnormalities and were damaged or detached within the microcolonies .
our study verifies c. spectabilis leaf extract as an effective anti - c .
albicans agent . | Introduction
Materials and methods
Results
Discussion
Conclusion
Conflicts of interest | this paper reports on the mechanism of action of standardized c. spectabilis leaf extract against a candidiasis - causing yeast , with the aim of providing a breakthrough for prediction of the medicinal effects of the extract so that it can be optimized for the eradication of candidiasis . the percentage cell viability ( cv )
was calculated using the following formula : cv ( % ) = average absorbance of drug wells / average absorbance of control wells 100 a dose response curve was plotted and the half maximal inhibitory concentration ( ic50 ) value was obtained with probit analysis using spss version 16.0 software ( spss inc . the mic concentration ( 6.25 mg / ml ) of the c. spectabilis leaf extract was added to the test tubes . amphotericin b ( ampb ; 10 g / ml ) was the positive control , and methanol was used as the negative control . the culture was divided into two upon which one sample was treated with 6.25 mg / ml of the c. spectabilis leaf extract for 10 hours at 37 c while methanol was added to the other sample and used as an untreated negative control . yyg medium ( 15 ml ) ( 1.17% yeast carbon base , 0.01% yeast extract , 0.27% glucose ) containing 6.25
mg / ml of the c. spectabilis leaf extract was inoculated with 106 cfu of c. albicans and incubated at 37 c for 48 hours while shaking at 60 rpm . the c. albicans cells were treated with 6.25 mg / ml ( the mic obtained from our previous study ) of the c. spectabilis leaf extract for 36 hours . 1c shows vero cells treated with 200 g / ml of the c. spectabilis leaf extract ; no viable cells are observed here . as for the extraction using naoh
, three major proteins were discovered to be absent in the cell walls of treated c. albicans compared to the untreated ones ( white arrows ) . 4 depicts the -glucosidase activity of the c. albicans cells treated with 6.25 mg / ml of the leaf extract . it was found that the cells treated with the extract possessed a significantly higher -glucosidase activity compared to the untreated control . it was observed that the cells treated with the c. spectabilis leaf extract ( 6.25 mg / ml ) exhibited a significantly higher proteinase activity compared to the control . 6b ) ; the cells seemed to develop small ruptures on the cell surface actuating in cell convulsion ; the treated cells were more elongated compared to the untreated cells . nevertheless , it was surprising that the toxic effect of c. spectabilis leaf extract was exhibited on c. albicans rather than on vero cells , suggesting that the extract displayed selective toxicity towards yeast cells . hence , it is concluded that the c. spectabilis leaf extract caused membrane damage in the c. albicans cells . the cell wall , being the first point of contact when subjected to the c. spectabilis leaf extract , pointedly provokes interruption in the organization or functions of the cell wall components . considering the fact that proteins are the ultimate gene products and representatives of various physiological and pathological processes of the living cell , the field of proteomics has made allowances for the influence of different conditions on the expression of proteins.41 , 42 for this reason , the cell wall protein expression was investigated to ratify the alteration involving the cell wall constituents caused by the c. spectabilis leaf extract . the presence of new proteins can be proposed as the action of c. spectabilis leaf extract in disintegrating cell wall moieties into smaller peptides . the enzymes -glucosidase and proteinase are culpable in the virulence of c. albicans and were found to be significantly higher in treated c. albicans cells in contrast with untreated control cells . in general terms
, the high -glucosidase and proteolytic activity of c. albicans is an indication of altered metabolism caused by the c. spectabilis leaf extract . c. albicans cells were exposed to the c. spectabilis leaf extract for 36 hours , and the morphological alterations were viewed through sem along with other untreated cells . the biofilm acts as a barrier , preventing penetration by immune system components or even antifungal agents.51 , 52 , 53 critically , sem revealed the after - effect of the c. spectabilis leaf extract on c. albicans biofilm construction . future studies will be necessary to identify the protein changes that occur within the cell wall during treatment , as well as the isolation and the characterization of active compound(s ) with antibiofilm activity . | [
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] |
the estimated incidence of mps i is 1 in every 100,000 live births . due to the chronic and progressive accumulation of glycosaminoglycans in the lysosomes of cells throughout the body
, patients affected by this devastating disease experience multiorgan dysfunction leading to considerable morbidity in most patients , and early mortality in those most severely affected . like most other metabolic inherited diseases ,
. this may be due to differences among patients in the severity of the underlying mutation(s ) and consequent degree of residual enzyme activity ; however , other factors may also contribute to the well - known phenotypic heterogeneity . historically , mps i has been classified into three syndromes hurler , hurler scheie , and scheie though it is now widely accepted that overlap in symptomatology exists among these subtypes .
hurler syndrome , the most severe form of mps i , typically involves significant developmental delay and cognitive decline , along with characteristic coarse facial features , joint stiffness and contractures , short stature , and respiratory , cardiac , and hepatic disease .
symptoms emerge shortly after birth and progress rapidly , such that most patients with hurler syndrome die within the first decade of life . at the other end of the mps i disease spectrum ,
while patients with the scheie phenotype usually develop significant disease - related morbidity , they show normal intelligence and survive into adulthood .
scheie syndrome represents an intermediate phenotype that is characterized by mild or no cognitive impairment but includes somatic symptoms that reduce life expectancy into the second or third decade of life .
delineation of the different mps i phenotypes can be challenging and is largely driven by consideration of the age of symptom onset and rate of disease progression as well as a patient 's genotype .
historically , treatment of mps i was restricted to palliative care and symptom - based interventions , including surgery ( e.g. , adenotonsillectomy , hernia repair , ventriculoperitoneal shunt , cardiac valve replacement , carpal tunnel release , and spinal decompression ) ; physical , occupational , and speech therapies ; respiratory support ; hearing aids ; and medications for pain and gastrointestinal disturbances . since 1981 , hematopoietic stem cell transplantation ( hsct ) has been used to treat mps i. when successful , it is a one - time procedure that can prolong survival , preserve cognitive function , and reduce some somatic features of the disease . however , due to its significant morbidity , hsct is reserved for patients with the most severe form of mps i , hurler syndrome .
hsct is typically recommended for patients under 2 years of age with normal cognition ( intelligence quotient > 70 ) because early intervention increases the likelihood of maintaining cognitive abilities . for patients with hurler
scheie and scheie syndromes , enzyme replacement therapy ( ert ) with laronidase ( recombinant human -l - iduronidase ; aldurazyme ) is the primary treatment option .
developmental outcomes are better when transplantation is performed before 24 months of age , and laronidase may also be more beneficial when started early in life , based on the suggestion from two case reports on siblings with hurler scheie syndrome .
unfortunately , early diagnosis ( and hence early initiation of treatment ) is limited by several factors , including the rarity of the disease , the wide variability in clinical presentation and disease course , and the nonspecific nature of some of the early manifestations of the disease . as is true of other rare genetic diseases , mps i awareness among physicians is also hampered by a general lack of familiarity with its symptoms and course . with the advent of several potentially lifesaving therapies ,
the mps i registry ( clinicaltrials.gov identifier : nct00144794 ) is a global , observational disease database established in 2003 by genzyme / biomarin and is maintained by genzyme , a sanofi company .
the collection of data in the registry is aimed at facilitating characterization of the course of disease and tracking clinical outcomes in patients with mps i. in addition , participating health - care professionals are provided access to aggregate data on mps i and can query the database for specific information to facilitate the care of their patients with mps i. analyses of data from the registry have provided insights about disease burden , genotype phenotype correlations , frequency and risks of surgical procedures , and changing treatment trends in mps i patients .
the intent of this analysis is to use data from the registry to describe the natural history of mps i by phenotype and across regions in an effort to improve the recognition and prompt diagnosis of this potentially fatal , but treatable , disease .
the global mps i registry was initiated in april 2003 as part of an effort to help health - care professionals involved in the diagnosis and treatment of mps i better understand the disease and its management and to help create mps i disease treatment monitoring guidelines .
each independent site is responsible for obtaining a patient 's informed consent for submitting his / her health information to the registry and for using and disclosing this information in subsequent aggregate analyses , such as journal articles , annual reports , educational materials , and public health reports .
historically , each independent site has been responsible for determining whether site - specific institutional review board or ethics committee review is required for participation in the registry in accordance with institutional policies and local laws and regulations . since december 2012
, the mps i registry protocol has required sites to submit to and receive approval from an institutional review board or ethics committee .
all patient data are anonymized and entered by participating sites in accordance with applicable privacy regulations . at enrollment ,
detailed medical histories and baseline data are collected and physicians are encouraged to follow a recommended schedule of clinical assessments and regularly report the results of their assessments . this report is based on registry data as of august 2013 , at which time information was available for 1,046 patients .
the registry includes comprehensive information for treated as well as untreated patients ; for the treated patients , data are available from the periods prior to , during , and/or subsequent to treatment .
therefore , a definition of the natural history period and the following patient inclusion criteria were used to select data for the analyses presented here : ( i ) known ert status , ( ii ) for patients receiving ert , known date of first infusion , ( iii ) known hsct status , and ( iv ) for patients who had received hsct , known date of transplant . from the initial cohort of 1,046 patients , 987 patients met the inclusion criteria . for patients who had never been treated ,
the natural history period was defined as the time from birth to the last follow - up recorded in the registry . among patients who had been treated with ert , hsct , or both ,
the natural history period was defined as the time from birth to first treatment ( either ert or hsct ) .
ages at first symptom , diagnosis , and treatment initiation ( where applicable ) were evaluated by region and mps i phenotype .
symptom prevalence and age of onset of symptoms relating to general appearance and neurologic , cardiac , respiratory , gastrointestinal , and musculoskeletal status were evaluated by phenotype .
only the symptoms observed during the predefined natural history period were considered . given that not all patients had data reported for every symptom during the natural history period , the assumption was made that symptoms not explicitly reported were not present , and frequencies were calculated as the number of patients with the symptom present divided by all patients in each region and/or phenotype .
this assumption means that the reported frequencies may underestimate the true frequency for each symptom .
descriptive statistics were computed for age of onset of each symptom ( as indicated by the earliest age each symptom was reported ) among patients who experienced the symptom . for those patients with
the same symptom reported multiple times , the earliest age was used , corresponding to onset or age first reported .
a total of 987 mps i patients with evaluable natural history information were enrolled in the registry as of august 2013 ( table 1 ) .
the largest proportion of patients was from europe ( 45.5% ) , with the next largest from north america ( 34.8% ) , followed by latin america ( 17.3% ) and asia pacific ( 2.4% ) .
the overall phenotypic distribution was 601 ( 60.9% ) for hurler , 227 ( 23.0% ) for hurler scheie , and 127 ( 12.9% ) for scheie .
another 32 ( 3.2% ) patients met the criteria for inclusion in this analysis , but their phenotypes were not reported in the registry and were therefore considered undetermined or missing . of note , north america ( 71.4% ) and europe ( 61.5% ) had higher proportions of patients with the severe hurler phenotype than latin america ( 42.7% ) or the asia pacific region ( 29.2% ) .
median ages at symptom onset , mps i diagnosis , and first treatment ( ert or hsct , if applicable ) are presented by region and phenotype in figure 1 . across all regions , patients with hurler syndrome , the most severe phenotype ,
the median age at symptom onset for these patients was 6 months , and diagnosis and treatment initiation followed quickly thereafter , at median ages of 12 and 18 months , respectively .
patients with hurler scheie and scheie syndromes , which have the more attenuated presentations , typically experienced initial symptoms sometime after infancy and exhibited a 2- to 4-year gap between onset of symptoms and diagnosis , respectively , and a 4- to 8-year gap between diagnosis and treatment initiation , respectively .
analyses of age of symptom onset , diagnosis , and treatment initiation based on phenotype and geographic region generally mirrored the results in the overall registry population , with a few notable exceptions . in north america , treatment initiation for patients with scheie syndrome was earlier than in the other regions . the median age for treatment initiation for patients diagnosed with scheie syndrome in north america was 11.7 years , whereas in europe and latin america it was 16.9 and 17.7 years , respectively .
the asia pacific region had an even higher median age of 31.5 years for treatment initiation .
scheie was also higher in the asia pacific region ( 23.8 years ) compared with the overall registry population ( 8 years ) .
another notable finding was the rate of patients with an undetermined or missing phenotype in latin america ( 11.1% ) , which was much higher compared with the other regions .
the median ages of symptom onset , diagnosis , and treatment initiation for patients in latin america with an undetermined phenotype were 0.7 , 1.3 , and 3.3 years , respectively , which is highly comparable to the median ages for patients in latin america with the hurler phenotype ( 0.7 , 1.7 , and 3.6 years , respectively ) , suggesting that some portion of the patients with undetermined phenotypes may , in fact , have hurler syndrome .
coarse facial features were the most predominant characteristic of all functional and anatomic abnormalities for both the hurler and the hurler
the symptoms occurring in at least 25% of patients are summarized by phenotype in figure 2 , and all symptoms , including those occurring in less than 25% of patients , are summarized by phenotype in supplementary table s1 online . among patients with the scheie phenotype , 48.0% presented with coarse facial features .
corneal clouding was noted in all three phenotypes at approximately the same rate : 70.9 , 68.3 , and 70.1% for hurler , hurler
hepatomegaly was present in the majority of patients with hurler ( 70.0% ) and hurler scheie ( 66.5% ) phenotypes and in approximately half ( 48.0% ) of those with the scheie phenotype .
similarly , splenomegaly was present in 50.9% of patients with hurler syndrome and 47.1% of patients with hurler scheie syndrome but in only 27.6% of those with the scheie phenotype .
the incidences of hernias were more evenly distributed among the phenotypic groups , with 58.9% in hurler , 59.9% in hurler
kyphosis / gibbus was the only abnormality present in the majority ( 70.0% ) of patients with hurler phenotype , and it was reported much less frequently in patients with hurler scheie ( 33.5% ) and scheie ( 21.3% ) phenotypes .
conversely , joint contractures and carpal tunnel syndrome were present in the majority of scheie patients ( 69.3 and 51.2% , respectively ) but were less common in hurler scheie patients ( 57.3 and 27.8% , respectively ) and even less frequent in hurler patients ( 37.9 and 7.8% , respectively ) .
cardiac valve abnormalities were observed in 67.7% of scheie patients , 59.0% of hurler scheie patients , and 48.9% of hurler patients .
airway - related symptoms , such as sleep disturbances / snoring were observed in 51.6% of hurler patients , 48.9% of hurler scheie patients , and 26.8% of scheie patients .
cognitive impairment was observed in 46.4 , 31.3 , and 9.4% of patients with hurler , hurler
as expected , first symptoms appeared earlier ( within the first 2 years of life ) in patients with the hurler phenotype ; whereas , in patients with the hurler scheie and scheie phenotypes , commonly occurring symptoms were first observed between 3 and 7 , and 5 and 13 years of age , respectively .
the median ages of onset of symptoms occurring in at least 25% of patients are presented by phenotype in figure 2 , and the median ages of onset of all symptoms , including those occurring in less than 25% of patients , are summarized by phenotype in supplementary table s2 online . across all phenotypes ,
hernias were the earliest reported symptom , which was noted at median ages of 0.8 , 3.2 , and 4.6 years of age in hurler , hurler
coarse facial features , the most prevalent symptom in patients with hurler and hurler scheie phenotypes , were also an early finding , with a median age at onset of 0.9 years in hurler patients and 3.4 and 8.7 years in patients with hurler scheie and scheie phenotypes , respectively .
corneal clouding , the most prevalent symptom in patients with the scheie phenotype , was first noted at a median age of 10.5 years in that subset of patients . among patients with the hurler phenotype
, kyphosis / gibbus had a median age at onset of 1.0 year ( figure 2a ) , and dysostosis multiplex , corneal clouding , and hepatomegaly had a median age at onset of 1.1 years .
several symptoms , including sleep disturbances / snoring , enlarged tongue , cognitive impairment , and splenomegaly had a median age at onset of 1.2 years , whereas cardiac valve abnormalities were noted at a median age of 1.3 years .
the symptoms that appeared latest in at least 25% of patients with hurler syndrome were enlarged tonsils and joint contractures , with a median age at onset of 1.5 and 1.6 years , respectively .
after hernias and coarse facial features , cognitive impairment was the earliest symptom observed , first occurring at a median age of 3.8 years .
the median ages of onset for enlarged tongue , sleep disturbances / snoring , enlarged tonsils , joint contractures , and dysostosis multiplex were between 4.0 and 4.2 years . both corneal clouding and hepatomegaly had a median age at onset of 4.4 years , whereas splenomegaly and kyphosis / gibbus had a median age at onset of 4.6 years .
scheie phenotype included cardiac valve abnormalities , hip dysplasia , and carpal tunnel syndrome , with median ages at onset of 5.7 , 6.2 , and 7.4 years , respectively .
symptoms reported in at least 25% of patients with scheie phenotype are shown in figure 2c .
after hernias , the earliest symptoms within this phenotype were joint contractures and dysostosis multiplex , with median ages at onset of 7.6 and 8.0 years , respectively .
hip dysplasia was first reported at a median age of 8.4 years . both sleep disturbances / snoring and coarse facial features
had a median age at onset of 8.7 years , and hepatomegaly was observed at a median age of 9.4 years .
symptoms appearing later included corneal clouding at 10.5 years , splenomegaly at 11.0 years , cardiac valve abnormalities at 11.7 years , and carpal tunnel syndrome at 12.5 years .
a total of 987 mps i patients with evaluable natural history information were enrolled in the registry as of august 2013 ( table 1 ) .
the largest proportion of patients was from europe ( 45.5% ) , with the next largest from north america ( 34.8% ) , followed by latin america ( 17.3% ) and asia pacific ( 2.4% ) .
the overall phenotypic distribution was 601 ( 60.9% ) for hurler , 227 ( 23.0% ) for hurler scheie , and 127 ( 12.9% ) for scheie .
another 32 ( 3.2% ) patients met the criteria for inclusion in this analysis , but their phenotypes were not reported in the registry and were therefore considered undetermined or missing . of note , north america ( 71.4% ) and europe ( 61.5% ) had higher proportions of patients with the severe hurler phenotype than latin america ( 42.7% ) or the asia pacific region ( 29.2% ) .
median ages at symptom onset , mps i diagnosis , and first treatment ( ert or hsct , if applicable ) are presented by region and phenotype in figure 1 . across all regions , patients with hurler syndrome , the most severe phenotype ,
the median age at symptom onset for these patients was 6 months , and diagnosis and treatment initiation followed quickly thereafter , at median ages of 12 and 18 months , respectively .
patients with hurler scheie and scheie syndromes , which have the more attenuated presentations , typically experienced initial symptoms sometime after infancy and exhibited a 2- to 4-year gap between onset of symptoms and diagnosis , respectively , and a 4- to 8-year gap between diagnosis and treatment initiation , respectively .
analyses of age of symptom onset , diagnosis , and treatment initiation based on phenotype and geographic region generally mirrored the results in the overall registry population , with a few notable exceptions . in north america , treatment initiation for patients with scheie syndrome was earlier than in the other regions . the median age for treatment initiation for patients diagnosed with scheie syndrome in north america was 11.7 years , whereas in europe and latin america it was 16.9 and 17.7 years , respectively .
the asia pacific region had an even higher median age of 31.5 years for treatment initiation .
scheie was also higher in the asia pacific region ( 23.8 years ) compared with the overall registry population ( 8 years ) .
another notable finding was the rate of patients with an undetermined or missing phenotype in latin america ( 11.1% ) , which was much higher compared with the other regions .
the median ages of symptom onset , diagnosis , and treatment initiation for patients in latin america with an undetermined phenotype were 0.7 , 1.3 , and 3.3 years , respectively , which is highly comparable to the median ages for patients in latin america with the hurler phenotype ( 0.7 , 1.7 , and 3.6 years , respectively ) , suggesting that some portion of the patients with undetermined phenotypes may , in fact , have hurler syndrome .
coarse facial features were the most predominant characteristic of all functional and anatomic abnormalities for both the hurler and the hurler scheie phenotypes , occurring in 86.4 and 72.7% of these patients , respectively .
the symptoms occurring in at least 25% of patients are summarized by phenotype in figure 2 , and all symptoms , including those occurring in less than 25% of patients , are summarized by phenotype in supplementary table s1 online . among patients with the scheie phenotype , 48.0% presented with coarse facial features .
corneal clouding was noted in all three phenotypes at approximately the same rate : 70.9 , 68.3 , and 70.1% for hurler , hurler
hepatomegaly was present in the majority of patients with hurler ( 70.0% ) and hurler scheie ( 66.5% ) phenotypes and in approximately half ( 48.0% ) of those with the scheie phenotype .
similarly , splenomegaly was present in 50.9% of patients with hurler syndrome and 47.1% of patients with hurler scheie syndrome but in only 27.6% of those with the scheie phenotype .
the incidences of hernias were more evenly distributed among the phenotypic groups , with 58.9% in hurler , 59.9% in hurler
kyphosis / gibbus was the only abnormality present in the majority ( 70.0% ) of patients with hurler phenotype , and it was reported much less frequently in patients with hurler scheie ( 33.5% ) and scheie ( 21.3% ) phenotypes .
conversely , joint contractures and carpal tunnel syndrome were present in the majority of scheie patients ( 69.3 and 51.2% , respectively ) but were less common in hurler
scheie patients ( 57.3 and 27.8% , respectively ) and even less frequent in hurler patients ( 37.9 and 7.8% , respectively ) .
cardiac valve abnormalities were observed in 67.7% of scheie patients , 59.0% of hurler scheie patients , and 48.9% of hurler patients .
airway - related symptoms , such as sleep disturbances / snoring were observed in 51.6% of hurler patients , 48.9% of hurler scheie patients , and 26.8% of scheie patients .
cognitive impairment was observed in 46.4 , 31.3 , and 9.4% of patients with hurler , hurler
as expected , first symptoms appeared earlier ( within the first 2 years of life ) in patients with the hurler phenotype ; whereas , in patients with the hurler scheie and scheie phenotypes , commonly occurring symptoms were first observed between 3 and 7 , and 5 and 13 years of age , respectively .
the median ages of onset of symptoms occurring in at least 25% of patients are presented by phenotype in figure 2 , and the median ages of onset of all symptoms , including those occurring in less than 25% of patients , are summarized by phenotype in supplementary table s2 online . across all phenotypes ,
hernias were the earliest reported symptom , which was noted at median ages of 0.8 , 3.2 , and 4.6 years of age in hurler , hurler
coarse facial features , the most prevalent symptom in patients with hurler and hurler scheie phenotypes , were also an early finding , with a median age at onset of 0.9 years in hurler patients and 3.4 and 8.7 years in patients with hurler scheie and scheie phenotypes , respectively .
corneal clouding , the most prevalent symptom in patients with the scheie phenotype , was first noted at a median age of 10.5 years in that subset of patients . among patients with the hurler phenotype ,
kyphosis / gibbus had a median age at onset of 1.0 year ( figure 2a ) , and dysostosis multiplex , corneal clouding , and hepatomegaly had a median age at onset of 1.1 years .
several symptoms , including sleep disturbances / snoring , enlarged tongue , cognitive impairment , and splenomegaly had a median age at onset of 1.2 years , whereas cardiac valve abnormalities were noted at a median age of 1.3 years .
the symptoms that appeared latest in at least 25% of patients with hurler syndrome were enlarged tonsils and joint contractures , with a median age at onset of 1.5 and 1.6 years , respectively .
after hernias and coarse facial features , cognitive impairment was the earliest symptom observed , first occurring at a median age of 3.8 years .
the median ages of onset for enlarged tongue , sleep disturbances / snoring , enlarged tonsils , joint contractures , and dysostosis multiplex were between 4.0 and 4.2 years .
both corneal clouding and hepatomegaly had a median age at onset of 4.4 years , whereas splenomegaly and kyphosis / gibbus had a median age at onset of 4.6 years .
scheie phenotype included cardiac valve abnormalities , hip dysplasia , and carpal tunnel syndrome , with median ages at onset of 5.7 , 6.2 , and 7.4 years , respectively .
symptoms reported in at least 25% of patients with scheie phenotype are shown in figure 2c .
after hernias , the earliest symptoms within this phenotype were joint contractures and dysostosis multiplex , with median ages at onset of 7.6 and 8.0 years , respectively .
both sleep disturbances / snoring and coarse facial features had a median age at onset of 8.7 years , and hepatomegaly was observed at a median age of 9.4 years .
symptoms appearing later included corneal clouding at 10.5 years , splenomegaly at 11.0 years , cardiac valve abnormalities at 11.7 years , and carpal tunnel syndrome at 12.5 years .
mps i is a rare panethnic genetic disorder characterized by a spectrum of disease with variable age of onset , progression , and organ involvement . if left untreated , patients with the most severe phenotype experience progressive deterioration of the musculoskeletal , cardiorespiratory , and central nervous systems and , typically , die before the age of 10 years .
although patients with the least severe phenotype usually have normal cognitive functioning and survive into adulthood , more than 50% may be affected by cardiac valve abnormalities , joint contractures , corneal clouding , hernias , and hepatomegaly .
the availability of improved disease - specific treatments , including hsct and ert , allows patients affected with mps i to obtain substantial clinical benefit for many disease manifestations , including hepatosplenomegaly , upper airway obstruction ( including sleep apnea ) , cardiac symptoms , and coarse facial features .
importantly , the chances of success from these treatments is improved when initiated prior to the onset of irreversible organ damage .
due to the rarity of the disease as well as the variability of clinical manifestations , mps i poses challenges for diagnosis .
the mps i registry provides the largest global data set for evaluating the natural history , clinical presentation , and potential treatment response of patients with mps i. the phenotypic distribution in the registry data set , with most patients ( 60.9% ) in the most severe phenotypic category , hurler , and the remaining more attenuated phenotypes , hurler scheie ( 23% ) and scheie ( 12.9% ) , is consistent with existing data , suggesting that ~5080% of patients have severe mps i. however , the observed rates of the various phenotypes within the registry may have been impacted by selection bias ; e.g. , more attenuated phenotypes could be underrepresented due to preferential identification and enrollment of patients with more severe clinical presentations . in an effort to minimize selection bias , the registry accepts all patients with a diagnosis of mps i , regardless of treatment status .
north america and europe had higher proportions of patients with the hurler phenotype than did latin america or the asia pacific region .
genetic landscape in different geographic regions or may be the result of regional differences in identification and enrollment of mps i patients in this voluntary registry .
some evidence exists for regional genetic differences , in that certain severe mutations ( common nonsense mutations ) identified in north america and europe have not been observed in the asia pacific region .
the profile of mps i mutations in brazil also differs from that found in other regions . based on the similarities in median ages of symptom onset , diagnosis , and treatment initiation
, the present study suggests that a substantial proportion of patients in latin america whose phenotypes were classified as undetermined / missing could have hurler syndrome .
early symptom recognition and diagnosis are essential to achieve the best long - term prognosis in patients with mps i. it is of paramount importance that not only pediatricians , but other specialists as well , are familiar with the clinical manifestations and consider an mps diagnosis , especially when symptoms are present in combination with each other .
therefore , the presence of inguinal or umbilical hernias in young children should raise suspicion of mps i. likewise , coarse facial features , which are early manifestations and the most prevalent symptom in patients with hurler and hurler scheie phenotypes , should be considered as an early sign of a potential mps i diagnosis .
corneal clouding is also a highly prevalent and relatively early manifestation of mps i across the phenotypes .
our findings indicate significant delays in diagnosis and treatment , particularly in patients with attenuated phenotypes .
regional differences were evident as well , with attenuated patients in asia pacific receiving treatment 1822 years following the onset of symptoms a 10- to 12-year delay compared with the 612 years between onset of symptoms and treatment in the overall registry population .
this delay may be due , in part , to a lack of access to ert in some countries .
the data presented here confirm findings from a previous analysis of registry data published in 2007 , shortly after the registry was established .
the current analyses are based on a much larger set of patients ( 987 vs. 302 ) , provide global and regional breakdowns of the data , and include data from the natural history period only .
regardless , and not surprisingly , there are similarities between the two reports in the age at symptom onset , age at diagnosis , and the rates of prevalence of common mps i symptoms .
however , the current report , based on data from a larger number of patients , provides a more detailed and refined analysis of the earlier findings . when interpreting results of data analyses from the mps i registry , certain limitations common to observational registries should be considered . as with any voluntary registry ,
incomplete or missing data may affect the results , though a recent analysis of the mps i registry using source document verification revealed an overall source - to - database error rate of < 4% , with no systematic errors .
it is also possible that assessment and data collection methods at the participating sites around the world may not be sufficiently standardized and/or that registry enrollment biases , perhaps based on regional disease classification , symptom severity , and symptom reporting practices , affected the results .
for example , patients who did not have a particular symptom may not have had any information reported for that symptom ( i.e. , rather than indicating that a patient did not have a symptom , the investigator did not provide information about that symptom ) .
this possibility led us to calculate symptom prevalence rates as the number of patients with a particular reported symptom divided by the total number of patients in each region and/or phenotype .
as a consequence , the true symptom prevalence rates are at least what we have calculated , and may be higher . in this study , a slightly higher than expected number of patients with attenuated mps i had cognitive impairment ( 31.3% of hurler scheie and 9.4% of scheie patients ) .
this likely reflects the fact that mps i presents as a disease continuum , making phenotypic classification of mps i somewhat subjective .
thus , it is possible that some hurler and hurler scheie patients may have been misclassified as hurler scheie and scheie phenotypes , respectively .
although the aforementioned potential limitations are not insignificant , the data compiled in the mps i registry are clearly valuable for drawing inferences and generating hypotheses .
the mps i registry is the largest global database of information from mps i patients and provides a useful tool for expanding knowledge about disease presentation , clinical status , and treatment outcomes .
greater understanding of the symptomatology of the disease can lead to earlier diagnosis and initiation of treatment , which may in turn lead to better patient outcomes .
scheie , and scheie , is associated with a characteristic constellation of symptoms and disease course .
this analysis of data from almost 1,000 patients facilitates definition of the natural history of mps i across the phenotypic spectrum , which will hopefully increase awareness of the disease and improve early diagnosis .
in addition , results from this investigation will be important in establishing benchmarks for future analyses of treatment interventions .
the mps i registry is the largest global database of information from mps i patients and provides a useful tool for expanding knowledge about disease presentation , clinical status , and treatment outcomes .
greater understanding of the symptomatology of the disease can lead to earlier diagnosis and initiation of treatment , which may in turn lead to better patient outcomes .
scheie , and scheie , is associated with a characteristic constellation of symptoms and disease course .
this analysis of data from almost 1,000 patients facilitates definition of the natural history of mps i across the phenotypic spectrum , which will hopefully increase awareness of the disease and improve early diagnosis .
in addition , results from this investigation will be important in establishing benchmarks for future analyses of treatment interventions .
m.b . received unrestricted grants , honoraria , and travel support from genzyme , shire , biomarin pharmaceutical , and actelion .
p.a . is the chair of the north american board of advisors and a member of the international board of advisors for the mps i registry .
r.g . received travel grants and/or speaker honoraria from genzyme to participate in scientific meetings and investigator fees for his participation in genzyme - sponsored clinical trials .
j.m . received honoraria , educational , and travel grants from janssen r&d , shire , and greencross and has participated in clinical trials with biomarin phamaceutical , genzyme , and shire . t.o . | purpose : in this study , we aimed to describe the natural history of mucopolysaccharidosis i.methods:data from 1,046 patients who enrolled in the mps i registry as of august 2013 were available for descriptive analysis . only data from untreated patients and data prior to treatment for patients who received treatment were considered .
age at symptom onset , diagnosis , and treatment initiation were examined by geographic region and phenotype ( from most to least severe : hurler , hurler
scheie , and scheie ) . for each symptom , frequency and age at onset were examined.results:natural history data were available for 987 patients .
most patients were from europe ( 45.5% ) , followed by north america ( 34.8% ) , latin america ( 17.3% ) , and asia pacific ( 2.4% ) .
phenotype distribution was 60.9% for hurler , 23.0% for hurler scheie , and 12.9% for scheie ( 3.2% undetermined ) syndromes .
median age at symptom onset for hurler , hurler
scheie , and scheie syndromes was 6 months , 1.5 years , and 5.3 years , respectively ; median age at treatment initiation was 1.5 years , 8.0 years , and 16.9 years , respectively .
coarse facial features and corneal clouding were among the most common symptoms in all three phenotypes.conclusion:a delay between symptom onset and treatment exists , especially in patients with attenuated mucopolysaccharidosis i. a better understanding of disease manifestations may help facilitate prompt diagnosis and treatment and improve patient outcomes . | Introduction
Materials and Methods
Results
Global occurrence of MPS I phenotypes
Chronology of symptom onset, diagnosis, and treatment by region
Natural history of MPS I by phenotype
Age of symptom onset by phenotype
Discussion
Conclusions
Disclosure
Supplementary Material | the largest proportion of patients was from europe ( 45.5% ) , with the next largest from north america ( 34.8% ) , followed by latin america ( 17.3% ) and asia pacific ( 2.4% ) . the overall phenotypic distribution was 601 ( 60.9% ) for hurler , 227 ( 23.0% ) for hurler scheie , and 127 ( 12.9% ) for scheie . across all regions , patients with hurler syndrome , the most severe phenotype ,
the median age at symptom onset for these patients was 6 months , and diagnosis and treatment initiation followed quickly thereafter , at median ages of 12 and 18 months , respectively . the median ages of symptom onset , diagnosis , and treatment initiation for patients in latin america with an undetermined phenotype were 0.7 , 1.3 , and 3.3 years , respectively , which is highly comparable to the median ages for patients in latin america with the hurler phenotype ( 0.7 , 1.7 , and 3.6 years , respectively ) , suggesting that some portion of the patients with undetermined phenotypes may , in fact , have hurler syndrome . corneal clouding was noted in all three phenotypes at approximately the same rate : 70.9 , 68.3 , and 70.1% for hurler , hurler
hepatomegaly was present in the majority of patients with hurler ( 70.0% ) and hurler scheie ( 66.5% ) phenotypes and in approximately half ( 48.0% ) of those with the scheie phenotype . the incidences of hernias were more evenly distributed among the phenotypic groups , with 58.9% in hurler , 59.9% in hurler
kyphosis / gibbus was the only abnormality present in the majority ( 70.0% ) of patients with hurler phenotype , and it was reported much less frequently in patients with hurler scheie ( 33.5% ) and scheie ( 21.3% ) phenotypes . across all phenotypes ,
hernias were the earliest reported symptom , which was noted at median ages of 0.8 , 3.2 , and 4.6 years of age in hurler , hurler
coarse facial features , the most prevalent symptom in patients with hurler and hurler scheie phenotypes , were also an early finding , with a median age at onset of 0.9 years in hurler patients and 3.4 and 8.7 years in patients with hurler scheie and scheie phenotypes , respectively . the largest proportion of patients was from europe ( 45.5% ) , with the next largest from north america ( 34.8% ) , followed by latin america ( 17.3% ) and asia pacific ( 2.4% ) . across all regions , patients with hurler syndrome , the most severe phenotype ,
the median age at symptom onset for these patients was 6 months , and diagnosis and treatment initiation followed quickly thereafter , at median ages of 12 and 18 months , respectively . the median ages of symptom onset , diagnosis , and treatment initiation for patients in latin america with an undetermined phenotype were 0.7 , 1.3 , and 3.3 years , respectively , which is highly comparable to the median ages for patients in latin america with the hurler phenotype ( 0.7 , 1.7 , and 3.6 years , respectively ) , suggesting that some portion of the patients with undetermined phenotypes may , in fact , have hurler syndrome . corneal clouding was noted in all three phenotypes at approximately the same rate : 70.9 , 68.3 , and 70.1% for hurler , hurler
hepatomegaly was present in the majority of patients with hurler ( 70.0% ) and hurler scheie ( 66.5% ) phenotypes and in approximately half ( 48.0% ) of those with the scheie phenotype . across all phenotypes ,
hernias were the earliest reported symptom , which was noted at median ages of 0.8 , 3.2 , and 4.6 years of age in hurler , hurler
coarse facial features , the most prevalent symptom in patients with hurler and hurler scheie phenotypes , were also an early finding , with a median age at onset of 0.9 years in hurler patients and 3.4 and 8.7 years in patients with hurler scheie and scheie phenotypes , respectively . the mps i registry provides the largest global data set for evaluating the natural history , clinical presentation , and potential treatment response of patients with mps i. the phenotypic distribution in the registry data set , with most patients ( 60.9% ) in the most severe phenotypic category , hurler , and the remaining more attenuated phenotypes , hurler scheie ( 23% ) and scheie ( 12.9% ) , is consistent with existing data , suggesting that ~5080% of patients have severe mps i. however , the observed rates of the various phenotypes within the registry may have been impacted by selection bias ; e.g. | [
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] |
from april 1999 to october 2003 , 1,300 liver cancers were treated with us - guided percutaneous rf ablation in our institution . of these ,
various kinds of rf devices manufactured by three different companies ( radiofrequency interstitial thermal ablation medical system , mountain view , cal ; radiotherapeutics corporation , mountain view , cal ; and radionics corporation ; burlington , mass ) were used ( 3 - 5 ) .
details concerning the preparation of the patients and the ablation techniques used have previously been reported ( 7 , 9 , 12 , 14 ) . immediately after the percutaneous rf ablation , all of the patients were evaluated by means of a gray - scale us examination , in order to detect whether any acute complications had occurred . for the early evaluation of the therapeutic response , we performed either contrast - enhanced us or ct or both .
the postprocedural contrast - enhanced ct examinations were performed with a helical scanner ( hispeed ; ge medical systems , milwaukee , wis ) immediately ( within 2 hours ) after the rf ablation .
images were obtained before , and 30 , 70 and 180 seconds after the initiation of the intravenous contrast material injection , representing the nonenhanced , hepatic arterial , portal venous and equilibrium phases , respectively . the following morning , contrast - enhanced us examinations were performed using a real - time us scanner ( hdi 5000 , advanced technology laboratories , bothell , wash ; logiq 9 or logiq 700 expert series , ge medical systems , milwaukee , wis ; or sequoia , siemens medical solutions , mountain view , cal ) , following the injection of a microbubble contrast agent .
the microbubble contrast agent that we used , sh u 508a , is a suspension of galactose in sterile distilled water . in this contrast agent ,
the tiny microbubbles ( 2 - 8 m ) are stabilized in the microparticle suspension .
this agent was prepared by vigorous shaking with 11 ml of water for 10 seconds .
a suspension of microbubbles and galactose microparticles was created by disaggregation of the granules . after standing for 2 minutes
, 12.5 ml of a 300 mg / ml suspension was administered through an antecubital vein .
all patients underwent follow - up four - phase helical ct - both nonenhanced and contrast - enhanced three - phase - scans 1 month after rf ablation as a baseline study for the evaluation of the therapeutic efficacy .
we used either contrast - enhanced us or ct or both for the early assessment of complications and/or macroscopic residual unablated tumors , since it was generally difficult to depict microscopic residual tumors , due to the presence of either reactive hyperemia or procedure - related arterioportal shunts . the final decision on the early therapeutic efficacy
was made on the basis of 1-month follow - up ct findings , since it is known that the reactive hyperemia is usually resolved by this time ( 7 ) .
when found , residual unablated tumors were usually treated with additional rf ablation . if the condition of residual tumor was not adequate for additional rf ablation , due to its poor conspicuity on us or the presence of multiple new lesions , transcatheter arterial chemoembolization ( tace ) was performed .
if there was any evidence of the technical success of the local treatment and no new hcc appeared in the other liver site on the 1-month follow - up ct , subsequent contrast - enhanced three - phase helical ct was repeated at 3-month intervals as our follow - up strategy . on the follow - up ct , we considered the tumor to be completely ablated when there was no enhancing portion within the ablation zone during either the hepatic arterial or portal venous phase .
similarly , we regarded the tumor as being completely ablated if there was neither vascularity nor enhancing focus within the ablation zone on contrast - enhanced us .
a residual unablated tumor was defined as an irregular peripheral - enhancing focus in the ablation zone on either the contrast - enhanced us or early follow - up ct , immediately or 1 month after rf ablation ( 7 , 15 ) .
local tumor progression was considered as the presence of growing enhancing tumors at the margin of the ablation zone on later follow - up ct , when there had been no previous evidence of a residual unablated tumor on the contrast - enhanced us and early follow - up ct ( 15 ) .
as described above , contrast - enhanced us has been used as the key modality in the imaging strategy used to assess the therapeutic efficacy following rf ablation in our institution ( fig .
during the first one year period ( between april 1999 and march 2000 ) , most patients treated with percutaneous rf ablation in our institution were evaluated with contrast - enhanced power doppler us after rf ablation .
these power doppler us examinations were performed before and after the injection of a microbubble contrast agent , and the power doppler us parameters were optimized .
we started to scan with power doppler us 20 seconds after initiation of the contrast agent injection , which was performed at a rate of 3 ml / min with an infusion pump ( ipx4 ; ivac medical systems , hampshire , u.k . ) .
we scanned intermittently to avoid early bubble destruction . with this technique , the enhancement effect lasted for more than 10 minutes .
focal areas with flow signals in the ablated lesions were considered as viable tumor portions ( 9 ) . in our previous report involving the treatment of 73 hepatocellular carcinomas ( hccs ) ( 12 ) ,
contrast - enhanced power doppler us showed no focal peripheral flow signals in 65 ( 89% ) ablation zones ( fig .
residual tumors were found on both the immediate follow - up ct and contrast - enhanced power doppler us .
the areas of the residual tumors on the power doppler us were well correlated with the enhancing portions on ct ( fig .
hence , 100% diagnostic agreement was achieved between the contrast - enhanced power doppler us and immediate follow - up ct .
of the 65 ablation zones without residual unablated tumors , however , 10 ( 15% ) had local tumor progression on follow - up ct .
pihi is one of the early - developed gray - scale harmonic techniques ( hdi 5000 , advanced technology laboratories , bothell , wash ) .
the transducer sends two pulses into the tissue in rapid succession ( one with a 180 phase change ) , and receives the sum of the echoes back from the two inverted pulses .
consequently , the signals from the harmonic component are boosted , and those from the fundamental component are suppressed ( 16 , 17 ) .
because microbubble agents produce nonlinear backscatter , they respond differently to phase inverted pulses and do not reflect identical inverted waveforms . according to some previous reports ( 10 , 16 , 17 ) , pihi is superior to conventional doppler us or second harmonic us in the depiction of tumor vascularity .
( 10 ) pointed out that contrast - enhanced pihi has increased the sensitivity of detecting residual tumors after rf ablation by up to 83% .
4 ) . pulse inversion harmonic imaging ( mechanical index , mi , 1.1 - 1.3 ) was performed at 4- to 6-sec intervals from 20 sec until 90 sec after initiation of the contrast agent injection , using a single - frame acquisition mode , rather than continuous imaging .
the residual unablated tumors were seen as focal enhancing structures in the periphery of the ablation zone .
cha is the other contrast - specific gray - scale harmonic us technique based on digitally encoding us technology ( logiq 9 or logiq 700 expert series , ge medical systems , milwaukee , wis ) .
this technique encodes the unwanted fundamental frequency component when transmitting pulses and then , through a decoding process when receiving the signals , removes the pre - tagged fundamental echoes without any loss or degradation of overlapping harmonic signals ( 18 - 20 ) .
this leaves only the desired wideband harmonic return signals , and allows for superior contrast and spatial resolution over conventional harmonic imaging techniques .
cha uses the b - flow technique , as well as digitally coded harmonic imaging . the b - flow technique , which is optimized for the direct visualization of blood cells on gray - scale images , uses codes to enhance weak blood echoes and equalize the non - moving tissue signal ( 19 ) thus
, the b - flow technique is free of doppler - related artifacts . by combining the benefits of these two techniques
, cha allows the harmonic signals resulting from tissue to be separated from those arising from contrast agents , and boosts the harmonic return from the contrast agents , while suppressing not only the fundamental , but also the harmonic signals from the background tissue ( 18 - 20 ) .
hence , cha is specifically sensitive to signals from the contrast agent , without producing any contrast - related artifacts .
this high sensitivity to the contrast agent creates a sufficiently stimulated acoustic emission effect at only medium mi ( 0.6 - 0.8 ) .
furthermore , such a condition , which is less destructive toward the microbubbles , provides a period of continuous scanning with excellent depiction of the mobile microbubbles in the intratumoral microvasculature . in interval - delay scanning ,
this allows the entire vascular volume , in which the microvessels are included , to become filled with the contrast agent .
when scanning begins after a short intermission , the accumulated microbubbles are destroyed , leading to the release of high - intensity , nonlinear echoes that are optimally detected using harmonic imaging .
stimulated acoustic emission does not rely on the movement of microbubbles and can be observed equally well with stationary microbubbles .
thus , this harmonic interval - delay method allows the detection of blood in the capillary bed , where the flow velocity is too low to be detected with conventional doppler us flow techniques . in this way
, gray - scale harmonic imaging can be used to detect echoes from contrast agents , when it is present in very low concentrations and distributed in microscopic vessels . before the injection of the contrast agent ,
a preliminary sweep was performed during suspended respiration in the optimal plane for the visualization of the lesion .
the machine settings , such as the focal zone and time - gain compensation , were optimized . following this preliminary sweep , we obtained serial contrast - enhanced cha images from 15 to 90 seconds after the initiation of the bolus contrast injection .
we evaluated the vascularity within the ablation zones with a continuous scan starting between 15 - 30 seconds after initiation of the contrast injection , and lasting for 3 - 5 seconds . to evaluate the perfusion of the ablation zones
, we also obtained intermittent stimulated acoustic emission imaging with a rapid sweeping technique every 10 seconds , from the end of the continuous scan to 90 seconds after initiation of the contrast injection .
we used intermittent interval - delay scanning with 10-second intervals , in order to avoid bubble destruction and to maximize the stimulated acoustic emission .
we froze the display in the interval between each scanning time , and then unfroze it for a very short period of time , during each scanning period .
thus , we obtained five or six intermittent stimulated acoustic emission imaging pieces during the perfusion scanning .
focal areas with irregular peripheral enhancement within the ablation zones were considered to be residual tumor portions ( 9 , 14 ) . in our recent report involving 81 hccs ( 14 ) , ten ( 12% ) showed irregular peripheral enhancement at the margin of the ablation zone on contrast - enhanced cha ( fig . 5 ) .
all of them were found to have the enhancing portion at the same location as that on the corresponding original image of the index tumor , during the hepatic arterial or portal venous phases on 1-month follow - up ct ( fig .
contrast - enhanced cha showed no evidence of enhancing foci , suggesting technical success in the remaining 71 ( 88% ) of the 81 hccs ( fig .
6 ) . among the 71 ablated hccs without residual unablated tumors , either on ct or contrast - enhanced cha , seven ( 10% ) had follow - up ct findings of local tumor progression in the ablation zones .
adi ( sequoia , siemens medical solutions , mountain view , cal ) is a multipulse technology that provides very high - resolution images , and uses the property of bubble emission to show regions where a contrast agent has localized ( 21 ) .
adi appears to be more useful for detecting microbubble contrast in tumors than other harmonic imaging techniques . because adi uses two pulses with the same polarity and subtracts
the signals from the two pulses , only those signals from the fundamental contrast agent and harmonic contrast agent remain . as such , adi
moreover , adi allows us to review either the functional contrast - enhanced images , the anatomic tissue images , or both at the same time , all from the same scan .
accordingly , this capability of providing a segmented display on contrast - enhanced adi is very useful for depicting the exact relationship between the index tumor ( on the anatomic tissue image ) and the enhanced residual tumor portion ( on the image displaying tissue and contrast ) .
the acoustic power of adi was set at the default ( maximal ) setting ( mi , 1.9 ) .
the line density and frame rate were set to a low level ( 9 hz ) with no frame averaging .
scanning was performed with a 4 - 1-mhz or 6 - 2-mhz convex array transducer .
we obtained serial contrast - enhanced adi images from 15 to 90 seconds after initiation of the bolus contrast injection .
we evaluated the vascularity within the ablation zones using a continuous scan with a duration of 5 - 8 seconds , between 15 and 30 seconds after initiation of the contrast injection . to evaluate the perfusion of the ablation zones
, we also obtained stimulated acoustic emission images with interval delay scanning and a rapid sweeping technique during the rest of the time .
each interval delay scanning was interrupted for at least 10 seconds . in our unpublished data concerning 52 hccs ,
neither contrast - enhanced adi the following morning nor 1-month follow - up ct showed evidence of enhancing foci within the 49 ( 94% ) ablation zones ( fig .
ablation zones , irregular peripheral - enhancing foci were found on contrast - enhanced adi ( fig .
all of these were found to have the enhancing portion at the same location as that of the corresponding lesions observed during the hepatic arterial or portal venous phases on the 1-month follow - up ct ( fig . 8) . among the 49 ablated hccs without residual unablated tumors , either on ct or contrast - enhanced adi , two ( 4% ) had follow - up ct findings of local tumor progression in the ablation zones . a more recent report ( 13 )
also suggested that contrast - enhanced adi was useful and as effective as ct for evaluating the therapeutic response to rf ablation and tace in patients with malignant hepatic tumors ( fig .
during the first one year period ( between april 1999 and march 2000 ) , most patients treated with percutaneous rf ablation in our institution were evaluated with contrast - enhanced power doppler us after rf ablation .
these power doppler us examinations were performed before and after the injection of a microbubble contrast agent , and the power doppler us parameters were optimized .
we started to scan with power doppler us 20 seconds after initiation of the contrast agent injection , which was performed at a rate of 3 ml / min with an infusion pump ( ipx4 ; ivac medical systems , hampshire , u.k . ) .
we scanned intermittently to avoid early bubble destruction . with this technique , the enhancement effect lasted for more than 10 minutes .
focal areas with flow signals in the ablated lesions were considered as viable tumor portions ( 9 ) . in our previous report involving the treatment of 73 hepatocellular carcinomas ( hccs ) ( 12 ) ,
contrast - enhanced power doppler us showed no focal peripheral flow signals in 65 ( 89% ) ablation zones ( fig .
residual tumors were found on both the immediate follow - up ct and contrast - enhanced power doppler us .
the areas of the residual tumors on the power doppler us were well correlated with the enhancing portions on ct ( fig .
hence , 100% diagnostic agreement was achieved between the contrast - enhanced power doppler us and immediate follow - up ct .
of the 65 ablation zones without residual unablated tumors , however , 10 ( 15% ) had local tumor progression on follow - up ct .
pihi is one of the early - developed gray - scale harmonic techniques ( hdi 5000 , advanced technology laboratories , bothell , wash ) .
the transducer sends two pulses into the tissue in rapid succession ( one with a 180 phase change ) , and receives the sum of the echoes back from the two inverted pulses .
consequently , the signals from the harmonic component are boosted , and those from the fundamental component are suppressed ( 16 , 17 ) .
because microbubble agents produce nonlinear backscatter , they respond differently to phase inverted pulses and do not reflect identical inverted waveforms . according to some previous reports ( 10 , 16 , 17 )
, pihi is superior to conventional doppler us or second harmonic us in the depiction of tumor vascularity .
( 10 ) pointed out that contrast - enhanced pihi has increased the sensitivity of detecting residual tumors after rf ablation by up to 83% .
4 ) . pulse inversion harmonic imaging ( mechanical index , mi , 1.1 - 1.3 ) was performed at 4- to 6-sec intervals from 20 sec until 90 sec after initiation of the contrast agent injection , using a single - frame acquisition mode , rather than continuous imaging .
the residual unablated tumors were seen as focal enhancing structures in the periphery of the ablation zone .
cha is the other contrast - specific gray - scale harmonic us technique based on digitally encoding us technology ( logiq 9 or logiq 700 expert series , ge medical systems , milwaukee , wis ) .
this technique encodes the unwanted fundamental frequency component when transmitting pulses and then , through a decoding process when receiving the signals , removes the pre - tagged fundamental echoes without any loss or degradation of overlapping harmonic signals ( 18 - 20 ) .
this leaves only the desired wideband harmonic return signals , and allows for superior contrast and spatial resolution over conventional harmonic imaging techniques .
cha uses the b - flow technique , as well as digitally coded harmonic imaging . the b - flow technique , which is optimized for the direct visualization of blood cells on gray - scale images , uses codes to enhance weak blood echoes and equalize the non - moving tissue signal ( 19 ) thus
, the b - flow technique is free of doppler - related artifacts . by combining the benefits of these two techniques
, cha allows the harmonic signals resulting from tissue to be separated from those arising from contrast agents , and boosts the harmonic return from the contrast agents , while suppressing not only the fundamental , but also the harmonic signals from the background tissue ( 18 - 20 ) .
hence , cha is specifically sensitive to signals from the contrast agent , without producing any contrast - related artifacts .
this high sensitivity to the contrast agent creates a sufficiently stimulated acoustic emission effect at only medium mi ( 0.6 - 0.8 ) .
furthermore , such a condition , which is less destructive toward the microbubbles , provides a period of continuous scanning with excellent depiction of the mobile microbubbles in the intratumoral microvasculature . in interval - delay scanning ,
this allows the entire vascular volume , in which the microvessels are included , to become filled with the contrast agent .
when scanning begins after a short intermission , the accumulated microbubbles are destroyed , leading to the release of high - intensity , nonlinear echoes that are optimally detected using harmonic imaging .
stimulated acoustic emission does not rely on the movement of microbubbles and can be observed equally well with stationary microbubbles .
thus , this harmonic interval - delay method allows the detection of blood in the capillary bed , where the flow velocity is too low to be detected with conventional doppler us flow techniques . in this way
, gray - scale harmonic imaging can be used to detect echoes from contrast agents , when it is present in very low concentrations and distributed in microscopic vessels . before the injection of the contrast agent ,
a preliminary sweep was performed during suspended respiration in the optimal plane for the visualization of the lesion .
the machine settings , such as the focal zone and time - gain compensation , were optimized . following this preliminary sweep , we obtained serial contrast - enhanced cha images from 15 to 90 seconds after the initiation of the bolus contrast injection .
we evaluated the vascularity within the ablation zones with a continuous scan starting between 15 - 30 seconds after initiation of the contrast injection , and lasting for 3 - 5 seconds . to evaluate the perfusion of the ablation zones
, we also obtained intermittent stimulated acoustic emission imaging with a rapid sweeping technique every 10 seconds , from the end of the continuous scan to 90 seconds after initiation of the contrast injection .
we used intermittent interval - delay scanning with 10-second intervals , in order to avoid bubble destruction and to maximize the stimulated acoustic emission .
we froze the display in the interval between each scanning time , and then unfroze it for a very short period of time , during each scanning period .
thus , we obtained five or six intermittent stimulated acoustic emission imaging pieces during the perfusion scanning .
focal areas with irregular peripheral enhancement within the ablation zones were considered to be residual tumor portions ( 9 , 14 ) . in our recent report involving 81 hccs ( 14 )
, ten ( 12% ) showed irregular peripheral enhancement at the margin of the ablation zone on contrast - enhanced cha ( fig .
all of them were found to have the enhancing portion at the same location as that on the corresponding original image of the index tumor , during the hepatic arterial or portal venous phases on 1-month follow - up ct ( fig . 5 ) .
contrast - enhanced cha showed no evidence of enhancing foci , suggesting technical success in the remaining 71 ( 88% ) of the 81 hccs ( fig .
6 ) . among the 71 ablated hccs without residual unablated tumors , either on ct or contrast - enhanced cha , seven ( 10% ) had follow - up ct findings of local tumor progression in the ablation zones .
adi ( sequoia , siemens medical solutions , mountain view , cal ) is a multipulse technology that provides very high - resolution images , and uses the property of bubble emission to show regions where a contrast agent has localized ( 21 ) .
adi appears to be more useful for detecting microbubble contrast in tumors than other harmonic imaging techniques . because adi uses two pulses with the same polarity and subtracts
the signals from the two pulses , only those signals from the fundamental contrast agent and harmonic contrast agent remain . as such , adi
moreover , adi allows us to review either the functional contrast - enhanced images , the anatomic tissue images , or both at the same time , all from the same scan . accordingly
, this capability of providing a segmented display on contrast - enhanced adi is very useful for depicting the exact relationship between the index tumor ( on the anatomic tissue image ) and the enhanced residual tumor portion ( on the image displaying tissue and contrast ) .
the acoustic power of adi was set at the default ( maximal ) setting ( mi , 1.9 ) .
the line density and frame rate were set to a low level ( 9 hz ) with no frame averaging .
scanning was performed with a 4 - 1-mhz or 6 - 2-mhz convex array transducer .
we obtained serial contrast - enhanced adi images from 15 to 90 seconds after initiation of the bolus contrast injection .
we evaluated the vascularity within the ablation zones using a continuous scan with a duration of 5 - 8 seconds , between 15 and 30 seconds after initiation of the contrast injection . to evaluate the perfusion of the ablation zones
, we also obtained stimulated acoustic emission images with interval delay scanning and a rapid sweeping technique during the rest of the time .
each interval delay scanning was interrupted for at least 10 seconds . in our unpublished data concerning 52 hccs ,
neither contrast - enhanced adi the following morning nor 1-month follow - up ct showed evidence of enhancing foci within the 49 ( 94% ) ablation zones ( fig .
ablation zones , irregular peripheral - enhancing foci were found on contrast - enhanced adi ( fig .
all of these were found to have the enhancing portion at the same location as that of the corresponding lesions observed during the hepatic arterial or portal venous phases on the 1-month follow - up ct ( fig . 8) . among the 49 ablated hccs without residual unablated tumors , either on ct or contrast - enhanced adi , two ( 4% ) had follow - up ct findings of local tumor progression in the ablation zones .
a more recent report ( 13 ) also suggested that contrast - enhanced adi was useful and as effective as ct for evaluating the therapeutic response to rf ablation and tace in patients with malignant hepatic tumors ( fig .
after rf ablation of liver cancer , it is very difficult to detect residual unablated tumors using conventional color doppler or power doppler us , because the internal blood flows of the patients are slow and the signals are weak .
as previously described , contrast - enhanced us can improve the detection of residual tumors in the ablation zone . during and immediately after rf ablation , hyperechogenicity in the ablation zone caused by microbubbles produced by the ablation procedure is almost always observed , which makes it difficult to obtain an accurate assessment of the therapeutic efficacy in the treated lesion ( fig .
thus , most physicians usually wait until this hyperechogenicity disappears from the ablation zone before performing contrast - enhanced harmonic us .
this transient hyperechoic zone persists for a long period of time , which has been reported to range from 15 minutes to 6 hours ( 9 , 22 ) .
it is difficult for physicians to wait for such a long period of time with the patient on the table , because of the inconvenience to the patient and the busy schedule in the us suite .
dodd et al . ( 1 ) asserted that the ideal goal of rf ablation was the ablation of a 1-cm cancer - free ablative margin .
however , it is practically impossible to obtain a satisfactory ablative margin when the tumor is located either in the subcapsular portion or in the vicinity of large vessels .
the shape of the ablation zone is variable , often being round or ovoid , and sometimes has a complex configuration .
after rf ablation , it is very difficult to accurately define a cancer - free margin on contrast - enhanced us .
this may be possible only when the index tumor is well circumscribed within the ablation zone ( fig .
the use of contrast - enhanced us after the rf ablation of liver cancer offers potential benefits over other imaging strategies .
when the residual unablated tumor is depicted on contrast - enhanced us , additional ablation can be applied with precise targeting ( guiding ) of the residual unablated tumor ( fig .
this real - time confirmation of the accurate and precise placement of the electrode can not be accomplished with ct .
also , contrast - enhanced us is more convenient , less time consuming and less expensive , as compared with immediate follow - up ct .
in addition to these considerations , early complications such as intraperitoneal hemorrhage can easily be found by means of us . in spite of these numerous strong points ,
contrast - enhanced us after rf ablation presents several potential drawbacks . in effect , despite the cooperative training required to reduce the interobserver variability and bring the reproducibility to an acceptable level , contrast - enhanced us is intrinsically operator - dependent .
this is especially true in interval - delay scanning in the same area , which is not easy for unskilled examiners to perform .
sufficient practice of interval - delay scanning must be acquired in the area that includes the ablation zone , before the injection of the contrast agent .
furthermore , each contrast - enhanced us technique has its own weak location in the liver .
thus , the use of contrast - enhanced power doppler us is limited in patients whose ablated zone is situated in the left hepatic lobe and is due to cardiac pulsation ( 12 , 14 ) .
although it is possible to limit the number of motion artifacts by decreasing the color gain , this adjustment may also diminish the sensitivity required to detect slow flow signals within the residual unablated tumors .
for contrast - enhanced gray - scale harmonic us , deep locations provide weak signals .
harmonic signals from a deep - seated lesion are often insufficient for acceptable imaging due to sound attenuation ( 18 ) . in this study , we could not perform contrast - enhanced harmonic us in the case of those ablation zones that were located deeper than 10 cm in the liver .
moreover , the anatomical detail can not be well demonstrated on contrast - enhanced cha .
adi has a narrow temporal window , because it is set to a high mi .
however , coupled with the development of newer us technologies , some new long - acting ( stabilized ) us contrast agents such as br1 ( sonovue ; bracco , milan , italy ) can improve the ability to detect smaller viable tumors in the ablated area using continuous scanning at a low mi ( 24 ) . in conclusion ,
contrast - enhanced gray - scale harmonic us , although it has some limitations , can constitute a reliable alternative to contrast - enhanced multiphase helical ct in the early assessment of the therapeutic response to rf ablation for liver cancer , when performed by experienced hands . | the early assessment of the therapeutic response after percutaneous radiofrequency ( rf ) ablation is important , in order to correctly decide whether further treatment is necessary .
the residual unablated tumor is usually depicted on contrast - enhanced multiphase helical computed tomography ( ct ) as a focal enhancing structure during the arterial and portal venous phases .
contrast - enhanced color doppler and power doppler ultrasonography ( us ) have also been used to detect residual tumors .
contrast - enhanced gray - scale us , using a harmonic technology which has recently been introduced , allows for the detection of residual tumors after ablation , without any of the blooming or motion artifacts usually seen on contrast - enhanced color or power doppler us . based on our experience and reports in the literature , we consider that contrast - enhanced gray - scale harmonic us constitutes a reliable alternative to contrast - enhanced multiphase ct for the early evaluation of the therapeutic response to rf ablation for liver cancer .
this technique was also useful in targeting any residual unablated tumors encountered during additional ablation . | POSTABLATION IMAGING STRATEGY
CONTRAST-ENHANCED US
Power Doppler US
Pulse Inversion Harmonic Imaging (PIHI)
Coded Harmonic Angio (CHA)
Agent Detection Imaging (ADI)
DISCUSSION | immediately after the percutaneous rf ablation , all of the patients were evaluated by means of a gray - scale us examination , in order to detect whether any acute complications had occurred . for the early evaluation of the therapeutic response , we performed either contrast - enhanced us or ct or both . all patients underwent follow - up four - phase helical ct - both nonenhanced and contrast - enhanced three - phase - scans 1 month after rf ablation as a baseline study for the evaluation of the therapeutic efficacy . we used either contrast - enhanced us or ct or both for the early assessment of complications and/or macroscopic residual unablated tumors , since it was generally difficult to depict microscopic residual tumors , due to the presence of either reactive hyperemia or procedure - related arterioportal shunts . a residual unablated tumor was defined as an irregular peripheral - enhancing focus in the ablation zone on either the contrast - enhanced us or early follow - up ct , immediately or 1 month after rf ablation ( 7 , 15 ) . as described above , contrast - enhanced us has been used as the key modality in the imaging strategy used to assess the therapeutic efficacy following rf ablation in our institution ( fig . during the first one year period ( between april 1999 and march 2000 ) , most patients treated with percutaneous rf ablation in our institution were evaluated with contrast - enhanced power doppler us after rf ablation . residual tumors were found on both the immediate follow - up ct and contrast - enhanced power doppler us . the areas of the residual tumors on the power doppler us were well correlated with the enhancing portions on ct ( fig . pihi is one of the early - developed gray - scale harmonic techniques ( hdi 5000 , advanced technology laboratories , bothell , wash ) . ( 10 ) pointed out that contrast - enhanced pihi has increased the sensitivity of detecting residual tumors after rf ablation by up to 83% . the residual unablated tumors were seen as focal enhancing structures in the periphery of the ablation zone . cha is the other contrast - specific gray - scale harmonic us technique based on digitally encoding us technology ( logiq 9 or logiq 700 expert series , ge medical systems , milwaukee , wis ) . among the 71 ablated hccs without residual unablated tumors , either on ct or contrast - enhanced cha , seven ( 10% ) had follow - up ct findings of local tumor progression in the ablation zones . a more recent report ( 13 )
also suggested that contrast - enhanced adi was useful and as effective as ct for evaluating the therapeutic response to rf ablation and tace in patients with malignant hepatic tumors ( fig . during the first one year period ( between april 1999 and march 2000 ) , most patients treated with percutaneous rf ablation in our institution were evaluated with contrast - enhanced power doppler us after rf ablation . residual tumors were found on both the immediate follow - up ct and contrast - enhanced power doppler us . ( 10 ) pointed out that contrast - enhanced pihi has increased the sensitivity of detecting residual tumors after rf ablation by up to 83% . the residual unablated tumors were seen as focal enhancing structures in the periphery of the ablation zone . cha is the other contrast - specific gray - scale harmonic us technique based on digitally encoding us technology ( logiq 9 or logiq 700 expert series , ge medical systems , milwaukee , wis ) . a more recent report ( 13 ) also suggested that contrast - enhanced adi was useful and as effective as ct for evaluating the therapeutic response to rf ablation and tace in patients with malignant hepatic tumors ( fig . after rf ablation of liver cancer , it is very difficult to detect residual unablated tumors using conventional color doppler or power doppler us , because the internal blood flows of the patients are slow and the signals are weak . as previously described , contrast - enhanced us can improve the detection of residual tumors in the ablation zone . during and immediately after rf ablation , hyperechogenicity in the ablation zone caused by microbubbles produced by the ablation procedure is almost always observed , which makes it difficult to obtain an accurate assessment of the therapeutic efficacy in the treated lesion ( fig . after rf ablation , it is very difficult to accurately define a cancer - free margin on contrast - enhanced us . when the residual unablated tumor is depicted on contrast - enhanced us , additional ablation can be applied with precise targeting ( guiding ) of the residual unablated tumor ( fig . for contrast - enhanced gray - scale harmonic us , deep locations provide weak signals . in conclusion ,
contrast - enhanced gray - scale harmonic us , although it has some limitations , can constitute a reliable alternative to contrast - enhanced multiphase helical ct in the early assessment of the therapeutic response to rf ablation for liver cancer , when performed by experienced hands . | [
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] |
conventional cancer chemotherapy including chemotherapy of breast cancer often results in
severe systemic toxicity at drug concentrations necessary for effective killing
of tumor cells .
this
obstacle can be overcome with the concept of gene - directed enzyme prodrug
therapy ( gdept ) that implies selective delivery into tumor cells and expression
of drug - metabolizing transgenes within them .
the oxazaphosphorine cyclophosphamide ( cpa ) and
its structural isomer ifosfamide ( ifa ) are dna - alkylating agents commonly used
in breast cancer chemotherapy .
these anticancer
agents are administered as prodrugs that are primarily activated by the hepatic
enzyme cytochrome p450 ( cyp ) . among the p450 enzymes , the subfamily 2b enzymes cyp2b1 ( from rat ) and
cyp2b6 ( from human ) have been shown to be the most active catalysts for this
enzymatic reaction [ 3 , 4 ] .
the generated anticancer
metabolites phosphoramide mustard ( from cpa ) or isophosphoramide mustard ( from
ifa ) as well as acrolein are systematically distributed throughout the body
eventually reaching the tumor but also causing undesired toxic side effects .
local activation of cyclophosphamide or ifosfamide at the site of the tumor
would allow to use lower concentrations of the prodrug resulting in lower
systemic toxicity with a still effective or , if using conventional prodrug
dosages , a much more potent cell killing effect on the tumor cells .
in addition ,
cyclophosphamide and ifosfamide suicide gene therapy has the advantage of also
exerting a bystander effect as it causes the death of not only the therapeutic
transgene - carrying cells but also of neighboring nontransgenic cells via passive diffusion of the
cytotoxic metabolites [ 5 , 6 ] .
gene therapy requires strong and , if possible , selective expression of the transgene
in the requested tissue or organ . for mammary gland - specific expression of
transgenes in mammals ,
a number of promoters from various sources have been
evaluated . among those are the promoters of milk protein - encoding genes such as the whey acidic
protein ( wap ) , -lactoglobulin , -s1-casein , -casein , or the c3(1 ) promoter . however , for mouse models of human
breast cancer , the long terminal repeat ( ltr ) of the mouse mammary tumor virus
( mmtv ) has emerged as the most potent and frequently used promoter to drive
transgene expression [ 812 ] .
it , therefore , also
represents one of the major candidate promoters for human breast cancer gene
therapy .
the mmtv promoter is most active during lactation due to induction by lactogenic
hormones such as prolactin [ 13 , 14 ] .
however , the most potent inducing effects
are due to the presence of hormone response elements ( hres ) within the u3
region of the viral ltr that respond to androgens , progestins ,
mineralocorticoids , and glucocorticoids . for in vitro and in vivo
transgene delivery , a number of techniques have been elaborated . among those ,
infection with retroviral vectors represents a very efficient method . due to their capacity to integrate
into the host genome , retroviral vectors are one choice if long - term gene
expression of a transgene is desired and thus they have been used in a variety
of gene therapy studies .
however , to date , several rounds of vector
delivery are necessary to achieve satisfactory transfer of a therapeutic gene in vivo .
this is mainly due to
unsatisfactory virus titers , rapid clearance of the vector by the liver and the
spleen , and , in the case of mlv - based retroviral vectors , the fact that
only dividing cells can be reached , thereby a priori limiting the number of accessible cells .
in addition , repeated delivery may cause the risk of the development
of an immune response against the vector , thereby impairing gene transfer .
the establishment of an in situ cell depot , constantly
producing therapeutic retroviral vectors where required , may overcome those
hurdles and may allow much more efficient delivery of the transgene . in previous experiments , for proof of principle , we have
encapsulated virus packaging cells that not only expressed a reporter gene from
a retroviral vector but also produced virus particles . upon
insertion into the mammary glands of mice ,
the virus particles were liberated
from the capsules and transferred the reporter gene to surrounding cells .
this type of delivery has also been shown by others to be functional using a
potentially therapeutic suicide gene for the treatment of glioblastoma ,
and similar long - term in vivo gene delivery could also be achieved using theracyte immuno - isolation devices
containing spleen necrosis retrovirus packaging cells .
we have also shown
previously that , when combined with ifosfamide - based chemotherapy , intratumoral
injection as well as instillation of capsules containing cyp2b1-expressing ( but
not virus - producing ) cells into tumor - supplying blood vessels leads to a
significantly increased tumoricidal effect on experimentally generated tumors
in mice [ 22 , 23 ] or on inoperable
pancreatic tumors in humans [ 24 , 25 ] .
in this study , we have constructed
mlv - based replication - deficient retroviral promoter conversion ( procon ) vectors
for the use in breast cancer gene therapy in combination with the in situ vector supply and delivery
system described above .
the vectors are designed to express the cyp2b1 gene
within the packaging cells to convert the prodrugs ifosfamide or
cyclophosphamide into their active forms .
in addition , the packaging cells
produce virus particles that are liberated from the cells and , by infection ,
can transfer the therapeutic gene into the surrounding target cells . in the vectors described in this
study , the original u3 region of the 3long terminal repeat ( ltr ) is replaced
with the heterologous mmtv promoter . in virus - packaging
cell lines ,
transcription of the egfp - encoding reporter gene or of the actual
therapeutic cyp2b1-encoding gene is driven by the mlv promoter / enhancer .
after infection , in the course of
reverse transcription , the heterologous mmtv promoter is duplicated and one
copy is translocated to the u3-region of the 5ltr .
this
rearrangement finally brings the expression of the transduced gene under
control of the mmtv promoter .
we show that the genes incorporated into the
vectors are efficiently expressed in virus packaging cells as well as in infected
cells .
virus titers obtained with the generated vectors are sufficiently high
to allow efficient infection of target cells both in vitro and in a murine tumor model in vivo .
we further show
that the generated therapeutic cyp2b1 protein is enzymatically active and exerts
a strong cell killing effect on infected breast cancer cells upon treatment
with ifosfamide or cyclophosphamide in vitro .
plasmids ppccmm1 and ppcemm1 ( see figure 1 ) are
identical in construction except for the fact that ppccmm1 carries the cyp2b1
gene and ppcemm1 carries the egfp gene .
ppccmm1 was constructed by replacing a
cytomegalovirus ( cmv ) promoter - harboring mlui
sacii
fragment of plasmid ppccmcmv.wpre ( harry holzmller , austrianova biotechnology
gmbh ) with an mlui
plasmid dna was isolated using a qiaprep spin miniprep kit ( qiagen , calif , usa ) or a qiagen - tip 100
plasmid midi kit ( qiagen ) .
linear dna fragments were purified using a qiaquick
gel extraction kit ( qiagen ) .
human 2gp19talf amphotropic retroviral
packaging cells , human 293 embryonic kidney cells ( atcc crl-1573 ) ,
and feline crfk kidney cells ( atcc ccl-94 ) were grown in dulbeccosmodified
eagle s medium ( dmem)/glutamax ( invitrogen life technologies , calif , usa)supplemented
with 10% fetal bovine serum ( fbs ; invitrogen life technologies ) .
human t-47d
( mammary gland ; ductal carcinoma ) cells ( atcc htb-133 ) were cultivated in
dmem / glutamax supplemented
with 10% fbs and 6.5 g / ml insulin ( sigma - aldrich , miss , usa ) .
transfections of 2gp19talf cells were performed by calcium phosphate coprecipitation according
to the instructions of the manufacturer ( amersham biosciences , nj , usa ) . for
stable transfections ,
the transfected cells were selected in medium containing
0.4 mg / ml geneticin(g418 ; invitrogen life technologies ) until mock - transfected
cells had died .
stably transfected cells were maintained as populations in the
presence of 0.4 mg / ml g418 .
culture supernatants from 2 10 virus - producing cells were usedto infect 4 10 target cells as described elsewhere . for titer calculation ,
dilutions of infected crfk cells were
trypsinized and replated 24 hours afterinfection in triplicates and
selected in medium containing 0.4 mg of g418/ml.after 10 to 14 days
of culture , drug - resistant colonies werecounted , and the number of colony - forming
units ( cfu ) per milliliter of vector supernatant wascalculated .
populations of stably infected t-47d and crfk cells were maintained in the presence
of 0.4 mg / ml g418 .
for stimulation of expression , cells were treated with 1 m
dexamethasone ( sigma - aldrich ) every 48 hours . for establishment of mixed tumors
2gp19talf / ppcemm1 cells and
t-47d / dsred or crfk / dsred cells were trypsinized
out of the culture flasks , washed three times with pbs , and mixed in a ratio of
1 : 5 or 1 : 10
. a total number of 6 10 mixed cells in a total volume
of 100 l rpmi medium containing 100 u / ml penicillin / streptomycin ( invitrogen life
technologies ) and 50% matrigel ( becton dickinson biosciences , nj , usa ) was
injected into the mammary fat pads of hsd : athymic nude - nu mice ( harlan
winkelmann , borchen , germany ) . for stimulation of tumor growth ,
slow - release estrogen
pellets ( 1.13 mg / pellet , 60 days release ; innovative research of america , fla ,
usa ) were implanted . at an average tumor size of 100200 mm , mice were treated
with 500 g dexamethasone / mouse ( voren , 1 mg / ml , boehringer ingelheim , ingelheim , germany )
intraperitoneally on 3 consecutive days . on day 4 , tumors were explanted ,
digested to single - cell suspension by incubation with 2 mg / ml collagenase
( sigma - aldrich ) in pbs at 37c for two hours , washed twice with dmem/10% fbs ,
and finally analyzed by fluorescence - activatedcell sorting ( facs )
and confocal laser - scanning microscopy . in vivo experiments were carried out
according to austrian law regulating animal experimentation ( gz 68.205/109-brgt/2003 ) . for detection of dsred- and egfp - expressing
tissue culture cells , those were trypsinized , washed twice with pbs/10% fbs , and
then 50,000 cells per sample were analyzed for fluorescencewith
an facs analyzer ( facscalibur ; becton dickinson ) .
the numbers and meanfluorescence
intensities ( mfis ) of positive cells were determined using the cellquest
software ( becton dickinson ) .
for confocal laser - scanning microscopy , a zeiss
lsm 510 inverted microscope equipped with a 40 x , 1.3 numerical aperture , oil
immersion objective ( plan neofluar , zeiss , gttingen , germany ) was used .
cells
analyzed by laser - scanning microscopy were prepared in the same way as for facs
analysis .
populations of cells expressing the fluorescent proteins egfp or dsred were facs sorted to exclude nonfluorescent
cells before their use in in vivo experiments . therefore , cells to be sorted were expanded to approximately 3 10 , harvested by trypsinization , resuspended in normal cell culture
medium , and pelleted by cetrifugation at 410 g for 5 minutes .
thereafter ,
cells were washed twice in pbs and resuspended in pbs containing 5% fbs .
then ,
the cell suspension was filtered through a sterile nylon gauze into an facs tube
and stored on ice until sorting .
facs sorting was performed using the
facsvantage se device ( becton dickinson ) which is controlled by the cell quest
pro software ( becton dickinson ) .
cyp2b1 was detected by western blotting using an anti - cyp2b1 antibody ( daiichi pure chemicals co. , ltd , nj ,
usa ) .
briefly , cells were washed twice with pbs , trypsinized out of
the culture flasks , and resuspended in dmem / glutamax medium lacking phenol red ( invitrogen life technologies )
supplemented with 10% fbs .
100 l of cell suspension containing a total of 4 10 cells was pipetted
into a black clear bottom 96-well plate in quadruplicates .
dmem / glutamax medium
containing 10% fbs but lacking phenol red was used as a blank .
5 l of substrate ( 0.3 mm benzylresorufin in dmso ; sigma - aldrich ) was
added to each well .
samples were fluorometrically analyzed for the presence
of generated resorufin in a plate reader ( tecan systems ) using an extinction
wavelength of 520 nm and an emission wavelength of 590 nm .
the cyp2b1 enzymatic activities of a
selected single cell clone ( 22p1 g ) served as a reference .
1 10 t-47d or
t-47d / ppccmm1 cells were seeded into 96 well plates in quadruplicates and
cultivated in dmem / glutamax medium lacking phenol red ( invitrogen life
technologies ) supplemented with 10% fbs for six days without changing the
medium .
24 hours after seeding of the cells , the medium was supplemented with
0 , 0.03 , 0.1 , 0.3 , 1 , or 3 mm ifosfamide ( baxter , ill , usa ) or cyclophosphamide
( baxter ) .
five days after incubation with ifosfamide or cyclophosphamide , the viabilities
of the cells were determined with an xtt assay according to the instructions of
the manufacturer ( promega , wiss , usa ) .
we have previously evaluated mlv - based procon
vectors ( ppcem and ppcemm1 ) containing the mammary gland - specific mmtv promoter
and the egfp gene as a reporter for their gene expression and infection capacities
in vitro . here , for in vivo
investigations of the efficacy of the therapeutic vectors , we chose the
following experimental setup : we wanted to determine if efficient infection of
target cells is ( i ) also achievable by cocultivation of virus - producing cells
and target cells in vitro instead of using
high - titer virus suspensions for in vitro infections and ( ii ) in a mixed tumor
model in vivo , thus mimicking a future therapeutic situation in which
viruses would be released from capsules . in 293-based virus - producing 2gp19talf
cells , vector ppcemm1 ( see figure 1 ) that was used in this study allows
the expression of the egfp reporter gene from an mlv promoter / cmv enhancer
cassette while in infected cells after promoter conversion , expression of the
egfp gene is driven by the heterologous mmtv promoter . for in vitro infection studies , 2gp19talf virus - producing cells that had stably been transfected with ppcemm1
were mixed in ratios of 1 : 10 or 1 : 5 with red fluorescent human t-47d breast
cancer cells and as a control with red fluorescent feline crfk kidney cells
that are known to effectively being infected by retroviruses .
red fluorescence of target cells was
achieved by stably transfecting the cells with the dsred protein - encoding
vector pcmv - dsred - express and enrichment of red fluorescent
cells was performed by cell sorting .
mixed cells were
cocultivated for five weeks and the numbers of green , red , and green / red cells
were monitored by facs ( see figure 2(a ) ) . after five weeks , about 74% of red
fluorescent t-47-d cells were also green fluorescent , indicating that they had
been infected with ppcemm1 .
the initial ratio between virus - producing cells and target cells did not
play a role in this setting .
the red fluorescent crfk
cells were infected to an extent of about 19% ( ratio 1 : 10 between virus - producing
cells and target cells ) and 40% ( ratio 1 : 5 between virus - producing cells and
target cells ) . in contrast ,
when green fluorescent nonvirus - producing 293 cells stably infected with ppcemm1 where
mixed with red fluorescent t-47d or crfk cells , only a very low number ( < 1% ) of false - positive red / green cells were detected by facs after six weeks of
cocultivation ( data not shown ) .
the obtained results were also verified by confocal laser - scanning
microscopy : no red / green double fluorescent cells could be detected as
exemplified in figure 2(b ) for a mix of 293 cells stably infected with ppcemm1
and crfk / dsred cells .
in contrast , when the cell mixes
of cocultivated red fluorescent t-47d or crfk cells and virus - producing green
fluorescent cells were analyzed , clearly a number of truly red / green double
fluorescent cells could be detected as demonstrated for a mix of crfk / dsred and
2gp19talf / ppcemm1cells ( see figure 2(c ) ) .
double fluorescence was not due to
red autofluorescence of 2gp19talf / ppcemm1 or green autofluorescence of
crfk / ppcemm1 and t-47d / ppcemm1 cells since no red fluorescent cells could be
detected in samples only consisting of green fluorescent 2gp19talf / ppcemm1
cells and no green fluorescence could be detected in samples of red fluorescent
crfk / dsred and t-47d / dsred cells ( data not shown ) . to investigate the in vivo infection capability of the mmtv promoter containing
procon vector , mixed tumors consisting of dsred - expressing t-47d or crfk cells
on the one hand and virus - producing 2gp19talf / ppcemm1 cells on the other hand
again ,
the ratios between virus - producing cells and target cells at the time point of
injection were 1 : 10 and 1 : 5 .
after 11 weeks of tumor growth , the tumors were
explanted and analyzed by facs ( see figure 3(a ) ) . of the red fluorescent t-47d
cells of six mixed tumors ,
22% to 55% were also green fluorescent , that is , had
been infected with ppcemm1 .
a range of 13%33% of red
fluorescent crfk cells isolated from mixed tumors also showed green
fluorescence , indicating infection by ppcemm1 .
in addition , cells were analyzed by confocal laser - scanning
microscopy ( see figures 3(b ) and 3(c ) ) .
clearly , cells
fluorescing red as well as green were visible in the samples derived from mixed
t-47d ( see figure 3(b ) ) as well as from
mixed crfk tumors ( see figure 3(c ) ) .
as expected , cells originating from
control tumors solely consisting of 2gp19talf / ppcemm1 cells showed only green
but no red fluorescence while cells stemming from control tumors exclusively
consisting of t-47d / dsred or crfk / dsred cells showed only red but no green
fluorescence .
cells derived from control tumors only consisting of 2gp19talf ,
t-47d , or crfk cells exhibited no fluorescence at all ( data not shown ) .
the egfp reporter gene was replaced with the therapeutic gene coding for
cytochrome p450 2b1 ( cyp2b1 ) .
the resulting vector ppccmm1 ( see figure 1 ) was
used to transfect 2gp19talf cells and vector harboring cells were selected with
geneticin in order to obtain a population of stably transfected cells .
western
blot analysis was used to prove that cyp2b1 was produced in 2gp19talf / ppccmm1
cells ( see figure 4(a ) ) .
to not only test for the presence of the protein but
also for its activity , resorufin formation as a consequence of cyp2b1 activity
was monitored using a 96-well format assay .
generation of resorufin in
2gp19talf / ppccmm1 cells in relation to 2gp19talf negative control cells after
incubation with pentoxyresorufin is shown in figure 4(b ) .
for infection of target tumor cells , sufficiently high numbers of
virus particles have to be generated by the virus - producing cells .
virus titers were determined by
infection of crfk cells with virus particles from the supernatant of
2gp19talf / ppccmm1 cells and followed by counting the numbers of g418 resistant
colonies .
titers were about 1.3 10 colony - forming units ( cfu)/ml of
cell supernatant ( see figure 4(c ) ) which is comparable to titers obtained with
the respective egfp gene - containing vector ppcemm1 .
as a model breast cancer cell line to study the efficacy of our vector system , the
t-47d cell line was chosen .
t-47d cells were infected with ppccmm1 and stable
populations were created by selection of infected cells with g418 . for
induction of gene expression from the mmtv promoter cells were stimulated with dexamethasone
for 48 hours and analyzed by western blotting for the presence of cyp2b1 .
while an anti - cyp2b1 antibody did
not detect any cyp2b1 in noninfected t-47d cells ( negative control ) , cyp2b1 was
clearly present in t-47d cells infected with ppccmm1 ( see figure 5(a ) ) .
resorufin assays revealed that the protein was also active in that
cell line ( figure 5(b ) ) .
enzyme activities were 2.5-fold higher in cells that
had been treated with dexamethasone compared to those in nontreated cells ( data
not shown ) . to evaluate the sensitizing effect of cyp2b1 on t-47d cells upon treatment with the anticancer prodrug ifosfamide ,
t-47d cells infected with ppccmm1 and noninfected t-47d control cells were
treated with increasing amounts of ifosfamide ( ranging from 0 to 3 mm ) for five
consecutive days after which cell viabilities were determined using an xtt
assay .
while noninfected t-47d cells remained largely unaffected up to an
ifosfamide concentration of 0.3 mm , t-47d / ppccmm1 cells expressing the cyp2b1
gene showed a marked decrease of 40% already at a concentration of 0.1 mm .
0.3 mm
ifosfamide was determined as the most effective concentration at which noninfected
t-47 cells were still largely unaffected but cyp2b1-producing t-47d / ppccmm1
cells showed a dramatic decrease in viability by 88% .
ic50 values
were determined as 1.6 mm and 0.1 mm for t-47d and t-47d / ppccmm1 , respectively .
this represents a decrease of the ic50 value by sixteen folds ,
underlining the potent cumulative cytotoxic effect of cyp2b1 and ifosfamide .
treatment of the cells with cyclophosphamide instead of ifosfamide led to
similar results ( see figure 6(b ) ) .
the procon vectors we have generated in this
study are a further development of an earlier , a -galactosidase reporter
gene - containing vector that was used for the generation of virus - producing
cells for encapsulation experiments .
the new vectors contain modifications
that have recently been shown by our laboratory to improve their overall
performance ( i.e. , a cmv enhancer to increase viral rna production in virus - producing cells , a strong
polyadenylation signal in the modified 3ltr to prevent read - through of viral
rna and to stabilize the mrna , and an elongated attachment site to increase the
integration efficiency of the provirus ) .
when transferred
into virus packaging cells , our newly generated vectors allow the production of
enzymatically active cyp2b1 that converts ifosfamide or cyclophosphamide into
their tumoricidal metabolites .
as demonstrated by western blotting and with
resorufin assays , a high concentration of cyp2b1 in virus - producing cells is
ascertained by the strong mlv promoter that drives the expression of the
therapeutic gene .
similarly , when incorporating the egfp reporter gene into the
vectors , high mfis in facs analyses can be achieved in virus - producing cells . in
contrast to
what was seen with similar vectors containing the wap promoter
, when incorporated into our new vectors , the woodchuck hepatitis virus
posttranscriptional regulatory element ( wpre ) enhanced the expression rate of
both the egfp and the cyp2b1 genes in virus packaging cells as well as in
infected cells ( data not shown ) .
therefore , vectors
ppccmm1 and ppcemm1 harboring the wpre were chosen for further experiments over
versions of those vectors lacking the wpre ( data not shown ) .
ppccmm1 and the reporter gene - harboring vector
ppcemm1 allow the generation of high virus titers in virus - packaging cells and
productive infection of target cells in
vitro .
in cocultivation experiments employing
virus packaging cells and target cells , the infection rates of t-47d and crfk
target cells reached 74% and 40% , respectively .
the lower infection rate of the crfk cells was
due to the higher growth rate of the crfk cells compared to that of the
2gp19talf / ppcemm1 virus - packaging cells , quickly leading to the displacement of
the latter ones .
as a consequence , virus particle numbers in the cell
supernatants decreased over time .
the infection rates
were also reflected by similar results obtained with confocal laser - scanning
microscopy , demonstrating true infection of target cells .
the small numbers of double
fluorescent cells ( < 1% ) obtained after six weeks of cocultivation of red
fluorescent target cells and green fluorescent nonvirus - producing cells
represented false - positive cells since no such double fluorescent cells were
observed in laser - scanning microscopy .
most likely , the
small numbers of false - positive red / green cells observed in facs experiments
were rather due to small numbers of red and green cells sticking together ,
thus , giving the impression of double - fluorescent cells .
this is also
underlined by the fact that small numbers of red / green fluorescent cells
were even detected when red fluorescent cells were mixed with green fluorescent
cells immediately prior to facs ( data not shown ) . since in vivo infection
may be less efficient compared to invitro
infection , we tested our vectors in a mixed tumor model in nude mice in vivo .
analysis of explanted tumors revealed that 22% to 55% of t-47d target cells and
13% to 33% of crfk target cells had been infected with vector ppcemm1 .
the
somewhat lower infection rate for crfk cells was probably due to their
excellent growth rate in vivo compared to the poor growth rate of the
293-based virus - producing cells and a subsequent shift in the ratio between
virus - producing cells and target cells in favor of the target cells .
this
probably led to a substantial decrease of virus titer within the tumor over
time .
however , this will not be an issue if vector - producing cells will be encapsulated . in
addition ,
an infection rate of 100% is not necessarily required during a
therapy employing encapsulated viral vector - producing cells because of the
bystander effect of cyclophosphamide or ifosfamide on neighboring noninfected cells
in gdept [ 6 , 33 ] .
incorporation of the mmtv promoter into the retroviral vectors allowed high transgene
expression of the egfp reporter gene or the therapeutic cyp2b1 gene in infected
cells .
this was not only seen for t-47d and crfk cells but also for a number of
other human or nonhuman cell lines ( mainly of mammary origin ; data not shown ) .
in most of the cell lines , transgene expression could be further boosted by
stimulation with the glucocorticoid dexamethasone .
transgene expression was
also increased in most of the cell lines when cells were treated with
progesterone , although the effect was less pronounced ( data not shown ) .
dexamethasone is frequently used in
cancer chemotherapy , either as a tumoricidal therapeutic [ 34 , 35 ] or to prevent
or diminish nausea and other side effects in patients ( including breast cancer
patients ) undergoing chemotherapy [ 36 , 37 ] .
thus , in a
gdept setting , dexamethasone would decrease the symptoms of the side effects of
the chemotherapy and at the same time increase the expression of the
therapeutic gene from the mmtv promoter . in this study
, we also showed that the level of
enzymatically active cyp2b1 generated in infected cells with the newly
developed vectors is pronounced enough to create a highly increased cell
killing effect on t-47d / ppccmm1 cells upon ifosfamide or cyclophosphamide treatment
in vitro .
the enhanced cytotoxic effect due to
infection with ppccmm1 became manifest in a sixteen fold - lowered ic50 value for ifosfamide in t-47d / ppccmm1 compared to noninfected t-47d cells .
effective concentrations of 0.10.5 mm
ifosfamide have been measured in the plasma of patients after administration of
typical dosages of ifosfamide during conventional chemotherapy .
these pharmacologically
active concentrations are identical to those we determined , which resulted in
efficient killing of cyp2b1-expressing t-47d cells while leaving t-47d cells
not expressing cyp2b1 largely unaffected . taken together , the newly generated vectors
constitute the basis for the development of a novel breast cancer gdept system :
the system is anticipated to employ the encapsulation of cells that ( i )
generate the therapeutic enzyme cyp2b1 and , ( ii ) in addition , produce viral
vector particles that transfer the therapeutic cyp2b1 gene directly into tumor
cells . | gene directed - enzyme prodrug therapy ( gdept ) is an approach for sensitization of tumor cells to an enzymatically activated , otherwise nontoxic , prodrug .
cytochrome p450 2b1 ( cyp2b1 ) metabolizes the prodrugs cyclophosphamide ( cpa ) and ifosfamide ( ifa ) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein .
we have constructed a retroviral promoter conversion ( procon ) vector for breast cancer gdept .
the vector allows expression of cyp2b1 from the mouse mammary tumor virus ( mmtv ) promoter known to be active in the mammary glands of transgenic animals .
it is anticipated to be used for the generation of encapsulated viral vector producing cells which , when placed inside or close to a tumor , will act as suppliers of the therapeutic cyp2b1 protein as well as of the therapeutic vector itself .
the generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active cyp2b1 in infected cells which sensitized them to killing upon treatment with both ifa and cpa .
determination of the respective ic50 values demonstrated that the effective ifa dose was reduced by sixteen folds .
infection efficiencies in vivo were determined using a reporter gene - bearing vector in a mammary cancer cell - derived xenograft tumor mouse model . | 1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION | this
obstacle can be overcome with the concept of gene - directed enzyme prodrug
therapy ( gdept ) that implies selective delivery into tumor cells and expression
of drug - metabolizing transgenes within them . the oxazaphosphorine cyclophosphamide ( cpa ) and
its structural isomer ifosfamide ( ifa ) are dna - alkylating agents commonly used
in breast cancer chemotherapy . the generated anticancer
metabolites phosphoramide mustard ( from cpa ) or isophosphoramide mustard ( from
ifa ) as well as acrolein are systematically distributed throughout the body
eventually reaching the tumor but also causing undesired toxic side effects . however , for mouse models of human
breast cancer , the long terminal repeat ( ltr ) of the mouse mammary tumor virus
( mmtv ) has emerged as the most potent and frequently used promoter to drive
transgene expression [ 812 ] . we have also shown
previously that , when combined with ifosfamide - based chemotherapy , intratumoral
injection as well as instillation of capsules containing cyp2b1-expressing ( but
not virus - producing ) cells into tumor - supplying blood vessels leads to a
significantly increased tumoricidal effect on experimentally generated tumors
in mice [ 22 , 23 ] or on inoperable
pancreatic tumors in humans [ 24 , 25 ] . in this study , we have constructed
mlv - based replication - deficient retroviral promoter conversion ( procon ) vectors
for the use in breast cancer gene therapy in combination with the in situ vector supply and delivery
system described above . we further show
that the generated therapeutic cyp2b1 protein is enzymatically active and exerts
a strong cell killing effect on infected breast cancer cells upon treatment
with ifosfamide or cyclophosphamide in vitro . here , for in vivo
investigations of the efficacy of the therapeutic vectors , we chose the
following experimental setup : we wanted to determine if efficient infection of
target cells is ( i ) also achievable by cocultivation of virus - producing cells
and target cells in vitro instead of using
high - titer virus suspensions for in vitro infections and ( ii ) in a mixed tumor
model in vivo , thus mimicking a future therapeutic situation in which
viruses would be released from capsules . in 293-based virus - producing 2gp19talf
cells , vector ppcemm1 ( see figure 1 ) that was used in this study allows
the expression of the egfp reporter gene from an mlv promoter / cmv enhancer
cassette while in infected cells after promoter conversion , expression of the
egfp gene is driven by the heterologous mmtv promoter . the egfp reporter gene was replaced with the therapeutic gene coding for
cytochrome p450 2b1 ( cyp2b1 ) . to evaluate the sensitizing effect of cyp2b1 on t-47d cells upon treatment with the anticancer prodrug ifosfamide ,
t-47d cells infected with ppccmm1 and noninfected t-47d control cells were
treated with increasing amounts of ifosfamide ( ranging from 0 to 3 mm ) for five
consecutive days after which cell viabilities were determined using an xtt
assay . the procon vectors we have generated in this
study are a further development of an earlier , a -galactosidase reporter
gene - containing vector that was used for the generation of virus - producing
cells for encapsulation experiments . as demonstrated by western blotting and with
resorufin assays , a high concentration of cyp2b1 in virus - producing cells is
ascertained by the strong mlv promoter that drives the expression of the
therapeutic gene . ppccmm1 and the reporter gene - harboring vector
ppcemm1 allow the generation of high virus titers in virus - packaging cells and
productive infection of target cells in
vitro . incorporation of the mmtv promoter into the retroviral vectors allowed high transgene
expression of the egfp reporter gene or the therapeutic cyp2b1 gene in infected
cells . in this study
, we also showed that the level of
enzymatically active cyp2b1 generated in infected cells with the newly
developed vectors is pronounced enough to create a highly increased cell
killing effect on t-47d / ppccmm1 cells upon ifosfamide or cyclophosphamide treatment
in vitro . taken together , the newly generated vectors
constitute the basis for the development of a novel breast cancer gdept system :
the system is anticipated to employ the encapsulation of cells that ( i )
generate the therapeutic enzyme cyp2b1 and , ( ii ) in addition , produce viral
vector particles that transfer the therapeutic cyp2b1 gene directly into tumor
cells . | [
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in sweden , the prevalence of diabetes mellitus has recently been estimated to be 4.4% among the adult population , with type 2 diabetes accounting for approximately 90% of all cases . as diabetes is a major cause of morbidity and premature mortality
, the disease has a significant impact on healthcare costs and quality of life ( qol ) [ 2 , 3 ] .
it is well established that adequate glycemic control [ glycosylated hemoglobin ( hba1c ) below 6.57% ] reduces the risk of diabetes - related complications , and this is therefore the key goal in management of type 2 diabetes .
insulin is one of the most effective hba1c - lowering interventions and , due to the progressive nature of type 2 diabetes at higher hba1c - levels , insulin may be the only treatment option for many patients . according to the swedish national board of health and welfare s clinical guidelines on management of type 2 diabetes ,
the first preventive measure to decrease hba1c is lifestyle intervention and later initiation of metformin .
when these measures fail to control glucose levels and the hba1c - level rises to 7.5% , treatment with a sulfonylurea ( su ) or insulin should be initiated . there are alternative approved treatment options that may be initiated before prescribing insulin , if hba1c levels are < 7.5% and the patient does not respond to metformin , including sitagliptins and glp-1 receptor antagonists .
however , many physicians choose to initiate insulin before the patient has reached an hba1c level of 7.5% .
insulin treatment has a number of disadvantages , such as weight gain , reaction from the injection and hypoglycemia , and in particular during the early period of initiating insulin , patients most likely show psychologic insulin resistance resulting in complications and increased healthcare costs .
although there are a number of published studies evaluating the costs of swedish patients with type 2 diabetes [ 911 ] , there are to our knowledge no swedish studies comparing the costs of pre- and post - initiation of insulin .
there is also little evidence when stratifying annual medical costs before and after initiation of insulin in patients with hba1c - level of < 7.5% , where other treatment options are still available , versus hba1c - level 7.5% .
the aims of this study were to evaluate the healthcare costs of patients with type 2 diabetes initiating insulin on top of metformin and/or su , and to understand if these costs differ if the patient has reached hba1c levels of 7.5% or not . if the results from this study indicate that treatment with insulin on top of metformin is related to substantially higher healthcare costs compared with treatment with metformin and/or su alone , this could be an indication that other oral anti - diabetic drugs ( oads ) apart from metformin could be favored while hba1c is still below critical levels .
this could have an impact both in terms of savings of healthcare resources and on patients qol .
this study is a register - based retrospective cohort study including swedish patients with type 2 diabetes who started treatment with metformin and/or su and were later prescribed insulin .
the register includes information on patients with type 2 diabetes receiving treatment within the county council from 2003 to 2004 onwards , as well as information on caregiver contacts , laboratory tests ( including hba1c values ) , diagnoses , procedures , prescribed drugs , and demographics .
information on filled prescriptions and devices were retrieved from the prescribed drug register , which is a national register held at the national board of health and welfare containing information on all prescribed medicines and pharmaceutical aids dispensed at swedish pharmacies since june 2005 .
information on mortality during the follow - up period was retrieved from the cause of death register at the national board of health and welfare .
the register includes nationwide data since 1961 and includes underlying and contributory causes of death according to the international classification of disease ( icd ) system .
this study included all patients in the diabetes register identified as being prescribed at least one prescription of metformin and/or su from 2003 to 2010 and later prescribed treatment with insulin .
patients were excluded from the study if they were being prescribed insulin before june 2005 , had an initial diagnosis of type 1 diabetes , had records of prescriptions of oads other than metformin or su prior to the index date , or if they had no records of metformin or su prior to the index date .
after approval from an ethical committee , data were extracted from the diabetes register based on the inclusion criteria and linked to the administrative registers at the national board of health and welfare through the patients national registration number .
all identifiable data were replaced with new study ids to de - identify the data . for each identified patient ,
the index date was defined as the date of the first insulin prescription in the diabetes register during the period january 1 , 2005 through december 31 , 2009 .
the hba1c level at initiation of insulin used in the stratified analysis was chosen as the last hba1c lab value measured prior to the index date .
the aim was to choose a value measured within 1 month and not longer than 3 months before insulin initiation .
hba1c was measured using the mono - s method . for conversion to measurements with the dcct / ngsp method ,
the following conversion formula may be used : hba1c ( ngsp ) ( % ) = 0.956 hba1c ( mono s ) ( % ) + 1.182 ( see e.g. , http://www.hba1c.nu/ ) .
annual medical costs were computed from the third party payer perspective for healthcare visits , treatment interventions , and procedures ( described as drg ) and prescriptions of anti - glycemic medications , diabetic devices , and aids . for the purpose of estimating costs incurred in the management before and after the initiation of insulin , unit costs for individual procedures
unit costs for healthcare resources were obtained through three different county council lists for costs and calculated as a mean of the three measures ( including skane county , vastragotaland region county council and norrlands lans county council ) according to swedish standards .
these unit costs varied by resource use , and were applied to the resource utilization items , which were used to calculate the overall medical costs of healthcare utilization . costs for prescribed drugs were primarily based on the information from the prescribed drug register including updated information on national costs from the latest reference year .
costs were calculated on an individual basis by summing the products of unit costs with quantities of different types of resources consumed and presented as means for all patients .
the latest reference year for unit costs was used ( sek 2012 ; 1 = 8.4 sek on march 25 , 2013 ) .
analyses were conducted using patient - level data , but all reporting was on an aggregated level . all data management and statistical analyses were performed using sas 9.2 ( sas institute inc . ,
the resource use items available within this study were visits to primary care , outpatient admissions to medical wards , surgeries and procedures through drg - codes , prescription pharmaceuticals , and devices for glucose - monitoring and insulin administration .
annual quantities of these resource items during the 12-month post - initiation of insulin were determined for each patient .
healthcare costs during the 12-month post - initiation of insulin were estimated by multiplying quantities of resource use by unit costs from published sources .
similarly , annual quantities of resource use during the year pre - initiation of insulin were determined for each patient , and corresponding healthcare costs were estimated by multiplying quantities of resource use with unit costs .
descriptive statistics on an aggregated level were used to present patient characteristics , resource utilization , and costs for all patients in the dataset . for continuous variables , the arithmetic mean , standard deviation ( sd ) , minimum , and maximum
are presented . for categorical variables , the proportions ( percentage ) in each category are presented .
the normality of the distributions was tested employing the shapiro wilks test and by plotting the data .
if found not to be normally distributed , means and sd were estimated using the bias corrected accelerated bootstrapping method with replacement . to test differences between costs before and after the index date
, a t test was used to measure if the difference between the two costs was separated from zero .
there were no missing values in the dataset and imputation of missing data was not necessary .
variables describing patient characteristics included age at the index date , sex , height , weight , co - morbidities , hba1c level at the index date , systolic blood pressure at the index date , and diastolic blood pressure at the index date .
the estimated healthcare costs were subsequently stratified by hba1c level at index date and statistical tests were used to compare costs pre- and post - initiation of insulin for each hba1c - group separately ( group 1 < 7.5% , group 2 7.5% ) .
the register includes information on patients with type 2 diabetes receiving treatment within the county council from 2003 to 2004 onwards , as well as information on caregiver contacts , laboratory tests ( including hba1c values ) , diagnoses , procedures , prescribed drugs , and demographics .
information on filled prescriptions and devices were retrieved from the prescribed drug register , which is a national register held at the national board of health and welfare containing information on all prescribed medicines and pharmaceutical aids dispensed at swedish pharmacies since june 2005 .
information on mortality during the follow - up period was retrieved from the cause of death register at the national board of health and welfare .
the register includes nationwide data since 1961 and includes underlying and contributory causes of death according to the international classification of disease ( icd ) system .
this study included all patients in the diabetes register identified as being prescribed at least one prescription of metformin and/or su from 2003 to 2010 and later prescribed treatment with insulin .
patients were excluded from the study if they were being prescribed insulin before june 2005 , had an initial diagnosis of type 1 diabetes , had records of prescriptions of oads other than metformin or su prior to the index date , or if they had no records of metformin or su prior to the index date .
after approval from an ethical committee , data were extracted from the diabetes register based on the inclusion criteria and linked to the administrative registers at the national board of health and welfare through the patients national registration number .
all identifiable data were replaced with new study ids to de - identify the data . for each identified patient ,
the index date was defined as the date of the first insulin prescription in the diabetes register during the period january 1 , 2005 through december 31 , 2009 .
the hba1c level at initiation of insulin used in the stratified analysis was chosen as the last hba1c lab value measured prior to the index date .
the aim was to choose a value measured within 1 month and not longer than 3 months before insulin initiation .
hba1c was measured using the mono - s method . for conversion to measurements with the dcct / ngsp method ,
the following conversion formula may be used : hba1c ( ngsp ) ( % ) = 0.956 hba1c ( mono s ) ( % ) + 1.182 ( see e.g. , http://www.hba1c.nu/ ) .
annual medical costs were computed from the third party payer perspective for healthcare visits , treatment interventions , and procedures ( described as drg ) and prescriptions of anti - glycemic medications , diabetic devices , and aids . for the purpose of estimating costs incurred in the management before and after the initiation of insulin , unit costs for individual procedures
unit costs for healthcare resources were obtained through three different county council lists for costs and calculated as a mean of the three measures ( including skane county , vastragotaland region county council and norrlands lans county council ) according to swedish standards .
these unit costs varied by resource use , and were applied to the resource utilization items , which were used to calculate the overall medical costs of healthcare utilization .
costs for prescribed drugs were primarily based on the information from the prescribed drug register including updated information on national costs from the latest reference year .
costs were calculated on an individual basis by summing the products of unit costs with quantities of different types of resources consumed and presented as means for all patients .
the latest reference year for unit costs was used ( sek 2012 ; 1 = 8.4 sek on march 25 , 2013 ) .
analyses were conducted using patient - level data , but all reporting was on an aggregated level .
all data management and statistical analyses were performed using sas 9.2 ( sas institute inc . , cary , nc , usa ) .
the resource use items available within this study were visits to primary care , outpatient admissions to medical wards , surgeries and procedures through drg - codes , prescription pharmaceuticals , and devices for glucose - monitoring and insulin administration .
annual quantities of these resource items during the 12-month post - initiation of insulin were determined for each patient .
healthcare costs during the 12-month post - initiation of insulin were estimated by multiplying quantities of resource use by unit costs from published sources .
similarly , annual quantities of resource use during the year pre - initiation of insulin were determined for each patient , and corresponding healthcare costs were estimated by multiplying quantities of resource use with unit costs .
descriptive statistics on an aggregated level were used to present patient characteristics , resource utilization , and costs for all patients in the dataset . for continuous variables , the arithmetic mean , standard deviation ( sd ) , minimum , and maximum are presented . for categorical variables , the proportions ( percentage ) in each category are presented .
the normality of the distributions was tested employing the shapiro wilks test and by plotting the data .
if found not to be normally distributed , means and sd were estimated using the bias corrected accelerated bootstrapping method with replacement . to test differences between costs before and after the index date
, a t test was used to measure if the difference between the two costs was separated from zero .
there were no missing values in the dataset and imputation of missing data was not necessary .
variables describing patient characteristics included age at the index date , sex , height , weight , co - morbidities , hba1c level at the index date , systolic blood pressure at the index date , and diastolic blood pressure at the index date .
the estimated healthcare costs were subsequently stratified by hba1c level at index date and statistical tests were used to compare costs pre- and post - initiation of insulin for each hba1c - group separately ( group 1 < 7.5% , group 2 7.5% ) .
in total , 667 patients initiated on insulin during the study period were identified and extracted from the diabetes register . after removing patients who had been treated with oads other than metformin or su prior to the index date
, had a primary diagnosis of type 1 diabetes , had been prescribed insulin prior to the index date , had not been prescribed metformin or su prior to the index date , and had < 365 days follow - up before or after the index date , 100 patients were eligible for the analysis.fig .
oad oral anti - diabetic drugs , met metformin , su sulfonylurea , t1 dm type 1 diabetes , t2 dm type 2 diabetes , dr diabetes register , pdr prescribed drug register patient exclusion .
oad oral anti - diabetic drugs , met metformin , su sulfonylurea , t1 dm type 1 diabetes , t2 dm type 2 diabetes , dr diabetes register , pdr prescribed drug register patient characteristics are demonstrated in table 1 .
the mean age of the patients was 64 ( sd 10 ) years , ranging from 35 to 80 years .
most patients were men ( n = 59 , 59%).table 1patient characteristics , all patients ( n = 100)patient characteristicsage ( years)64 ( 10 ) [ 3589]sex , female41%disease description co - morbidities hypertension49% cardiovascular disease14% hba1c level at initiation of insulin ( % ) 8 ( 1.7 ) [ 4.114 ] systolic blood pressure ( mmhg)141.1 ( 20.7 ) [ 105225 ] diastolic blood pressure ( mmhg)80.1 ( 10.7 ) [ 50110]data are expressed as mean ( sd ) [ range ] , unless otherwise indicated
sd standard deviation patient characteristics , all patients ( n = 100 ) data are expressed as mean ( sd ) [ range ] , unless otherwise indicated
sd standard deviation there were 41 patients who were initiated on insulin at hba1c levels < 7.5% , and 59 patients at hba1c levels 7.5% .
patient characteristics comparing patients with hba1c level < 7.5% and 7.5% at the index date are shown in table 2 .
patients with hba1c levels < 7.5% were older than those with hba1c levels 7.5% at the index date [ 67 ( sd 7 ) vs. 63 ( sd 11 ) years , p = 0.051 ] .
the mean hba1c level at the index date was 6.7% ( sd 0.7% ) among the hba1c level < 7.5% group and 8.9% ( sd 1.6% ) among the hba1c 7.5% group ( p = 0.0001 ) .
there was also a statistically significant difference between the two groups in terms of diastolic blood pressure [ 77.2 mmhg ( sd 9.1 ) vs. 82.1 mmhg ( sd 11.3 ) , p = 0.018 ] .
even though total number of co - morbidities did not differ between the two groups , patients with hba1c levels < 7.5% had a higher number of cardiovascular events ( see table 3).table 2patient characteristics comparing patients with hba1c levels < 7.5% and 7.5% at initiation of insulinhba1c < 7.5% ( n = 41)hba1c 7.5% ( n = 59 )
p valuepatient characteristics age67 ( 7)63 ( 11)0.051 female , n ( % ) 10 ( 24)24 ( 41)0.091 height172 ( 9)173 ( 12)0.576 weight85 ( 16)96 ( 27)0.477disease description years since index date2.2 ( 0.9)2.2 ( 1.2)0.69 years since diagnosis4.2 ( 0.9)4.2 ( 1.2)0.691 hba1c level at initiation of insulin , % ( sd)6.7 ( 0.7)8.9 ( 1.6)<0.0001 systolic blood pressure , mmhg ( sd)138.4 ( 17.9)142.9 ( 22.4)0.482 diastolic blood pressure , mmhg ( sd)77.2 ( 9.1)82.1 ( 11.3)0.018data are expressed as mean ( sd ) , unless otherwise indicated
sd standard deviationtable 3cardiovascular events stratified by hba1c level at index datepatients with event , n ( % ) hba1c level at index date<7.5% ( n = 41)7.5% ( n = 59)hypertension21 ( 51)39 ( 66)hypertensive heart and renal disease2 ( 5)2 ( 3)angina pectoris4 ( 10)9 ( 15)myocardial infarction2 ( 5)1 ( 2)ischemic heart disease8 ( 20)10 ( 17)heart failure9 ( 22)5 ( 8)intracerebral hemorrhage0 ( 0)3 ( 5)chronic kidney disease0 ( 0)2 ( 3)total4671 patient characteristics comparing patients with hba1c levels < 7.5% and 7.5% at initiation of insulin data are expressed as mean ( sd ) , unless otherwise indicated
sd standard deviation cardiovascular events stratified by hba1c level at index date total medical costs for the total cohort , and stratified by hba1c - level , are shown in tables 4 and 5 , respectively.table 4total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin ( n = 100)cost ( sek)pre - index datepost - index date
p value*meansdmeansdhealthcare visits11,79510,36618,28916,955<0.0001 visits to primary care6,8014,53910,4867,202<0.0001 visits to medical ward4,9949,5657,80316,2240.039procedures2,89511,6662,84512,6391.000filled prescriptions2,5401,92910,5225,624<0.0001total costs17,23017,22831,65624,331<0.0001
sd standard deviation * t testtable 5total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin stratified by hba1c - level at index datecost ( sek)hba1c < 7.5% ( n = 41)hba1c 7.5% ( n = 59)pre - index datepost - index datepre - index datepost - index datemeansdmeansd
p - valuemeansdmeansd
p value*healthcare visits13,80310,07220,02217,1540.11810,40010,42317,08416,856<0.0001 visits to primary care7,5194,12510,0016,1260.0816,3024,77610,8237,898<0.0001 visits to medical ward6,2849,95110,02116,0580.2484,0989,2686,26216,2960.711procedures8524,1115,99318,9720.4534,31514,6856583,5450.289filled prescriptions3,0242,3069,7324,657<0.00012,2031,55111,0716,187<0.0001total costs17,67812,94635,74730,411<0.000116,91819,76928,81318,779<0.0001
sd standard deviation * t test total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin ( n = 100 )
sd standard deviation total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin stratified by hba1c - level at index date
sd standard deviation the total medical healthcare costs for the entire cohort the year before initiation of insulin ( table 4 ) were sek 17,230 ( sd 17,228 ) , and the year after were sek 31,656 ( sd 24,331 ) ( p < 0.0001 ) . the highest proportion of costs before the index date was as a result of visits to primary care followed by costs due to visits to medical wards , procedures and medications , whilst the highest proportion after the index date were due to medications , followed by visits to primary care , visits to medical wards , and procedures . when stratifying total medical healthcare costs by hba1c group ( table 5 ) , the group with hba1c levels < 7.5% had total medical costs of sek 17,678 ( sd 12,946 ) the year before the index date , and sek 35,747 ( sd 30,411 ) the year after the index date ( p < 0.0001 ) .
the group with hba1c levels 7.5% had total medical costs of sek 16,918 ( sd 19,769 ) the year before the index date and sek 28,813 ( sd 18,779 ) the year after the index date ( p < 0.0001 ) . in patients with hba1c levels
< 7.5% , the year before initiation of insulin , the highest proportion of costs was due to visits to primary care , followed by costs due to visits to medical wards , medications , and procedures . the year after the index date , the highest costs were due to medications , followed by costs due to visits to primary care , visits to medical wards , and procedures . in the group with hba1c levels 7.5% at the index date , the year before initiation of insulin , the highest proportion of costs was due to visits to primary care , followed by costs due to visits to medical wards , procedures , and medications . the year after the index date
, the highest costs were due to medications , followed by costs due to visits to primary care , visits to medical wards , and procedures .
the total medical costs due to health care visits in the entire patient cohort were sek 11,795 ( sd 10,366 ) the year before the index date and sek 18,289 ( sd 16,955 ) the year after the index date ( p < 0.0001 ) .
the highest proportion of this was due to visits to primary care both before and after the index date [ sek 6,801 ( sd 4,539 ) pre - index ; sek 10,486 ( sd 7,202 ) post - index , p < 0.0001 ] .
the costs due to visits to medical wards were sek 4,994 ( sd 9,565 ) the year before the index date and sek 7,803 ( sd 16,224 ) the year after the index date ( p = 0.039 ) . even though not statistically significant
, the costs related to visits to medical wards included visits to physicians and nurses , both before and after the index date .
there were no statistically significant differences in mean costs the year before and after the index date due to procedures .
there were no statistically significant differences in costs due to health care visits before and after the index date compared with the year before the index date in patients with hba1c levels < 7.5% at the index date ( p = 0.118 ) .
the mean total medical costs due to health care visits were lower in the group with hba1c levels 7.5% at the index date compared with the group with hba1c levels < 7.5% , both the year before the index date [ sek 10,400 ( sd 10,423 ) ] , and the year after [ sek 17,084 ( sd 16,856 ) ] .
the total mean costs the year after the index date were statistically significantly higher than the year before ( p < 0.0001 ) .
the highest proportion of these costs were due to visits to primary care [ sek 6,302 ( sd 4,776 ) pre - index ; sek 10,823 ( sd 7,898 ) post - index , p < 0.0001 ] , whereas the costs due to visits to medical wards incurred sek 4,098 ( sd 9,268 ) the year before the index date and sek 6,262 ( sd 16,296 ) , the year after the index date ( p = 0.711 ) . in the total cohort , there was a statistically significant increase in mean total medical costs related to filled prescriptions the year after the index date [ sek 10,522 ( sd 5,624 ) ] compared with the year before the index date [ sek 2,540 ( sd 1,929 ) ; p < 0.0001 ] .
the highest mean costs related to insulin devices and aids were due to test sticks both before and after the index date [ sek 1,447 ( sd 1,554 ) pre - index ; sek 3,342 ( sd 3,290 ) post - index ; p < 0.0001 ] ( data not presented in the table ) . the total mean costs due to filled prescriptions among the hba1c < 7.5% group were statistically significantly higher the year after the index date compared with the year before the index date [ sek 3,024 ( sd 2,306 ) pre - index ; sek 9,732 ( sd 4,657 ) post - index ; p < 0.0001 ] .
the same statistically significant difference was seen in the group with hba1c levels 7.5% at the index date [ sek 2,203 ( sd 1,551 ) pre - index ; sek 11,071 ( sd 6,187 ) post - index ; p < 0.0001 ] .
the mean age of the patients was 64 ( sd 10 ) years , ranging from 35 to 80 years .
most patients were men ( n = 59 , 59%).table 1patient characteristics , all patients ( n = 100)patient characteristicsage ( years)64 ( 10 ) [ 3589]sex , female41%disease description co - morbidities hypertension49% cardiovascular disease14% hba1c level at initiation of insulin ( % ) 8 ( 1.7 ) [ 4.114 ] systolic blood pressure ( mmhg)141.1 ( 20.7 ) [ 105225 ] diastolic blood pressure ( mmhg)80.1 ( 10.7 ) [ 50110]data are expressed as mean ( sd ) [ range ] , unless otherwise indicated
sd standard deviation patient characteristics , all patients ( n = 100 ) data are expressed as mean ( sd ) [ range ] , unless otherwise indicated
sd standard deviation there were 41 patients who were initiated on insulin at hba1c levels < 7.5% , and 59 patients at hba1c levels 7.5% .
patient characteristics comparing patients with hba1c level < 7.5% and 7.5% at the index date are shown in table 2 .
patients with hba1c levels < 7.5% were older than those with hba1c levels 7.5% at the index date [ 67 ( sd 7 ) vs. 63 ( sd 11 ) years , p = 0.051 ] .
the mean hba1c level at the index date was 6.7% ( sd 0.7% ) among the hba1c level < 7.5% group and 8.9% ( sd 1.6% ) among the hba1c 7.5% group ( p = 0.0001 ) .
there was also a statistically significant difference between the two groups in terms of diastolic blood pressure [ 77.2 mmhg ( sd 9.1 ) vs. 82.1 mmhg ( sd 11.3 ) , p = 0.018 ] .
even though total number of co - morbidities did not differ between the two groups , patients with hba1c levels
< 7.5% had a higher number of cardiovascular events ( see table 3).table 2patient characteristics comparing patients with hba1c levels < 7.5% and 7.5% at initiation of insulinhba1c < 7.5% ( n = 41)hba1c 7.5% ( n = 59 )
p valuepatient characteristics age67 ( 7)63 ( 11)0.051 female , n ( % ) 10 ( 24)24 ( 41)0.091 height172 ( 9)173 ( 12)0.576 weight85 ( 16)96 ( 27)0.477disease description years since index date2.2 ( 0.9)2.2 ( 1.2)0.69 years since diagnosis4.2 ( 0.9)4.2 ( 1.2)0.691 hba1c level at initiation of insulin , % ( sd)6.7 ( 0.7)8.9 ( 1.6)<0.0001 systolic blood pressure , mmhg ( sd)138.4 ( 17.9)142.9 ( 22.4)0.482 diastolic blood pressure , mmhg ( sd)77.2 ( 9.1)82.1 ( 11.3)0.018data are expressed as mean ( sd ) , unless otherwise indicated
sd standard deviationtable 3cardiovascular events stratified by hba1c level at index datepatients with event , n ( % ) hba1c level at index date<7.5% ( n = 41)7.5% ( n = 59)hypertension21 ( 51)39 ( 66)hypertensive heart and renal disease2 ( 5)2 ( 3)angina pectoris4 ( 10)9 ( 15)myocardial infarction2 ( 5)1 ( 2)ischemic heart disease8 ( 20)10 ( 17)heart failure9 ( 22)5 ( 8)intracerebral hemorrhage0 ( 0)3 ( 5)chronic kidney disease0 ( 0)2 ( 3)total4671 patient characteristics comparing patients with hba1c levels < 7.5% and 7.5% at initiation of insulin data are expressed as mean ( sd ) , unless otherwise indicated
sd standard deviation cardiovascular events stratified by hba1c level at index date
total medical costs for the total cohort , and stratified by hba1c - level , are shown in tables 4 and 5 , respectively.table 4total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin ( n = 100)cost ( sek)pre - index datepost - index date
p value*meansdmeansdhealthcare visits11,79510,36618,28916,955<0.0001 visits to primary care6,8014,53910,4867,202<0.0001 visits to medical ward4,9949,5657,80316,2240.039procedures2,89511,6662,84512,6391.000filled prescriptions2,5401,92910,5225,624<0.0001total costs17,23017,22831,65624,331<0.0001
sd standard deviation * t testtable 5total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin stratified by hba1c - level at index datecost ( sek)hba1c < 7.5% ( n = 41)hba1c 7.5% ( n = 59)pre - index datepost - index datepre - index datepost - index datemeansdmeansd
p - valuemeansdmeansd
p value*healthcare visits13,80310,07220,02217,1540.11810,40010,42317,08416,856<0.0001 visits to primary care7,5194,12510,0016,1260.0816,3024,77610,8237,898<0.0001 visits to medical ward6,2849,95110,02116,0580.2484,0989,2686,26216,2960.711procedures8524,1115,99318,9720.4534,31514,6856583,5450.289filled prescriptions3,0242,3069,7324,657<0.00012,2031,55111,0716,187<0.0001total costs17,67812,94635,74730,411<0.000116,91819,76928,81318,779<0.0001
sd standard deviation * t test total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin ( n = 100 )
sd standard deviation total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin stratified by hba1c - level at index date
sd standard deviation the total medical healthcare costs for the entire cohort the year before initiation of insulin ( table 4 ) were sek 17,230 ( sd 17,228 ) , and the year after were sek 31,656 ( sd 24,331 ) ( p < 0.0001 ) .
the highest proportion of costs before the index date was as a result of visits to primary care followed by costs due to visits to medical wards , procedures and medications , whilst the highest proportion after the index date were due to medications , followed by visits to primary care , visits to medical wards , and procedures .
when stratifying total medical healthcare costs by hba1c group ( table 5 ) , the group with hba1c levels < 7.5% had total medical costs of sek 17,678 ( sd 12,946 ) the year before the index date , and sek 35,747 ( sd 30,411 ) the year after the index date ( p < 0.0001 ) .
the group with hba1c levels 7.5% had total medical costs of sek 16,918 ( sd 19,769 ) the year before the index date and sek 28,813 ( sd 18,779 ) the year after the index date ( p < 0.0001 ) . in patients with hba1c levels
< 7.5% , the year before initiation of insulin , the highest proportion of costs was due to visits to primary care , followed by costs due to visits to medical wards , medications , and procedures . the year after the index date , the highest costs were due to medications , followed by costs due to visits to primary care , visits to medical wards , and procedures . in the group with hba1c levels 7.5% at the index date , the year before initiation of insulin , the highest proportion of costs was due to visits to primary care , followed by costs due to visits to medical wards , procedures , and medications . the year after the index date , the highest costs were due to medications , followed by costs due to visits to primary care , visits to medical wards , and procedures
the total medical costs due to health care visits in the entire patient cohort were sek 11,795 ( sd 10,366 ) the year before the index date and sek 18,289 ( sd 16,955 ) the year after the index date ( p < 0.0001 ) .
the highest proportion of this was due to visits to primary care both before and after the index date [ sek 6,801 ( sd 4,539 ) pre - index ; sek 10,486 ( sd 7,202 ) post - index , p < 0.0001 ] .
the costs due to visits to medical wards were sek 4,994 ( sd 9,565 ) the year before the index date and sek 7,803 ( sd 16,224 ) the year after the index date ( p = 0.039 ) . even though not statistically significant
, the costs related to visits to medical wards included visits to physicians and nurses , both before and after the index date .
there were no statistically significant differences in mean costs the year before and after the index date due to procedures .
there were no statistically significant differences in costs due to health care visits before and after the index date compared with the year before the index date in patients with hba1c levels < 7.5% at the index date ( p = 0.118 ) .
the mean total medical costs due to health care visits were lower in the group with hba1c levels 7.5% at the index date compared with the group with hba1c levels < 7.5% , both the year before the index date [ sek 10,400 ( sd 10,423 ) ] , and the year after [ sek 17,084 ( sd 16,856 ) ] . the total mean costs the year after the index date were statistically significantly higher than the year before ( p < 0.0001 ) .
the highest proportion of these costs were due to visits to primary care [ sek 6,302 ( sd 4,776 ) pre - index ; sek 10,823 ( sd 7,898 ) post - index , p < 0.0001 ] , whereas the costs due to visits to medical wards incurred sek 4,098 ( sd 9,268 ) the year before the index date and sek 6,262 ( sd 16,296 ) , the year after the index date ( p = 0.711 ) . in the total cohort , there was a statistically significant increase in mean total medical costs related to filled prescriptions the year after the index date [ sek 10,522 ( sd 5,624 ) ] compared with the year before the index date [ sek 2,540 ( sd 1,929 ) ; p < 0.0001 ] .
the highest mean costs related to insulin devices and aids were due to test sticks both before and after the index date [ sek 1,447 ( sd 1,554 ) pre - index ; sek 3,342 ( sd 3,290 ) post - index ; p < 0.0001 ] ( data not presented in the table ) .
the total mean costs due to filled prescriptions among the hba1c < 7.5% group were statistically significantly higher the year after the index date compared with the year before the index date [ sek 3,024 ( sd 2,306 ) pre - index ; sek 9,732 ( sd 4,657 ) post - index ; p < 0.0001 ] .
the same statistically significant difference was seen in the group with hba1c levels 7.5% at the index date [ sek 2,203 ( sd 1,551 ) pre - index ; sek 11,071 ( sd 6,187 ) post - index ; p < 0.0001 ] .
this study aimed at estimating and comparing annual medical costs the year before and after initiation of insulin ( index date ) among swedish patients with type 2 diabetes .
the study also aimed at estimating these costs stratified by hba1c level at the date of initiation of insulin , based on the recommended cut - off level ( hba1c 7.5% ) .
our results demonstrated almost doubled , statistically significant increases in mean annual costs the year after the initiation of insulin compared with the year before .
the highest proportion of mean annual medical costs was due to visits to primary care the year before the index date , but shifted to costs due to filled prescriptions the year after .
this demonstrates that filled prescriptions of insulin have a significant impact on the total medical costs .
the increased costs of filled prescriptions were also due to increased filled packages of devices and aids such as glucose monitoring , injection needles , and lancets .
besides filled prescriptions , the increased costs the year after initiation of insulin were also due to increased visits to nurses and physicians , to both medical wards ( not statistically significant ) , and to primary care .
as can be expected in a diabetes population , many of the patients in this study had cardiovascular co - morbidities that might explain some of the increased costs the year after initiation of insulin .
there were no major differences in demographic and clinical characteristics at the index date when comparing patients with hba1c - levels < 7.5% or 7.5% at the index date .
both groups had statistically significant increased mean annual costs the year after initiation of insulin compared with the year before .
interestingly , patients with hba1c - levels < 7.5% at the index date had higher total medical costs , both the year before the index date and the year after , compared with the hba1c - level 7.5% group .
this might be explained by the higher proportion of major cardiovascular events in this group , but the major increase in costs the year before and after index date was still explained by an increase in filled prescriptions . for both groups ,
costs were mainly dominated by costs due to health care visits before the index date , whereas costs due to filled prescriptions were more prominent in both groups after the index date .
there are a number of published studies assessing the health - related costs in patients treated with insulin . in a german study published in 1997 , estimation of costs for insulin
treated patients was approximately six times as high as those for patients treated with oads , and 30 times as high as for patients treated with life - style interventions through specific diets .
the authors demonstrated that there was a significant increase in blood glucose devices during the 6 months after initiation of insulin , and that the mean 6-month costs increased from 579 to 961 .
a more recent study conducted in spain in 2011 reported that mean total healthcare costs per patient 6 months before and after insulin start were 639 and 1,110 , respectively .
mean total costs 6 months after insulin treatment was initiated included costs of hospitalization ( 31% ) , insulin ( 16% ) , primary care ( 14% ) , blood glucose monitoring ( 14% ) , specialized care ( 13% ) , oads ( 8% ) , and other diabetes - related treatments ( 4% ) . in a canadian cost - effectiveness study from 2011 , basic treatment with metformin
the average lifetime cost ( direct healthcare cost ) was reported to be $ 39,924 for the basic treatment with metformin .
the corresponding cost was $ 40,669 for metformin plus su , $ 47,191 for metformin plus dpp-4 inhibitor , $ 47,348 for metformin plus basal insulin , and $ 52,367 for metformin plus biphasic insulin .
hence , the incremental cost of adding basal insulin or biphasic insulin to the metformin treatment was $ 7,424 and $ 12,443 , respectively .
the results in our study demonstrate that increased medical healthcare costs the year before and after initiation of insulin are comparable with the results of previous studies [ 1619 ] . together
these data concur that the initiation of insulin treatment in type 2 diabetic patients increases medical costs , both in terms of increased costs due to filled prescriptions of medications and of devices , but also due to increased costs due to health care visits .
, our study demonstrated that the highest proportion of costs the year after the index date were due to filled prescriptions ( given that our study did not include information on hospitalizations ) .
studies have also demonstrated the relationship between costs and hba1c level . in a study by aagren et al . , from 2011 , the relationship between glycemic control , measured by hba1c - level , and short - term healthcare costs was assessed .
the population consisted of commercially insured diabetic patients ( hba1c level 6% ) in the united states ; 34,469 patients with type 2 diabetes and 1,837 with type 1 diabetes .
the study concluded that the hba1c - level ( and other factors ) significantly correlated with diabetes - related short - term medical costs for both patients with type 1 and type 2 diabetes .
specifically , a 1%-point increase in hba1c will , on average , lead to a 4.4% increase in diabetes - related medical costs for type 2 diabetes .
these results were not comparable with the results in our study , which is probably explained by the higher major cardiovascular co - morbidities in this group .
one reason for the difference in results compared to our study might be the selected study population . in our study
, costs were calculated for patients who had at least 365 days follow - up both before and after the index date . by using such an approach , patients who had < 365 days usage of metformin or su before the index date , and patients who died before 365 days after the index date ,
exclusion of these patients might therefore have biased the study population by allowing only patients with less severe disease to be included in the analysis .
first , the study sample is very small and limited to one county council and the results might therefore not be generalizable to other parts of sweden .
secondly , healthcare utilization is limited to procedures , outpatient visits at medical wards , and primary care visits , and does not account for inpatient and other outpatient care or emergency care .
there was also limited information on background data for co - morbidities and body mass index , limiting the possibilities to control for confounders .
also , when combining the two data sources , there were some differences between the different registers that led to some uncertainties in the data .
furthermore , it is important to recognize that the full clinical and economic benefits of an effective diabetes treatment , such as insulin , in the long run are not fully accounted for in our study .
in summary , despite the small sample size , this study demonstrates that mean annual medical costs almost double the year after patients are initiated on insulin .
this increase in costs is mainly due to increased visits to primary care and increased drug prescriptions .
this study also demonstrates that costs increase the year after initiation of insulin regardless of the hba1c level at initiation of insulin , which could be a rational for other treatment options when hba1c levels are still below the recommended threshold for initiation of insulin .
this register - based , retrospective cohort study was designed to evaluate the healthcare costs of patients with type 2 diabetes initiating insulin on top of metformin and/or sulfonylurea ( su ) in sweden , determine the glycosylated hemoglobin ( hba1c)-level at initiation of insulin , and stratify healthcare costs by this hba1c-level.patients were identified from the srmland county council diabetes register ; 100 patients being prescribed at least one prescription of metformin and/or su from 2003 - 2010 , and later prescribed insulin , were included.the mean age was 61 years , 59% of patients were male .
mean time since diagnosis was 4.1 years and since initiation of insulin was 2.2 years , and the mean hba1c level at index date was 8.0%.total mean costs for the whole cohort the year before initiation of insulin was sek 17,230 ( 17,228 ) and the year after was sek 31,656 ( 24,331 ) ( p < 0.0001).despite the small study sample , this study demonstrates that mean annual medical costs almost double the year after patients are initiated on insulin ; the costs increased regardless of the hba1c level at initiation of insulin , with the largest increase in costs due to increased filled prescriptions .
this register - based , retrospective cohort study was designed to evaluate the healthcare costs of patients with type 2 diabetes initiating insulin on top of metformin and/or sulfonylurea ( su ) in sweden , determine the glycosylated hemoglobin ( hba1c)-level at initiation of insulin , and stratify healthcare costs by this hba1c - level .
patients were identified from the srmland county council diabetes register ; 100 patients being prescribed at least one prescription of metformin and/or su from 2003 - 2010 , and later prescribed insulin , were included .
mean time since diagnosis was 4.1 years and since initiation of insulin was 2.2 years , and the mean hba1c level at index date was 8.0% .
total mean costs for the whole cohort the year before initiation of insulin was sek 17,230 ( 17,228 ) and the year after was sek 31,656 ( 24,331 ) ( p < 0.0001 ) . despite the small study sample
, this study demonstrates that mean annual medical costs almost double the year after patients are initiated on insulin ; the costs increased regardless of the hba1c level at initiation of insulin , with the largest increase in costs due to increased filled prescriptions .
johan lundberg is an employee of merck sharp & dohme , a subsidiary of merck & co. , inc . , owns stock options and stock in merck & co. , inc .
the analysis in this article is based on previously conducted studies , and does not involve any new studies of human or animal subjects performed by any of the authors .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . | introductionalthough insulin is one of the most effective interventions for the treatment of type 2 diabetes , its disadvantages incur substantial medical cost .
this study was designed to evaluate the medical costs of swedish type 2 diabetic patients initiating insulin on top of metformin and/or sulfonylurea ( su ) , and to evaluate if costs before and after insulin initiation differ for patients where insulin is initiated above or below the recommended glycosylated hemoglobin ( hba1c ) level ( 7.5%).methodsthis was a register - based retrospective cohort study in which patients were identified from the srmland county council diabetes register .
patients being prescribed at least one prescription of metformin and/or su from 2003 to 2010 , and later prescribed insulin , were included.resultsone hundred patients fulfilled the inclusion criteria and had at least 1 year of follow - up .
the mean age was 61 years and 59% of patients were male .
mean time since diagnosis was 4.1 years , and since initiation of insulin was 2.2 years .
the mean hba1c level at index date was 8.0% .
total mean costs for the whole cohort were sek 17,230 [ standard deviation ( sd ) 17,228 ] the year before insulin initiation , and sek 31,656 ( sd 24,331 ) the year after insulin initiation ( p < 0.0001 ) .
when stratifying by hba1c level , patients with hba1c
< 7.5% had total healthcare costs of sek 17,678 ( sd 12,946 ) the year before the index date and sek 35,747 ( sd 30,411 ) the year after ( p < 0.0001 ) .
patients with hba1c levels 7.5% had total healthcare costs of sek 16,918 ( sd 19,769 ) the year before the index date and sek 28,813 ( sd 18,779 ) the year after ( p < 0.0001).conclusiondespite the small sample size , this study demonstrates that mean annual medical costs almost double the year after patients are initiated on insulin .
the costs increased the year after insulin initiation , regardless of the hba1c level at initiation of insulin , and the largest increase in costs were due to increased filled prescriptions . | Introduction
Methods
Study Population
Statistical Considerations
Results
Patient Characteristics
Costs
Discussion
Conclusion
Key Summary Points
Conflict of interest
Compliance with ethics guidelines
Open Access | even though total number of co - morbidities did not differ between the two groups , patients with hba1c levels
< 7.5% had a higher number of cardiovascular events ( see table 3).table 2patient characteristics comparing patients with hba1c levels < 7.5% and 7.5% at initiation of insulinhba1c < 7.5% ( n = 41)hba1c 7.5% ( n = 59 )
p valuepatient characteristics age67 ( 7)63 ( 11)0.051 female , n ( % ) 10 ( 24)24 ( 41)0.091 height172 ( 9)173 ( 12)0.576 weight85 ( 16)96 ( 27)0.477disease description years since index date2.2 ( 0.9)2.2 ( 1.2)0.69 years since diagnosis4.2 ( 0.9)4.2 ( 1.2)0.691 hba1c level at initiation of insulin , % ( sd)6.7 ( 0.7)8.9 ( 1.6)<0.0001 systolic blood pressure , mmhg ( sd)138.4 ( 17.9)142.9 ( 22.4)0.482 diastolic blood pressure , mmhg ( sd)77.2 ( 9.1)82.1 ( 11.3)0.018data are expressed as mean ( sd ) , unless otherwise indicated
sd standard deviationtable 3cardiovascular events stratified by hba1c level at index datepatients with event , n ( % ) hba1c level at index date<7.5% ( n = 41)7.5% ( n = 59)hypertension21 ( 51)39 ( 66)hypertensive heart and renal disease2 ( 5)2 ( 3)angina pectoris4 ( 10)9 ( 15)myocardial infarction2 ( 5)1 ( 2)ischemic heart disease8 ( 20)10 ( 17)heart failure9 ( 22)5 ( 8)intracerebral hemorrhage0 ( 0)3 ( 5)chronic kidney disease0 ( 0)2 ( 3)total4671 patient characteristics comparing patients with hba1c levels < 7.5% and 7.5% at initiation of insulin data are expressed as mean ( sd ) , unless otherwise indicated
sd standard deviation cardiovascular events stratified by hba1c level at index date
total medical costs for the total cohort , and stratified by hba1c - level , are shown in tables 4 and 5 , respectively.table 4total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin ( n = 100)cost ( sek)pre - index datepost - index date
p value*meansdmeansdhealthcare visits11,79510,36618,28916,955<0.0001 visits to primary care6,8014,53910,4867,202<0.0001 visits to medical ward4,9949,5657,80316,2240.039procedures2,89511,6662,84512,6391.000filled prescriptions2,5401,92910,5225,624<0.0001total costs17,23017,22831,65624,331<0.0001
sd standard deviation * t testtable 5total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin stratified by hba1c - level at index datecost ( sek)hba1c < 7.5% ( n = 41)hba1c 7.5% ( n = 59)pre - index datepost - index datepre - index datepost - index datemeansdmeansd
p - valuemeansdmeansd
p value*healthcare visits13,80310,07220,02217,1540.11810,40010,42317,08416,856<0.0001 visits to primary care7,5194,12510,0016,1260.0816,3024,77610,8237,898<0.0001 visits to medical ward6,2849,95110,02116,0580.2484,0989,2686,26216,2960.711procedures8524,1115,99318,9720.4534,31514,6856583,5450.289filled prescriptions3,0242,3069,7324,657<0.00012,2031,55111,0716,187<0.0001total costs17,67812,94635,74730,411<0.000116,91819,76928,81318,779<0.0001
sd standard deviation * t test total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin ( n = 100 )
sd standard deviation total annual per patient medical healthcare costs ( sek ) before and after 1 year of initiation of insulin stratified by hba1c - level at index date
sd standard deviation the total medical healthcare costs for the entire cohort the year before initiation of insulin ( table 4 ) were sek 17,230 ( sd 17,228 ) , and the year after were sek 31,656 ( sd 24,331 ) ( p < 0.0001 ) . this register - based , retrospective cohort study was designed to evaluate the healthcare costs of patients with type 2 diabetes initiating insulin on top of metformin and/or sulfonylurea ( su ) in sweden , determine the glycosylated hemoglobin ( hba1c)-level at initiation of insulin , and stratify healthcare costs by this hba1c-level.patients were identified from the srmland county council diabetes register ; 100 patients being prescribed at least one prescription of metformin and/or su from 2003 - 2010 , and later prescribed insulin , were included.the mean age was 61 years , 59% of patients were male . mean time since diagnosis was 4.1 years and since initiation of insulin was 2.2 years , and the mean hba1c level at index date was 8.0%.total mean costs for the whole cohort the year before initiation of insulin was sek 17,230 ( 17,228 ) and the year after was sek 31,656 ( 24,331 ) ( p < 0.0001).despite the small study sample , this study demonstrates that mean annual medical costs almost double the year after patients are initiated on insulin ; the costs increased regardless of the hba1c level at initiation of insulin , with the largest increase in costs due to increased filled prescriptions . | [
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] |
shiga - toxin- ( stx- ) producing escherichia coli strains , including e. coli o157:h7 , are a worldwide cause
of human disease with a wide spectrum of symptoms ranging from mild diarrhea to
life - threatening hemolytic - uremic syndrome ( hus ) .
the stx - producing family of
human disease - associated e. coli is
also characterized by its diversity of toxin type ( i.e. , lysogenic either for
the stx1-encoding phage or the stx2-encoding phage , or with both lysogens ) and
by its o : h serotype range [ 1 , 2 ] . in spite of
diverse virulence characteristics , one common trait that emerges
very clearly
is that most of these strains have the ability to withstand gastric acidity [ 36 ] or the conditions in acidic foods [ 79 ] .
it is noteworthy that acid tolerance plays a vital role in the survival and
virulence of diarrheagenic e. coli strains [ 1012 ] .
the ability of e. coli o157:h7 strains to survive in
acidic conditions has been studied extensively [ 1316 ] , but there are few reports about
the tolerance of non - o157 stec serogroups to organic acids in foods [ 17 , 18 ] .
it is thought that acid resistance
and/or induction of acid tolerance may enable pathogens to survive
gastrointestinal acidity better and so , ultimately , cause disease [ 1922 ] .
cattle
are considered to be a major reservoir of e.
coli o157:h7 for human infection .
the pathogen has been isolated
from raw milk , and multiple outbreaks of e. coli o157:h7 linked to the ingestion
of raw milk and dairy products have been reported [ 25 , 26 ] .
several authors have studied the
ability of e. coli o157:h7 to grow
and survive in different types of cheese . in fresh cheese , e. coli o157:h7 increased by 2 log ( cfu per gram ) during cheese
manufacture , but total inactivation was
obtained during heat treatment ( 57c for more than 1.5 hours ) of the curd and whey .
reitsma and henning ( 1996 ) found that this microorganism was able to grow
during cheddar cheese manufacture , even with an initial inoculum in milk of
only 1 cfu ml .
e. coli o157:h7 also survived the manufacture and storage of camembert and feta cheeses
at 2 1c for 65 and 75 days , respectively .
moreover , this pathogen was able to
survive all stages of smear - ripened cheese production for up to 70 days
postmanufacture .
e. coli o157:h7 was not eliminated from goat 's milk lactic cheese , made with raw milk ,
because this organism tolerates the low ph and low temperatures [ 31 , 32 ] which characterize the
manufacturing and ripening process .
surprisingly , studies on contamination
of milk , or its products , with non - o157 e. coli have been limited .
two studies from france
have reported stec
prevalence in cheeses [ 33 , 34 ] , 3 studies reported results for
ewes ' and caprine milk cheeses [ 3537 ] and , very recently , a study
described the prevalence of stec in swiss raw milk cheeses .
moreover , there are no published
experiments evaluating the growth or survival of non - o157 stec in cheeses
. however , non - o157 e. coli infections are , within european
member states , considered to be at least as important as e. coli o157:h7 infections though they are thought to be generally
underdiagnosed .
for example , in italy , denmark , and germany , more than 40% of the confirmed
cases of stec - related hus were caused by non - o157 stec .
more recently , in 2005 ,
french raw milk camembert - type cheeses contaminated by escherichia coli o26 and o80 caused 16 hus cases and a national and
international recall of the entire production of cheeses .
other human infections with
non - o157 stec from dairy products are
well documented [ 4447 ] .
these outbreaks suggest that the
acid tolerance of the non - o157 stec strains , like the o157 stec strains ,
enables these bacteria to survive in moderately acidic food .
acid tolerance is
defined by the growth of log - phase cultures at a moderately low ph ( ph 5.5 to
6.0 ) inducing mechanisms of survival in the more extreme acid conditions of ph
2.5 . a similar phenomenon , termed the
acid tolerance response ,
the purpose
of the present work was to address the question of whether the acidic
resistance confers an ecological superiority .
this potential ecological
superiority of stec strains was evaluated by the ability of acid - resistant ( ar )
and non - acid - resistant ( nar ) stec strains to survive the fermentation process of
camembert - type cheeses . in a previous study
, we have
investigated the fate of e. coli o157:h7 during the manufacture and ripening of raw goat 's milk lactic cheeses
and noted that e. coli o157:h7 was
able to survive the cheese - making process .
the main objectives of the present
study were firstly to evaluate the growth and survival of ar and nar shiga - toxin - producing e. coli strains and , secondly , to
check whether microfiltered milk camembert cheeses could induce acid tolerance
in inoculated nar stec strains .
a collection of 62 stec strains were
isolated during previous french epidemiological studies , whose purpose was to
determine stec contamination prevalence in dairy products , in pork , and in the
environment [ 34 , 5254 ] . with the aim of evaluating the
ability of these bacteria to survive exposure to acid
these mechanisms are as follows : ar1 : oxidative
system ( glucose repressed system),ar2 : system
depending on the presence of glutamate , ar3 : system
depending on the presence of arginine .
it was tested according to the method previously
described by large et al . .
ar1 : oxidative
system ( glucose repressed system ) , ar2 : system
depending on the presence of glutamate , ar3 : system
depending on the presence of arginine .
four ar
stec and four nar stec strains were selected from a previous study ( data not
shown ) .
the survival rate of the strains in the presence of glucose and amino
acids is displayed in table 1 .
two
spontaneous nalidixic acid - resistant mutants were selected in vitro for each ar and nar stec
strain , previously selected by plating stec on bhi agar containing nalidixic
acid ( 0.1 to 10 g / ml ) in increasing concentrations , using the protocol
described by truong et al . .
the antibiotic
susceptibility of these ar or nar mutants is shown in table 1 . to follow the growth of these strains during the
manufacture and ripening of the camembert cheese , spontaneous
antibiotic - resistant derivatives ( nalidixic acid , rifampicin , and
spectinomycin )
we carefully checked that the chosen resistant
derivatives were neither affected in their growth rate nor in their
acid - resistance properties ( data not shown ) .
raw cow milk samples were collected
aseptically from the bulk storage tank after it had been cooled to < 5c
and maintained refrigerated at 3c for transportation to actilait .
raw milk was
microfiltered at 40c through a 1.4 m ceramic membralox membrane ( pall exekia , bazet , france ) .
the microfiltered cow milk was
analyzed , in order to check that it was not contaminated by stec , by performing
a polymerase chain reaction ( pcr ) for shiga - toxin coding genes ( stx genes ) after an enrichment step , as
described by fremaux et al . .
cultures of
antibiotic - resistant stec strains were maintained at 80c in
cryopreservative beads ( starlab , bagneux ,
france ) .
prior
to preparing the inoculation mixtures , each culture of antibiotic - resistant
stec strains was incubated overnight ( 18 hours ) in lb broth at 37c .
strains were grown
individually at 37c for 24 hours in lb broth , with or without antibiotics , to reaffirm that the
strains retained resistance .
cells were washed twice in 0.1% peptone water and suspended
in 0.1% peptone water to achieve the required inoculation ( 10 cfu ml ) level by standardization of the absorbance at a600 nm using a spectrophotometer ( biophotometer , eppendorf , le pecq , france ) .
they
were designed corresponding with the need to have 3 different antibiotic - resistant
or non - resistant phenotypes in the same mixture .
the 6 mixtures of stec strains
used for this experimentation are detailed in table 2 .
the stec population in each mixture
was confirmed by plating 100 l of the suspension onto lb - a plates supplemented
with the appropriate antibiotic to verify the initial inoculum levels .
six
millilitres of mixture were inoculated into 6.5 l of milk to obtain a final
concentration in the microfiltered milk of approximately 10 cfu ml of stec .
twelve batches of cheeses were
prepared ( two batches per mixture ) and 4 batches of uninoculated cheese samples
served as negative controls ( 1 batch per manufacturing day ) .
artisanal microfiltered cow 's milk
lactic cheeses were prepared following the industrial specifications of the
french institute of cheese ( actilait ) .
the milk
was inoculated ( 2 10 cfu per 100 kg ) with mesophilic
starters mm100 ( lactococcus lactis subsp .
lactis , cremoris , and lactis biovar diacetylactis ) , ( rhodia ,
dang - saint - romain , france ) as well as ripening flora penicillium camemberti ( pc p9 , 4 doses per 1000 kg cargill , st germain
en laye , france ) , geotrichum candidum ( gca , 2 doses per 1000 kg ,
cargill ) and 10.5 ml of a solution of cacl2 was also added per 100 kg
of milk .
the
inoculated milk was matured for 2 hours at 32c and renneting was
carried out using 30 ml of rennet per 100 l of
milk ( at 530 mgl of
chymosin , berthelot , abia s.a .
the
milk coagulated after 8 minutes and was left undisturbed until the ph decreased
to 6.15 .
the curd was cut into 3 cm cubes , healed for
30 minutes ( ph minimum = 6.00 ) ,
drained and transferred to 77 110 mm cylindrical plastic moulds .
cheeses weighing approximately 250 g were removed from the
mould , and the temperature of the cheese - making chamber was reduced to 2022c .
after 20 hours , the
cheeses were plunged into a saturated brine solution at 10c for 25 minutes .
cheeses
were dried at 13c for 5 hours and matured at 11c and 95% relative humidity for 20 days , then finally packaged and stored at 4c . the manufacturing protocol for lactic cheeses
made with microfiltered is outlined in figure 1 .
physicochemical and bacteriological
analyses were performed at the following steps.step 1 : microfiltered
milk before the maturation
stage.step 2 : curd during
draining ( 3 hours 20 minutes).step 3:cheese at the
end of the moulding stage ( 3 hours 45 minutes).step 4:cheese after
salting ( 1 day).step 5:cheese after
drying ( 1 day).step 6:cheese at the
middle of the ripening stage ( 10 days).step 7:cheese at the
end of ripening ( 20 days ) . microfiltered
milk before the maturation
stage
cheese at the
end of the moulding stage ( 3 hours 45 minutes ) .
cheese at the
end of ripening ( 20 days ) . during
cheese manufacture , 25 ml samples of microfiltered milk or
25 g of drained curd or cheese
( rind and core
) were sampled and homogenized with 225 ml of buffered peptone
water ( bpw , biomrieux , marcy l'etoile , france ) in a sterile bag filter
( bagsystem 400 ml model+ , interscience , saint nom la breteche , france ) and
stomached for 30 seconds ( stomacher mix1 , aes laboratory , bruz , france ) .
the filtered liquid was diluted in bpw and spread , using spiral plating ( wasp
spiral plating , aes laboratory , bruz , france ) , onto lb - a plates .
lb - a plates
were supplemented with rifampicin ( 100 g ml ) , spectinomycin ( 100 g ml ) , or nalidixic acid ( 40 g ml ) , when
appropriate , and incubated for 24 hours at 37c .
enumeration of the
colonies was performed with an automatic colony counter ec2 easy count 2 ( aes
laboratory , bruz , france ) .
the ph was measured for each cheese
( inoculated or not inoculated ) at each time of sampling . for the chemical
measurements ,
analyses were performed on the raw milk after microfiltration and
on the noninoculated ( negative controls ) cheeses after 1 day ( before brining )
and 20 days ( end of the ripening stage ) .
milk fat
content was determined by the acido - butyrometric
method of gerber , according to afnor
( nf v 04 - 210 ) , and the milk protein rate was obtained using the amido black
method ( afnor , nf
v 04 - 216 ) .
cheese moisture content was determined with an infrared - dryer
( prcisa xm60 ) , according to afnor ( nf v 04 - 282 ) , and the cheese fat content
was measured by the heiss butyrometric method
.
the ripened cheese salt content was determined using a chloride
analyzer ( afnor , nf v 04 - 288 ) .
cheese phs were measured using a
penetration electrode ( ph meter 330 , fisher bioblock scientific , f67403
illkirch cedex , france ) .
simulated gastric fluid was prepared accorded to the
protocol used by yuk and marshall
.
g l of proteose - peptone ( fluka - biochemika , switzerland ) , 3.5 g l of
d - glucose ( fluka - biochemika , switzerland ) , 2.05 g l of
nacl ( sigma - aldrich , lyon , france ) , 0.6 g l of kh2po4 ( merck sharp
& dohme , paris , france ) , 0.11
g l of cacl2 ( sigma - aldrich , lyon , france ) ,
0.37 g l of kcl ( sigma - aldrich , lyon , france ) , 0.05 g l of ox
bile ( sigma - aldrich , lyon , france ) , 0.1 g l of lysozyme
( sigma - aldrich , lyon , france ) , and 13.3 mg l of pepsin
( sigma - aldrich , lyon , france ) .
the final ph was adjusted to 1.5 using sterile
5.0 n hcl ( merck sharp & dohme , paris ,
france ) .
all
compounds were autoclaved separately , except for the ox bile , lysozyme , and
pepsin which were filter sterilized , followed by aseptic mixing . however , 90 ml of sgf ( ph 1.5 ) , at 37c , were added aseptically
to 10 g of inoculated cheese sampled at the end of the ripening stage . to obtain a
final inoculated sgf at ph 2.5 ,
the ph of each sample was lowered to 2.5 using
( 5.0 n ) hcl . for the enumeration of each stec
strain ( table 1 ) belonging to the different mixtures used for the inoculation
of the milk ,
viable cell densities were determined by spiral plating appropriate dilutions
in ts onto lb - a plates containing rifampicin ( 100 g ml ) ,
spectinomycin ( 100 g ml ) , or nalidixic acid ( 40 g ml ) .
lb - a plates were incubated at 37c for 24 hours and enumeration of the colonies was
performed with an automatic colony counter ec2 easy count 2 ( aes laboratory ,
bruz , france ) .
cheese
production was performed in a dairy laboratory in order to provide commercial
conditions during production .
the chemical composition of the cheese samples
produced from the microfiltered milks is shown in table 3 .
the fat and protein
content in the microfiltered milks were 37.00% and 32.58% w / w , respectively .
it declined slightly to 6.03 ( sd : 0.02 ) during the first 4 hours ( draining of
curdled milk ) .
then the ph decreased markedly from 6.03 to 4.65 ( sd : 0.05 ) at
the end of moulding step ( 24 hours ) .
the ph remained almost stable until the 10th
day ( 4.64 to 4.75 ) ( sd : 0.02 ) and increased slightly over the last 10 days of
the ripening period to a ph of 5.11 ( sd : 0.03 ) ( table 4 ) .
stec was never isolated from any of
the milk samples collected from the bulk storage tank nor from any of the
negative control cheeses .
there were no differences between
the counts of nar and ar stec strains during the manufacture and ripening of
the microfiltered milk lactic cheeses .
as an example , figure 2 shows the
survival of 4 stec mixtures : the ar2 ( 3 ar stec strains ) , as2 ( 3 nar stec
strains ) , arssb2 ( 2 nar and 1 ar stec strains ) , and asrra2 ( 2 ar and 1 nar stec
strains ) mixtures during cheese manufacture ( table 2 ) .
in general , whatever the mixture
used , stec counts increased by a range of 1 to 2 2 log cfu g during the first steps of the cheese manufacturing and remained relatively
stable after salting until the drying stage of the cheeses .
then , during
ripening ( 20 days ) , the counts of only one nar stec strain ( 346aspec )
decreased to 10 cfu g. the other ar or nar stec strains were all
counted at levels ranging from 10 to 10 cfu g
( figure 2 ) . at the end of ripening ( 20 days ) ,
samples of inoculated cheeses were placed in simulated gastric fluid where the
numbers of surviving stec cells were assessed at 5 , 10 , 20 , 30 , 40 , 50 , 60 , 90 ,
and 120 minutes .
exposure to
sgf ( ph : 2.5 ) reduced the number of nar stec strains to undetectable levels within
40 , 50 , and 60 minutes for anr 42anal , anr 418arif-346aspec , and 360brif , respectively .
in contrast to the nar stec
strains , all the ar stec strains survived an exposure of more than 120 minutes
in sgf at ph 2.5 ( table 5 ) .
multiple applications of low levels
of acid stress during the life cycles of escherichia
coli o157:h7 and other pathogens might increase their likelihood for
survival in foods and may enhance the development and establishment of
stress - adapted strains with potentially increased virulence in food
environments [ 6163 ] .
although acid tolerance in e. coli o157:h7 is normally transient ,
being induced at low ph [ 14 , 30 , 64 , 65 ] , some outbreak strains ( e.g. , atcc
43895 ) have attained a permanently high , ph - independent acid resistance , probably because of an
evolutionary response to severe acid stress . the objective of the present study
was to investigate the growth and survival of ar and nar stec strains in
camembert - type cheeses .
none of our findings have been subjected to a
statistical analysis since we have used mixtures of 3 stec strains for the
inoculation of the milk and , hence , some interaction between stec strains could
occur .
the use of mixtures , instead of a single strain , is explained by the
limited number of batches ( 16 ) allowed by actilait and the decision to study
the kinetics of 8 different stec strains .
moreover , it was not possible to
study pathogenic stec strains , such as e.
coli o157:h7 or e. coli o26 , due
to the lack of safety level / p3 laboratories .
the number
of stec increased from 1 to 2 log10 at the beginning of the cheese
manufacture , whilst the ph decreased slightly from 6.45 ( milk ph ) to 6.03
( first 4 hours ) .
much of this initial increase could be attributed to the
entrapment of stec in the curd during coagulation followed by further
concentration during whey drainage .
then we noted a plateau phase in the growth
of stec from the cheese at the end of moulding stage ( ph : 4.65 ) to the
cheese after drying stage ( ph : 4.66 ) . from the middle of ripening ( 10 days ,
ph : 4.75 ) to the end of ripening ( 20 days , ph 5.11 ) , the stec
population decreased markedly . in much the same way ,
after 24 hours of
manufacture and storage of camembert cheese inoculated at 10 cfu ml ,
ramsaran et al . observed an increase in e. coli o157:h7 counts of about 2 log10 .
this increase was followed by a decrease in the counts of e. coli o157:h7 in all of the cheeses throughout ripening and
storage at 2c .
. showed that significant numbers of
viable e. coli o157:h7 could be
detected in raw goat milk lactic cheeses even 42 days after processing . in cheddar cheeses , aged for 60 and 120 days , and
stored at 7c ,
the e.
coli o157:h7 population was reduced by less than 2 log .
marek et al . reported
that e. coli o157:h7 could persist in
unpasteurized cheddar cheese whey inoculated at 10 or 10 cfu ml for up to 2 - 3 weeks of storage at 4 , 10 , or 15c .
the results of maher et al . showed the presence of e. coli o157:h7 even after 90 days in
the rind and after 50 days in the core of smear ripened cheese produced from
raw milk .
the acid adaptation response is a
phenomenon by which microorganisms show an increased resistance to
environmental stress after exposure to a moderate acid environment . in this
study ,
the acid adaptation of 4 nar stec strains was not induced by the slow
and mild acid conditions ( 4.654.75 ) found during the lactic cheese process
because these strains were rapidly destroyed during the strong acid exposure in
simulated gastric fluid ( ph 2.5 ) .
hsin - yi and
chou noted the same effects in fermented
milk but a completely opposite effect in acid fruit juice .
the authors
explained that acid adaptation might lead to an increased susceptibility of the
test organism to antimicrobials , such as bacteriocins , hydrogen peroxide ,
ethanol , and diacetyl , produced by lactic acid bacteria in milk products .
the
effect of increasing susceptibility to these antimicrobials , due to acid
adaptation , may outweigh the effect of enhancing acid tolerance , reducing the
survival of acid - adapted e. coli o157:h7 in the milk products .
observed that e. coli o157:h7 is able to induce an adaptive tolerance response ( atr )
when exposed to mild acid conditions , thus conferring a higher resistance on
subsequent exposure to strong acid conditions .
bergholz and whittam studied the survival of enterohaemorrhagic escherichia coli of serotypes o157:h7 ,
o26:h11 , and o111:h8 in a simulated gastric environment .
their results
indicated that e. coli o157:h7
strains were better able to survive in a simulated gastric environment than the
stec strains belonging to the two other serogroups .
the authors indicated that
this difference was reduced when cultures were held at stationary phase for
longer periods of time , suggesting that e.
coli o157:h7 cells rapidly achieve an enhanced state of ar in the early
stationary phase , an ability that may underlie the low infectious dose of this
pathogen .
the present study indicates that ar
and nar stec strains , when initially present at 10 cfu ml in milk , would most likely survive artisanal camembert - type cheese manufacture
and ripening ( 20 days ) .
the biggest decrease was observed for
an
nar stec strain ( 346aspec ) whose counts reached 10 cfu g at 20 days . even if we must keep in mind that the inoculation levels were
certainly higher than those observed in naturally contaminated milk , the low
infectious dose associated with pathogenic stec suggests that the 20 day
ripening period of these cheeses may not guarantee a safe product for consumers
if stec are present in the raw milk
consequently , good milk hygiene is crucial
in order to reduce the risk of the presence of pathogens in the raw milk
cheeses .
moreover , acid adaptation
of nar stec strains during the manufacture of cheeses prior to their exposure
to simulated gastric fluid did not increase the acid resistance of the bacteria .
on the basis of these results , additional
investigations will be undertaken to evaluate the behavior of stec during the
manufacture of cheeses using a rapid curdling phase linked to a greater drop in
acidity than that employed in the present cheese technology . | growth and survival of acid - resistant ( ar ) and non - acid - resistant ( nar ) shiga - toxin - producing escherichia coli ( stec ) strains were investigated during the manufacture
and ripening of microfiltered milk camembert cheeses .
the induction of acid resistance of the stec strains in cheeses
was also studied .
six different mixtures of ar and/or nar stec strains were inoculated separately into microfiltered
milk at a level of 103 cfu ml1 . the stec counts ( ar and nar ) initially increased by 1 to 2 log10 cfu g1 during cheese - making .
thereafter , the populations stabilized during salting / drying and then
decreased during the early stages of ripening . exposing the stec strains in artificially inoculated cheeses
to simulated gastric fluid ( sgf - ph : 2.0 ) reduced the number of nar strains to undetectable levels within 40 minutes , versus 120 minutes for the ar stec strains .
ar and nar stec were able to survive during the manufacture and ripening of camembert cheese prepared from microfiltered milk with no evidence of induced acid tolerance in nar stec strains . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion | it is thought that acid resistance
and/or induction of acid tolerance may enable pathogens to survive
gastrointestinal acidity better and so , ultimately , cause disease [ 1922 ] . these outbreaks suggest that the
acid tolerance of the non - o157 stec strains , like the o157 stec strains ,
enables these bacteria to survive in moderately acidic food . this potential ecological
superiority of stec strains was evaluated by the ability of acid - resistant ( ar )
and non - acid - resistant ( nar ) stec strains to survive the fermentation process of
camembert - type cheeses . in a previous study
, we have
investigated the fate of e. coli o157:h7 during the manufacture and ripening of raw goat 's milk lactic cheeses
and noted that e. coli o157:h7 was
able to survive the cheese - making process . the main objectives of the present
study were firstly to evaluate the growth and survival of ar and nar shiga - toxin - producing e. coli strains and , secondly , to
check whether microfiltered milk camembert cheeses could induce acid tolerance
in inoculated nar stec strains . two
spontaneous nalidixic acid - resistant mutants were selected in vitro for each ar and nar stec
strain , previously selected by plating stec on bhi agar containing nalidixic
acid ( 0.1 to 10 g / ml ) in increasing concentrations , using the protocol
described by truong et al . to follow the growth of these strains during the
manufacture and ripening of the camembert cheese , spontaneous
antibiotic - resistant derivatives ( nalidixic acid , rifampicin , and
spectinomycin )
we carefully checked that the chosen resistant
derivatives were neither affected in their growth rate nor in their
acid - resistance properties ( data not shown ) . there were no differences between
the counts of nar and ar stec strains during the manufacture and ripening of
the microfiltered milk lactic cheeses . as an example , figure 2 shows the
survival of 4 stec mixtures : the ar2 ( 3 ar stec strains ) , as2 ( 3 nar stec
strains ) , arssb2 ( 2 nar and 1 ar stec strains ) , and asrra2 ( 2 ar and 1 nar stec
strains ) mixtures during cheese manufacture ( table 2 ) . in general , whatever the mixture
used , stec counts increased by a range of 1 to 2 2 log cfu g during the first steps of the cheese manufacturing and remained relatively
stable after salting until the drying stage of the cheeses . at the end of ripening ( 20 days ) ,
samples of inoculated cheeses were placed in simulated gastric fluid where the
numbers of surviving stec cells were assessed at 5 , 10 , 20 , 30 , 40 , 50 , 60 , 90 ,
and 120 minutes . exposure to
sgf ( ph : 2.5 ) reduced the number of nar stec strains to undetectable levels within
40 , 50 , and 60 minutes for anr 42anal , anr 418arif-346aspec , and 360brif , respectively . in contrast to the nar stec
strains , all the ar stec strains survived an exposure of more than 120 minutes
in sgf at ph 2.5 ( table 5 ) . the objective of the present study
was to investigate the growth and survival of ar and nar stec strains in
camembert - type cheeses . the number
of stec increased from 1 to 2 log10 at the beginning of the cheese
manufacture , whilst the ph decreased slightly from 6.45 ( milk ph ) to 6.03
( first 4 hours ) . in this
study ,
the acid adaptation of 4 nar stec strains was not induced by the slow
and mild acid conditions ( 4.654.75 ) found during the lactic cheese process
because these strains were rapidly destroyed during the strong acid exposure in
simulated gastric fluid ( ph 2.5 ) . their results
indicated that e. coli o157:h7
strains were better able to survive in a simulated gastric environment than the
stec strains belonging to the two other serogroups . the present study indicates that ar
and nar stec strains , when initially present at 10 cfu ml in milk , would most likely survive artisanal camembert - type cheese manufacture
and ripening ( 20 days ) . moreover , acid adaptation
of nar stec strains during the manufacture of cheeses prior to their exposure
to simulated gastric fluid did not increase the acid resistance of the bacteria . | [
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] |
in the past decade , there has been tremendous advance in the treatment of age - related macular degeneration ( amd ) , but the introduction of anti - angiogenic agents has not been without substantial costs .
the approved medications are expensive , and visits for intravitreal injections can be frequent , imposing an ever growing burden on healthcare systems , on physician practices , and on patients and their families .
thus , there is still considerable interest in preventing or slowing the progression of amd through interventions against modifiable risk factors .
epidemiological studies have shown that smoking and diet are two of the most consistently identified modifiable amd risk factors .
dietary modification through nutritional counseling is particularly appealing due to its universal applicability and its relatively low expense , but major dietary and lifestyle changes can be difficult to achieve in the elderly population at risk for visual loss .
this means that dietary supplements of vitamins , minerals and other nutritional factors are an attractive intervention for amd and other age - related eye diseases as long as a firm evidenced - based body of knowledge exists to support their use .
it is not unusual for patients and eye care providers to commonly refer to these supplements as eye vitamins , but many of their key components do not fit the strict definition of a vitamin , which is an organic compound required by an organism as a vital nutrient in limited amounts and for whom a deficiency state reproducibly results in a clinically defined pathological condition . by convention , there are thirteen universally recognized vitamins , but there are numerous other non - vitamin nutrients linked with improved ocular function and health including trace minerals , dietary lipids and plant pigments such as carotenoids and polyphenols that are typically included in supplements targeted for promotion of eye health and protection against eye disease .
prior to that time , all needed vitamins were obtained solely through food intake ; however , dietary modifications to meet ocular supplementation needs occurred long before when the ancient egyptians recognized that feeding a person liver , an excellent source of vitamin a essential for production of functional photoreceptor pigments , could cure night blindness .
today , dietary supplements are frequently used to ensure that adequate amounts of ocular nutrients are obtained on a daily basis but in some cases , the complex interactions between elevated levels of vitamins and other nutrients in the body are just now being elucidated .
unwanted effects can occur with taking high doses of even essential vitamins or if the person taking them has certain health conditions .
for example , a study published in 2009 found that antioxidant vitamins , c and e , which are often used in high doses in eye supplements , may actually decrease the benefits of exercise .
additionally , contradictory conclusions have been reached by different studies when a large , double - blind trial in 2011 found that vitamin e supplementation increased the risk of prostate cancer in healthy men , while a previous study in 1998 had shown a decreased risk of prostate cancer with vitamin e supplements .
today , more than ever , eye doctors are being asked to advise an increasingly aware patient population regarding nutrition and vitamin supplements related to vision .
most of the current recommendations regarding the use of eye vitamin supplementation to support macular health have been gleaned from a pair of large , randomized controlled studies known as the age - related eye disease study ( areds ) 1 and 2 .
these studies suggest that nutritional supplements are a promising means of delaying the leading cause of elderly blindness in developed countries , that is , advanced amd .
in addition , there is some evidence that vitamin supplementation may be beneficial in delaying cataract progression and treating dry eyes .
studies are currently underway evaluating their usefulness in treating glaucoma and diabetic retinopathy , the number one cause of blindness among the working population .
while it has been shown that extreme vitamin deficiencies can directly cause retinal dysfunction in animal experiments , large epidemiologic studies in humans are needed to determine if diet alone or modest nutritional supplementation can influence ocular diseases . for starters
, everyone consumes vitamins on some level , so the effect of supplementation depends on the amount of a vitamin already being consumed . while randomized trials with defined end - points are the gold standard , these results can be misleading .
one reason is that trial participants for the most part have good diets , and they may not show an effect of supplementation that might be exhibited in those with poorer diets .
additionally , trials on eyes may be too short for an effect to be demonstrated or may focus on persons at high risk for a disease or only those with an existing disease .
such trials may make it difficult to apply findings later to those with average risk .
generally , positive results of such trials are compelling , while negative results are difficult to interpret .
age - related macular degeneration remains the leading cause of elderly blindness in developed countries .
multiple genetic and environmental factors have been implicated in the pathogenesis of this complex disease .
age , smoking , genetics , diet , obesity , hypertension and hypercholesterolemia are the most recognized risk factors . among these , aging and smoking
increasing pack years of cigarettes smoked is directly correlated with an increasing risk of amd ; the risk is roughly doubled when smokers are compared to those who have never smoked . as such , smoking cessation
ethnicity also plays a role according to a 10 years longitudinal study , the multi - ethnic study of atherosclerosis ( mesa ) , reporting a lower prevalence of amd in blacks than in whites with the overall prevalence varying from 2.4% in african americans , 4.2% in hispanics , and 4.6% in chinese as compared to 5.4% in whites .
compared to other organs , the eye is uniquely susceptible to oxidative stress given its high consumption of oxygen , high content of polyunsaturated fatty acids , and exposure to visible light .
the formation of reactive oxygen species leads to the oxidation of docosahexaenoic acid ( dha ) which is thought be a major pathway of cellular damage and photoreceptor degeneration in amd .
this mechanistic understanding of amd has led to therapeutic strategies to reduce oxidative damage by cessation of smoking , limiting alcohol intake , avoiding obesity , regular exercise , and of course , the implementation of supplemental antioxidant eye vitamins .
additionally , there has been recent interest in supplementation with compounds possessing anti - inflammatory properties such as the omega-3 fatty acids , eicosapentaenoic acid ( epa ) and dha .
the age - related eye disease study ( areds ) , sponsored by the national eye institute , evaluated amd progression in participants supplemented over an average of 6.3 years with randomization at entry to 1 of 4 treatment categories of dietary supplements at levels well above recommended daily allowances : placebo ; antioxidants ( -carotene 15 mg , vitamin c 500 mg , and vitamin e 400 iu ) ; zinc ( 80 mg as zinc oxide and copper 2 mg ) ; and antioxidants and zinc combined .
these nutrients were chosen based on the best nutritional knowledge of eye disease in the 1980s when the areds study was conceived . a seminal study performed in utah
had recently shown a beneficial effect of zinc supplementation in amd patients , vitamins c and e were readily available antioxidants abundantly present in ocular tissues , and -carotene was a major commercially available vitamin a precursor which was known to be less toxic at high doses than vitamin a itself .
patients were characterized during enrollment with retinal images and varied from those with normal eyes to those with advanced amd .
disease level was then classified by investigators based on the category of amd in the patient 's worse eye : areds category 1 ( no amd ) consisted of fewer than 5 small ( < 63 m ) drusen ; category 2 ( mild amd ) , multiple small drusen , non - extensive intermediate ( 63124 m ) drusen , pigment abnormalities , or a combination ; category 3 ( intermediate amd ) , at least 1 large ( > 125 m ) druse , extensive intermediate drusen , or geographic atrophy not involving the center of the macula ; and category 4 ( advanced amd ) , central geographic atrophy or neovascular amd in 1 eye or visual loss resulting from amd , regardless of the lesion type .
the 5 years results of the areds study showed that supplementation with antioxidants and zinc combined , reduced the risk of progression to advanced amd by approximately 25% in those with intermediate amd or advanced amd in one eye .
the risk of losing three or more lines of vision was also reduced by 19% with this treatment .
it was concluded from this study that those with extensive intermediate drusen , at least one large druse , non - central geographic atrophy in one or both eyes , advanced amd or vision loss because of amd in one eye , and without contraindications such as smoking , should take an areds supplement of antioxidants plus zinc .
it should be noted however , that to date routine supplementation with antioxidant vitamins or minerals has not been demonstrated to prevent the onset of amd in patients who do not have areds category 3 or 4 disease . by the time the original areds study was published in 2001 ,
first it was recognized that the dose of -carotene used in the study was likely to present a significant risk of lung cancer development in smokers based on several large randomized trials published while areds was in progress .
second , ongoing biochemical studies clearly identified several common dietary constituents that were abundantly concentrated in the macula and whose dietary consumptions were epidemiologically linked with decreased risk of amd in the areds population and in other cohorts the xanthophyll carotenoids commonly found in dark green leafy vegetables and orange or yellow fruits and vegetables , lutein and zeaxanthin , and the omega-3 fatty acids abundantly present in fish oil , epa and dha
. out of over 600 carotenoids in nature , only lutein and zeaxanthin and their metabolites are present in the foveal region of the human eye where they form the yellow pigment of the macula lutea .
these natural blue - light screening antioxidants have been associated with decreased risk of amd in multiple epidemiological studies , and their unique localization to the fovea implies a potentially important physiological function in visual performance and in preservation of macular health . likewise , photoreceptor outer segments contain the highest percentage of omega-3 polyunsaturated fatty acids in the body .
based on the afore mentioned concerns about -carotene in smokers and the progression in knowledge of ocular nutrition , the national eye institute initiated the areds2 study which enrolled its first patient in 2006 and published its results in 2013 .
it assessed the effects on cataracts , amd , and moderate vision loss of oral supplementation with 10 mg lutein + 2 mg zeaxanthin , and/or 650 mg epa + 350 mg dha .
additionally , a secondary randomization was also performed in which study participants were given either : ) 1 ) the original areds formula , ( 2 ) areds formula minus -carotene , ( 3 ) areds formula with low dose zinc ( 25 mg ) , or ( 4 ) areds formula with no -carotene and low dose zinc .
this secondary randomization was included because high levels of zinc supplementation in the original areds formula was thought to be associated with significantly more hospitalizations due to genitourinary conditions and self - reported anemia even though overall mortality was not affected by zinc supplementation during the study .
80 mg was tested in the original areds formula because it was the dose used in an earlier trial that suggested benefit .
the areds2 study evaluated a lower 25 mg dose as more recent research had suggested this may be the maximal level absorbed by the gut .
first , two different randomized controlled clinical trials had demonstrated an increase in lung cancer rates and mortality in cigarette smokers supplemented with -carotene .
secondly , previous animal and human studies had suggested that simultaneous administration of high doses of -carotene and lutein + zeaxanthin may suppress serum and tissue levels of lutein + zeaxanthin because of competitive absorption of carotenoids .
the areds2 planners set an ambitious goal of achieving a 25% incremental improvement on the benefits of the already successful areds formula , and unfortunately , they did not achieve the pre - specified primary positive endpoint when each of the three active supplementation arms was compared individually with the control group , but pre - specified secondary analyses of the main effects of lutein and zeaxanthin produced statistically and clinically significant positive results . on the other hand , main effect analysis of the data from areds2 showed that the addition of omega-3 fatty acids was neither harmful nor beneficial . adding lutein + zeaxanthin to
the areds formula resulted in an additional beneficial effect of about 10% beyond the effects of the original areds formulation in reducing the risk of progressing to advanced amd , and when -carotene was removed , the incremental benefit increased to 18% , possibly due to amelioration of competitive absorption effects .
those who derived the most benefit from the addition of lutein + zeaxanthin were those in the lowest quintile of dietary lutein and zeaxanthin intake . furthermore , despite proscription against -carotene supplementation in current smokers , -carotene was still associated with a greater risk of lung cancer in areds2 participants ( 2% vs. 0.9% ) , especially in those who had previously been smokers .
this finding is clinically relevant , as 50% of participants in areds and areds2 with amd were former smokers , and 91% of those who developed lung cancer in areds2 were former smokers . on the other hand
finally , comparison of low - dose zinc versus high - dose zinc displayed no statistically significant effect .
the authors concluded that there was insufficient evidence to provide a clinical recommendation at this point regarding changing the dose to 25 mg .
given the results of this study , it should be expected that most supplement makers will soon remove -carotene from the eye vitamin formula and replace it with 10 mg of lutein and 2 mg of zeaxanthin certainly for smokers and former smokers , and for simplicity and uniformity of message this formulation can be recommended to nonsmokers as well .
areds2 did not find evidence to support the addition of omega-3 fatty acids to the formula at this point ; however , other studies have demonstrated a benefit from increased omega-3 intake , so clinicians are left to individually counsel patients regarding omega-3 supplementation , especially if these patients normally consume very little fish in their diets .
recent understanding regarding the genetics of heritable mutations associated with amd has shed much light on the pathogenesis of the disease and implicated several important biological pathways such as complement pathways , cholesterol and lipid metabolism pathways , extracellular / collagen matrix pathways , oxidative stress pathways and angiogenesis signaling pathways .
an international collaborative effort recently reviewed over 17,000 amd cases and compared them with 60,000 matched controls of european and asian ancestry and revealed 19 amd loci .
the question remains as to how many of these associated variants are causal , and further evaluation of the functional characterization of genes associated with these variants may provide biological relevance to our understanding of the pathogenesis of amd .
some of the known biological features of amd genetic risk factors predict that specific components of the areds formulation would be more beneficial . a recent study re - analyzed the areds results in conjunction with genotype data and evaluated the effectiveness of the original areds formula and concluded that the estimated potential benefit of using genotype specific nutritional therapy could have more than doubled the reduction in amd progression rate compared with treatment with the areds formula over a 10 years period .
they felt that patients with 1 or 2 complement factor h ( cfh ) risk alleles derived maximum benefit from antioxidants alone as zinc negated the benefits of antioxidants .
additionally , patients with age - related maculopathy sensitivity 2 ( arms2 ) risk alleles derived maximum benefit from zinc - containing regimens , with a deleterious response to antioxidants .
they also proposed that individuals homozygous for cfh and arms2 risk alleles derived no benefit from any category of areds treatment .
as possible explanations for this effect , they noted that patients with a known cfh mutation might be predicted to respond more poorly to an eye vitamin supplement containing zinc as cfh binds zinc , which can neutralize its ability to inactivate complement component 3b .
additionally , arms2 localizes to mitochondria , and might potentially affect oxidative phosphorylation and the generation of oxygen free radicals that could interact with antioxidants such as vitamins c and e. this post - hoc analysis must be interpreted with caution , however , as the genotype specific subgroups were often very small which necessitated complicated statistical modeling with wide confidence intervals , and their conclusions may not apply to newer areds2 recommendations . moreover , their biochemical explanations require additional in vitro and in vivo studies to prove their clinical relevance , and further confirmatory studies of the influence of amd risk genotypes on response to nutritional supplements are required before their recommendations can enter mainstream clinical practice . as additional studies are performed , it is likely that we will see improved outcomes from genotype - directed therapy in the future .
of course , this would also necessitate genetic testing becoming readily available for all patients with amd in order to categorize their genetic variants .
several previous studies have evaluated risk factors felt to be associated with cataract development such as : smoking , diabetes , sunlight exposure , educational level , body mass index , refraction , and estrogen replacement therapy ; however , most supplementation trials have tested the effect of high - dose antioxidants such as vitamin c , vitamin e , and -carotene . since this review has the goal of discussing the role of vitamin supplements in treating diseases of the eye
recently , areds report number 32 published results which demonstrated that centrum use amongst areds study patients was associated with a decreased risk of nuclear cataract .
these findings were consistent with an earlier report based on a propensity score analysis of cataract and centrum use in the areds population .
they also agreed with a large , randomized clinical trial recently performed in italy which showed a reduction in the development or progression of nuclear opacities ; however , this trial differed from other trials in that it also showed a significant increase in the development or progression of posterior subcapsular ( psc ) opacities .
interestingly , even though significant changes were noted in cataract progression in study participants , there were no significant effects on functional end - points such as visual acuity or cataract surgery .
identifying which individual supplements or combination of supplements within centrum vitamins contribute to the protective effect on nuclear cataract remains an area of investigation .
perhaps the study that showed the most promising benefits from vitamin supplementation on cataracts was performed in rural china where it demonstrated a 36% reduction in the prevalence of nuclear cataract in persons 6574 years old after 5 years .
study participants were divided into two arms with the study arm receiving 2 centrum tablets and 15 mg of -carotene daily compared with those assigned to a placebo formulation .
while these results were more statistically significant than other studies performed in western populations , the trend seems to be the same with a decrease in nuclear cataracts and a possible increase in psc opacities .
additionally , one may conclude that more nutritionally deprived populations seem to derive the most benefit from multivitamin supplementation , although further evaluation is needed .
interestingly , lutein and zeaxanthin are the only carotenoids that have been detected in the lens .
the areds2 results showed daily supplementation with lutein / zeaxanthin had no statistically significant overall effect on rates of cataract surgery or vision loss .
taking multivitamins may slow the development of age - related cataracts , but since this link in most appears weak , at best , patients should consider taking multivitamins on the basis of overall health benefits or risks .
dry eye syndrome ( des ) is one of the most prevalent ocular conditions in the world .
rapid tear evaporation , inadequate tear production and inflammation of the ocular surface have all been associated with dry eyes .
one school of thought for treatment of des is that meibum lipid composition can be influenced by increasing dietary lipid intake in an effort to manage meibomian gland dysfunction ( mgd ) .
therefore , it is recommended that oral supplementation with omega-3 essential fatty acids ( efas ) can be a therapeutic option for patients with mgd .
it has been demonstrated that breakdown of omega-3 efas leads to suppression of inflammation , and the breakdown of omega-6 efas promotes inflammation .
the first proposes that the breakdown of omega-3 efas competes with the same enzymes that are used to breakdown omega-6 efas and essentially inhibits the ability to breakdown omega-6 efas , thus leading to decreased inflammation along the eyelid margin .
the second hypothesis is that supplementation with omega-3 efas influences fatty acid composition and promotes tear stabilization while preventing blocked meibomian ducts .
current data support the use of systemic omega-3 fatty acid supplements for des , although there is a lack of large randomized , controlled , double - blinded studies evaluating their efficacy .
one such study on 71 patients with mild to moderate dry eye symptoms demonstrated a non - statistically significant improvement in schirmer test , tear break - up time , and fluorescein and lissamine green staining in patients who took oral polyunsaturated fatty acid supplements .
another study suggested that higher dietary intake of omega-3 fatty acids is associated with a decreased risk of dry eye syndrome in women .
there are no formal recommendations or fda approved formulations for dietary consumption of efas in the treatment of eye disease or the promotion of eye health , but many ophthalmologists currently recommend treatment with 1000 mg of flaxseed oil daily ( typically divided in three doses ) or another form of omega-3 efas .
additionally , the american heart association ( aha ) currently recommends at least two servings of fish high in omega-3 fatty acids per week for heart health .
it appears likely that many benefits are associated with a diet rich in omega-3 fatty acids including heart health , amd , and des .
future studies will hopefully provide outcome measures of the use of different types of efas compared in a standardized fashion .
the potential exists to modify ophthalmic preferred practice guidelines much the same way that the areds study has done for macular degeneration .
the limited studies to date suggest that a well - designed , multicenter , randomized , controlled trial of efas would be welcomed and could provide important insight on the benefits of using omega-3 efas as a supplement for des .
there have been a number of important studies affirming the relationship of diet and nutrition to the treatment , prevention , and/or slowing progression of a number of age - related ocular diseases .
it is important that any advice given to patients regarding lifestyle modifications and particularly recommendations on the benefits of nutritional supplementation be informed by the best available research evidence .
when we understand this evidence , we can help educate our patient populations to the link between nutrition and eye - health .
since nutrients are more conceptual , and thus invisible to consumers , a single page write - up or a stand - alone pamphlet can be very helpful to encourage the best diet / health practices for healthy vision .
they also can be valuable in initiating a discussion with patients on overall health beyond the eyes themselves . | there have been enormous advances in the past decade for the treatment of age - related macular degeneration ( amd ) ; however , these treatments are expensive and require frequent follow - up and injections which place a tremendous burden on both the healthcare system and patients .
consequently , there remains considerable interest in preventing or slowing the progression of amd requiring treatment .
epidemiological studies have shown that diet is a modifiable amd risk factor , and nutrient modification is a particularly appealing treatment for amd due to the perceived universal benefit and relatively low expense .
recently , the age - related eye disease study part two ( areds2 ) was concluded and demonstrated further benefit with the addition of lutein and zeaxanthin as a replacement for the -carotene of the previous generation formulation .
the addition of omega-3 essential fatty acids did not show an added benefit .
this review aims to highlight some of the evidenced based body of knowledge that has been accumulated from recent studies regarding the use of nutritional supplements and their effect on amd , cataracts , and dry eyes . | INTRODUCTION
EPIDEMIOLOGIC STUDIES
AGE-RELATED MACULAR DEGENERATION
AGE-RELATED MACULAR DEGENERATION (GENOTYPE SPECIFIC TREATMENT)
CATARACTS
DRY EYE SYNDROME | in the past decade , there has been tremendous advance in the treatment of age - related macular degeneration ( amd ) , but the introduction of anti - angiogenic agents has not been without substantial costs . thus , there is still considerable interest in preventing or slowing the progression of amd through interventions against modifiable risk factors . epidemiological studies have shown that smoking and diet are two of the most consistently identified modifiable amd risk factors . dietary modification through nutritional counseling is particularly appealing due to its universal applicability and its relatively low expense , but major dietary and lifestyle changes can be difficult to achieve in the elderly population at risk for visual loss . this means that dietary supplements of vitamins , minerals and other nutritional factors are an attractive intervention for amd and other age - related eye diseases as long as a firm evidenced - based body of knowledge exists to support their use . most of the current recommendations regarding the use of eye vitamin supplementation to support macular health have been gleaned from a pair of large , randomized controlled studies known as the age - related eye disease study ( areds ) 1 and 2 . the age - related eye disease study ( areds ) , sponsored by the national eye institute , evaluated amd progression in participants supplemented over an average of 6.3 years with randomization at entry to 1 of 4 treatment categories of dietary supplements at levels well above recommended daily allowances : placebo ; antioxidants ( -carotene 15 mg , vitamin c 500 mg , and vitamin e 400 iu ) ; zinc ( 80 mg as zinc oxide and copper 2 mg ) ; and antioxidants and zinc combined . disease level was then classified by investigators based on the category of amd in the patient 's worse eye : areds category 1 ( no amd ) consisted of fewer than 5 small ( < 63 m ) drusen ; category 2 ( mild amd ) , multiple small drusen , non - extensive intermediate ( 63124 m ) drusen , pigment abnormalities , or a combination ; category 3 ( intermediate amd ) , at least 1 large ( > 125 m ) druse , extensive intermediate drusen , or geographic atrophy not involving the center of the macula ; and category 4 ( advanced amd ) , central geographic atrophy or neovascular amd in 1 eye or visual loss resulting from amd , regardless of the lesion type . second , ongoing biochemical studies clearly identified several common dietary constituents that were abundantly concentrated in the macula and whose dietary consumptions were epidemiologically linked with decreased risk of amd in the areds population and in other cohorts the xanthophyll carotenoids commonly found in dark green leafy vegetables and orange or yellow fruits and vegetables , lutein and zeaxanthin , and the omega-3 fatty acids abundantly present in fish oil , epa and dha
. out of over 600 carotenoids in nature , only lutein and zeaxanthin and their metabolites are present in the foveal region of the human eye where they form the yellow pigment of the macula lutea . these natural blue - light screening antioxidants have been associated with decreased risk of amd in multiple epidemiological studies , and their unique localization to the fovea implies a potentially important physiological function in visual performance and in preservation of macular health . the areds2 planners set an ambitious goal of achieving a 25% incremental improvement on the benefits of the already successful areds formula , and unfortunately , they did not achieve the pre - specified primary positive endpoint when each of the three active supplementation arms was compared individually with the control group , but pre - specified secondary analyses of the main effects of lutein and zeaxanthin produced statistically and clinically significant positive results . on the other hand , main effect analysis of the data from areds2 showed that the addition of omega-3 fatty acids was neither harmful nor beneficial . adding lutein + zeaxanthin to
the areds formula resulted in an additional beneficial effect of about 10% beyond the effects of the original areds formulation in reducing the risk of progressing to advanced amd , and when -carotene was removed , the incremental benefit increased to 18% , possibly due to amelioration of competitive absorption effects . those who derived the most benefit from the addition of lutein + zeaxanthin were those in the lowest quintile of dietary lutein and zeaxanthin intake . areds2 did not find evidence to support the addition of omega-3 fatty acids to the formula at this point ; however , other studies have demonstrated a benefit from increased omega-3 intake , so clinicians are left to individually counsel patients regarding omega-3 supplementation , especially if these patients normally consume very little fish in their diets . there are no formal recommendations or fda approved formulations for dietary consumption of efas in the treatment of eye disease or the promotion of eye health , but many ophthalmologists currently recommend treatment with 1000 mg of flaxseed oil daily ( typically divided in three doses ) or another form of omega-3 efas . there have been a number of important studies affirming the relationship of diet and nutrition to the treatment , prevention , and/or slowing progression of a number of age - related ocular diseases . | [
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autism spectrum disorder ( asd ) is a developmental disorder characterized by abnormal communication , social impairment , and stereotyped restricted interests and behavior .
there has been a 10-fold increase in the incidence of asd over the last two decades , with asd now affecting 1 in 88 us children [ 1 , 2 ] .
asd has a 5 : 1 male to female gender bias and is usually diagnosed before 4 years of age .
other male biased conditions including dyslexia , specific language impairment , adhd , and oppositional defiant disorder ( odd ) have also increased during the last few decades [ 3 , 4 ] .
the cost of supporting people with asd in great britain , including the cost of lost productivity , is 2.7b / year for children and 25b / year for adults .
medical expenditures on individuals with asd in the us are 4.16.2 greater than for those without , with average annual healthcare expenditures of approximately $ 6000/child reported and precipitously rising [ 6 , 7 ] .
phenotypic analyses of male biased cognitive disorders suggest that genetic influences increase susceptibility to multiple cognitive deficits [ 810 ] .
the disease triggers and temporal window(s ) in susceptible individuals are unknown , although many have postulated exposure to environmental chemical and/or toxins as candidates .
fetal androgen exposure can alter responses to estrogens / androgens in later life with profound results .
individuals with asd have a cognitive empathy deficit , and women have higher scores in tests of cognitive empathy than do men .
masculinized is greatest in women who have a low 2d : 4d ratio , a marker of in utero androgen exposure .
proposed that the ratio of the second - to - fourth finger length , the 2d : 4d ratio , was a marker for prenatal androgen action .
thus , if the 2d : 4d ratio is a true proxy for fetal androgen exposure , then the masculinized
estrogen alters mitochondrial state 3 respiration , and many aspects of mitochondria function and biogenesis are under estrogenic control [ 14 , 15 ] , especially ( rat ) brain mitochondria .
breast lactate dehydrogenase- ( ldh ) is upregulated by 17-estradiol ( e2 ) , but in sertoli cells both e2 and dihydrotestosterone ( dht ) downregulate ldh expression and lactate generation while increasing glucose consumption , suggesting a switch from anaerobic to aerobic respiration .
with respect to asd , we should be able to observe a differential response to androgens / estrogens and perhaps to hormone mimetics in cells that have been presensitized in utero .
a number of groups have been attempting to understand the genetics of asd and the impact of rare copy number variants . in recent studies ,
the expression profiles of b - lymphocytes from asd individuals and age - matched controls were analyzed by cdna microarrays [ 23 , 24 ] .
further , steroid hormone and neurotransmitter pathways formed the bulk of the differentially expressed genes .
this is interesting , considering that in some studies the asd population has higher testosterone levels than their siblings .
blood samples from subjects with asd and their siblings are thus an important resource for examining genetic / environmental interactions on cell development .
b cells express both estrogen receptor- ( er ; both the 46 and 66 isoforms ) and estrogen receptor- ( er ) receptors , and androgens markedly alter igg / igm expression in mature b - lymphocytes .
the pesticide dichlorodiphenyltrichloroethane ( ddt ; banned in us since 1972 ) and its primary metabolite dichlorodiphenyldichloroethylene ( dde ) remain potential hazards due to their worldwide use , long half - life , and resultant accumulation in the food chain .
dde , which binds to both er and er , has both androgenic and antiandrogenic activities .
in addition , the levels of er and er in human breast cancer correlate with serum dde levels , indicating that cancer cells can be sensitized to estrogenic signals by dde .
dde also mimics the u-shaped e2 stimulation / repression titration curve of -hexosaminidase release in these mast cells over a 0.1100 nm range .
the eightfold increase in children born in california with asd since 1990 can not be attributed simply to changes in diagnostic criteria or record - keeping , suggesting a combination of genetic predisposition and environment .
anthropogenic endocrine disruptors are man - made chemicals that interfere with the body 's endocrine system and produce adverse effects in both humans and wildlife . a wide range of substances
are thought to cause endocrine disruption , including pharmaceuticals , dioxin / dioxin - like compounds , polychlorinated biphenyls , ddt and its primary metabolite dde , and plasticizers such as bisphenol a .
an investigation in sweden has also shown that asd appears to be linked to phthalate exposure early in development , resulting from the usage of pvc flooring . in this study ,
the responses to e2 , dht , and dde , and other hormone disruptors on immortalized b - lymphocytes from asd subjects and controls are compared .
lymphocytes are easy to obtain and culture but are clearly not present in brain under normal circumstances .
knowing very well that asd is a disorder with profound neurological sequelae and that neuronal and/or glial cells are almost certainly involved or affected , normal human astrocytes and cortical neurons were also examined in the presence of the aforementioned agents .
we postulated that b cells from asd individuals would have a different proliferative response to steroid hormones and other hormone disruptors , and normal human astrocytes and cortical neurons would have responses similar to control b - lymphocytes .
as we can not easily study astrocytes and cortical neurons from individuals with autism , we believe it is important to compare the results of responses of normal human astrocytes and neurons to normal control b - lymphocytes to see if the response is similar .
b - lymphocytes were obtained from 11 asd subjects ( aut ) from the autism genetic resource exchange ( agre ) tissue bank who had an unaffected fraternal twin and another unaffected sibling .
this included 10 male and 1 female aut , along with 11 brothers ( bro ) and 11 sisters ( sis ) .
the fraternal twins of the 10 male aut consisted of 4 brothers ( twin bro ) and 6 sisters ( twin sis ) .
b - lymphocytes of 11 individuals from a different cell bank ( coriell institute depository , controls for a longitudinal study of obesity ) were used as an external control ( con ) .
the con were sex and age matched to the aut subjects but had no known personal or family history of asd .
the growth of these cells was examined after 5 days of incubation with different concentrations of e2 , dht , and dde using two assay systems , lactate dehydrogenase and mitochondrial sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ( xtt ) reduction .
we measured total ldh levels as a proxy of cell numbers and xtt reduction as a proxy of mitochondrial reductase levels .
we validated that ldh levels correlated with cell numbers by selective counting using a countess cell counter ( invitrogen , carlsbad , ca , usa ) .
researchers were blinded to the identity of all cell families until the conclusion of all data acquisition .
normal human astrocytes ( nha ) were obtained from lonza ( walkersville , md , usa ) and human cortical neurons ( hcn ) from the atcc ( american type culture collection , manassas , va , usa ) and grown subject to their recommendations .
nha were grown in astrocyte cell basal medium supplemented with 3% fbs , glutamine , insulin , fhegf , ga-1000 , and ascorbic acid .
hcn were grown using atcc - formulated dulbecco 's modified eagle 's medium ( cat no .
nha were grown to confluency in the appropriate media on costar 96-well growth plates ( corning , nyc , ny , usa ) , and hcn were grown on 16-well lab - tek slide chambers ( nalge nunc , rochester , ny , usa ) .
cells were grown for 4 days in the presence of effectors at the following concentrations : 1.2 nm e2 , 12 nm dht , and 50 nm dde .
50 l of cells was assayed for the levels of lactate dehydrogenase activity in the presence of detergent [ 35 , 36 ] .
the final assay mixture comprised 110 mm lactate , 3.35 mm nad , 350 m resazurin , and 2.2 units / ml of diaphorase in 3 mm tris/30 mm hepes/10 mm nacl buffer ( ph 7.4 ) and 0.45% triton x-100 .
the resorufin formed was measured in a plate reader using 530/25 nm ex and 590/35 nm em .
. 50 l of cells was withdrawn to be assayed for mitochondrial function / number using the xtt ( 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ) mitochondrial and extramitochondrial dehydrogenases assay method [ 37 , 38 ] .
the cells were added to 50 l of 1 mg / ml xtt and diluted in growth media .
the xtt converts to formazan by mitochondrial reductase which is only active in metabolically intact cells .
after 60 minutes of incubation , 100 l of stop solution ( 40% sds in 1 : 1 water : ethanol ) was added , and the formazan product is measured at 570 minus 650 nm .
a cell - free media control was treated in the same manner to afford the baseline level of xtt reduction .
preliminary experiments demonstrated the importance of using charcoal stripped fetal calf serum in the growth of b cells and human cortical neurons ( hcn ) .
samples of stripped and unstripped serum were sent to two independent laboratories for hormonal ( texas veterinary medical diagnostic laboratory , college station , tx , usa ) and pesticide ( environmental micro analysis , inc . ,
pesticide levels in both unstripped and stripped fetal calf serum were below the detection threshold of the test procedures .
significant levels of steroidal hormones were found in unstripped media , and these were greatly lowered by charcoal stripping ( table 1 ) .
the differences in growth between stripped and unstripped media suggest that androgens and/or estrogens in the fetal calf serum may retard cell proliferation .
a large number of man - made compounds have been implicated as hormone disruptors , and we wished to examine if there was a differential sensitivity of auts to these compounds .
a range of compounds previously suspected of altering estrogen / androgen signaling in mammalian cells were screened .
di-2-ethylhexyl phthalate ( phthalate dehp ) , nonylphenol , bis - phenol a , and hydroxylated polychlorinated biphenyl ( ho - pcb ) were chosen based upon their wide usage and previous studies implicating these agents as hormone disruptors .
four randomly chosen male auts and their sex matched controls b - lymphocytes were exposed to these agents , and ldh and xtt assays were performed as described above .
b cells derived from individuals with asd ( aut ) react differently to the hormones / hormone mimetics tested ( i.e. , e2 , dht , and dde ) by exhibiting less growth suppression and less mitochondrial proliferation when compared to all other cell populations tested ( figures 1 and 2 ) . two - way anova analysis showed these differences to be statistically significant ( p < 0.0001 ) .
we found that b cells derived from individuals with asd have shallower u - shaped growth curves compared to the other b - cell populations when exposed to e2 , dht , and dde .
this indicates that aut b cells show less growth depression as compared to non - aut b cells in the presence of these agents .
all growth titrations show the classical u-shaped ( nonmonotonic dose response ) curves associated with estrogens , androgens , and pesticide hormone mimetics .
only a small proportion ( < 1% ) of the receptor pool need to be occupied by ligand to initiate a response , and so activation occurs at low hormone levels . at high hormone levels , signals may fall via receptor downregulation [ 39 , 40 ] .
the most significant effect was seen with dht , where aut cells showed the least growth suppression and mitochondrial upregulation ( see the middle columns of figures 1 and 2 as compared to the right and left columns ) . in terms of growth suppression , aut are significantly different from unaffected brothers and sisters and from age / sex matched cells taken from the general , unaffected , population . in all cases where
aut are compared with either internal or external controls , the cumulative sum plots actual observed slopes ' confidence intervals falling outside the theoretical critical slopes ' confidence intervals ( table 2 ) .
if there was no difference between the populations , we would expect to see overlap in the confidence intervals between the actual and theoretical slopes .
thus , there is a < 1% chance that the aut cell population is part of the general population of controls . in all cases where
internal and external controls are compared , there is overlap between the actual and theoretical slopes ' confidence levels .
thus , all internal and external controls appear to represent a single population . in table 3 , we compare the changes in the xtt / ldh ratio in the form of the cumulative sum plots shown in figure 2 .
aut are again statistically different from the external controls , this time with respect to their ability to upregulate mitochondrial numbers ( i.e. , xtt / ldh ratio ) in response to effectors . however , the response of the internal controls ( i.e. , bro and sis ) straddles the divide of the aut and con population . cells from the general population are able to increase their mitochondrial levels , while cells from autistics show a mitochondrial regulatory deficit , and the brothers and sisters of autistics represent a middle - ground between the two . in summary , tables 2 and 3 show that b - lymphocytes from autistic subjects can be characterized as having poor growth suppression in response to authentic hormones and dde and being less able to increase their mitochondrial numbers in response to e2 , dht , or dde .
based on the data we obtained using lymphocytes , we examined the effects of the three effectors on the growth and the mitochondrial numbers of normal human astrocytes ( nha ) and human cortical neurons ( hcn ) .
we find effects on both growth and in the xtt / ldh ratio in both cell types at concentrations which cause differential effects in aut / control b - lymphocytes ( figure 3 ) .
all three effectors cause a statistically significant ( p < 0.05 ) drop in the growth rate of both human astrocytes and neurons .
mitochondria numbers were increased in human cortical neurons by both e2 and dht , but not dde ( p < 0.05 ) .
conversely , upregulation of human astrocytic mitochondria numbers occurred following incubation with dht and dde , but not e2 , again at p < 0.05 .
long known to be neuroprotective , e2 stimulates astrocytes to release growth factors , at the expense of proliferation .
the neurons show a 20% increase in their mitochondrial numbers when exposed to e2 , which replicates the effects that e2 had on b cells .
androgens have been shown to induce mapk signaling in neurons that subsequently drives neuroprotection , protecting them from mitochondrial disruption [ 42 , 43 ] and amyloid peptide toxicity .
the two cell types have a differential response to dde : in astrocytes growth drops and mitochondrial levels rise , but neurons show no statistically significant changes to exposure .
it should be noted that these cells were not titrated with these effectors , and it is possible that astrocytes and neurons have different growth and mitochondrial response profiles to those observed in b - lymphocytes .
table 4 shows the effects of the other hormone disruptors on aut and con b cells along with e2 , dht , and dde .
the major difference between these four hormone disruptors and the authentic hormones or dde was the titration curve shape of both growth and the xtt / ldh ratio .
u-shaped , and instead of kd2 being 1020 times larger than kd1 , it was typically 10,000 to 100,000 times larger . in the titrations , there was always a slight upswing after the maximal change in amplitude , but the end point never returned to the control state .
autism is a neurodevelopmental disease , and this study was designed to examine hormonal signaling in cell growth rather than examining the mechanism of hormone action .
our principle finding is that b - lymphocytes derived from autistic individuals have a different response to e2 , dht , and dde when compared to control subjects .
we find that the biggest difference between the aut and the internal / external control cells is in their response to dht , where they show very little growth modulation and do not upregulate their mitochondrial levels ( see figures 1 and 2 ) .
aut cells had much smaller changes in the amplitude of both growth depression and mitochondrial number upregulation , and higher levels of dht were needed to change mitochondrial levels in aut .
we find this lack of response to dht by the aut b cells very interesting , as the same cells respond to e2 incubation by increasing their mitochondrial levels .
thus , their lack of response to dht is not due to an inability to increase their mitochondria numbers but some other unknown factor suppressing this response . such insensitivity to dht could represent a desensitization of the normal signaling pathway(s ) due to in utero exposure [ 45 , 46 ] .
the effects we observe in using dht are unlikely to be direct androgenic effects , given the resemblance of the e2 and dht u-shaped growth curves .
it is more likely that dht converts into an androgen with kds of 6 and 2 nm for the er and er receptors , respectively .
the hormone disruptors we used ( phthalate dehp , ho - pcb , and nonylphenol bis - phenol ) had similar effects as e2 , dht , and dde but at lower concentrations .
aut cells were less sensitive to the effects of these hormone disruptors than are the external controls .
however , the ability of aut to increase their mitochondria numbers , when challenged with low levels of these hormone disruptors , was always lower than the controls .
the extreme male brain ( emb ) theory of autism was initially formulated by hans asperger in 1944 .
he wrote the autistic personality is an extreme variant of male intelligence . even within the normal variation ,
baron - cohen forwarded a new theory of the psychology of sex differences in the 1990s ( the empathizing - systemizing theory ) and related this to asperger 's emb theory . the cambridge longitudinal foetal testosterone project examined in utero testosterone levels and child developmental parameters .
it showed that fetal testosterone ( ft ) is negatively correlated with social and language development but is positively correlated with a number of autistic traits , suggesting a role for high androgen levels in asd .
the 2d : 4d ratio in females appears to be a robust proxy of prenatal androgen exposure but is less robust in males .
children with asd and also children with adhd / odd have significantly lower 2d : 4d ratios than controls .
handedness is also tied to the 2d : 4d ratio , and in utero androgen exposure has long been believed to predispose to left - handedness .
recent evidence shows that ft levels in amniotic fluid predicts both language lateralization and handedness .
this adds further support for a role of ft in asd ; as in addition to having a low 2d : 4d ratio , the degree of non - right - handedness is 65% in asd [ 54 , 55 ] , compared to 1012% of the general population .
estrogen and androgen steroids are involved in the process of brain sculpting that generates sexual dimorphic male / female brains .
the largest known cognitive sex difference in human males and females is spatial ability , with 3d mental rotation tasks showing the greatest gender difference .
a recent study shows that asds significantly outperform matched controls in many complex 3d mental rotation tasks .
testosterone has direct effects via the androgen receptor and indirect effects following its enzymatic conversion to dihydrotestosterone ( dht ) and estradiol ( e2 ) , with both converting enzymes present in neural tissue .
dht is also a prohormone , and two enzymes generate the neurosteroids 5-androstan-3,17-diol and 3,17 -diols .
the 3-diol is an agonist for er , and thus some dht actions in brain are mediated by conversion to 3-diol and subsequent er activation .
sex hormone signaling disorders inform us of the extent that estrogen , testosterone , and dht have on converting the default female brain into that of the male brain .
46xy individuals with complete androgen insensitivity syndrome ( cais ) lack a functioning androgen receptor , and their tissues are unable to respond to testosterone / dht .
they are conventionally feminine across a range of psychological tests , and 2d : 4d digit ratios are feminized .
46xy individuals with complete 5-alpha - reductase-2-deficiency ( 5r2d ) have low or zero dht and slightly elevated t levels .
they present as female at birth ; however , the brain is masculinized even in individuals raised as girls .
complete aromatase deficiency ( ad ) is very rare , but 46xyad individuals who lack estrogen throughout development have brain masculinization .
21-hydroxylase deficiency ( 21hd ) results in testosterone / dht overexposure in utero and virilization of females .
46xx21hd individuals have a mildly masculinized spatial ability compared with unaffected control females , a low 2d : 4d ratio [ 67 , 68 ] , and a pronounced left - handedness bias .
it has long been known that men with androgenetic alopecia ( aga ) have high levels of 5-alpha - reductase ( 5ar ) , total androgens , unbound / free androgens , and a high dht : t ratio .
a genome scan looking for dht susceptibility loci in 95 families where at least two brothers had early - onset aga has been conducted .
the ar gene in the xp11-q25 region , long associated with aga , had an npl score of 2.67 , but a second locus , 3q26 , scored 2.69 .
a locus at 3q26 was found to be highly linked to these conditions in a finnish study of 38 families with high rates asd . in a very large
asd family there are 7 asd males , 11 unaffected males , and 10 unaffected females , and the 3q26.31q27.3 region was again an asd locus .
thus , at least one region on chromosome 3 is implicated in aga , a dht - sensitive condition , and asd , a putative androgenic sensitive condition .
this study was designed to give insight into the interaction of environment and genes with respect to asd .
a common womb constitutes part of the environment of the 10 aut , bro , and sis , but not the con .
if a gestational asd trigger is present in the 10 families , then by examining only unaffected brothers , we may be observing a survivor effect .
the statistically significant difference found only between aut and bro could possibly be explained by the following : aut represent a population susceptible to a familial gestational trigger , bro represent a risk resistant population immune to this theoretical insult , and the external controls represent an at risk population never exposed to the trigger as they were formed in another womb .
there is increasing evidence that individuals with autism are more sensitive to reactive oxygen species ( ros ) , and that changes in this sensitivity may be important in understanding the pathophysiology of autism . in this regard ,
we find it to be interesting and important that exposure to hormone disruptors influences mitochondrial function , as upregulation of mitochondria number and function are important responses to present damage from ros . in individuals with inherent genetic differential sensitivities to hormone disruptors
, early environmental exposure may play an important role in either the etiology of the disorder , or in the development of a number of cellular and metabolic deficits associated with it .
recent publications from our lab and others have implicated thimerosal in similar metabolic pathways examined in this paper [ 73 , 74 ] .
the possibility of thimerosal and/or other neurotoxins interacting with the hormones and hormone disruptors presented here to yield the asd phenotype is an area which we are currently investigating .
finally , it is quite clear that b - lymphocytes are not normally present in brain and that autism is a neurological disease .
thus , our b - cell data does not directly inform us if estrogens , androgens , and hormone disruptors have differential effects on the development and growth of the brain in autistic individuals .
however , we did examine the effects of e2 , dht , and dde on human astrocytes and cortical neurons at concentrations where there was a clear differential effect on autistic b cells .
we found that there were similar effects on cell growth and mitochondrial upregulation in astrocytes and neurons compared to those found in b - lymphocytes .
we therefore think it is reasonable to postulate that the disruption of hormonal signaling we found in b - lymphocytes from individuals with autism could also be present in neurons and glial cells of these individuals .
b - lymphocytes from people with asd exhibit a differential response to e2 , dht , and hormone disruptors in regard to cell growth , and mitochondrial number increases when compared to non - asd siblings and external controls .
specifically , asd b - lymphocytes show significantly less growth depression and less increase in mitochondrial number when exposed to these effectors .
normal human astrocytes and human cortical neurons exhibit responses similar to control b - lymphocytes with respect to cellular growth and mitochondrial response when exposed to these agents .
while astrocytes and neurons from individuals with asd were not tested in this study , the data here suggests that a response similar to what was seen in b - lymphocytes may occur in brain tissue in autistic individuals . | it has been postulated that androgen overexposure in a susceptible person leads to excessive brain masculinization and the autism spectrum disorder ( asd ) phenotype . in this study ,
the responses to estradiol ( e2 ) , dihydrotestosterone ( dht ) , and dichlorodiphenyldichloroethylene ( dde ) on b - lymphocytes from asd subjects and controls are compared .
b cells were obtained from 11 asd subjects , their unaffected fraternal twins , and nontwin siblings .
controls were obtained from a different cell bank .
lactate dehydrogenase ( ldh ) and sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ( xtt ) reduction levels were measured after incubation with different concentrations of e2 , dht , and dde .
xtt / ldh ratio , representative of mitochondria number per cell , was calculated .
e2 , dht , and dde all cause
u-shaped growth curves , as measured by ldh levels .
asd b cells show less growth depression compared to siblings and controls ( p < 0.01 ) .
they also have reduced xtt / ldh ratios ( p < 0.01 ) when compared to external controls , whereas siblings had values of xtt / ldh between asd and external controls .
b - lymphocytes from people with asd exhibit a differential response to e2 , dht , and hormone disruptors in regard to cell growth and mitochondrial upregulation when compared to non - asd siblings and external controls . specifically , asd b - lymphocytes show significantly less growth depression and less mitochondrial upregulation when exposed to these effectors . a mitochondrial deficit in asd individuals is implied . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion | breast lactate dehydrogenase- ( ldh ) is upregulated by 17-estradiol ( e2 ) , but in sertoli cells both e2 and dihydrotestosterone ( dht ) downregulate ldh expression and lactate generation while increasing glucose consumption , suggesting a switch from anaerobic to aerobic respiration . in recent studies ,
the expression profiles of b - lymphocytes from asd individuals and age - matched controls were analyzed by cdna microarrays [ 23 , 24 ] . in this study ,
the responses to e2 , dht , and dde , and other hormone disruptors on immortalized b - lymphocytes from asd subjects and controls are compared . we postulated that b cells from asd individuals would have a different proliferative response to steroid hormones and other hormone disruptors , and normal human astrocytes and cortical neurons would have responses similar to control b - lymphocytes . b - lymphocytes were obtained from 11 asd subjects ( aut ) from the autism genetic resource exchange ( agre ) tissue bank who had an unaffected fraternal twin and another unaffected sibling . the growth of these cells was examined after 5 days of incubation with different concentrations of e2 , dht , and dde using two assay systems , lactate dehydrogenase and mitochondrial sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ( xtt ) reduction . four randomly chosen male auts and their sex matched controls b - lymphocytes were exposed to these agents , and ldh and xtt assays were performed as described above . , e2 , dht , and dde ) by exhibiting less growth suppression and less mitochondrial proliferation when compared to all other cell populations tested ( figures 1 and 2 ) . we found that b cells derived from individuals with asd have shallower u - shaped growth curves compared to the other b - cell populations when exposed to e2 , dht , and dde . this indicates that aut b cells show less growth depression as compared to non - aut b cells in the presence of these agents . in summary , tables 2 and 3 show that b - lymphocytes from autistic subjects can be characterized as having poor growth suppression in response to authentic hormones and dde and being less able to increase their mitochondrial numbers in response to e2 , dht , or dde . we find effects on both growth and in the xtt / ldh ratio in both cell types at concentrations which cause differential effects in aut / control b - lymphocytes ( figure 3 ) . the neurons show a 20% increase in their mitochondrial numbers when exposed to e2 , which replicates the effects that e2 had on b cells . it should be noted that these cells were not titrated with these effectors , and it is possible that astrocytes and neurons have different growth and mitochondrial response profiles to those observed in b - lymphocytes . table 4 shows the effects of the other hormone disruptors on aut and con b cells along with e2 , dht , and dde . the major difference between these four hormone disruptors and the authentic hormones or dde was the titration curve shape of both growth and the xtt / ldh ratio . our principle finding is that b - lymphocytes derived from autistic individuals have a different response to e2 , dht , and dde when compared to control subjects . testosterone has direct effects via the androgen receptor and indirect effects following its enzymatic conversion to dihydrotestosterone ( dht ) and estradiol ( e2 ) , with both converting enzymes present in neural tissue . in this regard ,
we find it to be interesting and important that exposure to hormone disruptors influences mitochondrial function , as upregulation of mitochondria number and function are important responses to present damage from ros . however , we did examine the effects of e2 , dht , and dde on human astrocytes and cortical neurons at concentrations where there was a clear differential effect on autistic b cells . we found that there were similar effects on cell growth and mitochondrial upregulation in astrocytes and neurons compared to those found in b - lymphocytes . b - lymphocytes from people with asd exhibit a differential response to e2 , dht , and hormone disruptors in regard to cell growth , and mitochondrial number increases when compared to non - asd siblings and external controls . specifically , asd b - lymphocytes show significantly less growth depression and less increase in mitochondrial number when exposed to these effectors . normal human astrocytes and human cortical neurons exhibit responses similar to control b - lymphocytes with respect to cellular growth and mitochondrial response when exposed to these agents . while astrocytes and neurons from individuals with asd were not tested in this study , the data here suggests that a response similar to what was seen in b - lymphocytes may occur in brain tissue in autistic individuals . | [
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over the last few decades , metabolic syndrome has become a pervasive epidemic in the western hemisphere , and perhaps surprisingly , this growth is not restricted to developed countries.1 metabolic syndrome consists of a collection of risk factors for cardiovascular disease , encompassing such conditions as obesity , diabetes , and hypertension.1 epidemiologists are now witnessing a worldwide spread of metabolic syndrome due to the industrialization of developing countries in conjunction with the globalization of western dietary preferences.1 in the us , the national health and nutrition examination survey compiled during the years of 20032006 showed that 34% of adults over the age of 20 years met the criteria for metabolic syndrome , which is based on the clinical evaluation of abdominal obesity , plasma triglycerides , blood pressure , and fasting blood glucose levels.2 recently , nonalcoholic fatty liver disease ( nafld ) has been adopted as the hepatic manifestation of metabolic syndrome,3 and it is clinically defined by triacylglycerol ( tag ) storage exceeding 5% of the total liver mass.4 nafld affects an estimated 20%30% of the us population , and 20% of those affected will eventually develop nonalcoholic steatohepatitis ( nash ) .
once an individual has nash , fibrosis is visible in the liver tissue , and the continued presence of stressors may elicit the later emergence of cirrhosis or hepatocellular carcinoma.3,5 although the etiology of nafld is poorly understood , a two - hit hypothesis was proposed in 1998 by day and james6 to describe the development of nafld and its progression to nash . according to this hypothesis , the first
consists of hepatic insulin resistance or impaired -oxidation of fatty acids , both of which contribute to hepatic lipid accumulation .
the second hit consists of inflammation or oxidative stress , which may aggravate the existing steatosis and drive progression into nash.6,7 recently , it has been proposed that the first
hit can occur in utero due to a maternal diet that is high in fat .
indeed , the offspring of such a condition have been shown to have a three - fold increase in hepatic tag shortly after birth.4,8 this predisposes the offspring to the adult development of nafld , as only a second hit would be needed to spur the development of steatohepatitis .
the current literature regarding the contribution of prenatal nutrition to the development of nafld reveals several distinct phenotypic alterations .
this review seeks to connect these seemingly disconnected outcomes into a plausible sequence of events .
the underlying questions that this review addresses are : 1 ) in which ways does a lipid - rich nutritional environment furnished by the mother interact with the biochemical changes occurring within a developing fetus ; and 2 ) how may these interactions result in changes in gene expression that contribute to an adult phenotype amenable to tag accumulation in the liver ? as nafld continues to extend its reach worldwide , seeking answers to these questions is essential .
in maternal circulation , plasma concentrations of nonesterified fatty acids ( nefas ) bound to albumin and tag contained within chylomicrons gradually elevate throughout gestation.9 placental uptake of nefas occurs either by diffusion or through a variety of fatty acid transport proteins . for chylomicrons ,
the placenta expresses lipoprotein lipase , which cleaves the contained tag molecules into three nefas and a glycerol molecule.9 the nefas are then free to diffuse or be transported via a fatty acid transport protein into fetal circulation . upon reaching the developing liver ,
nefas diffuse into hepatocytes and they are rapidly converted into fatty acyl - coenzyme a ( coa).10 primarily , fatty acyl - coas enter the mitochondria for -oxidation .
this is mediated by the rate - limiting enzyme , carnitine palmitoyltransferase 1 ( cpt1 ) , which exchanges the bound coa for carnitine yielding acyl - carnitine which can then pass through the outer mitochondrial membrane.11 remaining nefas that do not undergo -oxidation will be stored by esterification at the endoplasmic reticulum ( er ) as tag.12 tag may be exported within very low - density lipoproteins ( vldl ) , or tag may be stored within the hepatocyte cytosol in lipid droplets.13,14 a lipid droplet is an organelle that functions to store tag within a phospholipid membrane comprised chiefly of phosphatidylcholine ( pc ) . since nefa accumulation is toxic to hepatocytes,15 vldl secretion and lipid droplet formation
can be regarded as protective measures against hepatotoxicity.16 if the liver as a whole attains more fatty acids than it eliminates via -oxidation or vldl secretion , tag accumulates in lipid droplets,10 and this represents the defining characteristic of nafld . in a model of lipid - rich prenatal nutrition , the generalized pathway followed by nefas , as described above , is perturbed in a manner that affects the offspring s physiology into adulthood , and induces a lifelong predisposition for nafld .
many of the studies reviewed here used mice as their model organism and the researchers apportioned a maternal prenatal diet to one group of dams with a higher composition of dietary lipids than that received by the control group .
although not uniform amongst all studies , control groups were fed a commercial standard chow
diet containing macronutrients distributed aptly for the nutritional needs of mice . increased consumption of dietary lipids is a frequent nutritional misallocation in the common western diet ; hence , what can be learned from these studies has the potential to be readily implemented and appreciated .
however , a significant and inherent limitation of this research is that increasing the quantity of one macronutrient requires decreasing that of another . if the total percentage of lipids increases , then the percentage of other nutrients falls relative to the total - it is difficult to know whether the experimental results can be attributed to an increase of one or a decrease in another , or the combination . additionally , it is unlikely that an average unbalanced western diet errs only in its total composition of lipids .
although the implications of a maternal high - fat ( hf ) diet explored in this review may apply to those mothers who , for example , additionally have a vitamin deficiency , whether there exists synergism or antagonism between certain dietary imbalances has yet to be explored thoroughly , especially in the context of prenatal nutrition .
in a study by bruce et al,17 the hepatic metabolic capacities of four groups of mice that received either a hf or control ( c ) prenatal and postnatal diet were analyzed .
the groups were designated as c / c , c / hf , hf / c , and hf / hf , in reference to their prenatal / postnatal nutrition.17 this study revealed that levels of cpt1 , which is the enzyme that mediates the transfer of fatty acyl - coa through the outer mitochondrial membrane in the initiation of -oxidation,11 did not differ among hf / c , c / hf , and hf / hf mice , although all three of these groups had marginally elevated cpt1 compared to the c / c group.17 this suggests that in the presence of increased cytosolic fatty acids , there is an upper limit to the amount of cpt1 that can be produced to accommodate the influx ; cpt1 is the rate - limiting enzyme in this process.11 the increase in nefas in fetal circulation , which is a result of the quantity of maternal dietary lipids , was met with a corresponding slight escalation in fetal -oxidation , as described by byrne et al.18 strikingly , this study determined that at 15 weeks of age , the hf / c and hf / hf groups exhibited a 3.7- and 3.2-fold respective decrease in electron transport chain ( etc ) complex i activity compared to the c / c group , and the c / hf group did not vary significantly from the c / c group in this regard.18 complex i of the etc is notorious for the leakage of reactive oxygen species ( ros ) ( figure 1).19 byrne et al18 hypothesize that increased ros leakage from complex i , which directly results from the increased rates of fetal -oxidation , causes mitochondrial deoxyribonucleic acid ( mtdna ) damage .
this could potentially reduce the postnatal mitochondrial capacity for -oxidation , as mtdna lacks the shielding histone structure that nuclear deoxyribonucleic acid ( dna ) is afforded.18 albeit reasonable , this explanation neglects to consider the c / hf group s unaffected complex i activity if higher -oxidation is the initiating event that results in downstream damage , as the authors assert , it remains unexplained why the c / hf group did not display decreased etc complex i activity comparable to that of the hf / c and hf / hf groups .
cytotoxic damage produced by sufficient ros is often referred to as oxidative stress . in several models of oxidative stress
, it has been demonstrated that a cell may selectively shunt metabolites from the methionine cycle ( figure 2 ) , down the transsulfuration pathway at the expense of s - adenosylmethionine in order to increase the synthesis of glutathione ( gsh ) , a scavenger of ros , in an effort to combat oxidative stress.20,21 independently , bravo et al22 showed that in rats fed a hf diet , expression of the enzymes cystathionine -synthase ( cbs ) and cystathionine -lyase , which participate in the transsulfuration pathway , were significantly downregulated.22 cbs is a necessary enzyme that is involved in the redirection of homocysteine from the methionine cycle toward gsh synthesis , which occurs in the first step of the transsulfuration pathway.21 in conjunction with pressure to generate gsh to neutralize ros , homocysteine levels are elevated ( figure 2 ) .
increased homocysteine levels lead to the downregulation of many antioxidant enzymes,23 including gsh peroxidase 1 , by directly interfering with translation.24 in 2011 , zhang et al25 determined that offspring of mice fed a hf diet during gestation had decreased expression of the antioxidant enzymes gsh peroxidase 1 , superoxide dismutase 1 , and paraoxonases 1 , 2 , and 3 .
although this group did not suggest a reason for this downregulation , they did expound upon the consequences of the resultant lowered threshold for oxidative stress in the absence of adequate endogenous defenses .
it is possible that mild hyperhomocysteinemia , due to the downregulation of cbs and the diversion of methionine cycle metabolites down the transsulfuration pathway , is responsible for the downregulation of antioxidant enzymes , as noted by zhang et al.25 in the study by byrne18 discussed earlier , the mice fed a c diet in utero and a hf diet postnatally ( c / hf ) did not display altered etc complex i activity , and it is possible that this is because their levels of ros - neutralizing antioxidant enzymes were not downregulated during development and , consequently , mtdna damage did not occur .
in hepatocytes , nefas that are not accommodated by cpt1 will be esterified to form tag , which may be packaged into a vldl particle and exported , or they may be stored within the cytosol of the hepatocyte in a lipid droplet.13,14 the storage of tag within a hepatocyte may be precipitated by four independent factors : 1 ) increased nefas in hepatocytes due to increased delivery of nefas or de novo lipogenesis ; 2 ) increased rates of tag synthesis ; 3 ) reduced levels of vldl export ; or 4 ) decreased capacity for -oxidation.11 given that tag accumulation exceeding 5% of the total liver weight is the defining criterion for the presence of nafld set forth by the american association for the study of liver diseases,4 presented here is a potential mechanism that connects excess hepatocyte cytosolic nefas with the derailing of lipid synthesis regulation by the er , resulting in the accumulation of tag within lipid droplets .
the er produces acyl - coa synthetase , which activates nefas in the cytosol by converting nefas into fatty acyl - coas to allow for entrance into the er , where the fatty acyl - coas will serve as substrates for lipid synthesis.12 furthermore , due to the increased delivery of cytoplasmic nefas that results from a maternal lipid - rich diet , some of the tag will be stored in lipid droplets adjacent to the er.14 for reasons incompletely understood , sustained increases in cytosolic nefa concentration and especially increases in saturated nefas
beget er stress and , subsequently , the induction of the unfolded protein response ( upr ) in both mice and in the yeast , saccharomyces cerevisiae.26 the existing discussion of nefa - induced er stress in the literature often elects to cover the downstream effects of er stress , rather than investigate the mechanism responsible for its appearance ; here , both will be addressed .
when tag is synthesized for storage in a lipid droplet , this presents to the hepatocyte the additional challenge of producing pc for the lipid droplet membrane.27 while the er membrane contains a specific ratio of pc and phosphatidylethanolamine ( pe ) , the preferential phospholipid coat for lipid droplets is pc ; due to its properties as a surfactant , pc maintains the integrity and prevents the coalescence of adjacent lipid droplets .
when pc is replaced with pe , neighboring lipid droplets rapidly merge.28 fu et al27 assert that in order to produce pc , the genes pemt and pcyt1a are activated .
pemt is a s - adenosylmethionine - dependent methyltransferase that converts existing pe into pc by way of sequential methylation , and it is only significantly expressed in the liver.27
pcyt1a encodes the more ubiquitous synthesizer of pc , ctp : phosphocholine cytidylyltransferase.29 membrane composition is a determinant of many biophysical properties of a cell or an organelle,30 and upon producing pc for lipid droplets , the specific pc / pe ratio in the er membrane is threatened and may rise.15,28 although the mechanism is incompletely understood , an elevated pc / pe ratio in the er membrane causes spontaneous dysfunction of sarco / er calcium adenosine triphosphatase ( serca ) , and returning the ratio to its initial state improves serca function.31 serca maintains a significant calcium gradient ; the concentration of calcium within the er is nearly 10,000 times greater than in the cytoplasm.15 proper serca function is crucial not only for maintaining low cytosolic calcium for the purpose of signaling events that may include a controlled release of calcium , but it is also important because many of the chaperone proteins in the er lumen such as binding immunoglobulin protein ( also known as grp78 ) , calreticulin , and calnexin are calcium - dependent.32,33 impaired serca function thus impedes the ability of calcium - dependent chaperones to properly fold proteins.34,35 additionally , the increase in cytosolic calcium concentration further aggravates mitochondria and contributes to additional oxidative stress.32 the accumulation of unfolded or misfolded proteins in the er lumen defines er stress and activates the upr , a series of intracellular signaling events that function to restore er homeostasis.36 these events culminate in reducing the ability of proteins to enter the er for posttranslational modifications , upregulation of chaperone proteins and , if homeostasis can not be restored , apoptosis.15 er stress and the upr have two implications that directly contribute to the predisposition of offspring to nafld : upon induction of the upr , activating transcription factor-6 ( atf6 ) one of the proteins that participates in upr intracellular signal transduction is activated by intramembrane proteolysis .
atf6 shares its proteolytic machinery with sterol response element binding protein 1c ( srebp-1c),36 so upon cleavage of atf6 , srebp-1c is also cleaved and is able to translocate to the nucleus.37 srebp-1c is a transcriptional activator for the lipogenic proteins , acetyl - coa carboxylase ( acc ) and glucokinase.10 er stress also activates the c - jun n - terminus kinase ( jnk ) pathway , which leads to the phosphorylation of certain serine residues in insulin receptor substrate-1 ( irs-1 ) .
given that the docking of irs-1 , the subsequent tyrosine phosphorylation , and the release of irs-1 is a necessary occurrence in successful insulin signaling , serine phosphorylation of irs-1 blocks signal transduction and contributes to hepatic insulin resistance.38,39 hepatic insulin resistance has been independently demonstrated to overactivate srebp-1c.40 srebp-1c activates the transcription of acc and glucokinase , which both contribute to postnatal fatty acid synthesis.10,37,40 however , the presence of both acc and glucokinase are complicated in a hf intrauterine environment .
acc converts acetyl - coa into malonyl - coa which , in addition to playing a key role in de novo lipogenesis , is a potent inhibitor of cpt1 and thus decreases the rate of -oxidation ( figure 3).10 additionally , it has been shown that the fetal liver is more sensitive than the liver of a newborn to the inhibitory effects of malonyl - coa.9 in fact , in a maternal hf diet experiment performed by ashino et al,4 82-day - old offspring of dams who consumed a diet deriving 45% of its caloric value from nondescript fats had higher levels of activated acc than did offspring of mice fed a control diet , which indicates that the elevation of acc persists in the postnatal state . since nagle et al11 included decreased -oxidation as one of the four factors that directly contributes to tag accumulation in the liver , it is probable that malonyl - coa contributes to the predisposition of offspring to nafld .
glucokinase participates in the first step of fatty acid synthesis by phosphorylating glucose , resulting in glucose-6-phosphate.41 trophoblasts ordinarily express numerous glucose transporters from maternal to fetal circulation , such that glucose is readily available for the fetus.9 additionally , it has been demonstrated in a rodent model that the placentas of mothers who consume a hf diet express a five - fold increase in glucose transporter-1 , which facilitates the diffusion of glucose into the fetal circulation.42 the increased expression of glucokinase in conjunction with an elevated influx of glucose may additionally contribute to de novo lipogenesis .
as nefas are produced by the de novo lipogenesis , in which both malonyl - coa and glucokinase participate , and since er stress was initiated with excessive nefas entering the er for lipid synthesis , this process can be perceived as cyclic .
certainly , the nefas produced by de novo lipogenesis could instead undergo -oxidation , but as their synthesis requires nicotinamide adenine dinucleotide phosphate,9 this would be more energetically expensive than to reserve -oxidation for exogenous , maternally - derived nefas .
additionally , the inhibition of -oxidation by malonyl - coa , and the potential mtdna damage caused by rampant ros , both encourage the continuation of nefas in the cycle .
given the multifaceted nature of tag accumulation borne in utero , it may seem likely that many opportunities exist for therapeutic interjection .
presently , omega-3 polyunsaturated fatty acids ( n-3 pufas ) and genistein have evidence to support their intake as part of preventative maternal prenatal nutrition .
novak et al43 performed a study whereby two groups of dams were fed the same total amount of dietary fat , but the composition of dietary fats differed between the groups .
each received different fats but both had the same total amount of dietary fat . on postnatal day 3 , the offspring of the group that received adequate n-3 pufas displayed higher levels of proteins that were integral to mitochondrial -oxidation , including cpt1 , and they downregulated genes associated with fatty acid synthesis , compared to the group that received inadequate n-3 pufas.43 it appears that supplementation of n-3 pufas as part of proper prenatal nutrition , which is reflective of this finding , may be a possible preventative route for nafld . however , in conflict with this proposition is evidence proposed by dennery in 2010,44 who claimed that maternal pufa supplementation causes oocyte mitochondria to increase their ros production , which leads to impaired embryonic development . in adults with nafld ,
supplementation with docosahexaenoic acid , an n-3 pufa , has been shown to reduce the rates of de novo lipogenesis via the negative regulation of srebp-1c transcription.45 perhaps a conciliatory solution lies in the timing of n-3 pufa supplementation ; questions with such therapeutic potential warrant additional investigation .
genistein , a soy isoflavone found in soybeans , has received more consistent favorable literature reviews than n-3 pufas . when provided concurrently with a maternal hf diet and comprising 0.2% ( by weight ) of total maternal nutrition ,
the offspring displayed significantly less hepatic tag accumulation and elevated levels of gsh when compared to offspring that solely received a maternal hf diet.46 as gsh production is regulated in response to ros levels , it is possible that genistein supplementation ameliorated these defenses and prevented the downstream hepatotoxic effects of uncontrolled ros . additionally , in an adult hf feeding study conducted by ji et al,47 genistein supplementation embodied the qualities of an anti - inflammatory compound and appreciably decreased the serum levels of the cytokines , tumor necrosis factor - alpha and interleukin 6 .
as inflammation provides the second hit that drives the transition from nafld into nash , the anti - inflammatory effects of genistein may prevent or attenuate the development of nash .
ji et al47 also noted that genistein is a tyrosine kinase inhibitor , and they implicated this characteristic as the cause of the marked inhibition of the jnk pathway observed in response to genistein supplementation .
activation of the jnk pathway is implicated in the development of hepatic insulin resistance and subsequent overactivation of srebp-1c.39,40 preventing these outcomes in utero by supplementing the prenatal diet with genistein may improve the offspring s hepatic phenotype .
it is also encouraging that these improvements in inflammation occurred in the postnatal environment , as they represent a potential degree of reversibility for those born at risk for the development of nafld . certainly with the number of factors that contribute to the development of nafld , additional therapeutic avenues for preventing tag accumulation have yet to be discovered .
an experimental approach of conjugating the lipophilic cation , triphenylphosphonium , to an antioxidant such as vitamin e or ubiquinone has been shown to permit the delivery of the antioxidant moiety through the mitochondrial membrane.48,49 when this approach was described by murphy and smith in 2007,48 it was their intention to employ the lipophilicity of triphenylphosphonium to ensure diffusion of the antioxidant into the mitochondria , where ros neutralization is most useful in relegating oxidative stress .
recently , finichiu et al49 confirmed that this form of antioxidant delivery does indeed reach mitochondria in the tissues with high levels of oxidative stress .
although this mechanism has only been investigated in general models of oxidative stress , given the role of ros in the development of nafld , future advances may render this an applicable method of antioxidant delivery for those affected by nafld .
there is a need for a clearly defined , ideal prenatal diet that considers the negative implications of the hf diet discussed here , and there is also the need for a diet for those affected by nafld .
the american dietetic association does not currently suggest a recommended daily allowance of fats for pregnant women.50 current research is exploring many different diet and exercise regimes for those living with nafld , with mixed results.51,52 future work needs to ascertain the maximum daily allowance for dietary fats when undesirable outcomes for the fetus arise .
this will allow for the development of dietary guidelines that could easily be followed by expecting mothers .
the diagnostic approach to nafld in childhood has recently been developed by the european society of paediatric gastroenterology , hepatology , and nutrition committee.53 the gold standard for the detection of nafld is through liver biopsy , but this should only be performed after less invasive procedures to rule out other conditions .
testing for appropriate biomarkers and imaging by magnetic resonance imaging or ultrasound are noninvasive and fairly reliable diagnostic tools for adults and older children , and these methods may also be developed for infants .
education aimed at promoting a balanced prenatal diet may be an effective intervention in convincing expecting mothers to consider nutrition as equally important to the health of a fetus as abstaining from tobacco use and alcohol consumption .
previous efforts to educate women on the harmful consequences of these activities during pregnancy have been met with success.9 a voluntary decrease in smoking and drinking alcohol by pregnant women was demonstrated following these efforts , which was indicative of a willingness by women to modify their lifestyle for the benefit of their babies.9 as the metabolic syndrome has garnered significant attention from public health organizations in western countries , the recent acceptance of nafld as the hepatic manifestation of metabolic syndrome will likely spur its inclusion in future public health educational efforts . of adults who present with nafld , most display significant mtdna abnormalities and mutations , and
it has been estimated that about 26% of hepatic tag in nafld patients is derived from de novo lipogenesis.10,21 in the context of elevated maternal dietary lipids , it is clear that both de novo lipogenesis and esterification of delivered nefas are encouraged in the fetus via multiple mechanisms discussed herein : damaged mtdna and associated reduced capacity for -oxidation ; the inhibition of cpt1 by malonyl - coa ; and the inadvertent activation of srebp-1c during the unfolded protein response .
the resultant phenotype of the offspring whose mothers consumed a lipid - rich diet during pregnancy shares many commonalities with that of adult nafld , and as illustrated in this review , it predisposes offspring for the later development of nafld . | nonalcoholic fatty liver disease ( nafld ) is the hepatic manifestation of metabolic syndrome .
it affects 20%30% of the us population , and it is increasing worldwide . recently , the role of lipid - rich maternal gestational nutrition in spurring the development of nafld among offspring has been indicated . fetal predisposition to nafld
involves numerous physiological reroutings that are initiated by increased delivery of nonesterified fatty acids to the fetal liver .
hampered -oxidation , uncontrolled oxidative stress , increased triacylglycerol synthesis , and the endoplasmic reticulum unfolded protein response are all implicated in sculpting a hepatic phenotype with a propensity to develop nafld in the postnatal state .
this review suggests a mechanism that integrates outcomes reported by a variety of studies conducted in an analysis of fetal hepatic metabolic capacity amid the maternal consumption of a high - fat diet .
potential preventive measures and therapies for use both as part of prenatal nutrition and for those at risk for the development of nafld are also discussed . | Introduction
Physiological background
Methods
Disruptions resulting from elevated -oxidation
Disruptions resulting from triacylglycerol storage
Management of NAFLD: prenatal and postnatal, present and future
Conclusion | over the last few decades , metabolic syndrome has become a pervasive epidemic in the western hemisphere , and perhaps surprisingly , this growth is not restricted to developed countries.1 metabolic syndrome consists of a collection of risk factors for cardiovascular disease , encompassing such conditions as obesity , diabetes , and hypertension.1 epidemiologists are now witnessing a worldwide spread of metabolic syndrome due to the industrialization of developing countries in conjunction with the globalization of western dietary preferences.1 in the us , the national health and nutrition examination survey compiled during the years of 20032006 showed that 34% of adults over the age of 20 years met the criteria for metabolic syndrome , which is based on the clinical evaluation of abdominal obesity , plasma triglycerides , blood pressure , and fasting blood glucose levels.2 recently , nonalcoholic fatty liver disease ( nafld ) has been adopted as the hepatic manifestation of metabolic syndrome,3 and it is clinically defined by triacylglycerol ( tag ) storage exceeding 5% of the total liver mass.4 nafld affects an estimated 20%30% of the us population , and 20% of those affected will eventually develop nonalcoholic steatohepatitis ( nash ) . the groups were designated as c / c , c / hf , hf / c , and hf / hf , in reference to their prenatal / postnatal nutrition.17 this study revealed that levels of cpt1 , which is the enzyme that mediates the transfer of fatty acyl - coa through the outer mitochondrial membrane in the initiation of -oxidation,11 did not differ among hf / c , c / hf , and hf / hf mice , although all three of these groups had marginally elevated cpt1 compared to the c / c group.17 this suggests that in the presence of increased cytosolic fatty acids , there is an upper limit to the amount of cpt1 that can be produced to accommodate the influx ; cpt1 is the rate - limiting enzyme in this process.11 the increase in nefas in fetal circulation , which is a result of the quantity of maternal dietary lipids , was met with a corresponding slight escalation in fetal -oxidation , as described by byrne et al.18 strikingly , this study determined that at 15 weeks of age , the hf / c and hf / hf groups exhibited a 3.7- and 3.2-fold respective decrease in electron transport chain ( etc ) complex i activity compared to the c / c group , and the c / hf group did not vary significantly from the c / c group in this regard.18 complex i of the etc is notorious for the leakage of reactive oxygen species ( ros ) ( figure 1).19 byrne et al18 hypothesize that increased ros leakage from complex i , which directly results from the increased rates of fetal -oxidation , causes mitochondrial deoxyribonucleic acid ( mtdna ) damage . pemt is a s - adenosylmethionine - dependent methyltransferase that converts existing pe into pc by way of sequential methylation , and it is only significantly expressed in the liver.27
pcyt1a encodes the more ubiquitous synthesizer of pc , ctp : phosphocholine cytidylyltransferase.29 membrane composition is a determinant of many biophysical properties of a cell or an organelle,30 and upon producing pc for lipid droplets , the specific pc / pe ratio in the er membrane is threatened and may rise.15,28 although the mechanism is incompletely understood , an elevated pc / pe ratio in the er membrane causes spontaneous dysfunction of sarco / er calcium adenosine triphosphatase ( serca ) , and returning the ratio to its initial state improves serca function.31 serca maintains a significant calcium gradient ; the concentration of calcium within the er is nearly 10,000 times greater than in the cytoplasm.15 proper serca function is crucial not only for maintaining low cytosolic calcium for the purpose of signaling events that may include a controlled release of calcium , but it is also important because many of the chaperone proteins in the er lumen such as binding immunoglobulin protein ( also known as grp78 ) , calreticulin , and calnexin are calcium - dependent.32,33 impaired serca function thus impedes the ability of calcium - dependent chaperones to properly fold proteins.34,35 additionally , the increase in cytosolic calcium concentration further aggravates mitochondria and contributes to additional oxidative stress.32 the accumulation of unfolded or misfolded proteins in the er lumen defines er stress and activates the upr , a series of intracellular signaling events that function to restore er homeostasis.36 these events culminate in reducing the ability of proteins to enter the er for posttranslational modifications , upregulation of chaperone proteins and , if homeostasis can not be restored , apoptosis.15 er stress and the upr have two implications that directly contribute to the predisposition of offspring to nafld : upon induction of the upr , activating transcription factor-6 ( atf6 ) one of the proteins that participates in upr intracellular signal transduction is activated by intramembrane proteolysis . | [
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probiotic bacteria have been shown to have positive effects on hosts with intestinal diseases such as clostridium difficile- , rotavirus- , and escherichia coli - induced diarrhea and also in the prevention of antibiotic - associated diarrhea and nosocomial infections [ 15 ] . according to the who , probiotics are defined as live organisms that , when ingested in sufficient amounts , have a beneficial effect on the overall health of the host . in animal nutrition , probiotics are used as feed additives with positive effects regarding health and growth performance traits [ 79 ] .
the probiotic enterococcus faecium ncimb 10415 ( e. faecium ) is licensed as a feed additive for sows and piglets and has been demonstrated to reduce diarrhea incidence and severity in weaning piglets [ 10 , 11 ] . in human medicine , e. faecium
is used successfully in the treatment of acute diarrheal diseases and in the prevention of antibiotic - associated diarrhea [ 12 , 13 ] .
however , the crosstalk between the probiotic and other microorganisms and between the probiotic and epithelial and immune cells of the intestinal wall is extremely complex [ 14 , 15 ] , and many of the underlying signaling mechanisms are still only partially understood .
in vivo models of gastrointestinal infections have demonstrated a positive effect of various probiotic feed additives on the functional barrier of the intestine [ 1618 ] .
these results are supported by data from in vitro cell culture infection models in which probiotic strains prevent or ameliorate damage to epithelial integrity by a pathogenic challenge [ 19 , 20 ] .
furthermore , the immunological response of the mucosa can be influenced by probiotic strains , which modulate the release of cytokines , amongst other effects [ 21 , 22 ] .
for example , culture supernatant of lactobacillus plantarum 2142 had a suppressive effect on the interleukin-8 ( il-8 ) and tumor necrosis factor- ( tnf- ) levels induced by oxidative stress in ipec - j2 cells .
the expression of heat shock proteins ( hsps ) , a set of proteins that are involved in many regulatory pathways and that serve as chaperones for preserving cellular proteins , can be influenced by the intestinal microbiota and by probiotics [ 2426 ] .
some of these hsps , such as the inducible form of hsp70 , are upregulated by several noxious conditions and can therefore be considered as cellular stress indicators .
hsps and cytokines , in turn , are able to regulate barrier properties by influencing tight junction proteins and the structure and function of the cytoskeleton or transport properties of the epithelium [ 24 , 28 ] . in previous studies , feed supplementation with e. faecium has been demonstrated to increase the absorptive and secretory capacity and improve barrier function of the small intestinal epithelium of piglets [ 29 , 30 ] .
furthermore the proinflammatory cytokine il-1 , which is expressed in higher amounts in peyer 's patches of e. faecium - treated piglets , increases chloride secretion in the small intestine of pigs .
the hypothesis of the current study is that intestinal epithelial cells play an important role in innate immune responses during enteric infections and that e. faecium , in turn , modulates the severity of enteric infections via the altered generation of proinflammatory cytokines and hsps in intestinal cells .
thus , the aim of the present study has been to investigate the influence of the probiotic e. faecium and two different pathogenic e. coli strains on the hsp and proinflammatory cytokine responses and the epithelial integrity of two intestinal epithelial cell lines .
we have further tested whether pre- and coincubation with e. faecium change the epithelial cell response to pathogenic e. coli strains .
the human epithelial intestinal cell line from colorectal adenocarcinoma , caco-2 ( atcc catalog number htb-37 , atcc , manassas , usa ; passages 3745 ) , was used as a model for the human small intestine .
the porcine intestinal epithelial cell line ( ipec - j2 ; passages 7379 ) was used as a model for the pig small intestine .
this cell line was established from the jejunum of a newborn pig and kindly provided by professor dr .
cells for the experiments were allowed to differentiate for 14 days ( ipec - j2 ) or 21 days ( caco-2 ) . on the day prior to experiments , the cells were fed with serum- and antibiotic - free media .
three different bacterial strains were used for the experiments : ( 1 ) the probiotic strain enterococcus faecium ncimb 10415 ( cultivated from cylactin , dsm , heerlen , the netherlands ) , ( 2 ) the enterotoxigenic e. coli imt4818 ( etec , isolated from a two - week - old piglet with enteritis , o149:k91:k88 ( f4 ) , and found to be positive for the presence of virulence genes est-1a , est-2 ( genes coding for heat stable enterotoxins i and ii ) and elt-1a / b ( gene coding for heat labile enterotoxin i ) by the polymerase chain reaction ( pcr ) ) , and ( 3 ) the human enteropathogenic e. coli e2348/69 ( epec , serotype o127:h6 , positive for the eae gene coding for the e. coli attaching - effacing factor ) .
the e. faecium ncimb 10415 strain was cultivated in brain - heart infusion ( bhi ) broth ( oxoid gmbh , wesel , germany ) and the e. coli strains in lb medium according to miller , containing 10 g / l tryptone ( oxoid gmbh , wesel , germany ) , 5 g / l yeast extract ( oxoid gmbh , wesel , germany ) , and 10 g / l nacl , at a ph of 7.0 . after overnight incubation of the cells at 37c , subcultures of bacteria
were grown for 3 to 4 h until mid - log phase and then centrifuged .
cell pellets were washed twice in phosphate - buffered saline ( pbs , biochrom , berlin , germany ) .
the bacteria were resuspended in antibiotic- and serum - free caco-2 or ipec - j2 cell culture medium to reach a concentration of 10 colony - forming units ( cfu)/ml .
the intestinal cells were infected with 10 bacteria per cell culture insert ( 1.12 cm ) or per well ( 1.91 cm ) , corresponding to a multiplicity of infection ( moi ) of about 10 bacteria per seeded cell .
the bacteria were added to the apical pole of the cells . for each experiment , the cell monolayers for the real - time quantitative pcrs ( rt - qpcr ) , for enzyme - linked immunosorbent assay ( elisa ) , and for the transepithelial electrical resistance ( ter ) measurements were incubated for 2 h with the respective bacterial strains ( etec , epec , or e. faecium ) at a concentration of 10 bacteria per cell culture insert or well ( see figure 1 ) .
two hours after the bacterial incubation , the cells were washed twice with gentamicin - supplemented media to wash out and kill the bacteria , and gentamicin - supplemented media were added .
gentamicin was added to the media at a concentration of 50 g / ml ( biochrom , berlin , germany ) .
the cells were incubated for various periods of time with regard to the different parameters measured , the exact time periods being given in figure 1 under the specific headings .
when the cells were incubated with the probiotic and either the etec or the epec together , the cells were preincubated with the e. faecium for 2 h , and then the pathogens were added .
the cells were in contact with the pathogens for the same amount of time as in the monoincubation with the etec or epec . in the following
, this experimental setup will be called coincubation and the incubation time will be given as the time that the cells were incubated with the pathogens .
for ter measurements , the cells were seeded at a density of 10 cells on cell culture inserts ( transwell , clear polyester membrane , 12 mm diameter , 1.12 cm area , 0.4 m pore size ; corning b.v .
the inserts for ipec - j2 cells were coated with rat tail collagen type i ( serva electrophoresis gmbh , heidelberg , germany ) .
ter was measured by a millicell - ers ( electrical resistance system ; millipore gmbh , schwalbach , germany ) .
ter values were measured every two hours and corrected for the resistance of blank filters and for the membrane area . for determining mrna expression , the cells were seeded on 24-well cell culture plates ( tpp , biochrom , berlin , germany ) at a density of 10 cells/1.91 cm .
after 4 h , cells were washed two times with pbs , stored in rnalater rna stabilization reagent ( qiagen gmbh , hilden , germany ) , and frozen at 20c .
the isolation of the total rna of the harvested cells , the assessment of the rna quality , and the cdna synthesis are described in detail in other publications [ 29 , 32 ] . the samples had to have a rna integrity number above 7 to be used for rt - qpcr .
for the synthesis of cdna , 100 ng total rna for the ipec - j2 cells and 500 ng total rna for the caco-2 cells were used .
three reference genes were selected for normalization ( actb , tbp , and ubp for the caco-2 cells and gapdh , tbp , and ywhaz for the ipec - j2 cells ) .
the primer information for the reference genes can be found in lodemann et al . .
rt - qpcr was performed and the relative amount of the target genes in the experimental groups was calculated by iq5 software ( bio - rad laboratories gmbh , mnchen , germany ) as described in klingspor et al . .
for the elisa experiments , the cells were seeded on 24-well cell culture plates ( tpp , biochrom , berlin , germany ) at a density of 10 cells/1.91 cm , and incubation with bacterial strains was as described above .
after 8 h , the supernatants of the cells were harvested , and species - specific il-8 elisas were performed on cell culture supernatants according to the manufacturers ' instructions ( invitrogen elisa kit , swine il-8 , invitrogen life technologies gmbh , darmstadt , germany , for ipec - j2 supernatants ; thermo scientific human il-8 elisa kit , pierce biotechnology , rockford , usa , for caco-2 supernatants ) .
the complete lysis - m reagent set ( roche diagnostics deutschland gmbh mannheim , germany ) was used , and the lysis buffer containing a mild detergent in bicine buffer and protease inhibitors was prepared following the manufacturers ' instructions .
the cells were washed with pbs , and 200 l lysis buffer was added to each well .
after 5 min of incubation at room temperature and gentle shaking , the cell lysate was collected .
elisa of the extracted protein was performed according to the manufacturers ' instructions ( porcine hsp70 elisa kit , bio - medical assay for ipec - j2 cell extracts and hsp70 elisa kit , stressmarq biosciences for caco-2 cell extracts ) .
statistical evaluations were carried out by means of the ibm spss - statistics program for windows , version 22 ( international business machines corp . , armonk , united states of america ) .
( incubation with medium ( control ) , e. faecium , etec , epec , etec or epec in coincubation with e. faecium ) .
an overall analysis of the data for each cell line and each parameter ( ter , mrna- , and protein expression ) was performed .
if a statistical significant difference occurred in the overall analysis each time point ( 4 h , 6 h , 8 h ) was analyzed separately . in the case of a significant difference between groups ( treatment of the cells ) , the fisher least significant difference post hoc test was performed .
the ter values of confluent cell monolayers were recorded as a measure of epithelial integrity .
the overall analysis revealed significant differences of ter between incubations with the various bacteria ( p < 0.001 ) .
monoincubation of the cells with the probiotic e. faecium caused an increase of ter compared with the control ( at 4 h ; figure 2 ) indicating a positive effect of e. faecium on barrier function .
exposure of the cells to etec significantly decreased ter ( at 4 h and 6 h ) , whereas the ter of epec - treated cells did not show significant alterations compared with the control .
cell monolayers coincubated with e. faecium and etec showed a significantly higher ter compared with cells incubated with etec alone . in ipec - j2 cells , significant differences between the various bacterial incubations could also be detected ( p = 0.01 ) .
in this cell line , the ter values were not affected in cells that were incubated with e. faecium alone compared with the control ( figure 3 ) .
however , the etec strain , again , caused a significant decrease in ter ( 4 h , 6 h ) , whereas the ter of cells incubated with epec did not differ significantly from the control .
pre- and coincubation with e. faecium reversed the decreased ter of etec - treated ipec - j2 cell monolayers at 4 h and , as a trend , at 6 h , while ter of epec - treated cells was not changed by pre- and coincubation with e. faecium .
as a stress indicator for the pathogenic challenge , hsp70 expression was tested in both cell lines at the mrna and protein levels . in the overall analysis ,
the differences between the treatment groups were statistically significant for the mrna expression of hsp70 ( p = 0.001 ) .
after four hours of incubation , no differences could be observed between control cells and cells incubated with epec or e. faecium ( alone or in combination with etec and epec ) ( figure 4(a ) ) .
however , the mrna expression of hsp70 was significantly higher in the cells that were incubated with the etec strain alone compared with all other treatments .
this implied that the coincubation of etec with e. faecium significantly reduced the increase in mrna expression observed during incubation with etec alone .
these results were reflected by the changes in hsp70 protein expression ( figure 4(b ) ) .
for ipec - j2 cells , the mrna and protein expression of hsp70 showed some numerical , but no statistically significant , differences between the treatment groups ( figure 5 ) .
the cytokine il-8 was chosen as a measure of the proinflammatory response of the cells .
the bacterial incubation significantly affected the mrna expression of il-8 ( p = 0.008 ) .
the mrna expression of il-8 did not differ between the cells incubated with e. faecium and the control cells .
however , it was significantly increased 4 h after incubation with the etec strain compared with the control and the e. faecium - treated cells .
although a numerical increase was seen in all groups incubated with bacteria , the mrna expression did not differ significantly between the other treatment groups and the control ( figure 6(a ) ) .
the changes in mrna expression were mirrored by similar changes in il-8 protein expression ( figure 6(b ) ) .
the results regarding il-8 expression by the ipec - j2 cell line were largely comparable with the results obtained for caco-2 cells . in the overall analysis ,
significant differences for il-8 mrna ( p = 0.001 ) and protein expression ( p = 0.001 ) were observed , and statistical differences between individual groups were as described for caco-2 cells ( figure 7 ) .
the aim of this study was to elucidate the effects of the probiotic e. faecium on barrier function and the inflammatory response of the intestinal epithelium , the latter effect being , in addition to other functions , an important part of the immune system of the gut . to examine whether the probiotic strain could modify the epithelial response to a pathogenic challenge
our hypothesis was that epithelial integrity might be improved , whereas the expression of hsp70 and proinflammatory cytokines might be reduced because of the action of e. faecium .
one approach is to treat the confluent cell monolayers directly with the pathogenic bacteria for the whole duration of the experiment .
the second is to incubate the epithelial cells with supernatants of the bacterial cultures [ 23 , 38 ] .
the advantage of using supernatants is the avoidance of ph decreases in the medium or nutrient competition by the rapidly growing bacteria .
however , supernatants or dead bacteria can not mimic the conditions of living bacteria and exclude potential direct interactions of living bacteria with the epithelial cells . to include such bacteria - epithelial interactions ,
the experimental design of the present study included an initial incubation with living bacteria for 2 h. thereafter , bacteria were killed by gentamicin , with the intention of diminishing secondary effects of the bacterial incubation .
two intestinal epithelial cell lines were used : porcine ipec - j2 and human caco-2 cells . the caco-2 cell line is well established and has been used for many years to investigate specific aspects of small intestinal function , including investigations on the effects of probiotic bacteria [ 40 , 41 ] . nonetheless , it has limitations because of its cancerogenic origin from the colon .
the porcine ipec - j2 cells seem to be a suitable model to mimic in vivo conditions of the small intestine , as they have no tumorous origin and were originally isolated from the small intestine [ 30 , 42 ] ; they have been recently used to study other probiotic microorganisms [ 23 , 39 ] .
the second aim of this study has therefore been to compare caco-2 and ipec - j2 cells under the same experimental conditions to provide further evidence for the use of ipec - j2 cells as a model for small intestinal simulation .
ter is a compound measure of paracellular and transcellular resistance and has been used to assess epithelial integrity in probiotic studies [ 43 , 44 ] . in the present study , the ter of both cell lines was slightly increased after 4 h of incubation with e. faecium alone , but significantly only in caco-2 cells .
these results are in agreement with data of earlier studies showing that various probiotic bacterial strains had either no or an enhancing effect on the ter of intestinal epithelial cells of various origins [ 4547 ] . in porcine ipec - j2 cells , no enhancing effect has been reported so far . the etec strain but not the epec strain significantly reduced ter in both cell lines compared with the control ; this indicates a change in the epithelial integrity as shown previously by other authors [ 48 , 49 ] .
this decrease was inhibited when cell monolayers were ( pre- and ) coincubated with the e. faecium strain .
similar results have been obtained for other probiotic strains in in vitro infection studies with various intestinal cell lines of human origin , such as t84 , ht29 , or caco-2 cells [ 43 , 44 , 50 ] . in a porcine cell model ( ipec-1 )
lactobacillus sobrius dsm 16698 also inhibited the decrease of ter caused by an etec strain . in former studies ,
a probiotic action to prevent the ter decrease induced by etec has been correlated with the inhibition of e. coli adhesion , which is the first step of etec infection .
another possibility by which e. faecium could prevent the decrease of ter after etec infection might be related to tight junction ( tj ) protein expression and localization or to the cytoskeletal organization .
these parameters have not been assessed here but will be the target of further studies .
hsps protect cells , tissues , and organs against various types of stress factors by reducing or avoiding the stress - induced denaturation of proteins .
gene expression of inducible hsp is triggered by many different stressors and is mainly regulated at the transcriptional level [ 5355 ] . in the gastrointestinal tract ,
the expression of inducible hsp is markedly influenced by the intestinal microbiota [ 56 , 57 ] .
the inducible form of hsp70 , which was assessed in the present study , is often considered to be cytoprotective as its induction by minor stressors can protect the tissue from major challenges [ 58 , 59 ] .
some probiotic bacteria obviously activate this cytoprotective mechanism as part of their mode of action [ 23 , 60 , 61 ] .
e. faecium alone , however , did not alter expression of hsp70 in the present study , indicating that different probiotics rely on different mechanisms and pathways to exert their effects on the host [ 15 , 60 ] .
however , in the caco-2 cells , the expression of hsp70 was significantly increased in the cells incubated with the etec .
this increase could be prevented by probiotic pre- and coincubation . in the ipec - j2 cells ,
although , as stated above , the upregulation of hsp70 is often interpreted as a positive effect because of its cytoprotective effects , evidence has also been presented suggesting that hsp70 expression is an indicator of pathological stress . in conjunction with the effects on ter and the proinflammatory cytokine response , the increased expression of hsp70 after pathogenic challenge indicates a stress reaction of the intestinal cells . as such , the reduced increase in hsp70 after pre- and coincubation with e. faecium in the present study suggests that cells are less damaged by etec . the proinflammatory cytokine response in the gastrointestinal tract
the cytokine expression of epithelial cells can be modulated by probiotic strains [ 21 , 40 , 62 ] .
altered cytokine release , in turn , can regulate the structure and function of tj and cytoskeleton [ 63 , 64 ] and the transport properties of epithelial cells [ 65 , 66 ] . in the present study , il-8 has been chosen as a representative cytokine of the epithelial proinflammatory response .
family and is reported to induce neutrophil and t - lymphocyte chemotaxis , neutrophil activation , and the enhanced expression of neutrophil adhesion molecules [ 67 , 68 ] .
one of the responses of intestinal epithelial cells as part of the innate immune system to various inflammatory stimuli is the production of il-8 . in the present study ,
il-8 expression was considerably increased when the cells were incubated with etec ; this increase was abrogated by concomitant incubation with e. faecium .
similar reductions of proinflammatory responses to a pathogen by probiotic strains have been observed in other in vivo and in vitro models [ 23 , 41 , 51 , 70 ] . in some cases ,
in porcine intestinal epithelial cells , various lactobacilli and bacilli strains counteract the increase in il-8 and other proinflammatory cytokines elicited by stimulation with etec , s. typhimurium , oxidative stress , or lipopolysaccharide . in some cases ,
this is associated with the protection of the epithelial barrier [ 23 , 39 , 71 ] .
although il-8 secretion is part of the innate immune response aimed at the elimination of pathogens , the persistent production of il-8 accompanied by the constant infiltration of neutrophils leads to massive epithelial cell damage , which is one cause of diarrhea .
several studies have demonstrated that epithelial damage can be prevented by interventions that suppress the il-8 levels in ibd [ 73 , 74 ] .
this could also be an approach for reducing epithelial damage and diarrhea in acute infections such as etec and , here , we consider it to be one of the positive effects of healthy microbiota . taking this into account ,
the effects of e. faecium observed in the present study indicate a protective effect of this probiotic in acute intestinal inflammation induced by etec .
the exact mechanisms by which e. faecium exerts its influence on cytokine secretion have to be further investigated .
preincubation with the probiotic e. faecium abrogates or reduces all examined effects induced by the etec such as the hsp70 stress response , the elevated expression of the proinflammatory cytokine il-8 , and the decrease in ter as a measure of epithelial integrity .
a key feature of the intestinal immune system is its ability to protect against pathogens while avoiding a destructive inflammatory response .
an exaggerated proinflammatory cytokine secretion such as il-8 leads to disease states and , in this case , a reduction of proinflammatory cytokines together with the reduction of hsp70 expression and the prevention of potential epithelial damage might alleviate symptoms , indicating a positive effect by the probiotic .
both cell lines react in a similar manner to incubation with pathogens and the probiotic .
therefore , the ipec - j2 cell line can be considered as a reliable model for studying the effects of probiotics on the protection of the intestinal epithelium from stressful conditions and inflammation .
furthermore , the parallel response in the two cell lines underlines the general transferability of the effects of e. faecium seen in the present study . | probiotics have shown positive effects on gastrointestinal diseases ; they have barrier - modulating effects and change the inflammatory response towards pathogens in studies in vitro .
the aim of this investigation has been to examine the response of intestinal epithelial cells to enterococcus faecium ncimb 10415 ( e. faecium ) , a probiotic positively affecting diarrhea incidence in piglets , and two pathogenic escherichia coli ( e. coli ) strains , with specific focus on the probiotic modulation of the response to the pathogenic challenge .
porcine ( ipec - j2 ) and human ( caco-2 ) intestinal cells were incubated without bacteria ( control ) , with e. faecium , with enteropathogenic ( epec ) or enterotoxigenic e. coli ( etec ) each alone or in combination with e. faecium .
the etec strain decreased transepithelial resistance ( ter ) and increased il-8 mrna and protein expression in both cell lines compared with control cells , an effect that could be prevented by pre- and coincubation with e. faecium .
similar effects were observed for the increased expression of heat shock protein 70 in caco-2 cells . when the cells were challenged by the epec strain , no such pattern of changes could be observed .
the reduced decrease in ter and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion | the probiotic enterococcus faecium ncimb 10415 ( e. faecium ) is licensed as a feed additive for sows and piglets and has been demonstrated to reduce diarrhea incidence and severity in weaning piglets [ 10 , 11 ] . thus , the aim of the present study has been to investigate the influence of the probiotic e. faecium and two different pathogenic e. coli strains on the hsp and proinflammatory cytokine responses and the epithelial integrity of two intestinal epithelial cell lines . we have further tested whether pre- and coincubation with e. faecium change the epithelial cell response to pathogenic e. coli strains . cells for the experiments were allowed to differentiate for 14 days ( ipec - j2 ) or 21 days ( caco-2 ) . three different bacterial strains were used for the experiments : ( 1 ) the probiotic strain enterococcus faecium ncimb 10415 ( cultivated from cylactin , dsm , heerlen , the netherlands ) , ( 2 ) the enterotoxigenic e. coli imt4818 ( etec , isolated from a two - week - old piglet with enteritis , o149:k91:k88 ( f4 ) , and found to be positive for the presence of virulence genes est-1a , est-2 ( genes coding for heat stable enterotoxins i and ii ) and elt-1a / b ( gene coding for heat labile enterotoxin i ) by the polymerase chain reaction ( pcr ) ) , and ( 3 ) the human enteropathogenic e. coli e2348/69 ( epec , serotype o127:h6 , positive for the eae gene coding for the e. coli attaching - effacing factor ) . the e. faecium ncimb 10415 strain was cultivated in brain - heart infusion ( bhi ) broth ( oxoid gmbh , wesel , germany ) and the e. coli strains in lb medium according to miller , containing 10 g / l tryptone ( oxoid gmbh , wesel , germany ) , 5 g / l yeast extract ( oxoid gmbh , wesel , germany ) , and 10 g / l nacl , at a ph of 7.0 . for each experiment , the cell monolayers for the real - time quantitative pcrs ( rt - qpcr ) , for enzyme - linked immunosorbent assay ( elisa ) , and for the transepithelial electrical resistance ( ter ) measurements were incubated for 2 h with the respective bacterial strains ( etec , epec , or e. faecium ) at a concentration of 10 bacteria per cell culture insert or well ( see figure 1 ) . when the cells were incubated with the probiotic and either the etec or the epec together , the cells were preincubated with the e. faecium for 2 h , and then the pathogens were added . ( incubation with medium ( control ) , e. faecium , etec , epec , etec or epec in coincubation with e. faecium ) . pre- and coincubation with e. faecium reversed the decreased ter of etec - treated ipec - j2 cell monolayers at 4 h and , as a trend , at 6 h , while ter of epec - treated cells was not changed by pre- and coincubation with e. faecium . as a stress indicator for the pathogenic challenge , hsp70 expression was tested in both cell lines at the mrna and protein levels . after four hours of incubation , no differences could be observed between control cells and cells incubated with epec or e. faecium ( alone or in combination with etec and epec ) ( figure 4(a ) ) . in the overall analysis ,
significant differences for il-8 mrna ( p = 0.001 ) and protein expression ( p = 0.001 ) were observed , and statistical differences between individual groups were as described for caco-2 cells ( figure 7 ) . the aim of this study was to elucidate the effects of the probiotic e. faecium on barrier function and the inflammatory response of the intestinal epithelium , the latter effect being , in addition to other functions , an important part of the immune system of the gut . the etec strain but not the epec strain significantly reduced ter in both cell lines compared with the control ; this indicates a change in the epithelial integrity as shown previously by other authors [ 48 , 49 ] . in conjunction with the effects on ter and the proinflammatory cytokine response , the increased expression of hsp70 after pathogenic challenge indicates a stress reaction of the intestinal cells . preincubation with the probiotic e. faecium abrogates or reduces all examined effects induced by the etec such as the hsp70 stress response , the elevated expression of the proinflammatory cytokine il-8 , and the decrease in ter as a measure of epithelial integrity . | [
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] |
one of the most challenging tasks in critical care medicine is the treatment of serious infection related multiple organ dysfunction , termed in general as sepsis , severe sepsis , and septic shock .
however , sepsis means a very heterogeneous patient population , which varies in etiology and severity ; therefore , universally applicable diagnostic criteria and treatment algorhythms are difficult to be defined .
this heterogeneity proved to be one of the most difficult hurdles that most prospective randomized trials could not concur ; hence , they failed to show either clear survival benefit or positive results of single center studies that were later contradicted by large multicenter trials .
nevertheless , sepsis has become a very important health economic issue all around the world .
early recognition and commencing initial steps of resuscitation are inevitable to give the best possible chance for survival , which has to be started on the primary care level : outside the hospital , in the emergency department or on the wards . in the absence of adequate initial management ,
although the results of prospective randomized clinical trials may be disappointing as far as survival is concerned , it is beyond doubt that we have learned a lot about the pathophysiology of sepsis during performing these studies over the last few decades . understanding the immunological background of the clinical picture is of utmost importance , which enables the clinician to interpret results of diagnostic tests and rationalize treatment modalities in the most appropriate way . to highlight
a few of the current novelties in sepsis pathophysiology and potential new perspectives is the purpose of this review .
this means that patients come in with a certain complaint , the physician after taking medical history , performing physical examination and diagnostic tests , defines the diagnosis and treat the patient accordingly . in the case of a well - defined disease more - or - less the same or similar diagnostic tests and therapeutic interventions are performed all around the world ( such as stroke and myocardial infarction ) .
the term we call sepsis syndrome was conceived in a hotel room in las vegas in 1980 , during the protocol writing of one of the first prospective randomized trials in sepsis , performed by a group of scientists led by the late roger bone [ 2 , 3 ] .
based on the inclusion criteria of this study a statement paper was later published by the same authors titled sepsis syndrome : a valid clinical entity
. however , these classical signs of the sepsis syndrome , such as fever / hypothermia , leukocytosis / leukopenia , tachycardia , and hypotension , meant a very large and nonspecific / noninfectious cohort of patients .
for this reason , a few years later a consensus conference was brought together and defined the so called consensus criteria of sepsis , which has also been recently questioned and criticized by vincent et al . .
in the most current surviving sepsis campaign guideline a more robust , more detailed definition has been created , in order to save the previous concept of the bone - criteria .
these efforts clearly show that finding the appropriate definition of sepsis has been a continuous challenge for more than 30 years .
the difficulty in defining sepsis originates from its pathophysiology , to be discussed in section 4 .
this has been recognized by international societies and currently an international task force has been working on a new , pathophysiology based sepsis definition .
nevertheless , in most specialties the disease itself is easily diagnosed by a laboratory or radiological test . however , in the case of sepsis it is different , which makes not just the diagnosis but the interpretation of the results of clinical trials and also epidemiological data very difficult .
according to recent surveys we treat several folds more critically ill patients on the intensive care units ( icu ) worldwide these days as compared to the figures from more than 10 years ago .
there seems to be an increase in the incidence of sepsis , with mortality rates of 2050% , and according to recent data from the united states , sepsis is the single most expensive reason for hospitalization at present [ 8 , 9 ] .
however , it is important to note that reported mortality shows considerable variation across the globe .
a recent retrospective analysis from australia and new zealand showed an increase in the number of critically ill and septic patients over the last 12 years , with a mortality reduction from more than 30% to less than 20% . in the process trial from the united states mortality was around 20% . according to these data outcome
however , results from europe , both retrospective and prospective , indicate greater mortality of 4555% , which was also accompanied by a 2- to 3-fold longer icu and hospital stay [ 11 , 12 ] , as compared to that reported by the two previously mentioned studies .
this raises the question of whether the care is better in those countries which reported lower mortality rate or is it the patient selection that causes this difference ?
although it is difficult to give a definitive answer , referring to our previous chapter , due to the difficulties in defining sepsis , severe sepsis , and septic shock , one can not exclude that this difference can be the result of the uncertainties in patient selection , and , in those countries reporting higher mortality rates , sicker patients were included in the
indeed , patients with the same diagnosis of septic shock could have completely different severity and prognosis .
insult in critical care , such as trauma , sterile inflammation ( acute pancreatitis ) , ischemia - reperfusion injury , major surgery , burns , and infection .
these conditions share the same feature in their pathophysiology , namely , that it is not the insult per se , but the host 's response , especially the immune response , which determines severity and outcome ( figure 1 ) .
the immune system is a team effort that involves many different players interacting with each other as an orchestra . the immune response to pathogens relies on both innate and adaptive components .
the first line of defense against invaders consists of physical barriers such as the skin [ 13 , 14 ] , the mucous membranes of our gastrointestinal , and respiratory and genitourinary tracts .
the second line is the rapid defense by the innate immune system ( including complement proteins , sentinel phagocyte cells , and natural killer cells ) , which plays an activator and a controller role of the adaptive immune system .
the innate system acts by broad recognition of antigens , mainly by sensing pathogen - associated molecular patterns ( pamp ) of carbohydrates and fatty acids located on the surfaces of common pathogens . by - and - large when a local response spread systemically the activation of several classes of pattern recognition receptors will generate a cytokine - chemokine storm .
however , very similar molecules are released due to cell injury after trauma , burns , ischemia - reperfusion , pancreatitis , major surgery , and so forth , derived from necrotic cells , mainly from the mitochondria . these are called damage - associated molecular patterns ( damp ) .
it was a very important recognition that after cellular injury similar proteins will be released during bacterial infection , because the genetic background of the bacteria and the mitochondria is very similar .
this highlights the fact that the bone - concept inevitably mixed patients who suffered insults due to pamp , damp , or the mixture of the two .
activation of neutrophils , macrophages , and monocytes by costimulatory molecules at the site of infection will turn the local adaptive immune system on and give
the aim of the innate response is the eradication of the damp and pamp , which is followed by the adaptive response with the resolution of the immunological process .
the adaptive immune response is based on maturation and proliferation , both influenced by the cytokine signature of the innate response .
these processes are well regulated maintaining an even balance between counteracting forces , hence keeping the inflammatory response localized . however
, in the case of an unbalanced ( proinflammatory and anti - inflammatory ) , dysregulated ( maturation and proliferation ) response , the localized process goes out of control and becomes systemic , in other words the disease of the whole body ; hence , it gives way for impairing the function of distant vital organs .
this makes the clinical manifestation of critical illness so similar regardless of the insult . to give an example , the same gravity of acute respiratory distress syndrome ( ards ) , shock , or deterioration in mental function
can occur in pancreatitis , just as well as after major surgery , or due to any type of infection ( figure 2 ) .
the adaptive immune system as the third level of defense is based on its memories .
cytokine signature of neutrophils and macrophages will give signals to the t and b lymphocytes via the dendritic cells , which after proliferation by maturation will express different cell surface receptors in soluble or membrane bound forms .
the adaptive immune response is a soluble matrix , which consists of the cascade - type activation of cytokines , coagulation factors , the release of acute phase proteins , stress hormones , and different chemokines and hormokines , forming a complex network .
the key factor of immune resolution is the balance between proinflammatory and anti - inflammatory forces , which is mainly determined by the balance between the relationship of th1 , th2 , th17 , and t to each other , namely , the maturation , magnitude , and the duration of their activity . based on the bone - criteria , systemic inflammatory response syndrome , invented on the consensus conference in 1991 , initially meant the classical sepsis syndrome criteria , without proven infection .
the sirs - criteria have also been criticized for similar reasons as the sepsis syndrome definition , but nevertheless this sirs - concept assumed that systemic inflammatory response can occur for an insult without infection . in the past sirs
was mainly thought to be related to the imbalance between the proinflammatory and anti - inflammatory responses .
however , it is more complex . in the context of the innate and adaptive immune responses
both proinflammatory and anti - inflammatory processes take place in a parallel fashion . when the proinflammatory and anti - inflammatory forces swing into action , the proinflammatory forces overwhelm the anti - inflammatory process at the beginning . in general
we can say that there is a short delay of the anti - inflammatory response as compared to the proinflammatory .
this proinflammatory dominance lasts for 2 to 4 days , but an oversized response , which means that the localized insult becomes systemic , will lead to different degree of tissue damage , shock , and eventually organ failure . during the course of disease the adaptive response
the proinflammatory process slowly turns itself off , while the adaptive response will switch to a th2 response .
in other words , this later phase helps to survive the proinflammatory process after the eradication of the insult with restitutio ad integrum .
however , a dysregulated , systemic form of the adaptive response could later induce immunoparalysis , jeopardizing the body 's defense , hence leaving it prone to further , even opportunistic infections .
there are many unanswered questions in this process , but discussing these issues in details goes well beyond the scope of this paper . in the later phase , in septic patients and in patients with severe noninfectious sirs ( such as burns , trauma , major surgery , hemorrhage , or ischemia - reperfusion after cardiac arrest ) , the anti - inflammatory process may overwhelm the proinflammatory forces .
immunodepression , but these are very general and simplified descriptions of what is really happening .
the term cellular reprogramming may be more accurate indicating the cellular changes during this process . in brief ,
cellular reprogramming means two contradictory parallel cellular processes : cells derived from hematopoietic compartments , such as bone marrow , spleen , lymph nodes , and blood , become hyporeactive . in contrast , cells derived from other tissues and solid organs ( like liver , kidney , lung , brain , or gastrointestinal tract ) can often be hyperreactive causing hyperinflammation in the particular organs , especially in the infected organ .
the inhibition of some signaling pathways parallel with others , which are maintained or enhanced , results in large variety of immune response .
unfortunately , tests able to measure the degree of immunosuppression are not available all around the clock ; hence , the clinician has nothing else to rely on at the bedside than the etiology , clinical picture , and biomarkers in order to detect the onset of a potentially devastating new infection as soon as possible [ 21 , 22 ] .
one of the most common misconceptions in sepsis diagnosis is that we have been searching for specific marker(s ) of sepsis .
however , there is not and there will never be one single marker which is able to diagnose sepsis , mainly due to the very colorful manifestation of sepsis and due to the heterogeneity of patients . recognizing the septic patient has two main elements . on the one hand
, we have to evaluate vital organ function and the degree of organ dysfunction via objective signs , such as hypotension , hypoperfusion , altered mental status , acid - base imbalance , hypoxemia , lactate levels , renal and liver dysfunction , and thrombocytopenia . based on these findings
we should start supportive therapy without any further delay , and if there is any suspicion of the possibility of an infection , empirical antibiotic therapy should also be started immediately ( figure 1 ) . in the meantime
in other words we have to decide whether critical illness is due to infection or not . because if it is due to infection antibiotics should be started as soon as possible , but if it is not related to a bacterial infection , antibiotics are not just a waste of time and money , but they may also do harm in short and long term .
clinical signs are the most important in recognizing critical illness , but they can not prove infection on their own .
conventional ( fever / hypothermia , leukocytosis / leukopenia , tachypnoe , tachycardia , and hypotension ) indicators , also listed in the classical sepsis - syndrome criteria , are very nonspecific , in fact poor indicators of infection . for microbiological proof of infection , although very important , unfortunately results become available 2448 hours at the earliest after sending the specimen to the laboratory . according to our current concept ,
it is of utmost importance to start adequate antibiotic therapy as soon as possible , but at least within an hour after the onset of infection caused hypotension ; otherwise chances for survival are reducing by the hour .
new molecular biology techniques are now available to define the presence of bacterial or fungal dna within the bloodstream of patients [ 24 , 25 ] .
highly sophisticated molecular biology based tests such as polymerase chain reaction ( pcr ) , matrix assisted laser desorption / ionization ( maldi / tof ) , and peptide nucleic acid fluorescence in situ hybridization ( pnafish ) based pathogen detection can theoretically shorten the recognition of the underlying pathogen to about 8 hours .
therefore , we need laboratory tests , which are sensitive and specific enough to show the onset and magnitude of bacterial invasion caused inflammatory response as soon as possible and may also be able to follow the progress of the disease within hours . these biologically active substances are called biomarkers
. there have been several biomarkers developed so far , but neither is suitable for all purposes .
they inevitably can support decision making but they will never be able to differentiate sepsis from sirs with a 100% sensitivity and specificity , mainly due to the problems we discussed earlier in details regarding the problems of defining sepsis , and also due to the complex , overlapping pathomechanism of pamp and damp .
nevertheless , there is still an ongoing search for better , new markers of inflammatory response and infection , with promising preliminary results .
there are almost 200 so - called sepsis markers ; therefore , discussing the features of those can not be integrated into the current review .
we will mainly focus on the two most commonly used markers : procalcitonin ( pct ) and c - reactive protein ( crp )
. however , briefly mentioning the main features of a few other new markers already applied in daily practice , such as soluble cd14 subtype ( presepsin ) and soluble urokinase - type plasminogen activator receptor ( supar ) , may be worthwhile .
higher presepsin concentrations in septic patients were associated with icu mortality in a recent large multicenter trial .
it was also suggested that changes in plasma concentrations may reflect the appropriateness of antibiotic therapy , but this have to be confirmed by future studies .
regarding the supar molecule it has been shown to be a very good indicator of severity of the acute disease and shows good correlation with the degree of organ dysfunction in the critically ill but can not be regarded as a sepsis marker due to its low specificity . any condition inducing damp or pamp could shed the endothelial glycocalyx layer .
it has been confirmed in several experimental studies in different septic models that damage of the endothelial glycocalyx layer is reflected in elevated serum syndecan-1 and syndecan-4 levels [ 4447 ] , which may be potentially a very interesting marker in the future , but again , it may be nonspecific for bacterial infection only .
finally , neutrophil - lymphocyte count ratio is a cheap , fast , and easily available tool to diagnose bacteremia and was found to improve bloodstream infection diagnostics in a recent study on the emergency ward .
nevertheless , the two most commonly used markers in infection / sepsis diagnostics and for guiding therapeutic interventions are pct and crp . despite their popularity
, there are still many pros and cons without clear answers regarding their usefulness and interpretation in guiding patient management .
procalcitonin is detectable in the serum within a few ( 46 ) hours after the onset of bacterial infection . during the normal course of an infection it reaches its peak within 24 hours and
then starts its decline in the case of adequate treatment with levels reducing by roughly 50% daily according to its half - life .
in contrast , crp moves slowly , and under similar circumstances it reaches its maximum value usually within 48 hours .
however , levels are generally elevated in most icu patients , making interpretation of crp very difficult .
the other major problem with crp on the icu is that it is lagging way behind the actual events of the inflammatory process .
procalcitonin differentiates bacterial infections from systemic inflammatory response of other etiologies with higher sensitivity and specificity compared to crp .
there is considerable evidence that pct supported decision making during antibiotic treatment has several beneficial effects .
it considerably reduced antibiotic use in lower respiratory tract infections without compromising survival , and it may also shorten the duration of antibiotic treatment on the icu .
although in the coming paragraphs we will mainly refer to studies investigating pct , the concept how to interpret these data is potentially applicable for any inflammatory marker and should be taken into account when evaluating biomarker levels at the bedside .
although sepsis is often referred to as a definitive disease ( see above ) , in a clinical trial published more than 10 years ago , pct levels were found to be several folds higher in surgical as compared to medical patients in septic shock despite the similar clinical manifestation and severity of the clinical picture .
indeed , there is increasing evidence that , in the case of massive cell injury , such as in severe trauma , after major surgery , and any ischemia - reperfusion type injury including cardiogenic shock , due to the mechanism of damp , unspecific elevations of pct levels can typically be seen even in the absence of a bacterial infection [ 57 , 58 ] .
theoretically , in surgical patients with sepsis damp and pamp take place at the same time leading to an overwhelming inflammatory response , whilst in medical patients it is primarily the activation of the pamp , resulting in a less extensive inflammatory response , hence lower biomarker levels . in the study by clec'h et al .
, the median pct value in sirs in medical versus surgical patients was 0.3 ( 0.11.0 ) versus 5.7 ( 2.78.3 ) , and in septic shock : 8.4 ( 3.676.0 ) versus 34.0 ( 7.176.0 ) ng / ml , respectively .
another very important addition to these findings was provided by a study by charles et al .
, in which they found different degree of inflammatory response in patients with the first as compared to those with the second septic insult .
they investigated patients with primary and secondary blood stream infections and found that the same gravity of infection was accompanied by a severalfold lower maximum pct level in patients during the second event of infection as compared to those with a primary event .
the receiver operating characteristic curve of serum pct for the diagnosis of blood stream infection in critically ill patients with primary sepsis with a cutoff value of 55.6 ng / ml was 0.934 , 95% ci : 0.8810.970 , and in patients with secondary sepsis with a pct cutoff 6.4 ng / ml it was 0.805 , 95% ci : 0.6990.879 .
this observation is in accord with what we have already discussed about immunoparalysis and cellular reprogramming in the previous paragraphs put into context with pct in figure 3 .
this shows that lower levels of pct should be taken seriously in the case of a leter ( secon or third ) onset of infection and this concept has been further supported by several recent reports [ 60 , 61 ] .
these studies clearly show that a given pct value should be interpreted differently based on etiology and the time course of the critically ill condition .
one size [ of biomarker ] does not fit all ; hence , careful evaluation of the given clinical scenario can not be neglected when interpreting a given laboratory result .
before we discuss the importance of kinetics of biomarkers , let us put the results of recently published clinical trials into this context first .
recent large clinical trials tested the effectiveness of pct - guided antibiotic strategies applying the
one size does fit all ; in other words predetermined absolute values ( e.g. , > 1 ng / ml ) as a concept and the results were either nonsignificant or patients required mechanical ventilation longer and the prolonged use of antibiotics in the pct - arm [ 62 , 63 ] .
however , the percentage of surgical patients was around 40% in both studies , and the pct value indicating the need for an intervention was chosen to be 1 ng / ml . based on the results of previous studies investigating pct levels in surgical and medical patients , as we discussed before , this 1 ng / ml cutoff value for intervention is a very low pct value in a specific , mainly high risk surgical population .
, in patients with a pct 1 ng / ml antibiotics were withheld only in 11% .
although data were not provided for this subgroup of surgical patients , one may assume that these patients received antibiotics unnecessarily in large proportion .
the same may hold true for the pass study that unnecessary antibiotic use , and antibiotic escalation , was inevitable in the pct - group due to the generally low
alert - pct levels ( 1 ng / ml ) in the study protocol
. however , if a biomarker 's half - life is short enough , taking kinetics into account , instead or in addition to their absolute values , may provide several theoretical benefits . although the absolute values of pct show substantial differences in different etiologies and the course of the disease , but the kinetics may be similar and more useful .
tsangaris et al . studied 50 patients who were in the icu for more than 10 days , free of infection and who presented with a new onset of fever .
procalcitonin showed a minimum of 2-fold increase in 27 patients from the day before to the day of fever onset , and in these patients infection was eventually proven . on the contrary ,
infection was not proven in 23 patients in whom pct remained persistently low and unchanged as compared to previous days .
their conclusion was that a twofold increase of pct between fever onset and the previous day was associated with proven infection .
furthermore , a normal pct value on the third day after the fever onset was associated with better survival .
it is important to note that the observed maximum pct values in patients with proven infection remained relatively low ( < 1.5 ng / ml ) , but it was not the absolute value but the severalfold increase , which indicated acute onset of infection .
were mainly tested to predict severity and outcome rather than to guide therapy [ 32 , 65 ] . in a recent pilot study in patients treated on the icu
, we found significant differences in the change of pct from the day before ( day1 ) to the day when new infection was suspected according to the clinical picture ( day0 ) .
on day1 pct levels were similar in patients in whom infection was eventually proven as compared to patients in whom we could not prove infection .
although on day0 absolute values of pct levels were elevated in both groups , levels were significantly higher in patients in whom infection was later proven .
most importantly , while there was no significant change in the levels of pct from day1 to day0 in the noninfectious group , the rate of increase was significant in the infection group .
it has also been shown that pct kinetics ( > 80% drop from its maximum value ) can be very useful in stopping antibiotic therapy early , hence reducing antibiotic consumption and length of treatment significantly , which is also recommended in the recent surviving sepsis guideline [ 6 , 52 ] .
these results suggest that therapy based on pct kinetics may be superior as compared to predefined absolute values , a hypothesis to be tested in the future .
recent studies show that fungal infections have an increasing tendency in critically ill patients [ 66 , 67 ] .
are the third or fourth most commonly isolated microorganism in the bloodstream of icu patients and its associated mortality is reported to be as high as 4060% [ 67 , 68 ] .
candidemia or invasive candidiasis is defined by positive blood cultures and presence of clinical signs of systemic infection .
fungal infections are difficult to diagnose from blood cultures because it takes a considerable amount of time to grow these organisms and it often remains negative [ 69 , 70 ] .
unfortunately , clinical features are very nonspecific to separate bacteria - related sepsis from candida sepsis .
a number of clinical trials have proposed the potential diagnostic value of pct in this context .
they have found that a low pct value ( 0.71 [ 0.51.1 ] ng / ml p = 0.001 ) in a critically ill septic patient is more likely to be related to candidemia than to bacteremia .
another trial by cortegiani et al . reported that pct could be a useful diagnostic tool to separate candida spp .
blood stream infection ( 0.99 ng / ml , 0.861.34 ) from blood stream infection caused by bacteria ( 16.7 ng / ml , 7.6550.2 ) or in mixed infections ( 4.76 ng / ml , 2.986.08 ) .
, by blood culture ( alive candida ) and real - time pcr ( killed candida ) in septic patients . in this study
determined the role of pct testing in patients with high risk for invasive fungal infection .
they included immunocompromised hematological patients undergoing chemotherapy or allogeneic hematopoietic stem cell transplantation and had bacterial or fungal infectious complications .
c - reactive protein and pct were prospectively assessed from the day following fever onset for four consecutive days .
they found increased crp combined with mildly or not elevated pct in immunocompromised patients probably due to fungal infection .
therefore , the complementary use of these biomarkers may help the diagnostic method . a recent systemic review and meta - analysis summarized current evidence on the role of pct in differentiating fungal infections from other infectious diseases .
the most frequently isolated causative organisms were candida albicans , candida parapsilosis , candida glabrata , candida tropicalis , aspergillus spp . , and penicillium zygomycota .
they found that pct has a good diagnostic power to separate invasive fungal and bacterial infection from noninfectious disease conditions .
another clinical trial investigated the differences between gram - negative ( g ) , gram - positive ( g+ ) , and fungal bloodstream infections .
they observed significantly higher pct levels in patients with g as compared to g+ infections and even lower levels in fungemia .
regarding viral infections , most of the published results agree that pct can differentiate between viral and bacterial infections , as levels will remain low in the latter case [ 7577 ] .
however , it is important to acknowledge that any condition , including sever viral infection , nonbacterial systemic inflammatory condition such as sterile acute pancreatitis or any ischemia - reperfusion injury , which is accompanied with significant hypotension / hypoperfusion of the tissues , can cause a damp - induced pct increase , which may complicate differential diagnosis [ 56 , 78 , 79 ] .
extracorporeal clearance of the plasma , via hemofiltration and plasma pheresis , has received major interest over the last decades in sepsis research . as the results were contradictory ,
nowadays the focus of interest has turned towards new alternatives , such as the targeted removal of toxins and mediators via specific adsorption .
polymyxin - b ( pmx - b ) is a cyclic cationic polypeptide antibiotic originated from bacillus polymyxa .
studies have shown that pmx - b blunts the tnf- response to endotoxin , which is due to the high binding affinity of pmx
however , when polymyxin b is linked covalently to a polystyrene - derived fiber in a hemoperfusion cartridge , it can be used to remove circulating endotoxins without exerting its undesired effects systematically .
the surface area of the column is extremely large , so it can clear up a large amount of circulating endotoxins in a relatively short period of time .
another potentially beneficial effect of pmx - b hemoperfusion is the removal of certain inflammatory cells .
this device has been used and tested in many patients with a very low incidence of adverse events ( < 1% ) , such as thrombocytopenia , allergic reactions , and transient hypotension .
as pmx - b hemofiltration has been available for several years , it can not be regarded as a
treatment alternative per se ; nevertheless , it is far from routine use in the everyday practice ; hence , future studies are warranted .
the cartridge contains biocompatible , greatly porous polymer beads capable of absorbing molecules in the ~1050 kda range [ 8587 ] .
cytokine overproduction is a common feature in many life - threatening conditions in addition to sepsis , such as trauma , major surgery in high risk patients , viral infections , acute respiratory distress syndrome ( ards ) , serious burn injury , and acute pancreatitis , liver failure just to name a few .
several case reports have been published about the use of cytosorb treatment over the last couple of years .
these include -hemolytic streptococcus - induced necrotizing fasciitis , septic shock with multiorgan dysfunction , and rhabdomyolysis .
elevated cytokine levels have been reported during donor conditioning for organ transplantation , which were associated with dysfunction of donor organs before and after transplantation [ 91 , 92 ] . in a recent clinical trial
it was found that , in addition to conventional treatment , attenuating the inflammatory response by cytokine absorption , graft survival can be prolonged .
the late phase of sepsis and the late phase of cancer by - and - large share similar immune suppression mechanism .
one of the similarities is based on the presence of negative costimulatory molecules , such as pd-1 ( programmed cell death-1 ) .
its expression is induced primarily on t cells ' cd4 and cd8 surface proteins , the signaling via which pd-1 inhibits t cell proliferation , cytokine production , and cytotoxic ability .
persistent antigen exposure ( damp - pamp ) causes increased levels of pd-1 consequently t - cell depletion [ 93 , 94 ] . theoretically ,
blocking the pd-1 receptor or its ligand by antibodies could reverse t cell dysfunction and inhibit the pathogen or tumor cells initiated immunosuppression .
inhibition of the pd-1 pathway in animal models resulted in clinically significant survival benefit in bacterial and fungal sepsis [ 95 , 96 ] . in a recent clinical trial
, patients with lung cancer , melanoma , and small - cell renal cancer patients responded to anti - pd-1 antibody treatment in 20 to 25% .
based on the similar immune - pathomechanism of cancer and sepsis , testing the effect of anti - pd-1 or anti - pd - l1 in the future certainly makes sense .
furthermore , since septic patients do not require long - term anti - pd-1 or anti - pd - l1 therapy the potential adverse effects of certain autoimmune reactions or other serious complications should be very rare .
therefore , future studies are warranted to confirm safety and efficacy issues of anti - pd-1 , anti - pd-1l treatment in immunoparalysed septic patients ; furthermore , evaluating pd-1 or pd - l1 expression in immune cells may be a useful biomarker for immunomodulatory therapy .
bone marrow - derived multipotent mesenchymal stem cells ( mscs ) are already in the clinical use in multiple clinical disorders including myocardial infarction , diabetes , hematological malignancies , hepatic , and renal failure .
recent animal experiments suggested that bone marrow - derived mscs may also have a potential role in the treatment of acute renal failure , ards , and sepsis [ 104106 ] .
in several recent animal models in mice , investigating drug- and ischemia - reperfusion - induced acute kidney injury , mcss therapy was found to enhance recovery and prolong survival [ 104 , 107 , 108 ] .
in other animal models circulating mscs were able to help to regenerate new renal tubular cells in acute kidney injury [ 109 , 110 ] .
mscs can also be potentially used in ards by attenuating proinflammatory response by regulating both the innate and adaptive immune systems and modulation of macrophages .
they can influence activated cd4 and cd8 t cells via the inhibition of the inflammatory cytokine production and stimulate the regulatory t cells .
mscs can directly affect sepsis , one of the most common causes of ards , by enhancing macrophage phagocytosis and increasing antimicrobial peptide secretion , thereby increasing bacterial clearance [ 106 , 111 ] .
another animal experiment showed that mscs can also help to repair the injured lung following ventilation - induced lung injury .
there is increasing evidence about the potential mechanisms via which mscs act in the injured lung .
there is one ongoing multicenter clinical trial on the effects of allogeneic mscs therapy in patients with moderate to severe ards , in which patient recruitment has already started [ clinicaltrials.gov , nct01775774 ] .
as patients respond differently for seemingly same infectious insults , genetic variants are likely to explain the differential susceptibility in the risk of severe sepsis .
it is obvious that host genetics can influence sepsis outcomes but no specific loci have yet been confirmed .
this year , the first genome - wide study reported significant correlation between certain single nucleotide polymorphisms and 28-day mortality in intensive care patients with sepsis , severe sepsis , or septic shock .
after the exact clarification of some responsible loci and its role in the background , mechanism , and course of sepsis , genetic manipulation may be another potential therapeutic approach of sepsis therapy in the future .
understanding the underlying pathology in sepsis and critical illness in general is inevitable in order to evaluate clinical signs and biomarkers in the right context at the bedside .
in - depth analysis of recent research shed light on several important issues including the immunological background of host response for different insults summarized in the damp and pamp concept , which also explains why biomarker levels should be interpreted differently based on etiology and why their kinetics may carry more appropriate information than the absolute values . furthermore , this understanding may lead us to a completely different strategy in treatment where the major role will be played by adsorption techniques and cellular reprograming .
however , this knowledge also revealed that in the complex condition of sepsis nothing will ever replace the well trained , experienced , thinking physician , who takes all of the available information into consideration at the bedside and then makes a decision .
finally , even if this decision will be proved to be wrong retrospectively , it should not be interpreted as a failure , but rather as an important source of our experience .
this experience , which contradicted our expectations and disappointed us at the time , leads to the design of several research projects and more importantly it already helped us to understand more about sepsis and changed the way we thought about it 30 years ago , completely . | sepsis has become a major health economic issue , with more patients dying in hospitals due to sepsis related complications compared to breast and colorectal cancer together . despite extensive research in order to improve outcome in sepsis over the last few decades , results of large multicenter studies were by - and - large very disappointing .
this fiasco can be explained by several factors , but one of the most important reasons is the uncertain definition of sepsis resulting in very heterogeneous patient populations , and the lack of understanding of pathophysiology , which is mainly based on the imbalance in the host - immune response .
however , this heroic research work has not been in vain .
putting the results of positive and negative studies into context , we can now approach sepsis in a different concept , which may lead us to new perspectives in diagnostics and treatment .
while decision making based on conventional sepsis definitions can inevitably lead to false judgment due to the heterogeneity of patients , new concepts based on currently gained knowledge in immunology may help to tailor assessment and treatment of these patients to their actual needs . summarizing where we stand at present and what the future may hold are the purpose of this review . | 1. Introduction
2. Sepsis Is Not a Definitive Disease
3. Epidemiology
4. Pathophysiology
5. Diagnostic Challenges
6. Interpreting PCT
7. Treatment: What the Future Holds?
8. Conclusion | one of the most challenging tasks in critical care medicine is the treatment of serious infection related multiple organ dysfunction , termed in general as sepsis , severe sepsis , and septic shock . however , sepsis means a very heterogeneous patient population , which varies in etiology and severity ; therefore , universally applicable diagnostic criteria and treatment algorhythms are difficult to be defined . in the absence of adequate initial management ,
although the results of prospective randomized clinical trials may be disappointing as far as survival is concerned , it is beyond doubt that we have learned a lot about the pathophysiology of sepsis during performing these studies over the last few decades . understanding the immunological background of the clinical picture is of utmost importance , which enables the clinician to interpret results of diagnostic tests and rationalize treatment modalities in the most appropriate way . to highlight
a few of the current novelties in sepsis pathophysiology and potential new perspectives is the purpose of this review . however , in the case of sepsis it is different , which makes not just the diagnosis but the interpretation of the results of clinical trials and also epidemiological data very difficult . there seems to be an increase in the incidence of sepsis , with mortality rates of 2050% , and according to recent data from the united states , sepsis is the single most expensive reason for hospitalization at present [ 8 , 9 ] . although it is difficult to give a definitive answer , referring to our previous chapter , due to the difficulties in defining sepsis , severe sepsis , and septic shock , one can not exclude that this difference can be the result of the uncertainties in patient selection , and , in those countries reporting higher mortality rates , sicker patients were included in the
indeed , patients with the same diagnosis of septic shock could have completely different severity and prognosis . activation of neutrophils , macrophages , and monocytes by costimulatory molecules at the site of infection will turn the local adaptive immune system on and give
the aim of the innate response is the eradication of the damp and pamp , which is followed by the adaptive response with the resolution of the immunological process . the adaptive immune response is a soluble matrix , which consists of the cascade - type activation of cytokines , coagulation factors , the release of acute phase proteins , stress hormones , and different chemokines and hormokines , forming a complex network . the key factor of immune resolution is the balance between proinflammatory and anti - inflammatory forces , which is mainly determined by the balance between the relationship of th1 , th2 , th17 , and t to each other , namely , the maturation , magnitude , and the duration of their activity . one of the most common misconceptions in sepsis diagnosis is that we have been searching for specific marker(s ) of sepsis . however , there is not and there will never be one single marker which is able to diagnose sepsis , mainly due to the very colorful manifestation of sepsis and due to the heterogeneity of patients . it has been confirmed in several experimental studies in different septic models that damage of the endothelial glycocalyx layer is reflected in elevated serum syndecan-1 and syndecan-4 levels [ 4447 ] , which may be potentially a very interesting marker in the future , but again , it may be nonspecific for bacterial infection only . based on the results of previous studies investigating pct levels in surgical and medical patients , as we discussed before , this 1 ng / ml cutoff value for intervention is a very low pct value in a specific , mainly high risk surgical population . the same may hold true for the pass study that unnecessary antibiotic use , and antibiotic escalation , was inevitable in the pct - group due to the generally low
alert - pct levels ( 1 ng / ml ) in the study protocol
. in a recent pilot study in patients treated on the icu
, we found significant differences in the change of pct from the day before ( day1 ) to the day when new infection was suspected according to the clinical picture ( day0 ) . however , it is important to acknowledge that any condition , including sever viral infection , nonbacterial systemic inflammatory condition such as sterile acute pancreatitis or any ischemia - reperfusion injury , which is accompanied with significant hypotension / hypoperfusion of the tissues , can cause a damp - induced pct increase , which may complicate differential diagnosis [ 56 , 78 , 79 ] . studies have shown that pmx - b blunts the tnf- response to endotoxin , which is due to the high binding affinity of pmx
however , when polymyxin b is linked covalently to a polystyrene - derived fiber in a hemoperfusion cartridge , it can be used to remove circulating endotoxins without exerting its undesired effects systematically . the late phase of sepsis and the late phase of cancer by - and - large share similar immune suppression mechanism . however , this knowledge also revealed that in the complex condition of sepsis nothing will ever replace the well trained , experienced , thinking physician , who takes all of the available information into consideration at the bedside and then makes a decision . | [
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] |
since its introduction in 1973 , x - ray ct has
revolutionized radiography and become a cornerstone of all the
modern hospitals and clinics .
with development of sources , detectors , computers , and algorithms ,
x - ray ct is in a rapid transition from fan - beam to cone - beam
geometry . on the daily basis
, the state - of - the - art medical ct
scanners routinely produce a huge amount of 2d , 3d , 4d , and even
5d ( multiple energies ) images of anatomy and functions with sub - mm
spatial resolution , a few thousandth contrast resolution , and
subsecond temporal resolution . on the other hand ,
the rapid
development of small animal models , especially those with
genetically engineered mice , has generated the critical needs for
preclinical imaging . with refined ccd cameras and microfocus x - ray
tubes ,
a number of micro - ct systems were constructed since the
1990s , reaching image resolutions between 10100 m .
nevertheless , important and immediate biomedical studies still
demand significantly better ct / micro - ct performance , so do
industrial , homeland security , and other applications .
a public concern with x - ray ct is that the radiation
dose is delivered to the patient during the ct scan .
annually ,
over 6 000 000 ct scans were performed in the us with 600 000
of those done on pediatric patients .
the ct dose is the primary
component in the radiation exposure to the us population .
while ct
studies account for only 4% of radiological procedures , they
contribute nearly 40% of the average medical radiation dose .
the contribution of ct to the average medical radiation dose level
is expected to grow as the ct technology improves with multirow
detectors and cone - beam designs .
therefore , there is a serious and
increasing public concern over ct dose , particularly in the
context of mass screening and pediatric imaging .
the radiation
dose to children from ct procedures is a particular concern since
their risk of radiation - induced cancer is higher than that of
adults , they have a longer lifetime for the cancer to be expressed
and the effective dose they receive is typically larger than that
received by adults for a comparable study [ 2 , 3 ] . because the
radiation detriment is conservatively assumed to be linearly
related to dose
, there should be substantial health benefits on
the overall us population from low - dose ct .
as of this date , the
dose reduction potential has not been systematically investigated
in terms of algorithmic optimization , which we believe is an
urgent issue we must address .
similar negative arguments can be made for micro - ct studies of small
animals , especially mice and rats .
micro - ct has been widely
used as a most valuable imaging tool in this regard . the nature of such
small animal studies such as mouse studies requires higher spatial , contrast ,
and temporal resolution to be delivered periodically and even continuously .
as a result
, the increment in radiation dose becomes a major factor
preventing more effective applications of micro - ct in this area .
for
example , to evaluate the heart and lungs , we need to depict the boarders of
the lungs , lobes , sublobar segments , airways , vessels , as well as cardiac
chambers , myocardium and dynamics
. however , even the best micro - ct protocols
and systems clearly fall behind our expectations , not only the involved
radiation dose but also slow data acquisition . technically speaking
, the limited data reconstruction strategy
holds the promise to enhance the ct / micro - ct performance
significantly .
this strategy may reduce the x - ray radiation
exposure and improve the data acquisition speed at the same time .
the importance of performing exact image reconstruction from the
minimum account of data has been recognized for long time .
the
first landmark achievement is the well - known fan - beam half - scan
formula .
a relatively recent milestone is the fan - beam
super - short - scan formula developed by noo et al . .
let
(r )
be a smooth function on a compact support
, with
r = ( r1 , r2 ) and the 2d real space .
define the line integral
( 1)p(s,)=(su()+tu())dt
for s and 0 < < ,
where
u ( ) = ( cos , sin ) and u
( ) = ( sin , cos ) .
p(s , ) can be extended to by p(s, + ) = p(s , ) . for a fixed
0 , by gel'fand and graev and noo
et al .
, the backprojection data
( 2)b(r0)=1200+p(s,)ss = r0u()d
can be expressed as the hilbert transform of along the line
l
through r0 which is parallel to
n
=
( sin 0 , cos 0 :
( 3)b(r0)=1pvr(r0tn)dtt=(hl)(r0 ) ,
where pv represents the principal value . by the inversion
formula of the finite hilbert transform
, the
backprojection data can be inverted to reconstruct the function
. in , noo et al .
proposed a sufficient condition for
exact and stable roi reconstruction from 2d limited data , which
can be summarized as : the function can be exact reconstructed at a point
r0 if one can find a unit vector
n
= ( sin 0
, cos 0 ) and a simply connected segmented l
l of the line l parallel to n through r0 such that ( i )
the segment
l
includes r0
and covers the whole support of along l ,
that is , ( r ) = 0for r
l\ l ;
and ( ii ) for each r
l and each angle [ 0 ,
0 + ]
the line integral p(s , ) are known for a
neighborhood of s =
r
u( ) . in the
cone - beam geometry , the groundbreaking work by katsevich allows
exact image reconstruction from truncated helical cone - beam data
of less than two turns
[ 1012 ] .
his results were further improved by a backprojection - filtration
formulation in the helical cone - beam case and its
generalization
[ 1422 ] , which permit
transversely truncated data as well .
further strengthened their above - quoted sufficient condition
by modifying ( i ) as the segment l contains
r0 and at least one of its
end points is outside the convex hull of the support of
along l .
this latest finding represents the
up - to - date record in the area of limited data reconstruction . in this paper , we present a general roi / voi reconstruction approach using a
truly truncated hilbert transform on a line - segment inside a compactly
supported object aided by partial knowledge on one or both neighboring
intervals of that segment . as a result , the most flexible roi / voi
reconstruction can be exactly performed in the fan - beam / cone - beam geometry .
we are excited by numerous practical possibilities and associated benefits
in image quality improvement and radiation dose reduction . in
section 2
, we will study the inverse problem of the truncated hilbert
transform and establish the uniqueness and stability of the solution . in
section 3
, we will formulate a new sufficient condition for exact
reconstruction of an roi from limited data and propose a generalized
reconstruction approach . in section 4
finally , in section 5 , we will
discuss the relevant issues and conclude the paper .
in reference to , let us denote the 2d ( r)on certain
line l as f(x ) , where x is the one - dimensional ( 1d ) coordinate along
the line l. without loss of generality , we further assume that the support
of f(x ) on l is [ 1,1 ] .
denote by
( 4)g(x)=(hf)(x)=1pv11f(y)dyxy
the hilbert transform of f(x ) . by tricomi ,
f(x ) can be recovered
from its hilbert transform g(x ) by
( 5)1x2f(x)=cf+1pv11g(y)1y2dyyx ,
where
( 6)cf=111f(x)dx
is a known quantity .
a , b , c are three real numbers with 1 < a < b < c < 1 ( see
figure 1 ) . a function f(x ) supported on [ 1,1 ] can be exactly reconstructed on [ b , c ) if ( i ) f(x ) is known on ( a , b ) ; ( ii ) g(x ) is known on ( a , c ) , and ( iii ) the constant cf is known ( see figure 1 ) . by ( 5 ) , we have the inversion formula
( 7)1x2f(x)=cf+1pv1ag(y)1y2dyyx + 1pvabg(y)1y2dyyx + 1pvbcg(y)1y2dyyx + 1pvc1g(y)1y2dyyx .
denote by h1 ( x ) , h2 ( x ) ,
h3 ( x ) , and h4 ( x ) the four integrals on
the right - hand side of ( 7 ) , respectively .
x < b , the left - hand side of ( 8) is known . by our assumptions ,
h2 ( x ) and h3 ( x ) are known for any x. therefore ,
( 9)h1(x)+h4(x)=1x2f(x)cfh2(x)h3(x )
is known for a < x < b. note that for a < x < c ,
( 10)h1(x)=11ag(y)1y2dyyx , h4(x)=1c1g(y)1y2dyyx
are given by ordinary integrals , because y x 0 for 1 < y < a and
c y < 1 . let us define complex functions h1 ( z ) and h4 ( z ) for
z as
( 11)h1(z)=11ag(y)1y2dyyz , h4(z)=1c1g(y)1y2dyyz .
by the cauchy integral theorem , h1 ( z ) , h4 ( z ) , and hence h1 ( z ) +
h4
( z ) are analytic on the complex plane with cuts along
the real axis from to a and from c to
+ . in
particular , h1 ( z ) + h4 ( z ) is analytic on the real interval ( a , c ) . from
( 9 ) ,
h1 ( x ) + h4 ( x ) is known on ( a , b ) , and the right - hand side of
( 9 ) is also analytic on ( a , b ) .
note that f(x ) is not an analytic
function but f1(x ) = 1x2
f(x ) cf h2 ( x ) h3 ( x ) can
be extended to an analytic function f1(z ) in a neighborhood of ( a , b ) .
since h1 ( z ) + h4 ( z ) is an analytic function on ( a , c ) , the known
analytic function f1 ( z ) can be analytically continued from ( a , b ) to
( a , c ) . in other words ,
( x ) + h4 ( x ) is now known on ( a , c ) . using
( 8) , f(x )
can now be uniquely reconstructed since h2 ( x ) and h3
( x ) are known on ( a , c ) as well .
now let us study the stability of this reconstruction approach and estimate
its error bound .
suppose that the function f(x ) is measured as
f ( x ) with a measurement noise f ( x ) by
( 12)f(x)=f(x)+f(x ) for a < x < b ,
with
( 13)|f(x ) | < for a < x < b ,
where > 0 is a small number .
we also assume that the
backprojection ( 2 ) produces an error bounded by . in terms
of the hilbert transform ,
( 14)g(x)=g(x)+g(x ) for 1<x<1 ,
with
( 15)|g(x ) | < for 1<x<1 .
we expect that the variation rate of the error term
g ( x ) is
small .
this can be seen from the fact that g(x ) as a backprojection in
( 2 ) is defined by an integral and hence by an averaging process .
the data sampling will lead
to a small variation rate of g ( x ) in a stochastic sense .
recall that
( 16)h2(x)+h3(x)=1pvacg(y)1y2dyyx .
rewriting the pv integral in ( 15 ) , we have
( 17)h2(x)+h3(x)=1pvacg(y)1y2g(x)1x2yxdy + g(x)1x2pvacdyyx = 1pvacg(y)1y2g(x)1x2yxdy + g(x)1x2log(cxxa ) .
. then h2 ( x ) + h3
( x ) will become
( 18)h23(x)=h2(x)+h3(x)+h23(x ) for a < x < c ,
where the error term is bounded by
( 19)|23h(x)|<c+|log(cxxa)| ,
where the relationship in ( 17 ) and the bound in
( 15 ) have been used .
note that
this error bound becomes large when x is close to c. this
suggests that one should only seek to reconstruct f(x ) on
[ b , c ] with c < c appropriately . the right - hand
side of ( 19 ) also becomes large when x is close to a.
this will not cause any problem since f(x ) is known on ( a , b ) . to determine the stability of the analytic continuation of f1
( z ) = h1 ( z ) + h
4 ( z ) from ( a , b ) to ( a , c )
, we point out that ,
different from f1 ( x ) , the measured function with error term
f1 ( x ) ,
( 20)f1(x)=1x2f(x)cfh23(x)=f1(x)+f1(x ) ,
with f ( x ) and
h23 ( x ) as in
( 12 ) and ( 18 ) , can not be extended to an
analytic function .
the stability of the analytic continuation of
f1 ( z ) thus depends on the numerical method used . in
section 4
, we will use the projection onto the
convex sets ( pocs ) method to compute the analytic continuation
and f(x ) from the measured data f1 ( x ) .
the
stability of our algorithm therefore follows from that of pocs . in
view of ( 20 ) , ( 12 ) , ( 13 ) ,
( 18 ) , and ( 19 ) , the reconstruction error is
bounded by
( 21)1x2|f(x)f(x)|c1+c2|log(cxxa)| .
the following comments are in order on the above theorem : first ,
no information on f(x ) and g(x ) is needed on [ 1,a ] and
[ c , 1 ] , hence we are truly dealing with a truncated hilbert
transform .
second , the method employed in can be adapted
to reconstruct f(x ) on [ b , c ) directly , and more sophisticated
algorithms may be designed in the future .
third , although
( r ) is assumed to be a 2d function ,
theorem 1 can be readily applied in the 3d case .
fourth , for practical implementation , both f(x ) and
g(x ) are
discretized at fine steps . regarding the assumption of the
finite - length interval ( a , b )
, it can be as small as the sampling
step so that f(x ) can only be known on one sampling point inside
the interval ( a , b ) ! from theorem 1 , we have the following corollaries .
let a , b , c , d be four real numbers with 1 < a < b < c < d < 1 . a function f(x ) supported on [ 1,1 ] can be exactly reconstructed on
( a , b ] and [ c , d ) if ( i ) f(x ) is known on ( b , c ) ; ( ii ) g(x ) is known on ( a , d ) , and ( iii ) the constant cf is known .
let a , b , c , d be four real numbers with 1 < a < b < c < d < 1 .
a function f(x ) supported on [ 1,1 ] can be exactly reconstructed on [ b , c ] if ( i ) f(x ) is known on ( a , b ) and ( c , d ) , ( ii ) g(x ) is known on ( a , d ) , and ( iii ) the constant cf is known .
the proofs and stability analysis of corollaries 1 and 2 can be made similar to that for theorem 1 . under
the same assumption ,
the reconstructed error of corollary 2 is
bounded by
( 22)1x2|f(x)f(x)|c3.
in fact , for b x c in
corollary 2 , the corresponding term of
|log((cx)/(xa))|
in ( 21 ) is
bounded .
this better
control of reconstruction error is a main advantage of this reconstruction
scheme with f(x ) being known on two intervals .
from theorem 1 , we immediately have the following new data sufficient
condition for exact and stable reconstruction of an roi from limited
projection data .
the function can be exact reconstructed at
a point r0 if one can find a unit vector n =
( sin 0 , cos 0 ) and a simply connected
segmented l l of the line
l
parallel to n through r0
such that ( i ) the segment l
contains
r0 and a segment l0 l
on which the function
is known , and ( ii )
for each r l and each angle
[ 0 ,
0 + ] the line integral p(s, )
are known for a neighborhood of
s = r
u( ) . to illustrate our above condition ,
let us define the field of view
( fov ) as follows : for any
r and [ 0, ) , there exists an s
satisfying p(s , ) through the point r. it is well
known that a necessary condition for exact reconstruction of an
roi is that the roi must be contained in the fov of a ct scan .
now , we consider circular fovs as shown in figure 2 .
traditionally , to reconstruct an roi exactly , all the lines going
through the compact support of the object function should be
measured , which indicates that the recoverable region is
empty for all the cases .
allows that (r ) at any point r
inside the
fov is recoverable if there exists a line through r and
the intersection between the line and compact support of
( r ) is completely contained in the fov .
hence , we can have
a small recoverable roi as shown in figure 2(a ) .
claims that ( r )
at any point r inside the fov is recoverable if there
exists a line segment in the fov through r and at least
one of its ends is outside the convex hull of the object
support .
in contrast to the condition by noo et al . , the
recoverable roi is greatly enlarged as in figure 2(b ) .
our new data sufficient condition states that ( r ) at
any point r inside the fov is recoverable if there exists
a line segment in the fov through r and the function
is known on part of that line segment .
clearly , the condition of
defrise et al . is a special case of ours .
require
that the known part , which equals to zero , should be outside of
the convex hull of the compact support .
the
known part can be inside the convex hull with
( r ) = 0
or even inside the compact support with ( r ) is known ,
as shown in figures 2(c ) and 2(d ) ,
respectively .
it should be pointed out that the above analysis can
be directly extended to the 3d case for voi . to reconstruct the object function inside an fov satisfying our data
sufficient condition ,
a general reconstruction approach is given in the
following steps :
construct a group of line segments each of which goes
through both known and unknown regions;reconstruct the unknown region based on
theorem 1
in section 2 ; repeat steps ( i ) and ( ii ) until the object function at all eligible
points inside the fov is reconstructed .
our approach works like a water stream flowing from a known region to all
the connected unknown zones subject to our data sufficiency condition .
note that using our
approach there are multiple ways to perform exact roi / voi reconstructions ,
suggesting opportunities for further theoretical and numerical studies .
construct a group of line segments each of which goes
through both known and unknown regions ; reconstruct the unknown region based on
theorem 1
in section 2 ; repeat steps ( i ) and ( ii ) until the object function at all eligible
points inside the fov is reconstructed .
similar to what defrise et al . did in , we computed the
inversion of the
truncated hilbert transform as used in theorem 1 using the
projection onto convex sets ( pocs ) method . using the notation in
section 2 , our goal is to determine a second - order
continuous function f(x)
l( ) in the intersection of the following five convex sets :
c1 = { f l( ) ( hf)(x ) = g(x ) , x ( a , c ) } , c2 = { f l( ) f(x ) = f0 ( x ) , x ( a , b)},c3 = { f l( ) ( 1/ ) 1 f(x)dx = cf } ,
c4 = { f l( ) f(x ) 0 , x [ 1,1 ] } , c5 = { f l( ) f(x ) fmax , x [ 1,1 ] } ,
where f0 ( x ) is the known part , and fmax is the upper bound of
f(x ) . with an initial guess of the unknown function , which can be
constructed over the known object support , the pocs algorithm iteratively
projects an intermediate solution to each of the above five convex sets
until it converges to a satisfactory result .
c1 = { f l( ) ( hf)(x ) = g(x ) , x ( a , c ) } , c2 = { f l( ) f(x ) = f0 ( x ) , x ( a , b ) } , c3 = { f l( ) ( 1/ ) 1 f(x)dx = cf } ,
c4 = { f l( ) f(x ) 0 , x [ 1,1 ] } , c5 = { f l( ) f(x ) fmax , x [ 1,1 ] } , the above pocs method was numerically implemented in matlab to
demonstrate the correctness of our data sufficient condition and
generalized reconstruction framework . as illustrated in
figure 4 ,
the function
(r ) is an axial
slice of the forbild thorax phantom with two small
ellipses added to the heart to make it more challenging for
reconstruction , which was also used in the paper by defrise
et al . .
nontruncated fan - beam projection data of 1200
directions were analytically computed over a full - scan range .
hence , the backprojection function g at any point can be
calculated along any line to simulate different fov
configurations .
first , we repeated the work by defrise
et al . to reconstruct a rectangular roi indicated in
figure 4(a ) from noise - free projection data .
then , we
reconstructed two cross - shaped rois in figures
4(b ) and
4(c ) using our approach proposed in
section 3 .
while in figure 4(b ) we used the prior information that the reconstructed function was zeros
outside its compact support , we assumed that the central part of
the cross - shaped roi was known in figure 4(c ) .
to test
the stability of our method , the above results were repeated from
noisy data with 2 10 photons per incident ray .
the
representative images were presented in figures 5 and
6 . as compared to the results in , our
reproduced image quality in figure 5 seemed better .
the possible reasons include ( a ) the condition f(x ) fmax was used for the pocs method with fmax = 2 , and ( b ) 400
iterations was executed , which is twice that in .
as seen
in figures 5 and
6 , the reconstructed image
quality in the cross - shaped rois is very comparable to that in the
rectangular roi .
while our work has been presented in the context of x - ray ct and micro - ct ,
we underline that the significance and implication of our results are far
beyond what has been described above .
the same or similar techniques can be
applied for x - ray phase - contrast imaging and tomography , emission tomography
including pet and spect , and other modalities that rely on a projective
imaging model .
our proposed approach can be used not only for exact
reconstruction of an roi / voi but also for approximate reconstruction of
various types .
furthermore , new lambda tomography techniques may be
developed based on the truncated hilbert transform theory proposed in this
paper and will be further refined in the future .
the conventional wisdom has
been that the exact and stable reconstruction of an roi / voi inside an object
support is generally impossible from truly truncated data that go only
through the roi / voi .
however , according to our new data sufficiency
condition , such an exact and stable reconstruction becomes feasible if a
small subregion is known inside the roi / voi , even though the projection
data remain truly truncated ! in conclusion , we have presented a general roi / voi reconstruction approach
using a truly truncated hilbert transform on a segment of a chord inside a
compactly supported object aided by partial knowledge on one or both
neighboring intervals of that segment .
our approach and associated new data
sufficient condition allows the most flexible roi / voi image reconstruction
from the minimum account of data in both the fan - beam and cone - beam
geometry .
we are actively working along this direction to realize major
theoretical potentials and enable innovative practical applications . | exact image reconstruction from limited projection data has been a central topic in the computed tomography ( ct ) field . in this paper , we present a general region - of - interest / volume - of - interest ( roi / voi ) reconstruction approach using a truly truncated
hilbert transform on a line - segment inside a compactly supported object aided by partial knowledge on one or both neighboring
intervals of that segment . our approach and associated new data sufficient condition allows the most flexible roi / voi image
reconstruction from the minimum account of data in both the fan - beam and cone - beam geometry
. we also report primary numerical
simulation results to demonstrate the correctness and merits of our finding .
our work has major theoretical potentials
and innovative practical applications . | 1. INTRODUCTION
2. TRUNCATED HILBERT TRANSFORM WITH PARTIAL
NEIGHBORING INFORMATION
3. DATA SUFFICIENT CONDITION AND
RECONSTRUCTION APPROACH
4. SIMULATION RESULTS
5. DISCUSSIONS AND CONCLUSIONS | with development of sources , detectors , computers , and algorithms ,
x - ray ct is in a rapid transition from fan - beam to cone - beam
geometry . the contribution of ct to the average medical radiation dose level
is expected to grow as the ct technology improves with multirow
detectors and cone - beam designs . the importance of performing exact image reconstruction from the
minimum account of data has been recognized for long time . in the
cone - beam geometry , the groundbreaking work by katsevich allows
exact image reconstruction from truncated helical cone - beam data
of less than two turns
[ 1012 ] . his results were further improved by a backprojection - filtration
formulation in the helical cone - beam case and its
generalization
[ 1422 ] , which permit
transversely truncated data as well . in this paper , we present a general roi / voi reconstruction approach using a
truly truncated hilbert transform on a line - segment inside a compactly
supported object aided by partial knowledge on one or both neighboring
intervals of that segment . as a result , the most flexible roi / voi
reconstruction can be exactly performed in the fan - beam / cone - beam geometry . in
section 3
, we will formulate a new sufficient condition for exact
reconstruction of an roi from limited data and propose a generalized
reconstruction approach . from theorem 1 , we immediately have the following new data sufficient
condition for exact and stable reconstruction of an roi from limited
projection data . our new data sufficient condition states that ( r ) at
any point r inside the fov is recoverable if there exists
a line segment in the fov through r and the function
is known on part of that line segment . to reconstruct the object function inside an fov satisfying our data
sufficient condition ,
a general reconstruction approach is given in the
following steps :
construct a group of line segments each of which goes
through both known and unknown regions;reconstruct the unknown region based on
theorem 1
in section 2 ; repeat steps ( i ) and ( ii ) until the object function at all eligible
points inside the fov is reconstructed . note that using our
approach there are multiple ways to perform exact roi / voi reconstructions ,
suggesting opportunities for further theoretical and numerical studies . c1 = { f l( ) ( hf)(x ) = g(x ) , x ( a , c ) } , c2 = { f l( ) f(x ) = f0 ( x ) , x ( a , b ) } , c3 = { f l( ) ( 1/ ) 1 f(x)dx = cf } ,
c4 = { f l( ) f(x ) 0 , x [ 1,1 ] } , c5 = { f l( ) f(x ) fmax , x [ 1,1 ] } , the above pocs method was numerically implemented in matlab to
demonstrate the correctness of our data sufficient condition and
generalized reconstruction framework . nontruncated fan - beam projection data of 1200
directions were analytically computed over a full - scan range . while our work has been presented in the context of x - ray ct and micro - ct ,
we underline that the significance and implication of our results are far
beyond what has been described above . furthermore , new lambda tomography techniques may be
developed based on the truncated hilbert transform theory proposed in this
paper and will be further refined in the future . the conventional wisdom has
been that the exact and stable reconstruction of an roi / voi inside an object
support is generally impossible from truly truncated data that go only
through the roi / voi . however , according to our new data sufficiency
condition , such an exact and stable reconstruction becomes feasible if a
small subregion is known inside the roi / voi , even though the projection
data remain truly truncated ! in conclusion , we have presented a general roi / voi reconstruction approach
using a truly truncated hilbert transform on a segment of a chord inside a
compactly supported object aided by partial knowledge on one or both
neighboring intervals of that segment . our approach and associated new data
sufficient condition allows the most flexible roi / voi image reconstruction
from the minimum account of data in both the fan - beam and cone - beam
geometry . we are actively working along this direction to realize major
theoretical potentials and enable innovative practical applications . | [
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fibroblasts represent a heterogeneous population of mesenchymal cells that play important roles in the production and maintenance of extracellular matrix ( raghow 1994 ; ohnishi et al . 1998 ; phan 2008 ) .
in addition , fibroblasts have important regulatory roles modulating the function of many other cell types ( knight 2001 ; nanki et al . 2001 ; rennard 2001 ; hay 2005 ) .
while fibroblast heterogeneity is clearly recognized , progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined .
this is , to a significant degree , complicated by a lack of surface markers to define differentiated fibroblast phenotypes .
fibroblasts , therefore , are , at the present time , best characterized by morphology , ultrastructure , and function supported by molecular marker expression ( powell et al .
the ultimate progenitor cell is the embryonic stem cell ( esc ) , which in in vitro culture systems , ecss can differentiate in to cells of many lineages .
interestingly , the culture of escs is most commonly accomplished by co - culture with fibroblast feeder layers , which provide undefined but necessary cofactors .
because xenogenic fibroblasts present a number of theoretical and technical problems , several investigators have described methods to prepare autogenic and syngenic fibroblasts from escs to use as feeder layers for escs ( xu et al .
however , these studies have not demonstrated that the fibroblast - like cells function like fibroblasts , which would be a necessary step toward an experimental system to delineate the differentiation pathways leading to heterogeneous populations of fibroblasts .
the current study , therefore , was designed to develop a reliable methodology that would permit in vitro differentiation of fibroblasts from human and murine escs .
the development of this methodology provides a means for delineating the mechanisms that control fibroblast differentiation and that lead to functionally heterogeneous mature cell populations .
native type i collagen ( rat tail tendon collagen [ rttc ] ) was extracted from rat - tail tendons by a previously published method ( elsdale and bard 1972 ) .
commercially available reagents were obtained as follows : transforming growth factor ( tgf)-1 was from r&d systems ( minneapolis , mn ) ; prostaglandin e2 ( pge2 ) , monoclonal anti--smooth muscle actin ( sma ) , anti - pan cytokeratin monoclonal , anti - vimentin monoclonal antibodies , anti - mouse igg fitc ( fluorescein isothiocyanate stain - green immunofluorescence ) conjugate , propidium iodide and 2-mercaptoethanol were from sigma ( st . louis , mo ) ; esgro ( leukemia inhibitory factor ; lif ) , anti - stage specific embryonic antigen ( ssea)-1 and 4 monoclonal antibodies were from chemicon international ( temecula , ca ) ; dulbecco s modified eagle s medium ( dmem ) , fetal calf serum ( fcs ) , dmem / f12 [ 1:1 mixture ] , knockout serum replacement , knockout dmem , non - essential amino acids , l - glutamine , basic fibroblast growth factor ( bfgf ) , collagenase type iv , and 0.05% trypsin - edta were from invitrogen ( carlsbad , ca ) .
the national institutes of health - approved human embryonic stem cell line h9.2 ( passages 4565 ; wicell research institute , madison , wi ) was used in this study with the approval of the institutional review board and embryonic stem cell research oversight committee of the university of nebraska medical center .
undifferentiated human escs were cultured on irradiated mouse embryonic fibroblasts ( mef ) in six - well plates with human esc culture medium containing 80% dmem / f12 , 20% knockout serum replacement , 1% non - essential amino acids , 1 mmol / l l - glutamine , 0.1 mmol / l 2-mercaptoethanol , and 4 ng / ml bfgf .
colonies were mechanically dissected with finely pulled glass micropipettes ( 1.0 mm od ; clark electromedical instruments , reading , uk ) every 7 d and transferred to a freshly prepared mef layer .
culture of embryoid bodies in type i collagen gels . to prepare embryoid bodies ( ebs ) , human escs from four- to five - wells of a six - well plate were treated with 1 mg / ml collagenase and cells were collected by centrifugation at 200g for 2 min .
the pellet was resuspended in differentiation medium containing 90% dmem / f12 , 10% knockout serum replacement , 1% non - essential amino acids , and 1 mmol / l l - glutamine without 2-mercaptoethanol and bfgf ( schuldiner et al .
cells were then placed into a petri dish ( sarstedt , nmbrecht , germany ) and cultured for 45 d. floating ebs from the petri dish were collected into a 50 ml polypropylene conical tube ( falcon ; becton - dickinson labware , franklin lakes , nj ) and precipitated without centrifugation.collagen gels were prepared as described previously ( mio et al .
, rttc , distilled water and 4 concentrated dmem were combined so that the final mixture resulted in 0.75 mg / ml collagen , with a physiologic ionic strength of 1 dmem at ph 7.4 .
aliquots ( 1.0 ml / well ) of the mixture of ebs in collagen were then cast into each well of a 12-well tissue culture plate ( falcon ) and allowed to polymerize .
after polymerization was completed , normally within 20 min at room temperature , basal medium ( 1:1 mixture of differentiation medium and dmem / f12 ) was added on the top of the gels in a 12-well plate ( 1.0 ml / well ) .
the basal medium was changed every 23 d and ebs were cultured for 21 d in type i collagen gels .
knockout dmem with 20% knockout serum replacement , 1% non - essential amino acid , 1 mmol / l l - glutamine , 0.1 mmol / l 2-mercaptoethanol and 10 units / ml lif was used for culture medium , and knockout dmem with 2% fcs for basal medium .
the gels in a 12-well culture plate were dissolved with 1 mg / ml collagenase at 37c in a 5% co2 atmosphere for 1 h. the resulting cells were resuspended with dmem containing 10% fcs ( 10% fcs - dmem ) and centrifuged at 200g for 5 min .
the cells , containing ebs , were cultured in a 100 mm tissue culture dish ( falcon ) with dmem containing 10% fcs , 45 units / ml penicillin , 45 g / ml streptomycin , and 1 g / ml amphotericin b. when near confluent , the cells were trypsinized gently to prevent ebs from detaching and the cells were passaged in 10% fcs - dmem ( fig . 9 ) .
cultures were routinely inspected using phase contrast microscopy and cells were assessed after 45 passages . collagen gel contraction assay .
differentiated fibroblasts were trypsinized and mixed with the neutralized collagen solution so that the final cell density in the collagen solution was 3 10 cells / ml . aliquots ( 0.5 ml / well ) of the mixture of cells in collagen were cast into each well of 24-well tissue culture plates ( falcon ) and the mixture was allowed to polymerize .
after polymerization was completed , the gels were gently released from the 24-well tissue culture plates and transferred into 60-mm tissue culture dishes ( three gels in each dish ) which contained 5 ml of freshly prepared serum - free dmem ( sf - dmem ) with or without 10 mol / l tgf-1 or 10 mol / l pge2 .
the gels were then incubated at 37c in a 5% co2 atmosphere for 5 d. gel contraction was quantified using an optomax v image analyzer ( optomax , burlington , ma ) daily .
briefly , 26 l of sf - dmem containing human fibronectin ( 20 g / ml ) was placed into the bottom wells .
eight - micrometer pore polycarbonate membranes ( neuroprobe inc . ) , which were precoated with 5 g / ml gelatin in 0.1% acetic acid , were employed .
cells were then pelletted and re - suspended in sf - dmem at a density of 1 10/ml .
fifty microliters of the cell suspension supplemented with or without tgf-1 ( 10 mol / l ) or pge2 ( 10
cells were allowed to migrate at 37c in a 5% co2 atmosphere for 12 h. cells that had not migrated were scraped off the upper surface of the membrane , and the membranes were air - dried .
cells were then stained with protocol ( fisher scientific , swedesboro , nj ) and mounted on a glass microscope slide .
chemotaxis was assessed by counting the number of cells in five high - power fields .
cells were plated into 12-well plates ( 10 cells per each well ) in 10% fcs - dmem with or without 10 mol / l tgf-1 or 10 mol / l pge2 .
cells were fed with fresh 10% fcs - dmem every 2 d. cell numbers from three separate wells were determined after 24 , 72 , and 120 h using a coulter electronic cell counter ( beckman coulter inc . , fullerton , ca ) .
differentiated ebs in type i collagen gels were fixed in 4% paraformaldehyde for 30 min .
differentiated fibroblasts were cultured until sub - confluent in eight chamber slides ( nunc inc , naperville , il ) in 10% fcs - dmem and fixed in 4% paraformaldehyde for 30 min at passage 4 .
cells were washed briefly with phosphate buffered saline followed by permeabilization with 0.1% triton in sodium citrate buffer at 4c for 5 min . after blocking with horse serum , the cells were incubated with monoclonal anti--sma ( 1:200 dilution ) , anti - pan cytokeratin ( 1:200 ) , anti - vimentin ( 1:200 ) , anti - ssea-1 ( 1:100 ) , or anti - ssea-4 ( 1:100 ) antibodies at 4c overnight .
after washing , cells were then incubated with fitc - conjugated anti - mouse igg antibody followed by nuclear staining with propidium iodide .
stained cells were visualized and photographed using a nikon eclipse te300 microscope ( nikon , tokyo , japan ) equipped with a dp71 digital camera ( olympus , tokyo , japan ) .
cells were cultured on thermanox coverslips ( thermo fisher scientific , rochester , ny ) and fixed in 2% glutaraldehyde , 2% paraformaldehyde , and 0.5% acrolein . after washing with 0.1 mol / l sorenson s phosphate buffer , samples were post - fixed in 1% osmium tetroxide .
thin sections were stained with 2% uranyl acetate and reynolds lead citrate , and examined using a philips 410ls transmission electron microscope ( philips electronics , eindhoven , the netherlands ) operated at 60kv .
images were acquired with an advanced microscopy techniques digital imaging system ( danvers , ma ) . statistical analysis .
data were expressed as means standard error of the mean ( sem ) .
experiments with multiple comparisons were evaluated using one - way analysis of variance followed by bonferroni s test .
differentiation of human escs into fibroblast - like cells in three - dimensional type i collagen gel culture .
generally , spindle - shaped cells appeared surrounding human ebs after 710 d of culture in three - dimensional type i collagen gels , and were increasingly prominent with further culture to day 21 ( fig . 1 ) .
the spindle - shaped cells were collected following collagenase treatment and re - plated into tissue culture plates in 10% fcs - dmem .
the ebs were also released from the collagen gel , but remained floating and were lost with serial feeding and passaging .
ebs were cast into type i collagen gels and allowed to differentiate for 21 d. ( a ) day 0 , ( b ) day 10 , ( c ) day 21 ( original magnification , 40 ) , ( d ) higher magnification ( 100 ) demonstrating spindle - shaped cells surrounding differentiated ebs ( arrows).to evaluate the differentiation of human ebs in three - dimensional type i collagen gels , we first assessed several markers of cell differentiation by immunocytochemistry .
ebs in collagen gels were ssea-4 positive , a marker for undifferentiated embryonic stem cells , but ssea-1 negative ( fig .
spindle - shaped cells surrounding the ebs were positive for vimentin and negative for cytokeratin ( fig . 2c ,
the leading edge of the spindle - shaped cells showed positive staining for -sma ( fig .
in contrast , fibroblast - like cells in monolayer culture showed positive staining for vimentin in 96% of cells ( fig .
we further assessed the ultra - structural feature of the fibroblast - like cells derived from human escs by transmission electron microscopy .
cells showed a characteristic spindle - shaped morphology with prominent rough endoplasmic reticulum and stress fibers , which were consistent with fibroblasts and myofibroblasts ( figs .
ebs were cast into three - dimensional collagen gels and allowed to differentiate . on day 21
( a ) ssea-4 , ( b ) ssea-1 , ( c ) vimentin , ( d ) cytokeratin , ( e ) -sma ( original magnification , 200).figure 3.immunocytochemistry of differentiated fibroblasts in monolayer culture .
collagen gels in which ebs had been cultured for 21 d were dissolved with collagenase and the cells were passaged into monolayer culture . at passage 4 ,
( a ) ssea-4 , ( b ) ssea-1 , ( c ) vimentin , ( d ) cytokeratin , ( e ) -sma ( original magnification , 200).figure 4.ultra-structure of fibroblasts derived from human escs .
cells derived from human escs showed a characteristic spindle - shaped morphology with a prominent rough endoplasmic reticulum and stress fibers ( a : magnification , 2,400 ; ( b ) magnification 14,000 ) .
ebs were cast into type i collagen gels and allowed to differentiate for 21 d. ( a ) day 0 , ( b ) day 10 , ( c ) day 21 ( original magnification , 40 ) , ( d ) higher magnification ( 100 ) demonstrating spindle - shaped cells surrounding differentiated ebs ( arrows ) .
ebs were cast into three - dimensional collagen gels and allowed to differentiate . on day 21
( a ) ssea-4 , ( b ) ssea-1 , ( c ) vimentin , ( d ) cytokeratin , ( e ) -sma ( original magnification , 200 ) . immunocytochemistry of differentiated fibroblasts in monolayer culture .
collagen gels in which ebs had been cultured for 21 d were dissolved with collagenase and the cells were passaged into monolayer culture . at passage 4 ,
( a ) ssea-4 , ( b ) ssea-1 , ( c ) vimentin , ( d ) cytokeratin , ( e ) -sma ( original magnification , 200 ) .
cells derived from human escs showed a characteristic spindle - shaped morphology with a prominent rough endoplasmic reticulum and stress fibers ( a : magnification , 2,400 ; ( b ) magnification 14,000 ) .
characteristics of differentiated fibroblast - like cells . to assess the functional features of the fibroblast - like cells , we evaluated cell proliferation , chemotaxis , and contraction of three - dimensional type i collagen gels mediated by the differentiated fibroblasts .
in addition , we investigated the ability of exogenous tgf-1 or pge2 to modulate each function.differentiated fibroblasts slowly grew in 10% fcs - dmem with longer than 48 h of doubling time ( fig . 5 ) .
exogenous tgf-1 significantly stimulated cell proliferation , whereas pge2 significantly suppressed proliferation ( fig .
ability of cell migration was assessed by the chemotaxis assay using fibronectin as a chemoattractant .
consistent with our previous reports on lung fibroblast chemotaxis ( kohyama et al . 2002 ) , esc - differentiated fibroblasts migrated towards fibronectin .
furthermore , exogenous pge2 significantly inhibited , while tgf-1 slightly but ( not significant ) augmented chemotaxis of these cells towards fibronectin ( fig . 6 ) .
these differentiated fibroblasts could also contract type i collagen gels when they were cast into the gels and cultured in sf - dmem , which is considered as an in vitro model of tissue repair .
collagen gel contraction was significantly augmented by tgf-1 but inhibited by pge2 at all time points assessed ( fig .
7 ) .
figure 5.effects of tgf-1 and pge2 on proliferation of fibroblasts derived from human escs .
fibroblasts were cultured in monolayers in 10% fcs - dmem with or without pge2 ( 10 mol / l ) or tgf-1 ( 10 mol / l ) .
cells were detached with trypsin / edta and cell numbers were determined using a coulter electronic cell counter .
vertical axis cell number ( 10 cells / ml ) ; horizontal axis , time ( d ) .
each point shows mean sem of three separate experiments , each of which included triplicated wells .
circles control , triangles pge2 , squares tgf-1 ; * p < 0.05 , compared with control group .
sems are not evident as they are within the plot symbols.figure 6.chemotaxis of fibroblasts derived from human escs .
fibroblasts derived from differentiated ebs were trypsinized and chemotaxis toward fibronectin ( 10 g / ml ) was assessed in the presence or absence of either 10 mol / l tgf-1 or 10 mol / l pge2 .
each point represents the mean sem of three replicates in four separate experiments ; * p < 0.05 , compared to fibronectin alone ( control group).figure 7.contraction of three - dimensional collagen gels by fibroblasts derived from human escs .
the gels were released into sf - dmem supplemented with or without either 10 mol / l tgf-1 or 10 mol / l pge2 .
vertical axis gel size ( percentage of initial area ) , horizontal axis time ( d ) .
each point represents mean sem of five separate experiments , each performed in triplicate gels .
circles control , triangles pge2 , squares tgf-1 ; * p < 0.01 , compared with control group .
fibroblasts were cultured in monolayers in 10% fcs - dmem with or without pge2 ( 10 mol / l ) or tgf-1 ( 10
cells were detached with trypsin / edta and cell numbers were determined using a coulter electronic cell counter .
vertical axis cell number ( 10 cells / ml ) ; horizontal axis , time ( d ) .
each point shows mean sem of three separate experiments , each of which included triplicated wells .
circles control , triangles pge2 , squares tgf-1 ; * p < 0.05 , compared with control group .
fibroblasts derived from differentiated ebs were trypsinized and chemotaxis toward fibronectin ( 10 g / ml ) was assessed in the presence or absence of either 10 mol / l tgf-1 or 10 mol / l pge2 .
each point represents the mean sem of three replicates in four separate experiments ; * p < 0.05 , compared to fibronectin alone ( control group ) .
the gels were released into sf - dmem supplemented with or without either 10 mol / l tgf-1 or 10 mol / l pge2 .
vertical axis gel size ( percentage of initial area ) , horizontal axis time ( d ) .
each point represents mean sem of five separate experiments , each performed in triplicate gels .
circles control , triangles pge2 , squares tgf-1 ; * p < 0.01 , compared with control group .
having demonstrated that culture of human escs in three - dimensional type i collagen gels led to differentiation of fibroblast - like cells , we next sought to determine if similar results would be obtained with murine escs.spindle-shaped cells appeared after 1014 d of culture of murine ebs in three - dimensional collagen gels ( fig .
8a d ) . to evaluate the phenotype of these fibroblast - like cells , we performed immunocytochemical staining for vimentin and cytokeratin in monolayer culture . as expected , these cells showed positive staining for vimentin , but were negative for cytokeratin ( fig .
it was also demonstrated that exogenous tgf-1 significantly augmented chemotactic activity toward fibronectin and collagen gel contraction of the fibroblast - like cells derived from murine escs , whereas pge2 significantly inhibited both chemotaxis and gel contraction ( fig .
figure 8.differentiation of murine escs into fibroblasts in three - dimensional collagen gel culture .
( a ) murine escs in monolayer culture with feeder layer ( original magnification , 400 ) .
( b ) murine ebs were ready to cast into gels ( original magnification , 200 ) .
( c ) murine ebs in three - dimensional collagen gels ( original magnification , 200 ) .
( d ) spindle - shaped cells surrounding murine ebs as indicated by the arrows ( original magnification , 400 ) .
( e and f ) immunocytochemistry of differentiated murine fibroblasts in monolayer culture stained for vimentin ( e ) and cytokeratin ( f ) ( original magnification , 200 ) .
chemotaxis of murine fibroblasts toward fibronectin was assessed in the presence or absence of either 5 10 mol / l tgf-1 or 10 mol / l pge2 .
( h ) contraction of three - dimensional collagen gels by fibroblasts derived from murine escs .
collagen gels containing fibroblasts were floated in the media with or without 10 mol / tgf-1 or 10 mol / l pge2 .
circles control , triangles pge2 , squares tgf-1 ; * p < 0.05 , compared with control group . differentiation of murine escs into fibroblasts in three - dimensional collagen gel culture . ( a ) murine escs in monolayer culture with feeder layer ( original magnification , 400 ) .
( b ) murine ebs were ready to cast into gels ( original magnification , 200 ) .
( c ) murine ebs in three - dimensional collagen gels ( original magnification , 200 ) .
( d ) spindle - shaped cells surrounding murine ebs as indicated by the arrows ( original magnification , 400 ) .
( e and f ) immunocytochemistry of differentiated murine fibroblasts in monolayer culture stained for vimentin ( e ) and cytokeratin ( f ) ( original magnification , 200 ) .
chemotaxis of murine fibroblasts toward fibronectin was assessed in the presence or absence of either 5 10 mol / l tgf-1 or 10 mol / l pge2 .
( h ) contraction of three - dimensional collagen gels by fibroblasts derived from murine escs .
collagen gels containing fibroblasts were floated in the media with or without 10 mol / tgf-1 or 10 mol / l pge2 .
circles control , triangles pge2 , squares tgf-1 ; * p < 0.05 , compared with control group .
fibroblasts represent a heterogeneous population of cells present in mesenchymal connective tissues ( fries et al . 1994 ; powell et al .
these cells are thought to be the major cells responsible for the production and maintenance of extracellular matrix ( raghow 1994 ; ohnishi et al .
in addition , fibroblasts produce mediators that regulate epithelial and endothelial cell proliferation and functions ( roberts and sporn 1989 ; vignola et al .
fibroblasts , moreover , can also regulate inflammatory cell recruitment and activation ( glaros et al . 2009 ) .
tgf- , for example , induces the expression of -sma and increases fibroblast production of extracellular matrix ( ronnov - jessen and petersen 1993 ; desmouliere and gabbiani 1994 ) .
myofibroblasts as the increased expression of -sma containing fibers resembles the fibers present in smooth muscle cells ( hinz et al .
in addition to mediator - induced modulation of structure and function , which may be transient , heterogeneous populations of fibroblasts with stable phenotypes have been described in many tissues ( fries et al .
alterations in populations of differentiated fibroblasts , moreover , have been described in a number of disease states ( kahari 1993 ; fireman et al .
2001 ; holz et al . 2004 ; sugiura et al . 2007 ; togo et al . 2008 ; sato et al . in press ) .
recent evidence suggests that circulating cells may contribute as progenitors of tissue fibroblasts ( lama and phan 2006 ; hinz et al .
little is known , however , about the mechanisms that control differentiation of stem / progenitor cells into stable populations of fibroblasts .
the current study provides a method for the evaluation of fibroblast differentiation from embryonic stem cells as illustrated in fig .
figure 9.schematic illustration of the method for differentiation of escs into fibroblasts in three - dimensional type i collagen gel culture .
cells are then placed into a petri dish and cultured for 45 d to allow formation of ebs .
ebs are cast into type i collagen gels in a 12-well plate ( 1.0 ml / well ) and cultured for 21 d three - dimensionally in collagen gels with basal medium .
gels are dissolved by collagenase and the cells are suspended in 10% fcs - dmem .
schematic illustration of the method for differentiation of escs into fibroblasts in three - dimensional type i collagen gel culture .
cells are then placed into a petri dish and cultured for 45 d to allow formation of ebs .
ebs are cast into type i collagen gels in a 12-well plate ( 1.0 ml / well ) and cultured for 21 d three - dimensionally in collagen gels with basal medium .
gels are dissolved by collagenase and the cells are suspended in 10% fcs - dmem .
several other investigators have reported the derivation of fibroblast - like cells from escs ( xu et al .
2008 ) , as described in the current report , and direct differentiation from ecs were used .
all these studies were designed to prepare non - xenogenic fibroblast - like cells to use as feeder layers .
the utility of the various types of derived cells for this purpose was well established .
much less attention has been given to demonstrating that the derived cells function as fibroblasts .
( 2005 ) described their cells as being keratin negative and positive for the enzyme prolyl hydroxylase , which plays a role in collagen biosynthesis .
assessed a number of histochemical markers , but the derived cells differed from foreskin fibroblasts , which are a specific type of differentiated cell .
several lines of evidence support describing the fibroblast - like cells prepared by the method described in the current report as fibroblasts .
in addition , the ultrastructure of the cells is consistent with that of fibroblasts / myofibroblasts
. finally , cells cultured in the present study expressed the cytoskeletal protein vimentin , which is characteristically present in fibroblasts , and lacked the cytoskeletal protein cytokeratin , which is characteristically present in epithelial cells .
a major distinction between fibroblasts on the one hand and epithelial cells and endothelial cells on the other are their responses to pge2 and tgf- ( liu et al .
similarly , pge2 characteristically stimulates proliferation and migration of epithelial cells while it inhibits proliferation and migration of fibroblasts ( zhu et al .
cells cultured in the current study responded to pge2 and tgf- in a manner characteristic of fibroblasts .
in addition , fibroblasts cultured in three - dimensional collagen gels attached to collagen and exerted mechanical tension , resulting in gel contraction .
this property is thought to be a model of tissue reorganization ( mio et al . 1996 ) .
characteristically , tgf- augments and pge2 inhibits this process in fibroblasts ( campbell et al .
2004 ; togo et al . 2008 ) as was observed in the cells prepared in the current study .
in contrast , the ability of endothelial and epithelial cells to contract collagen gels differs markedly .
2000 ) and epithelial cells contract gels , but only when plated on the surface and pge has no effect ( liu et al .
, the current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine escs .
the development of this methodology provides a means to define the mechanisms that regulate fibroblast differentiation . | fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix .
although their heterogeneity is recognized , progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined .
the current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast - like cells from human and murine embryonic stem cells ( escs ) .
undifferentiated escs were differentiated into embryoid bodies ( ebs ) with differentiation media .
ebs were then cast into type i collagen gels and cultured for 21 d with basal media .
the spindle - shaped cells that subsequently grew from the ebs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum .
differentiated cells showed a characteristic spindle - shaped morphology and had ultrastructural features consistent with fibroblasts .
immunocytochemistry showed positive staining for vimentin and alpha - smooth muscle actin but was negative for stage - specific embryonic antigens and cytokeratins .
assays of fibroblast function , including proliferation , chemotaxis , and contraction of collagen gels demonstrated that the differentiated cells , derived from both human and murine escs , responded to transforming growth factor-1 and prostaglandin e2 as would be expected of fibroblasts , functions not expected of endothelial or epithelial cells .
the current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine escs .
this methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation . | Introduction
Materials and Methods
Results
Discussion | fibroblasts represent a heterogeneous population of mesenchymal cells that play important roles in the production and maintenance of extracellular matrix ( raghow 1994 ; ohnishi et al . while fibroblast heterogeneity is clearly recognized , progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined . however , these studies have not demonstrated that the fibroblast - like cells function like fibroblasts , which would be a necessary step toward an experimental system to delineate the differentiation pathways leading to heterogeneous populations of fibroblasts . the current study , therefore , was designed to develop a reliable methodology that would permit in vitro differentiation of fibroblasts from human and murine escs . the development of this methodology provides a means for delineating the mechanisms that control fibroblast differentiation and that lead to functionally heterogeneous mature cell populations . the basal medium was changed every 23 d and ebs were cultured for 21 d in type i collagen gels . generally , spindle - shaped cells appeared surrounding human ebs after 710 d of culture in three - dimensional type i collagen gels , and were increasingly prominent with further culture to day 21 ( fig . ebs were cast into type i collagen gels and allowed to differentiate for 21 d. ( a ) day 0 , ( b ) day 10 , ( c ) day 21 ( original magnification , 40 ) , ( d ) higher magnification ( 100 ) demonstrating spindle - shaped cells surrounding differentiated ebs ( arrows).to evaluate the differentiation of human ebs in three - dimensional type i collagen gels , we first assessed several markers of cell differentiation by immunocytochemistry . spindle - shaped cells surrounding the ebs were positive for vimentin and negative for cytokeratin ( fig . 2c ,
the leading edge of the spindle - shaped cells showed positive staining for -sma ( fig . in contrast , fibroblast - like cells in monolayer culture showed positive staining for vimentin in 96% of cells ( fig . cells showed a characteristic spindle - shaped morphology with prominent rough endoplasmic reticulum and stress fibers , which were consistent with fibroblasts and myofibroblasts ( figs . cells derived from human escs showed a characteristic spindle - shaped morphology with a prominent rough endoplasmic reticulum and stress fibers ( a : magnification , 2,400 ; ( b ) magnification 14,000 ) . ebs were cast into type i collagen gels and allowed to differentiate for 21 d. ( a ) day 0 , ( b ) day 10 , ( c ) day 21 ( original magnification , 40 ) , ( d ) higher magnification ( 100 ) demonstrating spindle - shaped cells surrounding differentiated ebs ( arrows ) . cells derived from human escs showed a characteristic spindle - shaped morphology with a prominent rough endoplasmic reticulum and stress fibers ( a : magnification , 2,400 ; ( b ) magnification 14,000 ) . to assess the functional features of the fibroblast - like cells , we evaluated cell proliferation , chemotaxis , and contraction of three - dimensional type i collagen gels mediated by the differentiated fibroblasts . these differentiated fibroblasts could also contract type i collagen gels when they were cast into the gels and cultured in sf - dmem , which is considered as an in vitro model of tissue repair . having demonstrated that culture of human escs in three - dimensional type i collagen gels led to differentiation of fibroblast - like cells , we next sought to determine if similar results would be obtained with murine escs.spindle-shaped cells appeared after 1014 d of culture of murine ebs in three - dimensional collagen gels ( fig . to evaluate the phenotype of these fibroblast - like cells , we performed immunocytochemical staining for vimentin and cytokeratin in monolayer culture . it was also demonstrated that exogenous tgf-1 significantly augmented chemotactic activity toward fibronectin and collagen gel contraction of the fibroblast - like cells derived from murine escs , whereas pge2 significantly inhibited both chemotaxis and gel contraction ( fig . the current study provides a method for the evaluation of fibroblast differentiation from embryonic stem cells as illustrated in fig . ebs are cast into type i collagen gels in a 12-well plate ( 1.0 ml / well ) and cultured for 21 d three - dimensionally in collagen gels with basal medium . ebs are cast into type i collagen gels in a 12-well plate ( 1.0 ml / well ) and cultured for 21 d three - dimensionally in collagen gels with basal medium . , the current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine escs . the development of this methodology provides a means to define the mechanisms that regulate fibroblast differentiation . | [
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] |
people with intellectual disabilities ( i d ) have a wide range of needs and most exhibit behavioral problems . around 7 - 15% of people with i d have severely challenging behavioral problems .
the nature and severity of these behavioral problems vary with the degree of i d . in children with i
d , the social environment in which they live and interact also shapes their behavior . having a child with i d is stressful for families and the child 's behavioral problems can create additional stress and frustration for parents and caretakers .
furthermore , behavioral problems also impede the child 's learning in a number of settings , including at school and at home .
many children with i d in rural communities are isolated from their peers and are therefore deprived of interaction and play because of their behavioral issues .
this isolation limits their opportunities to learn through observation and interaction with other children , as reported in a previous study in india .
due to a lack of awareness and knowledge , such behavioral problems are mistakenly considered manifestations of mental illness . however , in people with i d , behavioral problems do increase the likelihood of mental illness and can lead to serious life - threatening situations if not treated .
managing behavioral problems is a major concern in the comprehensive rehabilitation of people with i d .
children with i d that attend schools receive some form of behavioral management , irrespective of the nature of school ( special or regular ) . in rural india , where the majority of children with i d do not attend school
, there is no institutional support in place to help children with their behavioral problems .
in addition , the outreach activities performed by rehabilitation institutions in rural communities are poor .
insufficient awareness , misinformation , malpractice and social issues negatively affect the management of behavioral problems in children with i d in rural communities . in the absence of institutional support , parents apply various methods of handling such behavioral problems .
three approaches are prominent in rural communities : first , parents often ignore the behavior .
according to the principles of behavior modification , children 's undesired behaviors get stronger and more when behavior management involves inconsistent or inadequate reinforcement .
there is an unmet need for studies that focus on behavioral interventions for children with i d that live in low- and middle - income countries .
for example , we do not yet know which i d benefit more from behavioral intervention or if there is any relationship between a child 's intelligence quotient ( iq ) ( a child who has i d ) and their behavioral improvement after an intervention .
there are limited resources available for people with i d who live in poor rural areas in india because most of the government rehabilitation institutions in india are in cities and they do not often reach out to the people in poor rural areas .
rural populations in india are primarily served by non - governmental organizations ( ngos ) that are not well - equipped because of little financial support from the government and infrastructures that are inadequate for serving most of india 's population ( 68.84% ) , which is located in rural areas .
most of these ngos are adapting a community - based rehabilitation ( cbr ) approach because it is cost - effective , feasible and empowers people with disabilities and the communities in which they reside .
a cbr approach allows people with i d to receive comprehensive rehabilitation in their own environment . due to
the lack of government rehabilitation institutions , ashagram trust ( agt ) , an ngo started in 1983 , successfully implemented a cbr program for rehabilitating chronically and severely mentally ill people in the barwani district of madhya pradesh , which is one of the poorest districts in india .
the population of this district is made up of tribal ( 68% ) and non - tribal ( 32% ) groups .
the rural population is mostly tribal and is severely deprived of health care , education and other government programs .
only 8.3% of the people in this district receive safely piped drinking water , whereas only 4.3% have access to a toilet .
the majority of the tribal population lives in small villages that are not well connected to cities because of poor or non - existent roads and limited transportation .
the similarly impoverished tribal districts of khargoon , dhar and jhabua surround barwani and these populations were also provided access to medical and rehabilitation services through the clinic and rehabilitation center located at the agt campus in barwani .
do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with id?does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ?
do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with i d ?
does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ?
do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with id?does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ?
do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with i d
? does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ?
children with i d from 3 to 18 years of age ( 9.57 3.57 ) and iq ( 43.83 15.62 ) received behavioral intervention by agt . while taking case histories ,
often parents reported that children were stubborn , did not listen , cried all the time , bit people , fought , etc . , the behavioral assessment scale for indian children with mental retardation ( basic - mr ) was administered on every child who was described as having a behavioral problem .
the basic - mr lists 75 behavioral problems in 10 domains based on their nature .
peshawaria and venkatesan at the national institute for mentally handicapped , in india , developed this tool . in most settings , this tool is applicable for evaluating and treating behavioral problems in persons with i d .
a decline in score is considered to be an improvement in behavior ( reduced behavioral problems ) . participants
behavioral progress was monitored and recorded periodically , at least every month by cbr workers and every 3 months by professionals .
behavioral interventions were performed in two settings : a cbr setting and a clinical setting , both described in detail below .
children in the cbr group received medication for attention deficit hyperactivity disorder , epilepsy and mental illness , but they were not given any psychotropic medications for their behavioral problems .
behavioral outcomes were measured on each child using the basic - mr after 1 year of intervention was completed . consequently , most parents did not have information about their child 's i d condition .
because of their behavioral problems and issues functioning in day - to - day life , the children in the study were not enrolled in school .
many children were also isolated within their own families , which resulted in neglect and in some cases even being beaten and chained .
children may have benefited if they were allowed to play with other children in community and participate in social activities , but most were shunned because of stigma : many people in the community believe that children with i d can transfer their disabilities to other children . some of the children 's behavioral problems may have resolved if they were placed in schools .
most of children with i d who participated in the study had not subsequently received any kind of rehabilitation service .
many of the study participants had been treated in private or government hospitals for medical needs , but they had not been provided information on their i d . in the cbr setting , 262 children with i d were served . out of this group
of these 211 children , 128 were excluded from the study because they had the least severe form of i d ( borderline ) , incomplete information in their file , poor parental support , frequent absence from the community , insignificant behavioral problems , or they had died .
children with borderline i d were excluded because their problems were mild and their parents viewed their problems either as an adjustment or as a result of academics . for this group of children ,
thus , the level of intervention for children with borderline i d was different than the rest of children with i d .
behavioral intervention in a clinical setting was provided for a total of 95 children with i d ( from 2005 to 2007 ) . out of these 95 children , 64 had some form of behavioral problem and 43 of these 64 children were excluded for similar reasons to those described above the breakdown of study participants can be seen in figure 1 .
the cbr project was implemented by the barwani - based ngo agt from 2000 to 2010 .
this project aimed to provide rehabilitation services to people with all types of disabilities in a community setting .
conversely , the clinic was operated on the agt campus twice per week exclusively for populations who were not supported by any of the agt projects .
a specialist in mental retardation ( rl ) and his colleague ( sb ) served in the clinic once per week ; rl and sb were employed by agt but financially supported through action aid . in both the cbr and clinical setting , written or oral ( because of poor literacy ) informed consent was obtained from every child and parent in order to anonymously use data obtained in the study for research purposes .
the populations treated in both settings were similar in characteristics and demographics , but the mode of behavioral intervention was different between settings .
two professionals specializing in i d collectively provided behavioral intervention in both treatment settings ( one professional is author of this paper ) .
the behavioral interventions were divided between both professionals and these professionals often substituted for each other .
agt was responsible for providing behavioral intervention to children with i d in the cbr setting .
interventions were designed by professionals but were carried out by cbr workers ( cbrws ) and parents in the home and at non - formal education ( nfe ) centers .
every month children received 1 - 2 1-h sessions of intervention by cbrws and at least 1 1-h session by a therapist , either at home or in a camp setting .
nfes were operated by the project in certain villages in order to teach basic academic skills and motivate disabled and non - disabled children to return to or join school .
a variety of community awareness activities were arranged , such as street plays , community meetings , parent meetings , musical nights , visits to other rehabilitation centers and distributions of informational material .
parents of children with i d were given a 1-week training course at the agt campus to teach them how to handle and manage their children 's behavioral problems .
children with i d were linked to various schemes to receive their disability ( social security ) , school pension and employment opportunity in national rural employment guarantee scheme .
the cbr program only served participants from 63 villages in the barwani district . in the clinical setting , interventions were offered by professionals and also explained by cbr workers .
parents were provided information on various behavioral intervention schemes and were encouraged to practice them . in the clinical setting , the community from with the participant hailed
follow - up of participants in the clinical setting involved parents bringing the children back to the clinic when it was convenient .
however , children were asked to come back every 6 weeks for follow - up . on average , every child received a session of approximately h by professionals and cbr workers every 2 months .
participants who underwent clinical intervention came from the districts of dhar , jhabua , kargoon and khandwa .
all children were administered at least two diagnostic tests : the developmental screening test ( dst ) and the vinland social maturity scale ( vsms ) , where dst can be used to determine the development quotient ( dq ) and vsms can be used to find the social quotient ( sq ) .
the average of dq and sq was taken to be the iq used for diagnosis and icd-10 classification .
this is the standard practice for obtaining iq scores at the national institute for mentally handicapped in secunderabad , india
. other intelligences tests , such as the indian adaptation of the stanford binet kamat test , malin 's intelligence scale for indian children and bhatia battery , were used as needed .
behavioral problems reported by parents were recorded in the study participants ' case files and behavioral goals were chosen during parental consultations .
a behavior modification plan was prepared after conducting the functional behavior assessment , which provides a clinical function of behavior . in behavior modification , the function
is considered to be the cause of the behavior and it is necessary to address the function of the behavior in order to address it .
these techniques were selected on the basis of the participant 's specific behavioral problem , its function , the severity of the problem and the ability of parents to carry out and conform to the technique .
approximately 3 - 4 techniques were applied to each study participant at a time and the techniques used varied on a case - by - case basis .
the behavioral techniques used are follows : ( a ) restructuring the environment we tried to prevent the behavior from occurring by changing the setting ; ( b ) extinction the regulating function of behavior was removed on the occurrence of the behavior ; ( c ) token economy tokens , such as a star or cards , were given to participants , when they exhibited desired behavior , which could be redeemed for edible items at local shop ; ( d ) over correction
for example , if an item was thrown by a child , the child was instructed to bring the item back and fix the damage or disturbance that occurred as a result of the undesired behavior ; ( e ) response cost earned privileges , such as tokens , were withhold if the participant exhibited the behavior ; ( f ) differential reinforcement for incompatible / alternate behavior
behaviors that prevented the occurrence of the problem behavior were encouraged ; ( g ) differential reinforcement for low - frequency behavior positive behaviors that did not occur often were rewarded ; ( h ) differential reinforcement for other any positive behavior in place of an undesired behavior was rewarded ;
( i ) physical restraining the child was physically restrained to stop the undesired behavior ; ( j ) time out the child was removed from the location where the unacceptable behavior was displayed .
in addition to being exposed to behavioral interventions , study participants also underwent training on personal care skills , given a daily schedule to follow and were involved in household activities wherever possible . statistical package for social science software ( spss version 21 , manufacturer - ibm ) was used for analyses .
the baseline scores were compared with the post - intervention scores of each behavioral domain using wilcoxon matched - pairs signed - rank test .
the was used to determine differences between different age intervals , genders , population types , poverty levels , severity of i d , number of disabilities and interventional setting .
non - parametric tests were preferred over parametric tests because of non - homogeneity , skewedness and kurtosis ( > 0 ) of the data .
consequently , most parents did not have information about their child 's i d condition .
because of their behavioral problems and issues functioning in day - to - day life , the children in the study were not enrolled in school .
many children were also isolated within their own families , which resulted in neglect and in some cases even being beaten and chained .
children may have benefited if they were allowed to play with other children in community and participate in social activities , but most were shunned because of stigma : many people in the community believe that children with i d can transfer their disabilities to other children .
some of the children 's behavioral problems may have resolved if they were placed in schools .
most of children with i d who participated in the study had not subsequently received any kind of rehabilitation service .
many of the study participants had been treated in private or government hospitals for medical needs , but they had not been provided information on their i d . in the cbr setting , 262 children with i d were served . out of this group
of these 211 children , 128 were excluded from the study because they had the least severe form of i d ( borderline ) , incomplete information in their file , poor parental support , frequent absence from the community , insignificant behavioral problems , or they had died .
children with borderline i d were excluded because their problems were mild and their parents viewed their problems either as an adjustment or as a result of academics .
thus , the level of intervention for children with borderline i d was different than the rest of children with i d .
behavioral intervention in a clinical setting was provided for a total of 95 children with i d ( from 2005 to 2007 ) . out of these 95 children , 64 had some form of behavioral problem and 43 of these 64 children were excluded for similar reasons to those described above the breakdown of study participants can be seen in figure 1 .
in the cbr setting , 262 children with i d were served . out of this group , 211 had some form of behavioral problem .
of these 211 children , 128 were excluded from the study because they had the least severe form of i d ( borderline ) , incomplete information in their file , poor parental support , frequent absence from the community , insignificant behavioral problems , or they had died . children with borderline i d were excluded because their problems were mild and their parents viewed their problems either as an adjustment or as a result of academics . for this group of children
thus , the level of intervention for children with borderline i d was different than the rest of children with i d .
behavioral intervention in a clinical setting was provided for a total of 95 children with i d ( from 2005 to 2007 ) . out of these 95 children ,
64 had some form of behavioral problem and 43 of these 64 children were excluded for similar reasons to those described above the breakdown of study participants can be seen in figure 1 .
the cbr project was implemented by the barwani - based ngo agt from 2000 to 2010 .
action aid india financially supported the project . this project aimed to provide rehabilitation services to people with all types of disabilities in a community setting .
conversely , the clinic was operated on the agt campus twice per week exclusively for populations who were not supported by any of the agt projects .
a specialist in mental retardation ( rl ) and his colleague ( sb ) served in the clinic once per week ; rl and sb were employed by agt but financially supported through action aid . in both
the cbr and clinical setting , written or oral ( because of poor literacy ) informed consent was obtained from every child and parent in order to anonymously use data obtained in the study for research purposes .
the populations treated in both settings were similar in characteristics and demographics , but the mode of behavioral intervention was different between settings .
two professionals specializing in i d collectively provided behavioral intervention in both treatment settings ( one professional is author of this paper ) .
the behavioral interventions were divided between both professionals and these professionals often substituted for each other .
agt was responsible for providing behavioral intervention to children with i d in the cbr setting .
interventions were designed by professionals but were carried out by cbr workers ( cbrws ) and parents in the home and at non - formal education ( nfe ) centers .
every month children received 1 - 2 1-h sessions of intervention by cbrws and at least 1 1-h session by a therapist , either at home or in a camp setting .
nfes were operated by the project in certain villages in order to teach basic academic skills and motivate disabled and non - disabled children to return to or join school .
a variety of community awareness activities were arranged , such as street plays , community meetings , parent meetings , musical nights , visits to other rehabilitation centers and distributions of informational material .
parents of children with i d were given a 1-week training course at the agt campus to teach them how to handle and manage their children 's behavioral problems .
children with i d were linked to various schemes to receive their disability ( social security ) , school pension and employment opportunity in national rural employment guarantee scheme .
the cbr program only served participants from 63 villages in the barwani district . in the clinical setting , interventions were offered by professionals and also explained by cbr workers .
parents were provided information on various behavioral intervention schemes and were encouraged to practice them . in the clinical setting ,
the community from with the participant hailed was not involved in any community awareness activities .
follow - up of participants in the clinical setting involved parents bringing the children back to the clinic when it was convenient .
however , children were asked to come back every 6 weeks for follow - up . on average , every child received a session of approximately h by professionals and cbr workers every 2 months .
participants who underwent clinical intervention came from the districts of dhar , jhabua , kargoon and khandwa .
agt was responsible for providing behavioral intervention to children with i d in the cbr setting .
interventions were designed by professionals but were carried out by cbr workers ( cbrws ) and parents in the home and at non - formal education ( nfe ) centers .
every month children received 1 - 2 1-h sessions of intervention by cbrws and at least 1 1-h session by a therapist , either at home or in a camp setting .
nfes were operated by the project in certain villages in order to teach basic academic skills and motivate disabled and non - disabled children to return to or join school .
a variety of community awareness activities were arranged , such as street plays , community meetings , parent meetings , musical nights , visits to other rehabilitation centers and distributions of informational material .
parents of children with i d were given a 1-week training course at the agt campus to teach them how to handle and manage their children 's behavioral problems .
children with i d were linked to various schemes to receive their disability ( social security ) , school pension and employment opportunity in national rural employment guarantee scheme .
in the clinical setting , interventions were offered by professionals and also explained by cbr workers . some behavior modification techniques used in the interventions
parents were provided information on various behavioral intervention schemes and were encouraged to practice them . in the clinical setting , the community from with the participant hailed
follow - up of participants in the clinical setting involved parents bringing the children back to the clinic when it was convenient .
however , children were asked to come back every 6 weeks for follow - up . on average , every child received a session of approximately h by professionals and cbr workers every 2 months .
participants who underwent clinical intervention came from the districts of dhar , jhabua , kargoon and khandwa .
all children were administered at least two diagnostic tests : the developmental screening test ( dst ) and the vinland social maturity scale ( vsms ) , where dst can be used to determine the development quotient ( dq ) and vsms can be used to find the social quotient ( sq ) .
the average of dq and sq was taken to be the iq used for diagnosis and icd-10 classification .
this is the standard practice for obtaining iq scores at the national institute for mentally handicapped in secunderabad , india .
other intelligences tests , such as the indian adaptation of the stanford binet kamat test , malin 's intelligence scale for indian children and bhatia battery , were used as needed .
behavioral problems reported by parents were recorded in the study participants ' case files and behavioral goals were chosen during parental consultations .
a behavior modification plan was prepared after conducting the functional behavior assessment , which provides a clinical function of behavior . in behavior modification , the function
is considered to be the cause of the behavior and it is necessary to address the function of the behavior in order to address it .
these techniques were selected on the basis of the participant 's specific behavioral problem , its function , the severity of the problem and the ability of parents to carry out and conform to the technique .
approximately 3 - 4 techniques were applied to each study participant at a time and the techniques used varied on a case - by - case basis .
the behavioral techniques used are follows : ( a ) restructuring the environment we tried to prevent the behavior from occurring by changing the setting ; ( b ) extinction the regulating function of behavior was removed on the occurrence of the behavior ; ( c ) token economy tokens , such as a star or cards , were given to participants , when they exhibited desired behavior , which could be redeemed for edible items at local shop ; ( d ) over correction
for example , if an item was thrown by a child , the child was instructed to bring the item back and fix the damage or disturbance that occurred as a result of the undesired behavior ; ( e ) response cost earned privileges , such as tokens , were withhold if the participant exhibited the behavior ; ( f ) differential reinforcement for incompatible / alternate behavior
behaviors that prevented the occurrence of the problem behavior were encouraged ; ( g ) differential reinforcement for low - frequency behavior positive behaviors that did not occur often were rewarded ; ( h ) differential reinforcement for other any positive behavior in place of an undesired behavior was rewarded ;
( i ) physical restraining the child was physically restrained to stop the undesired behavior ; ( j ) time out the child was removed from the location where the unacceptable behavior was displayed .
in addition to being exposed to behavioral interventions , study participants also underwent training on personal care skills , given a daily schedule to follow and were involved in household activities wherever possible .
statistical package for social science software ( spss version 21 , manufacturer - ibm ) was used for analyses .
the baseline scores were compared with the post - intervention scores of each behavioral domain using wilcoxon matched - pairs signed - rank test .
the was used to determine differences between different age intervals , genders , population types , poverty levels , severity of i d , number of disabilities and interventional setting .
non - parametric tests were preferred over parametric tests because of non - homogeneity , skewedness and kurtosis ( > 0 ) of the data .
table 1 categorizes the study participants using variety characteristics and shows statistical differences between groups .
we found that the majority of the study participants were impoverished , highlighted by the fact that only 8.7% of children in the study came from a family with middle socio - economic status , while all others either very poor ( 55.8% ) or poor ( 35.6% ) . families that were considered very poor did not have any source of income other than seasonal manual work , while poor families also depended on seasonal manual work but had a few cattle .
categorizing participants by the severity of their i d showed that 31 children ( 29.8% ) had mild i d , 46 ( 44.2% ) had moderate i d , 18 ( 17.3% ) had severe i d and 9 ( 8.7% ) had profound i d respectively .
the presence of an additional disability along with i d , such as cerebral palsy , epilepsy , mental illness , or down syndrome was present in 39 children ( 37.5% ) .
although their primary disability was intellectual , children with such additional disabilities were considered to have multiple disabilities .
it is also important to note that the majority of study participants ( 79.8% ) received behavioral intervention in a cbr setting , while only 22.2% underwent intervention in a clinic [ table 1 ] .
characteristics of study participants and ( n=104 ) the baseline and post - intervention scores ( overall scores and scores separated by domain ) were compared using wilcoxon matched - pairs signed - rank test .
post - intervention scores for each domains and the overall final basic - mr score were significantly lower than corresponding baseline scores ( p 0.001 ) , representing statistically significant behavioral improvements across the board [ table 2 ] .
wilcoxon matched - pairs signed - rank test - participants baseline and post - intervention scores of the basic - mr ( median of differences between baseline and post - intervention equals zero ) the number of behavioral problems ranged from 2 to 16 .
parents of children in the clinical group had a 75% follow - up rate , while children in the cbr group achieved a follow - up rate of almost 100% .
however , in many cases ( approximately 20% ) parents were not able to perform the follow - up tasks . in those cases
cbrws spent more time and connected those children with village volunteers ( not paid by the project ) and nfe teachers to help families with the tasks .
approximately 15% of children were found to be sleeping close to livestock , such as goats and chicken .
behavioral intervention was found to be effective in both interventional settings : in the cbr and in the clinic .
however , improvements varied according to the level of i d , the presence of additional disabilities ( multiple disabilities group ) . the age of participants did not affect behavioral outcomes in this study , which is inconsistent with some studies and consistent with others .
the improvement level was found to be different depending on the severity of i d , which can be attributed to the different behaviors that are associated with different severities of i d . while the needs of indian parents have been fount not to vary with the severity of their child 's i d , in this study we observed , in both the cbr and clinical setting , that parents having children with mild and moderate i d were more concerned about their behavioral problems .
this concern was likely one reason that these particular parents focused more on their child 's behavioral management than parents having children with severe and profound i d .
less dramatic behavioral improvements occurred in children with severe and profound i d and in children who had multiple disabilities .
one reason for this finding may relate to the nature of their behavioral problems ; these children exhibit more self - injurious behaviors and they tend to have higher chances of genetic disorders or medical conditions . it is possible that their undesired behavior may arise from the pain they are experiencing from such medical or genetic conditions .
in addition , many children with severe and profound i d were living in much poorer and unhygienic conditions than children who had moderate and mild i d .
such unhygienic conditions may have affected the development of skin infections and resulted in tissue damage and self - injurious behavior . in parent meetings and trainings , we observed that parents having children with severe or profound i d were somewhat withdrawn and less hopeful about the prognosis of their child 's condition compared with other parents .
they were more concerned about their child 's personal needs , such as helping with eating , toileting , brushing and dressing , sitting , standing , walking and talking .
such parents were more immediately concerned with their child 's basic survival , which is related to how well the child can take care of his or her basic personal needs independently .
children with profound and severe i d who are unable of taking care of their personal needs have shortened life spans .
baseline and post - intervention basic - mr scores across all behavior domains of the scale declined significantly .
behavioral intervention was found to be clinically significant for reducing the frequency , magnitude and duration of poor behaviors .
these findings are consistent with several studies that have demonstrated the positive effects of behavioral intervention .
intervention in the cbr setting was effective and would be consistent with a large - scale community - based study in a developed county focusing on children with developmental disabilities .
most studies of this nature are conducted with small sample sizes , mostly involve a single subject and apply few behavioral modification techniques .
our study had a large sample size and included longitudinal research that employed a range of behavior modification techniques .
in addition , the study included a remote , impoverished and highly uneducated population but was able to involve parents , families and community members .
parental involvement was the key element of success in this program . in both settings , other than behavioral management , we encouraged parents to acquire behavioral skills , change their negative attitudes toward their children and develop better adjustment and coping abilities .
psycho education and involvement of parents in the delivery of behavioral management has been suggested previously .
there was limited data from participants in the clinical setting ; these participants had significantly different characteristics than those in the cbr setting .
in addition , in cbr behavioral interventions , parents and cbr workers along with professionals were involved in implementing and monitoring progress of the behavioral intervention , while in the clinical setting , parents were only provided with information about how to perform behavioral interventions .
other possible confounding factors associated with the cbr setting may have been the relocation of key cbr workers within the cbr area , frequent migration of families , sickness of participants , myths , misbeliefs , cultural practices , parental attitudes , parental cooperation and ( to some extent ) the language barrier between professionals and parents .
furthermore , because of the many activities and interactions between parents and the professionals or cbr workers built close , trusting relationships .
improvement did not occur equally between children who had additional disabilities versus those who did not have any additional disabilities ( beyond i d ) .
improvements were also equal between children who received intervention in the cbr setting and those who received it in the clinic .
the findings of this study are relevant to various rehabilitation and educational settings where people with i d get help managing behavioral problems and undergo educational placement . | background : management of behavioral problems in children with intellectual disabilities ( i d ) is a great concern in resource - poor areas in india .
this study attempted to analyze the efficacy of behavioral intervention provided in resource - poor settings.objective:this study was aimed to examine the outcome of behavioral management provided to children with i d in a poor rural region in india.materials and methods : we analyzed data from 104 children between 3 and 18 years old who received interventions for behavioral problems in a clinical or a community setting . the behavioral assessment scale for indian children with mental retardation ( basic - mr )
was used to quantify the study subjects behavioral problems before and after we applied behavioral management techniques ( baseline and post - intervention , respectively ) .
the baseline and post - intervention scores were analyzed using the following statistical techniques : wilcoxon matched - pairs signed - rank test for the efficacy of intervention ; 2 for group differences.results:the study demonstrated behavioral improvements across all behavior domains ( p < 0.05 ) .
levels of improvement varied for children with different severities of i d ( p = 0.001 ) , between children who did and did not have multiple disabilities ( p = 0.011).conclusion : the outcome of this behavioral management study suggests that behavioral intervention can be effectively provided to children with i d in poor areas . | INTRODUCTION
Research questions
MATERIALS AND METHODS
Participants and families
Selection of participants
Interventional settings
Community setting
Clinical setting
Diagnosis and classification
Behavioral interventions
Statistical analysis
RESULTS
DISCUSSION
LIMITATIONS AND CONFOUNDING FACTORS
CONCLUSIONS | people with intellectual disabilities ( i d ) have a wide range of needs and most exhibit behavioral problems . children with i d that attend schools receive some form of behavioral management , irrespective of the nature of school ( special or regular ) . insufficient awareness , misinformation , malpractice and social issues negatively affect the management of behavioral problems in children with i d in rural communities . there are limited resources available for people with i d who live in poor rural areas in india because most of the government rehabilitation institutions in india are in cities and they do not often reach out to the people in poor rural areas . do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with id?does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ? do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with i d ? does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ? do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with id?does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ? do gender , age , population type , socioeconomic status , category of i d , interventional settings and associated conditions affect the outcome of behavioral intervention in children with i d
? does improvement take place across all domains of behavioral problems in children with i d who have received behavioral intervention ? children with i d from 3 to 18 years of age ( 9.57 3.57 ) and iq ( 43.83 15.62 ) received behavioral intervention by agt . , the behavioral assessment scale for indian children with mental retardation ( basic - mr ) was administered on every child who was described as having a behavioral problem . out of this group
of these 211 children , 128 were excluded from the study because they had the least severe form of i d ( borderline ) , incomplete information in their file , poor parental support , frequent absence from the community , insignificant behavioral problems , or they had died . behavioral intervention in a clinical setting was provided for a total of 95 children with i d ( from 2005 to 2007 ) . agt was responsible for providing behavioral intervention to children with i d in the cbr setting . the baseline scores were compared with the post - intervention scores of each behavioral domain using wilcoxon matched - pairs signed - rank test . out of this group
of these 211 children , 128 were excluded from the study because they had the least severe form of i d ( borderline ) , incomplete information in their file , poor parental support , frequent absence from the community , insignificant behavioral problems , or they had died . thus , the level of intervention for children with borderline i d was different than the rest of children with i d . behavioral intervention in a clinical setting was provided for a total of 95 children with i d ( from 2005 to 2007 ) . of these 211 children , 128 were excluded from the study because they had the least severe form of i d ( borderline ) , incomplete information in their file , poor parental support , frequent absence from the community , insignificant behavioral problems , or they had died . for this group of children
thus , the level of intervention for children with borderline i d was different than the rest of children with i d . behavioral intervention in a clinical setting was provided for a total of 95 children with i d ( from 2005 to 2007 ) . agt was responsible for providing behavioral intervention to children with i d in the cbr setting . agt was responsible for providing behavioral intervention to children with i d in the cbr setting . the baseline scores were compared with the post - intervention scores of each behavioral domain using wilcoxon matched - pairs signed - rank test . characteristics of study participants and ( n=104 ) the baseline and post - intervention scores ( overall scores and scores separated by domain ) were compared using wilcoxon matched - pairs signed - rank test . post - intervention scores for each domains and the overall final basic - mr score were significantly lower than corresponding baseline scores ( p 0.001 ) , representing statistically significant behavioral improvements across the board [ table 2 ] . wilcoxon matched - pairs signed - rank test - participants baseline and post - intervention scores of the basic - mr ( median of differences between baseline and post - intervention equals zero ) the number of behavioral problems ranged from 2 to 16 . the improvement level was found to be different depending on the severity of i d , which can be attributed to the different behaviors that are associated with different severities of i d . less dramatic behavioral improvements occurred in children with severe and profound i d and in children who had multiple disabilities . baseline and post - intervention basic - mr scores across all behavior domains of the scale declined significantly . improvement did not occur equally between children who had additional disabilities versus those who did not have any additional disabilities ( beyond i d ) . | [
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spherical nucleic acid
( sna ) conjugates represent an emerging class
of bionanomaterials that typically consist of two types of fundamental
building blocks , oligonucleotides and inorganic nanoparticle cores . due to their ability to naturally enter multiple
mammalian cell types without the aid of lipid or cationic transfection
agents , snas are extremely potent and useful in a variety of biomedical
applications .
snas have been shown to be effective for intracellular
diagnostic assays and as novel gene regulation agents .
we previously postulated a mechanism for endocytosis of snas , which
involves the specific recognition of their dense oligonucleotide shell
by class a scavenger receptors on the cell membrane as well as their
subsequent uptake via the lipid - raft / caveolae pathway .
unfortunately , very little is known about the intracellular
events of snas following cell entry .
previous work by others
on different classes of nanoparticles has
suggested that particle size , shape , density , and surface
chemistry can impact cellular entry .
however ,
investigations of the intracellular events that happen to nanoparticles
subsequent to their cellular entry are scarce and do not seem to reach
a universal consensus . indeed , it is likely that there is no universal
description of biotrafficking for nanoparticles and that all variables
must be considered . for example , polymeric nanoparticles can access
multiple intracellular compartments , such as the golgi apparatus ,
cytosol , and endoplasmic reticulum .
protein - coated gold nanoparticles ( aunps ) often
get trapped inside endosomes but can be directed elsewhere in the
cell through the addition of cell - penetrating peptides or a liposome
exterior , or even recycled out of the cell .
similarly , lipid nanoparticles containing sirna
have been shown to be largely limited to the endocytic pathway , resulting
in recycling . due to their three - dimensional
( 3d ) oligonucleotide shells ,
snas represent a fundamentally different
class of nanomaterial with the ability to engage receptors that other
classes of particles often do not .
therefore , snas may exhibit a unique
profile of intracellular fate , a topic worthy of exploration . in this work ,
we synthesized a new class of sna consisting of a
quantum dot ( qd ) core and a fluorescent oligonucleotide shell ( fl - qd - sna )
to support our investigation of the intracellular events that occur
following the cellular uptake of our classic gold sna ( au - sna ) construct .
specifically , we studied the intracellular trafficking of snas in
c166 mouse endothelial cells , the signals which dictate trafficking
routes , the rate and origins of sna degradation , and the recycling
potential of the material .
based upon the results of these studies ,
we propose a mechanism by which snas are recognized , processed , and
distributed within the cell .
we first studied the intracellular locations
of snas as a function of incubation time by using c166 cells ( mouse
endothelial ) as a model cell line , under conditions where cells were
continuously incubated ( no change in medium ) with snas ( 10 nm ) . for
this purpose ,
we prepared cyanine 5 ( cy5)-labeled snas ( cy5-snas )
made from 5 cy5-labeled , single - stranded dna oligonucleotides
( cy5-ssdnas ) covalently attached to the surface of 13 nm aunps ( 80
5 oligonucleotides per particle ) .
in addition to the cy5 moiety ,
these oligonucleotides bear a nontargeting sequence that is antisense
to the mrna transcript of the green fluorescent protein ( gfp ) ( sequence
information listed in table s1 ) .
we chose
the gfp sequence for our studies because it is irrelevant and non - targeting
for the c166 cell - line , which does not contain the gene .
subsequent
discussion of snas will refer to this sequence unless otherwise specified .
by examining whether the fluorescent signals of cy5-snas colocalized
with the immunofluorescence of specific protein markers for intracellular
compartments with strong literature precedent for nanoparticle accumulation
,
we were able to delineate the route of trafficking of snas inside
cells .
these protein markers include early endosome antigen 1 ( eea1 ) ,
ras - related protein 9 ( rab9 ) , lysosomal - associated membrane protein
1 ( lamp1 ) , and giantin .
their corresponding intracellular compartments
are early endosome , late endosome , lysosome , and the
trans - golgi network , respectively .
confocal microscopy
of cy5-labeled au - snas ( red ) and immunofluorescence
staining of organelle markers ( green ) .
markers are eea1 ( early endosome ) ,
rab9 ( late endosome ) , lamp1 ( lysosomes ) , and giantin ( trans - golgi
network . )
most snas colocalize with late endosomes during continuous
incubation in c166 cells . some sna colocalization with early endosomes
snas do not colocalize at any time with lysosomes or
the trans - golgi network .
mander s colocalization coefficients
are displayed in yellow ( > 0.6 indicates substantial colocalization . )
our previous work has shown that snas are primarily localized
in
early endosomes after 12 h of incubation with cells . in these experiments ,
the snas carry a cy5 dye
( red ) , and the cells are stained with a complementary dye - labeled
( green ) marker of interest ( in figure 1a , the
marker of interest is eea1 , a protein that localizes exclusively in
early endosomes ) . here
, we demonstrate that snas show moderate colocalization
with early endosomes from the fourth to 24th hour of incubation ( figure 1a ) .
this continual association with early endosomes
suggests that uptake and trafficking are continuous processes .
importantly ,
strong colocalization of snas with rab9 ( protein localizes in late
endosomes ) was observed after the fourth hour of incubation , and colocalization
persisted 24 h post - incubation .
experimentally , we observe a typical
cell doubling time of 18 h for c166 cells , and we believe that snas
largely reside within late endosomes after their departure from early
endosomes ( figure 1b , note the orange - yellow
color , indicating colocalization of snas and rab-9 marker ) .
significantly ,
we did not observe appreciable colocalization between the fluorescent
signals of snas and markers for lysosomes ( lamp-1 ) or trans - golgi
network ( giantin ) over the entire incubation period of 24 h ( figure 1c , d ) . from these data , we conclude that the probability
of snas trafficking from early endosomes to the lysosome or trans - golgi
network is low .
it is also interesting to note that our data suggest
that snas do not fully progress through the typical route of the endolysosomal
pathway for the degradation of biological entities . instead , over
this time period
, snas stall inside of late endosomes and do not traffic
extensively beyond this point .
transmission electron microscopy
( tem ) images for cells collected
after continuous treatments of au - snas further reinforce the confocal
imaging data . at most time points ,
the overwhelming majority of snas
is observed inside perinuclear vesicles measuring from 400 nm to 1
m in diameter ( figure 2a , c ) , a size
range consistent with the reported average dimension of late endosomes
( 700 nm ) .
only a tiny portion of these
particles escape the endosomes and are found in the cytosol ( figure s1 ) .
moreover , high - magnification tem
images reveal a key defining ultrastructural feature of late endosomes ,
the presence of numerous luminal vesicles that resemble multivesicular
bodies ( mvbs ) encapsulated by the late endosomes ( figure 2b ) .
these luminal vesicles mostly measure 50100
nm across ( figure 2c ) , fitting literature reports
for luminal vesicles characteristic of late endosomes .
structures such as cisternae and electron dense
lumens were not seen in association with snas , indicating no intracellular
trafficking to the trans - golgi network and mature lysosome , respectively .
moreover , tem micrographs reveal nanoparticle clusters of increasing
sizes as well as decreasing interparticle distances inside late endosomes
as a function of incubation time . after 4 h of incubation , snas in
clusters of 2030 particles
are localized in late endosomes
without apparent contact with each other . by contrast , snas typically
manifest as clusters of 300500 particles accumulated
inside late endosomes after 16 h of incubation . in short , upon cellular
entry , au - snas that exist in early endosomes as individual particles
gradually traverse to late endosomes as clusters of particles in close
proximity to one another .
au - sna comprises of a 13 nm
diameter aunp core and 3 thiol terminated ssdna adsorbed to
the surface .
qd - sna comprises of a 7 nm diameter cdse / zns 630 emission
qd coated with a layer of azide bearing amphiphilic polymer , which
surrounds the surface of aliphatic - ligand protected cdse / zns qd by
hydrophobic interactions .
ssdna containing dbco groups on the 5
end are attached to the amphiphilic polymer shell by copper - free click
chemistry .
hollow snas are composed of a t - alkyne crosslinked cage
on the 3 end of ssdna .
hydrodynamic radii ( r ) of each construct
were measured using dynamic light scattering .
tem micrographs
show intracellular au - snas collect inside increasingly
larger and more perinuclear compartments over time .
( a ) most au - snas traffic through increasingly
larger , membrane - bound vesicles and remain inside these compartments
over 24 h of continuous incubation in c166 cells .
the bottom panel contains magnified images
of the boxed area of the top panel .
( b ) high magnification of large
vesicles inside cells after 16 h incubation .
the
right panel shows the magnified image of the boxed area of the left
panel .
( c ) size distribution of ( top ) endosomes containing au - snas
and ( bottom ) luminal vesicles in these endosomes after long incubations
( 1624 h ) .
following identification of intracellular compartments
which house our conventional au - snas after their cellular entry , we
set out to preliminarily determine whether trafficking to as well
as accumulation in late endosomes without further departure was a
phenomenon generalizable to other snas with different nanoparticle
cores .
to do so , we treated cells with two structural variants of
the au - sna , namely a hollow sna and an sna with a cdse / zns qd core
( scheme 1 ) .
hollow snas are 3d oligonucleotide - based
nanoconstructs obtained by cross - linking multiple alkyne - terminated
cy5-ssdnas on the surface of an aunp core that is subsequently dissolved
by kcn .
quantum dot snas ( qd - snas ) were
first synthesized by our group in 1999 by covalently attaching oligonucleotides directly to the qd core .
subsequent to that work , we reported a method for non - covalently immobilizing
ssdna on the surface of aliphatic - ligand protected cdse / zns quantum
dots by reacting them with amphiphillic polymers , functionalized with
dna .
significantly , we have used qd - snas and
the hollow snas to show that the composition and even absence of the
nanoparticle core have no appreciable effect on the intracellular
fate of snas ; both sna structure variants were found to be within
late endosomes after 4 h of incubation ( figure 3a , compare figure 1b ) .
next , we studied
two snas composed of different oligonucleotide sequences to determine
if there was sequence - specific sorting within the cell . for these
experiments , we functionalized aunp cores with two distinct ssdna
sequences , namely a repeated thymidine sequence ( t30 ) and
a sequence antisense to the transcript of the survivin oncogene .
t30 is not gene targeting , and the survivin sequence exists
but is not overexpressed in this cell line .
therefore , these two types
of snas allow one to probe if sequence makes a significant difference
with regard to intracellular sna fate .
confocal immunofluorescence
studies show that these snas behave analogously to our gfp sequence
snas , also trafficking into late endosomes after 4 h of continuous
incubation ( figure 3b ) .
tem micrographs also
show the presence of t30 snas inside of large membrane - bound
vesicles ( figure s2 ) .
therefore , the sheer
3d architecture of the oligonucleotides , rather than the sequence
information encoded by them , is likely the primary attribute which
governs the intracellular trafficking behavior of snas .
we hypothesized that sna trafficking into the
late endosome could potentially be driven by subsequent waves of sna
uptake . to investigate the effect of continuous uptake on the trafficking
of snas
, we utilized a pulse - chase setup to follow a small window
of uptake events .
cells were treated with au - snas ( 10 nm ) for 1 h
only and then placed into clean , fresh sna - free media for different
durations of time .
immunofluorescence analysis shows colocalization
of snas with the early endosomes at the end of the 1 h treatment ,
but strong colocalization with late endosomes 24 h after the initial
treatment with snas has ended ( figure 3c ) .
tem images support this conclusion as au - snas are in small , featureless
vesicles as isolated entities at the end of 1 h treatment but collect
inside of larger , multivesicular endosomes after the 24 h treatment - free
incubation period ( figure 3d ) . treating cells
with au - snas for only 4 h also gives similar results ( figure s3 ) .
in other words , c166 cells
will naturally sort an overwhelming majority of snas from early endosomes
to late endosomes unbiased by the stimulus induced by the uptake of
subsequent waves of snas .
immunofluorescence rab9 staining ( green ) of cells treated
with different sna constructs ( red ) for 4 h shows that ( a ) hollow
snas ( no core ) , qd - snas with a cdse - zns core , ( b ) snas consisting
of a repeated thymidine sequence ( sna : t30 ) , and snas consisting
of a sequence antisense to the transcript that encodes the survivin
oncogene ( sna : sv ) also colocalize strongly with late endosomes .
( c ) pulse - chase experiments
show cy5-labeled au - snas that have already entered the cell 1 h postincubation
progress in the endocytic cycle .
immunofluorescence staining shows
that snas colocalize with early endosomes ( eea-1 ) but not late endosomes
( rab9 ) after 1 h incubation .
they then colocalize with late endosomes
but not early endosomes 24 h after the initial incubation of 1 h.
mander s colocalization coefficients are displayed in yellow .
( d ) representative tem micrographs show rare occurrences of au - snas
within small compartments as isolated entities after 1 h incubation .
( e ) large clusters of au - snas ( > 200 particles per cluster ) are
found
abundantly in large , perinuclear compartments 24 h after the initial
incubation of 1 h. for ( d ) and ( e ) , the bottom panel contains magnified
images of the boxed area of the top panel .
the late endosome represents an integral part
of the cellular degradation pathway and has been shown to eventually
fuse with lysosomes .
the lumen of the
late endosome is known to be an environment that facilitates degradation
of biomacromolecules .
acidic ph , presence of catabolic enzymes , and
redox active species are just a few characteristic features of the
late endosome .
proteins and oligonucleotides
have been shown to be extensively degraded due to this environment .
we were interested in probing the susceptibility
of snas to degradation due to their prolonged accumulation inside
the late endosomes . from our tem imaging data in figure 2 ,
the intracellular aggregation of the aunp core becomes increasingly
prevalent as a function of incubation time , suggesting the possibility
of intracellular degradation of the oligonucleotide shell that originally
provided steric stabilization to and electrostatic repulsion between
sna nanostructures . to address the issue of degradation ,
we incubated
cy5-labeled au - snas ( also used for the confocal imaging studies in
figure 1 ) under different chemical conditions
and measured how many cy5-ssdna strands remain on the surface of the
aunp core after the treatment .
briefly , we centrifuged the chemically
treated sna solution to recover the sna pellet , dissolved the aunp
core , and quantified the cy5 fluorescence of the solution against
a standard calibration curve .
we first subjected cy5-labeled au - snas
to various buffers with ph values ranging from 7.5 to 4.5 , a window
that covers the essential intracellular compartments expected to be
traversed by a sna along the endolysosomal pathway , including extracellular
fluid ( ph = 7.4 ) , early endosomes ( ph = 5.56.0 ) , and late
endosomes / lysosomes ( ph = 4.55.0 ) .
after 3 d of incubation , cy5-snas did not show reduction in oligonucleotide
loading .
we next added cy5-snas to a degassed pbs solution that contains
intracellular concentrations of glutathione ( 110 mm ) to analyze whether the surplus of thiol groups
from glutathione ( gsh ) in the cell would displace thiolated ssdna
strands off the aunp surface .
for this experiment , the pbs was thoroughly
degassed by repeated freeze thaw cycles to prevent the oxidation
of gsh to form dimers ( gssg ) in the cell - free environment . again ,
after incubation for 1 d , the fluorescence associated with the solution
of cy5-snas did not significantly increase , indicating no appreciable
oligonucleotide displacement from the particle surface ( figure s4 ) .
thus , change in ph and thiol displacement
by glutathione can not account for the aggregation of au - snas inside
the cell .
finally , we investigated if dna nucleases natively
found in late endosomes or lysosomes may contribute to the degradation
of snas .
two common nucleases pertinent to dna degradation are deoxyribonuclease
i ( dnase i ) and deoxyribonuclease ii ( dnase ii ) .
dnase i has been
implicated in dna degradation in the serum , extracellular space , and
also in the cytosol of cells .
an acidic
endonuclease , dnase ii is usually found within intracellular compartments ,
most notably lysosomes . since late endosomes
are able to fuse with other late endosomes or lysosomes , we hypothesize that dnase ii is responsible
for dna degradation when snas are shuttled to and stalled in the late
endosomes . to test this hypothesis , we introduced cy5-labeled au - snas
into a cell - free solution that contains the same concentration of
either dnase i or dnase ii , each buffered at the appropriate ph required
for its proper functioning .
after 4 h of incubation , cy5-labeled au - snas
treated with dnase ii lost 60% of their original oligonucleotide
loading , whereas those treated with dnase i lost only 25%
( figure 4a ) .
we further showed that , by contrast
to the sna architecture , more than 80% of cy5-ssdna of the same nucleotide
sequence was degraded after incubation with both enzymes for 4 h.
for this assay , free dna was synthesized with a 3 molecular
quencher of the 5 dye ( see supporting
information for sequence information ) in order to allow for
similar percentage degradation calculations as those done with snas .
thus , the arrangement of dna oligonucleotides in the form a dense
3d shell can endow snas with additional stability against enzymatic
degradation , but snas are less resistant to enzymatic attack by dnase
ii than dnase i , a conclusion supported by early , less comprehensive
work .
note that the time points in these
studies may or may not be relevant to the cellular studies since the
intracellular concentration of these enzymes has not been reported
to date .
buffer tests of ( a ) au - snas and ( b ) qd - snas show that both structure
variants on the sna architecture offer protection against dnase i
and ii compared to free , single - stranded dna ( ssdna ) .
dnase degradation
profiles for both au - sna and qd - sna are sufficiently similar to suggest
that they behave similarly under intracellular enzymatic environments .
note that , in buffer , the sna architecture is more prone to degradation
by dnase ii , an enzyme commonly found in late endosomes or lysosomes ,
than dnase i , which is usually found in extracellular fluids or the
cytosol .
we wish to further visualize how
intracellular nucleases like dnase
ii may disassemble the sna architecture . to achieve this goal
, we
need to independently track the movement of both the np core and dna
strands in the cell .
while dna strands can be labeled fluorescently ,
the innate lack of fluorescence of aunps precluded us from visualizing
how the dna strands are falling off the np core for our classic au - snas
by confocal imaging . to circumvent this bottleneck , we synthesized
fl - qd - snas , a new class of sna nanostructure that consists of a cdse / zns
core ( with an emission wavelength of 630 nm ) covalently functionalized
with fluorescein - labeled ssdnas .
we believe that the fl - qd - sna serves
as a reasonable proxy for the classic au - sna due to its similar hydrodynamic
diameter ( 20 nm ) and oligonucleotide loading ( 70 ssdna 4/particle )
as previously described .
indeed , dnases
exhibit similar activity profiles for both constructs , whereby incubation
in dnase ii led to more significant reduction in oligonucleotide loading
than dnase i ( figure 4a , b ) .
we then treated
c166 cells continuously with fl - qd - snas and imaged them at various
time points .
confocal microscopy shows the fl - qd - snas remain largely
intact for up to 16 h , as evidenced by overlapping fluorescence signals
of the qd core and fl - ssdnas ( figure 5 ) .
after
16 h , the fluorescein signal from the oligonucleotides separates from
that of the qd core , with an even larger effect after 24 h. this separation
is likely due to dna cleavage from the sna by enzymes in the late
endosomes , particularly dnase ii , which has high activity at low ph .
it is important to note that the treatment in this study is continuous ,
and any newly uptaken qd - snas likely influence the true time scale
of degradation . in this case
, degradation products must build up for
a time before they are observable by confocal microscopy .
once we utilized
qd - snas to establish that the sna architecture disassemble on the
time scale of roughly 1624 h after entering c166 cells , we
returned to au - snas and explored whether their individual components
are expelled from the cell . using the same pulse - chase setup previously
described in figure 3 , we tracked the gold
content in cells using inductively coupled plasma mass spectrometry
( icp - ms ) and quantified the cy5 fluorescence in cells by spectroscopic
analysis .
after a 4 h pulse treatment of cells with cy5-snas , we washed
out the nanoparticles , grew the treated cells in clean medium , and
then divided cell samples for icp - ms and fluorescence analysis .
to
exclude the possibility that any observed reduction in the intracellular
gold or dna amount is due to cell division but not actual exocytosis ,
we treated the cell sample as a population rather than on a single
cell basis by ensuring a near - constant density of c166 cells plated
for this experiment .
snas composed of a cdse / zns qd core and fluorescein - tagged
dna
strands ( fl - qd - snas ) are used as a proxy to monitor and visualize
the degradation of snas in c166 cells . by confocal imaging , the qd
core ( red ) and the fluorescein - tagged oligonucleotides ( green )
have
visibly separated at 16 h and beyond , likely due to enzymatic cleavage .
before 16 h , overlapping signals of qd and fl - dnas ( yellow ) indicate
that the sna architecture is largely intact .
coefficients
are indicated in yellow for merged images , showing gradual loss of
colocalization between the qd and the fl - dna over time .
after the 4 h pulse incubation , intracellular gold
content remains
relatively constant in the cell population over the course of 24 h
of growth , indicating no net exocytosis of the aunp core ( figure 6a ) .
given our tem data that reveal increased clustering
of aunp cores , this near - perfect mass balance of intracellular gold
content across the entire period of incubation time suggests that
the aunp core of snas is continuously sorted from multiple early endosomes
of smaller sizes to significantly fewer late endosomes of much larger
sizes .
the lack of recycling of the aunp core may stem from the degradation
of a large portion of the oligonucleotide shell by intracellular nucleases
( most likely , dnase ii ) .
such degradation likely leads to the loss
of biological recognition of the sna architecture and renders the
aunp core susceptible to colloidal aggregation in the presence of
intracellular amounts of salt .
ultimately , these aunp clusters may
not traffic efficiently due to there being no known cellular receptors
for this material . by contrast
, cy5 fluorescence is observed to rapidly
decrease in the cell lysates if the sna treatment is discontinued
after 4 h ( figure 6b ) .
this decline in fluorescence
likely results from degradation products of dnase cleavage that are
expelled from the cell either by active transport or diffusion , but
not via any recycling pathway based on our confocal imaging data presented
in figure 1 .
diffusion seems unlikely as the
confocal images presented in figure 5 reveal
punctate spots rather than homogeneous patches of fluorescence , which
would be indicative of release into the cytoplasm .
analysis of material
retention inside c166 cells as a function
of growth time post-4 h incubation with cy5-labeled au - snas .
( a ) gold
content in a single cell population ( pellet ) remains fairly constant
over time .
slight increases may be due to uptake of residual snas
loosely adhered to the plastic of the tissue culture plate .
( b ) fluorescence
in a single population of cells sharply decreases after the incubation ,
indicating the expulsion of cy5 or dna fragments into the extracellular
space .
moreover , we measured
the fluorescence of the culture medium collected
from the same pulse - chase experiment at different time points after
the initial incubation of 4 h. the fluorescence of the medium steadily
increases over time after the removal of cy5-snas , thus adding credence
to the notion that there is net exocytosis of free cy5 moieties or
processed dna fragments to the medium ( figure
s5 ) .
from our studies , we
have shown that snas enter into the endocytic
pathway after entry into the cell .
snas progress largely into late
endosomes , which is their final intracellular location over the 24
h time frame considered . a small , unquantifiable portion of these
particles
these
are the entities that are likely responsible for knockdown in both
antisense and rnai mediated gene regulation pathways . both immunofluorescence
and tem ultrastructural analysis support this conclusion
. moreover ,
snas traffic along this route and reach the late endosomes , independent
of the surface oligonucleotide sequences and the core compositions
studied here .
our pulse - chase experiments also show that the intracellular
fate of snas is guided only by the single - entity agent alone and seems
invariant to the quantity of snas uptaken .
finally , we show that snas
are partially broken down by nucleases within the late endosome , and
degradation products are differentially processed by the cellular
transport machinery : the core is retained in the late endosome , while
the dye or oligonucleotide fragments are cleared from the cell , at
least for this cell line .
the sna architecture can function
successfully as a gene regulation
construct , in part due to the increased stability of the oligonucleotides
on snas as compared to their free forms .
the observation that the vast majority
of snas are tied up in the endosome suggests that they particularly
potent gene regulation agents .
indeed , a small amount escapes the
endosome , which accounts for their activity in antisense and likely
sirna pathways .
any increased availability of snas to the cytosol
will further boost the therapeutic activity of snas , and we pose the
design and synthesis of snas capable of more efficient endosomal escape
as a challenge to the community .
it brings to light the importance of realizing next
generation snas that can take advantage of their location inside late
endosomes , which may include introducing functionalities to modulate
processes such as immune activation and exosome packaging .
it also underscores the importance of designing
synthesizing hollow snas or structures with biodegradable cores to
avoid the unanticipated consequences of the core materials on cellular
function .
dnas were synthesized
on an mm48 oligonucleotide synthesizer ( bioautomation ) using standard
solid - phase synthesis and reagents ( glen research ) .
all dnas were
purified using a prostar high - performance liquid chromatography ( hplc )
instrument ( varian ) with a microsorb c18 column ( varian ) .
table s1 contains detailed sequence information
on the dnas . for gold - sna nanoconjugates ,
thiolated dnas were added to 13 nm citrate - capped aunps at a concentration
of 1 od of dna per ml of 10 nm aunps supplemented with 0.5% tween-20 .
after stirring at rt for 1 h , the solution was aged with gradual additions
of nacl over 6 h to bring the final nacl concentration to 0.5 m. functionalized
aunps were separated from free dna strands via dialysis against nanopure
water using a 50 kda amicon molecular weight cutoff membrane ( millipore ) .
aunp and dna concentrations were determined by measuring their extinction
at 524 and 260 nm , respectively , on a cary 5000 uv vis spectrophotometer
( agilent ) .
quantum dot sna nanoconjugates were also prepared
as detailed previously using cdse - zns quantum dots ( ocean nanotech ) .
all
cell experiments
described in this work employ c166 cells ( mouse endothelial ) , which
were cultured at 37 c and 5% co2 in dmem supplemented
with 10% fbs and 1% streptomycin / penicillin . to measure the extent
of cellular association by icp - ms
, cells were first seeded in a 24-well
plate at a population of 5 10 cells per well 24
h in advance and incubated with 0.3 ml of snas ( 10 nm in dmem ) per
well . to visualize the extent of cellular uptake by tem , cells were
seeded in a 6-well plate at a population of 5 10 cells per well and then incubated with 1.5 ml of snas ( 10 nm in
dmem ) per well . for both icp - ms and tem studies , snas were removed
at different time points , followed by dmem rinses , trypsinization
for counting using a hemacytometer and centrifugation at 8000 rpm
for 5 min to form a cell pellet . for pulse - chase experiments ,
cells
were first treated with 10 nm sna for either 1 or 4 h , washed twice
with dmem , and replenished with fresh dmem .
the cells were then incubated
for the designated duration of time before harvesting them for icp - ms
and tem studies .
cell pellets were digested
with 0.3 ml of 3%
hcl in concentrated hno3 at rt overnight . after adding
5 l of 5 ppm indium ( internal standard ) and 5 ml of matrix
solution ( 2% hcl and 2% hno3 ) , the au-197 content of the
resultant solution was measured by an x series ii icp - ms ( thermo fisher )
after subtracting the background gold content of untreated cells .
unless otherwise mentioned ,
cell pellets were fixed by resuspension in 3.7%
paraformaldehyde ( pfa ) in pbs for 15 min .
cells were then pelleted
again by centrifugation at 6000 rpm for 5 min and enrobed in molten
2% agarose at 37 c .
molten agarose cell mixtures were expressed
into water at rt to produce noodle - shaped
the cell - containing noodle gels
were fixed in 2.5% glutaraldehyde in 100 mm sodium cacodylate buffer
( ph = 7.4 ) , stained by 1% oso4 and by 0.9% oso4 and 0.3% k4fe(cn)6 , with all steps carried
out at 4 c for 2 h. after gradual dehydration with ethanol and
propylene oxide , the cell - containing noodle gels were embedded in
epon 812 resins ( electron microscopy sciences ) and further polymerized .
we deposited 80 nm - thick sections on 200-mesh copper grids ( electron
microscopy sciences ) and stained with 2% uranyl acetate ( spi supplies )
and reynolds lead citrate for visualization under a jem 1230 microscope
( jeol ) using a beam voltage of 80 kv . an orius sc 1000 ccd camera
( gatan )
endosome and luminal vesicle
diameter measurements were taken using imagej and freehand blob identification .
data were binned into bins 5%
of average measurement . seeded in
a 35 mm fluorodish ( world precision instruments )
, cells were incubated
with 10 nm of cy5-snas or cy5-hollow - sna in complete dmem for different
time points .
cells were rinsed with pbs , fixed in 3.7% pfa in pbs
for 15 min , and imaged under a zeiss lsm 510 inverted confocal scanning
microscope .
the excitation wavelength of cy5-snas was 633 nm , and
the corresponding emission filter was 660710 nm . to track
the colocalization of snas with intracellular proteins , after incubation
with 10 nm cy5-snas for different durations of time
, cells were rinsed
with pbs , fixed in 3.7% pfa in pbs , and permeated with 0.1% triton
x-100 for 10 min . after blocking with 2% bsa in pbs for 1 h ,
cells
were stained with a primary antibody against the protein marker of
interest at 5 g / ml ( 1% bsa in pbs ) overnight at 4 c .
if necessary , after rinses with 0.05% tween-20 in pbs , cells were
stained with an alexafluor 488-labeled secondary antibody ( invitrogen
alexa fluor 488 goat anti - rabbit igg ( h+l ) ) at 1 g / ml ( 1% bsa
in pbs ) for 1 h at rt .
the excitation wavelength of the secondary
antibody was 488 nm , and the corresponding emission filter was 500550
nm .
the primary antibodies include rabbit against eea1 ( abcam ab2900 ) ,
rabbit against rab9 ( santa cruz biotechnology fl-201 ) , rabbit against
lamp1 ( abcam ab24170 ) , and rabbit against giantin ( abcam ab24586 ) .
to measure the extent of colocalization between the fluorescence signals
of snas and protein markers , the zen digital imaging ( zeiss ) software
allows for the calculation of the manders overlap coefficient .
an overlap coefficient higher than 0.6 indicates
strong colocalization . to probe the intracellular
fate of the individual components of qd - snas ( i.e. , the qd ) core and
the fluorescein - labeled oligonucleotides )
, cells were incubated with
10 nm qd - snas in dmem for different durations of time . following the
same rinsing , fixation , and permeation procedures
the excitation
wavelengths for qd and fluorescein are 633 and 488 nm , respectively .
the corresponding emission filters for qd and fluorescein are 660710
nm and 500550 nm , respectively .
1012 nmoles
of cy5-labeled snas were treated with dnase i ( new england biolabs
m0303s ) or dnase ii ( sigma d4138 ) ( 5u / rxn ) for set time points .
all snas
were pelleted at 10 000 g and washed
with water .
pellets were resuspended in a cleaving buffer ( 100 mm
kcn and 100 mm dtt ) to dissolve the gold core .
after solution had
cleared , the cy5 fluorescence of the oligonucleotides was read on
a synergy h4multimode microplate reader ( bio - tek ) to determine the
quantity of oligo lost due to dnase activity .
free oligos were assayed
in a similar manner in which a quencher ( dabycl ) was conjugated to
the opposite ( 3 ) end of the oligo to suppress fluorescence .
oligo cleavage was calculated through increase of fluorescence as
quencher and fluorophore are separated . for qd - sna
, 0.5% hcl was used
to dissolve the qd core and dismantle the sna . following acid treatment ,
| spherical
nucleic acid ( sna ) nanoparticle conjugates are a class
of bionanomaterials that are extremely potent in many biomedical applications .
their unique ability to enter multiple mammalian cell types as single - entity
agents arises from their novel three - dimensional architecture , which
consists of a dense shell of highly oriented oligonucleotides chemically
attached typically to a gold nanoparticle core
. this architecture
allows snas to engage certain cell surface receptors to facilitate
entry . here
, we report studies aimed at determining the intracellular
fate of snas and the trafficking events that occur inside c166 mouse
endothelial cells after cellular entry .
we show that snas traffic
through the endocytic pathway into late endosomes and reside there
for up to 24 h after incubation .
disassembly of oligonucleotides from
the nanoparticle core is observed 16 h after cellular entry , most
likely due to degradation by enzymes such as dnase ii localized in
late endosomes .
our observations point to these events being likely
independent of core composition and treatment conditions , and they
do not seem to be particularly dependent upon oligonucleotide sequence .
significantly and surprisingly , the snas do not enter the lysosomes
under the conditions studied . to independently track the fate of the
particle core and the fluorophore - labeled oligonucleotides that comprise
its shell , we synthesized a novel class of quantum dot snas to determine
that as the sna structures are broken down over the 24 h time course
of the experiment , the oligonucleotide fragments are recycled out
of the cell while the nanoparticle core is not .
this mechanistic insight
points to the importance of designing and synthesizing next - generation
snas that can bypass the degradation bottleneck imposed by their residency
in late endosomes , and it also suggests that such structures might
be extremely useful for endosomal signaling pathways by engaging receptors
that are localized within the endosome . | Introduction
Results and Discussion
Conclusions
Experimental Section | spherical nucleic acid
( sna ) conjugates represent an emerging class
of bionanomaterials that typically consist of two types of fundamental
building blocks , oligonucleotides and inorganic nanoparticle cores . due to their ability to naturally enter multiple
mammalian cell types without the aid of lipid or cationic transfection
agents , snas are extremely potent and useful in a variety of biomedical
applications . however ,
investigations of the intracellular events that happen to nanoparticles
subsequent to their cellular entry are scarce and do not seem to reach
a universal consensus . due to their three - dimensional
( 3d ) oligonucleotide shells ,
snas represent a fundamentally different
class of nanomaterial with the ability to engage receptors that other
classes of particles often do not . in this work ,
we synthesized a new class of sna consisting of a
quantum dot ( qd ) core and a fluorescent oligonucleotide shell ( fl - qd - sna )
to support our investigation of the intracellular events that occur
following the cellular uptake of our classic gold sna ( au - sna ) construct . specifically , we studied the intracellular trafficking of snas in
c166 mouse endothelial cells , the signals which dictate trafficking
routes , the rate and origins of sna degradation , and the recycling
potential of the material . experimentally , we observe a typical
cell doubling time of 18 h for c166 cells , and we believe that snas
largely reside within late endosomes after their departure from early
endosomes ( figure 1b , note the orange - yellow
color , indicating colocalization of snas and rab-9 marker ) . significantly , we have used qd - snas and
the hollow snas to show that the composition and even absence of the
nanoparticle core have no appreciable effect on the intracellular
fate of snas ; both sna structure variants were found to be within
late endosomes after 4 h of incubation ( figure 3a , compare figure 1b ) . finally , we investigated if dna nucleases natively
found in late endosomes or lysosomes may contribute to the degradation
of snas . note that , in buffer , the sna architecture is more prone to degradation
by dnase ii , an enzyme commonly found in late endosomes or lysosomes ,
than dnase i , which is usually found in extracellular fluids or the
cytosol . to achieve this goal
, we
need to independently track the movement of both the np core and dna
strands in the cell . to circumvent this bottleneck , we synthesized
fl - qd - snas , a new class of sna nanostructure that consists of a cdse / zns
core ( with an emission wavelength of 630 nm ) covalently functionalized
with fluorescein - labeled ssdnas . after
16 h , the fluorescein signal from the oligonucleotides separates from
that of the qd core , with an even larger effect after 24 h. this separation
is likely due to dna cleavage from the sna by enzymes in the late
endosomes , particularly dnase ii , which has high activity at low ph . once we utilized
qd - snas to establish that the sna architecture disassemble on the
time scale of roughly 1624 h after entering c166 cells , we
returned to au - snas and explored whether their individual components
are expelled from the cell . the lack of recycling of the aunp core may stem from the degradation
of a large portion of the oligonucleotide shell by intracellular nucleases
( most likely , dnase ii ) . snas progress largely into late
endosomes , which is their final intracellular location over the 24
h time frame considered . our pulse - chase experiments also show that the intracellular
fate of snas is guided only by the single - entity agent alone and seems
invariant to the quantity of snas uptaken . finally , we show that snas
are partially broken down by nucleases within the late endosome , and
degradation products are differentially processed by the cellular
transport machinery : the core is retained in the late endosome , while
the dye or oligonucleotide fragments are cleared from the cell , at
least for this cell line . it brings to light the importance of realizing next
generation snas that can take advantage of their location inside late
endosomes , which may include introducing functionalities to modulate
processes such as immune activation and exosome packaging . | [
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mantle cell lymphoma constitutes about 6% of non - hodgkin lymphoma in the western world .
it shows a characteristic clinical behavior with initial response to therapy followed by relapses and death in 35 years .
aggressive therapeutic regimens are promising , but to - date mcl is still considered incurable , with the exception of a small group of long - surviving patients . in recent years , many high - throughput techniques have been applied to mcl in order to gain insights into its pathogenesis , and to discover suitable therapeutic targets [ 29 ] .
these studies have clarified a role for increased proliferation and resistance to apoptosis in mcl , involving several signaling pathways . a few studies from our group and also from others have attempted the study of mcl from a proteomic point of view [ 1018 ] .
some of these studies were mainly focused on the diagnostic implementation of proteomic techniques [ 1416 ] , while other were more focused on the pathogenetic mechanisms [ 12 , 1820 ] .
these studies have identified single up - regulated proteins in mcl compared to tonsil b - cells , or several proteins up - regulated in mcl cell lines compared to fl - derived cell lines followed by network analysis .
more recently , a proteomic study focused on the plasma membrane proteome of leukemic mcl and compared it to normal b - cells ; this study identified several alterations in the proteome of mcl lipid rafts that might have therapeutic potential . in a previous study
we have focused our attention on the phospho - proteome of several mcl cell lines by using immobilized metal affinity chromatography ( imac ) pre - fractionation ( a column - based technique that selectively enriches all phospho - proteins by a reversible binding to metal oxydes ) , followed by 2d - page / ms . in that study we identified several activated pathways that might reflect the biology of mcl , including the alternative nf - kb pathway , the mtor pathway and the mitochondrial pathway . in the present work
we have used a complementary approach , called phosphoscan , that involves an immunoprecipitation of peptides by using an anti - phosphotyrosine antibody followed by capillary electrophoresis and ms identification of eluted peptides . compared to imac
this approach has already been successfully used in the phospho - profiling of primary and metastatic lung cancer , in acute leukemia samples , in bcr / abl positive cell lines , aml cell lines and hodgkin lymphoma cell lines . since tyrosine phosphorylation regulates the activity of many proteins , the phosphoscan approach can provide a picture of the most active pathways in the analyzed samples . moreover , since many kinases are themselves activated by tyrosine phosphorylation , the profile of active kinases is also provided ; often these kinases are driving cell proliferation and survival , and are therefore candidate targets for therapy . in the current work
, we used this approach to recognize the most represented activated pathways in mcl cell lines .
we then verified the presence of these activated molecules in mcl cell lines and tumor tissues .
we subsequently explored the functional role of the most activated pathway in mcl cell lines by inhibition experiments .
in the current paper we describe the results obtained from the application of phosphoscan to mcl cells .
this is to our knowledge the first use of this technique in mcl ; we report a large amount of data with precise identification of phosphorylation sites for each protein , some of which novel .
phosphoscan analysis identified 421 unique tyrosine - phosphorylated peptides , corresponding to 341 proteins , ranked by overall abundance in table 1 ( for a complete list of peptides and complete ms information see supplemental table 1 ) .
interestingly , several identified proteins mapped to cytogenetic loci that have been reported to be altered in mcl [ 2931 ] . among these
table 1top 50 phospho - peptides identified ( number of phospho peptides corresponding to the specific protein is reported . for complete list and exact phosphorylation sites ,
table 1)protein namegranta-519jeko-1maver-1rec-1bcr pathwaykynasecdc217381626*cdk210341418*cdk310331317*syk919243**ship7181010*pag91448*prp431982*lyn41097**ig - beta02220*hck3853**wasp2743*shp-14741*btk01141**cofilin 12751sgk2236144*lanp - l0653pkcd4324**gcet25710*hsp90b5502gsk3a2343**gsk3b2343**p38-alpha2361**hnrnp a15231grf-11424sptbn10541sptbn1 iso20541plcg22430*dyrk1a1431*lck4212**hsp90a2411blk2321**irap1430ddx34300pl104300cbl - b1311*bcap1410dok30600*anxa21221vim2220l - plastin0420eno10222pik3r11221**dyrk1b1221*src1212**fyn1212**yes1212*hnrnp 2h92310hnrnp u2310hnrnp u iso22310pai - rbp12220fig .
1list of protein kinases identified by the phosphoscan approach , ranked by overall abundance in the four mcl cell lines analyzed .
color intensity proportional to the number of peptides ( see figure for color corresponding to the average number of peptides per cell line ) top 50 phospho - peptides identified ( number of phospho peptides corresponding to the specific protein is reported . for complete list and exact phosphorylation sites ,
. table 1 ) list of protein kinases identified by the phosphoscan approach , ranked by overall abundance in the four mcl cell lines analyzed .
color intensity proportional to the number of peptides ( see figure for color corresponding to the average number of peptides per cell line ) the three most represented phospho - peptides are cyclin - dependent kinases cdc2 , cdk2 and cdk3 .
cdc2 ( cdk1 ) is actually part of the proliferation signature able to predict prognosis in mcl and has also been validated at the immunohistochemical level .
cdk2 gains have been detected in mcl in a variable proportion of cases [ 30 , 31 , 33 ] , and have been associated with a poor prognosis .
cdk3 is less studied compared to other cyclin - dependent kinases , but its locus has also been reported as subject to gains [ 31 , 34 ] .
its involvement in mcl is a novel finding , although the role of cdk3 as an oncogene has been demonstrated in other models [ 35 , 36 ] .
many kinases belonging to this pathway ( e.g. syk , lyn , hck , btk , pkc - delta ) , were among the most represented phospho - peptides identified by the phosphoscan approach , and this was confirmed when looking for non - random enrichment of kyoto encyclopedia of genes and genomes ( kegg ) pathways compared to the hypothesis of random distribution .
this analysis showed that ( using a cutoff value of more than five total peptides ) , the most enriched pathway was hsa04662:b cell receptor signaling pathway ( 17.16-fold enrichment ) ( for complete data see supplemental table 2 ) .
many of the proteins belonging to the most enriched pathways were either present in different pathways ( redundant ) or are functionally connected .
manual annotation by literature search revealed that several of these , in the topmost part of the ranking , were connected to bcr signaling , even if not present in the canonical kegg bcr pathway ( table 1 and fig . 2 ) .
2simplified diagram showing some of the identified bcr pathway members . in red , activating members ; in green , inhibiting members ; for some members the precise final effect is not clear ; in grey , other proteins known to be present but not identified .
full arrows : direct interaction / activation ; dotted arrows : indirect interaction / activation .
data derived from kyoto encyclopedia of genes and genomes ( kegg ) pathway and from published literature .
kegg is a widely used annotated database of pathways , ligands and genes ( http://www.genome.jp/kegg/ ) simplified diagram showing some of the identified bcr pathway members . in red , activating members ; in green
, inhibiting members ; for some members the precise final effect is not clear ; in grey , other proteins known to be present but not identified .
full arrows : direct interaction / activation ; dotted arrows : indirect interaction / activation .
data derived from kyoto encyclopedia of genes and genomes ( kegg ) pathway and from published literature .
kegg is a widely used annotated database of pathways , ligands and genes ( http://www.genome.jp/kegg/ ) to further verify the functional role of bcr signaling in mcl , we analyzed the phosphorylation status of syk , lyn , btk , blnk and gsk3alfa / beta in mcl cell lines .
although not present in the top 50 phospho - peptides , blnk was investigated because it is considered a classical downstream molecule of the bcr pathway .
the presence of basal levels of phospho - syk y525 and y323 , as well as of phospho - blnk ( y84 ) was verified by flow cytometry ( fig .
in addition basal levels of phospho - lyn ( y396 and y507 ) and also of downstream effectors phospho - btk ( s180 ) and phospho - gsk3alfa / beta ( s9/21 ) were demonstrated by flow cytometry ( supplemental figure 1 ) .
bcr pathway activation in cell lines is somehow intriguing since it is present in absence of an appropriate antigen stimulation , and is therefore probably self - sustained by tumor cells , either by side - by - side activation or by auto - activation .
3analysis of basal levels of phosphorylated syk and blnk residues by flow cytometry . in grey ,
isotypic control ; in red , basal levels . on the x axis , arbitrary fluorescence units ( log scale ) ; on the y axis , cell count analysis of basal levels of phosphorylated syk and blnk residues by flow cytometry . in grey , isotypic control ; in red , basal levels . on the x axis ,
arbitrary fluorescence units ( log scale ) ; on the y axis , cell count in order to verify whether we could find this activation in mcl tumors as well , we resorted to western blotting analysis of phosphorylated forms of bcr pathway members .
this analysis showed that the activated forms of syk ( in 5/6 cases , 83% ) , lyn ( in 6/6 cases , 100% ) , and blnk ( in 6/6 cases , 100% ) were present also in mcl tumor tissues ( fig .
4 ) , therefore supporting the in vivo role of active bcr signaling ; as far as we know , this is the first report of the presence of active ( phosphorylated ) bcr pathway members in mcl tissues .
the presence of phospho - syk ( y525 ) , phospho - lyn ( y396 ) and phospho - blnk ( y84 ) is shown in six mcl tumor tissues .
cases 1 , 2 , 5 and 6 were classical mcl , while cases 3 and 4 were blastoid variants western blotting analysis of mcl tissues .
the presence of phospho - syk ( y525 ) , phospho - lyn ( y396 ) and phospho - blnk ( y84 ) is shown in six mcl tumor tissues .
cases 1 , 2 , 5 and 6 were classical mcl , while cases 3 and 4 were blastoid variants the activation of the bcr pathway in mcl has been hypothesized in a previous paper based on cytogenetic and rna studies , but to our knowledge this is the first protein - based and data - driven study that supports this hypothesis .
another proteomic study focusing only on the plasma membrane showed an abnormal association of pkcbeta to the cell membrane in mcl leukemic cells , indirectly supporting an active bcr signaling .
recent studies have shown the importance of tonic bcr signaling in dlbcl [ 38 , 39 ] and b - cll , with a basal activation of phospho - syk residue y352 , while y525 was detected only after bcr cross - linking .
the presence of significant basal levels of phospho - syk y525 and y323 , with no detectable phospho - syk y352 in basal conditions in mcl cells are not concordant with what has been reported in b - cll and dlbcl , and suggest a different pattern of activation of bcr signaling in mcl .
a recent report of a phase 1/2 clinical trial of fostamatinib disodium , the first clinically available oral syk inhibitor , in patients with recurrent b - cell non - hodgkin lymphoma , showed that only 1 in 9 mcl showed some response .
second , relapsed lymphomas might have evolved into bcr - independent clones ( such as the cell line rec-1 ) .
third , since our data support the hypothesis that the activation pattern of syk in mcl is different from b - cll and dlbcl , it is possible that this phenomenon influences the response to fostamatinib . since the proteins belonging to the bcr signaling pathway were shown to be active , we tested the effect of the blockade of this pathway on mcl cells . for this purpose , syk activity was inhibited by a widely used inhibitor , piceatannol [ 4245 ] , a natural stilbene also resulting from the hepatic metabolism of resveratrol , a compound found to be pro - apoptotic for the mcl cell line jeko-1 .
the rec-1 cell line showed a marked hypersensitivity to dmso , and it was deemed unfit for further experimental work , which was conducted on less sensitive cell lines .
piceatannol induced apoptosis in mcl cell lines jeko-1 , upn-1 , mino , maver-1 and granta-519 , as evidenced by annexin v staining , with a ld50 varying from 9.50 m to 28.91 m at 24 h and 5.51 m and 23.54 m at 48 h ( supplemental table 3 and fig .
upper panel shows the percentage of live cells ( y axis ) in function of the piceatannol concentration ( x axis ) at 24 h. lower panel shows the same variables at 48 h of treatment .
annexin v staining was used to discriminate apoptotic cells induction of apoptosis in mcl cell lines .
upper panel shows the percentage of live cells ( y axis ) in function of the piceatannol concentration ( x axis ) at 24 h. lower panel shows the same variables at 48 h of treatment .
annexin v staining was used to discriminate apoptotic cells interestingly , jeko-1 , which is very sensitive to piceatannol , also shows the highest number of phospho - peptides belonging to the bcr pathway ( supplemental table 4 ) .
apoptosis was dose and time dependent being visible after 4 h of treatment in jeko-1 , but only after 16 h in maver-1 ( data not shown ) .
cyclin d1 protein levels were massively down - regulated in treated cells , as densitometric analysis showed a drop by 85 to 90% compared to untreated cells ( supplemental figure 2 ) .
the corresponding ccnd1 mrna was down - regulated after piceatannol treatment , showing 22% to 71% decrease ( after normalization ) in five tested cell lines ( supplemental figure 3 ) .
this decrease was however significant only in three cell lines , namely upn-1 ( 54% decrease ; p = 0.047 ) , granta-519 ( 71% decrease ; p = 0.0078 ) and maver-1 ( 69% decrease ;
this finding is interesting , considering that the ccnd1 gene is translocated under the control of a strong enhancer such as the ig enhancer .
a hypothetical scenario could be hypothesized in which bcr signaling and ccnd1 gene transcription are connected , possibly via syk .
cyclin d1 has been shown to be a syk target gene in breast cancer and other cells .
it is possible that ccnd1 gene transcription is directly repressed by syk , in accordance to the fact that normal b cells show very low levels of this protein and at the same time nuclear localization of syk .
as previously stated , in support of this hypothesis syk acts as a transcriptional repressor of the ccnd1 gene in breast carcinoma cells , and this activity is necessary for its tumor - suppressor function .
one hypothesis is that in mcl cells syk might act via stat3 , a known ccnd1 gene transcription inducer , as suggested by a recent publication . in support of this hypothesis ,
the phosphorylation of stat3 was reduced by piceatannol in mcl cell lines ( supplemental figure 4 ) , while total stat3 was reduced in jeko-1 and upn-1 , but not significantly in maver-1 and granta-519 . according to these data , while the phosphorylation of stat3 was reduced in all cell lines , the total level is reduced only in sensitive ones .
several publications support a role for stat3 in the pathogenesis of mcl [ 4852 ] . according to our data
the down - regulation of ccnd1 mrna can not explain the whole picture , since it was significant only in three cell lines after normalization using an unrelated transcript .
an increase in cyclin d1 degradation is another possible explanation , and might operate alongside transcription repression . in support of this complementary hypothesis
is the reduction of gsk3 phosphorylation on negative regulatory serine residues , which increases its kinase activity with subsequent phosphorylation and degradation of cyclin d1 .
syk mrna was significantly down - regulated by piceatannol treatment in upn-1 and maver-1 ( 59% and 75% reduction ; p = 0.047 and p = 0.01 respectively ) .
it was also reduced in granta-519 , but the values did not reach statistical significance ( 49% reduction ; p = 0.11 ) .
syk mrna was increased after treatment in jeko-1 and mino ( by 7% and 40% respectively ) although values were not statistically significant ( p = 0.8 and p = 0.32 respectively ) .
modifications of mrna levels of syk and ccnd1 showed a good correlation with an r = 0.88 ( supplemental figure 3 ) .
apoptosis was triggered trough the intrinsic pathway , involving caspase 9 cleavage , as shown by western blotting experiments .
cleaved caspase 8 levels were actually lower in the treated samples than in the untreated ones ( not shown ) .
several apoptosis ( bcl-2 , bcl - xl , bax , p53 , p21 ) , cell - cycle related ( p53 , p21 , p27 ) molecules were investigated by western blotting .
all investigated proteins showed the same behavior in all cell lines with the exception of bax ( supplemental figure 5 ) . in detail , p53 levels were increased after treatment ( jeko-1 resulted completely p53-negative due to the presence of a truncating mutation , while other cell lines with the exception of granta-519 bear a missense mutation ) ; p21 was strongly down - regulated ; p27 was cleaved with a decrease of the 27 kda isoform and increase of the 23kda isoform ; bcl-2 was slightly increased , bcl - xl was decreased , while bax was increased in jeko-1 and decreased in other cell lines .
the mechanism inducing the increase of bax ( a pro - apoptotic bcl-2 family member ) in jeko-1 is not clear , but this phenomenon might indicate a more pronounced perturbation of mitochondrial outer membrane potential .
the only clear - cut genomic abnormality that might suggest a stronger syk - dependance of jeko-1 is the presence of the amplification of the syk locus , although this cell line also bears a peculiar missense mutation of tp53 ( in addition to a deletion of the other allele ) that causes a premature stop codon with degradation of its mrna , and at variance with other cell lines shows no p53 at all ; this condition might cause an abnormal regulation of bax , that is a known p53 target gene . to verify the effects of piceatannol on the pattern of phosphorylation of syk and other bcr pathway members , we resorted to flow cytometry using phospho - specific antibodies .
these experiments showed that piceatannol induced a marked decrease in phospho - syk tyrosine residues y525 and y323 , while unexpectedly the levels of y352 , which were almost negative in basal conditions , increased after treatment ( fig .
this residue resides in the linker region of syk , in a stretch that is essential for nuclear localization , since a mutant lacking residues 332359 was unable to enter the nucleus , as reported by a previous article .
these modifications were detectable after 6 h of treatment , but not after two ( data not shown ) .
as previously stated , b - cll and dlbcl cells show a basal activation of syk y352 , but no information is available for comparison about how this phosphorylation might be changed by syk inhibition .
fig .
after treatment , phosphorylation of residues y525 and y323 is reduced , while that of residue y352 is increased .
red , untreated cells ; blue , treated cells ; solid grey , isotypic control modification of syk phosphorylation profile following piceatannol treatment . after treatment , phosphorylation of residues y525 and y323 is reduced , while that of residue y352 is increased .
red , untreated cells ; blue , treated cells ; solid grey , isotypic control the levels of the downstream effectors phospho - blnk and phospho - btk were also reduced by piceatannol treatment in jeko-1 and to a lesser degree in maver-1 cells ( supplemental figure 6 ) . to obtain independent information about the modifications of phospho - syk after treatment , we used immunofluorescence microscopy .
these experiments showed that in basal conditions phospho - syk ( y525 ) is present in all cell lines tested , and is apparently confined to the cytoplasmic compartment of mcl cell lines ( with the exception of granta-519 , that showed also a partial nuclear localization ) .
after piceatannol treatment phospho - syk ( y525 ) was down - regulated in the cytoplasm , but its presence could be demonstrated in the nucleus of mcl cell lines ( jeko-1 , upn-1 , mino , granta-519 , maver-1 ) ( fig . 7 ) .
this finding is in accordance with the reduction of phospho - syk ( y525 ) detected by antibodies used in flow cytometry experiments , which were able to enter the cytoplasm but not the nucleus in our experimental conditions .
p - syk y525 ( shown in green pseudo - color ) disappears from the cytoplasm and appears in the nucleus after treatment in all cell lines .
nuclei counter stained in blue pseudo - color ( dapi staining ) localization of phospho - syk following piceatannol treatment .
p - syk y525 ( shown in green pseudo - color ) disappears from the cytoplasm and appears in the nucleus after treatment in all cell lines .
nuclei counter stained in blue pseudo - color ( dapi staining ) one possible hypothesis to explain this phenomenon is the fact that syk has two known splice variants , of which only the longest is able to enter the nucleus .
western blotting experiments in the five mcl cell lines jeko-1 , granta-519 , maver-1 , upn-1 and rec-1 showed that only one band was identifiable in the expected molecular weight range .
a proteolytic 36 kda isoform of syk was also detected ( data not shown ) , although the significance of this isoform described in erythrocytes , is still not clear in our cells , because it might be either a true functional variant or an experimental artifact .
the absence of the shortest splice variant of syk was also demonstrated by isoform - specific syk rt - pcr experiments ( supplemental figure 7 ) .
the nuclear exclusion phenomenon therefore is probably not related to syk differential splicing , but more probably to functional modifications of the protein , such as phosphorylation .
phosphoscan analysis identified 421 unique tyrosine - phosphorylated peptides , corresponding to 341 proteins , ranked by overall abundance in table 1 ( for a complete list of peptides and complete ms information see supplemental table 1 ) .
interestingly , several identified proteins mapped to cytogenetic loci that have been reported to be altered in mcl [ 2931 ] . among these
table 1top 50 phospho - peptides identified ( number of phospho peptides corresponding to the specific protein is reported . for complete list and exact phosphorylation sites ,
table 1)protein namegranta-519jeko-1maver-1rec-1bcr pathwaykynasecdc217381626*cdk210341418*cdk310331317*syk919243**ship7181010*pag91448*prp431982*lyn41097**ig - beta02220*hck3853**wasp2743*shp-14741*btk01141**cofilin 12751sgk2236144*lanp - l0653pkcd4324**gcet25710*hsp90b5502gsk3a2343**gsk3b2343**p38-alpha2361**hnrnp a15231grf-11424sptbn10541sptbn1 iso20541plcg22430*dyrk1a1431*lck4212**hsp90a2411blk2321**irap1430ddx34300pl104300cbl - b1311*bcap1410dok30600*anxa21221vim2220l - plastin0420eno10222pik3r11221**dyrk1b1221*src1212**fyn1212**yes1212*hnrnp 2h92310hnrnp u2310hnrnp u iso22310pai - rbp12220fig .
1list of protein kinases identified by the phosphoscan approach , ranked by overall abundance in the four mcl cell lines analyzed .
color intensity proportional to the number of peptides ( see figure for color corresponding to the average number of peptides per cell line ) top 50 phospho - peptides identified ( number of phospho peptides corresponding to the specific protein is reported . for complete list and exact phosphorylation sites ,
. table 1 ) list of protein kinases identified by the phosphoscan approach , ranked by overall abundance in the four mcl cell lines analyzed .
color intensity proportional to the number of peptides ( see figure for color corresponding to the average number of peptides per cell line ) the three most represented phospho - peptides are cyclin - dependent kinases cdc2 , cdk2 and cdk3 . cdc2 ( cdk1 ) is actually part of the proliferation signature able to predict prognosis in mcl and has also been validated at the immunohistochemical level .
cdk2 gains have been detected in mcl in a variable proportion of cases [ 30 , 31 , 33 ] , and have been associated with a poor prognosis .
cdk3 is less studied compared to other cyclin - dependent kinases , but its locus has also been reported as subject to gains [ 31 , 34 ] .
its involvement in mcl is a novel finding , although the role of cdk3 as an oncogene has been demonstrated in other models [ 35 , 36 ] .
many kinases belonging to this pathway ( e.g. syk , lyn , hck , btk , pkc - delta ) , were among the most represented phospho - peptides identified by the phosphoscan approach , and this was confirmed when looking for non - random enrichment of kyoto encyclopedia of genes and genomes ( kegg ) pathways compared to the hypothesis of random distribution .
this analysis showed that ( using a cutoff value of more than five total peptides ) , the most enriched pathway was hsa04662:b cell receptor signaling pathway ( 17.16-fold enrichment ) ( for complete data see supplemental table 2 ) .
many of the proteins belonging to the most enriched pathways were either present in different pathways ( redundant ) or are functionally connected .
manual annotation by literature search revealed that several of these , in the topmost part of the ranking , were connected to bcr signaling , even if not present in the canonical kegg bcr pathway ( table 1 and fig . 2 ) .
2simplified diagram showing some of the identified bcr pathway members . in red , activating members ; in green , inhibiting members ; for some members the precise final effect is not clear ; in grey , other proteins known to be present but not identified .
full arrows : direct interaction / activation ; dotted arrows : indirect interaction / activation .
data derived from kyoto encyclopedia of genes and genomes ( kegg ) pathway and from published literature .
kegg is a widely used annotated database of pathways , ligands and genes ( http://www.genome.jp/kegg/ ) simplified diagram showing some of the identified bcr pathway members . in red ,
activating members ; in green , inhibiting members ; for some members the precise final effect is not clear ; in grey , other proteins known to be present but not identified .
full arrows : direct interaction / activation ; dotted arrows : indirect interaction / activation .
data derived from kyoto encyclopedia of genes and genomes ( kegg ) pathway and from published literature .
kegg is a widely used annotated database of pathways , ligands and genes ( http://www.genome.jp/kegg/ )
to further verify the functional role of bcr signaling in mcl , we analyzed the phosphorylation status of syk , lyn , btk , blnk and gsk3alfa / beta in mcl cell lines .
although not present in the top 50 phospho - peptides , blnk was investigated because it is considered a classical downstream molecule of the bcr pathway .
the presence of basal levels of phospho - syk y525 and y323 , as well as of phospho - blnk ( y84 ) was verified by flow cytometry ( fig .
in addition basal levels of phospho - lyn ( y396 and y507 ) and also of downstream effectors phospho - btk ( s180 ) and phospho - gsk3alfa / beta ( s9/21 ) were demonstrated by flow cytometry ( supplemental figure 1 ) .
bcr pathway activation in cell lines is somehow intriguing since it is present in absence of an appropriate antigen stimulation , and is therefore probably self - sustained by tumor cells , either by side - by - side activation or by auto - activation .
3analysis of basal levels of phosphorylated syk and blnk residues by flow cytometry . in grey , isotypic control ;
in red , basal levels . on the x axis , arbitrary fluorescence units ( log scale ) ; on the y axis , cell count analysis of basal levels of phosphorylated syk and blnk residues by flow cytometry . in grey , isotypic control ; in red , basal levels . on the x axis , arbitrary fluorescence units ( log scale ) ; on the y axis , cell count in order to verify whether we could find this activation in mcl tumors as well , we resorted to western blotting analysis of phosphorylated forms of bcr pathway members .
this analysis showed that the activated forms of syk ( in 5/6 cases , 83% ) , lyn ( in 6/6 cases , 100% ) , and blnk ( in 6/6 cases , 100% ) were present also in mcl tumor tissues ( fig .
4 ) , therefore supporting the in vivo role of active bcr signaling ; as far as we know , this is the first report of the presence of active ( phosphorylated ) bcr pathway members in mcl tissues .
the presence of phospho - syk ( y525 ) , phospho - lyn ( y396 ) and phospho - blnk ( y84 ) is shown in six mcl tumor tissues .
cases 1 , 2 , 5 and 6 were classical mcl , while cases 3 and 4 were blastoid variants western blotting analysis of mcl tissues .
the presence of phospho - syk ( y525 ) , phospho - lyn ( y396 ) and phospho - blnk ( y84 ) is shown in six mcl tumor tissues .
cases 1 , 2 , 5 and 6 were classical mcl , while cases 3 and 4 were blastoid variants the activation of the bcr pathway in mcl has been hypothesized in a previous paper based on cytogenetic and rna studies , but to our knowledge this is the first protein - based and data - driven study that supports this hypothesis .
another proteomic study focusing only on the plasma membrane showed an abnormal association of pkcbeta to the cell membrane in mcl leukemic cells , indirectly supporting an active bcr signaling .
recent studies have shown the importance of tonic bcr signaling in dlbcl [ 38 , 39 ] and b - cll , with a basal activation of phospho - syk residue y352 , while y525 was detected only after bcr cross - linking . the presence of significant basal levels of phospho - syk y525 and y323 , with no detectable phospho - syk y352 in basal conditions in mcl cells are not concordant with what has been reported in b - cll and dlbcl , and suggest a different pattern of activation of bcr signaling in mcl
. a recent report of a phase 1/2 clinical trial of fostamatinib disodium , the first clinically available oral syk inhibitor , in patients with recurrent b - cell non - hodgkin lymphoma , showed that only 1 in 9 mcl showed some response .
second , relapsed lymphomas might have evolved into bcr - independent clones ( such as the cell line rec-1 ) .
third , since our data support the hypothesis that the activation pattern of syk in mcl is different from b - cll and dlbcl , it is possible that this phenomenon influences the response to fostamatinib .
since the proteins belonging to the bcr signaling pathway were shown to be active , we tested the effect of the blockade of this pathway on mcl cells . for this purpose ,
syk activity was inhibited by a widely used inhibitor , piceatannol [ 4245 ] , a natural stilbene also resulting from the hepatic metabolism of resveratrol , a compound found to be pro - apoptotic for the mcl cell line jeko-1 .
the rec-1 cell line showed a marked hypersensitivity to dmso , and it was deemed unfit for further experimental work , which was conducted on less sensitive cell lines .
piceatannol induced apoptosis in mcl cell lines jeko-1 , upn-1 , mino , maver-1 and granta-519 , as evidenced by annexin v staining , with a ld50 varying from 9.50 m to 28.91 m at 24 h and 5.51 m and 23.54 m at 48 h ( supplemental table 3 and fig .
upper panel shows the percentage of live cells ( y axis ) in function of the piceatannol concentration ( x axis ) at 24 h. lower panel shows the same variables at 48 h of treatment .
annexin v staining was used to discriminate apoptotic cells induction of apoptosis in mcl cell lines .
upper panel shows the percentage of live cells ( y axis ) in function of the piceatannol concentration ( x axis ) at 24 h. lower panel shows the same variables at 48 h of treatment .
annexin v staining was used to discriminate apoptotic cells interestingly , jeko-1 , which is very sensitive to piceatannol , also shows the highest number of phospho - peptides belonging to the bcr pathway ( supplemental table 4 ) .
apoptosis was dose and time dependent being visible after 4 h of treatment in jeko-1 , but only after 16 h in maver-1 ( data not shown ) .
cyclin d1 protein levels were massively down - regulated in treated cells , as densitometric analysis showed a drop by 85 to 90% compared to untreated cells ( supplemental figure 2 ) .
the corresponding ccnd1 mrna was down - regulated after piceatannol treatment , showing 22% to 71% decrease ( after normalization ) in five tested cell lines ( supplemental figure 3 ) .
this decrease was however significant only in three cell lines , namely upn-1 ( 54% decrease ; p = 0.047 ) , granta-519 ( 71% decrease ; p = 0.0078 ) and maver-1 ( 69% decrease ; p = 0.008 ) .
this finding is interesting , considering that the ccnd1 gene is translocated under the control of a strong enhancer such as the ig enhancer .
a hypothetical scenario could be hypothesized in which bcr signaling and ccnd1 gene transcription are connected , possibly via syk .
cyclin d1 has been shown to be a syk target gene in breast cancer and other cells .
it is possible that ccnd1 gene transcription is directly repressed by syk , in accordance to the fact that normal b cells show very low levels of this protein and at the same time nuclear localization of syk .
as previously stated , in support of this hypothesis syk acts as a transcriptional repressor of the ccnd1 gene in breast carcinoma cells , and this activity is necessary for its tumor - suppressor function .
one hypothesis is that in mcl cells syk might act via stat3 , a known ccnd1 gene transcription inducer , as suggested by a recent publication . in support of this hypothesis ,
the phosphorylation of stat3 was reduced by piceatannol in mcl cell lines ( supplemental figure 4 ) , while total stat3 was reduced in jeko-1 and upn-1 , but not significantly in maver-1 and granta-519 . according to these data , while the phosphorylation of stat3 was reduced in all cell lines , the total level is reduced only in sensitive ones .
several publications support a role for stat3 in the pathogenesis of mcl [ 4852 ] .
according to our data the down - regulation of ccnd1 mrna can not explain the whole picture , since it was significant only in three cell lines after normalization using an unrelated transcript .
an increase in cyclin d1 degradation is another possible explanation , and might operate alongside transcription repression . in support of this complementary hypothesis
is the reduction of gsk3 phosphorylation on negative regulatory serine residues , which increases its kinase activity with subsequent phosphorylation and degradation of cyclin d1 .
syk mrna was significantly down - regulated by piceatannol treatment in upn-1 and maver-1 ( 59% and 75% reduction ; p = 0.047 and p = 0.01 respectively ) .
it was also reduced in granta-519 , but the values did not reach statistical significance ( 49% reduction ; p = 0.11 ) .
syk mrna was increased after treatment in jeko-1 and mino ( by 7% and 40% respectively ) although values were not statistically significant ( p = 0.8 and p = 0.32 respectively ) . modifications of mrna levels of syk and ccnd1 showed a good correlation with an r = 0.88 ( supplemental figure 3 ) .
apoptosis was triggered trough the intrinsic pathway , involving caspase 9 cleavage , as shown by western blotting experiments .
cleaved caspase 8 levels were actually lower in the treated samples than in the untreated ones ( not shown ) .
several apoptosis ( bcl-2 , bcl - xl , bax , p53 , p21 ) , cell - cycle related ( p53 , p21 , p27 ) molecules were investigated by western blotting .
all investigated proteins showed the same behavior in all cell lines with the exception of bax ( supplemental figure 5 ) . in detail
, p53 levels were increased after treatment ( jeko-1 resulted completely p53-negative due to the presence of a truncating mutation , while other cell lines with the exception of granta-519 bear a missense mutation ) ; p21 was strongly down - regulated ; p27 was cleaved with a decrease of the 27 kda isoform and increase of the 23kda isoform ; bcl-2 was slightly increased , bcl - xl was decreased , while bax was increased in jeko-1 and decreased in other cell lines .
the mechanism inducing the increase of bax ( a pro - apoptotic bcl-2 family member ) in jeko-1 is not clear , but this phenomenon might indicate a more pronounced perturbation of mitochondrial outer membrane potential . the only clear - cut genomic abnormality that might suggest a stronger syk - dependance of jeko-1 is the presence of the amplification of the syk locus , although this cell line also bears a peculiar missense mutation of tp53 ( in addition to a deletion of the other allele ) that causes a premature stop codon with degradation of its mrna , and at variance with other cell lines shows no p53 at all ; this condition might cause an abnormal regulation of bax , that is a known p53 target gene .
to verify the effects of piceatannol on the pattern of phosphorylation of syk and other bcr pathway members , we resorted to flow cytometry using phospho - specific antibodies .
these experiments showed that piceatannol induced a marked decrease in phospho - syk tyrosine residues y525 and y323 , while unexpectedly the levels of y352 , which were almost negative in basal conditions , increased after treatment ( fig .
this residue resides in the linker region of syk , in a stretch that is essential for nuclear localization , since a mutant lacking residues 332359 was unable to enter the nucleus , as reported by a previous article .
these modifications were detectable after 6 h of treatment , but not after two ( data not shown ) .
as previously stated , b - cll and dlbcl cells show a basal activation of syk y352 , but no information is available for comparison about how this phosphorylation might be changed by syk inhibition .
fig .
after treatment , phosphorylation of residues y525 and y323 is reduced , while that of residue y352 is increased .
red , untreated cells ; blue , treated cells ; solid grey , isotypic control modification of syk phosphorylation profile following piceatannol treatment . after treatment , phosphorylation of residues y525 and y323
red , untreated cells ; blue , treated cells ; solid grey , isotypic control the levels of the downstream effectors phospho - blnk and phospho - btk were also reduced by piceatannol treatment in jeko-1 and to a lesser degree in maver-1 cells ( supplemental figure 6 ) . to obtain independent information about the modifications of phospho - syk after treatment , we used immunofluorescence microscopy .
these experiments showed that in basal conditions phospho - syk ( y525 ) is present in all cell lines tested , and is apparently confined to the cytoplasmic compartment of mcl cell lines ( with the exception of granta-519 , that showed also a partial nuclear localization ) .
after piceatannol treatment phospho - syk ( y525 ) was down - regulated in the cytoplasm , but its presence could be demonstrated in the nucleus of mcl cell lines ( jeko-1 , upn-1 , mino , granta-519 , maver-1 ) ( fig . 7 ) .
this finding is in accordance with the reduction of phospho - syk ( y525 ) detected by antibodies used in flow cytometry experiments , which were able to enter the cytoplasm but not the nucleus in our experimental conditions .
p - syk y525 ( shown in green pseudo - color ) disappears from the cytoplasm and appears in the nucleus after treatment in all cell lines .
nuclei counter stained in blue pseudo - color ( dapi staining ) localization of phospho - syk following piceatannol treatment .
p - syk y525 ( shown in green pseudo - color ) disappears from the cytoplasm and appears in the nucleus after treatment in all cell lines .
nuclei counter stained in blue pseudo - color ( dapi staining ) one possible hypothesis to explain this phenomenon is the fact that syk has two known splice variants , of which only the longest is able to enter the nucleus .
western blotting experiments in the five mcl cell lines jeko-1 , granta-519 , maver-1 , upn-1 and rec-1 showed that only one band was identifiable in the expected molecular weight range . a proteolytic 36 kda isoform of syk was also detected ( data not shown ) , although the significance of this isoform described in erythrocytes , is still not clear in our cells , because it might be either a true functional variant or an experimental artifact .
the absence of the shortest splice variant of syk was also demonstrated by isoform - specific syk rt - pcr experiments ( supplemental figure 7 ) .
the nuclear exclusion phenomenon therefore is probably not related to syk differential splicing , but more probably to functional modifications of the protein , such as phosphorylation .
our study has identified a large number of phosphorylated proteins and several activated pathways in mcl cells , which deserve a more thorough investigation by functional validation experiments .
our experimental data suggest that active bcr signaling is present in mcl cell lines and tumors , and that it plays a role in the survival of mcl cells ; syk is possibly one of the key molecules in this event , and it might have a double - face role in the cytoplasmic and nuclear compartments .
our data also suggest that piceatannol , resveratrol or their analogues could represent a therapeutic option for patients with mcl if in vitro data will be confirmed in vivo .
cell lines mcl cell lines jeko-1 , granta-519 and rec-1 were purchased from dsmz ( braunschweig , germany ) .
elias campo ( barcelona , spain ) . due to over - sensitivity to dmso - induced apoptosis
tumor samples tumor samples were collected from the archives of the department of pathology and diagnostics of the university of verona .
informed consent had been collected for all patients and procedures were carried out according to the ethical guidelines of the university hospital of verona with the approval of an ethics committee , in compliance with the helsinky declaration .
phosphoscan analysis phosphoscan analysis was performed as previously described on mcl cell lines maver-1 , granta-519 , jeko-1 , and rec-1.the phospho - proteins identified using this analysis were categorized into kegg pathways using the david ease web framework . for p - value and fold enrichment calculation the complete list of human genes was considered as a background .
the calculation was repeated for lists obtained by selecting genes above the abundance cut - offs of 3 , 4 and 5.illustrations were produced using the r statistical software package ( http://www.r-project.org ) .
flow cytometry flow cytometric analysis of several tyrosine - phosphorylated forms of syk ( y323 , y352 and y525 ) and downstream target phospho - blnk was performed as previously described using antibodies shown in table 2 .
table 2antibodies used in the studyprimary antibodyclonesourcedilutionusebaxpolyclonalcell signaling1:1000western blottingbcl - xl54h6cell signaling1:1000western blottingbcl-2124dakocytomation1:4000western blottingcaspase 9polyclonalcell signaling1:1000western blottingcyclin d1sp4labvision1:1000western blottingp21sx118dakocytomation1:200western blottingp27sx53g8dakocytomation1:200western blottingp53do-7novocastra1:500western blottingsykpolyclonalcell signaling1:1000western blottingp - syk ( y525/526)c87c1cell signaling1:200western blottingstat3124h6cell signaling1:4000western blottingp - stat3(y705)d3a7cell signaling1:500western blottingpe - p - syk ( y352)17a / p - zap70bd biosciences1:100flow cytometryp - syk ( y525/526)polyclonalcell signaling1:100flow cytometry / immunofluorescencep - syk ( y323)ep573yabcam1:100flow cytometryp - blnk ( y84)j117 - 1278bd biosciences1:100/1:1000flow cytometry / western blottingp - btk ( s180)3d3cell signaling1:100flow cytometryp - lyn ( y507)polyclonalcell signaling1:100flow cytometryp - lyn ( y396)ep503yabcam1:100/1:200flow cytometry / western blottingp - gsk3/ ( s9/21)37f11cell signaling1:100flow cytometry antibodies used in the study rna isolation and cdna synthesis rna isolation was performed using the allprep dna / rna / protein mini kit ( qiagen gmbh , hilden , germany ) , and rna quality was assessed by using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca , usa ) and rna 6000 nano chips ( agilent technologies ) . each cdna was synthesized from 1 g total rna using random primers and the superscript iii first - strand synthesis system ( invitrogen , carlsbad , ca , usa ) according to the manufacturer s instructions .
reaction products were analyzed by the agilent 2100 bioanalyzer and the dna1000 chip ( agilent technologies ) .
quantitative rt - pcr quantitative rt - pcr mrna expression analysis was performed on abi prism 7900ht fast real - time pcr system ( applied biosystems , foster city , ca ) using power sybr green pcr master mix ( applied biosystems ) .
oligonucleotide primers used were : ccnd1-f aactacctggaccgcttcct , and ccnd1-r ggggatggtctccttcatct ; tbp - f , gcacaggagccaagagtgaa , and tbp - r , tcacagctccccaccatatt.tata box binding protein ( refseq i d nm_003194.3 ) transcript level was used to normalize syk expression.calibration curves for each couple of primers were obtained by serial dilution of cdna .
expression data were analyzed by the comparative threshold cycle ( ct ) method accordingly to user bulletin no .
the statistical significance of the data was investigated by student s t - test .
syk inhibition experiments piceatannol ( sigma - aldrich chemie gmbh , steinheim , germany ) was resuspendend in dmso as a 100 mm stock .
cells were diluted at 500 * 10/ml in rpmi 1640 ( 1% fetal calf serum ) and let grow for 16 h. piceatannol was then added at concentrations between 1 m and 80 m ( depending on the sensitivity of cell lines ) .
cells were harvested at 24 and 48 h and apoptosis levels analyzed by annexin v ( annexin v fitc apoptosis detection kit i , bd ) as previously described .
western blotting immunoblotting was performed by standard methodology , using the antibodies and dilutions indicated table 2 .
protein content of samples was measured using a colorimetric method ( dc protein assay , bio - rad ) .
all lanes were loaded with the same amount of total protein ( in duplicate or triplicate ) , and were also visually verified by ponceau red staining after electroblotting .
immunofluorescence immunofluorescence was performed using the antibodies shown in table 2 ( both primary and secondary antibodies were diluted 1:100 ) .
briefly , cells were washed in pbs , deposited on charged slides by gravity , fixed in cold methanol ( 20c ) for 20 min and then air - dried .
cells were then re - hydrated , incubated with protein blocking solution for 10 , then incubated serially with primary and secondary antibodies ( with three washes in pbs in - between ) .
images were acquired using appropriate filters via a monochromatic camera cooled ccd camera ( iai , japan ) and analyzed using the olympus cytovision software .
basal levels of phospho - lyn ( y396 and y507 ) , phospho - btk ( s180 ) and phospho - gsk3alfa / beta ( s9/21 ) as analyzed by flow cytometry in mcl cells . in grey , isotypic control ; in red , basal levels . on the x axis , arbitrary fluorescence units ( log scale ) ; on the y axis , cell count .
( jpeg 423 kb ) western blotting analysis of cyclin d1 protein levels in piceatannol - treated cells .
( jpeg 96 kb ) modifications of ccnd1 and syk mrna after piceatannol treatment ( normalized ) .
a decrease of one ct unit corresponds to a 50% reduction of rna levels . a correlation plot between modifications of mrna levels is also shown .
( jpeg 536 kb ) western blotting analysis of modifications of levels of phospho - stat3 ( y705 ) and stat3 after piceatannol treatment in cell lines jeko-1 , upn-1 , maver-1 and granta-519 .
( jpeg 571 kb ) western blotting analysis of apoptosis - related and cell cycle - related molecules after exposure to piceatannol by western blotting .
upn-1 was totally bcl2-negative , while jeko-1 showed no p53 protein due to a truncating mutation .
( jpeg 963 kb ) levels of phospho - blnk and phospho - btk after piceatannol treatment in cell lines jeko-1 and maver-1 analyzed by flow cytometry . in grey , isotypic control ; in red , basal levels , in blue , treated cells . on the x axis ,
arbitrary fluorescence units ( log scale ) ; on the y axis , cell count .
( jpeg 354 kb ) isoform - specific syk rt - pcr experiments . pseudo - gel image ( upper panel ) and two representative electropherograms ( lower panel ) of rt - pcr products are shown ( images produced by microfluidic analysis ) .
( xls 487 kb ) list of kyoto encyclopedia of genes and genomes ( kegg ) pathways enriched in mcl cell lines compared to random distribution .
( xls 28 kb ) ld50 of piceatannol at 24 and 48 h for mcl cell lines .
( xls 6 kb ) number of identified phospho - peptides corresponding to members of the kegg bcr pathway in the four tested mcl cell lines .. ( xls 10 kb ) | backgroundmantle cell lymphoma ( mcl ) is currently an incurable entity , and new therapeutic approaches are needed .
we have applied a high - throughput phospho - proteomic technique to mcl cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues.methodsphosphoscan analysis was performed on mcl cell lines .
results were validated by flow cytometry and western blotting .
functional validation was performed by blocking the most active pathway in mcl cell lines.resultsphosphoscan identified more than 300 tyrosine - phosporylated proteins , among which many protein kinases .
the most abundant peptides belonged to proteins connected with b - cell receptor ( bcr ) signaling .
active bcr signaling was demonstrated by flow cytometry in mcl cells and by western blotting in mcl tumor tissues .
blocking bcr signaling by syk inhibitor piceatannol induced dose / time - dependent apoptosis in mcl cell lines , as well as several modifications in the phosphorylation status of bcr pathway members and a collapse of cyclin d1 protein levels.conclusionour data support a pro - survival role of bcr signaling in mcl and suggest that this pathway might be a candidate for therapy .
our findings also suggest that syk activation patterns might be different in mcl compared to other lymphoma subtypes.electronic supplementary materialthe online version of this article ( doi:10.1007/s13402 - 011 - 0019 - 7 ) contains supplementary material , which is available to authorized users . | Electronic supplementary material
Introduction
Results and discussion
PhosphoScan analysis identifies the tyrosine-activated phosphoproteome of MCL cell lines and identifies B-cell receptor signaling as the most active pathway
The presence of active B-cell receptor signaling is validated in MCL cell lines and tissues
Inhibition of Syk induces apoptosis in MCL cell lines
Inhibition of Syk down-regulates Cyclin D1 protein levels
Piceatannol-induced apoptosis involves the activation of the intrinsic pathway, and is accompanied by the modification of several regulators of cell cycle and apoptosis
Piceatannol induces complex modifications of the Syk phosphorylation pattern and alterations of downstream effectors of the B-cell receptor pathway
Conclusions
Methods
Electronic supplementary material | these studies have identified single up - regulated proteins in mcl compared to tonsil b - cells , or several proteins up - regulated in mcl cell lines compared to fl - derived cell lines followed by network analysis . compared to imac
this approach has already been successfully used in the phospho - profiling of primary and metastatic lung cancer , in acute leukemia samples , in bcr / abl positive cell lines , aml cell lines and hodgkin lymphoma cell lines . in the current work
, we used this approach to recognize the most represented activated pathways in mcl cell lines . we then verified the presence of these activated molecules in mcl cell lines and tumor tissues . kegg is a widely used annotated database of pathways , ligands and genes ( http://www.genome.jp/kegg/ ) to further verify the functional role of bcr signaling in mcl , we analyzed the phosphorylation status of syk , lyn , btk , blnk and gsk3alfa / beta in mcl cell lines . the presence of basal levels of phospho - syk y525 and y323 , as well as of phospho - blnk ( y84 ) was verified by flow cytometry ( fig . on the x axis ,
arbitrary fluorescence units ( log scale ) ; on the y axis , cell count in order to verify whether we could find this activation in mcl tumors as well , we resorted to western blotting analysis of phosphorylated forms of bcr pathway members . 4 ) , therefore supporting the in vivo role of active bcr signaling ; as far as we know , this is the first report of the presence of active ( phosphorylated ) bcr pathway members in mcl tissues . the presence of significant basal levels of phospho - syk y525 and y323 , with no detectable phospho - syk y352 in basal conditions in mcl cells are not concordant with what has been reported in b - cll and dlbcl , and suggest a different pattern of activation of bcr signaling in mcl . piceatannol induced apoptosis in mcl cell lines jeko-1 , upn-1 , mino , maver-1 and granta-519 , as evidenced by annexin v staining , with a ld50 varying from 9.50 m to 28.91 m at 24 h and 5.51 m and 23.54 m at 48 h ( supplemental table 3 and fig . kegg is a widely used annotated database of pathways , ligands and genes ( http://www.genome.jp/kegg/ )
to further verify the functional role of bcr signaling in mcl , we analyzed the phosphorylation status of syk , lyn , btk , blnk and gsk3alfa / beta in mcl cell lines . on the x axis , arbitrary fluorescence units ( log scale ) ; on the y axis , cell count in order to verify whether we could find this activation in mcl tumors as well , we resorted to western blotting analysis of phosphorylated forms of bcr pathway members . 4 ) , therefore supporting the in vivo role of active bcr signaling ; as far as we know , this is the first report of the presence of active ( phosphorylated ) bcr pathway members in mcl tissues . the presence of significant basal levels of phospho - syk y525 and y323 , with no detectable phospho - syk y352 in basal conditions in mcl cells are not concordant with what has been reported in b - cll and dlbcl , and suggest a different pattern of activation of bcr signaling in mcl
. piceatannol induced apoptosis in mcl cell lines jeko-1 , upn-1 , mino , maver-1 and granta-519 , as evidenced by annexin v staining , with a ld50 varying from 9.50 m to 28.91 m at 24 h and 5.51 m and 23.54 m at 48 h ( supplemental table 3 and fig . our experimental data suggest that active bcr signaling is present in mcl cell lines and tumors , and that it plays a role in the survival of mcl cells ; syk is possibly one of the key molecules in this event , and it might have a double - face role in the cytoplasmic and nuclear compartments . phosphoscan analysis phosphoscan analysis was performed as previously described on mcl cell lines maver-1 , granta-519 , jeko-1 , and rec-1.the phospho - proteins identified using this analysis were categorized into kegg pathways using the david ease web framework . basal levels of phospho - lyn ( y396 and y507 ) , phospho - btk ( s180 ) and phospho - gsk3alfa / beta ( s9/21 ) as analyzed by flow cytometry in mcl cells . | [
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] |
perinatal asphyxia is a significant cause of brain damage in the human newborns and can result in long - term neurodevelopmental disability .
neonatal seizures are important risk factors for impaired neurodevelopment in neonatal hypoxic - ischemic encephalopathy ( hie ) .
most studies of neonatal seizures depend on clinically diagnosed seizures . however , increased use of continuous electroencephalographic ( eeg ) monitoring has clarified the fact that seizures in newborns are often subclinical and that neonatologists have difficulties in distinguishing seizures from nonseizure spells .
there had been conflicts as to the importance of seizures that are electrographic without clinical manifestations as compared with those that manifest obvious clinical signs .
previous studies found a clear relationship between seizure load and injury on magnetic resonance imaging ( mri ) .
however , little is known regarding frequency of seizure , its distribution , its time of onset and seizure expression , and their relationship to mri brain injury during therapeutic hypothermia ( th ) .
our aim was to determine the value of video - eeg monitoring in newborns with hie treated by hypothermia through determination of eeg background , prevalence of seizures during th , determination of eeg patterns that can predict mri brain injury and value of detection , and treatment of subclinical seizures .
this work was conducted on 39 full - term newborns with hie admitted to the neonatal intensive care unit ( nicu ) of al hada and taif military hospitals , saudi arabia .
their gestational ages ranged from 37 to 40 weeks with a mean of 38 1.8 .
their weights ranged from 3.1 to 3.8 kg with a mean of 3.32 0.56 , 46% were males and 54% were females .
all cases were subjected to th with whole - body cooling according to standard protocols .
inclusion criteria for th included :
gestational age at birth 36 weeks.any of the following :
ph < 7.0 of cord or first blood gasbase deficit > 12 of cord or first blood gas10-min agar score < 5 .
gestational age at birth 36 weeks . any of the following :
ph < 7.0 of cord or first blood gasbase deficit > 12 of cord or first blood gas10-min agar score < 5 .
ph < 7.0 of cord or first blood gas base deficit > 12 of cord or first blood gas 10-min agar score < 5 .
exclusion criteria included premature babies known or suspected cases of metabolic disorders and congenital anomalies .
th was initiated as early as possible after birth with whole - body cooling ( target temperature 33.5c ) for 72 h followed by rewarming over 6 h. antiepileptic drugs were used to control seizures according to guidelines .
sedation was done for all patients using continuous infusion of morphine in a dose of ( 1025 g / kg / h ) all over cooling to prevent any abnormal movement .
newborns with seizure were subdivided into two groups : group 1 included newborns with a treatment of both types of ( clinical and subclinical ) seizures ( 12 cases ) .
group 2 included newborns with the treatment of clinical seizures only ( 7 cases ) .
a nicoletone eeg monitor was used to record continuous video - eeg recordings for 72 h. it started early after birth .
eeg background at the onset of the recording was classified into one of five patterns : ( 1 ) transient discontinuity for less than one - half of the recording is considered normal for gestational age .
( 2 ) excessively discontinuous , with an existence of discontinuity for more than one - half of the recording . ( 3 ) depressed and undifferentiated , there is an absence of faster frequencies with persistence of a low background activity .
there is also often a suppression of amplitude ( 515 v ) , and the tracing may be more discontinuous that would be expected for gestational age .
( 4 ) burst suppression ( bs ) pattern is characterized by periods of excessively suppressed background ( 5 v ) interrupted by bursts of abnormal activity .
( 5 ) extremely low voltage , with amplitude < 5 v or with no perceptible cerebral activity .
a repetitive rhythmic activity of > 10 s duration identifies an electrographic seizure , with a sharp beginning , middle , and end with clear evolution .
these features are important in differentiating of ictal rhythmic discharges from artifacts [ figure 2 ] .
when electrographic seizure activity was continued for at least half an hour or recurs for at least one - half of 13 h recording time , status epilepticus ( se ) was identified .
burst suppression pattern in severe birth asphyxia showing severely suppressed background activity with intermittent bursts of normal activity seizure pattern showing a repetitive , rhythmic , and stereotyped activity of > 10 s duration with a sharp beginning , middle , and end with clear evolution neonates were imaged with conventional t1-weighted , t2-weighted , and diffusion - weighted imaging sequences .
basal ganglia / thalamus score < 2 and watershed score <3 identify normal to mild mri injury .
basal ganglia / thalamus score 2 or watershed pattern 3 identifies moderate to severe mri injury .
data were analyzed with spss , and variables were analyzed with t - test and chi - square .
specificity and sensitivity were used to assess the prognostic value of eeg background patterns at different intervals .
linear regression analysis was used to test the relationship between the duration of seizure patterns and mri scores .
a nicoletone eeg monitor was used to record continuous video - eeg recordings for 72 h. it started early after birth .
eeg background at the onset of the recording was classified into one of five patterns : ( 1 ) transient discontinuity for less than one - half of the recording is considered normal for gestational age .
( 2 ) excessively discontinuous , with an existence of discontinuity for more than one - half of the recording . ( 3 ) depressed and undifferentiated , there is an absence of faster frequencies with persistence of a low background activity .
there is also often a suppression of amplitude ( 515 v ) , and the tracing may be more discontinuous that would be expected for gestational age .
( 4 ) burst suppression ( bs ) pattern is characterized by periods of excessively suppressed background ( 5 v ) interrupted by bursts of abnormal activity .
( 5 ) extremely low voltage , with amplitude < 5 v or with no perceptible cerebral activity .
a repetitive rhythmic activity of > 10 s duration identifies an electrographic seizure , with a sharp beginning , middle , and end with clear evolution .
these features are important in differentiating of ictal rhythmic discharges from artifacts [ figure 2 ] .
when electrographic seizure activity was continued for at least half an hour or recurs for at least one - half of 13 h recording time , status epilepticus ( se ) was identified .
burst suppression pattern in severe birth asphyxia showing severely suppressed background activity with intermittent bursts of normal activity seizure pattern showing a repetitive , rhythmic , and stereotyped activity of > 10 s duration with a sharp beginning , middle , and end with clear evolution
neonates were imaged with conventional t1-weighted , t2-weighted , and diffusion - weighted imaging sequences .
basal ganglia / thalamus score < 2 and watershed score <3 identify normal to mild mri injury .
basal ganglia / thalamus score 2 or watershed pattern 3 identifies moderate to severe mri injury .
data were analyzed with spss , and variables were analyzed with t - test and chi - square .
linear regression analysis was used to test the relationship between the duration of seizure patterns and mri scores .
all clinical characteristics were not significantly associated with brain injury in mri [ table 1 ] .
clinical characteristics of newborns in relation to brain injury at the beginning of cooling , no one of newborns whom background was normal had moderate to severe injury . in 12 newborns whom background pattern was excessively discontinuous , 9 ( 75% ) had normal or mild injury and 3 ( 25% ) had moderate to severe injury .
bs or extremely low - voltage pattern was found in 16 newborns , 10 ( 62.5% ) had moderate to severe injury and 6 ( 37.5% ) newborns had a normal mri or only a mild injury .
eeg improved in all six newborns with bs or extremely low - voltage pattern by 1218 h of recording and background normalized by the middle of cooling [ table 2 ] .
electroencephalographic background during cooling in relation to magnetic resonance imaging brain injury during this interval , one newborn whom eeg improved from a discontinuous pattern to a normal one at the beginning of cooling had moderate to severe mri injury . during the middle of cooling , no one of the remaining newborns with a normal eeg had moderate to severe injury . among the 11 infants with a discontinuous eeg pattern , the background had moderate to severe injury in ( 2/11 , 18% ) .
all newborns ( 7/7 , 100% ) with bs or extremely low - voltage patterns had moderate to severe injury [ table 2 ] . among the 11 infants whom background was normal
furthermore , among the 15 infants with an excessively discontinuous pattern , 2 ( 13% ) of them showed moderate to severe mri injury .
compared to all five infants ( 100% ) whom background had bs or extremely low voltage [ table 2 ] .
the same two newborns whom background was normal ( 13% ) at this time point showed moderate to severe injury .
after rewarming , all six infants with bs or extremely low - voltage patterns had moderate to severe injury [ table 2 ] .
seizures recurred during the middle of cooling in two infants and during rewarming in another two . among the 12 infants with electrographic seizures , 5 infants ( 42% )
did not show a clinical correlate ( subclinical seizure ) . among the five infants with subclinical seizure , three of them showed subclinical se .
all three infants with se showed bs or extremely low - voltage patterns with the start of cooling .
patients with moderate to severe mri injury had isolated or recurrent seizures more frequently than those with no or mild injury ( 47% vs. 21% , p = 0.05 ) , and the se was seen only in infants with moderate to severe injury ( p = 0.01 ) [ table 3 ] .
background of electroencephalographic , seizures , status epilepticus , and outcome of magnetic resonance imaging brain outcome a normal eeg background at the start of cooling was more predictive of a favorable mri outcome than at later time points with a specificity of 100% and 80% , respectively .
bs pattern or extremely low - voltage background is of a prognostic value for moderate to severe injury that increased from the start of cooling ( 75% specificity ) to the middle of cooling and later ( 100% specificity ) [ table 4 ] .
electroencephalographic background sensitivity and specificity during and after cooling for moderate to severe magnetic resonance imaging brain injury there were no differences between groups regarding clinical characteristics .
the duration ( median standard deviation ) of seizure patterns was 42 83 min in group 1 , compared with 86 134 min in group 2 [ figure 3 ] .
no significant difference in duration was found between the groups using linear regression . there was a significant relationship between the duration of seizure patterns and mri scores in linear regression analysis in group 1 [ figure 4 ] .
duration of seizure patterns for the clinical and subclinical seizure treatment group 1 and the clinical seizure treatment group 2 .
the vertical lines indicate the limit lines : ranges relationship between duration of seizure patterns and magnetic resonance imaging scores ( linear regression ) in group 1
at the beginning of cooling , no one of newborns whom background was normal had moderate to severe injury . in 12 newborns whom background pattern was excessively discontinuous , 9 ( 75% ) had normal or mild injury and 3 ( 25% ) had moderate to severe injury .
bs or extremely low - voltage pattern was found in 16 newborns , 10 ( 62.5% ) had moderate to severe injury and 6 ( 37.5% ) newborns had a normal mri or only a mild injury .
eeg improved in all six newborns with bs or extremely low - voltage pattern by 1218 h of recording and background normalized by the middle of cooling [ table 2 ] .
during this interval , one newborn whom eeg improved from a discontinuous pattern to a normal one at the beginning of cooling had moderate to severe mri injury . during the middle of cooling , no one of the remaining newborns with a normal eeg had moderate to severe injury . among the 11 infants with a discontinuous eeg pattern , the background had moderate to severe injury in ( 2/11 , 18% ) .
all newborns ( 7/7 , 100% ) with bs or extremely low - voltage patterns had moderate to severe injury [ table 2 ] .
among the 11 infants whom background was normal , 2 ( 18% ) of them showed moderate to severe mri injury .
furthermore , among the 15 infants with an excessively discontinuous pattern , 2 ( 13% ) of them showed moderate to severe mri injury .
compared to all five infants ( 100% ) whom background had bs or extremely low voltage [ table 2 ] .
the same two newborns whom background was normal ( 13% ) at this time point showed moderate to severe injury .
after rewarming , all six infants with bs or extremely low - voltage patterns had moderate to severe injury [ table 2 ] .
seizures recurred during the middle of cooling in two infants and during rewarming in another two . among the 12 infants with electrographic seizures , 5 infants ( 42% )
did not show a clinical correlate ( subclinical seizure ) . among the five infants with subclinical seizure , three of them showed subclinical se .
all three infants with se showed bs or extremely low - voltage patterns with the start of cooling .
patients with moderate to severe mri injury had isolated or recurrent seizures more frequently than those with no or mild injury ( 47% vs. 21% , p = 0.05 ) , and the se was seen only in infants with moderate to severe injury ( p = 0.01 ) [ table 3 ] .
background of electroencephalographic , seizures , status epilepticus , and outcome of magnetic resonance imaging brain outcome a normal eeg background at the start of cooling was more predictive of a favorable mri outcome than at later time points with a specificity of 100% and 80% , respectively .
bs pattern or extremely low - voltage background is of a prognostic value for moderate to severe injury that increased from the start of cooling ( 75% specificity ) to the middle of cooling and later ( 100% specificity ) [ table 4 ] .
electroencephalographic background sensitivity and specificity during and after cooling for moderate to severe magnetic resonance imaging brain injury there were no differences between groups regarding clinical characteristics .
the duration ( median standard deviation ) of seizure patterns was 42 83 min in group 1 , compared with 86 134 min in group 2 [ figure 3 ] .
there was a significant relationship between the duration of seizure patterns and mri scores in linear regression analysis in group 1 [ figure 4 ] .
duration of seizure patterns for the clinical and subclinical seizure treatment group 1 and the clinical seizure treatment group 2 .
the vertical lines indicate the limit lines : ranges relationship between duration of seizure patterns and magnetic resonance imaging scores ( linear regression ) in group 1
some complicating factors are encountered during th in the form of sedation and clinical changes that occur during cooling .
brain mri provides structural details only and its sensitivity for hie in the first few days of life is of a limited value .
video - eeg monitoring is considered the gold standard test for assessment of brain functions and for detection of subclinical seizure in neonatal hie .
previous studies for prediction of seizures were based on clinically diagnosed seizures , confirmed by intermittent eeg recordings .
subclinical seizures constitute about two - thirds of neonatal seizures and require video - eeg monitoring to be diagnosed .
continuous video - eeg monitoring is considered the most accurate test for neonatal seizure detection .
the potential of neuroprotective therapies , such as hypothermia , has raised the importance of accurate prediction of outcome in the first hours of life .
our study showed that none of the clinical characteristics were associated with brain injury in mri .
these data were concordant with that reported by murray et al . , 2009 , who reported neither
the condition at birth nor the degree of metabolic acidosis reliably predicts the occurrence of seizures . throughout all treatment periods ,
a normal eeg was associated with no or mild mri brain injury . at the start of cooling ,
a normal eeg background had better predictive value ( 100% specificity ) than at later time points ( 80% specificity ) . over the first 24 h of monitoring ,
only one newborn with moderate to severe mri injury showed improvement in the eeg background from excessively discontinuous to normal .
, 1982 , holmes and lombroso , 1993 , and nash et al . , 2011 , who reported that normal eeg background at the start of cooling had better predictive value than at later time points with a specificity of 100% and 93% , respectively .
however , bs pattern or extremely low - voltage background is of a prognostic value for moderate to severe injury that increased from the start of cooling ( 75% specificity ) to middle of cooling and later ( 100% specificity ) , reflecting six newborns with these patterns at the start of recording who were protected from further injury by middle of cooling .
similar results were reported by nash et al . , 2011 and biagioni et al .
abnormal eeg background activity continuous for 24 h or more or getting worse denotes poor prognosis .
good prognostic features include improvement of eeg background activity in the form of increase in voltage , decrease in discontinuity , or appearance of sleep - wake cycles within 1224 h after birth . in our study , by the 2 day of life coinciding with the time of middle of cooling , a bs or extremely low - voltage eeg becomes of high predictive value for detection of moderate to severe mri injury .
2010 , studied the effect of cooling on amplitude - integrated eeg in infants with asphyxia .
he found that a severely abnormal eeg background pattern in the cooled infants was not specific for abnormal developmental outcome until the 2 day of life .
serial eegs are preferred to single recordings so that persistence of abnormalities can be determined .
single recordings may have little significance ; however , if the pattern persists for several weeks , it may be of more prognostic value . as an example ,
an eeg obtained soon after birth can show significant abnormalities due to stress of birth ; these abnormalities may disappear completely within a few days and thus have little clinical significance .
severity of neonatal eeg abnormalities correlates with the severity of neurological insult to the neonate .
this makes eeg in this age group a valuable tool in predicting outcome of at - risk neonates .
markedly abnormal and normal eegs have the greatest reliability in predicting poor and good outcomes , respectively . between these two extremes
, there are many background patterns with different prognostic values that are difficult to use with different studies giving varying results . in such conditions , other neuroimaging studies and
previous studies concluded that during the first 24 h of life , the eeg background had a poor prognostic value .
previous studies in noncooled infants with hie are in favor of our results . in our study , the majority of infants ( 75% ) with an excessively discontinuous pattern after rewarming had no or only mild mri injury .
, 2002 , who found that a discontinuous eeg pattern early in life is sometimes associated with poor outcome . the discrepancy from our results can be attributed to different methods used , different definitions in the literature for excessively discontinuous background , and effect of hypothermia .
improvement of neurological sequelae necessitates rapid detection and treatment of seizures , which will depend on continuous eeg monitoring . in our study ,
furthermore , wusthoff et al . , 2011 , found 65% of participants had electrographic seizures during or immediately after treatment with hypothermia .
these findings are consistent with the prehypothermia literature , which describes seizures in 22%64% , suggesting that hypothermia as employed for hie does not substantially affect the incidence of seizures . in our study ,
continuous video - eeg revealed 5 of 12 patients ( 42% ) with seizure activity did not show clinical seizure . among those five patients , three patients had subclinical se .
in a previous study conducted by murray et al . , 2008 , on 51 full - term neonates with hie , he showed that only 34% of the electrographic seizures on video - eeg monitoring had clinical evidence .
high incidence of seizures had been reported in our patients despite the effect of hypothermia in reduction of seizures in experimental studies .
furthermore , srinivasakumar et al . , 2013 , stated that th was associated with a reduced seizure burden in infants with mild and moderate injury but not in those with severe injury .
this discrepancy may be attributed to the different nature of cooling in animal models that is deep and early . in many studies , detection of seizure in newborns
however , most seizures are subclinical and can be missed without continuous eeg monitoring . however , clinical observation alone can not differentiate between actual seizures and movements mimic seizures in infants . in our study , all newborns with se had severely abnormal mri while not all newborns with isolated or recurrent seizures were associated with moderate to severe brain injury .
these findings are in concordance of other authors who reported that newborns with se had a significantly worse outcome in comparison to newborns with recurrent seizures .
furthermore , we were able to show that immediate treatment of clinical and subclinical seizure patterns detected in eeg results in a reduction of total duration of seizure patterns , and there is a significant association between duration of seizure patterns detected in the eeg and severity of brain injury as seen on mri .
the finding that treatment of clinical and subclinical seizures results in a reduction of total seizure duration supports the hypothesis that subclinical seizures should be treated . in most
nicus doing eeg monitoring , treatment of subclinical seizures is recommended . whether this policy has a positive impact on prognosis
has not yet proven , but there is some evidence for the best outcome for treating infants .
there are some limitations to our study ; we used mri as a short - term outcome measure and long - term outcome is not known in our patients . although cooling does not affect the prognostic value of mri in newborns with hie , long - term developmental follow - up of our patients to confirm our results is recommend finally , we did not include a control group in our study as the protocol in our unit to subject any case fulfilling the criteria of th to early cooling .
this was based on safety and benefits of th as well as the lack of other effective therapies .
further studies are recommended to clarify that eeg patterns are attributed to either brain injury or therapeutic intervention ( hypothermia and medications ) .
we can conclude that eeg monitoring in newborns with hie is very crucial in seizure management and prognosis .
a trend was found for a reduction in the duration of seizure patterns when clinical and subclinical seizures were treated .
this trend and the significant association of seizure duration and severity of brain injury found on mri scans suggest that recognition and treatment of neonatal seizures ( clinical and subclinical ) in infants with hie can reduce brain injury . there are no conflicts of interest . | background : the values of electroencephalography ( eeg ) in neonatal hypoxic - ischemic encephalopathy ( hie ) during therapeutic hypothermia ( th ) are still uncertain.aims:the aim of this study is to detect eeg background , the prevalence of seizures during cooling , and to determine different eeg patterns that can predict brain injury in magnetic resonance imaging ( mri).patients and methods : thirty - nine newborns with hie were subjected to th .
continuous monitoring by video - eeg was carried out throughout cooling and during rewarming .
mri was done for all newborns after rewarming .
the predictive value of eeg background for mri brain injury was evaluated at 6-h intervals during cooling and rewarming.results:at all - time intervals , normal eeg was associated with no or mild mri brain injury . at the beginning of cooling , normal background was more predictive of a favorable mri outcome than at later time points .
after 24 h of monitoring , diffuse burst suppression and depressed patterns had the greatest prognostic value . in most patients , a discontinuous pattern was not associated with poor prognosis .
thirty - one percent developed electrical seizures , and 8% developed status epilepticus .
seizures were subclinical in 42% .
there is a significant association between duration of seizure patterns detected on the eeg and severity of brain injury on mri.conclusions:continuous eeg monitoring in newborns with hie under cooling has a prognostic value about early mri brain injury and identifies electrographic seizures , approximately 50% of which are subclinical .
treatment of clinical and subclinical seizure results in a reduction of the total duration of seizure pattern supports the hypothesis that subclinical seizures should be treated . | Introduction
Patients and Methods
Video-electroencephalographic monitoring
Brain magnetic resonance imaging
Statistical analysis
Results
Beginning of cooling
Middle of cooling
End of cooling
After cooling
Electrographic seizures
Discussion
Conclusions
Financial support and sponsorship
Conflicts of interest | neonatal seizures are important risk factors for impaired neurodevelopment in neonatal hypoxic - ischemic encephalopathy ( hie ) . however , little is known regarding frequency of seizure , its distribution , its time of onset and seizure expression , and their relationship to mri brain injury during therapeutic hypothermia ( th ) . our aim was to determine the value of video - eeg monitoring in newborns with hie treated by hypothermia through determination of eeg background , prevalence of seizures during th , determination of eeg patterns that can predict mri brain injury and value of detection , and treatment of subclinical seizures . clinical characteristics of newborns in relation to brain injury at the beginning of cooling , no one of newborns whom background was normal had moderate to severe injury . electroencephalographic background during cooling in relation to magnetic resonance imaging brain injury during this interval , one newborn whom eeg improved from a discontinuous pattern to a normal one at the beginning of cooling had moderate to severe mri injury . background of electroencephalographic , seizures , status epilepticus , and outcome of magnetic resonance imaging brain outcome a normal eeg background at the start of cooling was more predictive of a favorable mri outcome than at later time points with a specificity of 100% and 80% , respectively . bs pattern or extremely low - voltage background is of a prognostic value for moderate to severe injury that increased from the start of cooling ( 75% specificity ) to the middle of cooling and later ( 100% specificity ) [ table 4 ] . duration of seizure patterns for the clinical and subclinical seizure treatment group 1 and the clinical seizure treatment group 2 . the vertical lines indicate the limit lines : ranges relationship between duration of seizure patterns and magnetic resonance imaging scores ( linear regression ) in group 1
at the beginning of cooling , no one of newborns whom background was normal had moderate to severe injury . during this interval , one newborn whom eeg improved from a discontinuous pattern to a normal one at the beginning of cooling had moderate to severe mri injury . background of electroencephalographic , seizures , status epilepticus , and outcome of magnetic resonance imaging brain outcome a normal eeg background at the start of cooling was more predictive of a favorable mri outcome than at later time points with a specificity of 100% and 80% , respectively . duration of seizure patterns for the clinical and subclinical seizure treatment group 1 and the clinical seizure treatment group 2 . the vertical lines indicate the limit lines : ranges relationship between duration of seizure patterns and magnetic resonance imaging scores ( linear regression ) in group 1
some complicating factors are encountered during th in the form of sedation and clinical changes that occur during cooling . video - eeg monitoring is considered the gold standard test for assessment of brain functions and for detection of subclinical seizure in neonatal hie . throughout all treatment periods ,
a normal eeg was associated with no or mild mri brain injury . at the start of cooling ,
a normal eeg background had better predictive value ( 100% specificity ) than at later time points ( 80% specificity ) . over the first 24 h of monitoring ,
only one newborn with moderate to severe mri injury showed improvement in the eeg background from excessively discontinuous to normal . , 2011 , who reported that normal eeg background at the start of cooling had better predictive value than at later time points with a specificity of 100% and 93% , respectively . however , bs pattern or extremely low - voltage background is of a prognostic value for moderate to severe injury that increased from the start of cooling ( 75% specificity ) to middle of cooling and later ( 100% specificity ) , reflecting six newborns with these patterns at the start of recording who were protected from further injury by middle of cooling . in such conditions , other neuroimaging studies and
previous studies concluded that during the first 24 h of life , the eeg background had a poor prognostic value . , 2008 , on 51 full - term neonates with hie , he showed that only 34% of the electrographic seizures on video - eeg monitoring had clinical evidence . in our study , all newborns with se had severely abnormal mri while not all newborns with isolated or recurrent seizures were associated with moderate to severe brain injury . furthermore , we were able to show that immediate treatment of clinical and subclinical seizure patterns detected in eeg results in a reduction of total duration of seizure patterns , and there is a significant association between duration of seizure patterns detected in the eeg and severity of brain injury as seen on mri . the finding that treatment of clinical and subclinical seizures results in a reduction of total seizure duration supports the hypothesis that subclinical seizures should be treated . we can conclude that eeg monitoring in newborns with hie is very crucial in seizure management and prognosis . a trend was found for a reduction in the duration of seizure patterns when clinical and subclinical seizures were treated . this trend and the significant association of seizure duration and severity of brain injury found on mri scans suggest that recognition and treatment of neonatal seizures ( clinical and subclinical ) in infants with hie can reduce brain injury . | [
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data used in the preparation of this article were obtained from the alzheimer 's disease neuroimaging initiative ( adni ) database ( http://adni.loni.usc.edu ) .
adni was launched in 2003 by the national institute on aging ( nia ) , the national institute of biomedical imaging and bioengineering , the us food and drug administration , private pharmaceutical companies , and nonprofit organizations , as a $ 60-million , 5-year , public private partnership .
the primary goal of adni has been to test whether serial mri , pet , other biological markers , and clinical and neuropsychological assessment can be combined to measure clinical progression in mild cognitive impairment ( mci ) and early ad .
determination of sensitive and specific markers of very early ad progression is intended to aid researchers and clinicians to develop new treatments and monitor their effectiveness , as well as lessen the time and cost of clinical trials .
the principal investigator of this initiative is michael w. weiner , md , va medical center and university of california , san francisco .
adni is the result of efforts of many coinvestigators from a broad range of academic institutions and private corporations , and participants have been recruited from more than 50 sites across the united states and canada .
the initial goal of adni was to recruit 800 adults , aged 55 to 90 , to participate in the research
approximately 200 cognitively normal older individuals to be followed for 3 years , 400 people with mci to be followed for 3 years , and 200 people with early ad to be followed for 2 years . for up - to - date information ,
the diagnosis of ad was based on the national institute of neurological and communicative disorders and stroke and the ad and related disorders association ( nincds - adrda ) criteria .
participants with ad were required to have mini - mental state examination ( mmse ) scores between 20 and 26 and a clinical dementia rating ( cdr ) score of 0.5 to 1 at baseline .
qualifying mci participants had memory complaints but no significant functional impairment , scored between 24 and 30 on the mmse , had a global cdr score of 0.5 , a cdr memory score of 0.5 or greater , and objective memory impairment on wechsler memory scale logical memory ii test .
healthy control participants had mmse scores between 24 and 30 , a global cdr score of 0 , and did not meet criteria for mci and ad .
participants were excluded if they refused or were unable to undergo mri ; had other neurologic disorders , active depression , or a history of psychiatric diagnosis , alcohol , or substance dependence within the past 2 years ; had less than 6 years of education ; or were not fluent in english or spanish .
the full list of inclusion / exclusion criteria may be accessed on pages 2329 of the online adni protocol ( see http://www.adni-info.org/scientists/adniscientistshome.aspx ) .
the subset of 18 ad , 52 mci , and 3 cognitively normal adni participants who received [ c]-pib have been included in this study .
we downloaded all available pib adni1 scans from the adni web site ( http://adni.loni.usc.edu ) in october 2008 .
[ c]-pib with minimum 90% radiochemical purity and minimum specific activity of 300 ci / mmol was synthesized .
dynamic acquisition frames were obtained on a pet scanner 5070 minutes after injection . a pib standardized uptake value ratio ( suvr ) image was obtained by averaging the individual 5070 minutes after injection frames .
pib scans were normalized to the mean pib retention value of the cerebellar cortex ( suvrcbgm ) .
after normalization , the pib suvrcbgm data were spatially coregistered to the participants ' baseline mri scan using the minctracc algorithm and 9-parameter ( 9p ) transformation ( 3 translations , 3 rotations , 3 scales ) .
the pib data were smoothed with a 15-mm kernel and convected onto the 3d hemispheric models derived with the cortical pattern matching technique as previously described .
genotypic data were extracted from the publicly accessible gwas data from the adni web site ( http://adni.loni.usc.edu ) .
briefly , single nucleotide polymorphism ( snp ) genotyping was completed on all adni participants for more than 620,000 target snps using a total of 7 ml of blood from all participants .
genomic dna was extracted using the qiaamp dna blood maxi kit ( qiagen , inc , valencia , ca ) and analyzed using human 610-quad beadchip ( illumina , inc , san diego , ca ) according to the manufacturer 's protocols ( infinium hd assay ; super protocol guide ; rev .
apoe genotyping was carried out by pcr amplification , hhai restriction enzyme digestion , and subsequent standard gel resolution and visualization processes .
we classified participants as carriers and noncarriers for the following ad risk snps : bin1 rs744373 and rs7561528 ; clu rs11136000 , rs2279590 , and rs9331888 ; picalm rs3851179 and rs541458 ; cr1 rs3818361 ; abca7 rs3764650 ; ms4a6a rs610932 ; cd33 rs3826656 and rs3865444 ; ms4a4e rs670139 ; cd2ap rs9349407 and rs3865444 . the following snps had balanced carrier / noncarrier status and were further analyzed : cd2ap rs9349407 ( 40 carriers , 33 noncarriers ) , cr1 rs3818361 ( 30 carriers , 43 noncarriers ) , bin1 rs744373 ( 42 carriers , 31 noncarriers ) , cd33 rs3826656 ( 31 carriers , 42 noncarriers ) , clu rs9331888 ( 44 carriers , 29 noncarriers ) , and clu rs2279590 ( 47 carriers , 26 noncarriers ) .
the plasma samples were then sent to rules - based medicine ( rbm , austin , tx ) for measurement of 190 protein analytes with an immunoassay panel developed on the luminex xmap ( austin ) platform .
each plate was run with 3 levels of quality control , and each analyte had a validation report ; samples from the total adni plasma cohort were run on 15 plates .
the 3d association between apoe protein plasma levels and cortical pib suvrcbgm was studied using linear regression in the pooled sample using age and sex as covariates .
next , we split the pooled sample based on the presence or absence of disease - associated alleles for cd2ap rs9349407 , cr1 rs3818361 , bin1 rs744373 , cd33 rs3826656 and clu rs9331888 and studied the associations of the plasma apoe levels and pib suvrcbgm separately in minor snp carriers and noncarriers while adjusting for age and sex .
the 3d associations between cortical pib suvrcbgm and risk genotypes were studied using linear regression with age and sex as covariates .
our linear regression 3d statistical maps were subjected to multiple comparison correction by permutation analyses ( permuting the predictor variables ) . using a set - level inference approach
, we defined a single corrected p value for each map based on the number of points surviving a particular a priori threshold ( set to 0.01 in our analyses ) .
this approach tends to be more sensitive for detecting a distributed pattern of weak effects as opposed to the peak height of the maximum statistic , which is best for detecting a spatially highly concentrated effect .
data used in the preparation of this article were obtained from the alzheimer 's disease neuroimaging initiative ( adni ) database ( http://adni.loni.usc.edu ) .
adni was launched in 2003 by the national institute on aging ( nia ) , the national institute of biomedical imaging and bioengineering , the us food and drug administration , private pharmaceutical companies , and nonprofit organizations , as a $ 60-million , 5-year , public private partnership .
the primary goal of adni has been to test whether serial mri , pet , other biological markers , and clinical and neuropsychological assessment can be combined to measure clinical progression in mild cognitive impairment ( mci ) and early ad .
determination of sensitive and specific markers of very early ad progression is intended to aid researchers and clinicians to develop new treatments and monitor their effectiveness , as well as lessen the time and cost of clinical trials .
the principal investigator of this initiative is michael w. weiner , md , va medical center and university of california , san francisco .
adni is the result of efforts of many coinvestigators from a broad range of academic institutions and private corporations , and participants have been recruited from more than 50 sites across the united states and canada .
the initial goal of adni was to recruit 800 adults , aged 55 to 90 , to participate in the research
approximately 200 cognitively normal older individuals to be followed for 3 years , 400 people with mci to be followed for 3 years , and 200 people with early ad to be followed for 2 years . for up - to - date information ,
the diagnosis of ad was based on the national institute of neurological and communicative disorders and stroke and the ad and related disorders association ( nincds - adrda ) criteria .
participants with ad were required to have mini - mental state examination ( mmse ) scores between 20 and 26 and a clinical dementia rating ( cdr ) score of 0.5 to 1 at baseline .
qualifying mci participants had memory complaints but no significant functional impairment , scored between 24 and 30 on the mmse , had a global cdr score of 0.5 , a cdr memory score of 0.5 or greater , and objective memory impairment on wechsler memory scale logical memory ii test .
healthy control participants had mmse scores between 24 and 30 , a global cdr score of 0 , and did not meet criteria for mci and ad .
participants were excluded if they refused or were unable to undergo mri ; had other neurologic disorders , active depression , or a history of psychiatric diagnosis , alcohol , or substance dependence within the past 2 years ; had less than 6 years of education ; or were not fluent in english or spanish .
the full list of inclusion / exclusion criteria may be accessed on pages 2329 of the online adni protocol ( see http://www.adni-info.org/scientists/adniscientistshome.aspx ) .
the subset of 18 ad , 52 mci , and 3 cognitively normal adni participants who received [ c]-pib have been included in this study .
we downloaded all available pib adni1 scans from the adni web site ( http://adni.loni.usc.edu ) in october 2008 .
[ c]-pib with minimum 90% radiochemical purity and minimum specific activity of 300 ci / mmol was synthesized .
dynamic acquisition frames were obtained on a pet scanner 5070 minutes after injection . a pib standardized uptake value ratio ( suvr )
pib scans were normalized to the mean pib retention value of the cerebellar cortex ( suvrcbgm ) .
after normalization , the pib suvrcbgm data were spatially coregistered to the participants ' baseline mri scan using the minctracc algorithm and 9-parameter ( 9p ) transformation ( 3 translations , 3 rotations , 3 scales ) .
the pib data were smoothed with a 15-mm kernel and convected onto the 3d hemispheric models derived with the cortical pattern matching technique as previously described .
genotypic data were extracted from the publicly accessible gwas data from the adni web site ( http://adni.loni.usc.edu ) . the detailed gwas genotyping protocol has been previously described . briefly , single nucleotide polymorphism ( snp ) genotyping was completed on all adni participants for more than 620,000 target snps using a total of 7 ml of blood from all participants .
genomic dna was extracted using the qiaamp dna blood maxi kit ( qiagen , inc , valencia , ca ) and analyzed using human 610-quad beadchip ( illumina , inc , san diego , ca ) according to the manufacturer 's protocols ( infinium hd assay ; super protocol guide ; rev .
apoe genotyping was carried out by pcr amplification , hhai restriction enzyme digestion , and subsequent standard gel resolution and visualization processes .
we classified participants as carriers and noncarriers for the following ad risk snps : bin1 rs744373 and rs7561528 ; clu rs11136000 , rs2279590 , and rs9331888 ; picalm rs3851179 and rs541458 ; cr1 rs3818361 ; abca7 rs3764650 ; ms4a6a rs610932 ; cd33 rs3826656 and rs3865444 ; ms4a4e rs670139 ; cd2ap rs9349407 and rs3865444 . the following snps had balanced carrier / noncarrier status and were further analyzed : cd2ap rs9349407 ( 40 carriers , 33 noncarriers ) , cr1 rs3818361 ( 30 carriers , 43 noncarriers ) , bin1 rs744373 ( 42 carriers , 31 noncarriers ) , cd33 rs3826656 ( 31 carriers , 42 noncarriers ) , clu rs9331888 ( 44 carriers , 29 noncarriers ) , and clu rs2279590 ( 47 carriers , 26 noncarriers ) .
the plasma samples were then sent to rules - based medicine ( rbm , austin , tx ) for measurement of 190 protein analytes with an immunoassay panel developed on the luminex xmap ( austin ) platform .
each plate was run with 3 levels of quality control , and each analyte had a validation report ; samples from the total adni plasma cohort were run on 15 plates .
the 3d association between apoe protein plasma levels and cortical pib suvrcbgm was studied using linear regression in the pooled sample using age and sex as covariates .
next , we split the pooled sample based on the presence or absence of disease - associated alleles for cd2ap rs9349407 , cr1 rs3818361 , bin1 rs744373 , cd33 rs3826656 and clu rs9331888 and studied the associations of the plasma apoe levels and pib suvrcbgm separately in minor snp carriers and noncarriers while adjusting for age and sex . the 3d associations between cortical pib suvrcbgm and risk genotypes were studied using linear regression with age and sex as covariates .
our linear regression 3d statistical maps were subjected to multiple comparison correction by permutation analyses ( permuting the predictor variables ) . using a set - level inference approach
, we defined a single corrected p value for each map based on the number of points surviving a particular a priori threshold ( set to 0.01 in our analyses ) .
this approach tends to be more sensitive for detecting a distributed pattern of weak effects as opposed to the peak height of the maximum statistic , which is best for detecting a spatially highly concentrated effect .
all demographic variables and comparisons are shown in table 1 . we found no significant differences in age , sex distribution , or level of education between carriers and noncarriers for each gene .
there were significantly more mci than ad and healthy control subjects among cd2ap noncarriers ( p = 0.03 ) .
bin1 and cd2ap carriers had lower mmse scores than noncarriers ( bin1 mean mmse 25.7 vs 26.5 , p = 0.005 ; cd2ap mean mmse 25.3 vs 27.0 p = 0.047 ) . of note , there were no significant differences in apoe 4 status between carriers and noncarriers for any risk gene .
demographic statistics of participant pool , indicated by the pooled sample and genotype none of the ad risk genotypes showed significant association with pib suvrcbgm in the pooled sample .
plasma apoe levels showed an association with pib suvr throughout the brain with the exception of the sensorimotor and entorhinal cortex ( figure 1 ) across the pooled sample ( left pcorr = 0.004 , right pcorr = 0.008 ) .
plasma apoe levels showed an association with pib suvrcbgm only in cd2ap rs9349407 and cr1 rs3818361 minor allele noncarriers ( cd2ap rs9349407 noncarriers left pcorr = 0.003 , right pcorr = 0.004 ; cr1 rs3818361 noncarriers left pcorr = 0.008 , right pcorr = 0.01 , figure 1 and table 2 ) .
these results remained unchanged after correcting for diagnosis ( cd2ap rs9349407 noncarriers left pcorr = 0.009 , right pcorr = 0.008 ; cr1 rs3818361 noncarriers left pcorr = 0.014 , right pcorr = 0.025 , table 2 and figure e-1 at neurology.org/ng ) . after correcting for apoe 4 ,
the association between apoe protein plasma levels and pib binding remained for cd2ap ( left pcorr = 0.03 , right pcorr = 0.03 ) but not cr1 ( table 2 and figure e-1 ) .
plasma apoe showed an association with pib suvrcbgm in bin1 rs744373 minor allele carriers ( left pcorr = 0.006 , right pcorr = 0.01 , figure 1 and table 2 ) .
this bin1 association remained after correcting for diagnosis ( left pcorr = 0.007 , right pcorr = 0.017 , figure e-1 ) and apoe 4 genotype ( left pcorr = 0.028 , right pcorr = 0.038 , table 2 and figure e-1 ) .
significant p values of the association between plasma apoe levels and cortical pib binding stratified by bin1 , cd2ap , and cr1 carrier status
all demographic variables and comparisons are shown in table 1 . we found no significant differences in age , sex distribution , or level of education between carriers and noncarriers for each gene .
there were significantly more mci than ad and healthy control subjects among cd2ap noncarriers ( p = 0.03 ) .
bin1 and cd2ap carriers had lower mmse scores than noncarriers ( bin1 mean mmse 25.7 vs 26.5 , p = 0.005 ; cd2ap mean mmse 25.3 vs 27.0 p = 0.047 ) . of note , there were no significant differences in apoe 4 status between carriers and noncarriers for any risk gene .
demographic statistics of participant pool , indicated by the pooled sample and genotype none of the ad risk genotypes showed significant association with pib suvrcbgm in the pooled sample .
plasma apoe levels showed an association with pib suvr throughout the brain with the exception of the sensorimotor and entorhinal cortex ( figure 1 ) across the pooled sample ( left pcorr = 0.004 , right pcorr = 0.008 ) .
plasma apoe levels showed an association with pib suvrcbgm only in cd2ap rs9349407 and cr1 rs3818361 minor allele noncarriers ( cd2ap rs9349407 noncarriers left pcorr = 0.003 , right pcorr = 0.004 ; cr1 rs3818361 noncarriers left pcorr = 0.008 , right pcorr = 0.01 , figure 1 and table 2 ) .
these results remained unchanged after correcting for diagnosis ( cd2ap rs9349407 noncarriers left pcorr = 0.009 , right pcorr = 0.008 ; cr1 rs3818361 noncarriers left pcorr = 0.014 , right pcorr = 0.025 , table 2 and figure e-1 at neurology.org/ng ) . after correcting for apoe 4 ,
the association between apoe protein plasma levels and pib binding remained for cd2ap ( left pcorr = 0.03 , right pcorr = 0.03 ) but not cr1 ( table 2 and figure e-1 ) .
plasma apoe showed an association with pib suvrcbgm in bin1 rs744373 minor allele carriers ( left pcorr = 0.006 , right pcorr = 0.01 , figure 1 and table 2 ) .
this bin1 association remained after correcting for diagnosis ( left pcorr = 0.007 , right pcorr = 0.017 , figure e-1 ) and apoe 4 genotype ( left pcorr = 0.028 , right pcorr = 0.038 , table 2 and figure e-1 ) .
significant p values of the association between plasma apoe levels and cortical pib binding stratified by bin1 , cd2ap , and cr1 carrier status
our data show that bin1 rs744373 , cd2ap rs9349407 , and cr1 rs3818361 genotypes modulate the association between apoe protein plasma levels and brain amyloidosis . in the pooled sample
, we saw the expected association between plasma apoe levels and pib suvr throughout the brain . yet , when stratified by ad risk genotypes , plasma apoe showed a significant association with pib suvrcbgm only in noncarriers of the minor allele of cd2ap rs9349407 and cr1 rs3818361 and only in carriers of the bin1 rs744373 minor allele .
these findings imply a downstream interaction between these genes and the apoe disease - associated pathways and suggest a direct or modulatory role on a accumulation and clearance .
brain and plasma apoe4 levels are believed to be lower than apoe3 levels because of a difference in their fate after they are endocytosed and routed to multivesicular bodies that function as sorting endosomes .
apoe3 is packaged into recycling endosomes and retroendocytosed back to the surface and secreted , whereas apoe4 is typically trafficked on through to the lysosome and degraded .
bridging integrator 1 , or bin1 , encodes a nucleocytoplasmic adaptor protein abundantly expressed in the cns that is involved in synaptic vesicle endocytosis , immune response , calcium homeostasis , and apoptosis .
the protein encoded by bin1 has been shown to link the microtubule cytoskeleton and cellular membrane and has been implicated in amyloid precursor protein turnover and hence a production through clathrin - mediated amyloid precursor protein endocytosis .
bin1 rs744373 minor allele carriers have been reported to have an increased risk of ad .
postmortem , no association with amyloid plaque burden has been observed for bin1 rs7561528 , and bin1 rs744373 , consistent with our results in the pooled sample .
an association of bin1 rs744373 minor allele with in vivo amyloid deposition measured with f - florbetapir has been reported , but this effect disappeared in the presence of the protective picalm minor allele at rs3851179 .
bin1 rs744373 also failed to show an association with csf a levels . in our study , the presence of the bin1 rs744373 risk variant was associated with preserved and potentially enhanced association between apoe protein plasma levels and pib binding , lending evidence for a potential downstream functional interaction between the bin1 and apoe genes .
bin1 rs744373 increases mrna expression and protein expression . as mentioned above , apoe levels are strongly related to the classic clathrin - dependent receptor - mediated endocytosis with impaired recycling of apoe4 isoforms .
these complexes have been shown to regulate the rab5/7 switch that influences the transition from early to late endosomes and controls traffic to the lysosomes .
a and apoe are known for trafficking through rab5/rab7 to lysosomes and to rab11 recycling endosomes for degradation .
dependent manner in which apoe3 promotes greater a and apoe traffic to the lysosome for clearance .
therefore , bin1 is ideally positioned to regulate both a and apoe influx into lysosomes and trafficking toward degradation or recycling through retroendocytosis .
thus higher bin1 expression can be hypothesized to increase apoe recycling and elevate protein levels notably , and this would be linked to faster amyloid clearance and less deposition associated with higher apoe levels .
cd2ap , the cd2-associated protein gene , codes for a scaffolding protein that plays a role in the formation of tight junctions , endocytosis , cellular waste management , and immune response .
cd2ap has an established role in endocytosis , vesicle trafficking within the cell , and formation of the cytoskeleton .
cd2ap is involved in endosome trafficking , binds directly to rab4 , and regulates recycling endosomes , which play an essential role in the retroendocytosis of apoe that controls apoe levels .
consistent with this , cd2ap - knockout mice have deficits in multivesicular body ( mvb ) formation and endosomal lysomal trafficking .
however , studies have failed to find a direct association between this snp and pib binding in adni .
we found that the association between apoe protein plasma levels and pib binding is no longer present in minor allele carriers , indicating that cd2ap influences amyloid pathology .
since cd2ap rs9349407 trends toward reducing cd2ap expression in human brain , one might hypothesize that decreased cd2ap ( or altered interaction with binding partners ) could reduce apoe 's isoform - dependent recycling and endosomal - lysosomal traffic that regulates both apoe levels and a clearance in lysosomes .
the latter has been implicated as a cause for both low plasma apoe4 and defects in a clearance .
since cd2ap rs9349407 trends toward reducing cd2ap expression in human brain , one might hypothesize that decreased cd2ap ( or altered interaction with binding partners ) could reduce apoe 's isoform - dependent recycling and endosomal lysosomal traffic that regulates both apoe levels and a clearance in lysosomes .
the cr1 gene encodes complement component 3b/4b receptor 1a membrane glycoprotein found on erythrocytes , leukocytes , glomerular podocytes , and splenic follicular dendritic cells .
cr1 serves to mediate clearance of immune complexes and phagocytosis by neutrophils and monocytes and plays a role in antigen presentation to b lymphocytes .
the presence of the cr1 rs3818361 risk variant is associated with lower pib binding in 2 separate cognitively normal cohorts including an adn1 subsample but not in the full adni1 sample . at the same time , 2 other snps , cr1 rs6656401 and rs6701713 , showed an association with increased amyloid plaque burden postmortem . in this study
we observed that cr1 rs3818361 minor allele carrier status leads to a loss of the association between apoe protein plasma levels and pib binding , which disappeared in the presence of apoe 4 as covariate .
adni uses unified subject assessment , mri , pib - pet , csf , and peripheral blood collection protocols and meticulous data quality control across all study sites .
one of the major limitations of our study is its small sample size limited by pib availability in adni1 .
significant disproportion between carriers and noncarriers for the remaining adni risk snps led to the exclusion of these variants , allowing us to test our hypothesis for only 6 snps across 5 ad risk genes .
last but not least , adni uses rigorous exclusion criteria typical of clinical trials and the study population is not representative of the general population , which may negatively affect the generalizability of our results .
the adni pet core chose cerebellar gray matter as the reference region for intensity normalization of adni1 pib data .
other regions that have been used in the amyloid pet literature include whole cerebellum , periventricular white matter , whole brainstem , and composite regions derived from more than one of these measures .
recently , a group of pet experts compared the performance of 4 of these normalization regions : whole cerebellum , cerebellar gray matter , pons , and whole cerebellum plus brainstem .
the authors concluded that normalization to the pons performed worse than normalization strategies including the cerebellum .
the 2 regions that performed best ( i.e. , had lowest suvr variance ) were whole cerebellum and whole cerebellum plus brainstem .
our study uses the cerebellar gray matter , which performed better than pons but resulted in more noise ( higher suvr variance ) in the normalized data . yet despite this potentially suboptimal signal - to - noise ratio , we were able to find significant associations .
overall , our findings imply an interaction between several ad risk gene minor alleles and apoe genotype driven brain amyloidosis . in the absence of a direct association with brain amyloidosis , we found that several ad risk genes nonetheless exert a modulatory effect on one of the most fundamental disease - associated pathophysiologic events
these findings lend the basis for further exploration of the exact ad - related pathophysiologic mechanisms of these genes and their products , which might ultimately lead to new therapeutic strategies .
andreas lazaris is a co - first author of the manuscript , responsible for study design , data processing , statistical analyses , and drafting of the manuscript .
kristy s. hwang is a co - first author of the manuscript , assisting with study design , data processing , and the completion of integral statistical analyses .
naira goukasian completed some of the final analysis of our data and took part in revising of the manuscript .
leslie m. ramirez completed some of the initial analyses of our data and took part in revising of the manuscript .
jennifer eastman completed some of the initial analyses of our data and took part in revising of the manuscript .
anna e. blanken completed some of the final analyses of our data and took part in revising of the manuscript .
edmond teng took part in some analysis and interpretations of the data and participated in revising of the manuscript .
karen gylys provided critical insights for interpretation of our results and participated in revising of the manuscript .
greg cole provided critical insights for interpretation of our results and participated in revising of the manuscript .
andrew saykin contributed to the overall adni study design , data collection , and genetic analyses .
he also provided critical insights for interpretation of our results and participated in revising of the manuscript .
leslie shaw contributed to the overall adni study design , data collection , and proteomic analyses .
he also provided critical insights for interpretation of our results and participated in revising of the manuscript .
john q. trojanowski contributed to the overall adni study design , data collection , and proteomic analyses .
he also provided critical insights for interpretation of our results and participated in revising of the manuscript .
william j. jagust contributed to the overall adni study design , data collection , and amyloid pet data analyses .
he also provided critical insights for interpretation of our results and participated in revising of the manuscript .
he has contributed to the overall adni study design , data collection , and data analyses .
he provided critical insights for interpretation of our results and participated in revising of the manuscript .
liana g. apostolova is the senior author of this manuscript and is responsible for the study concept and design .
she provided major oversight over all analyses , interpretation of results , and participated in writing of the manuscript .
data collection and sharing for this project was funded by the alzheimer 's disease neuroimaging initiative ( adni ) ( nih grant u01 ag024904 ) and dod adni ( department of defense award w81xwh-12 - 2 - 0012 ) .
adni is funded by the national institute on aging , the national institute of biomedical imaging and bioengineering , and through generous contributions from the following : alzheimer 's association ; alzheimer 's drug discovery foundation ; bioclinica , inc ; biogen idec , inc ; bristol - myers squibb company ; eisai , inc ; elan pharmaceuticals , inc ; eli lilly and company ; f. hoffmann - la roche ltd and its affiliated company genentech , inc ; ge healthcare ; innogenetics , n.v . ;
ixico ltd ; janssen alzheimer immunotherapy research & development , llc ; johnson & johnson pharmaceutical research & development llc ; medpace , inc ; merck & co , inc ; meso scale diagnostics , llc ; neurorx research ; novartis pharmaceuticals corporation ; pfizer , inc ; piramal imaging ; servier ; synarc , inc ; and takeda pharmaceutical company .
the canadian institutes of health research is providing funds to support adni clinical sites in canada .
the grantee organization is the northern california institute for research and education , and the study is coordinated by the alzheimer 's disease cooperative study at the university of california , san diego .
adni data are disseminated by the laboratory for neuro imaging at the university of southern california .
the csf biomarker and pib suvr analyses reported in this manuscript were funded by the easton consortium for alzheimer 's drug discovery and biomarker development , nia r01 ag040770 , nia k02 ag048240 , and nia p50 ag16570 .
andreas lazaris , kristy s. hwang , naria goukasian , leslie m. ramirez , jennifer eastman , and anna e. blanken report no disclosures .
edmond teng owns stock in general electric and cerner corporations ; has served on the editorial board for dementia and geriatric cognitive disorders ; and has received research support from eli lilly , biogen , genentech , merck , and k08 ag 34628 ( jointly sponsored by nia , afar , the john a. hartford foundation , the atlantic philanthropies , the starr foundation , and an anonymous donor ) .
karen gylys has served on the editorial board for acta neuropahtologica communications ; and has received research support from nih r01 ag027465 - 01a2 , p50 ag16570 , ucla alzheimer 's disease research center , and the lincy foundation .
greg cole holds patents for an fddnp pet probe for protein aggregates , curcumin formulation for enhanced bioavailability with lipidation and antioxidant stabilization , and medical food for brain health , provides unpaid advice to neurovision imaging , which is using his and coholders ' patent pending curcumin formulation for retinal imaging ; has received research support from nih nccih at006816 , greater la va , and grecc ; receives royalties for the following : fddnp ucla patent royalties , curcumin formulation / ucla / va patent royalties , and medical food ucla / va patent royalties ; and was a an expert witness for legal proceedings regarding whitewave ( makers of horizon milk ) .
andrew saykin serves as adni genetics core leader ; receives research support from siemens medical solutions and welch allyn , and from the following alzheimer 's disease related nih grants : r01 ag19771 , r01 ca101318 , r01 lm011360 , u01 ag032984 , rc2 ag036535 , and p30 ag10133 ; has served as a consultant to siemens healthcare , eli lilly , and arkley biotek ; has served on the advisory board for siemens healthcare and eli lilly ; has received honoraria from siemens healthcare ; and has served on the editorial board of brain imaging and behavior ( a springer journal ) .
leslie shaw has been a consultant to innogenetics and collaborates on quality assessment activities as part of the alzheimer 's disease neuroimaging initiative ; has been a consultant for janssen and novartis ; serves as consultant , member of advisory board and has received speaker fees and travel expenses from eli lilly and company ; has served on the editorial board for therapeutic drug monitoring ; and has received research support from eisai , nih , and the alzheimer 's disease neuroimaging initiative .
john q. trojanowski has served on the editorial board of alzheimer 's & dementia ; may accrue revenue on patents submitted by the university of pennsylvania wherein he is inventor including : modified avidin
biotin technique ; method of stabilizing microtubules to treat alzheimer 's disease ; method of detecting abnormally phosphorylated tau ; method of screening for alzheimer 's disease or disease associated with the accumulation of paired helical filaments ; compositions and methods for producing and using homogeneous neuronal cell transplants ; rat comprising straight filaments in its brain ; compositions and methods for producing and using homogeneous neuronal cell transplants to treat neurodegenerative disorders and brain and spinal cord injuries ; diagnostic methods for alzheimer 's disease by detection of multiple mrnas ; methods and compositions for determining lipid peroxidation levels in oxidant stress syndromes and diseases ; compositions and methods for producing and using homogenous neuronal cell transplants ; method of identifying , diagnosing , and treating alpha - synuclein positive neurodegenerative disorders ; mutation - specific functional impairments in distinct tau isoforms of hereditary frontotemporal dementia and parkinsonism linked to chromosome-17 : genotype predicts phenotype ; microtubule stabilizing therapies for neurodegenerative disorders ; and treatment of alzheimer 's and related diseases with an antibody ; is coinventor on patents submitted the university of pennsylvania wherein he is inventor that have generated income he has received from the sale of avid to eli lily including : amyloid plaque aggregation inhibitors and diagnostic imaging agents ; and has received research support from the marian s. ware alzheimer program and benaroya .
william j. jagust has served as a consultant to banner alzheimer institute , genentech , inc , synarc , janssen alzheimer immunotherapy , f. hoffman la roche , novartis , and siemens ; has served on the scientific advisory boards of genentech , inc and novartis ; has served on the editorial boards of frontiers in human neuroscience , annals of neurology , brain imaging and behavior , alzheimer 's disease and associated disorders , and neuroimage : clinical ; and has received research support from avid radiopharmaceuticals f - av-45-a14 , nih grants ag034570 , ag025303 , ag044292 , ag012435 , ag021028 , ag031563 , ag019724 , ag030048 , ag032306 , and ag024904 , and the tau consortium ( rainwater foundation ) .
michael w. weiner has served on the scientific advisory boards for pfizer , bolt international , neurotrope bioscience , alzheon , university of pennsylvania 's neuroscience of behavior initiative , national brain research center ( nbrc ) , india , learn program at university of north carolina , dolby family ventures , lp , adni , and eli lilly ; has provided consulting to synarc , pfizer , janssen , klj associates , easton associates , harvard university , university of california , los angeles ( ucla ) , alzheimer 's drug discovery foundation ( addf ) , neurotrope bioscience , avid radiopharmaceuticals , clearview healthcare partners , perceptive informatics , smartfish as , decision resources , inc , araclon , merck , defined health , howard university , bogen idec , boclinica , and genentech ; has received travel funding from pfizer , paul sabatier university , mci group france , travel edreams , inc , neuroscience school of advanced studies ( nsas ) , danone trading , bv , ctad ant congres , kenes , intl , adrc , ucla , ucsd , adcs , sanofi - aventis groupe , university center hospital , toulouse , araclon , ac immune , eli lilly , new york academy of sciences ( nyas ) , national brain research center , northwestern university , fidelity biosciences research initiative , university of pennsylvania , the alzheimer 's association , merck , adpd , alzheimer 's drug discovery foundation ( addf ) , tokyo university , kyoto university , cornell - weill university , rockafeller university , memorial sloan - kettering cancer center , biogen , and india for johns hopkins medicine ; has received honoraria from pfizer , tohoku university , consortium for multiple sclerosis centers ( cmsc ) , and danone trading , bv ; has received research support from merck and avid ; has been an employee of the university of california , san francisco and the san francisco va medical center ; has received research support from merck , avid , lilly , alzheimer 's diseases discovery foundation ( addf ) , the veterans administration ( va ) , department of defense , from the following grants : 2u01ag024904 , w81xwh-13 - 1 - 0259 , w81xwh-12 - 2 - 0012 , r01 ag10897 , 1p41 eb015904 , p01 ag19724 , r01 ag032306 , r01a g03879 , adni 2 - 12 - 233036 , 20110506 , r01 mh098062 - 01 , and from nih / nia / national institute of mental health , dod , alzheimer 's association , alzheimer 's drug discovery foundation , merck , avid , and the veterans administration ( va ) .
liana g. apostolova has served as a consultant to lilly and ge healthcare ; has received speaker honoraria from eli lilly ; has served on the editorial boards of alzheimer and dementia : diagnosis , assessment and disease monitoring ; has served on the speakers bureaus of eli lilly and ge healthcare ; and has received research support from ge healthcare and nia . | objective : we investigated the association between apoe protein plasma levels and brain amyloidosis and the effect of the top 10 alzheimer disease ( ad ) risk genes on this association.methods:our dataset consisted of 18 ad , 52 mild cognitive impairment , and 3 cognitively normal alzheimer 's disease neuroimaging initiative 1 ( adni1 ) participants with available [ 11c]-pittsburgh compound b ( pib ) and peripheral blood protein data .
we used cortical pattern matching to study associations between plasma apoe and cortical pib binding and the effect of carrier status for the top 10 ad risk genes.results:low plasma apoe was significantly associated with high pib suvr , except in the sensorimotor and entorhinal cortex . for bin1 rs744373 ,
the association was observed only in minor allele carriers . for cd2ap rs9349407 and cr1 rs3818361 ,
the association was preserved only in minor allele noncarriers .
we did not find evidence for modulation by clu , picalm , abca7 , bin1 , and ms4a6a.conclusions:our data show that bin1 rs744373 , cd2ap rs9349407 , and cr1 rs3818361 genotypes modulate the association between apoe protein plasma levels and brain amyloidosis , implying a potential epigenetic / downstream interaction . | METHODS
Standard protocol approvals, registrations, and patient consents.
PiB analyses.
Genetic analyses.
Plasma biomarkers.
Statistical analyses.
RESULTS
Demographic comparisons.
DISCUSSION
Supplementary Material
AUTHOR CONTRIBUTIONS
STUDY FUNDING
DISCLOSURE | the subset of 18 ad , 52 mci , and 3 cognitively normal adni participants who received [ c]-pib have been included in this study . the 3d association between apoe protein plasma levels and cortical pib suvrcbgm was studied using linear regression in the pooled sample using age and sex as covariates . next , we split the pooled sample based on the presence or absence of disease - associated alleles for cd2ap rs9349407 , cr1 rs3818361 , bin1 rs744373 , cd33 rs3826656 and clu rs9331888 and studied the associations of the plasma apoe levels and pib suvrcbgm separately in minor snp carriers and noncarriers while adjusting for age and sex . the subset of 18 ad , 52 mci , and 3 cognitively normal adni participants who received [ c]-pib have been included in this study . the 3d association between apoe protein plasma levels and cortical pib suvrcbgm was studied using linear regression in the pooled sample using age and sex as covariates . next , we split the pooled sample based on the presence or absence of disease - associated alleles for cd2ap rs9349407 , cr1 rs3818361 , bin1 rs744373 , cd33 rs3826656 and clu rs9331888 and studied the associations of the plasma apoe levels and pib suvrcbgm separately in minor snp carriers and noncarriers while adjusting for age and sex . plasma apoe levels showed an association with pib suvrcbgm only in cd2ap rs9349407 and cr1 rs3818361 minor allele noncarriers ( cd2ap rs9349407 noncarriers left pcorr = 0.003 , right pcorr = 0.004 ; cr1 rs3818361 noncarriers left pcorr = 0.008 , right pcorr = 0.01 , figure 1 and table 2 ) . after correcting for apoe 4 ,
the association between apoe protein plasma levels and pib binding remained for cd2ap ( left pcorr = 0.03 , right pcorr = 0.03 ) but not cr1 ( table 2 and figure e-1 ) . significant p values of the association between plasma apoe levels and cortical pib binding stratified by bin1 , cd2ap , and cr1 carrier status
all demographic variables and comparisons are shown in table 1 . plasma apoe levels showed an association with pib suvrcbgm only in cd2ap rs9349407 and cr1 rs3818361 minor allele noncarriers ( cd2ap rs9349407 noncarriers left pcorr = 0.003 , right pcorr = 0.004 ; cr1 rs3818361 noncarriers left pcorr = 0.008 , right pcorr = 0.01 , figure 1 and table 2 ) . after correcting for apoe 4 ,
the association between apoe protein plasma levels and pib binding remained for cd2ap ( left pcorr = 0.03 , right pcorr = 0.03 ) but not cr1 ( table 2 and figure e-1 ) . significant p values of the association between plasma apoe levels and cortical pib binding stratified by bin1 , cd2ap , and cr1 carrier status
our data show that bin1 rs744373 , cd2ap rs9349407 , and cr1 rs3818361 genotypes modulate the association between apoe protein plasma levels and brain amyloidosis . yet , when stratified by ad risk genotypes , plasma apoe showed a significant association with pib suvrcbgm only in noncarriers of the minor allele of cd2ap rs9349407 and cr1 rs3818361 and only in carriers of the bin1 rs744373 minor allele . in our study , the presence of the bin1 rs744373 risk variant was associated with preserved and potentially enhanced association between apoe protein plasma levels and pib binding , lending evidence for a potential downstream functional interaction between the bin1 and apoe genes . we found that the association between apoe protein plasma levels and pib binding is no longer present in minor allele carriers , indicating that cd2ap influences amyloid pathology . in this study
we observed that cr1 rs3818361 minor allele carrier status leads to a loss of the association between apoe protein plasma levels and pib binding , which disappeared in the presence of apoe 4 as covariate . leslie shaw has been a consultant to innogenetics and collaborates on quality assessment activities as part of the alzheimer 's disease neuroimaging initiative ; has been a consultant for janssen and novartis ; serves as consultant , member of advisory board and has received speaker fees and travel expenses from eli lilly and company ; has served on the editorial board for therapeutic drug monitoring ; and has received research support from eisai , nih , and the alzheimer 's disease neuroimaging initiative . | [
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] |
acetabular orientation refers to the direction of the acetabular cup relative to the axial and coronal planes .
this is an important factor in the determination of the appropriate type and extent of operations on the hips , such as an osteotomy of the pelvis in development dysplasia of the hip ( ddh ) , or total hip arthroplasty for osteoarthritis or trauma to the acetabulum .
some investigators have developed methods to evaluate the acetabular orientation , including x - ray examination , computed tomography ( ct ) , and magnetic resonance imaging ( mri ) .
when evaluating acetabular orientation , 2 important angles are widely used , including the acetabular anteversion angle ( aaa ) and the acetabular inclination angle ( aia ) .
they were measured on cadavers , x - ray films , 2-dimensional ct images , mri , and 3-dimensional ct reconstruction systems .
on ct imaging , the aaa is formed by a reference line perpendicular to the line connecting the posterior iliums and a line connecting the posterior and anterior margins of the acetabulum .
the aia is the angle between the face of the acetabular cup and the transverse axis .
however , there are still some shortcomings because the cartilage of the acetabulum and the femoral head can not be distinguished on ct and the information regarding the cartilaginous acetabulum is lost . in recent years , more and more studies focusing on mri evaluation of acetabular orientation are being reported .
mri overcomes the shortcomings of ct , since it shows the cartilage and bone more clearly and may provide more useful information than does ct when evaluating acetabular orientation .
douira - khomsi et al conducted a prospective study about mri evaluation of acetabular residual dysplasia in ddh , they demonstrated that mri promoted more accurate selection of patients for pelvic osteotomy and aided in the choice of the most appropriate type of osteotomy .
unfortunately , there are no mri data regarding the measurement of aaa and aia in normal children , making it difficult to differentiate normal from abnormal hips .
our study aims to investigate the change of aaa and aia with age in normal chinese children .
a retrospective study of the medical records of children in our hospital who underwent mri examination ( including the hip joint ) from january 2009 to december 2015 was performed .
this study was approved by the ethical committee of guangzhou women and children 's medical center ( no .
2015020904 ) . generally , these patients underwent mri examination because of diseases pertaining to proximal femur or pelvis , or lower limb pain , or congenital abnormality of the urogenital system .
the patients with a normal mri reading of the hip joint or diagnosis of mild synovitis of the hip were included in this study .
we excluded the patients with nonstandard positioning during mri examination or with other diseases of the hip such as femoral head necrosis , ddh , septic arthritis of the hip , fractures of acetabulum or femoral neck , or tuberculosis of the hip .
a total of 180 patients ( 81 girls and 99 boys ) aged from 6 months to 16 years met our criteria for this study and were eligible for final analysis .
mri examination was performed using a 1.5 t philips gyroscan achieva mri system ( philips , best , the netherlands ) with the standard examination including the whole pelvis and proximal femur with axial , sagittal , and coronal plane sequences .
the mri examination was carried out under sedation or general anesthesia with the patients placed supine , legs in a neutral position , and with a body array coil placed anterior and posterior to the pelvis .
the sequences included t1-weighted ( t1-w ) spin - echo ( se ) images ( repetition time / echo time [ tr / te ] 372/7 milliseconds ; time of acquisition 4 minutes 28 seconds ) in the coronal and sagittal planes and t2-weighted ( t2-w ) fast field - echo ( ffe ) ( repetition time 369 milliseconds ; echo time 14 milliseconds ; time of acquisition 3 minutes 16 seconds ; flip angle 25 ) in coronal and sagittal planes .
all sequences used a 22 cm field of view , 3 mm slice thickness , 0.4 mm slice gap , 384 - 384 matrix , and 2 for the number of excitations . using the mri images ,
the following bony and cartilage parameters were measured : the aaa , aia , the acetabular index ( ai ) , the axial section total femoral head coverage angle ( a - tca ) , and the coronal section total femoral head coverage angle ( c - tca ) .
the angles of a - tca and c - tca are parameters of the relationship of acetabulum and femoral head ; as the angle increases , the femoral head is covered by the acetabulum to a larger extent .
the method to measure aaa and aia were similar to the methods used for ct by stem et al .
for measuring bony angles , we used the bony margins of the acetabulum , whereas for cartilage angles , we used the cartilage margins of the acetabulum . figures 1 and 2
show the method . on t1-w se sequences in the axial plane , at the level of the center of the hip joint ( with a largest femoral head diameter or deepest point of the acetabulum ) , aaa was measured as the angle formed by a reference line perpendicular to a line connecting the posterior iliac wings or triradiate cartilage and a line connecting the posterior and anterior margins of the acetabulum .
the a - tca is formed by 2 lines connecting the center of the femoral head to the posterior and anterior margins of the acetabulum .
on t2-w ffe sequences in the coronal plane at the level of the center of the hip joint ( using images with the largest femoral head diameter or greatest depth of the acetabulum ) , the aia was measured as the angle formed by a reference line connecting the triradiate cartilage and a line connecting the superior and inferior margins of the acetabulum .
the ai was measured as the angle formed by the same reference line connecting the triradiate cartilages and a line connecting the center of the acetabulum and the superior acetabulum .
the c - tca is formed by the 2 lines connecting the center of the femoral head to the superior and inferior margins of the acetabulum . the measurement of bony and cartilage acetabular anteversion angle and axial section total femoral head coverage angle on axial section imaging of magnetic resonance imaging in an 8-year - old male .
the measurement of bony and cartilage acetabular inclination angle , acetabular index , and coronal section total femoral head coverage angle on coronal section imaging of magnetic resonance imaging in a 6-year - old male .
two persons ( orthopedic doctors ) measured these angles independently , and both of them were trained by a radiologist in our hospital before measuring , to make sure all measurements were accurate and repeatable . paired t tests
were used to compare the difference between 2 measurers , differences between bony and cartilage angles , as well as the difference between the values of left and right side .
one - way analysis of variance ( anova ) and multiple comparisons were used to compare the angles among the different age groups .
we show the results of the measurements of acetabular orientation angles in tables 1 and 2 .
sample means of bony aaa , aia , ai , a - tca , and c - tca in different age groups ( n = 180 ) .
sample means of cartilage aaa , aia , ai , a - tca , and c - tca in different age groups ( n = 180 ) .
interobserver agreement was high between the 2 orthopedic surgeons ( p = 0.352 ) .
. there were no differences ( p = 0.503 ) between the left and right side , and thus we combined the data and used the sample mean values .
there was no difference between male and female in bony and cartilage aaa , aia , ai , a - tca , and c - tca ( p > 0.05 ) .
the sample means of the bony aaa in the 6-month age group and the 14- to 16-year age group were 12.3 0.9 and 13.8 1.8 , respectively .
the total sample mean bony aaa was 12.2 2.5 , and there was no significant ( n.s . )
difference among the 14 different age groups ( p = 0.169 ) with analysis of variance .
the sample means of the bony aia in the 6-month age group and in the 14- to 16-year age group were 51.1 2.1 and 49.8 3.5 , respectively .
the total sample mean bony aia was 50.9 2.5 ; again there was no difference between the 14 age groups ( p = 0.103 ) .
the mean bony ai decreased significantly with age ( p < 0.01 ) .
the mean bony ai in the 6-month age group was 24.1 2.4 , decreasing to 12.5 2.3 in the 12- to 13-year age group .
the total sample mean cartilage aaa was 12.1 2.5 and there were n.s .
similar results were found for the cartilage aia ( p = 0.272 ) and cartilage ai ( p = 0.627 ) .
the total sample means of the cartilage aia and ai were 41.2 3.8 and 5.9 1.7 , respectively .
these results indicated that both cartilage aaa , aia , and ai remained unchanged with age .
no differences were found between bony and cartilage aaa ( p = 0.250 ) ; however , bony aia was significantly larger than cartilage aia ( p < 0.001 ) .
the bony a - tca and c - tca significantly change with age ( p < 0.001 ) .
the mean bony a - tca at 6 months was 117.0 5.8 , increasing to 144.5 4.6 in the 14- to 16-year age group .
the mean c - tca at 6 months was 127.5 5.1 , increasing to 140.7 2.5 at 14- to 16-year age group .
no differences were found in a - tca between the 6-month , 1- , 2- , 3- , 4- , and 5-year age groups ( n.s . ) .
however , the a - tca in the 6-month , 1- , 2- , 3- , and 4-year age groups were significantly lower than the a - tca in the remaining groups , and similar results were found for age - specific c - tca values .
the total sample means of cartilage a - tca and c - tca were 145.2 7.2 and 154.1 5.7 , both of them remained unchanged with age ( n.s . ) . in the 14- to 16-year age group ,
cartilage c - tca was significantly larger than bony c - tca ( p < 0.001 ) .
however , cartilage a - tca was similar when compared to bony a - tca ( p = 0.599 ) .
figures 3 and 4 are the comparisons of 0.5- and 12-year - old patients on bony and cartilage aaa , aia , ai , a - tca , and c - tca
comparisons of 0.5- and 12-year - old patients on bony acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle .
comparisons of 0.5- and 12-year - old patients on cartilage acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle .
they are the same patients of fig . 3 , and both of them are males .
the sample means of the bony aaa in the 6-month age group and the 14- to 16-year age group were 12.3 0.9 and 13.8 1.8 , respectively .
the total sample mean bony aaa was 12.2 2.5 , and there was no significant ( n.s . )
difference among the 14 different age groups ( p = 0.169 ) with analysis of variance .
the sample means of the bony aia in the 6-month age group and in the 14- to 16-year age group were 51.1 2.1 and 49.8 3.5 , respectively .
the total sample mean bony aia was 50.9 2.5 ; again there was no difference between the 14 age groups ( p = 0.103 ) .
the mean bony ai decreased significantly with age ( p < 0.01 ) .
the mean bony ai in the 6-month age group was 24.1 2.4 , decreasing to 12.5 2.3 in the 12- to 13-year age group .
the total sample mean cartilage aaa was 12.1 2.5 and there were n.s .
similar results were found for the cartilage aia ( p = 0.272 ) and cartilage ai ( p = 0.627 ) .
the total sample means of the cartilage aia and ai were 41.2 3.8 and 5.9 1.7 , respectively .
these results indicated that both cartilage aaa , aia , and ai remained unchanged with age .
no differences were found between bony and cartilage aaa ( p = 0.250 ) ; however , bony aia was significantly larger than cartilage aia ( p < 0.001 ) .
the bony a - tca and c - tca significantly change with age ( p < 0.001 ) .
the mean bony a - tca at 6 months was 117.0 5.8 , increasing to 144.5 4.6 in the 14- to 16-year age group .
the mean c - tca at 6 months was 127.5 5.1 , increasing to 140.7 2.5 at 14- to 16-year age group .
no differences were found in a - tca between the 6-month , 1- , 2- , 3- , 4- , and 5-year age groups ( n.s . ) .
however , the a - tca in the 6-month , 1- , 2- , 3- , and 4-year age groups were significantly lower than the a - tca in the remaining groups , and similar results were found for age - specific c - tca values .
the total sample means of cartilage a - tca and c - tca were 145.2 7.2 and 154.1 5.7 , both of them remained unchanged with age ( n.s . ) . in the 14- to 16-year age group ,
cartilage c - tca was significantly larger than bony c - tca ( p < 0.001 ) .
however , cartilage a - tca was similar when compared to bony a - tca ( p = 0.599 ) .
figures 3 and 4 are the comparisons of 0.5- and 12-year - old patients on bony and cartilage aaa , aia , ai , a - tca , and c - tca .
comparisons of 0.5- and 12-year - old patients on bony acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle .
comparisons of 0.5- and 12-year - old patients on cartilage acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle .
they are the same patients of fig . 3 , and both of them are males .
in this study , we investigated the changes of acetabular orientation using mri in children .
this is not a cross - sectional population study , and thus there is potential bias in the selection of patients .
we believe that our study is the first to measure the acetabular orientation on mri in normal children during the course of skeletal maturation .
the results of our study provide important information in understanding the normal growth of the hip joint and the variation between individuals .
it will assist us to evaluate the hip with what appears to be an abnormal acetabular anteversion and acetabular inclination on bony and cartilage .
in addition , it will also help us to make operation plans pertaining to the hip , providing more accurate information for the selection of patients for pelvic osteotomy and the choice of the most appropriate type of osteotomy . in our study ,
the overall sample mean bony and cartilage aaa were 12.2 and 12.1 , respectively , and both of them did not change among different age groups in normal chinese children .
so far , few studies have been conducted on the acetabular orientation in normal children .
mckibbin measured the aaa on 15 pelvises from fresh infant cadavers , and their sample means for the aaa in normal male and female neonatal hips were 6 and 9 , respectively .
jacquemier et al measured the aaa on ct scans in 143 normal children aged from 1 to 15 years and reported a mean aaa of 13 which remained constant during growth .
our bony and cartilage aaa measurements agree well with those reported by jacquemier et al and weiner et al , but differ from those reported by mckibbin .
the mean bony and cartilage aaa measured in our sample of children at 6 months were 12.3 and 13.6 on mri , respectively .
in addition , no differences were observed between the 6-month and the 1- or 2-year age groups .
first , in the study of mckibbin , they measured aaa on cadavers . although it may reflect the cartilaginous aaa as measured in our study , it probably may be different from an aaa measured by either ct or mri .
in addition , mckibbin measured aaa on fresh newborn cadavers ( died within the first 2 weeks of life ) .
their measurements may be different from our measured aaa at the age of about 6 months .
although we did not measure the bony aaa in patients older than 16 years , some studies indicated a significantly increased bony aaa in adults ( table 3 ) .
furthermore , our results are more similar to the results of jacquemier et al and weiner et al , indicating that there is probably no difference between chinese children and other ethnicities regarding aaa .
our study also showed that the bony and cartilage aia remained constant during skeletal maturation in normal chinese children until 14 to 16 years of age .
the mean bony and cartilage aia in our sample of apparently normal children were 50.9 and 41.2 on mri , respectively .
to our knowledge , there are no studies focusing on aia in normal children using mri measurements . however , there are some studies reporting on the bony aia in adults by other imaging methods . in the study by stem
et al , the mean aia in their adult sample was 39. suo et al evaluated acetabular orientation on x - ray films of 40 hips , and the average aia was 44.1. in a study by fukui et al , 25 cadavers ( 31 hips ) were used to examine the orientation of the cup component aligned with the transverse acetabulum ligament .
the average radiographic aia was 43.5. the mean bony aia in our sample of apparently normal children was 50.9 and somewhat larger than the results reported by fukui et al , but the cartilage aia in our study ( 41.2 ) is similar to them . considering the adult studies mentioned , we hypothesize that there may be a continued decrease in the aia after skeletal maturity , this may be attributed to the ossification of acetabular cartilage at the outer margin of acetabulum , and therefore the final bony aia resembles the cartilage aia .
our study also indicates that cartilage ai remains unchanged with age , but the bony ai decreases with age , in similarity to the results of other studies . in the study of huber
et al , who analyzed 115 hips , the bony ai decreased until the age group of 4 to 6 years and then remained relatively constant around 15 , and the cartilage ai ( around 5 ) stayed relatively constant until the age of 13 years .
tonnis measured the ai on x - ray films in a large number of normal hips , and the mean ai was about 30 at the time of birth and 15 at the age of 5 to 7 years .
shi et al measured the ai on 2333 apparently normal x - ray films of the pelvis , and the mean ai was 28.39 in the newborn period and 12.80 at the age of 10 years . in our study , the bony ai measured by mri in normal children at the age of 6 months was 24.1 and decreased to 12.5 in the 12- to 13-year - old age group , in agreement with the results of tonnis and shi . some factors may contribute to the stable value of the aaa and the aia in our sample of children .
the acetabulum is formed by 3 bones : ilium ( superiorly ) , ischium ( inferiorly ) , and pubis ( anteriorly ) .
the triradiate cartilage is the center of the acetabulum and connects the 3 bones , subsequently forming the acetabular cup .
the growth of acetabulum is closely associated with the process of enchondral ossification in the acetabulum .
it has been demonstrated that biomechanical loading regulates enchondral ossification , and thus biomechanical factors play a role in the development of acetabulum , including acetabular orientation . in normal hips
, there is a congruent relationship of the acetabulum with the femoral head , and compressive stress is distributed across the acetabulum during endochondal ossification .
this may lead to a very balanced growth of ilium , ischium , and pubis within the cartilaginous acetabulum which maintains a stable acetabular orientation .
their results indicated that changes in the ossification of the acetabular anterior and posterior walls were minimal and resulted in only minor variations in acetabular anteversion with growth .
we believe that the ossification of the acetabular labral cartilage may account for the changes that appear to occur in the aaa and aia after skeletal maturity .
our study showed that the bony femoral head coverage increased with age , but the cartilage femoral head coverage remained unchanged from 6 months to 16 years of age .
generally , as the acetabulum is growing , the cup becomes larger in diameter and deepens , and a larger portion of the femoral head is thus covered by the acetabulum .
however , this increase of coverage area may not change the rate of femoral head coverage . from our study
, we consider that the increase of bony femoral head coverage rate may be attributed to the ossification of the acetabular cartilage at the acetabulum margin , as the bony a - tca at 14 to 16 years ( 144.5 ) was similar to cartilage a - tca ( 145.2 ) .
interestingly , our study also indicated that the bony femoral head coverage angles ( a - tca and c - tca ) remained stable until 4 to 5 years of age , but significantly increased with age afterward .
although the patients with a normal mri reading of the hip joint were included , they were not healthy children and had diseases pertaining to proximal femur or pelvis , or lower limb pain , or congenital abnormality of the urogenital system .
in fact , it would be more clear if measured on t2-w ffe . both of them might influence the results of measurements .
we found a mean bony and cartilage aaa of 12.2 2.5 and 12.1 2.5 ; and a mean bony and cartilage aia of 50.9 2.5 and 41.2 3.8 in an apparently normal sample of 180 chinese children .
the bony coverage of the femoral head by the acetabulum increases with age , but the cartilage coverage of the femoral head remains stable up to 14 to 16 year of age . | abstractthere are no data regarding the acetabular orientation on magnetic resonance imaging ( mri ) ; this study investigates the changes of acetabular orientation with age in normal chinese children.we retrospectively analyzed the medical records of children who underwent hip mri examination at our hospital from january 2009 to december 2015 .
a total of 180 patients with normal mri reading of the hip joints were included and were divided into 14 groups according to age : from 6 months of age and then for each year from 1 to 16 years .
the bony and cartilage acetabular anteversion angle ( aaa ) , acetabular inclination angle ( aia ) , and acetabular index ( ai ) were measured .
total bony and cartilage femoral head coverage angles were measured on axial section total femoral head coverage angle ( a - tca ) and coronal section total femoral head coverage angle ( c - tca).the mean bony aaa and aia were 12.2 2.5 and 50.9 2.5 , respectively ; both of them stayed constant from the age of 6 months to 16 years .
similar results were found in cartilage aaa ( 12.1 2.5 ) and aia ( 41.2 3.0 ) .
there was no difference between bony and cartilage aaa , but bony aia was significantly larger than cartilage aia .
bony ai was 24.1 2.4 at the age of 6 months , decreasing to 12.5 2.3 by 12 to 13 years of age ; cartilage ai ( 5.9 1.7 ) maintained a steady value with age .
the mean bony a - tca and c - tca at 6 months were 117.0 5.8 and 127.5 5.1 , increasing to 144.5 4.6 and 140.7 2.5 at the age of 16 years .
however , the cartilage a - tca ( 145.2 7.2 ) and c - tca ( 154.1 5.7 ) did not change significantly with age.both bony and cartilage aaa and aia remain constant up to the age of 16 years in normal chinese pediatric population . although the cartilage coverage of femoral head by the acetabulum remains unchanged with age , the bony coverage of femoral head increases . | Introduction
Materials and methods
Results
Results of bony and cartilage AAA, AIA, and AI
Result of bony and cartilage total femoral head coverage angle
Discussion
Conclusion | using the mri images ,
the following bony and cartilage parameters were measured : the aaa , aia , the acetabular index ( ai ) , the axial section total femoral head coverage angle ( a - tca ) , and the coronal section total femoral head coverage angle ( c - tca ) . the measurement of bony and cartilage acetabular inclination angle , acetabular index , and coronal section total femoral head coverage angle on coronal section imaging of magnetic resonance imaging in a 6-year - old male . no differences were found between bony and cartilage aaa ( p = 0.250 ) ; however , bony aia was significantly larger than cartilage aia ( p < 0.001 ) . the mean bony a - tca at 6 months was 117.0 5.8 , increasing to 144.5 4.6 in the 14- to 16-year age group . the mean c - tca at 6 months was 127.5 5.1 , increasing to 140.7 2.5 at 14- to 16-year age group . the total sample means of cartilage a - tca and c - tca were 145.2 7.2 and 154.1 5.7 , both of them remained unchanged with age ( n.s . ) figures 3 and 4 are the comparisons of 0.5- and 12-year - old patients on bony and cartilage aaa , aia , ai , a - tca , and c - tca
comparisons of 0.5- and 12-year - old patients on bony acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle . comparisons of 0.5- and 12-year - old patients on cartilage acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle . no differences were found between bony and cartilage aaa ( p = 0.250 ) ; however , bony aia was significantly larger than cartilage aia ( p < 0.001 ) . the mean bony a - tca at 6 months was 117.0 5.8 , increasing to 144.5 4.6 in the 14- to 16-year age group . the mean c - tca at 6 months was 127.5 5.1 , increasing to 140.7 2.5 at 14- to 16-year age group . the total sample means of cartilage a - tca and c - tca were 145.2 7.2 and 154.1 5.7 , both of them remained unchanged with age ( n.s . ) comparisons of 0.5- and 12-year - old patients on bony acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle . comparisons of 0.5- and 12-year - old patients on cartilage acetabular anteversion angle , acetabular inclination angle , acetabular index , axial section total femoral head coverage angle , and coronal section total femoral head coverage angle . in our study ,
the overall sample mean bony and cartilage aaa were 12.2 and 12.1 , respectively , and both of them did not change among different age groups in normal chinese children . from our study
, we consider that the increase of bony femoral head coverage rate may be attributed to the ossification of the acetabular cartilage at the acetabulum margin , as the bony a - tca at 14 to 16 years ( 144.5 ) was similar to cartilage a - tca ( 145.2 ) . interestingly , our study also indicated that the bony femoral head coverage angles ( a - tca and c - tca ) remained stable until 4 to 5 years of age , but significantly increased with age afterward . we found a mean bony and cartilage aaa of 12.2 2.5 and 12.1 2.5 ; and a mean bony and cartilage aia of 50.9 2.5 and 41.2 3.8 in an apparently normal sample of 180 chinese children . the bony coverage of the femoral head by the acetabulum increases with age , but the cartilage coverage of the femoral head remains stable up to 14 to 16 year of age . | [
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the study was approved by the danish data protection agency ( 2007 - 58 - 0015/local j.nr .
register - based studies in which persons can not be identified do not need ethics approval in denmark .
all danish citizens are medically covered by a tax - financed system that provides each citizen with equal health care free of copayment . for administrative purposes ,
the government has kept records of all hospitalizations ( in - hospital and outpatient visits ) since 1978 ( in the danish national patient registry ) and records of all prescription claims from danish pharmacies since 1995 ( in the danish register of medicinal product statistics ) .
furthermore , registration of all births and deaths , including dates of occurrence , has been complete for all citizens since 1968 ( in the national population register ) .
because all danish citizens are given a permanent personal civil registration number at time of birth or immigration , they can be followed up by individual - level linkage of the registers . for the present study
, we included all persons in denmark between 1997 and 2011 who were aged 70 years at the time when they claimed their first prescription of antipsychotic medication and who had previously been treatment - nave ( ie , allowing for a look back to 1995 , when the danish register of medicinal product statistics was established ) .
we identified comorbidities throughout the whole observational period by continuous screening of the danish national patient register .
because most diagnoses were for chronic conditions , people were classified as having the disease from the first day of diagnosis .
for all diagnoses but dementia , schizophrenia , and parkinson s disease , only validated in - hospital diagnoses were considered.16 in addition to previously validated chronic comorbid diagnoses , we retrieved information on acute delirium based on in - hospital diagnoses .
because the diagnoses of dementia , parkinson s disease , and schizophrenia are often based on outpatient visits , diagnoses from both in - hospital and outpatient visits were considered for these comorbidities .
the primary outcomes were mace , comprising the first occurring nonfatal acute myocardial infarction ( international classification of diseases , 10th revision [ icd-10 ] code i21 ) or nonfatal ischemic stroke ( icd-10 codes i63 and i64 [ i64 denotes unspecified stroke , of which the majority are of ischemic origin])17 and cardiovascular mortality ( icd-10 codes i00 to i99 ) and non cardiovascular - related mortality ( causes of mortality were obtained from the danish causes of death register ) . from the national register of medicinal product statistics , we identified exposure to the individual aps outlined in table1 . for these agents and for vitamin k antagonists ( b01aa03 ) , clopidogrel ( b01ac04 ) , low - dose aspirin ( b01ac06 ) , and loop diuretics ( c03ca01 , c03ca02 ) , treatment was updated continuously , that is , patients were considered to be exposed only while covered by claimed prescriptions . to determine treatment length and average daily dosages ,
we created an algorithm for each agent in which minimum , maximum , and typical daily dosages of used medication were defined .
for patients first claimed prescriptions , the typical daily dosage was assigned , and treatment length was calculated by dividing the amount of claimed medications by that daily dosage . for patients who were covered by a previous prescription at the time of claiming a new prescription ,
the daily dosage was reset , and a new daily dosage was calculated as the amount of claimed medications during the previous period divided by time between prescription claims ( based on up to 3 previous prescriptions without treatment breaks ) .
if calculated dosages exceeded the predefined highest daily dosages , patients were assigned the maximum dosages , and exceeding tablets were assumed to be stored and consumed during the immediate period following the end of the last prescription .
classification of the different antipsychotic agents for other medications ( ie , dementia medications [ atc code n06da ] , beta blockers [ c07 ] , thiazides [ c03a ] , calcium channel blockers [ c08 ] , digoxin [ c01aa ] , renin - angiotensin system inhibitors [ c09 ] , aldosterone blockers [ c03d ] , and statins [ c10aa ] ) , treatment status was updated every 30 days for the whole observational period .
persons were considered to be in treatment with the different medications if they claimed at least 1 prescription during the preceding 4 months ( this pragmatic method was applied because of computational limitations ) .
all patients were followed from the date of first claimed prescription until date of death , emigration , or december 31 , 2011 .
the incidence rate ratios ( irrs ) associated with exposure to aps were analyzed using multivariable poisson regression models .
the following variables were included as time - dependent variables : treatment with antipsychotic medications ; all comorbidities ( schizophrenia , parkinson s disease , dementia , acute delirium , prior cerebrovascular disease , heart failure , acute myocardial infarction , peripheral vascular disease , chronic obstructive pulmonary disease , atrial fibrillation , moderate or severe renal disease , diabetes , cancer , and cancer with metastases ; for icd-10 codes , see table2 ) ; age ; and use of warfarin , aspirin , clopidogrel , loop diuretics , thiazides , renin - angiotensin system blockers , aldosterone antagonists , statins , beta blockers , calcium channel blockers , or digoxin .
all models were adjusted for the aforementioned variables and for actual calendar year and sex .
use of antipsychotic medications was included as a time - dependent multilevel categorical variable for which people using > 1 medication for a given time were grouped in a separate use of multiple medication category .
people who were not covered by a claimed prescription were assigned to a no treatment category .
for all comparisons , use of risperidone as monotherapy served as the referent group ( because this group was the largest quantitatively ) .
, we created 3 different categorical variables for antipsychotic use according to time elapsed since first treatment because time dependency for risks was present ( p for interaction between time elapsed and associated risks < 0.0001 ; use of medications was split into the first 30 days , 31 to 365 days , and > 365 days after treatment initiation ) . after having explored the irrs among different medications in different time periods
, the differences appeared to be rather small between the periods ; therefore , in subsequent models , we included a variable reflecting only time since first treatment initiation to account for time dependency in risks . in the second models , we tested for effect modification between the different treatment groups and prevalent dementia ( diagnosis or use of dementia medications ) and cvd ( myocardial infarction , congestive heart failure , stroke , or peripheral vascular disease ) by inclusion of an interaction term between the categorical treatment variable and cvd or dementia , respectively .
because these were highly significant , we created a categorical dummy variable stratifying use of medications by the prevalence of the different diseases .
icd-10 codes for various medical conditions icd-10 indicates international classification of diseases , 10th revision .
for the present study , we included all persons in denmark between 1997 and 2011 who were aged 70 years at the time when they claimed their first prescription of antipsychotic medication and who had previously been treatment - nave ( ie , allowing for a look back to 1995 , when the danish register of medicinal product statistics was established ) .
we identified comorbidities throughout the whole observational period by continuous screening of the danish national patient register .
because most diagnoses were for chronic conditions , people were classified as having the disease from the first day of diagnosis .
for all diagnoses but dementia , schizophrenia , and parkinson s disease , only validated in - hospital diagnoses were considered.16 in addition to previously validated chronic comorbid diagnoses , we retrieved information on acute delirium based on in - hospital diagnoses . because the diagnoses of dementia , parkinson s disease , and schizophrenia are often based on outpatient visits , diagnoses from both in - hospital and outpatient visits were considered for these comorbidities .
the primary outcomes were mace , comprising the first occurring nonfatal acute myocardial infarction ( international classification of diseases , 10th revision [ icd-10 ] code i21 ) or nonfatal ischemic stroke ( icd-10 codes i63 and i64 [ i64 denotes unspecified stroke , of which the majority are of ischemic origin])17 and cardiovascular mortality ( icd-10 codes i00 to i99 ) and non cardiovascular - related mortality ( causes of mortality were obtained from the danish causes of death register ) .
from the national register of medicinal product statistics , we identified exposure to the individual aps outlined in table1 . for these agents and for vitamin k antagonists ( b01aa03 ) , clopidogrel ( b01ac04 ) , low - dose aspirin ( b01ac06 ) , and loop diuretics ( c03ca01 , c03ca02 ) , treatment was updated continuously , that is , patients were considered to be exposed only while covered by claimed prescriptions . to determine treatment length and average daily dosages ,
we created an algorithm for each agent in which minimum , maximum , and typical daily dosages of used medication were defined .
for patients first claimed prescriptions , the typical daily dosage was assigned , and treatment length was calculated by dividing the amount of claimed medications by that daily dosage . for patients who were covered by a previous prescription at the time of claiming a new prescription ,
the daily dosage was reset , and a new daily dosage was calculated as the amount of claimed medications during the previous period divided by time between prescription claims ( based on up to 3 previous prescriptions without treatment breaks ) . if calculated dosages exceeded the predefined highest daily dosages , patients were assigned the maximum dosages , and exceeding tablets were assumed to be stored and consumed during the immediate period following the end of the last prescription .
classification of the different antipsychotic agents for other medications ( ie , dementia medications [ atc code n06da ] , beta blockers [ c07 ] , thiazides [ c03a ] , calcium channel blockers [ c08 ] , digoxin [ c01aa ] , renin - angiotensin system inhibitors [ c09 ] , aldosterone blockers [ c03d ] , and statins [ c10aa ] ) , treatment status was updated every 30 days for the whole observational period .
persons were considered to be in treatment with the different medications if they claimed at least 1 prescription during the preceding 4 months ( this pragmatic method was applied because of computational limitations ) .
all patients were followed from the date of first claimed prescription until date of death , emigration , or december 31 , 2011 . the incidence rate ratios ( irrs ) associated with exposure to aps
the following variables were included as time - dependent variables : treatment with antipsychotic medications ; all comorbidities ( schizophrenia , parkinson s disease , dementia , acute delirium , prior cerebrovascular disease , heart failure , acute myocardial infarction , peripheral vascular disease , chronic obstructive pulmonary disease , atrial fibrillation , moderate or severe renal disease , diabetes , cancer , and cancer with metastases ; for icd-10 codes , see table2 ) ; age ; and use of warfarin , aspirin , clopidogrel , loop diuretics , thiazides , renin - angiotensin system blockers , aldosterone antagonists , statins , beta blockers , calcium channel blockers , or digoxin .
all models were adjusted for the aforementioned variables and for actual calendar year and sex .
use of antipsychotic medications was included as a time - dependent multilevel categorical variable for which people using > 1 medication for a given time were grouped in a separate use of multiple medication category .
people who were not covered by a claimed prescription were assigned to a no treatment category .
for all comparisons , use of risperidone as monotherapy served as the referent group ( because this group was the largest quantitatively ) .
in the first , we created 3 different categorical variables for antipsychotic use according to time elapsed since first treatment because time dependency for risks was present ( p for interaction between time elapsed and associated risks < 0.0001 ; use of medications was split into the first 30 days , 31 to 365 days , and > 365 days after treatment initiation ) . after having explored the irrs among different medications in different time periods
, the differences appeared to be rather small between the periods ; therefore , in subsequent models , we included a variable reflecting only time since first treatment initiation to account for time dependency in risks . in the second models , we tested for effect modification between the different treatment groups and prevalent dementia ( diagnosis or use of dementia medications ) and cvd ( myocardial infarction , congestive heart failure , stroke , or peripheral vascular disease ) by inclusion of an interaction term between the categorical treatment variable and cvd or dementia , respectively .
because these were highly significant , we created a categorical dummy variable stratifying use of medications by the prevalence of the different diseases .
icd-10 codes for various medical conditions icd-10 indicates international classification of diseases , 10th revision .
we identified 91 774 persons ( mean age 827 years , 35 474 [ 39% ] were men ) .
numbers of those ever exposed to the different aps are presented in table3 , along with the baseline characteristics for different exposure groups .
the different exposure groups had comparable age , sex , and comorbidity burdens , although patients receiving levomepromazine and haloperidol had higher prevalence of cancers ( 30% versus 10% for the other groups ) .
numbers are presented as counts ( % ) for discrete variables and means ( sd ) for continuous variables .
total exposure time , mace , and noncardiovascular mortality for each treatment group are given in tables4 and 5 .
crude event rates were lowest for flupentixol and chlorprothixen compared with the other ap groups . as seen in the tables , for all agents , we observed a highly increased incidence rate of mace and noncardiovascular mortality during the first month after initiation , with a subsequent decline for longer treatment durations .
associated adjusted irrs were increased for treatment with levomepromazine or haloperidol and treatment with multiple medications , whereas treatment with flupentixol , ziprasidone , chlorprothixen , or quetiapine was associated with a significantly lower irr compared with treatment with risperidone , as shown in figure1 ( mace ) and figure2 ( noncardiovascular mortality ) . for longer treatment duration , levomepromazine and use of multiple medications
were no longer associated with increased risks compared with risperidone monotherapy ( figures1c and 2c ) .
the risks associated with use of different antipsychotic medications differed for patients with and without cvd and were greater among patients with established cvd ( p for interaction between treatment group and cvd < 0.00001 ) ( figure3 .
similarly , the risks were slightly higher among patients without established dementia compared with patients with dementia ( p for interaction between treatment group and dementia < 0.0001 ) ( figure4 .
numbers of major adverse cardiovascular events , exposure time , and crude incidence rates for different antipsychotic agents numbers of noncardiovascular mortality , exposure time , and crude incidence rates for different antipsychotic agents multivariable - adjusted risks of mace for different treatment regimens in different time periods after treatment initiation : ( a ) first 30 days , ( b ) 31 to 365 days , ( c ) > 365 days .
multivariable - adjusted risks of noncardiovascular mortality for different treatment regimens in different time periods after treatment initiation : ( a ) first 30 days , ( b ) 31 to 365 days , ( c ) > 365 days .
multivariable - adjusted risks of ( a ) mace and ( b ) noncardiovascular mortality stratified by prevalent cvd .
cv indicates cardiovascular ; cvd , cardiovascular disease ; mace , major adverse cardiovascular events .
multivariable - adjusted risks of ( a ) mace and ( b ) noncardiovascular mortality stratified by prevalent dementia .
in this danish study of all persons without prior use of aps who were aged 70 years , we investigated the association between different aps and risks of mace and noncardiovascular mortality . in general , we observed that use of haloperidol or levomepromazine and treatment with multiple medications were associated with similar or greater irrs than use of risperidone , whereas treatment with flupentixol , ziprasidone , chlorprothixen , or quetiapine was associated with significantly lower irrs compared with treatment with risperidone .
the absolute event rates were highest shortly after treatment initiation and declined with long - term use .
after findings of increased risk of cerebrovascular events associated with use of second - generation aps,10,18,19 the us food and drug administration ( fda ) issued a warning in 2005 against use of second - generation aps in dementia.18,20 in 2008 , the fda extended this warning to include use of first - generation aps in dementia.21 several studies have since demonstrated increased mortality with use of aps in dementia,8,10,22 and some studies have shown increased mortality in elderly patients regardless of dementia status.9,11,19 previous studies have also suggested that the use of aps is associated with increased risk of myocardial infarction among older patients with treated dementia13 and that exposure to aps may be a trigger for stroke.11 furthermore , a cochrane review estimated the risk of cerebrovascular events to be > 3-fold higher in risperidone- versus placebo - treated elderly patients with dementia.23 our observations suggest that medications belonging to the second- and first - generation classes of aps may be associated with more or less comparable risk in elderly patients with or without dementia , although flupentixol , chlorprothixen , and quetiapine may be particularly associated with lower mace and mortality rates than risperidone .
for all drugs , we found that the incidence rates of mace and noncardiovascular mortality were highest during the first 30 days of treatment and declined with longer exposure time .
this finding is consistent with the findings of other studies.11,22,24 when elderly patients are prescribed aps , it may be due to neuropsychiatric symptoms in dementia or to delirium as part of a somatic disease that carries a high mortality risk .
because we were unable to accurately control for several diseases in our study , it can not be excluded that the higher risks observed , especially among nondemented elderly people who were treated , may be partly due to confounding by indication .
delirium , however , is an acute disease with high short - term mortality ; therefore , associations with long - term use may be less prone to confounding than the analyses of short - term use .
worth emphasizing in this context is that the relative risk of mortality with levomepromazine and haloperidol shortly after treatment initiation was higher than that observed for other aps .
a very high proportion of patients using these drugs had diagnosed cancer ; therefore , we can not rule out that the irr for the first period after treatment initiation was driven by confounding by indication .
even when excluding patients with a preexisting cancer , haloperidol has been shown to be associated with the highest risk of death among aps users in nursing homes.24 second - generation aps have been promoted for having a low risk of extrapyramidal symptoms compared with first - generation aps .
previous studies suggest that both first- and second - generation aps can lower blood pressure , leading to increased risk of falls and to a varying extent to prolonged qt interval , which might lead to arrhythmia and death.2528 another problem related to second - generation aps is adverse influence on glucose metabolism , diabetes , and blood lipid composition , leading to increased cardiovascular risks with long - term use.29,30 several prior observational studies have compared the safety of first- versus second - generation aps6,8,9,11,22,19,3133 and generally have indicated that the risk of death and/or mace when using second - generation aps is at least as high as that for first - generation aps.21 at least 1 retrospective cohort study has suggested that in older adults with dementia , second- and first - generation aps were associated with comparable risks of ischemic stroke.31 a larger retrospective cohort study in 2009 also suggested a comparable dose - related increased risk of sudden cardiac death for first- and second - generation aps.12 the main strength and novelty of this study is that we compared mace and noncardiovascular mortality risk among individual aps across the first and second generations in a rather large group of patients aged 70 years . moreover , because medical care is offered to all danish citizens without copayment and because medications are reimbursed by the government , selection bias related to socioeconomic status and , for example , prior participation in the labor market is likely less pronounced compared with many other observational studies .
another strength of our study was the sample size , which allowed us to examine the effect of individual drugs , although it must be acknowledged that we might still have lacked power to firmly conclude anything about some of the individual drugs ( particularly ziprasidone ) .
we lacked data on several clinical variables including blood pressure , hematological profiles , electrocardiograms , and indication for treatment .
all of these variables may have influenced clinicians decisions of whether to prescribe a particular agent .
after findings of increased risk of cerebrovascular events associated with use of second - generation aps,10,18,19 the us food and drug administration ( fda ) issued a warning in 2005 against use of second - generation aps in dementia.18,20 in 2008 , the fda extended this warning to include use of first - generation aps in dementia.21 several studies have since demonstrated increased mortality with use of aps in dementia,8,10,22 and some studies have shown increased mortality in elderly patients regardless of dementia status.9,11,19 previous studies have also suggested that the use of aps is associated with increased risk of myocardial infarction among older patients with treated dementia13 and that exposure to aps may be a trigger for stroke.11 furthermore , a cochrane review estimated the risk of cerebrovascular events to be > 3-fold higher in risperidone- versus placebo - treated elderly patients with dementia.23 our observations suggest that medications belonging to the second- and first - generation classes of aps may be associated with more or less comparable risk in elderly patients with or without dementia , although flupentixol , chlorprothixen , and quetiapine may be particularly associated with lower mace and mortality rates than risperidone .
for all drugs , we found that the incidence rates of mace and noncardiovascular mortality were highest during the first 30 days of treatment and declined with longer exposure time .
this finding is consistent with the findings of other studies.11,22,24 when elderly patients are prescribed aps , it may be due to neuropsychiatric symptoms in dementia or to delirium as part of a somatic disease that carries a high mortality risk .
because we were unable to accurately control for several diseases in our study , it can not be excluded that the higher risks observed , especially among nondemented elderly people who were treated , may be partly due to confounding by indication .
delirium , however , is an acute disease with high short - term mortality ; therefore , associations with long - term use may be less prone to confounding than the analyses of short - term use .
worth emphasizing in this context is that the relative risk of mortality with levomepromazine and haloperidol shortly after treatment initiation was higher than that observed for other aps .
a very high proportion of patients using these drugs had diagnosed cancer ; therefore , we can not rule out that the irr for the first period after treatment initiation was driven by confounding by indication .
even when excluding patients with a preexisting cancer , haloperidol has been shown to be associated with the highest risk of death among aps users in nursing homes.24
second - generation aps have been promoted for having a low risk of extrapyramidal symptoms compared with first - generation aps .
previous studies suggest that both first- and second - generation aps can lower blood pressure , leading to increased risk of falls and to a varying extent to prolonged qt interval , which might lead to arrhythmia and death.2528 another problem related to second - generation aps is adverse influence on glucose metabolism , diabetes , and blood lipid composition , leading to increased cardiovascular risks with long - term use.29,30 several prior observational studies have compared the safety of first- versus second - generation aps6,8,9,11,22,19,3133 and generally have indicated that the risk of death and/or mace when using second - generation aps is at least as high as that for first - generation aps.21 at least 1 retrospective cohort study has suggested that in older adults with dementia , second- and first - generation aps were associated with comparable risks of ischemic stroke.31 a larger retrospective cohort study in 2009 also suggested a comparable dose - related increased risk of sudden cardiac death for first- and second - generation aps.12
the main strength and novelty of this study is that we compared mace and noncardiovascular mortality risk among individual aps across the first and second generations in a rather large group of patients aged 70 years .
moreover , because medical care is offered to all danish citizens without copayment and because medications are reimbursed by the government , selection bias related to socioeconomic status and , for example , prior participation in the labor market is likely less pronounced compared with many other observational studies .
another strength of our study was the sample size , which allowed us to examine the effect of individual drugs , although it must be acknowledged that we might still have lacked power to firmly conclude anything about some of the individual drugs ( particularly ziprasidone ) .
we lacked data on several clinical variables including blood pressure , hematological profiles , electrocardiograms , and indication for treatment .
all of these variables may have influenced clinicians decisions of whether to prescribe a particular agent .
there is an important but limited role for antipsychotic treatment of severe neuropsychiatric symptoms.23 our study demonstrated high incidence rates of mace and noncardiovascular mortality associated with use of individual aps in elderly persons with or without dementia and with and without cvd .
this underscores that aps should be used with caution in elderly patients , regardless of dementia status , at the lowest possible dose and for shortest possible time . a particular focus on risks and benefits
is warranted among people with prevalent cvd because these patients seem to have the highest risks associated with aps .
our study further suggested some diversity in risks associated with individual aps but no systematic differences between first- and second - generation aps .
randomized placebo - controlled studies are warranted to confirm our findings and to identify the safest agents . until then
, the antipsychotic benefit in people with and without dementia must be considered against the risks of adverse events . finally ,
in denmark , restrictions on the use of aps are focused predominantly on people with dementia .
dr andersson was funded by an independent research grant from the danish agency for science , technology and innovation ( grant number fss-11 - 120873 ) .
none of the funding sources had any influence on design and conduct of the study ; collection , management , analysis , and interpretation of the data ; and preparation , review , or approval of the manuscript .
sahlberg ( none ) , holm ( none ) , gislason ( none ) , torp - pedersen ( reports grants and personal fees from cardiome , grants and personal fees from merck , grants and personal fees from sanofi , grants and personal fees from daiichi , grants from bms , outside the submitted work ) , kber ( none ) , andersson ( reports grants from danish agency for science , technology and innovation , grants from astrazeneca , during the conduct of the study ) . | backgrounddata from observational studies have raised concerns about the safety of treatment with antipsychotic agents ( aps ) in elderly patients with dementia , but this area has been insufficiently investigated .
we performed a head - to - head comparison of the risk of major adverse cardiovascular events and noncardiovascular mortality associated with individual aps ( ziprasidone , olanzapine , risperidone , quetiapine , levomepromazine , chlorprothixen , flupentixol , and haloperidol ) in danish treatment - nave patients aged 70 years.methods and resultswe followed all treatment - nave danish citizens aged 70 years that initiated treatment with aps for the first time between 1997 and 2011 ( n=91 774 , mean age 827 years , 35 474 [ 39% ] were men ) .
incidence rate ratios associated with use of different aps were assessed by multivariable time - dependent poisson regression models . for the first 30 days of treatment , compared with risperidone ,
incidence rate ratios of major adverse cardiovascular events were higher with use of levomepromazine ( 3.80 , 95% ci 3.43 to 4.21 ) and haloperidol ( 1.85 , 95% ci 1.67 to 2.05 ) and lower for treatment with flupentixol ( 0.54 , 95% ci 0.45 to 0.66 ) , ziprasidone ( 0.31 , 95% ci 0.10 to 0.97 ) , chlorprothixen ( 0.76 , 95% ci 0.61 to 0.95 ) , and quetiapine ( 0.68 , 95% ci 0.58 to 0.80 ) .
relationships were generally similar for long - term treatment .
the majority of agents were associated with higher risks among patients with cardiovascular disease compared with patients without cardiovascular disease ( p for interaction < 0.0001 ) .
similar results were observed for noncardiovascular mortality , although differences in associations between patients with and without cardiovascular disease were small.conclusionsour study suggested some diversity in risks associated with individual aps but no systematic difference between first- and second - generation aps .
randomized placebo - controlled studies are warranted to confirm our findings and to identify the safest agents . | Methods
Population and Comorbidities
Outcomes
Pharmacotherapy
Statistics
Results
Discussion
Safety of APs in Elderly Persons
First- Versus Second-Generation APs
Strengths and Limitations of the Study
Conclusion and Clinical Implications
Sources of Funding
Disclosures | after findings of increased risk of cerebrovascular events associated with use of second - generation aps,10,18,19 the us food and drug administration ( fda ) issued a warning in 2005 against use of second - generation aps in dementia.18,20 in 2008 , the fda extended this warning to include use of first - generation aps in dementia.21 several studies have since demonstrated increased mortality with use of aps in dementia,8,10,22 and some studies have shown increased mortality in elderly patients regardless of dementia status.9,11,19 previous studies have also suggested that the use of aps is associated with increased risk of myocardial infarction among older patients with treated dementia13 and that exposure to aps may be a trigger for stroke.11 furthermore , a cochrane review estimated the risk of cerebrovascular events to be > 3-fold higher in risperidone- versus placebo - treated elderly patients with dementia.23 our observations suggest that medications belonging to the second- and first - generation classes of aps may be associated with more or less comparable risk in elderly patients with or without dementia , although flupentixol , chlorprothixen , and quetiapine may be particularly associated with lower mace and mortality rates than risperidone . previous studies suggest that both first- and second - generation aps can lower blood pressure , leading to increased risk of falls and to a varying extent to prolonged qt interval , which might lead to arrhythmia and death.2528 another problem related to second - generation aps is adverse influence on glucose metabolism , diabetes , and blood lipid composition , leading to increased cardiovascular risks with long - term use.29,30 several prior observational studies have compared the safety of first- versus second - generation aps6,8,9,11,22,19,3133 and generally have indicated that the risk of death and/or mace when using second - generation aps is at least as high as that for first - generation aps.21 at least 1 retrospective cohort study has suggested that in older adults with dementia , second- and first - generation aps were associated with comparable risks of ischemic stroke.31 a larger retrospective cohort study in 2009 also suggested a comparable dose - related increased risk of sudden cardiac death for first- and second - generation aps.12 the main strength and novelty of this study is that we compared mace and noncardiovascular mortality risk among individual aps across the first and second generations in a rather large group of patients aged 70 years . after findings of increased risk of cerebrovascular events associated with use of second - generation aps,10,18,19 the us food and drug administration ( fda ) issued a warning in 2005 against use of second - generation aps in dementia.18,20 in 2008 , the fda extended this warning to include use of first - generation aps in dementia.21 several studies have since demonstrated increased mortality with use of aps in dementia,8,10,22 and some studies have shown increased mortality in elderly patients regardless of dementia status.9,11,19 previous studies have also suggested that the use of aps is associated with increased risk of myocardial infarction among older patients with treated dementia13 and that exposure to aps may be a trigger for stroke.11 furthermore , a cochrane review estimated the risk of cerebrovascular events to be > 3-fold higher in risperidone- versus placebo - treated elderly patients with dementia.23 our observations suggest that medications belonging to the second- and first - generation classes of aps may be associated with more or less comparable risk in elderly patients with or without dementia , although flupentixol , chlorprothixen , and quetiapine may be particularly associated with lower mace and mortality rates than risperidone . previous studies suggest that both first- and second - generation aps can lower blood pressure , leading to increased risk of falls and to a varying extent to prolonged qt interval , which might lead to arrhythmia and death.2528 another problem related to second - generation aps is adverse influence on glucose metabolism , diabetes , and blood lipid composition , leading to increased cardiovascular risks with long - term use.29,30 several prior observational studies have compared the safety of first- versus second - generation aps6,8,9,11,22,19,3133 and generally have indicated that the risk of death and/or mace when using second - generation aps is at least as high as that for first - generation aps.21 at least 1 retrospective cohort study has suggested that in older adults with dementia , second- and first - generation aps were associated with comparable risks of ischemic stroke.31 a larger retrospective cohort study in 2009 also suggested a comparable dose - related increased risk of sudden cardiac death for first- and second - generation aps.12
the main strength and novelty of this study is that we compared mace and noncardiovascular mortality risk among individual aps across the first and second generations in a rather large group of patients aged 70 years . | [
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over the course of evolution , the catabolic pathways of l - tryptophan ( trp ) and of l - arginine ( arg ) have evolved to be primary regulatory nodes in the control of immune responses ( grohmann and bronte , 2010 , murray , 2016 ) .
trp , an essential amino acid for mammals , is a substrate for indoleamine 2,3-dioxygenase 1 ( ido1 ) , which catalyzes the first , rate - limiting step in the kynurenine pathway , leading to trp depletion and the production of a series of immunoregulatory molecules collectively known as kynurenines ( grohmann et al .
, 2003b , mellor and munn , 2004 , puccetti and grohmann , 2007a ) . both effects
namely , trp starvation and kynurenine production are involved in the conversion of naive cd4 t cells into foxp3 regulatory t ( treg ) cells ( fallarino et al . , 2006 ,
moreover , the main ido1 catalytic product , l - kynurenine ( kyn ) , has immunoregulatory effects in the absence of trp starvation , via activation of the aryl hydrocarbon receptor ( ahr ) ( grohmann and puccetti , 2015 , platten et al . , 2012 ) .
high ido1 expression and catalytic activity occur in dendritic cells ( dcs ) in response to the cytokine interferon- ( ifn- ) ( grohmann et al .
, 2003b ) . in dcs stimulated with transforming growth factor ( tgf- ) , ido1 becomes instead phosphorylated in its immune - based inhibitory tyrosine motifs ( itims ) , so to mediate intracellular signaling events in a self - sustaining feedforward loop that leads to durable immunoregulatory effects ( bessede et al .
, 2014 , pallotta et al . , 2011 , volpi et al . , 2016 ) .
the composite mode of action of ido1 may well explain its being recognized as an authentic regulator of immunity under several physiopathologic conditions ( orabona and grohmann , 2011 , puccetti and grohmann , 2007a ) .
arg is a semi - essential amino acid , i.e. , it is required by mammals only under special circumstances , such as immune responses . in immune cells ,
arg is actively metabolized by arginase 1 ( arg1 ) or arg2 to produce urea and l - ornithine ( orn ) or it is used for protein biosynthesis ( wu and morris , 1998 ) . moreover
, nitric oxide synthases use arg to make nitric oxide a key anti - microbial gas and signaling molecule and l - citrulline ( bronte and zanovello , 2005 ) .
arg consumption by arg1 , rather than arg2 or nitric oxide synthases , represents a well - known immunoregulatory mechanism exploited by m2 macrophages ( murray , 2016 , sica and mantovani , 2012 ) , as well as myeloid - derived suppressor cells ( mdscs ) in tumoral settings ( gabrilovich and nagaraj , 2009 , marigo et al . ,
conversely , the immunoregulatory role of catalytic products via arg1 activity ( urea and orn ) has been unclear ( grohmann and bronte , 2010 ) .
t helper 2 ( th2 ) cytokines , such as interleukin-4 ( il-4 ) and il-13 , represent efficient inducers of arg1 expression ( gabrilovich and nagaraj , 2009 , marigo et al .
, 2008 , sica and mantovani , 2012 ) , although tgf- has also been shown to upregulate the enzyme , at least in rat peritoneal macrophages ( boutard et al . , 1995 ) .
besides its involvement in mouse dendritic cell ( dc ) differentiation ( yang et al . , 2015 ) , the biology of arg1 in dcs is still obscure . as a whole , the bulk of the data on arg1 and ido1 would suggest that the two amino - acid metabolic enzymes might operate in quite distinct spatial ( i.e. , cells ) and mechanistic modes , namely via either amino acid starvation itself ( as is the case for arg1 ) or via the combined effects of immunoregulatory kyn and signaling activity ( ido1 ) .
this suggests that the enzymes have separate functions ( to possibly cope with distinct environmental needs ) and/or have evolved to complement , rather than integrate , each other .
dcs integrate multiple signals and mechanisms in order to promote either adaptive immunity or immune tolerance in response to specific conditions , such as distinct types of cytokinic milieu ( banchereau and steinman , 1998 , macagno et al . , 2007 ) .
by using the main cytokines capable of inducing those metabolic enzymes ( i.e. , ifn- , il-4 , and tgf- ) , we here investigated whether arg1 and ido1 were co - expressed in dcs and what the functional meaning if any of their co - expression would be .
we found that ( 1 ) tgf- , but neither il-4 , ifn- , nor combination thereof , will induce both arg1 and ido1 in dcs , with arg1 being upregulated before ido1 ; ( 2 ) arg1 activity is absolutely required for ido1-dependent signaling events as initiated by tgf- ; ( 3 ) a polyamine spermidine , resulting from orn decarboxylation can replace tgf- in the activation of the src kinase that phosphorylates ido1 and trigger immunosuppressive ido1 signaling ; and ( 4 ) dcs can be conditioned by arg1 mdscs to express an ido1-dependent immunosuppressive phenotype .
we investigated the expression and catalytic activity of arg1 and ido1 in splenic cd11c dcs stimulated with il-4 ( i.e. , the main arg1 inducer ) , ifn- , or tgf- , both of which are ido1 inducers .
combinations of tgf- and il-4 or ifn- were also tested . in accordance with previous data ( pallotta et al . , 2011 ) , transcript analysis at 3 and 18 hr of cytokine incubation revealed that both ifn- and tgf- upregulated ido1 expression , with ifn- inducing the ido1-encoding gene to a higher extent and more rapidly than tgf- ( figure 1a ) .
by contrast , il-4 did not increase ido1 , but it did induce arg1 expression ( > 7-fold increase at 3 hr of stimulation ) .
in contrast , tgf- upregulated arg1 in a more intense fashion and with faster kinetics than ido1 .
negligible transcript expression for arg2 and nos2 ( coding for the inducible nitric oxide synthase ) could be observed in either unstimulated or tgf--stimulated dcs ( data not shown ) .
dc incubation with tgf- plus il-4 resulted in a synergistic effect on arg1 upregulation ( > 12- and 20-fold increase at 3 and 18 hr , respectively ) but in no induction of ido1 . on combining tgf- with ifn- , no incremental effect over that of ifn- alone
was observed at 3 hr and impaired upregulation of ido1 expression by tgf- would instead occur at 18 hr . we next evaluated whether the differential modulation of arg1 and ido1 could also be observed at the level of protein expression and catalytic activity .
immunoblot analyses with anti - arg1 and anti - ido1 antibodies on lysates of dcs subjected to the same treatments as in figure 1a but for 24 hr confirmed the real - time pcr experiments , i.e. , il-4 and ifn- induce only arg1 and ido1 , respectively , whereas tgf- alone , but not in combination with il-4 or ifn- , upregulated protein expression of both enzymes ( figure 1b ) .
similarly , the enzymic activities of both arg1 ( measured in terms of urea contents in cell lysates ; figure 1c ) and ido1 ( in terms of kyn concentrations in culture supernatants ; figure 1d ) were significantly upregulated at 24 hr by tgf- but not il-4 , ifn- , or combinations thereof .
therefore , these data showed that arg1 can be expressed in dcs , specifically in response to il-4 and/or tgf- , and that arg1 expression precedes that of ido1 in dcs exposed to tgf- alone .
perhaps more importantly , these data unveiled that the arg and trp catabolic pathways can be co - activated in dcs . to better appreciate the temporal relationship between arg1 and ido1 expressions in tgf--stimulated dcs
i.e . , from 0.5 to 72 hr of cytokine incubation of transcripts coding for the two amino - acid catabolic enzymes .
the results confirmed the earlier induction of arg1 ( 34 hr ; figures 1a and 2a ) as compared to ido1 transcripts ( 1618 hr ; figures 1a and 2a ) , as well as a higher extent of arg1 expression that peaked at 2448 hr ( > 7 fold increase as compared to unstimulated cells ) of tgf- stimulation .
nevertheless , in agreement with our previous data ( pallotta et al . , 2011 ) , the extent of ido1 expression remained stable throughout the experiment , i.e. , up to 72 hr , after which dcs could no longer be analyzed because of poor viability .
the sequential expressions of arg1 and ido1 prompted us to investigate whether arg1 was required for ido1 induction by tgf- in dcs .
we examined ido1 transcript amounts over time using dcs purified from the spleens of itgax - cre;arg1 mice , lacking arg1 expression in cd11c ( i.e. , dcs ) but not in cd11b cells ( figure s1 ) .
we found that arg1 deficiency abrogated ido1 induction by tgf- but not by ifn- in dcs ( figure 2b ) .
analysis of ido1 activity ( figure 2c ) at 24 hr of tgf- or ifn- stimulation also indicated that lack of arg1 expression in dcs allowed ifn- but not tgf- to upregulate ido1 .
thus these data identified a cytokine milieu whereby dcs exploit arg1 expression to increase ido1 . at variance with ido1 , which exerts immunoregulatory additional effects also via signaling activity ( pallotta et al .
2016 ) , arg1-mediated mechanisms have been shown to occur only via arg catalytic degradation .
we thus investigated whether upregulation of ido1 transcripts in dcs by tgf- could be modulated by n - hydroxy - nor - arg ( nor - noha ) , an inhibitor of arginases .
pre - incubation with 50 m or 100 m nor - noha for 1 hr prior to the addition of tgf- significantly impaired the ido1-inducing ability of the cytokine ( figure 3a ) , indicating a functional role for arg1 catalytic activity in ido1 modulation .
we next investigated whether orn , an arg1 product , was involved in ido1 upregulation .
incubation of dcs with orn at 50 or 100 m ( figure 3b ) significantly increased ido1 expression , similar to what observed with the cytokine alone ( figure 3b ) .
in contrast , no modulatory effect in ido1 expression could be observed in dcs stimulated with ifn- in the presence of the arg1 inhibitor or orn , both at 100 m ( figure 3c ) . to evaluate whether autocrine and/or paracrine tgf- could be involved in the ido1-upregulating effects of orn
, wt dcs were incubated with the arg metabolite for different times after a 1 hr pretreatment with an inhibitor of tgf- receptor signaling , namely sb-431542 , ly2109761 , or ly2157299 ( also known as galunisertib ) ( figure 3d ) .
dcs purified from transgenic cd11c mice ( expressing a truncated form of tgf- receptor ii subunit in cd11c cells ) ( laouar et al . , 2005 )
both pharmacologic and genetic means of tgf- signaling inhibition indicated that orn effects do require autocrine and/or paracrine effects of tgf- in both early inducing and late maintaining phase of ido1 expression ( see also supplemental text ) and that the cytokine could be produced constitutively by dcs in basal conditions , in accordance to our previous data ( belladonna et al . , 2008 ) .
in contrast to orn , media deficient in arg ( 4 and 40 m in the place of standard 400 m ; figure 3f ) did not significantly increased ido1 expression . moreover , orn at 100 m but not the use of an arg - deficient medium ( i.e. , arg at 4 m ) significantly increased ido1 activity to an extent comparable to that observed with tgf- ( figure 3 g ) .
therefore , our data indicated that orn , a main arg1 product , but not arg starvation , can condition dcs to upregulate ido1 expression and activity if autocrine and/or paracrine tgf- is present .
ido1 immunosuppressive effects include non - enzymic functions , namely intracellular signaling events that , initiated by itim phosphorylation in the enzyme , are involved in reprogramming gene expression and in the induction of a stably regulatory phenotype in dcs ( orabona et al . , 2012 , pallotta et al . , 2011 ) ,
capable of controlling t cell - mediated autoimmune responses ( pallotta et al .
in particular , ido1 s itim phosphorylation is triggered in dcs by tgf- via a pathway that requires phosphatidylinositide 3-kinase ( pi3k ) and a tyrosine kinase of the src family ( bessede et al .
, 2016 ) , which phosphorylates ido1 itims . in turn , phosphorylated ido1 itims work as docking sites for tyrosine protein phosphatases , such as shp-1 and shp-2 , which are concomitantly upregulated by tgf- ( orabona et al . , 2012 ,
these events lead to the activation of an immunoregulatory signaling pathway in dcs that promotes endogenous production of tgf- and induction of the ido1 gene , perpetuating ido1 signaling events and associated immunosuppressive effects over the long term . because orn was capable of upregulating ido1 in dcs in a sustained fashion comparable to that of tgf- ( figure 3 ) , we investigated whether orn could replace the cytokine in activating the ido1 s signaling and immunosuppressive effects . by means of an antibody specific for the phosphorylated form of the enzyme ( bessede et al .
2016 ) , we found that the dc incubation with orn did induce ido1 phosphorylation , with a peak at 15 min , i.e. , an effect earlier than that triggered by tgf- ( at 3060 min ) ( figure 4a ) .
moreover , similarly to tgf- , stimulation with the arg1 product upregulated transcript expression of ptpn6 ( coding for shp-1 ) and tgfb1 at 18 hr in wt dcs .
in contrast , no or less upregulation of ptpn6 and tgfb1 , respectively , was induced by tgf- in itgax - cre;arg1 dcs ( figure 4b ) , further confirming the important role of arg1 in triggering and maintaining the ido1 signaling . to appreciate the immunosuppressive potential of dcs conditioned by orn
, we used the skin test assay , an established protocol for measuring the in vivo induction of antigen - specific immunoreactivity versus tolerance in dcs ( grohmann et al . , 2002 , grohmann et al .
, we sensitized wild - type ( wt ) mice with the hy peptide ( containing the h-2d epitope of male minor transplantation antigen ) presented by wt cd8 dcs ( constituting an immunostimulatory , splenic dc subset ) ( grohmann et al . , 2003a ) administered alone or in combination with a minority fraction of the same cells ( 5% ) purified from either wt or ido1 animals after conditioning with tgf- , orn , or medium alone for 24 hr . after priming the mice , we assessed immune reactivity at two weeks by intrafootpad challenge with the hy peptide in the absence of dcs , as described ( grohmann et al . , 2002 , grohmann et al . , 2007 ,
as expected , the default priming ability of immunostimulatory dcs was not affected by the presence of untreated cells .
yet , sensitization together with tgf-- but also orn - pretreated wt dcs caused suppression of hy - specific reactivity , an effect not detectable in mice sensitized with ido1 dcs , regardless of whichever type of conditioning molecule had been used ( figure 4c ) . as a whole
, our data indicated that the mere incubation of dcs with an arg metabolite can activate molecular events traceable to ido1 signaling as well as ido1-dependent immunosuppressive outcomes detectable in vivo .
orn can be further metabolized into putrescine , a polyamine that , in turn , can be transformed into spermidine and then spermine ( figure s2 ) , by orn decarboxylase ( odc ) . in tumor cells , where it is often highly expressed , odc favors proliferative events .
more recently , odc has been shown to be likewise involved in immunoregulatory mechanisms that oppose anti - tumor immunity ( hayes et al . ,
administration of -difluoromethylornithine ( dfmo ) , an odc inhibitor , will inhibit tumor growth via impairment of mdsc - mediated suppressive effects ( ye et al .
whether dfmo - promoted immunity might also depend on impairment of ido1 activity in dcs is currently unknown .
to evaluate odc expression in dcs , we conducted a meta - analysis of public microarray data restricted to the expression of the odc1 gene in several mouse dc subsets purified from both lymphoid and nonlymphoid organs .
the results showed that high amounts of odc1 transcripts were detectable in all dc subsets analyzed so far , including splenic , conventional cd11c dcs , the subject of the current study ( figure 5a ) .
prompted by these data , we investigated the effects of odc inhibition on orn actions in dcs . because of the obligate requirement for odc in many cell types ( cervelli et al .
we found that co - incubation of splenic dcs with orn and dfmo abrogated the capacity of orn alone to upregulate ido1 expression ( figure 5b ) .
moreover , by using the skin test assay as in figure 4c , we found that no immunosuppressive effects could be conferred by orn on wt dcs upon co - incubation of cells with dfmo ( figure 5c ) . because stimulation with orn induced significant upregulation of the ido1 gene in human dcs as well , and dfmo negated this effect ( figure s3 )
, our data suggest that the odc catalytic activity basally expressed in dcs exerts important immunoregulatory effects and that metabolites downstream of odc function might represent the most proximal inducers of ido1 signaling and ido1-mediated immunosuppression in dcs .
polyamines , i.e. , the diamine putrescine , the triamine spermidine , and the tetra - amine spermine , are highly bioactive polycations capable of binding nucleic acids and proteins and of modulating several signaling pathways .
polyamine functions have been studied most extensively in tumors , where they are often required for cell transformation and proliferation ( gerner and meyskens , 2004 ) . whether polyamines can be produced by , or exert effects on , dcs is currently unknown .
real - time pcr analyses revealed that dcs expressed high amounts of srm and sms genes , coding for spermidine and spermine synthase , respectively , and that such expression was not modulated by either tgf- or il-4 ( figure s4a ) . to evaluate the effective production of polyamines by dcs , we assessed the profile of arg metabolites along with that of trp over time in culture supernatants of cells incubated with orn ( figure 6a ) . following orn incubation ,
an increase was observed in dc release of all polyamines , with putrescine and spermidine production being evident at 4 hr and spermine at 16 hr . moreover , orn incubation also led to upregulation of ido1 products , i.e. , l - formylkynurenine at 4 hr , followed by l - kynurenine at 16 hr .
an increase in other orn metabolites ( i.e. , proline , 1-pyrroline 4-hydroxy-2-carboxylate , -l - glutamyl - putrescine , and -l - glutamyl - amino - butyraldehyde ) and trp metabolites ( serotonin , tryptamine , 5-hydroxy - l - tryptophan , and indole-3-acetaldehyde ) could also be observed upon dc incubation with orn .
the results showed that dc incubation with either putrescine or spermidine for 24 hr significantly increased expression of the ido1 gene to an extent comparable to those induced by orn ( figure 6b ) , whereas induction of the ido1 protein could be observed only for spermidine ( figure s4b ) .
in contrast , spermine did not upregulate ido1 but rather showed a tendency to downregulate the enzyme expression ( figure 6b and s4 ) . in pro - inflammatory microenvironments ,
ido1 is subjected to regulatory proteolysis mediated by the immunoproteasome in dcs ( orabona et al . , 2008 ) .
we therefore investigated whether lack of ido1 protein upregulation by putrescine could be due to a concomitant increase in immunoproteasome activity .
we found that putrescine but not spermidine significantly upregulated psmb8 , psmb9 , and psmb10 transcripts , coding for 5i , 1i , and 2i immunoproteasome subunits , respectively , in dcs ( figure s4c ) .
moreover , we analyzed ido1 protein expression in lysates from dcs exposed to cycloheximide ( an inhibitor of protein synthesis ) prior to incubation with putrescine alone or in combination with mg132 , a proteasome inhibitor .
these results revealed that , in the presence of cycloheximide , putrescine alone rather promoted a reduction in ido1 protein expression , which was opposed by the co - presence of mg132 ( figure s4d ) .
the combination of mg132 and putrescine was accompanied by the appearance of proteins of higher molecular weight than 42 kda , possibly representing polyubiquitinated ido1 species , as described ( orabona et al . , 2008 ) .
in tumor cells , putrescine and spermidine but not spermine promote the phosphorylation and consequent activation of mapk , src , and pi3k ( normally followed by akt ) kinases , via still undefined mechanisms ( hltt et al . , 1993 ) .
conversely , spermine has been shown to restrain immune responses in activated macrophages by inhibiting gene expression of nos2 ( zhang et al . , 1997 ) .
in intestinal epithelial cells , spermine negatively modulates the activity of src kinase via direct binding of the src sh2 domain ( ray et al . , 2012 ) . because src is involved in the activation of ido1 signaling in dcs ( bessede et al .
2016 ) , we evaluated the ability of polyamines in modulating src phosphorylation over time .
putrescine and spermidine , but not spermine , increased src phosphorylation in dcs , as soon as at 5 min of cell incubation ( figure 6c ) . to conduct a more comprehensive evaluation of the potential of putrescine and spermidine in modulating signaling pathways in dcs
, we performed a kinomic analysis using a microarray of peptides phosphorylable by tyrosine , serine , and threonine kinases ( van baal et al . , 2006 ) and cell extracts from dcs incubated with the polyamines for 15 or 30 min .
we found that putrescine is a potent activator of several kinases in dcs ( figure s4e ) , including src , fyn , hck , and lck tyrosine kinases ( table s1 ) , and that it also promotes phosphorylation of a peptide from ikk , a kinase involved in the activation of the pro - inflammatory nf-b pathway ( table s4 ) .
although less potent when considering the overall effect ( figure s4e ) , spermidine did promote tyrosine phosphorylation of peptides from protein kinase c ( type ) and -catenin , known substrates of src and fyn kinases ( table s1 ) , respectively , but not from ikk ( table s4 ) . in a skin test assay ,
dc incubation with spermidine ( figure 6d ) led to immunosuppressive effects detectable in vivo .
moreover , as observed for orn ( figure 4c ) , spermidine effects were lost in ido1 dcs ( figure 6d ) .
importantly , the immunosuppressive effects were also impaired by coincubating wt dcs with spermidine and pp2 ( a src kinase inhibitor ) but not pp3 ( a negative control ) ( figure 6d ) .
overall , our data suggest that spermidine and its metabolic precursor putrescine are produced by dcs and represent major modulators of their own signaling pathways and function , including the activation of src kinase
. however , only spermidine triggered immunosuppressive ido1 signaling , an effect possibly lost when using putrescine for dc conditioning because of the concomitant activation of several kinases other than those belonging in the src family , including proinflammatory ikk , and because of the increased expression of proteasome subunits possibly degrading the ido1 protein . as evident from their name , the defining feature of mdscs is their ability to suppress immune cell functions .
main factors implicated in mdsc - mediated immune suppression include high expression of arg1 , among others ( marvel and gabrilovich , 2015 ) .
in addition to their inherent immunosuppressive activity , mdscs might amplify regulatory properties of other immune cells , particularly in tumor microenvironments . although some mechanisms underlying mdsc - macrophage interaction have been established ( ugel et al . , 2015 ) , the cross - talk between mdscs and dcs is still unclear ( ostrand - rosenberg et al . ,
prompted by the finding that an extracellular polyamine can condition dcs toward an ido1-dependent , immunosuppressive phenotype , we investigated whether mdscs could also exert similar effects on dcs via the arg1 pathway .
arg1 mdscs were obtained from the bone marrow of wt mice as described ( youn et al . ,
2008 ) , pre - treated with nor - noha , dfmo ( 100 m and 1 mm , respectively ) , or medium alone for 1 hr , extensively washed , and cocultured with dcs in separate chambers of transwells ( figure 7a ) . after 24 hr , dcs were recovered and analyzed for ido1 transcript expression by real - time pcr as well as immunosuppressive potential in vivo by skin test assay as in figure 4c and figure 6d .
controls included dcs cultured in transwells without mdscs ( medium ; figure 7a ) .
we found that the separate coculture with untreated mdscs significantly upregulated ido1 expression ( figure 7b ) and conferred in vivo immunosuppressive properties on dcs ( figure 7c ) .
however , both effects were lost when either arg1 or odc catalytic activity had been inhibited in mdscs before their co - culturing with dcs . moreover , no in vivo immunosuppressive phenotype could be acquired by ido1 dcs by conditioning with mdscs not subjected to arg1 or odc inhibitor treatment ( figure 7c ) .
thus our data confirmed that arg and trp immunoregulatory pathways are functionally integrated and that this integration can occur both intra- ( i.e. , dcs ) and inter - cellularly ( mdscs and dcs ) ( figure s5a and figure 7 ) .
an effective communication networking represents the basis of life of evolved multicellular organisms . as a matter of fact , evidence for critical cross - talk mechanisms between metabolism and immunity the main systems involved in maintaining and defending a constant internal milieu ( odegaard and chawla , 2013 , pearce and pearce , 2013)are emerging . in establishing adaptive immunometabolic networks over evolution , functional repurposing of ancestral proteins ( originally only metabolic or immune in nature ) may have represented a powerful strategy .
such evolved structures defined as moonlighting proteins when they maintain their original function often derive from gene duplication , a major driving force that renders biological systems more robust to environmental perturbations ( espinosa - cant et al . , 2015 ) .
immune regulation is a highly evolved form of biologic response that controls immunity to self but also dampens exaggerated inflammation ( fazekas de st groth , 1998 , flajnik and kasahara , 2010 , grohmann and bronte , 2010 ) .
metabolism of arg and trp and their consequent starvation in cell microenvironments still represent a survival strategy in phylogenetically ancient organisms . yet , arg and trp catabolisms have been co - opted by immune regulation in mammals ( murray , 2016 ) . in this regard
, the bulk of available information would suggest that the arg1 enzyme , known to catabolize arg mainly in immune cells such as mdscs and macrophages , might have acquired immunoregulatory functions by simply extending the ancient mechanism of starvation to the control of activation and proliferation of t lymphocytes .
this is even more true if one considers that , for instance , mouse macrophages stimulated with il-4 will upregulate arg1 expression by 100- to 1,000-fold ( pauleau et al . , 2004 ) .
in contrast , in our study , the increase in arg1 transcripts in dcs incubated with il-4 was no higher than 8-fold , suggesting that the arg1 function in dcs might be considerably different as compared to the profound arg starvation determined by arg1 macrophages . nevertheless , the co - presence of il-4 and tgf- further upregulated arg1 transcripts more than 20-fold , suggesting that maximal expression of arg catabolism would require multiple signals in dcs .
in contrast , ido1 , the trp catabolizing enzyme mainly operating in immune cells such as dcs , has been shown to exert immunoregulatory effects via trp starvation but also via its catalytic products , i.e. , kynurenines , and , perhaps most importantly , via a non - enzymic signaling activity ( chen , 2011 , grohmann et al .
in fact , by means of its itim domains ( phosphorylable by src tyrosine kinases activated in the presence of tgf- ) , ido1 establishes an intracellular signaling network in dcs that leads to the long - term expression of ido1 itself and subsequent , sustained control of adaptive immunity . at variance with arg1 , mouse ido1 , and human ido1 as well , might be the result of duplication of the more ancient ido2 ( ido2 in humans ) gene , an ido1 ( ido1 ) paralog characterized by low efficiency in trp degradation and inability to act as a signaling molecule ( orabona et al .
tryptophan 2,3-dioxygenase ( tdo ) , another ancient enzyme mainly expressed in the liver and responsible for the degradation of trp entered by diet , does not contain itims .
we here showed that , in the presence of tgf- but not il-4 , ifn- , or combinations thereof , arg1 and ido1 expressions co - exist in dcs .
if anything , the co - presence of other cytokines rather impaired the tgf- upregulating action on both amino - acid catabolizing enzymes , an effect possibly due the antagonistic effects of il-4 ( musso et al . , 1994 ) and ifn- ( bronte and zanovello , 2005 ) ( g. natoli , personal communication ) on ido1 and arg1 gene expressions , respectively .
the apparently transient , yet intense , induction of arg1 enzymatic activity by tgf- was mandatory for the subsequent ido1 upregulation in terms of both catalytic and signaling mechanisms .
these arg1 effects were not mediated by arg deprivation ( as one might have expected , due to the poor or absent proliferative capacity of dcs ) but rather by its downstream enzymatic catabolites , namely , the polyamine spermidine , generated downstream of decarboxylation of orn , one of the direct arg1 catalytic products .
moreover , our data indicated that this polyamine can promote ido1 phosphorylation and signaling events in dcs ( bessede et al .
spermidine might therefore represent a two - sided node responsible for an intersection between the immunometabolic pathways of arg1 and ido1 .
perhaps most importantly , this relay pathway would allow the immune system to translate an initial short - term ( arg1-mediated ; typical of early - acting mdscs and regulatory macrophages ; goldszmid et al . , 2014 ) into a sustained regulatory response ( via ido1 ; typical of long - term acting tolerogenic dcs ; morelli and thomson , 2007 ) .
tgf- is a cytokine that appeared in metazoans and , by virtue of its marked pleiotropy and multiple effects , plays a prominent role in the logic of communicative networks in multicellular organisms ( massagu and gomis , 2006 ) .
although the output of a tgf- response is highly contextual , the presence of this cytokine in microenvironments often favors local immunosuppression , inhibiting anti - tumor immunity ( gorelik and flavell , 2002 , pickup et al . , 2013 , tu et al . , 2014 ) . the tgf- potential for inducing both enzymes , i.e. , arg1 in macrophages ( boutard et al . , 1995 ) and
ido1 in dcs ( belladonna et al . , 2009 , pallotta et al . ,
however , we here demonstrated that the immunoregulatory effects of tgf- in dcs would go beyond the mutually exclusive upregulation of the arg1 and ido1 enzymes ( observable in the presence of il-4 and ifn- , respectively ) by allowing the establishment of a network involving both arg1 and ido1 in dcs , further underlining the functional plasticity of these cells .
moreover , because mdscs themselves are an abundant source of tgf- production ( bierie and moses , 2010 ) , the network established by the triad constituted by tgf- , arg1 , and ido1 might be highly relevant to establishing potent immunosuppressive environments if also dcs are present as well . in conclusion , our data , besides further underlining the importance of critical pathways linking metabolism and immunity in multicellular animals , suggest that the appearance of a highly evolved form of biologic response such as immune regulation would rely on a network based on the co - option of two ancient pathways , i.e. , arg and trp catabolisms ( anderson et al . ,
2016 ) as reinforced by tgf-. a consequence of this could be that tumors , considered to be the result of an evolutionary process ( billaud and santoro , 2011 ) , have become particularly apt to co - opt metabolic and immunosuppressive networks to propel their generation and progression . thus our data might predict that the simultaneous inhibition of two immune checkpoints such as arg1 ( https://clinicaltrials.gov/show/nct02903914 ) and ido1 ( buqu et al .
eight- to ten - week - old female c57bl/6 mice were obtained from charles river breeding laboratories .
ido1 mice were purchased from the jackson laboratory and bred at charles river . to obtain itgax - cre;arg mice ( i.e. , lacking arg1 expression in cd11c cells )
, c57bl/6-arg1j were bred to the strain c57bl/6j - tg ( itgax - cre ,- egfp)4097ach / j ( the jackson laboratory ) , with inducible cre recombinase expression in the cd11c cells ( figure s1 ) .
cd11c mice on c57/bl6 background ( laouar et al . , 2005 ) were bred and maintained at the animal facility of the university of michigan school of medicine .
splenic dcs were purified using cd11c microbeads ( miltenyi biotec ) , as described ( grohmann et al . , 2002 ) .
mdscs were obtained from bone marrow cells , as previously described ( youn et al . , 2008 ) .
details of mdsc purification and treatments can be found in supplemental experimental procedures . for all in vitro studies , cd11c or cd8 dcs were cultured at 1 10 cells per well in 24-well plates in iscove s modified dulbecco s medium ( imdm , thermo fisher scientific ) or , in selected experiments , in dulbecco s modified eagle medium ( dmem , thermo fisher scientific ) , containing low or standard arg levels ( 4 , 40 , or standard 400 m ) and completed by adding l - asparagine ( 120 m ) and l - lysine ( 790 m ) .
recombinant human tgf- ( r&d system ) , orn , and polyamines were used at the final concentration of 20 ng / ml , 50100 m , and 20 m , respectively . in specific experiments , dcs were conditioned by co - culture for 24 hr with mdscs ( either such or pretreated for 1 hr with inhibitors ) using transwell cell culture inserts ( nunc ) .
arg1 protein expression was investigated in dcs by immunoblot with a goat polyclonal anti - mouse arg1 antibody ( abcam ) .
ido1 and pido1 expressions were investigated with a rabbit monoclonal anti - mouse ido1 antibody ( cv152 ) ( romani et al . , 2008 ) or a rabbit polyclonal antibody to the phosphorylated itim1 motif of ido1 ( pallotta et al . , 2011 ) , respectively .
a skin test assay was used for measurements of major histocompatibility complex class i restricted delayed - type hypersensitivity ( dth ) responses to the hy peptide ( wmhhnmdli ) in c57bl/6 female recipient mice , as described ( pallotta et al . , 2011 ) .
for in vivo immunization , 3 10 peptide - loaded cd8 dcs , combined with a minority fraction ( 5% ) of peptide - loaded c57bl/6 or ido1 cd8 dcs , were injected subcutaneously into recipient mice .
two weeks later , a dth response was measured to intrafootpad challenge with the eliciting peptide , and results were expressed as the increase in footpad weight of peptide - injected footpads over that of vehicle - injected ( internal control ) counterparts .
the minority cell fraction , constituted by wt or ido1 cd8 dcs , was left untreated or treated overnight with specific reagents as above .
unpaired student s t test was used for in vitro analyses , using at least three values from 23 experiments per group , whereas for the skin test assay paired student s t test was used ( using at least six mice per group ) .
g.m . performed the majority of in vitro experiments and prepared most of the figures .
performed and supervised , respectively , meta - analyses of odc1 expression . u.g . wrote the manuscript | summaryarginase 1 ( arg1 ) and indoleamine 2,3-dioxygenase 1 ( ido1 ) are immunoregulatory enzymes catalyzing the degradation of l - arginine and l - tryptophan , respectively , resulting in local amino acid deprivation .
in addition , unlike arg1 , ido1 is also endowed with non - enzymatic signaling activity in dendritic cells ( dcs ) . despite considerable knowledge of their individual biology , no integrated functions of arg1 and ido1
have been reported yet .
we found that ido1 phosphorylation and consequent activation of ido1 signaling in dcs was strictly dependent on prior expression of arg1 and arg1-dependent production of polyamines .
polyamines , either produced by dcs or released by bystander arg1 + myeloid - derived suppressor cells , conditioned dcs toward an ido1-dependent , immunosuppressive phenotype via activation of the src kinase , which has ido1-phosphorylating activity . thus our data indicate that arg1 and ido1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings . | Introduction
Results
Discussion
Experimental Procedures
Author Contributions | over the course of evolution , the catabolic pathways of l - tryptophan ( trp ) and of l - arginine ( arg ) have evolved to be primary regulatory nodes in the control of immune responses ( grohmann and bronte , 2010 , murray , 2016 ) . trp , an essential amino acid for mammals , is a substrate for indoleamine 2,3-dioxygenase 1 ( ido1 ) , which catalyzes the first , rate - limiting step in the kynurenine pathway , leading to trp depletion and the production of a series of immunoregulatory molecules collectively known as kynurenines ( grohmann et al . in immune cells ,
arg is actively metabolized by arginase 1 ( arg1 ) or arg2 to produce urea and l - ornithine ( orn ) or it is used for protein biosynthesis ( wu and morris , 1998 ) . arg consumption by arg1 , rather than arg2 or nitric oxide synthases , represents a well - known immunoregulatory mechanism exploited by m2 macrophages ( murray , 2016 , sica and mantovani , 2012 ) , as well as myeloid - derived suppressor cells ( mdscs ) in tumoral settings ( gabrilovich and nagaraj , 2009 , marigo et al . , cells ) and mechanistic modes , namely via either amino acid starvation itself ( as is the case for arg1 ) or via the combined effects of immunoregulatory kyn and signaling activity ( ido1 ) . we found that ( 1 ) tgf- , but neither il-4 , ifn- , nor combination thereof , will induce both arg1 and ido1 in dcs , with arg1 being upregulated before ido1 ; ( 2 ) arg1 activity is absolutely required for ido1-dependent signaling events as initiated by tgf- ; ( 3 ) a polyamine spermidine , resulting from orn decarboxylation can replace tgf- in the activation of the src kinase that phosphorylates ido1 and trigger immunosuppressive ido1 signaling ; and ( 4 ) dcs can be conditioned by arg1 mdscs to express an ido1-dependent immunosuppressive phenotype . , 2012 ,
these events lead to the activation of an immunoregulatory signaling pathway in dcs that promotes endogenous production of tgf- and induction of the ido1 gene , perpetuating ido1 signaling events and associated immunosuppressive effects over the long term . in contrast , no or less upregulation of ptpn6 and tgfb1 , respectively , was induced by tgf- in itgax - cre;arg1 dcs ( figure 4b ) , further confirming the important role of arg1 in triggering and maintaining the ido1 signaling . because stimulation with orn induced significant upregulation of the ido1 gene in human dcs as well , and dfmo negated this effect ( figure s3 )
, our data suggest that the odc catalytic activity basally expressed in dcs exerts important immunoregulatory effects and that metabolites downstream of odc function might represent the most proximal inducers of ido1 signaling and ido1-mediated immunosuppression in dcs . because src is involved in the activation of ido1 signaling in dcs ( bessede et al . we found that putrescine is a potent activator of several kinases in dcs ( figure s4e ) , including src , fyn , hck , and lck tyrosine kinases ( table s1 ) , and that it also promotes phosphorylation of a peptide from ikk , a kinase involved in the activation of the pro - inflammatory nf-b pathway ( table s4 ) . overall , our data suggest that spermidine and its metabolic precursor putrescine are produced by dcs and represent major modulators of their own signaling pathways and function , including the activation of src kinase
. however , only spermidine triggered immunosuppressive ido1 signaling , an effect possibly lost when using putrescine for dc conditioning because of the concomitant activation of several kinases other than those belonging in the src family , including proinflammatory ikk , and because of the increased expression of proteasome subunits possibly degrading the ido1 protein . ,
prompted by the finding that an extracellular polyamine can condition dcs toward an ido1-dependent , immunosuppressive phenotype , we investigated whether mdscs could also exert similar effects on dcs via the arg1 pathway . ,
however , we here demonstrated that the immunoregulatory effects of tgf- in dcs would go beyond the mutually exclusive upregulation of the arg1 and ido1 enzymes ( observable in the presence of il-4 and ifn- , respectively ) by allowing the establishment of a network involving both arg1 and ido1 in dcs , further underlining the functional plasticity of these cells . | [
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] |
pancreatic ductal adenocarcinoma ( pda ) is a highly lethal malignancy with a 5-year survival rate of 7%1 .
it is expected that by 2020 pda will surpass breast and colorectal cancer to become the second most common cause of cancer - related deaths2 .
currently , there are no active screening methods to detect pda at early stages , and patients with localized disease exhibit no overt symptoms .
thus , pda is often diagnosed too late and the few available therapeutic options have little durable activity .
in fact , if evidence of the disease is detected early , the only potentially curative option for pancreatic cancer is surgery , followed by adjuvant chemotherapy .
nevertheless , early recurrence and disease progression after surgery is evident in a large proportion of patients .
though the underlying cause(s ) of recurrence or disease progression remain largely unknown , key culprits are undetected micrometastases and cellular drug resistance mechanisms . in the metastatic setting
, two recent randomized - controlled trials demonstrated the advantage of combination therapies over single agent gemcitabine , a nucleoside analogue that had been the standard of care since 19973 . to this extent , combining folinic acid , 5-fluorouracil ( 5-fu ) , irinotecan , and oxaliplatin ( folfirinox ) provided patients with a 4.3 month increase in overall survival when compared to gemcitabine alone ( hazard ratio for death , 0.57 ; 95% confidence interval [ ci ] , 0.45 to 0.73 ; p<0.001)4 , 5 .
similarly , gemcitabine plus nab - paclitaxel ( abraxane ) increased overall survival by approximately 2 months when compared to single - agent gemcitabine ( hazard ratio for death , 0.72 ; 95% confidence interval [ ci ] , 0.62 to 0.83 ; p<0.001)6 . despite the recent success of these trials ,
patients who respond to these therapies inevitably recur and succumb to their disease . in this perspective , we survey current therapeutic resistance mechanisms both extrinsically including the tumor microenvironment and also intrinsically within the cellular machinery of pda cells ( fig .
we refer the reader to other outstanding reviews and publications on elements of the tumor microenvironment , drug availability and honing issues7 - 10 .
we will focus on the classical view of therapeutic resistance mechanisms that are most likely intact in the majority of pdas . in brief , we are aware that there are distinct differences between innate / acute and acquired resistance therapeutic mechanisms .
the somatic mutation section is clearly describing a putative acquired resistance event that would take at least months to develop , compared to other described mechanisms herein that may take only hours to disrupt the biology enough to affect drug efficacy .
however , we disclose upfront that for the purpose of this review we do not label each aspect of resistance as acute or acquired .
1 . an element of the microenvironment : hypoxia - induced resistance : tumor formation and the tumor microenvironment pose a unique set of challenges to the neoplastic cell including decreased glucose concentrations , oxidative stress , poor vascularization , low partial pressures of oxygen and low intratumoral perfusion ( fig .
1 ) . this nutrient and oxygen poor environment imparts a selective pressure , favoring the growth of the most aggressive and fit pda cells11 - 13 .
it is these cells which tend to be the most elusive to cytotoxic chemotherapeutic agents11 , 14 , 15 . in order to overcome the harsh stress imposed by chronic hypoxia ( e.g. : low oxygen pressure [ po2 ] and intratumoral perfusion ) pda cells orchestrate a multi - faceted response by activating hypoxia - inducible factors ( hifs ; e.g. : hif-1 , pim1 , caix , pdk1 , ccnd1)16
this potent acute cellular reprogramming activates pathways responsible for regulating cell motility , intracellular ph , mitochondrial function , angiogenesis , cellular metabolism , dna repair , and cell survival17 - 19 .
our mechanistic understanding of hypoxia - induced chemoresistance is limited , particularly in pda models .
many previous studies focused on the role of hif-1 and its downstream effectors in this process . in various tumor models , hif-1 is implicated in the upregulation of vegf , mdr / p - gp , caix , glut1 , wsb-1 , shh , ldh - a and bcl220 - 25 .
this transcriptional response is pleiotropic in nature and involved in apoptosis , cellular senescence , cellular metabolism , dna repair , drug resistance , and oxidative stress20 - 25 .
demonstrated under hypoxic conditions that sirna inhibition of either hif-1 or nfb sensitized pda cells to gemcitabine and increased apoptosis26 .
maftouh et.al . examined the role of ldh - a , an enzyme involved in anaerobic glycolysis and known target of hif-1 , in hypoxic chemoresistance25 .
they demonstrated increased gemcitabine chemosensitivity utilizing both sirna as well as novel ldh - a inhibitors .
this study provides rationale for targeting cellular metabolism in converting cells to a chemosensitive state under hypoxic stress .
examined pim1 , a serine / threonine kinase with pleiotropic effects on cell survival , apoptosis , and metabolism27 .
they used a dominant negative pim1 to rescue sensitivity to 5-fu , gemcitabine and cisplatin in hypoxia .
they were further able to show that pim1 acted to stabilize the mitochondrial membrane and inhibit apoptosis in a hif-1-independent manner .
our lab has delineated the mechanism by which pim1 is upregulated in hypoxia and implicated the rna binding protein hur in hypoxic chemoresistance ( blanco et al , oncogene in press ) .
this work demonstrates upon hypoxic stimuli , hur ( elavl1 ) is translocated to the cytoplasm where it stabilizes the 3 ' utr of the pim1 transcript . using sirna as well as a novel pharmacologic inhibitor of hur
, the pim1-hur axis was demonstrated to be necessary for hypoxic chemoresistance to 5-fu and oxaliplatin .
finally , multiple studies by onishi et.al have elucidated the importance of the sonic hedgehog ( shh ) pathway in hypoxic chemoresistance22 , 23 .
the shh pathway is involved in pancreatic development and is upregulated under hypoxic conditions23 , 28 . utilizing cyclopamine ,
an shh pathway inhibitor , the investigators rescued 5-fu and gemcitabine resistance in hypoxic conditions .
accordingly , using sirna towards hif-1 , they showed that shh mediated hypoxic chemoresistance was hif-1 independent .
translational researchers have recently attempted to use the hypoxic tumor environment as a therapeutic advantage .
th-302 ( evofosfamide ) is a 2-nitroimidazole prodrug of the cytotoxin bromo - isophosphoramide mustard . under hypoxic conditions , the prodrug undergoes reduction and preferentially releases the active drug in the hypoxic tumor environment29 .
a recent phase ii study reported that co - administering th-302 with gemcitabine resulted in an increase in progression - free survival in patients with advanced pda30 . while current studies are being done in the backbone of gemcitabine - abraxane ,
these results indicate that targeting tumor hypoxia may be a viable approach to overcome resistance in pda .
although the studies outlined above have begun to elucidate the specific mechanisms as well as future avenues for targeting hypoxic chemoresistance , there is still much work to be done .
it is unknown how many of these mechanisms are tumor cell type specific or whether they represent more universal mechanisms of hypoxic chemoresistance . furthermore , there has been no comprehensive evaluation using novel techniques to delve into this important mechanism of resistance . with the advent of gene editing ,
next generation sequencing , and proteomic advances over the past few years , unbiased screens may serve to answer these questions in the future .
cancer somatic mutations as drivers of resistance : classical progression of pancreatic carcinogenesis has been classified into early ( telomere shortening and activating mutations in kras ) , intermediate ( inactivating mutations or epigenetic silencing of cdkn2a2 ) and late ( inactivating mutations of tp53 and smad4 ) events31 .
high throughput sequencing and copy number studies of several pda genomes have identified and validated genes such as kras , tp53 , cdkn2a , smad4 , etc .
as well as identifying novel gene mutations that may be involved in cell growth , dna repair , invasiveness and angiogenesis15 , 32 - 34 ( fig .
however , this extensive understanding of the somatic genetic landscape of pda has yet to substantially contribute to an improvement in prognosis and treatment strategies .
the unmet need can be potentially bridged by shifting focus to downstream events including , metabolic reprogramming , angiogenesis alterations , cell cycle abnormalities , overcoming stromal - microenvironment reaction , immune pathways and inflammation . while folfirinox and gemcitabine plus abraxane form the backbone of current frontline therapies for pda , targeted therapeutics based on the above sequencing studies are being widely explored to combat and overcome acquired resistance in metastatic disease .
a review of recent modern chemotherapy trials reveal that front - line therapy may cause a recist response ( or 30% shrinkage ) in 1/3 of patients4 , 14 and stabilize disease in another 1/3 , virtually all patients will ultimately progress , with a median time to progression of roughly 5 months .
it is fascinating to consider that the mechanism of pda drug resistance to chemotherapy is essentially unknown .
our group has focused on an understudied aspect molecular adaptation that occurs within hours of chemotherapy exposure , and is governed by regulatory proteins that rapidly change the expression of numerous pro - survival proteins by affecting their rna stability ( see section below)35 .
however , it is tempting to speculate that pro - survival somatic mutations are acquired in response to the intense selection pressure imposed by chemotherapy , and these heritable molecular events predominate in resistance clones .
this molecular pattern has been well documented in numerous cancer genes , as ' secondary mutations ' that occur in response to targeted therapies ( e.g. , kit and imatinib 36 , gefitinib and egfr 37 , crizotinib and alk 38 , vemurafanib and braf 39 , vismodegib and smo 40 ) .
interestingly , this theory has never been thoroughly investigated in pancreatic cancers that are resistant to standard chemotherapies .
a clinical trial funded by the pancreatic cancer action network ( pi brody ) will , in part , directly address this question if ex vivo modeling allows for sampling of pre- and post- treated specimens ( fig .
an alternative pathway to resistance : post - transcriptional gene regulation ( ptgr ) is the modulation of rna stability and expression , primarily mediated by rna binding proteins ( rbps ) and micrornas ( fig .
downstream effects of such modulation include alternative splicing , rna processing and nuclear export and redirecting the rna towards storage , translation or degradation .
for instance , muc4 , a protein that plays a major role in pancreatic tumorigenesis , undergoes post - transcriptional regulation and alternative splicing to generate a variant muc4/4 , which has been linked to increased malignancy and resistance to apoptosis41 . because of their ability to globally affect varied regulatory networks , rbps regulate several cellular processes such as cancer initiation and progression , immunological responses , and neurological processes .
specifically in the context of tumorigenesis , post - transcriptional modulation contributes to changes in tumor cell growth and proliferation , angiogenesis , invasion and metastasis , drug responses and ultimately cancer prognosis .
of particular importance , rbps such human antigen r ( hur ) , tristetraproline ( ttp ) , sam68 , eif4e , la , auf1 , play a significant role in regulating tumor responses .
hur 's function is largely dependent on its overall expression and cellular localization in response to cancer - associated stimuli .
stress - induced cytoplasmic translocation of hur and its subsequent stabilization of specific pro - survival transcripts have been linked to drug resistance in solid tumors .
for instance , hur - mediated up - regulation of proteins in the mapk and jnk signaling pathways has been shown to cause tamoxifen resistance in breast cancer mcf-7 cells42 .
several reports indicate the interplay between hur and microrna 's resulting in chemotherapeutic resistance in ovarian43 , prostate44 , breast45 , colon46 , 47 and pancreatic cancer48 , 49 .
a recent study indicated that cancer- associated stressors such as a highly oxidative environment , starvation and dna - damaging agents correlate with a selective increase in expression of hur , which in turn affects the phosphorylation of the initiation factor eif4e and results in chemotherapeutic resistance50 . in pda
, hur expression regulates the stability and expression of enzyme dck , which metabolizes the prodrug gemcitabine , thereby modulating response to the standard of care51 .
another study pioneered the role of hur in supporting a drug resistance phenotype through its stabilization of the mitotic kinase inhibitor , wee1 . in response to dna damaging agents such as mitomycin c and platinum agents , hur translocates to the cytoplasm wherein it stabilizes wee1 mrna , hence allowing cancer cells to pause at g2/m checkpoint , repair damaged dna and evade apoptosis35 .
micrornas ( mirs ) which can rapidly and effectively alter gene expression likely play a role in treatment efficacy52 - 54 . for example , it has been reported that mir-22 regulates sensitivity of colorectal cancer cells to 5-fu by post - transcriptionally regulating crucial target genes which , in turn , facilitate autophagy55 .
several studies have also indicated a link between deregulated mirna expression profiles and chemoresistance in ewing 's sarcoma56 - 59 .
similarly , microrna signatures also influence resistance to trail60 and kinase inhibitors 61 in non - small cell lung and pancreatic cancer . recently ,
poly - adp ribose polymerase ( parp ) inhibitors ( parpi ) , which target the dna damage repair ( ddr ) pathway , have delivered promising preclinical and clinical results .
mechanistically , parpi work through the concept of synthetic lethality , by specifically targeting the achilles ' heel of cancer cells that are already carrying mutations in major dna repair genes .
cells deficient in these enzymes encoded by tumor - suppressor genes , brca1 and brca2are therefore heavily dependent of parp for dna damage repair .
parp1 , an enzyme with a native function to repair single - stranded breaks ( ssbs ) by base excision repair ( ber ) is now solely responsible for hr- function to repair dsbs arising from exposure to chemotherapy ( alkylating agents , topoisomerase inhibitors , etc . ) .
therefore , inhibition of parp1 via parpi has proven to be effective towards treating patients with tumors that harbor mutations in dna repair - related genes62 , 63 .
this concept of synthetic lethality is a promising therapeutic strategy , which brings the medical oncology community closer to a ' personalized ' approach towards treating a subset of pda patients .
approximately 10% of pda patients carry mutations in dna repair genes such as brca1 , brca2 , fanconi anemia genes , palb2 , etc . which makes parp inhibition the best personalized approach for treating this particular subset of pda patients65 , 66 .
a recent study found that the sensitivity of parp1 inhibitor , additional to brca - deficiency , was influenced by expression profiles of dna damage repair ( ddr ) pathway genes ( ercc3 , rad17 , sumo1 , mutyh , cry1 , hsp90b1 , cdc37 , rxra , and usp5 ) 67 .
additionally , a screen identified the deubiquitylating enzyme usp11 as a participant in hr repair of dsbs .
the loss of usp11 caused impaired recruitment of a subset of dsb repair proteins such as rad51 and 53bp1 to the repair foci 68 .
this suggests that , apart from brca and fa genes , parp1 demonstrates synthetic lethality with other ddr genes .
parp1 is known to be hyperactive in brca1- or brca2-deficient cells and p53 also plays a role as a regulator of dna repair pathways .
therefore , patients carrying a gene defect in the dsb repair pathways together with p53 mutations are selected for parp inhibitor therapy69
. currently , parp inhibitors are in different phases of clinical trials , either as single agent ( olaparib ) or in combination ( olaparib , veliparib , rucaparib , bmn673 ) with standard of care gemcitabine or other chemotherapeutic agents for treating patients with locally advanced , unresectable or metastatic pancreatic cancer .
however , de novo and acquired resistance to parp inhibitors poses a significant clinical problem 70 , 71 . to date
a ) targeted mutation reversion : successful targeted therapies in cancer can induce reversion mutations in a cell with a hypermutable state ( e.g. , gleevec giving rise to bcr - abl mutations ) 73 .
secondary , reversion mutations that restore deleterious brca2 mutant function have been the predominantly described mechanism for parp inhibitor resistance74 , and have been shown to be a result of acquired , not de novo mechanisms75 .
these findings were reproduced in cancer cells exposed to platinum - based therapies 76 , 77 .
a thorough study of independently derived parpi- resistant brca2- mutant capan1 cell lines indicated that brca2 function is not typically restored upon prolonged exposure to parpi .
instead , deletion events in brca2 dna either restored the orf that encodes the c - terminal rad51 binding domain or resulted in small tracts of dna sequence homology arising from error - prone repair due to brca2 deficiency 77 .
perhaps the best evidence of a targeted mutation reversion came from a parpi ( olaparib ) study that performed dna sequencing on treatment - naive and post- treatment biopsies .
however , only two patients harbored brca2 reversion mutations in olaparib - resistant metastases that restored brca2 function 78 .
b ) hypermorphic / unclassified brca - alleles : not all identified mutated brca1/2 patients respond to parpi therapy and thus understanding all disease - related brca - alleles will be critical in predicting which patients will respond best to parpi - based therapy .
c ) ineffective parpi uptake or drug export : early pharmacodynamics studies have demonstrated that parpi uptake is effective 79 , 80 , yet a p - glycoprotein - mediated drug resistance mechanism has been demonstrated in a mouse model 81 .
elevated expression of abcb1 , a p - glycoprotein efflux pump has been shown to cause olaparib resistance ; this can be abrogated by treatment with a pgp inhibitor , tariquidar81 .
d ) rewiring of the dna damage response : a more likely but complicated resistance mechanism is the compensation of dna repair network , which will result in negating the ' synthetic lethal ' setting in hr - deficient pancreatic cancer cells .
compensatory loss of another dna repair factor , p53-binding protein 1 ( 53bp1 ) reduces non - homologous end joining ( nhej ) efficiency and is one of the best described ddr rewiring causing parpi resistance 82 , 83 .
however , neither the validation of this ddr rewiring , nor the involvement of these molecules as biomarkers , have been fully established in human tumor samples from clinical trials . an alternative post - transcriptional parpi resistance mechanism ( revisiting hur ) : recently , our group showed evidence that strongly suggests that pda cells develop resistance to dna damaging agents through post - transcriptional gene regulation84 through the rna - binding protein , hur .
primarily localized in the nucleus , hur translocates to the cytoplasm in response to cellular stress ( e.g. , dna damage , nutrient depletion , and hypoxia)85 , where it becomes functionally active as a stress response protein . as an rbp , hur binds to the au - rich elements ( ares ) typically in the 3'-untranslated regions ( utrs ) of specific , survival target genes involved in cell proliferation , evading apoptosis 86 , and mitotic inhibition ( e.g. , the mitotic kinase inhibitor wee1 ) 84 , 87 - 90 .
importantly , we have shown that chemical and genetic silencing of hur results in modulation of key cell cycle regulator , wee1 and efficacy of chemotherapy 84 .
indeed , a seminal study demonstrated that wee1 inhibition enhances accumulation of lethal dna damage and apoptosis , thereby enhancing sensitivity to parp inhibitors in pancreatic cancer cells91 .
we have preliminary data demonstrating that parpi : 1 ) induces hur translocation from the nucleus to the cytoplasm and 2 ) hur can regulate pro - survival transcripts that diminish the efficacy of parpi and ultimately maybe a critical factor in causing pda resistance to this targeted therapy ( chand et al . , unpublished ) .
future studies will determine the significance that hur biology has on the clinical effectiveness of parpi - based therapies .
because of their ability to globally affect varied regulatory networks , rbps regulate several cellular processes such as cancer initiation and progression , immunological responses , and neurological processes
. specifically in the context of tumorigenesis , post - transcriptional modulation contributes to changes in tumor cell growth and proliferation , angiogenesis , invasion and metastasis , drug responses and ultimately cancer prognosis .
of particular importance , rbps such human antigen r ( hur ) , tristetraproline ( ttp ) , sam68 , eif4e , la , auf1 , play a significant role in regulating tumor responses .
hur 's function is largely dependent on its overall expression and cellular localization in response to cancer - associated stimuli .
stress - induced cytoplasmic translocation of hur and its subsequent stabilization of specific pro - survival transcripts have been linked to drug resistance in solid tumors .
for instance , hur - mediated up - regulation of proteins in the mapk and jnk signaling pathways has been shown to cause tamoxifen resistance in breast cancer mcf-7 cells42 .
several reports indicate the interplay between hur and microrna 's resulting in chemotherapeutic resistance in ovarian43 , prostate44 , breast45 , colon46 , 47 and pancreatic cancer48 , 49 .
a recent study indicated that cancer- associated stressors such as a highly oxidative environment , starvation and dna - damaging agents correlate with a selective increase in expression of hur , which in turn affects the phosphorylation of the initiation factor eif4e and results in chemotherapeutic resistance50 . in pda
, hur expression regulates the stability and expression of enzyme dck , which metabolizes the prodrug gemcitabine , thereby modulating response to the standard of care51 .
another study pioneered the role of hur in supporting a drug resistance phenotype through its stabilization of the mitotic kinase inhibitor , wee1 . in response to dna damaging agents such as mitomycin c and platinum agents , hur translocates to the cytoplasm
wherein it stabilizes wee1 mrna , hence allowing cancer cells to pause at g2/m checkpoint , repair damaged dna and evade apoptosis35 .
micrornas ( mirs ) which can rapidly and effectively alter gene expression likely play a role in treatment efficacy52 - 54 .
for example , it has been reported that mir-22 regulates sensitivity of colorectal cancer cells to 5-fu by post - transcriptionally regulating crucial target genes which , in turn , facilitate autophagy55 .
several studies have also indicated a link between deregulated mirna expression profiles and chemoresistance in ewing 's sarcoma56 - 59 .
similarly , microrna signatures also influence resistance to trail60 and kinase inhibitors 61 in non - small cell lung and pancreatic cancer .
recently , poly - adp ribose polymerase ( parp ) inhibitors ( parpi ) , which target the dna damage repair ( ddr ) pathway , have delivered promising preclinical and clinical results .
mechanistically , parpi work through the concept of synthetic lethality , by specifically targeting the achilles ' heel of cancer cells that are already carrying mutations in major dna repair genes .
cells deficient in these enzymes encoded by tumor - suppressor genes , brca1 and brca2are therefore heavily dependent of parp for dna damage repair .
parp1 , an enzyme with a native function to repair single - stranded breaks ( ssbs ) by base excision repair ( ber ) is now solely responsible for hr- function to repair dsbs arising from exposure to chemotherapy ( alkylating agents , topoisomerase inhibitors , etc . ) .
therefore , inhibition of parp1 via parpi has proven to be effective towards treating patients with tumors that harbor mutations in dna repair - related genes62 , 63 .
this concept of synthetic lethality is a promising therapeutic strategy , which brings the medical oncology community closer to a ' personalized ' approach towards treating a subset of pda patients .
approximately 10% of pda patients carry mutations in dna repair genes such as brca1 , brca2 , fanconi anemia genes , palb2 , etc . which makes parp inhibition the best personalized approach for treating this particular subset of pda patients65 , 66 .
a recent study found that the sensitivity of parp1 inhibitor , additional to brca - deficiency , was influenced by expression profiles of dna damage repair ( ddr ) pathway genes ( ercc3 , rad17 , sumo1 , mutyh , cry1 , hsp90b1 , cdc37 , rxra , and usp5 ) 67 .
additionally , a screen identified the deubiquitylating enzyme usp11 as a participant in hr repair of dsbs .
the loss of usp11 caused impaired recruitment of a subset of dsb repair proteins such as rad51 and 53bp1 to the repair foci 68 .
this suggests that , apart from brca and fa genes , parp1 demonstrates synthetic lethality with other ddr genes .
parp1 is known to be hyperactive in brca1- or brca2-deficient cells and p53 also plays a role as a regulator of dna repair pathways .
therefore , patients carrying a gene defect in the dsb repair pathways together with p53 mutations are selected for parp inhibitor therapy69
. currently , parp inhibitors are in different phases of clinical trials , either as single agent ( olaparib ) or in combination ( olaparib , veliparib , rucaparib , bmn673 ) with standard of care gemcitabine or other chemotherapeutic agents for treating patients with locally advanced , unresectable or metastatic pancreatic cancer .
however , de novo and acquired resistance to parp inhibitors poses a significant clinical problem 70 , 71 . to date , four parpi resistant mechanisms have been highlighted in the literature 72 .
a ) targeted mutation reversion : successful targeted therapies in cancer can induce reversion mutations in a cell with a hypermutable state ( e.g. , gleevec giving rise to bcr - abl mutations ) 73 .
secondary , reversion mutations that restore deleterious brca2 mutant function have been the predominantly described mechanism for parp inhibitor resistance74 , and have been shown to be a result of acquired , not de novo mechanisms75 .
these findings were reproduced in cancer cells exposed to platinum - based therapies 76 , 77 .
a thorough study of independently derived parpi- resistant brca2- mutant capan1 cell lines indicated that brca2 function is not typically restored upon prolonged exposure to parpi . instead , deletion events in brca2 dna either restored the orf that encodes the c - terminal rad51 binding domain or resulted in small tracts of dna sequence homology arising from error - prone repair due to brca2 deficiency 77 .
perhaps the best evidence of a targeted mutation reversion came from a parpi ( olaparib ) study that performed dna sequencing on treatment - naive and post- treatment biopsies .
however , only two patients harbored brca2 reversion mutations in olaparib - resistant metastases that restored brca2 function 78 .
b ) hypermorphic / unclassified brca - alleles : not all identified mutated brca1/2 patients respond to parpi therapy and thus understanding all disease - related brca - alleles will be critical in predicting which patients will respond best to parpi - based therapy .
c ) ineffective parpi uptake or drug export : early pharmacodynamics studies have demonstrated that parpi uptake is effective 79 , 80 , yet a p - glycoprotein - mediated drug resistance mechanism has been demonstrated in a mouse model 81
. elevated expression of abcb1 , a p - glycoprotein efflux pump has been shown to cause olaparib resistance ; this can be abrogated by treatment with a pgp inhibitor , tariquidar81 .
d ) rewiring of the dna damage response : a more likely but complicated resistance mechanism is the compensation of dna repair network , which will result in negating the ' synthetic lethal ' setting in hr - deficient pancreatic cancer cells .
compensatory loss of another dna repair factor , p53-binding protein 1 ( 53bp1 ) reduces non - homologous end joining ( nhej ) efficiency and is one of the best described ddr rewiring causing parpi resistance 82 , 83 .
however , neither the validation of this ddr rewiring , nor the involvement of these molecules as biomarkers , have been fully established in human tumor samples from clinical trials . an alternative post - transcriptional parpi resistance mechanism ( revisiting hur ) : recently , our group showed evidence that strongly suggests that pda cells develop resistance to dna damaging agents through post - transcriptional gene regulation84 through the rna - binding protein , hur .
primarily localized in the nucleus , hur translocates to the cytoplasm in response to cellular stress ( e.g. , dna damage , nutrient depletion , and hypoxia)85 , where it becomes functionally active as a stress response protein . as an rbp , hur binds to the au - rich elements ( ares ) typically in the 3'-untranslated regions ( utrs ) of specific , survival target genes involved in cell proliferation , evading apoptosis 86 , and mitotic inhibition ( e.g. , the mitotic kinase inhibitor wee1 ) 84 , 87 - 90 .
importantly , we have shown that chemical and genetic silencing of hur results in modulation of key cell cycle regulator , wee1 and efficacy of chemotherapy 84 .
indeed , a seminal study demonstrated that wee1 inhibition enhances accumulation of lethal dna damage and apoptosis , thereby enhancing sensitivity to parp inhibitors in pancreatic cancer cells91 .
we have preliminary data demonstrating that parpi : 1 ) induces hur translocation from the nucleus to the cytoplasm and 2 ) hur can regulate pro - survival transcripts that diminish the efficacy of parpi and ultimately maybe a critical factor in causing pda resistance to this targeted therapy ( chand et al . , unpublished ) .
future studies will determine the significance that hur biology has on the clinical effectiveness of parpi - based therapies .
the above described cellular and molecular mechanisms either discuss how the milieu and/or the molecular machinery contribute to therapeutic resistance .
the three - year survival for even the most effective modern chemotherapeutic regimens is 5% or less 4 , 14 .
in contrast , the 5-year survival in large surgical series is close to 20% , and roughly 1/3 of patients survive over 3 years 92 .
nevertheless , it is justifiable to question the role of surgery for the treatment of pancreatic cancer at all .
the recurrence rate after resection , which is a pre - selected group of patients with a relatively favorable prognosis , is in excess of 90% 92 .
it stands to reason that occult residual disease ( distant sites , regional metastases , or at the resection margin ) , are present in the majority of patients .
thus , why is resection a viable option , if the disease is so often systemic ?
the strongest evidence in favor of surgery for localized disease is not biologic , but actually empiric . in japan , forty - two patients with localized and resectable disease were randomized to resection and no adjuvant therapy vs. exploratory laparotomy and biopsy only , followed by chemoradiation .
there were no long - term survivors in the group of patients who did not undergo resection , while 20% of patients in the resection group lived more than 3 years .
therefore , unlike most other cancers , the benefit of resection to treat pancreatic cancer is supported by randomized data . from a biologic standpoint , resection is able to functionally achieve a ' complete response ' in one day 's work ( that is , render the patient with no radiographic evidence of disease ) .
in contrast , the most effective chemotherapy regimens are unable to achieve this result over the course of 6 months of treatment . if patients with pancreatic cancer ultimately die from a cancer syndrome when
a critical volume of disease burden has been reached , then a complete resection can lengthen life by debulking 99% of the cancer cells . assuming a cancer doubling time of 100 days , six doublings , or two years , are required to return to the pre - resection disease burden 93 .
rather than render surgery obsolete , it is more likely that improvements in chemotherapy in the future will expand the number of patients who are eligible or will likely benefit from resection .
simplistically , as treatments to control systemic disease improve , control of local disease by resection and/or radiation will likely become more important .
prospective , single - institution studies ( albeit non - randomized ) with modern multi - agent therapy reveal that the majority of patients with locally advanced disease may be converted from ' unresectable ' to resectable , which far exceeds historical figures 94 . despite the isolated , non - randomized studies surfacing now ,
the fact remains that roughly 20% of patients in the general population who develop pancreatic adenocarcinoma will have localized and resectable disease95 .
pancreatic cancer is staged according to the ajcc tnm , 7 ed . and based on the presence of regional lymph node metastases , distant metastases , and cancer involvement of major visceral vessels96 .
however , from a practical perspective , pancreatic cancer is surgically staged to determine resectability .
cancers localized to the pancreas are typically staged for resection using a multislice ct scan using three contrast phases ( early arterial , late arterial , and venous ) to carefully examine the relationship of the tumor to nearby vessels 97 .
localized cancers that do not invade and distort the superior mesenteric vein or portal vein , and do not abut the superior mesenteric or celiac arteries , are considered resectable and patients are typically offered resection ( although neoadjuvant chemotherapy is an acceptable option ) .
when the veins are distorted , or the arteries abutted by the tumor , the cancer is considered ' borderline resectable , ' and most surgeons favor a neoadjuvant approach , using chemotherapy ( with or without the addition or chemoradiation ) .
invasion of the veins with a technically reconstruction option , or encasement of the arteries indicate that the cancer is locally advanced , and unlikely to be treatable by resection .
modern chemotherapy regimens have increased the percentage of patients who are able to undergo an attempt at resection , after a course of neoadjuvant treatment for borderline or locally advanced pancreatic cancer .
considerations : although surgery is probably ' the best ' option for a patient diagnosed with pda and better adjuvant therapies need to be developed in order to improve outcomes , a few considerations remain about resectional therapy ( fig .
1 ) : 1 ) identification of biomarkers of early recurrence or progression , in order to spare patients with particularly aggressive cancers unnecessary surgery .
3 ) determining whether surgery , in some instances , cause a milieu in which growth factors or tissue dissection activate the spreading of micrometastateses .
4 ) identifying whether neoadjuvant therapy , although relevant for down staging for resection , ultimately adds a selection process on distant metastatic cells and eventually strengthens the metastatic clones .
immunotherapies : tumor immunotherapy has become an effective tool in the treatment of many diseases , most notably melanoma98 .
a recent phase i study which evaluated the combination of an agonist cd40 monoclonal antibody in combination with gemcitabine in patients with metastatic chemotherapy - naive pda indicated promise , though marginal 99 .
although it appears that like most promising therapeutics , pda appears to be unresponsive to several new and established immunotherapeutic strategies .
we do acknowledge the progress made and pursuit for a successful vaccine for use as a therapy 100 .
considerations : the mutational and cellular heterogeneity of pda makes this a difficult disease to bolster the immune system to fight against . thus , although tumor immunotherapy is an extremely promising strategy to combat cancer , 1 ) it remains to be determined if it is an effective treatment option for pda cells .
2 ) further studies are required to define if there are too few tumor infiltrating b lymphocytes that can circulate and find their way to pda tumor cells in order for a t - cell based therapeutic strategy to work .
3 ) future investigation should identify whether depleting the cellular elements of the tumor microenvironment will allow for proper honing and targeting of immuno- and chemotherapy .
4 ) recognizing if pda cells produce factors or enzymes , such as ido2 , that need to be targeted in order to truly combat the immune blockage against pda.101 targeted and personalized ( and combination therapies ) : combination therapies and even cytotoxic therapies along with new targeted approaches are being evaluated in clinical trials . even in the best ' personalized approach ' scenario
, these targeted or combinatory approaches will most likely yield resistant pda cells . as the therapies become better and we establish a better understanding of a molecularly tailored approach to treating pda patients
, we may need to think about other aspects of combination therapy and personalized approaches to treating this disease .
considerations : 1 ) determining the importance of the ' timed sequencing ' of drug administration in combination treatment strategies in an effort to slow down and overcome resistance mechanisms .
for instance , if one drug induces the subcellular localization of hur ( see above ) , that induces a survival network for another drug , it is crucial to determine if these drugs should be given separately or spaced out over days versus hours .
2 ) additional elements of the cell machinery ( not discussed here ) include the adaptable phosphoproteome . as technologies advance , following
the phosphoproteomic signature may be very relevant to drug resistance and optimal drug selection for a patient who recurs for 2 and 3 line therapies(fig .
3 ) evaluate if targeting a specific disrupted pathway could either be trumped or further enhanced by the addition of a cytotoxic agent ( e.g. , a generic dna damaging agent could potentially set the stage for a targeted approach against a dna repair pathway such as parp inhibition ) .
we are hopeful that an era of better drug selections and therapeutic options are around the corner for pda patients . yet , until ' personalized medicine ' and better targeted therapies are ready for the clinic , optimizing current therapeutic strategies that have activity in patients will be critical .
similarly , even if ' personalized therapy ' becomes a reality , we will need to better understand how pda cells become resistant to the best matched , available therapies ( fig .
3 ) . monitoring every patient 's tumor in real time ( i.e. , biopsying and sequencing the tumor as a moving target ) is what we may need to do to manage this disease effectively ( fig . | pancreatic cancer ( pancreatic ductal adenocarcinoma , pda ) is infamously moving to the top of the list as one of the most lethal cancers with an overall 5 year survival rate of 7% .
multiple genomic - based and molecular characterization studies of pda specimens and established animal models have provided the field with multiple targets and a progression model of this disease . still , to date , the best therapeutic options are surgery and combination cytotoxic therapies . in general , even in the best case scenario ( i.e. , an early stage diagnosis and a response to a specific therapy ) , most of these fortunate patients ' pda cells acquire or exert resistance mechanisms and eventually kill the patient . herein , we touch on a growing field of investigation that focuses on pda cell therapeutic resistance mechanisms .
we examine extrinsic elements ( i.e. , the tumor microenvironment , hypoxia ) to the intrinsic processes within the cell ( i.e. , post - transcriptional gene regulation and somatic mutations ) that are important for therapeutic efficacy and resistance . even as better targeted and personalized approaches move through the clinical trial pipeline the discussed resistance mechanisms
will most likely play a role in the management of this deadly disease . | Overview of Pancreatic Cancer
Prelude
RNA-Binding proteins in cancer prognosis or treatment responses
HuR and chemotherapeutic resistance
Role of MicroRNAs in cancer prognosis or treatment responses
PARP Inhibition in PDA
Non-cellular mechanisms affecting therapeutic resistance
Future directions | pancreatic ductal adenocarcinoma ( pda ) is a highly lethal malignancy with a 5-year survival rate of 7%1 . in this perspective , we survey current therapeutic resistance mechanisms both extrinsically including the tumor microenvironment and also intrinsically within the cellular machinery of pda cells ( fig . we refer the reader to other outstanding reviews and publications on elements of the tumor microenvironment , drug availability and honing issues7 - 10 . we will focus on the classical view of therapeutic resistance mechanisms that are most likely intact in the majority of pdas . an element of the microenvironment : hypoxia - induced resistance : tumor formation and the tumor microenvironment pose a unique set of challenges to the neoplastic cell including decreased glucose concentrations , oxidative stress , poor vascularization , low partial pressures of oxygen and low intratumoral perfusion ( fig . this work demonstrates upon hypoxic stimuli , hur ( elavl1 ) is translocated to the cytoplasm where it stabilizes the 3 ' utr of the pim1 transcript . an alternative pathway to resistance : post - transcriptional gene regulation ( ptgr ) is the modulation of rna stability and expression , primarily mediated by rna binding proteins ( rbps ) and micrornas ( fig . for instance , muc4 , a protein that plays a major role in pancreatic tumorigenesis , undergoes post - transcriptional regulation and alternative splicing to generate a variant muc4/4 , which has been linked to increased malignancy and resistance to apoptosis41 . specifically in the context of tumorigenesis , post - transcriptional modulation contributes to changes in tumor cell growth and proliferation , angiogenesis , invasion and metastasis , drug responses and ultimately cancer prognosis . micrornas ( mirs ) which can rapidly and effectively alter gene expression likely play a role in treatment efficacy52 - 54 . compensatory loss of another dna repair factor , p53-binding protein 1 ( 53bp1 ) reduces non - homologous end joining ( nhej ) efficiency and is one of the best described ddr rewiring causing parpi resistance 82 , 83 . an alternative post - transcriptional parpi resistance mechanism ( revisiting hur ) : recently , our group showed evidence that strongly suggests that pda cells develop resistance to dna damaging agents through post - transcriptional gene regulation84 through the rna - binding protein , hur . primarily localized in the nucleus , hur translocates to the cytoplasm in response to cellular stress ( e.g. as an rbp , hur binds to the au - rich elements ( ares ) typically in the 3'-untranslated regions ( utrs ) of specific , survival target genes involved in cell proliferation , evading apoptosis 86 , and mitotic inhibition ( e.g. specifically in the context of tumorigenesis , post - transcriptional modulation contributes to changes in tumor cell growth and proliferation , angiogenesis , invasion and metastasis , drug responses and ultimately cancer prognosis . micrornas ( mirs ) which can rapidly and effectively alter gene expression likely play a role in treatment efficacy52 - 54 . compensatory loss of another dna repair factor , p53-binding protein 1 ( 53bp1 ) reduces non - homologous end joining ( nhej ) efficiency and is one of the best described ddr rewiring causing parpi resistance 82 , 83 . an alternative post - transcriptional parpi resistance mechanism ( revisiting hur ) : recently , our group showed evidence that strongly suggests that pda cells develop resistance to dna damaging agents through post - transcriptional gene regulation84 through the rna - binding protein , hur . primarily localized in the nucleus , hur translocates to the cytoplasm in response to cellular stress ( e.g. cancers localized to the pancreas are typically staged for resection using a multislice ct scan using three contrast phases ( early arterial , late arterial , and venous ) to carefully examine the relationship of the tumor to nearby vessels 97 . 3 ) future investigation should identify whether depleting the cellular elements of the tumor microenvironment will allow for proper honing and targeting of immuno- and chemotherapy . 4 ) recognizing if pda cells produce factors or enzymes , such as ido2 , that need to be targeted in order to truly combat the immune blockage against pda.101 targeted and personalized ( and combination therapies ) : combination therapies and even cytotoxic therapies along with new targeted approaches are being evaluated in clinical trials . even in the best ' personalized approach ' scenario
, these targeted or combinatory approaches will most likely yield resistant pda cells . as the therapies become better and we establish a better understanding of a molecularly tailored approach to treating pda patients
, we may need to think about other aspects of combination therapy and personalized approaches to treating this disease . similarly , even if ' personalized therapy ' becomes a reality , we will need to better understand how pda cells become resistant to the best matched , available therapies ( fig . , biopsying and sequencing the tumor as a moving target ) is what we may need to do to manage this disease effectively ( fig . | [
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] |
despite the prevalence
of respiratory infections and deaths relating
to furanoterpenoids on global health of cattle and possibly humans ,
the concentration levels of these compounds in diseased and healthy - looking
parts of infected sweetpotatoes is poorly understood and insufficiently
documented .
sweetpotato contains several phytoalexins , collectively
known as furanoterpenoids , including ipomeamarone , 1 ;
dehydroipomeamarone , 2 ; 4-ipomeanol , 3 ;
and 1,4-ipomeadiol , 4 ( figure 1 ) .
biotic factors such as fungi are reported to elicit
varying levels of furanoterpenoids , which
cause hepatoxicity , pneumonia , lung edema , and cattle deaths .
although information on furanoterpenoids is available
concerning isolation methods and presence
in sweetpotatoes , there is no documented or accessible
information on variation of the concentrations from inoculation point
on toward healthy - looking parts of an infected sample .
this is important
because in sub - saharan africa ( ssa ) , sweetpotato is a food crop for
many rural poor families whereby both healthy and infected sweetpotato
storage roots are harvested together .
the infected parts are usually
removed and fed to farm animals , while the remaining apparently healthy
parts are consumed by the farm households , typically in western kenya
( dr . robert mwanga , sweetpotato breeder , personal communication ) .
furanoterpenoids
in sweetpotatoes : ipomeamarone , 1 ; dehydroipomeamarone , 2 ; 4-ipomeanol , 3 ; and 1,4-ipomeadiol , 4 .
sweetpotato is ranked the third
most important root and tuber crop
after potato and cassava in ssa . as a food crop , sweetpotato
is used
by many poor families because of the higher yield in dry matter content
per unit area compared to cereal cultivation on an equivalent piece
of land .
in addition , sweetpotatoes are
particularly important during dry periods of the year when cereal
crops are unavailable .
unfortunately ,
it is during the same dry period that the crop is attacked by insects
and microbes .
it is also known that farmers
in ssa practice piece - meal harvesting , which exposes the storage roots
left in the soil during dry seasons to insects and microbes . farmers who harvest
sweetpotato storage roots
for sale are faced with postharvest infection because microbes attack
the storage roots owing to the favorable warm conditions and bruising
caused by handling .
storage roots respond
to attack by eliciting production of furanoterpenoids to fight off
the infection within the sweetpotato tissues .
the total furanoterpenoid
level of three compounds was documented between 25 and 67,000 mg / kg
in harvested storage root samples irrespective of the microbe that
elicited their accumulation .
the use of radioactivity
to analyze ipomeamarone levels has been done , but more sensitive techniques ,
such as coupled liquid chromatography - quadruple time - of - flight mass
spectrometry ( lc qtof ms ) and coupled gas chromatography mass
spectrometry ( gc ms ) , are needed .
it is not known how far into the healthy - looking storage root
samples
that these furanoterpenoids are found and at what levels .
since this
could potentially expose both humans and farm animals to either chronic
or acute toxicity , we ( i ) isolated and identified a fungus from field
samples that elicited furanoterpenoid production ; ( ii ) characterized
the furanoterpenoids using gc ms and lc qtof ms ;
( iii ) evaluated the furanoterpenoid concentration levels produced
for different sweetpotato cultivars after infection by the isolated
fungus ; and ( iv ) determined the furanoterpenoid levels within the
apparently healthy part of infected storage roots .
two types of sweetpotato
( ipomoea batatas l. lam ) samples were
used to analyze
for ipomeamarone concentrations : weevil - infected sweetpotatoes for
fungal isolation and healthy sweetpotatoes for inoculation experiments
with the isolated fungus .
healthy sweetpotatoes were purchased from
markets in nairobi , kenya and were classified by the vendors as kemb ,
naspot , bungoma , and nyawo cultivars .
weevil - infested sweetpotato
storage roots were sampled from a farm at the kenya agricultural research
institute ( kari ) , marigat , kenya during the dry season when infestation
was high .
sampled storage roots were carefully placed in covered but
aerated plastic jars ( 20.5 cm 10.5 cm ) immediately after harvest
and then incubated at 2528 c for 5 days to promote growth
of microbes .
isolation was carried out from fully infected
storage roots in jars by using a pin and plated on potato dextrose
agar ( pda ) ( oxoid , hampshire , england ) media supplemented with 50
g / ml streptomycin sulfate ( sigma , st .
louis , mo ) and chloramphenicol
( 25 g / ml ) ( sigma , st .
there was a colony that was consistently
found on all the pda plates , and then named marigat isolate-1 ( mi-1 ) .
a sterile glass coverslip was placed
in an oblique position on the culture , and plates were incubated at
25 c for 46 days . from day two onward , coverslips were
removed daily and mounted on a slide stained with lactophenol aniline
blue ( sigma - aldrich , st .
the coverslips were examined
using a light microscope ( leica , wetzlar , germany ) at the magnifications
8 and 40 .
molecular characterization of mi-1 was
initiated by isolation of genomic dna using ctab method from mycelia
grown on pda plates .
polymerase chain
reaction ( pcr ) was conducted using internal transcribed spacers ( its )
primer pairs .
the pcr comprised a 25 l reaction that consisted
of pcr buffer ( 1x ) , its1 primer ( 0.2 m ) , its4
primer ( 0.2 m ) , dntps ( 0.06 mm ) , mgcl2 ( 2 mm ) , and
taq polymerase ( 0.5 unit ) ( thermo scientific , wyman , ma ) .
the primer
sequences for its1 and its4 were 5 tccgtaggtgaacctgcgg
3 and 5 tcctccgcttattgatatgc 3 , respectively .
this reaction mix was subjected to the following
pcr program : initial denaturation 94 c for 5 min , followed by
40 cycles of denaturation 94 c for 45 s , annealing temperature
60 c for 30 s , elongation 72 c for 90 s , and a final elongation
72 c for 6 min .
pcr was conducted on several other microbes
to verify that the amplicon generated from mi-1 was not present in
other microbes or if present , it was not of a similar size .
the microbes
included were as follows : mi-1 ; metarhizium anisopliae ic30 from icipe ; ceratocystis fimbriata f. sp . platani ( cbs 127659 ) ; clavibacter michiganensis ssp .
michiganensis ; catharanthus roseus containing udinese - stolbur
phytoplasma from cbs - knaw fungal biodiversity centre ( utrecht , the
netherlands ) ; and an unknown fungus culture collected from potato
fields and were grown on pda media containing streptomycin sulfate .
pcr products were resolved on
1% agarose gel in tbe for 1 h at 100 v. pcr products were purified
using qiaquick pcr purification kit ( qiagen , hilden , germany ) and
gel extraction kit ( qiagen , hilden , germany ) .
purified pcr products
were sequenced using gs - flx 454 platform ( 454 life sciences / roche ,
bradford , ct ) .
the storage root samples were prepared using
a previously described method with few
modifications .
healthy storage roots from sweetpotato cultivars namely
kemb , naspot , nyawo , and bungoma were washed , surface sterilized for
5 min using 0.5% sodium hypochlorite , and rinsed three times in sterile
distilled water in a laminar flow hood . in the first experiment , the
storage root samples were cut into halves and placed in clean sterile
plastic containers and then inoculated with actively growing fungal
isolate ( mi-1 ) from agar plugs , while the controls were not inoculated .
the sweetpotato samples ( both inoculated and noninoculated ) were replicated
three times for each cultivar .
the second experiment consisted of
inoculating kemb and naspot cultivars using mi-1 , and storage roots
tied with polythene and elastic bags to restrict growth of the fungus .
this second experiment consisted of two cultivars ,
kemb and naspot , with seven samples .
each sample was chopped into
1 cm slices to analyze furanoterpenoid levels from infection point
to the healthy - looking tissue .
the storage roots were then
weighed and blended in 100 ml of methanol and 3 g of nacl for 3 min .
for isolation and purification of dehydroipomeamarone and ipomeamarone ,
268 g of infected sweetpotato was extracted with 550 ml of methanol .
4 , poured
in conical flasks , and then concentrated in a rotary evaporator to
remove methanol and water .
the volume of concentrated crude extract
was estimated as 10 ml , and the same volume of 10 ml of dichloromethane
( sigma , st .
louis , mo ) was added to effectively double the volume
of the extract for each flask .
the organic phases were combined , concentrated
to dryness in a rotary evaporator to remove any remaining solvents
from the crude extract , weighed , and then purified by chromatography .
fifteen grams of the extracted materials was chromatographed on
3263 m silica gel ( riedel - de haen , seelze , germany )
using a hexane ethyl acetate gradient .
the furanoterpenoids
fraction was eluted with 90% hexane : ethyl acetate , which was further
cleaned by column chromatography using a hexane
further purification of the eluent
was carried out on a shimadzu vp series using a 205 mm 10 mm
i.d .
the
mobile phase used an isocratic program ( a : b ) , 60:40 with a flow rate
of 1 ml / min , and run time was 20 min .
detection was by uv absorption
at 270 nm to obtain 18 mg of ipomeamarone and 15 mg of dehydroipomeamarone
with percentage yield being 0.12% and 0.10% , respectively ( figure 2 ) .
structures of the isolated furanoterpenoids were
determined by means of chromatographic and spectroscopic techniques
( lc qtof ms , gc ms , and 1- and 2-d nmr ) and by
comparison with spectroscopic literature data .
hplc profile
of the furanoterpenoid mix isolated from column chromatography
with ipomeamarone and dehydroipomeamarone indicated as 16.43 min and
11.52 min , respectively .
for gc ms analyses , the furanoterpenoids fractions
was analyzed
by split / splitless injection using a model 7890 gas chromatograph
coupled to a 5975c inert xl ei / ci mass spectrometer ( agilent technologies ,
palo alto , ca ) ( gc ms ) equipped with a 30 m 0.25 mm
i.d .
0.25 m film thickness hp-5 column ( el 70 ev ) ( agilent
technologies , palo alto , ca ) .
helium was used as the carrier gas at
a flow rate of 1.2 ml / min .
the oven temperature was held at 35 c
for 3 min and then programmed to increase at 10 c / min to 280
c and maintain this temperature for 10 min .
the target peaks
were identified through comparison of their mass spectra with adams2.l ,
chemecol.l , and nist05a.l library data ( figure 3 ) .
ms total ion chromatogram of purified
furanoterpenoids : ipomeamarone , 1 ; and dehydroipomeamarone , 2 . for lc
qtof ms analyses ,
the crude extract was concentrated
in vacuo to dryness , redissolved in 3 ml of lc
louis , mo ) , and centrifuged at 14,000
rpm for 5 min , after which 0.5 l was automatically injected
into lc
the chromatographic separation was
achieved on a waters acquity uplc ( ultraperformance liquid chromatography )
i - class system ( waters corporation , maple street , ma ) fitted with
a 2.1 mm 100 mm , 1.7-m particle size waters acquity
uplc beh c18 column ( waters corporation , dublin , ireland ) heated to
40 c and an autosampler tray cooled to 15 c .
mobile phases
of water ( a ) and acetonitrile ( b ) , each with 0.01% formic acid were
employed .
the following gradient was used : 01.5 min , 10% b ;
1.52 min , 1050% b ; 26 min , 50100%
b ; 69 min , 100% b ; 910 min , 9010% b ; 1012
min , 10% b. the flow rate was held constant at 0.4 ml / min .
the uplc
system was interfaced by electrospray ionization ( esi ) to a waters
xevo qtof
data were acquired in resolution mode over the m / z range of 1001200 with a scan time of
1 s using a capillary voltage of 0.5 kv , sampling cone voltage of
40 v , source temperature of 100 c , and desolvation temperature
of 350 c .
the nitrogen desolvation flow rate was 500 l / h . for
the high - energy scan function ,
a collision energy ramp of 2545
ev was applied in the t - wave collision cell using ultrahigh purity
argon ( 99.999% ) as the collision gas .
a continuous lock spray
reference compound ( leucine enkephalin ; [ m + h ] = 556.2766 )
was sampled at 10 s intervals for centroid data mass correction .
the
mass spectrometer was calibrated across the 501200 da mass
range using a 0.5 mm sodium formate solution prepared in 90:10 2-propanol / water
( v / v ) .
masslynx version 4.1 scn 712 ( waters corporation , maple street ,
ma ) was used for data acquisition and processing .
potential assignments were calculated
using monoisotopic masses with specifications of a tolerance of 10
ppm deviation and both odd- and even - electron states possible .
the
number and types of expected atoms were set as follows : carbon , 100 ;
hydrogen , 100 ; oxygen , 50 ; nitrogen , 6 ; sulfur ,
6 .
ms data acquisition and analysis
were based on the following defined parameters : mass accuracy ( ppm )
= 1,000,000 ( calculated mass accurate mass)/calculated
mass ; fit conf % is the confidence with which accurate mass ( measured
data ) matches the theoretical isotope models of the elemental composition
in the list ; elemental composition is a suggested formula for the
specified mass .
this is a summation of the quantities of elements ,
isotopes , or superatoms that can compose the measured data , calculated
using the following atomic masses of the most abundant isotope of
the elements : c = 12.0000000 , h = 1.0078250 , n = 14.0030740 , o = 15.9949146 ,
and s = 31.9720718 .
the empirical formula generated was used to predict
structures that were proposed based on the online database , fragmentation
pattern , and literature . to verify the identity of these peaks ,
nuclear magnetic resonance
( nmr )
the isolated and purified ipomeamarone and dehydroipomeamarone
samples ( 5 mg each ) were each dissolved in cdcl3 ( cambridge
isotope laboratories , tewksbury , ma ) and placed in 2.5 mm 100
mm match nmr tubes ( norell , landisville , nj ) .
1d and 2d h and c nmr spectroscopy , including correlation spectroscopy
( cosy ) , heteronuclear single - quantum coherence ( hsqc ) , and heteronuclear
multiple - bond correlation ( hmbc ) spectroscopy , were used for verification .
for the dehydroipomeamarone ,
2d nuclear overhauser effect spectroscopy
( noesy ) data were collected and analyzed for additional structural
verification ( see supporting information ) .
all nmr spectra were
acquired at 22 c using a 5 mm txi cryoprobe ( bruker corporation ,
billerica , ma ) and a bruker avance ii 600 console ( 600 mhz for h and 151 mhz for c ) except for 2d noesy data ,
which was collected at 25 c .
residual chcl3 was used
to reference chemical shifts to 7.26 ppm for h ,
and c1 of ipomeamarone is referenced to 72.6 ppm for c in the hsqc spectrum by a previous report for consistency
with the literature .
we also checked
the residual chcl3 in cdcl3 for c in the hsqc spectra to confirm that it was properly referenced
according to a previous study .
nmr spectra
were processed using bruker topspin 2.0 and mestrenova ( mestrelab
research ) software packages .
ms in full scan ms in positive
mode was used to detect furanoterpenoids in extracts based on accurate
mass measurement , retention time , fragmentation pattern , and reference
spectra database published online of isolated dehydroipomeamarone
and ipomeamarone ( standards ) .
dehydroipomeamarone
and ipomeamarone were quantitated using generated standard calibration
curves prepared from the isolated compounds .
serially diluted solutions
of isolated standards ( 0.01200 g/l ) were analyzed
by lc
ms to generate linear calibration curves
( peak area vs concentration ) for ipomeamarone [ y =
583,064x + 642,221 ( r = 0.9991 ) ] and dehydroipomeamarone [ y = 324,980x + 177,617 ( r = 0.9995 ) ] ,
which served as the bases for external quantitation .
identification of mi-1
fungus isolated from the field - collected
sweetpotato storage roots was conducted because different microbes
elicit varying furanoterpenoid responses . on the basis of morphological features ( sporangia , sporangiophores ,
spores , collumella , rhizoids , and stolons ) ; sporangiophores that arise
from intersections with rhizoids and stolons ; the dome - shaped columella ;
and not falling off
when the sporangium dried out , mi-1 was identified
as belonging to the genus rhizopus .
the major finding was
the collumella being dome - shaped , which differed from other rhizopus species as previously reported as follows : r. oryzae has an ellipsoidal collumella ; r. sexualis , a conical - cylindrical shape ; and r. microspores , a subglobose to conical shape . from these observations
the morphological features of rhizopus further confirmed by molecular evidence enabled us identify the
species .
the its primer pair generated an amplicon of 950 bp for mi-1
fungus ; none of the other species in this study had a similar band
size to mi-1 .
sequencing generated a number of reads ,
but searches were conducted using sequences longer than 200 bp , based
on nonredundant database of ncbi , and produced 100 hits .
these hits
were mainly its1 , its2 , 5.8s , 18s , and 28s fragments with partial
or complete length sequences ; the top 19 hits based on e - value were rhizopus stolonifer .
sequence
similarity was of 9199% with e - values between
0 and 9 10 .
the best alignment to mi-1
was isolate am933546.1 , which had a sequence identity of 98% and an e - level of 0 .
the r. stolonifer isolate had a partial sequence of 18s rrna gene , its1 ; 5.8s rrna
gene , its2 ; and partial sequence of 28s rrna gene .
molecular characterization
using its primers confirmed that mi-1 fungus was r.
stolonifer ; such primers have previously been used
in r. stolonifer identification .
ms ,
lc qtof ms , and 1d and 2d nmr techniques by comparing
their resonances to published data . the hplc analysis ( figure 2 ) showed six peaks with ipomeamarone and dehydroipomeamarone
eluting at 16.4 and 11.6 min , respectively .
gc ms analysis
also revealed six peaks and tentatively identified two of these peaks
as ipomeamarone , 1 , at a retention time ( rt ) of 20.8 min and dehydroipomeamarone , 2 , at an rt of 21.4 min ( figure 3 ) .
ms analysis also identified
the two peaks as ipomeamarone ( with elemental composition , c15h23o3 ; m / z 251.1647 , 0.0 ppm error to theoretical value ; and a fit conf % of
99.96 ) at a rt of 2.87 min and dehydroipomeamarone
( with elemental composition , c15h21o3 ; m / z 249.1491 , 2.0 ppm error to
theoretical value ; and a fit conf % of 98.78 ) at a rt of 3.04 min .
the hplc purified peaks analyzed by nmr
confirmed the presence of ipomeamarone and dehydroipomeamarone .
h chemical shifts of ipomeamarone were in agreement with literature ,
while dehydroipomeamarone was a mixture based on lc qtof
ms
and nmr data . for dehydroipomeamarone , we observed
the h chemical shifts at positions c-7 as 6.15 ppm ( 1h ) ,
c-9 as 2.13 ppm ( 3h ) , and c-10 as 1.85 ppm ( 3h ) , which were the only h chemical shifts as provided by previous workers .
these workers reported the h chemical
shifts for positions c-7 as 6.11 ppm ( 1h ) , c-9 as 2.09 ppm ( 3h ) , c-10
as 1.81 ppm ( 3h ) , and all of the other h chemical shifts
were similar to those of ipomeamarone .
unfortunately , these three h chemical shifts differed
0.04 ppm between our spectrum and previously reported data .
since the dehydroipomeamarone sample was a mixture ,
having just h 1d nmr was not satisfactory to confirm the
presence of dehydroipomeamarone . unfortunately , previous workers did
not provide any information about nmr solvent or c chemical
shift data for the synthetic dehydroipomeamarone .
therefore , we acquired a 2d nmr ( cosy , hsqc , hmbc , noesy )
spectrum for dehydroipomeamarone and ipomeamarone samples to compare
the c chemical shifts , since dehydroipomeamarone is a
derivative of ipomeamarone , which we confirmed using h
nmr .
both h and c chemical shifts of dehydroipomeamarone were
similar to ipomeamarone for carbon positions from 14
and from 16 , but differed at positions 7 , 8 , 9 , and 10 : consistent
with the structure .
the main furanoterpenoids produced were ipomeamarone , 1 , and dehydroipomeamarone , 2 , found in all samples
and noninoculated controls .
the concentration of furanoterpenoid ranged
between 50.6 and 2,330 mg / kg for inoculated samples and 12.4144.5
mg / kg for the controls .
these high levels exceeded those reported
previously for r. stolonifer of 2001,100
mg / kg but were comparable to high ipomeamarone elicitors such as c. fimbriata and fusarium solani with levels from 1,1009,300 mg / kg .
similar results have been reported on noninfected sweetpotatoes
with low ipomeamarone concentrations between 40 and 325 mg / kg for
cultivars in the united kingdom .
generally ,
mechanical injury or damaged but noninfected sweetpotato is reported
to elicit low furanoterpenoid levels , while infected samples have
high levels due to the increase in enzyme activity , which correlates
to furanoterpenoid production . the low ipomeamarone
levels in storage roots of negative control samples from this study
confirm that injury or bruising of sweetpotato roots occurs , but the
ipomeamarone levels are low .
concentration levels for ipomeamarone
ranged between 50.6 and 2,126.7 mg / kg , while they ranged from 39.32,230.4
mg / kg for dehydroipomeamarone . the higher dehydroipomeamarone levels
could possibly be because it is a precursor of ipomeamarone , which
means that it was yet to be enzymatically converted to ipomeamarone ,
or dehydroipomeamarone might have inhibited production of ipomeamarone
to some extent as reported previously . in preliminary experiments ( not shown ) , ipomeamarone levels were
high in inoculated samples , probably due to long incubation time of
28 days compared to the current study where inoculation ranged between
7 and 14 days .
variations in mean ipomeamarone levels by cultivar were as follows :
kemb had the highest mean of 1,476.2 mg / kg , followed by nyawo with
1,089.9 mg / kg ; naspot had a mean of 833.7 mg / kg , while bungoma had
a mean of 676.5 mg / kg ( table 1 ) .
high concentrations
of dehydroipomeamarone were also observed in almost all samples in
this study : kemb had the highest with 1,462.6 mg / kg , nyawo had 1,459.7
mg / kg , naspot had 1,153.2 mg / kg , and bungoma had 910 mg / kg ( table 1 ) .
in addition , there were differences
in ipomeamarone levels within the same cultivar , for example , nyawo
had 602 mg / kg in one sample , while the other had 2,126 mg / kg .
similar
variations were observed for different cultivars for ipomeamarone
in other studies ranging from 112,000 mg / kg .
such variations have been speculated to be associated with moisture
loss , root maturity , and susceptibility or resistance to fungal infection .
other causes of variation include environmental
factors such as postharvest practices by different farmers before
arrival at the markets .
differences in concentrations for these cultivars
also suggest that they vary in resistance to r. stolonifer attack with kemb being the most resistant , followed by nyawo , naspot ,
and bungoma .
these results concur with reports from other studies
where the differences in ipomeamarone levels were attributed to rhizopus soft rot resistance .
we were successful in confining mi-1 fungus
in the controlled infection experiment of naspot and kemb cultivars .
we observed visually the infection to be high at the inoculation place
while decreasing progressively with increased distance from the inoculation
site ( figure 4 ) .
by contrast , evaluation of
the 1 cm slices recorded high ipomeamarone levels for regions with
visibly low infection , while low levels were observed for regions
with visibly high infection ( figure 4 ) .
this
trend was observed for all samples evaluated , which showed high ipomeamarone
levels at the border between the visibly infected and the healthy - looking
site .
similar results have been reported that phytoalexins are produced
in healthy tissues surrounding wounded parts but terminate in the
necrotic tissue .
the highest levels of
ipomeamarone were observed in both kemb and naspot samples of 1,671.2
and 1,708.4 mg / kg , respectively , at the border between the visible
infection and healthy - looking tissues ( figure 4 ) .
we also observed differences in ipomeamarone levels in some samples ,
which could have been due to variations in infection on the fungus
along the storage root ( figure 4 ) .
in another study , it was reported that elimination of 310
mm of disease portions beyond infected regions and also cooking removes
most ipomeamarone .
this could be true
for root samples with high levels only at the border , but in the current
study , some samples had high levels even in subsequent layers .
this
suggests that removal of 1 cm from the infection area will still leave
behind most of ipomeamarone depending on how much the subsequent layers
had accumulated .
in other studies , cooking was reported to destroy
about 80% of ipomeamarone , but this depends on the initial concentration
level found in the storage root before cooking .
controlled
infection of sweetpotato samples by rhizopus
stolonifer that enabled ipomeamarone analysis of 1
cm slices for kemb ( a , b ) and naspot ( c , d ) cultivar samples . since rural poor farmers in ssa
keep 12 cows , and these
farm animals contribute 3080% of farm income , if they feed them with diseased sweetpotatoes with exceeding
toxicity , their fragile revenues will be at threatened by high costs
of treatment or even total low of the intoxicated cow .
this information
is important for farmers knowledge since they harvest infected
roots , cut off the infected part to feed the farm animals , and then
consume the healthy - looking parts ( dr . robert mwanga , sweetpotato
breeder , personal communication ) . although there are no reports on
how far into the visibly healthy - looking part the farmers cut off
these storage roots , either the farmers household consume
the parts with high ipomeamarone concentration , or the animals are
fed with it .
results drawn from this study suggest that at least 23
cm away from the border between the healthy - looking and the infected
part of the damaged sweetpotato storage root should be cut off and
disposed of , and the infected part should not be fed to the farm animals
because of the presence of toxic phytoalexins . in conclusion ,
ipomeamarone accumulation in the apparently healthy
parts of damaged sweetpotato storage roots should be considered as
a potential economic problem and health threat to both the rural poor
farm families and large - scale farmers worldwide .
our results reveal
the importance of this problem , but more research should be directed
to obtaining information on ( i ) furanoterpenoids present in infested
and infected sweetpotatoes harvested from the sweetpotato fields ;
( ii ) furanoterpenoid levels in sweetpotatoes from farmers
households ; ( iii ) varietal response to fungal infection resulting
in ipomeamarone accumulation ; ( iv ) degradation of ipomeamarone after
processing infested roots and accumulation in metabolism in animal
and human organisms ; and ( v ) management of the problem by the rural
poor . | furanoterpenoid accumulation in response
to microbial attack in
rotting sweetpotatoes has long been linked to deaths and lung edema
of cattle in the world . however , it is not known whether furanoterpenoid
ipomeamarone accumulates in the healthy - looking parts of infected
sweetpotato storage roots .
this is critical for effective utilization
as animal feed and assessment of the potential negative impact on
human health .
therefore , we first identified the fungus from infected
sweetpotatoes as a rhizopus stolonifer strain and then used it to infect healthy sweetpotato storage roots
for characterization of furanoterpenoid content .
ipomeamarone and
its precursor , dehydroipomeamarone , were identified through spectroscopic
analyses , and detected in all samples and controls at varying concentrations .
ipomeamarone concentration was at toxic levels in healthy - looking
parts of some samples .
our study provides fundamental information
on furanoterpenoids in relation to high levels reported that could
subsequently affect cattle on consumption and high ipomeamarone levels
in healthy - looking parts . | Introduction
Materials and Methods
Results
and Discussion | despite the prevalence
of respiratory infections and deaths relating
to furanoterpenoids on global health of cattle and possibly humans ,
the concentration levels of these compounds in diseased and healthy - looking
parts of infected sweetpotatoes is poorly understood and insufficiently
documented . although information on furanoterpenoids is available
concerning isolation methods and presence
in sweetpotatoes , there is no documented or accessible
information on variation of the concentrations from inoculation point
on toward healthy - looking parts of an infected sample . this is important
because in sub - saharan africa ( ssa ) , sweetpotato is a food crop for
many rural poor families whereby both healthy and infected sweetpotato
storage roots are harvested together . it is not known how far into the healthy - looking storage root
samples
that these furanoterpenoids are found and at what levels . since this
could potentially expose both humans and farm animals to either chronic
or acute toxicity , we ( i ) isolated and identified a fungus from field
samples that elicited furanoterpenoid production ; ( ii ) characterized
the furanoterpenoids using gc ms and lc qtof ms ;
( iii ) evaluated the furanoterpenoid concentration levels produced
for different sweetpotato cultivars after infection by the isolated
fungus ; and ( iv ) determined the furanoterpenoid levels within the
apparently healthy part of infected storage roots . the second experiment consisted of
inoculating kemb and naspot cultivars using mi-1 , and storage roots
tied with polythene and elastic bags to restrict growth of the fungus . the target peaks
were identified through comparison of their mass spectra with adams2.l ,
chemecol.l , and nist05a.l library data ( figure 3 ) . this is a summation of the quantities of elements ,
isotopes , or superatoms that can compose the measured data , calculated
using the following atomic masses of the most abundant isotope of
the elements : c = 12.0000000 , h = 1.0078250 , n = 14.0030740 , o = 15.9949146 ,
and s = 31.9720718 . the main furanoterpenoids produced were ipomeamarone , 1 , and dehydroipomeamarone , 2 , found in all samples
and noninoculated controls . these high levels exceeded those reported
previously for r. stolonifer of 2001,100
mg / kg but were comparable to high ipomeamarone elicitors such as c. fimbriata and fusarium solani with levels from 1,1009,300 mg / kg . by contrast , evaluation of
the 1 cm slices recorded high ipomeamarone levels for regions with
visibly low infection , while low levels were observed for regions
with visibly high infection ( figure 4 ) . this
trend was observed for all samples evaluated , which showed high ipomeamarone
levels at the border between the visibly infected and the healthy - looking
site . we also observed differences in ipomeamarone levels in some samples ,
which could have been due to variations in infection on the fungus
along the storage root ( figure 4 ) . this information
is important for farmers knowledge since they harvest infected
roots , cut off the infected part to feed the farm animals , and then
consume the healthy - looking parts ( dr . although there are no reports on
how far into the visibly healthy - looking part the farmers cut off
these storage roots , either the farmers household consume
the parts with high ipomeamarone concentration , or the animals are
fed with it . results drawn from this study suggest that at least 23
cm away from the border between the healthy - looking and the infected
part of the damaged sweetpotato storage root should be cut off and
disposed of , and the infected part should not be fed to the farm animals
because of the presence of toxic phytoalexins . in conclusion ,
ipomeamarone accumulation in the apparently healthy
parts of damaged sweetpotato storage roots should be considered as
a potential economic problem and health threat to both the rural poor
farm families and large - scale farmers worldwide . our results reveal
the importance of this problem , but more research should be directed
to obtaining information on ( i ) furanoterpenoids present in infested
and infected sweetpotatoes harvested from the sweetpotato fields ;
( ii ) furanoterpenoid levels in sweetpotatoes from farmers
households ; ( iii ) varietal response to fungal infection resulting
in ipomeamarone accumulation ; ( iv ) degradation of ipomeamarone after
processing infested roots and accumulation in metabolism in animal
and human organisms ; and ( v ) management of the problem by the rural
poor . | [
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] |
worldwide , cervical carcinoma is the fourth largest cause of death from cancer in women and overall gynecological cancer responsible for more than 14% of female cancer deaths . that is more than breast cancer or female lung cancer . in italy ,
the incidence of gynecological cancers is about 10% with about 16,600 cases occurring every year , of which 27% of cases are recorded in northern italy .
the collective population of piedmont ( 4,500,000 ) , liguria ( 1,600,000 ) , and valle d'aosta ( 128,000 ) is of about 6,200,000 inhabitants , which represents an important catchment area for radiotherapy ( rt ) centers in these regions . in several countries ,
the documentation of use of rt to treat gynecological malignancies has produced interesting researches [ 3 , 4 , 5 , 6 , 7 , 8 ] ( table 1 ) . however in italy , the oncology network of piedmont and valle d'aosta does not currently produce guidelines for gynecological tumors , and the two italian documents of the airo ( italian association of radiation oncology ) brachytherapy group are outdated [ 9 , 10 ] .
gynecological surveys of the last five years in 2015 , at the tri - regional annual meeting of the airo , we focused our attention on rt practices adopted in these regions for the treatment of gynecological tumors .
we designed a survey , which would investigate the standard practices for gynecological cancer management adopted in the last three years in order to obtain current data about rt treatment with modern techniques and technologies .
the main purpose of the survey was to produce a snapshot of the current use of brachytherapy ( brt ) and external beam radiotherapy ( ebrt ) in the treatment of gynecological tumors in the three regions concerned .
the second goal was to produce guidelines , based on the latest evolution of brt and ebrt in gynecology cancer treatment ( image guided adaptive brachytherapy and radiotherapy igabt and igrt ) .
the third and most important objective was to improve the quality of care with the aim to offer the best treatment to all patients in this area and to optimize local resources .
a 30-questions survey was designed by the host of the tri - regional annual meeting of the airo , approved by the scientific council of the tri - regional airo and proposed to the centers with a large amount of gynecological cancer cases .
the survey included questions about the organization of patient care , clinical choices , and types of treatments in the years 2012 , 2013 , and 2014 .
we know that 19 regional centers of radiotherapy ( 1 in valle d'aosta , 5 in liguria , and 13 in piedmont ) deal with gynecological diseases : 14 general hospitals ; 3 university hospitals ( 1 reserved to gynecological and pediatric cancers only ) ; 1 cancer institute , and 1 private clinic .
all these centers responded to the survey . to cover the entire process of treatment management ,
the first section concerned patient management and multidisciplinary approaches , which includes ebrt and brt ( the number of patients , computed tomography [ ct]-simulation , contouring , dose prescription , planning , delivery , and treatment verification ) .
the second group of questions regarded follow - up ( assessment of outcome and toxicity ) , and the third area of inquiry was scientific productivity .
twelve of the centers involved in the research program had a multidisciplinary team dedicated to gynecological tumors . in five cases ,
the same group was also involved in breast cancer with the constant presence of the surgeon , oncologist , and radiation oncologist .
other complementary specialists were present in some centers : a pathologist in nine , a radiologist in eight , a nuclear medicine specialist in seven , a pain therapist in four , a psychologist in two , and lastly a nurse and a specialist in palliative care only in one center . only in 2 hospitals all the patients
radiotherapy approach is different according to the anatomical sites ( endometrium , cervix , vagina , and vulva ) , except for ovarian cancer .
in fact , for ovarian cancer , the use of rt has been traditionally limited in italy with a stronger preference for surgical and oncological options .
chemotherapy was performed in all centers by a medical oncologist , except in one center where chemotherapy was directly performed by a radiation oncologist .
number of centers treating different pathologies one thousand nine hundred seventy - eight patients affected by gynecological malignancies received rt in the last three years .
the trend was quite stable : 665 cases treated in 2012 , 678 in 2013 , and 635 in 2014 .
post - operative treatment was mainly used in endometrial ( 802 cases ) and vulvar cancer ( 70 cases ) , while in cervical cancer the radiotherapy intent was equally distributed ( 279 cases , 259 radical cases ) , and the palliative intent was the preeminent indication in ovarian cancer ( 39 cases ) ( table 3 ) . finally , the number of patients treated according to multicenter and/or international clinical protocols was less than 10% and is carried out in only six of nineteen centers .
figure 1 shows the trend of gynecological rt during the period 2012 - 2014 .
trend of gynecological radiotherapy ( during 2012 - 2014 ) patients number according to tumor site and intent of treatment in eight centers , gross tumor volume ( gtv ) delineation was performed with diagnostic imaging ( ct and/or magnetic resonance imaging [ mri ] contrast agents and/or positron emission tomography [ pet]-ct ) in the treatment position fused with ct simulation without contrast enhancement ( ce ) : rigid fusion in 5 cases , elastic in 3 cases . in eleven centers ,
contouring was performed with diagnostic imaging ( ct and/or mri contrast agents and/or pet - ct ) but not in the treatment position , and fusion with ct simulation was rigid in 5 cases and elastic in 6 cases .
eleven centers also performed ce - ct simulation , and fusion with ce - mri and/or pet - ct in the treatment position .
five centers used ct simulation alone . regarding the use of the margins around the gtv to the clinical target volume ( ctv ) and from ctv to planning target volume ( ptv ) , it was preferred in both cases to adopt margins between 5 and 10 mm . in six centers ,
the adoption of the internal target volume ( itv ) minimized these margins under 5 mm .
the more frequent dose delivery techniques were conformal rt ( 3dcrt ) and static imrt by linear accelerator ( linac ) with a slight prevalence of the second .
the dose range of ebrt for most of the centers was between 50 gy and 60 gy ( table 4 ) , using typically a boost with brt .
radiotherapy by centers according to dose and tumor sites the treatment verification method most frequently used was 2d electronic portal imaging device ( epid ) with mv beam ( 12/19 ) , or kv beam ( 5/19 ) .
the volumetric methods of igrt were common and were employed in ten of nineteen centers .
the cases treated with brt were essentially stable over time : 200 in 2012 , 222 in 2013 , and 228 in 2014
six of those were equipped with high - dose - rate ( hdr ) machines , one center with hdr , low - dose - rate ( ldr ) , and pulsed - dose - rate ( pdr ) devices and one center with a pdr machine .
one center recently acquired the equipment to perform a combined intracavitary and interstitial gynecological brt , to boost parametrial disease or to treat patients with unsatisfactory dose distribution with a standard applicator .
four centers fixed the cylinder applicators , three centers using plate , and one using bandages . the brt was performed both with radical intent and as a boost ( table 5 ) mostly by hdr .
the exclusive brt was performed prescribing doses between 20 - 30 gy in 4 - 6 gy / fraction . for unfit patients doses of 21 gy in 3 fractions
the brt boost was administrated with doses between 10 - 20 gy in 2 - 4 sessions .
radiotherapy techniques according to tumor sites 3dcrt 3 dimensional radiation therapy , imrt intensity - modulated radiotherapy , vmat volumetric arc therapy , hdr
high - dose - rate , pdr pulsed - dose - rate range of doses of brachytherapy according to technique and tumor sites hdr high - dose - rate , pdr pulsed - dose - rate brachytherapy boost was usually administered in two sessions a week , not concurrent with ebrt , except in one center .
the interval between ebrt and brt was 6.6 days on average ( range 3 - 10 ) .
a single rt department employed a two daily hyperfractionated treatments for endometrial cancers unfit for surgery .
all brt plans with tandem applicator were calculated on ct scan , except one that used mri .
two centers still used the traditional planning by radiographs for brt on vaginal cuff ; no center used mri for brt planning with cylinder . in all centers
were used rectum preparation and bladder filling to control organ and/or tumor motion in ebrt . in brt , rectum preparation
only one center performed in vivo dosimetry by means of rectal tandem diodes . for radical treatments , 4 centers used a control imaging each brt session and 2 centers performed a re - planning . for adjuvant brt ,
a program of follow - up treatment was offered to assess the treatment response and possible side effects , evaluated according to rtog ( radiation therapy oncology group)/eortc ( european organization for research and treatment of cancer ) scale .
the follow - up was widely shared with gynecologists and clinical oncologists ( in 10 centers ) : only in three centers follow - up was performed by a radiation oncologist with a frequency , for the majority of cases , of about four months for a duration of one year in one center , five years in eight centers , and ten years or more in the six remaining centers .
one center published in medical journals in the last three years [ 11 , 12 , 13 , 14 , 15 ] and nine centers produced abstracts for various events . finally , five centers were involved in clinical trials regarding gynecologic oncology .
twelve of the centers involved in the research program had a multidisciplinary team dedicated to gynecological tumors . in five cases ,
the same group was also involved in breast cancer with the constant presence of the surgeon , oncologist , and radiation oncologist .
other complementary specialists were present in some centers : a pathologist in nine , a radiologist in eight , a nuclear medicine specialist in seven , a pain therapist in four , a psychologist in two , and lastly a nurse and a specialist in palliative care only in one center . only in 2 hospitals all the patients
radiotherapy approach is different according to the anatomical sites ( endometrium , cervix , vagina , and vulva ) , except for ovarian cancer .
in fact , for ovarian cancer , the use of rt has been traditionally limited in italy with a stronger preference for surgical and oncological options .
chemotherapy was performed in all centers by a medical oncologist , except in one center where chemotherapy was directly performed by a radiation oncologist .
one thousand nine hundred seventy - eight patients affected by gynecological malignancies received rt in the last three years .
the trend was quite stable : 665 cases treated in 2012 , 678 in 2013 , and 635 in 2014 .
post - operative treatment was mainly used in endometrial ( 802 cases ) and vulvar cancer ( 70 cases ) , while in cervical cancer the radiotherapy intent was equally distributed ( 279 cases , 259 radical cases ) , and the palliative intent was the preeminent indication in ovarian cancer ( 39 cases ) ( table 3 ) .
finally , the number of patients treated according to multicenter and/or international clinical protocols was less than 10% and is carried out in only six of nineteen centers .
figure 1 shows the trend of gynecological rt during the period 2012 - 2014 .
trend of gynecological radiotherapy ( during 2012 - 2014 ) patients number according to tumor site and intent of treatment in eight centers , gross tumor volume ( gtv ) delineation was performed with diagnostic imaging ( ct and/or magnetic resonance imaging [ mri ] contrast agents and/or positron emission tomography [ pet]-ct ) in the treatment position fused with ct simulation without contrast enhancement ( ce ) : rigid fusion in 5 cases , elastic in 3 cases . in eleven centers ,
contouring was performed with diagnostic imaging ( ct and/or mri contrast agents and/or pet - ct ) but not in the treatment position , and fusion with ct simulation was rigid in 5 cases and elastic in 6 cases .
eleven centers also performed ce - ct simulation , and fusion with ce - mri and/or pet - ct in the treatment position .
five centers used ct simulation alone . regarding the use of the margins around the gtv to the clinical target volume ( ctv ) and from ctv to planning target volume ( ptv ) , it was preferred in both cases to adopt margins between 5 and 10 mm . in six centers ,
the adoption of the internal target volume ( itv ) minimized these margins under 5 mm .
the more frequent dose delivery techniques were conformal rt ( 3dcrt ) and static imrt by linear accelerator ( linac ) with a slight prevalence of the second .
the dose range of ebrt for most of the centers was between 50 gy and 60 gy ( table 4 ) , using typically a boost with brt .
radiotherapy by centers according to dose and tumor sites the treatment verification method most frequently used was 2d electronic portal imaging device ( epid ) with mv beam ( 12/19 ) , or kv beam ( 5/19 ) .
the volumetric methods of igrt were common and were employed in ten of nineteen centers .
the cases treated with brt were essentially stable over time : 200 in 2012 , 222 in 2013 , and 228 in 2014 .
six of those were equipped with high - dose - rate ( hdr ) machines , one center with hdr , low - dose - rate ( ldr ) , and pulsed - dose - rate ( pdr ) devices and one center with a pdr machine .
one center recently acquired the equipment to perform a combined intracavitary and interstitial gynecological brt , to boost parametrial disease or to treat patients with unsatisfactory dose distribution with a standard applicator .
four centers fixed the cylinder applicators , three centers using plate , and one using bandages . the brt was performed both with radical intent and as a boost ( table 5 ) mostly by hdr .
the exclusive brt was performed prescribing doses between 20 - 30 gy in 4 - 6 gy / fraction . for unfit patients doses of 21 gy in 3 fractions
the brt boost was administrated with doses between 10 - 20 gy in 2 - 4 sessions .
radiotherapy techniques according to tumor sites 3dcrt 3 dimensional radiation therapy , imrt intensity - modulated radiotherapy , vmat volumetric arc therapy , hdr high - dose - rate , pdr pulsed - dose - rate range of doses of brachytherapy according to technique and tumor sites hdr high - dose - rate , pdr pulsed - dose - rate brachytherapy boost was usually administered in two sessions a week , not concurrent with ebrt , except in one center .
the interval between ebrt and brt was 6.6 days on average ( range 3 - 10 ) .
a single rt department employed a two daily hyperfractionated treatments for endometrial cancers unfit for surgery .
all brt plans with tandem applicator were calculated on ct scan , except one that used mri .
two centers still used the traditional planning by radiographs for brt on vaginal cuff ; no center used mri for brt planning with cylinder . in all centers
were used rectum preparation and bladder filling to control organ and/or tumor motion in ebrt . in brt ,
rectum preparation was used in 4 centers . in all centers , patients were contoured and planned with the oncentra brachy treatment planning system .
only one center performed in vivo dosimetry by means of rectal tandem diodes . for radical treatments , 4 centers used a control imaging each brt session and 2 centers performed a re - planning . for adjuvant brt ,
a program of follow - up treatment was offered to assess the treatment response and possible side effects , evaluated according to rtog ( radiation therapy oncology group)/eortc ( european organization for research and treatment of cancer ) scale .
the follow - up was widely shared with gynecologists and clinical oncologists ( in 10 centers ) : only in three centers follow - up was performed by a radiation oncologist with a frequency , for the majority of cases , of about four months for a duration of one year in one center , five years in eight centers , and ten years or more in the six remaining centers .
one center published in medical journals in the last three years [ 11 , 12 , 13 , 14 , 15 ] and nine centers produced abstracts for various events .
multidisciplinarity is a hot topic due to the proliferation of highly specialized structures that involve different specialists as surgeons , clinicians , and radiation oncologists .
this synergy has resulted in excellent therapeutic strategies [ 16 , 17 , 18 , 19 ] .
however , the possibility to perform formal multidisciplinary meeting with different specialists is a difficult challenge due to a lack of staff time .
the incidence of treated patients was shown to be stable over time ( figure 1 ) and rt was an established option for all different intents of care .
however , the low number of patients treated in clinical trials does not allow a comparison among different centers limiting offered treatments .
gynecological radiotherapy is only now beginning to take advantage of the technological innovations available [ 20 , 21 , 22 ] .
the use of new morphological and functional diagnostic imaging ( multiparametric mri , 18-fdg pet ) allowed not only to optimize the disease staging and to contour with precision the target volumes , integrating multimodality imaging information by rigid or elastic co - registration algorithms . for target delineation ,
however , these methods did not allow the adoption of reduced margins , which is one of the goals for a better target definition .
technological development also introduced the use of new irradiation techniques such as imrt , which allows , thanks to the possibility of a better dose conformation , to deliver high doses preserving healthy tissue close to the target .
therefore , the new control system of the images ( igrt ) allowed for safe delivery high hypofractionated doses [ 23 , 24 , 25 ] . the results showed that linac - imrt was almost used as 3dcrt , and the adoption of volumetric techniques ( tomotherapy and vmat ) also increased ( table 5 ) . despite the fact that imrt appeared to be a well - established technique for the complex volumes irradiation , such as the pelvic and/or lumbar - aortic volume , different verification modalities ( traditional portal verification and modern image guidance ) coexisted .
it could be due to the equipment modernization with the coexistence ( double control ) of both modalities , or to the use of different imaging modalities for different treatments .
therefore , the implementation of these new techniques did not modify the use of conventional fractionation ( table 5 ) or the dose range ( table 4 and 6 ) .
even if in italy brt is a technique which is gradually falling out of use due to the lack of dedicated radiation oncologists , in the north - west italian regions brt is still widely used and the treated cases were essentially stable as displayed in figure 1 . in most gynecological cancers , brt remains a powerful weapon even in comparison to the sophisticated techniques of ebrt .
intensity modulated radiation therapy with igrt can hardly compete with an optimal brt implant in terms of organ motion management and dose distribution .
historically , our brt centers were located far from each other ( the mean distance between each center is more than 90 km ) , rather than concentrated in a few specialized institutions ( figure 2 ) .
recent studies evaluated the effect of distance on the possibility of access to rt care but there are no papers dedicated to brt .
a future goal would be to assess how many patients do not receive brt due to the distances and the center distribution within the catchment area .
distribution of brachytherapy centers on the territory the hdr technique was the most commonly used , because it is fast , cheap , acceptable to patients , and does not require hospitalization .
furthermore , only two centers had care protected rooms , in which ldr and pdr were performed . over half of the centers
used mri compatible applicators but contouring and planning were performed on ct in most cases , since it was very difficult to have the availability of mri equipment .
in few cases where a dedicated ct was not present , adjuvant brt planning was performed using orthogonal radiographs . in order to understand the current practice in our regions ,
for this reason , the study shows several limitations due to the lack of more detailed analysis for each topic : ebrt , brt , contouring , planning and diagnostic pathway , and other related therapies .
the final results will allow us to create a treatment guideline , which will be proposed to the oncology network for our regions .
this survey , following the example of other countries [ 3 , 4 , 5 , 6 , 7 , 8 ] , is the first to asses practice patterns for rt treatments in liguria - piedmont - valle d'aosta .
it shows that in the last three years , the rt practice shifted towards multidisciplinary cares with a uniformity of performance as regards techniques , doses , and treatment volumes .
the use of imrt is almost comparable to the more traditional 3d practices , and the more modern simulation approaches and volumes identification seem to result , in many cases , in traditional control treatment systems , fractionation schedules , and doses .
there are an appropriate numbers of brt centers that are well distributed in the territory .
computed tomography planning is almost a standard , and the introduction of mri is starting , but up to now is limited by the availability of resources .
high - dose - rate brachytherapy is the most common technique because it is easy and does not require hospitalization . the efforts in implementation of technology made by different centers , in this hardship time ,
still do not translate effectively into a scientific production , which could help us to evaluate the effectiveness of gynecological tumor treatment in our region .
human resources , shared data , and time dedicated to research may allow a more complete analysis .
the purpose of this survey is to inform all radiation oncologists of the results achieved in order to define goals for the future in the context of multidisciplinary cooperation , and the creation of a set of common guidelines . in this way
, we aim to improve the treatment offered to all patients of the three regions .
this manuscript is based on data presented at the tri - regional annual meeting of the italian association of radiation oncologist ( airo ) in candiolo , may 23 , 2015 . | purposewe focused the attention on radiation therapy practices about the gynecological malignancies in piedmont , liguria , and valle d'aosta to know the current treatment practice and to improve the quality of care.material and methodswe proposed a cognitive survey to evaluate the standard practice patterns for gynecological cancer management , adopted from 2012 to 2014 by radiotherapy ( rt ) centers with a large amount of gynecological cancer cases . there were three topics : 1 . taking care and multidisciplinary approach , 2 .
radiotherapy treatment and brachytherapy , 3 .
follow-up.resultsnineteen centers treated gynecological malignancies and 12 of these had a multidisciplinary dedicated team .
radiotherapy option has been used in all clinical setting : definitive , adjuvant , and palliative .
in general , 1978 patients were treated .
there were 834 brachytherapy ( brt ) treatments .
the fusion between diagnostic imaging ( magnetic resonance imaging mri , positron emission tomography pet ) and computed tomography ( ct ) simulation was used for contouring in all centers .
conformal rt and intensity modulated radiation therapy ( imrt ) were the most frequent techniques .
the image guided radiation therapy ( igrt ) was used in 10/19 centers .
there were 8 active brt centers .
brachytherapy was performed both with radical intent and as boost , mostly by hdr ( 6/8 centers ) .
the doses for exclusive brt were between 20 to 30 gy .
the doses for brt boost were between 10 and 20 gy .
four centers used ct - mri compatible applicators but only one used mri for planning .
the brt plans on vaginal cuff were still performed on traditional radiographies in 2 centers .
the plan sum was evaluated in only 1 center .
only 1 center performed in vivo dosimetry.conclusionsin the last three years , multidisciplinary approach , contouring , treatment techniques , doses , and control systems were similar in liguria - piedmont and valle d'aosta .
however , the technology implementation did n't translate in a real treatment innovation so far . | Purpose
Material and methods
Results
Patient management and multidisciplinary approach
Radiotherapy
Brachytherapy
Follow-up
Discussion
Conclusions
Meeting presentations
Disclosure | the collective population of piedmont ( 4,500,000 ) , liguria ( 1,600,000 ) , and valle d'aosta ( 128,000 ) is of about 6,200,000 inhabitants , which represents an important catchment area for radiotherapy ( rt ) centers in these regions . however in italy , the oncology network of piedmont and valle d'aosta does not currently produce guidelines for gynecological tumors , and the two italian documents of the airo ( italian association of radiation oncology ) brachytherapy group are outdated [ 9 , 10 ] . we designed a survey , which would investigate the standard practices for gynecological cancer management adopted in the last three years in order to obtain current data about rt treatment with modern techniques and technologies . the main purpose of the survey was to produce a snapshot of the current use of brachytherapy ( brt ) and external beam radiotherapy ( ebrt ) in the treatment of gynecological tumors in the three regions concerned . the third and most important objective was to improve the quality of care with the aim to offer the best treatment to all patients in this area and to optimize local resources . a 30-questions survey was designed by the host of the tri - regional annual meeting of the airo , approved by the scientific council of the tri - regional airo and proposed to the centers with a large amount of gynecological cancer cases . we know that 19 regional centers of radiotherapy ( 1 in valle d'aosta , 5 in liguria , and 13 in piedmont ) deal with gynecological diseases : 14 general hospitals ; 3 university hospitals ( 1 reserved to gynecological and pediatric cancers only ) ; 1 cancer institute , and 1 private clinic . to cover the entire process of treatment management ,
the first section concerned patient management and multidisciplinary approaches , which includes ebrt and brt ( the number of patients , computed tomography [ ct]-simulation , contouring , dose prescription , planning , delivery , and treatment verification ) . trend of gynecological radiotherapy ( during 2012 - 2014 ) patients number according to tumor site and intent of treatment in eight centers , gross tumor volume ( gtv ) delineation was performed with diagnostic imaging ( ct and/or magnetic resonance imaging [ mri ] contrast agents and/or positron emission tomography [ pet]-ct ) in the treatment position fused with ct simulation without contrast enhancement ( ce ) : rigid fusion in 5 cases , elastic in 3 cases . in eleven centers ,
contouring was performed with diagnostic imaging ( ct and/or mri contrast agents and/or pet - ct ) but not in the treatment position , and fusion with ct simulation was rigid in 5 cases and elastic in 6 cases . the brt was performed both with radical intent and as a boost ( table 5 ) mostly by hdr . for unfit patients doses of 21 gy in 3 fractions
the brt boost was administrated with doses between 10 - 20 gy in 2 - 4 sessions . trend of gynecological radiotherapy ( during 2012 - 2014 ) patients number according to tumor site and intent of treatment in eight centers , gross tumor volume ( gtv ) delineation was performed with diagnostic imaging ( ct and/or magnetic resonance imaging [ mri ] contrast agents and/or positron emission tomography [ pet]-ct ) in the treatment position fused with ct simulation without contrast enhancement ( ce ) : rigid fusion in 5 cases , elastic in 3 cases . in eleven centers ,
contouring was performed with diagnostic imaging ( ct and/or mri contrast agents and/or pet - ct ) but not in the treatment position , and fusion with ct simulation was rigid in 5 cases and elastic in 6 cases . the brt was performed both with radical intent and as a boost ( table 5 ) mostly by hdr . for unfit patients doses of 21 gy in 3 fractions
the brt boost was administrated with doses between 10 - 20 gy in 2 - 4 sessions . only one center performed in vivo dosimetry by means of rectal tandem diodes . the use of new morphological and functional diagnostic imaging ( multiparametric mri , 18-fdg pet ) allowed not only to optimize the disease staging and to contour with precision the target volumes , integrating multimodality imaging information by rigid or elastic co - registration algorithms . over half of the centers
used mri compatible applicators but contouring and planning were performed on ct in most cases , since it was very difficult to have the availability of mri equipment . this survey , following the example of other countries [ 3 , 4 , 5 , 6 , 7 , 8 ] , is the first to asses practice patterns for rt treatments in liguria - piedmont - valle d'aosta . it shows that in the last three years , the rt practice shifted towards multidisciplinary cares with a uniformity of performance as regards techniques , doses , and treatment volumes . | [
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] |
since the discovery in the late 1980s that t helper cells exhibit distinct cytokine profiles , the unique immunological profile associated with helminth infection has been explained by the activation of the th2 cell pathway .
in particular , the dramatic increase in numbers of eosinophils , mast cells , and ige could be directly explained by cytokines produced by the th2 subset .
more recently , we have come to appreciate that in addition to eosinophils and mast cells ,
alternatively - activated macrophages ( aam ) are a characteristic feature of the polarized th2 response .
macrophages at helminth infection sites were termed aam because they exhibited specific characteristics , such as arginase 1 production , that had been observed when macrophages were exposed in vitro to il-4 [ 13 ] .
siamon gordon and colleagues described this as an alternative activation pathway that contrasted with the classical activation by lps and ifn [ 4 , 5 ] . with further exploration of the in vivo phenotype ,
it has become apparent that aam express a whole range of molecules that distinguish them from classically activated macrophages ( cam ) [ 69 ] .
what has been remarkable is the consistency of the findings between the diverse laboratories and infectious models in which these cells have been studied , with identification of the same key molecules ( arginase 1 , resistin - like molecule [ relm ] , ym1 , etc . ) expressed during helminth infection ( see table in reyes & terrazas review ) .
this is remarkable because helminths represent an enormously diverse range of pathogens with entirely different phylogenetic origins and life histories .
indeed , the aam phenotype seems to occur in any strong th2 environment including allergy and some chronic microbial infections [ 1012 ] .
the commonality of these finding has , perhaps erroneously , suggested to us that we could define a broad function for these cells analogous to microbial killing for cam. however , despite an ever - broadening definition of aam and their associated markers and characteristics , we are still essentially ignorant of their in vivo function .
perhaps it is time to explore the many differences between the models used to study aam and consider that these cells may function differently depending on context .
helminth parasites are routinely used as models to study t helper cell polarization and as a result , our understanding of th2 subset development and control has become increasingly sophisticated [ 1317 ] . however , it is important to appreciate that to draw broad conclusions from experiments with schistosomes and then apply these to nematode parasites ( or vice versa ) , is potentially misleading . despite the common terminology , the only shared biological features of many helminthes are their metazoan origins and the ability to infect mammals .
schistosomes are part of the platyhelminths that include the cestodes ( tapeworms ) and other trematodes ( flukes or flatworms ) .
the split that led to platyhelminthes and nematoda occurred over 1 billion years ago , long predating the split between vertebrates and invertebrates .
nematodes are the most abundant animal on earth both in terms of total numbers and numbers of species . within this group of animals
, parasitism has independently evolved many times and parasitic nematodes represent an enormous burden in terms of human , animal , and even plant health . in terms of human disease , platyhelminths infect fewer numbers but are responsible for higher levels of morbidity and mortality .
the utility of helminths as models to study th subset bias stems from the striking feature that , despite their phylogenetic diversity , they all induce profound th2 responses , characterized by cd4 + t cells producing il-4 , il-5 , il-9 , il-10 , and il-13 among others .
however , recently it has become apparent that even the th2 subset itself is enormously complex , with t cells that specifically function to provide b cell help and produce il-4 but not the other signature th2 cytokines ( follicular helper cells ) , specific il-9 producing cells , and other t helper subsets that produce th2 cytokines such as the recent discovery that il-10 is produced by th1 cells . in an adaptive immune response
, macrophages ultimately respond to t cell derived cytokines , and a sustained alternative activation phenotype absolutely requires cd4 + th2 cells .
thus , differences in th cell cytokine profiles in different tissues and in response to distinct parasites will determine differences in macrophage activation .
although virtually all helminths induce th2 cytokines , the pattern and magnitude of these responses differ widely due to not only the vast differences in the biology of the pathogens as mentioned above , but also their broadly different migration and eventual host niche .
indeed , within hours of infection innate activation of the th2 pathway can be detected [ 2326 ] . however , even within the nematode phyla the intensity of this early type 2 response varies , perhaps reflecting the differential ability of nematodes to inhibit the type 1 inducing cytokine , il-12 .
eggs released by the schistosome parasites are believed to be the strongest known inducers of th2 cytokines in mammals , and yet the invasive cercariae induces only a moderate th2 response that is matched by a th1 response of similar magnitude . this initial mixed response
is swamped by the extraordinarily high th2 response generated to the eggs produced when the adult pairs reach sexual maturity .
because of its importance as a cause of human morbidity and the availability of excellent mouse models , far more is known about the immunology of schistosome infection than other trematodes . however
, fasciola hepatica , a liver fluke that predominantly infects sheep and cattle , has also been studied in mouse models .
consistent with indicators of type 2 immunity in cattle , f. hepatica infection of mice results in a dominant th2 response that has the capacity to suppress th1 responses .
cestodes are also dramatically understudied despite their capacity to cause severe disease in animals and people .
nonetheless , the data is consistent with the general helminth literature in that cestodes by and large have a strong propensity to drive th2 immune responses . similar to responses to schistosomes , peritoneal implantation of balb / c mice with taenia crassiceps metacestodes results in an initially weak mixed th1/th2 response that becomes strongly th2 dominated as infection progresses to the chronic phase [ 31 , 32 ] . however , there are resistant mouse strains that expel the parasite in the acute phase due to the dominance of ifn production .
reponses to peritoneal infection with echinococcus granulosus protoscoleces are unusual in that the initial th2 responses that dominate early ( week 1 ) become more mixed , with emergence of ifn production as infection progresses ( week 4 ) .
in addition to the differences in the kinetics and magnitude of th2 induction , the role of th2 immunity in host protection varies substantially between these different parasites .
indeed , the paradigm that th2 immunity is acting to destroy or expel worms is by no means universal .
the scenario in which there is an absolute requirement for th2 immunity in host protection is that of the gastro - intestinal nematodes .
however , the specific cytokines ( il-4 , il-5 , il-9 , il-10 , il-13 among others ) and the effector cells on which they act ( epithelial , smooth muscle , mast cell , macrophage , nerve ) vary tremendously depending on the location of the parasite , as well as its invasive properties .
the situation with nematodes , such as the filariae , that live entirely in the tissues is somewhat different .
although th2 responses are required for worm killing , th1 immunity and particularly ifn , rather than inhibiting the anti - parasite th2 response , act synergistically with il-5 to kill the adult stage of the parasite . in the case of these tissue - dwelling nematodes , our increased understanding of the cytokine pathways required for parasite destruction has still left us in the relative dark as to the actual killing mechanism(s ) . during schistosome infection ,
th2 responses are essential for host survival but this has little to do with detrimental effects on the parasite .
this is largely due to the fact that pathology is the result of the egg deposition stage and it appears that in the absence of a th2 response ( or a th1 response for that matter ) , the egg is highly tissue destructive .
death can occur either due to overwhelming gut inflammation and sepsis or liver damage . additionally , th2 responses to the egg promote fibrosis that itself can be a major cause of morbidity .
cestodes provide an unusual perspective , in that despite inducing a potent th2 response , protective immunity can require th1 cells [ 33 , 39 ] . indeed ,
dominance of th2 responses during infection with t. crassiceps leads to susceptibility to infection mainly through the suppression of th1-driven nitric oxide ( no ) production , a key effector molecule against these parasites [ 32 , 39 ] .
however , cestodes are also unusual in that there is little consistency in immunological mechanisms that protect against infections within this group . in this respect ,
resistance to e. granulosus actually increases in the absence of no production , possibly due to the absence of the considerable suppressive effects on proliferation that this molecule exerts in echinococcosis .
aam are becoming increasingly recognised as a key effector arm of the th2 immune response , but their actual function in different helminth infections has yet to be unravelled ( see below ) and is likely to be as diverse as the role of th2 immunity itself .
the concept of alternative macrophage activation was introduced by siamon gordon and colleagues in the early 1990s to describe the in vitro response of macrophages to the th2 cytokines il-4 and il-13 [ 4 , 5 ] .
significantly , the term aam was coined to highlight the activated nature of these cells that distinguished them not only from macrophages classically activated by microbial products and th1 cytokines ( cam ) , but from deactivated macrophages in which costimulatory molecules and class ii expression are suppressed by down - regulatory cytokines such as il-10 .
the two features that distinguished aam in vitro were the expression of arginase 1 and the mannose receptor .
the requirement for il-4 and/or il-13 was subsequently confirmed in vivo , using gene - deficient mice [ 6 , 11 , 42 , 43 ] .
realization that the aam described in vitro were a feature of helminth infection came from studies of schistosoma mansoni and brugia malayi [ 6 , 43 ] . both studies verified il-4 dependent arginase 1 expression by macrophages in vivo , but the b. malayi study additionally identified novel il-4 dependent genes associated with this phenotype including ym1 and relm/fizz1 . the highly unique profile was rapidly confirmed across the full range of helminth infections [ 7 , 4448 ] .
although ym1 and relm were discovered in vivo , the direct induction of these genes by il-4 and/or il-13 was also demonstrated in vitro [ 2 , 3 ] .
it should be noted here that il-4 and il-13 both utilize the same signal transducing receptor chain , the il-4 receptor ( il-4r ) , which explains the considerable overlap in function of these cytokines . which of these cytokines is more important for alternative activation of macrophages in vivo remains to be fully determined , however , a recent report using mice deficient for the il-13 receptor 1 subunit suggests that il-13 is dispensable for expression of ym1 and relm but not arginase in the liver during s. mansoni infection .
a molecular signature for aam ( defined here as an il-4/il-13 dependent phenotype ) has arisen that is represented by the three most abundant il-4/il-13 dependent gene products : ym1 , relm , and arginase 1 .
ym1 is a member of the family 18 chitinases but has no chitinolytic activity and is thus referred to as a chitinase - like molecule .
other members of this family in mice include ym2 and acidic mammalian chitinase ( amcase ) , the later functioning as a true chitinase .
ym2 and amcase are also il-4/il-13 inducible proteins and the similarity between ym1 and ym2 is so high that most studies do not actually distinguish between them .
all antibodies to date recognize both , and most pcr methods do not distinguish them , although this is possible with careful primer design . thus ,
unless a study clearly identifies a specific ym protein , it might be appropriate to use the more ambiguous designation ym1/2 .
relm was first described in a lung asthma model , where it was described as fizz1 , but was subsequently identified as a member of a family of cysteine - rich molecules related to resistin , a hormone involved in glucose metabolism .
arginase 1 is the best studied of these proteins and has well - established roles in regulating no production by competing with inos for their common substrate l - arginine , as well as inhibition of t cell responses through l - arginine depletion .
the arginase pathway additionally leads to the production of proline and polyamines , which contribute to tissue repair and fibrosis .
subsequently there has been identification of numerous other markers associated with the alternative activation phenotype [ 7 , 9 ] and this number is likely to grow as more extensive transcriptomic and proteomic analyses are undertaken .
macrophages with an aam phenotype characterized mainly by arginase 1 production also arise in protozoan ( reviewed in ) and certain bacterial infections .
in cutaneous leishmaniasis ( leishmania major ) , this aam phenotype is dependent on signaling through the il-4r chain as in helminth infection models [ 6 , 11 , 42 ] .
however , a stat6-independent pathway also leads to arginase 1 expression during mycobacterium tuberculosis and toxoxplasma gondii infections , which in the former is dependent upon tlr signaling .
the main effect of arginase 1 expression in all of these settings appears to be an increase in susceptibility to infection through diversion of l - arginine from production of the reactive nitrogen intermediates that kill these pathogens [ 10 , 56 ] . as interest in these cells grew , the term alternatively - activated came to include any cell displaying an alternate phenotype to cam. subdivision of the m1 and m2 terminology has helped to address this issue with m1 equating with cam while m2 includes m2a , m2b , and m2c .
m2a most closely reflects the il-4/13 dependent phenotype originally associated with aam , while m2b includes activation by other modulators such as immune complexes that lead to high il-10 production and m2c reflecting the more deactivated phenotype associated with il-10 treatment in vitro . nonetheless , the difficulty in finding appropriate terms is a reflection of the enormous diversity in macrophage phenotype found both in vivo and in vitro , as well as their capacity to rapidly alter their expression profile in response to a new set of environmental signals .
a current difficulty in delineating the functions of aam is that many of the signature aam molecules are not restricted to macrophages .
the availability of good antibodies for intracellular staining and fluorescence microscopy , the creation of mice that report gene expression , and the ability to sort cell subsets prior to gene expression analysis have greatly increased our knowledge of the range of cells that show an alternative - activation
phenotype , as well as the comparative breadth of expression of the different aam markers .
for example , in liver granulomas from mice infected with s. mansoni , the main producer of relm appears to be eosinophils rather than macrophages , whilst in lung granulomas induced by i.v .
injection of schistosome eggs , relm cells are comprised of macrophages , eosinophils , and airway epithelial cells . in the serous cavities of mice infected with litomosoides sigmodontis or b. malayi ,
we have observed a similarly broad pattern of relm expression , with mature macrophages ( f4/80 siglec - f ) , eosinophils ( siglec - f f4/80 ) , and f4/80 siglec - f cells that include dc , all capable of making this protein ( figure 1(a ) and data not shown ) .
expression of ym1/2 is markedly different , being almost exclusively restricted to f4/80 siglec - f mature macrophages ( 90% ) , and with no expression detectable in eosinophils ( figure 1(b ) ) .
however , ym1/2 , like relm can also be expressed by epithelial cells in the lung [ 61 , 63 , 64 ] and both ym1/2 and relm appear to be a feature of many types of antigen presenting cell found in the lymph nodes draining helminth infection sites .
thus , it is now apparent that many cell types can show an alternative - activation
epithelial cells in particular express not only ym1/2 and relm during th2 immune responses but related family members including the true chitinase , amcase and relm . of the three most abundant aam markers
this was demonstrated by reese et al . using mice that contain an ires - yfp knock - in allele that reports arginase 1 expression , in which extra - hepatic arginase 1 expression was macrophage - restricted in the lung or peritoneum of nippostrongylus brasiliensis infected or chitin injected mice , respectively .
another major difficulty has been efforts to translate our understanding of murine aam to humans , not only because some of the mouse defined aam markers are not present in the human genome , but because the relevant tissues can not be readily accessed .
an example of this problem has been the argument over whether human macrophages express arginase 1 , strongly reminiscent of earlier arguments about no production .
it may be that arginase 1 expression is more limited in human macrophages or that we have just not yet identified the right tissues .
indeed , arginase 1 can be induced in human macrophages by il-4 and can be observed in monocytes of filiarial - infected individuals .
even more problematic has been the realization that ym1 is not even present in the human genome . however , distribution of the family 18 chitinases ( including amcase and ym1/2 ) between different mammalian species is a fascinating puzzle in itself .
mammals have two genes encoding active chitinases that represent an ancient gene duplication event and show high sequence homology to chitinases of lower organisms .
the mammalian chitinase - like proteins ( clps ) that include ym1 , appear to represent more recent gene duplication events with subsequent loss - of - function mutations .
thus all mammals express the highly conserved active enzymes , chitotriosidase and acidic mammalian chitinase ( amcase ) but additionally express a broad range of diverse clps , with each mammalian species exhibiting a different complement of clps . in mice
these include ym1 , ym2 , and ykl-40/brp-39 , which have all been strongly implicated in th2 conditions [ 50 , 71 ] .
humans express ykl-40 but also a distinct clp , ykl-39 . because no two mammals express the same set of these proteins and clps appear to be undergoing remarkably active evolution , no animal model can fully represent the human genes .
studying mice should nonetheless be informative as one can presume that despite species differences a common theme lies behind the evolutionary forces driving the divergence of clps .
as the molecular definition of aam becomes more refined , our hope has been that an understanding of function would follow .
however , the functions of gene products associated with alternative activation , such as relm and ym1 remain elusive and our full understanding of the contribution of macrophages during helminth infection is an increasingly active area of investigation . considering the diversity of helminth infection and the complexity of the associated th2 response , a single well - defined role for aam is unlikely to emerge .
the depletion of macrophages using clodronate - loaded liposomes has provided a powerful tool by which to analyse the function of these cells during helminth infection .
this technique has provided evidence that macrophages play a central role in nematode expulsion during intestinal infection , both in the memory response to a secondary infection with heligmosomoides polygyrus , and in expulsion of primary n. brasiliensis infection . in both these settings
, parasite clearance is dependent upon a strong th2 response , which acts to rapidly recruit immune cells including macrophages to the infection site and to stimulate their expression of arginase 1 , relm , and ym1/2 in a stat-6 dependent manner .
critically , blocking recruitment of macrophages via depletion of monocytes resulted in prevention of worm expulsion , whilst the th2 response and recruitment of other inflammatory cell populations were left intact .
however , observations that worm survival during murine peritoneal infection with either brugia pahangi or malayi l3 larvae is enhanced following injection of carbon particles or carrageenan [ 74 , 75 ] imply an effector function for peritoneal macrophages .
consistent with a role in filarial killing , macrophages make up significant proportion of the granulomas that encase dying b. malayi and l. sigmodontis worms but the conundrum is : do granulomas cause worm damage or form because the worms are already damaged ? while there is evidence for macrophage effector function during nematode infections , it is still unknown whether this occurs via direct or indirect mechanisms .
macrophages greatly increase the hypercontractility of intestinal smooth muscle during n. brasiliensis infection , providing a potentially indirect effector mechanism .
because filarial nematodes are restricted to tissue sites during infection , it is likely a distinct though overlapping array of effector mechanisms is required to act against these nematodes .
perhaps a more likely role for macrophages in these infections is to recruit other th2 effector cells important in nematode attrition . in this respect ,
eosinophils have a well - documented role in vivo , acting against larval stages of both b. malayi and l. sigmodontis [ 76 , 77 ] , and recent data demonstrates that recruitment of eosinophils to the peritoneal cavity following n. brasiliensis infection or injection of chitin is dependent on macrophages [ 66 , 78 ] . an attractive possibility for a direct anti - nematode effector function
is the association of aam with chitinases and chitinase - like molecules , which in principle have the capacity to act on chitin - containing stages of the parasite .
however , as of yet , there is no direct evidence to support this . whilst macrophages can perform as anti - nematode effector cells , the question remains whether they need to alternatively activate to exert this function .
anthony et al . , showed that , like macrophage depletion , an inhibitor of arginase , s-(2-boronethyl)-l - cysteine , could impair worm expulsion during secondary h. polygyrus infection .
using the same technique , arginase i expression was also implicated as mediating expulsion of n. brasiliensis , although experiments were inconclusive since treatment only prevented worm expulsion in 60% of the mice despite parasite egg production and host smooth muscle hyper contractility being greatly impaired . the broad - acting nature of this treatment ( it blocks both arginase i and ii and could potentially act directly on worms in addition to other non macrophage host cell sources ) makes it hard to draw firm conclusions .
a stronger case against alternative activation driving these macrophage effector mechanisms , is provided by two earlier studies both of which used mice on the same resistant balb / c background as zhao et al . .
these demonstrated that il-4r need not be expressed on macrophages / neutrophils or indeed any hematopoetic population in order for efficient expulsion of n. brasiliensis [ 79 , 80 ] . using the same macrophage / neutrophil - specific il-4r-deficient mice , it has also been shown that alternative activation of macrophages
th2-associated macrophages may also be independent of alternative activation state , as for example , macrophage - dependent - recruitment of eosinophils in response to chitin injection is stat6-independent .
it is quite conceivable that in th2 infections , macrophage effector function could be completely independent of aam-associated molecules or that expression of arginase 1 or other aam-associated molecules could be induced by an il-4r-independent mechanism , for example via a tlr - dependent event [ 10 , 56 ] such as exposure to gut flora . unfortunately , the expression of either arginase 1 or other aam associated markers was not investigated in the intestinal tissues of n. brasiliensis or t. spiralis infected m/neutrophil - specific il-4r-deficient mice [ 79 , 81 ] .
comparative analysis of the susceptibly of mice which lack , in macrophages specifically , either il-4r or alternative activation
proteins such as arginase 1 would help considerably to resolve the issue of the function of alternative activation
interestingly , a study with such mice has shown that arginase 1 expression by aam has no host protective effect against primary infection with the trematode s. mansoni .
in contrast to the ambiguity surrounding alternative activation in immunity to nematodes , it is clear that the reactive oxygen or nitrogen species can damage most types of helminth parasites [ 39 , 8386 ] . however , only in cestode infection do reactive nitrogen species and cam appear to function against the parasite in vivo .
in murine cysticercosis ( t. crassiceps ) blocking of inos using the inhibitor l - ng - monomethyl arginine leads to increased parasite burdens .
consistent with this , induction of th2 responses and stat-6 signaling underlie susceptibility to infection , whilst th1 responses and stat-4 signaling underlie resistance [ 32 , 33 ] .
however , as mentioned above , this is not a requirement in immunity to all cestodes .
indeed , nos2 deficient mice , which are incapable of making inos , are actually less susceptible to infection with the cestode parasite e. multilocularis . in this infection cam appear to have a pathological effect , most likely due to the direct immunosuppressive effect of no on cell proliferation . given the divergence of the helminth parasite phyla and the host tissue sites they have chosen to infect , it is perhaps unsurprising that diverse effector mechanisms are required for immunity to different infections [ 44 , 88 ] .
however , a common thread is that macrophages can act against both nematode and platyhelminth infections , and there is still no published evidence of any infection in which macrophages can be dispensed at no cost to resistance .
as discussed below , aam do play an important role in protecting the host in schistosomiasis by limiting parasite - mediated tissue damage rather than mediating killing .
indeed as we struggle to identify direct antihelminth effects of aam , the evidence builds that the macrophage products most associated with alternative activation such as arginase 1 and relm have profound inhibitory effects on host immunity , including the th2 response itself [ 61 , 62 , 82 ] .
this raises the possibility that the alternative activation state of macrophages does not function primarily as an effector arm but has critical regulatory or parasite disposal ( rather than killing ) roles .
one property of activated macrophages that is consistently observed in a wide variety of systems is the ability to block the proliferation of cells with which they are cocultured .
this feature has been well described for cam in which the antiproliferative properties of no are responsible .
myeloid cells derived from helminth infected animals also exhibit similar antiproliferative properties [ 60 , 8991 ] .
importantly , it can be replicated in vitro by treatment of macrophages with il-4 or il-13 [ 2 , 60 ] and in vivo is reliant on il-4 and/or il-13 in certain settings .
indeed , the ability to inhibit cellular proliferation is a defining characteristic of aam. despite the near - universal finding that aam suppress cellular proliferation ex vivo , the in vivo significance is not known .
understanding the relevance of this proliferative suppression has been complicated by the fact that , unlike cam , a single mechanism for proliferative inhibition has not been identified . instead
a multitude of pathways have been found that differ depending on the infection context ( reviewed in ) and include programmed death ligand ( pd - l ) interactions [ 92 , 93 ] , tgf- production , lipid mediator release , il-10 production [ 96 , 97 ] , and l - arginine depletion .
there appear to be three categories of proliferative suppression generally observed during helminth infection : contact and il-4 dependent , contact dependent and il-4 independent , and finally il-4 dependent and contact - independent .
no doubt the target cells will also differ depending on the pathways involved , with some mechanisms , such as the pd - l pathway seen during infection with the platyhelminths , t. crassiceps , and s. mansoni [ 92 , 93 ] , affecting predominantly t cells .
other mechanisms have a broader target including even tumor cells that typically have no restriction on cell division .
identified as critical for aam-mediated suppression of t cells , there is another study that finds that mechanism dispensable .
this disparity could be due to the distinct biological mediators released by these vastly different parasites , which presumably all favour an immuno - suppressive environment .
however , many other factors could account for this diversity , from differences in the magnitude and bias of the th cell response to tissue localization . of interest ,
proliferative suppression is also a feature of myeloid - derived suppressor cells ( mdsc ) , which share many features with aam but are associated with cancer and other immune suppressive environments rather than helminth infection .
t cell suppression by mdsc is mediated by both inos - driven production of no and arginase 1-driven depletion of l - arginine .
l - arginine is essential for t cell activation but l - arginine depletion could also lead to production of suppressive reactive oxygen intermediates [ 95 , 100 ] .
this is similar to recent data showing that macrophage - derived arginase 1 is required to suppress the proliferation of t cells from s. mansoni - infected mice but also during non - healing leishmania major infection , which is associated with aam .
although arginase 1 is emerging as one of the most important mediators of proliferative suppression , it is not the full story .
chemical blockade of arginase 1 had only a small impact on suppression mediated by aam from the peritoneal cavity of b. malayi implanted mice , and full il-4-dependent suppressive capacity was maintained when arginase expression was reduced by lps / ifn treatment .
finally , it is important to consider that no mediated suppression , although most strongly associated with microbial infection , also has a role to play during helminth infection .
as already mentioned , no can act as an effector molecule during infection with the cestode t. crassiceps .
however , within the same infection model , and infection with e. multilocularis , no mediated suppression by peritoneal cells has been observed . even in filariasis , where the il-4 dependent aam suppressive phenotype has been well described , no - mediated suppression can play a role . in line with the immuno - suppressive effects of aam described above , one of the most consistent findings in human studies is that individuals infected with helminth parasites exhibit profound defects in lymphocyte proliferation [ 102105 ] .
one popular hypothesis has been that monocytes or macrophages from infected individuals were somehow defective in their antigen presentation capacity .
however , as the discovery of alternative activation emerged and their capacity to actively block cellular proliferation was revealed the expectation shifted somewhat .
further , by definition aam are activated and thus might be expected to express good levels of class ii and costimulatory molecules . not surprisingly , the analysis of macrophage apc activation state during helminthiases has been shown to vary considerably with infection .
however , expression of antigen presentation - associated molecules is frequently intact or elevated , consistent with an activation profile .
mice carrying schistosome infections show marked up - regulation of mhcii but not cd80 or cd86 by splenic macrophages .
transient up - regulation of co - stimulatory molecule and mhcii expression on lung macrophages occurs during the period n. brasiliensis larvae migrate through the lung but is quickly lost thereafter .
following peritoneal implant of adult b. malayi , macrophages exhibit relatively high levels of mhcii , cd80 , and cd86 expression compared to thioglycollate elicited m , but not compared to lps - stimulated cells .
perhaps the strongest activation is seen in t. crassiceps infected mice , where mhcii , cd40 , and cd86 but not cd80 are greatly up - regulated over an 8-week period . however , this is by no means a feature of cestode infection , since the one documented parasitic helminth that leads to a reduction in activation state compared to nave m is e. multilocularis although only expression of cd40 is reduced whilst cd80 and cd86 remained unchanged .
a number of labs have investigated m expression of b7 family members pd - l1 and pd - l2 , with a diversity of findings in nematode , trematode , and cestode models .
defined pd - l2 as a marker for aam , specifically up - regulated by il-4 in a il-4r/stat-6 dependent manner and pd - l1 as a th1-associated ligand .
however , neither pd - l1 or pd - l2 are up - regulated on peritoneal aam elicited by the nematode b. malayi .
in contrast , both ligands are up - regulated in the lung following but not during n. brasiliensis larval migration .
similar dichotomy exists in the response to platyhelminths , with only pd - l1 up - regulation in response to s. mansoni infection , yet pd - l1 and pd - l2 up - regulation in response to t. crassiceps . significantly in these two settings , pd - l1 and/or pd - l2 act to potently block the proliferation of t cells and are thus at least in part responsible for the contact - dependent proliferative suppressive effect of aam discussed above .
how then do aam perform as apc ? given that aam exhibit a profound ability to suppress cell division and fail to induce nave t - cell proliferation , it was a surprise when initial experiments showed that aam from b. malayi infected mice were strong inducers of th2 cytokine production when cocultured with naive t - cells .
this ability is also shared with aam from chronic late - stage t. crassiceps infection .
interestingly , the capacity to drive th2 cytokine production correlated with alternative activation , as adherent peritoneal cells from early - stage infection induce more of a mixed th1/th2 response while showing much lower expression of relm and ym1/2 . it remains to be determined whether the ability to drive th2 cytokine production is a shared function of aam from all helminth infections .
the difficulty in extracting aam in sufficient quantity from tissues , such as the gut lamina propria , has so far prohibited analysis of apc function in many settings , particularly intestinal infections .
it is tempting to draw a parallel to dendritic cells ( dc ) obtained from schistosome infected mice or exposed to helminth products in vitro .
these exhibit a muted activation phenotype , with little change in expression of costimulatory molecules , and limited up - regulation of mhcii .
however , they also efficiently promote th2 polarisation and cytokine production [ 110112 ] .
furthermore , dc can exhibit an alternative activation phenotype in vitro [ 46 , 113 ] and during infection or allergy , up - regulating expression of ym1/2 and relm in an il-4/il-13 dependent manner [ 46 , 113 , 114 ] .
indeed , experiments looking at the ability of alternatively activated dc to drive th2 responses in vitro and in vivo have identified ym1/2 as a key molecule involved in the process [ 113 , 114 ] .
ym1/2 appears to exert this effect by binding to 12/15-lipoxygenase and blocking production of ppar ligands , which are thought to have immunoregulatory effects on macrophages and t cells .
given the large quantities of ym1/2 produced by aam it is quite possible they also influence th2 priming via this molecule .
the discovery that two novel proteins ( ym1 and relm ) were secreted in abundance by macrophages activated during helminth infection led rapidly to the speculation that these would be effector molecules against the metazoan invaders .
this was supported by the realisation that ym1 was a member of a family of chitinases with presumed defensive roles against chitin - containing pathogens such as nematodes .
more direct ( but still circumstantial ) evidence came with the recognition that relm , another resistin family member , was abundantly secreted by epithelial cells in the intestines of nematode infected mice and bound directly to the chemosensory structures of the parasite .
the expectation naturally followed that similar anti - parasite roles would be identified for macrophage - derived relm. however , two recent papers utilizing relm-deficient mice have turned that idea on its head and instead identified relm as a critical regulator of th2 immunity [ 61 , 62 ] . using models of s. mansoni and n. brasiliensis infection , and schistosome egg - induced lung granuloma formation
, relm was shown to limit th2-mediated immune pathologies by suppressing th2 but not th1 cytokine production .
importantly , this was mediated at least in part by a direct suppressive effect of relm on cytokine production by th2 cells , as relm bound to th2 cells and could exert this suppressive effect on t cells cultured alone in vitro .
relm could also be detected bound to other cells , including macrophages and dc ( but not th1 cells ) suggesting other non t cell mediated functions for this molecule .
it is worth noting that macrophages appeared to be only a minor source of relm in the lung and liver in these studies , perhaps explaining why th2 responses remain normal during s. mansoni and n. brasiliensis infections in macrophage / neutrophil - specific il-4r deficient mice
. given that one of the downstream products of arginase - mediated l - arginine catabolism is a major component of collagen , it has been widely assumed that aam would promote the fibrotic pathologies associated with chronic th2 stimuli .
however , a recent elegant study using mice in which macrophages were deficient in arginase 1 expression has demonstrated that in fact , arginase 1 negatively regulates th2 responses and actually suppresses th2-mediated fibrosis .
, arginase 1 expression by macrophages impaired ifn- production by t cells in addition to down regulating output of th2 cytokines .
t cell proliferation in the draining lymph node was also exaggerated in the absence of arginase 1 expression by macrophages .
importantly , this data confirms an in vivo role for arginase 1 in proliferative suppression mediated by macrophages , but extends this to show that macrophages also exert an inhibitory effect on cytokine production .
critically , they demonstrate that macrophages exhibit an overall inhibitory effect on fibrosis during schistosomiasis via their production of arginase 1 .
one caveat to the conclusion that aam have a critical function in the regulation of th2 cytokines in both nematode and platyhelminth infections is the fact that th2 generation in both the secondary lymphoid organs and the infection site ( in which aam are present in greatest numbers ) appeared unaffected by either the absence of il-4r signaling in macrophages or the depletion of macrophages , during s. mansoni , n. brasiliensis , and t. spiralis infection , and h. polygyrus and n. brasiliensis infection , respectively , [ 72 , 73 , 79 , 81 ] .
it can not be ignored that because the role of macrophages in th2 generation was not the main focus of these studies , the methodology for assessing the quality and quantity of the responses was not as thorough as that described for the studies on the function of relm [ 61 , 62 ] , arginase 1 , and ym1/2 [ 113 , 114 ] .
it is possible though , that the removal of macrophages , or their alternative activation state , takes away both negative ( relm and arginase 1 ) and positive ( ym1/2 ) regulatory signals such that the net effect on th2 responses is nil .
whilst an in vivo role for arginase 1 production specifically by macrophages in th2 regulation during schistosome infection can not be denied , we await confirmation that relm and ym1/2 production by these cells plays a major role in regulation of th2 cytokine production in vivo .
alternatively activated dc and cells such as basophils play the greater role in th2 response induction , maintenance , and regulation . with the recent recognition that basophils are a critical apc in promoting th2 cell activation [ 14 , 116 ] , it would be of interest to know whether ym1/2 is produced by these cells .
much of the data described above suggests that aam act as anti - inflammatory down - regulatory cells , consistent with previously proposed functions for macrophages during helminth infection [ 117 , 118 ] .
additionally aam are important sources of tgf- and il-10 [ 60 , 109 , 119 ] , as well pge2 and the il-1 receptor antagonist [ 119 , 120 ] .
the chemokine expression profile is also strongly associated with a noninflammatory role and with specific down - regulation of key proinflammatory cytokines by il-4 [ 6 , 120 ] .
it may seem counter - intuitive that an activated cell population manifests such profoundly suppressive features
. however , this could be in part explained if one sees tissue repair or wound healing as a fundamental function associated with aam. effective tissue repair can only proceed if inflammation has been stopped [ 119 , 121 ] and thus all these anti - inflammatory features may contribute to their role in repair .
early reviews on aam ascribed them a wound healing phenotype based on the production of arginase 1 and angiogenic factors as well as extracellular matrix components and fibronectin . however , the specific role of il-4/il-13 in this healing phenotype versus glucocorticoids or il-10 , which the authors also considered alternative activators , was not immediately apparent . furthermore , the relevance to helminth infection was not obvious .
two recent papers have provided evidence that there is indeed very strong relevance to helminth infection . while investigating the kinetics of alternative activation in a model whereby b. malayi parasites are surgically implanted into the peritoneal cavity of mice
, we noted that control animals who underwent only sham surgery exhibited transient up - regulation of ym1/2 , relm , and arginase 1 in a strictly il-4r manner .
however , only when both the nematode and th2 cells were present was this alternative activation response sustained .
this suggested that the induction of the signature molecules of aam was in fact an innate response to direct injury .
one feature all these helminths have in common is the capacity to injure tissue in the course of their migration through the host , providing a possible evolutionary explanation for the association of th2 immunity and wound healing .
the strongest evidence to date from helminth models that aam have a combined anti - inflammatory / wound healing function is in a study of s. mansoni infection in mice that lack the il-4r specifically on macrophages and neutrophils and thus completely lack aam but have otherwise intact th2 responses .
following s. mansoni infection , these mice died from overwhelming inflammatory responses in the intestine and leakage of bacteria into the blood .
although not conclusive evidence , the data strongly suggests that in the absence of aam , these mice were unable to repair the damage caused by egg migration through the intestinal wall . further supporting a direct role for aam in wound healing , relm has angiogenic properties and ym1/2 has the ability to bind extracellular matrix .
the specific roles these proteins play in the complex orchestra for tissue repair and remodeling are still to be established .
mast cells , basophils and eosinophils have long been considered the serious cellular players in the host response to helminth infection . previously ignored
, the macrophage is now taking center stage in this cellular family as one of the most important targets of th2 immunity .
this is fully appropriate when we consider that macrophages are frequently the most abundant cell type recruited to the site of helminth infection .
however , it is only since the discovery of aam in vivo less than 10 years ago that a focus on these cells in helminth infection has begun . as a result
, we have a long ways to go before we attain the extensive knowledge associated with cam. the challenge is to define key roles for aam while accepting that these may differ radically depending on infection stage , site , and parasite species .
macrophages are the workhorse of the immune system , and as such , can radically alter their phenotype to adapt to environmental signals [ 55 , 59 , 60 ] . in turn , they can actively regulate the inflammatory environment to which they are recruited or the tissues in which they reside . using the tools available to modern scientists we can now begin to define the environmental codes that alter the aam expression profile , understand the function of the products they produce , and decipher their communication with other cells .
recent discoveries that aam are central to the regulation of host metabolism mean this cross - talk is not only between cells of the immune system but with the entire organism . unravelling this amazing complexity | this review summarizes current knowledge of macrophages in helminth infections , with a focus not only on delineating the striking similarities in macrophage phenotype between diverse infections but also on highlighting the differences .
findings from many different labs illustrate that macrophages in helminth infection can act as anti - parasite effectors but can also act as powerful immune suppressors .
the specific role for their alternative ( th2-mediated ) activation in helminth killing or expulsion versus immune regulation remains to be determined .
meanwhile , the rapid growth in knowledge of alternatively activated macrophages will require an even more expansive view of their potential functions to include repair of host tissue and regulation of host metabolism . | 1. Introduction
2. Helminths and Th2 Immunity
3. The Molecular Profile of AAM
4. Functional Roles of AAM
5. Summary | although virtually all helminths induce th2 cytokines , the pattern and magnitude of these responses differ widely due to not only the vast differences in the biology of the pathogens as mentioned above , but also their broadly different migration and eventual host niche . in addition to the differences in the kinetics and magnitude of th2 induction , the role of th2 immunity in host protection varies substantially between these different parasites . significantly , the term aam was coined to highlight the activated nature of these cells that distinguished them not only from macrophages classically activated by microbial products and th1 cytokines ( cam ) , but from deactivated macrophages in which costimulatory molecules and class ii expression are suppressed by down - regulatory cytokines such as il-10 . which of these cytokines is more important for alternative activation of macrophages in vivo remains to be fully determined , however , a recent report using mice deficient for the il-13 receptor 1 subunit suggests that il-13 is dispensable for expression of ym1 and relm but not arginase in the liver during s. mansoni infection . nonetheless , the difficulty in finding appropriate terms is a reflection of the enormous diversity in macrophage phenotype found both in vivo and in vitro , as well as their capacity to rapidly alter their expression profile in response to a new set of environmental signals . however , ym1/2 , like relm can also be expressed by epithelial cells in the lung [ 61 , 63 , 64 ] and both ym1/2 and relm appear to be a feature of many types of antigen presenting cell found in the lymph nodes draining helminth infection sites . however , the functions of gene products associated with alternative activation , such as relm and ym1 remain elusive and our full understanding of the contribution of macrophages during helminth infection is an increasingly active area of investigation . considering the diversity of helminth infection and the complexity of the associated th2 response , a single well - defined role for aam is unlikely to emerge . whilst macrophages can perform as anti - nematode effector cells , the question remains whether they need to alternatively activate to exert this function . however , a common thread is that macrophages can act against both nematode and platyhelminth infections , and there is still no published evidence of any infection in which macrophages can be dispensed at no cost to resistance . there appear to be three categories of proliferative suppression generally observed during helminth infection : contact and il-4 dependent , contact dependent and il-4 independent , and finally il-4 dependent and contact - independent . it remains to be determined whether the ability to drive th2 cytokine production is a shared function of aam from all helminth infections . it is worth noting that macrophages appeared to be only a minor source of relm in the lung and liver in these studies , perhaps explaining why th2 responses remain normal during s. mansoni and n. brasiliensis infections in macrophage / neutrophil - specific il-4r deficient mice
. importantly , this data confirms an in vivo role for arginase 1 in proliferative suppression mediated by macrophages , but extends this to show that macrophages also exert an inhibitory effect on cytokine production . it can not be ignored that because the role of macrophages in th2 generation was not the main focus of these studies , the methodology for assessing the quality and quantity of the responses was not as thorough as that described for the studies on the function of relm [ 61 , 62 ] , arginase 1 , and ym1/2 [ 113 , 114 ] . whilst an in vivo role for arginase 1 production specifically by macrophages in th2 regulation during schistosome infection can not be denied , we await confirmation that relm and ym1/2 production by these cells plays a major role in regulation of th2 cytokine production in vivo . alternatively activated dc and cells such as basophils play the greater role in th2 response induction , maintenance , and regulation . much of the data described above suggests that aam act as anti - inflammatory down - regulatory cells , consistent with previously proposed functions for macrophages during helminth infection [ 117 , 118 ] . however , the specific role of il-4/il-13 in this healing phenotype versus glucocorticoids or il-10 , which the authors also considered alternative activators , was not immediately apparent . furthermore , the relevance to helminth infection was not obvious . the specific roles these proteins play in the complex orchestra for tissue repair and remodeling are still to be established . however , it is only since the discovery of aam in vivo less than 10 years ago that a focus on these cells in helminth infection has begun . recent discoveries that aam are central to the regulation of host metabolism mean this cross - talk is not only between cells of the immune system but with the entire organism . | [
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the online version of this article ( doi:10.1007/s00134 - 010 - 1924 - 3 ) contains supplementary material , which is available to authorized users .
the underlying physiology of hyperglycemia that drives mortality , independently of glucose control ( gc ) , has been described [ 3 , 4 ] .
gc aiming at normoglycemia [ i.e. , blood glucose level ( bgl ) of 80110 mg / dl , frequently referred to as tight glycemic control ] decreased morbidity and mortality of critically ill patients in two randomized controlled trials [ 5 , 6 ] .
recent published meta - analysis [ 7 , 8 ] could not confirm the benefits of gc , but there are some important methodological concerns about this study [ 9 , 10 ] and many intensive care units ( icus ) still use gc in their routine clinical practice . although some studies showed that the implementation of gc with insulin came with the risk of hypoglycemia [ 5 , 6 , 1113 ] , others did not [ 1418 ] .
some studies therefore advise maintaining a bgl < 150 mg / dl [ 1921 ] . another recent randomized controlled trial showed that maintaining bgl < 180 mg / dl is superior to adhering to the tight glycemic control in terms of mortality and morbidity .
however , this result should be interpreted in the context of the specific implemented guideline and the level of compliance with it .
this context is important because the quality of gc and outcome are directly linked [ 1 , 23 , 24 ] .
determining how and for whom gc is safe and effective remains quite elusive [ 2527 ] .
implementing gc as well as the performance of the gc process itself is of great importance in intensive care medicine . to measure gc performance one has to determine and define adequate performance indicators and analyze these indicators over time
this implies monitoring the implementation process and determining whether gc is truly applied and functioning at a consistent and acceptable level .
statistical process control ( spc ) is a powerful tool for quality measurement of phenomena over time and the improvement of processes .
our objective was to study gc performance over time during implementation of this strategy in three different icus .
first , we described and analyzed the performance of gc in the early stage of gc but before implementation of local guidelines , and after the implementation of a written guideline in each center .
second , we analyzed differences between simple and complex guidelines , and between non - computerized and computerized gc .
in addition , we described the influence of concerns of clinicians and nurses regarding the safety of gc .
collection of data was performed in three closed - format mixed medical - surgical icus in the netherlands .
icu - c is an 18-bed icu of a nonacademic teaching hospital . in icu - a and icu - c
icu - a ( march 2002 ) and icu - c ( april 2001 ) were equipped with a patient data management system ( pdms ) . the pdms can display and process all bgls directly after their measurement with a maximum delay of 1 min .
tables 1 and 2 show the guideline characteristics and changes in them over time .
note that for severe hypoglycemia events , the guidelines recommend stopping insulin infusion as well as injecting glucose.table 1short description of changes in glucose control protocol over timeicuicu - aicu - bicu - cchange in glucose controliiiiiiiiiiiiiiiiiiglucose control protocol characteristics type of protocolsimple set of rulessliding scalessliding scalessimple set of rulessliding scalessimple set of rulessliding scalessliding scalessliding scales present in what form(s)writtenwrittenwrittenwrittenwrittenwrittenwrittenwrittenwritten decision support presentnonononononononoyeswho is responsible for glucose control start of insulin : icu nurse or physicianphysiciannursenursenursenursenursenursenursenurse dosing of insulin : icu nurse or physiciannurse + physiciannursenursenurse + physiciannurse + physiciannursenursenursenurse correction of hypoglycemia : icu nurse or physiciannurse + physiciannursenursenurse + physiciannurse + physiciannurse + physiciannursenursenurseprotocol thresholds and targets start of insulin ( mg / dl)>144>144>144>144>144>110>126>126>126 bgl targets ( mg / dl)801448014490144901448014480110721267212672126 timing of bgl measurements described in or mandated by the protocolnoyesyesnoyesnoyesyesyesrules on stopping insulin threshold to stop insulin infusion<80<63 or 6380 with > 50% reduction in bgl<63 or 63144 with > 50% reduction in bgl < 90<54 or 5480 with > 50% reduction in bgl<54<80<80<80 other reason for stopping insulin feeding stoppedfeeding stopped feeding stoppedfeeding stoppedaction in case of hypoglycemia bgl < 40 mg / dl50 ml 20% glucose50 ml 20% glucose50 ml 50% glucose20 ml 50% glucose20 ml 30% glucose20 ml 30% glucose20 ml 30% glucosein all three centers , change
i indicates the initiation of glucose control ( directly or shortly after the publication of the first randomized control trial showing beneficial effects of tight glycemic control ) ; major changes ii and iii in icu - a were in august 2004 and may 2005 , respectively ; major changes ii and iii in icu - b were in september 2004 and may 2005 , respectively ; major change ii and iii in icu - c were in january 2003 and may 2003 ; see text for details on changes .
mainly arterial ( and only occasionally venous ) blood samples were used for bgl measurements . in icu
- a , for almost all bgl measurements blood gas analyzers ( rapidlab 865 , bayer , germany ) were used . in icu - b two types of glucose analyzers were used ( hitachi 917 , roche diagnostics , and accutrend sensor , roche diagnostics ) . in icu - c ,
the term sliding scale here refers to a dynamic protocol for intravenous insulin infusionicu intensive care unit , bgl blood glucose level , no advice given in the protocoltable 2description of guidelines revisions in three studied icusicudate of changedescriptionabefore november 2001for a long time , hyperglycemia was considered an adaptive response to critical illness .
therefore before publication of the first randomized controlled trial showing benefit of tight glycemic control , only bgl > 200 mg / dl was a reason to start insulin infusion.november 2001 ( major change i)a simple guideline on glucose control was implemented involving all icu patients . the bgl target in this first written protocol was 80144 mg / dl .
glucose control was considered a combined icu physician and icu nurse activity ; initiation of insulin infusion was by the attending icu physician ( and never the icu nurse ) , changes in insulin infusion were by icu physician and/or icu nurse .
the protocol did not make recommendations on timing of bgl measurements.august 2004 ( major change ii)a more strict guideline was implemented .
this protocol was completely nurse - driven ; initiation of and all changes in insulin infusion were done only by the attending icu nurse ( and never the icu physician ) .
in addition , there were now also recommendations for treatment of hypoglycemia and for the frequency of bgl measurements after hypoglycemia.may 2005 ( major change iii)the guideline was slightly revised to decrease the risk for severe hypoglycemia . from then on the guideline recommendations strived for bgl of 90144 instead of 80144 mg / dl.bnovember 2001 ( major change i)a written guideline on glucose control was introduced .
the guideline was similar to the one used in icu - a , with the exception that this guideline aimed at bgl of 90144 instead of 80144 mg / dl.september 2004 ( major change ii)a more strict glucose control guideline was implemented , aiming at bgl between 80 and 144 mg / dl .
similar to the first version , in this guideline glucose control was also a combined icu physician and icu nurse activity .
, there were recommendations for treatment of hypoglycemia and for the frequency of bgl measurements after hypoglycemia.may 2005 ( major change iii)the icu team concluded that the guideline was too strict and rigid ; in particular it was considered to cause too many hypoglycemic events .
it was decided to no longer use the guideline and a simple order was added to the chart by the attending icu physician stating that the bgl should be between 80 and 110 mg / dl .
it was left to the icu nurses to decide whether or not to use the previous guideline and to start and adapt insulin infusion whenever necessary . the same held for the bgl measurements , i.e.
, they were taken whenever icu nurses considered that to be necessary . only in case of difficulties in making decisions pertaining to glucose control
november 2001bgls were considered acceptable between 180 and 216 mg / dl.november 2001 ( major change i)it was simply recommended that bgl should be more strictly controlled.april 2002a simple written guideline aiming at glucose control with bgl targets of 72126 mg / dl was introduced .
this guideline was completely nurse-driven.january 2003 ( major change ii)the guideline was evaluated and found insufficient .
this led to the development of a new written guideline , introduced at the bedside .
compared to the 1-page sliding scale used in icu - a and icu - b , this 4-page diagram was far more complex .
it had many extra steps and more detailed rules , with recommendations on timing of bgl measurements .
there were also recommendations for treatment of hypoglycemia and for the frequency of bgl measurements after hypoglycemia.may 2003 ( major change iii)this elaborate guideline was transformed into a computerized decision support system ( cdss ) , a custom - made visual basic application integrated within the pdms .
this cdss was introduced at 50% of the beds ( as part of a study ) .
the application displayed glucose and insulin data and suggested adjustments in insulin dose and the interval to the next bgl measurement.september 2003the cdss is used for all icu patients .
short description of changes in glucose control protocol over time in all three centers , change i indicates the initiation of glucose control ( directly or shortly after the publication of the first randomized control trial showing beneficial effects of tight glycemic control ) ; major changes ii and iii in icu - a were in august 2004 and may 2005 , respectively ; major changes ii and iii in icu - b were in september 2004 and may 2005 , respectively ; major change ii and iii in icu - c were in january 2003 and may 2003 ; see text for details on changes .
mainly arterial ( and only occasionally venous ) blood samples were used for bgl measurements . in icu - a , for almost all bgl measurements blood gas analyzers ( rapidlab 865 , bayer , germany ) were used . in icu - b
two types of glucose analyzers were used ( hitachi 917 , roche diagnostics , and accutrend sensor , roche diagnostics ) . in icu - c ,
the term sliding scale here refers to a dynamic protocol for intravenous insulin infusion icu intensive care unit , bgl blood glucose level , no advice given in the protocol description of guidelines revisions in three studied icus all adult critically ill patients who stayed for > 24 h between 1999 and 2007 were included .
the hospital information system and pdms were searched for all records on bgl for these patients .
the first bgls directly after icu admittance were excluded from the final analysis because we considered them not to be influenced by any icu regimen .
the most common performance indicators were selected to show the quality of glucose regulation in the three icus .
the indicators , described in table s1 in the electronic supplementary material ( esm ) , were categorized in three groups : effectiveness / efficiency - related indicators ( mean bgl , bgl within predefined targets , and time to reach target ) , safety - related indicators ( severe hypoglycemia , hyperglycemia , and hyperglycemia index ) , and protocol - related indicators ( sampling frequency ) .
we used the spc technique , described in the esm , and the xmr control chart to construct and analyze the processes . due to big subgroup size and the increasing chance of a false - positive result
, we used the xmr chart in place of attribute charts [ 31 , 32 ] .
the quality indicators that we chose were calculated per quarter and plotted as points on the xmr chart .
the mean of the points before gc implementation was calculated along with the 3 sigma limits . to determine whether a change in the process occurs further along the time axis , the mean and process control limits were extrapolated over the entire study period . because the time of intervention is known and because the process is stable ( i.e. , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits were recalculated in the intervention period .
the kruskal - wallis h , mann - whitney u , and chi - squared tests were used to assess the statistical significance of differences among pre- and post - intervention periods and to compare these results with those of the spc analysis .
collection of data was performed in three closed - format mixed medical - surgical icus in the netherlands .
icu - c is an 18-bed icu of a nonacademic teaching hospital . in icu - a and icu - c
icu - a ( march 2002 ) and icu - c ( april 2001 ) were equipped with a patient data management system ( pdms ) . the pdms can display and process all bgls directly after their measurement with a maximum delay of 1 min .
tables 1 and 2 show the guideline characteristics and changes in them over time .
note that for severe hypoglycemia events , the guidelines recommend stopping insulin infusion as well as injecting glucose.table 1short description of changes in glucose control protocol over timeicuicu - aicu - bicu - cchange in glucose controliiiiiiiiiiiiiiiiiiglucose control protocol characteristics type of protocolsimple set of rulessliding scalessliding scalessimple set of rulessliding scalessimple set of rulessliding scalessliding scalessliding scales present in what form(s)writtenwrittenwrittenwrittenwrittenwrittenwrittenwrittenwritten decision support presentnonononononononoyeswho is responsible for glucose control start of insulin : icu nurse or physicianphysiciannursenursenursenursenursenursenursenurse dosing of insulin : icu nurse or physiciannurse + physiciannursenursenurse + physiciannurse + physiciannursenursenursenurse correction of hypoglycemia : icu nurse or physiciannurse + physiciannursenursenurse + physiciannurse + physiciannurse + physiciannursenursenurseprotocol thresholds and targets start of insulin ( mg / dl)>144>144>144>144>144>110>126>126>126 bgl targets ( mg / dl)801448014490144901448014480110721267212672126 timing of bgl measurements described in or mandated by the protocolnoyesyesnoyesnoyesyesyesrules on stopping insulin threshold to stop insulin infusion<80<63 or 6380 with > 50% reduction in bgl<63 or 63144 with > 50% reduction in bgl < 90<54 or 5480 with > 50% reduction in bgl<54<80<80<80 other reason for stopping insulin
feeding stoppedfeeding stopped feeding stoppedfeeding stoppedaction in case of hypoglycemia bgl < 40 mg / dl50 ml 20% glucose50 ml 20% glucose50 ml 50% glucose20 ml 50% glucose20 ml 30% glucose20 ml 30% glucose20 ml 30% glucosein all three centers , change i indicates the initiation of glucose control ( directly or shortly after the publication of the first randomized control trial showing beneficial effects of tight glycemic control ) ; major changes ii and iii in icu - a were in august 2004 and may 2005 , respectively ; major changes ii and iii in icu - b were in september 2004 and may 2005 , respectively ; major change ii and iii in icu - c were in january 2003 and may 2003 ; see text for details on changes .
mainly arterial ( and only occasionally venous ) blood samples were used for bgl measurements . in icu - a , for almost all bgl measurements blood gas analyzers ( rapidlab 865 , bayer , germany ) were used . in icu - b
two types of glucose analyzers were used ( hitachi 917 , roche diagnostics , and accutrend sensor , roche diagnostics ) . in icu - c ,
the term sliding scale here refers to a dynamic protocol for intravenous insulin infusionicu intensive care unit , bgl blood glucose level , no advice given in the protocoltable 2description of guidelines revisions in three studied icusicudate of changedescriptionabefore november 2001for a long time , hyperglycemia was considered an adaptive response to critical illness .
therefore before publication of the first randomized controlled trial showing benefit of tight glycemic control , only bgl > 200 mg / dl was a reason to start insulin infusion.november 2001 ( major change i)a simple guideline on glucose control was implemented involving all icu patients .
glucose control was considered a combined icu physician and icu nurse activity ; initiation of insulin infusion was by the attending icu physician ( and never the icu nurse ) , changes in insulin infusion were by icu physician and/or icu nurse .
the protocol did not make recommendations on timing of bgl measurements.august 2004 ( major change ii)a more strict guideline was implemented .
this protocol was completely nurse - driven ; initiation of and all changes in insulin infusion were done only by the attending icu nurse ( and never the icu physician ) .
in addition , there were now also recommendations for treatment of hypoglycemia and for the frequency of bgl measurements after hypoglycemia.may 2005 ( major change iii)the guideline was slightly revised to decrease the risk for severe hypoglycemia . from then on the guideline recommendations strived for bgl of 90144 instead of 80144 mg / dl.bnovember 2001 ( major change i)a written guideline on glucose control was introduced .
the guideline was similar to the one used in icu - a , with the exception that this guideline aimed at bgl of 90144 instead of 80144 mg / dl.september 2004 ( major change ii)a more strict glucose control guideline was implemented , aiming at bgl between 80 and 144 mg / dl .
similar to the first version , in this guideline glucose control was also a combined icu physician and icu nurse activity .
in addition , there were recommendations for treatment of hypoglycemia and for the frequency of bgl measurements after hypoglycemia.may 2005 ( major change iii)the icu team concluded that the guideline was too strict and rigid ; in particular it was considered to cause too many hypoglycemic events .
it was decided to no longer use the guideline and a simple order was added to the chart by the attending icu physician stating that the bgl should be between 80 and 110 mg / dl .
it was left to the icu nurses to decide whether or not to use the previous guideline and to start and adapt insulin infusion whenever necessary . the same held for the bgl measurements , i.e.
, they were taken whenever icu nurses considered that to be necessary . only in case of difficulties in making decisions pertaining to glucose control
thus from then on the guideline was considered to be merely icu nurse based.cbefore november 2001bgls were considered acceptable between 180 and 216 mg / dl.november 2001 ( major change i)it was simply recommended that bgl should be more strictly controlled.april 2002a simple written guideline aiming at glucose control with bgl targets of 72126 mg / dl was introduced .
this guideline was completely nurse-driven.january 2003 ( major change ii)the guideline was evaluated and found insufficient .
this led to the development of a new written guideline , introduced at the bedside .
compared to the 1-page sliding scale used in icu - a and icu - b , this 4-page diagram was far more complex .
it had many extra steps and more detailed rules , with recommendations on timing of bgl measurements .
there were also recommendations for treatment of hypoglycemia and for the frequency of bgl measurements after hypoglycemia.may 2003 ( major change iii)this elaborate guideline was transformed into a computerized decision support system ( cdss ) , a custom - made visual basic application integrated within the pdms .
this cdss was introduced at 50% of the beds ( as part of a study ) .
the application displayed glucose and insulin data and suggested adjustments in insulin dose and the interval to the next bgl measurement.september 2003the cdss is used for all icu patients .
short description of changes in glucose control protocol over time in all three centers , change i indicates the initiation of glucose control ( directly or shortly after the publication of the first randomized control trial showing beneficial effects of tight glycemic control ) ; major changes ii and iii in icu - a were in august 2004 and may 2005 , respectively ; major changes ii and iii in icu - b were in september 2004 and may 2005 , respectively ; major change ii and iii in icu - c were in january 2003 and may 2003 ; see text for details on changes .
mainly arterial ( and only occasionally venous ) blood samples were used for bgl measurements . in icu - a , for almost all bgl measurements blood gas analyzers ( rapidlab 865 , bayer , germany ) were used . in icu - b
two types of glucose analyzers were used ( hitachi 917 , roche diagnostics , and accutrend sensor , roche diagnostics ) . in icu - c ,
the term sliding scale here refers to a dynamic protocol for intravenous insulin infusion icu intensive care unit , bgl blood glucose level , no advice given in the protocol description of guidelines revisions in three studied icus
all adult critically ill patients who stayed for > 24 h between 1999 and 2007 were included .
the hospital information system and pdms were searched for all records on bgl for these patients .
the first bgls directly after icu admittance were excluded from the final analysis because we considered them not to be influenced by any icu regimen .
the most common performance indicators were selected to show the quality of glucose regulation in the three icus .
the indicators , described in table s1 in the electronic supplementary material ( esm ) , were categorized in three groups : effectiveness / efficiency - related indicators ( mean bgl , bgl within predefined targets , and time to reach target ) , safety - related indicators ( severe hypoglycemia , hyperglycemia , and hyperglycemia index ) , and protocol - related indicators ( sampling frequency ) .
we used the spc technique , described in the esm , and the xmr control chart to construct and analyze the processes . due to big subgroup size and the increasing chance of a false - positive result
, we used the xmr chart in place of attribute charts [ 31 , 32 ] .
the quality indicators that we chose were calculated per quarter and plotted as points on the xmr chart .
the mean of the points before gc implementation was calculated along with the 3 sigma limits . to determine whether a change in the process occurs further along the time axis , the mean and process control limits were extrapolated over the entire study period . because the time of intervention is known and because the process is stable ( i.e. , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits were recalculated in the intervention period .
the kruskal - wallis h , mann - whitney u , and chi - squared tests were used to assess the statistical significance of differences among pre- and post - intervention periods and to compare these results with those of the spc analysis .
in total 9,392 , 2,968 , and 4,751 admissions from icu - a and icu - b ( from 1 january 1999 to 31 september 2007 ) and icu - c ( from 1 january 2001 to 31 september 2007 ) , respectively , were extracted and analyzed .
table s2 in the esm shows the patient baseline characteristics including age , gender , apache iii score , admission type , icu mortality , and length of stay in each year of study .
spc showed that they were stable and did not change significantly during the study period .
figure 1 shows the quality process control charts for the effectiveness / efficiency - related indicators of gc in the three icus .
( i.e. , a change was detected ) after implementing the gc guideline in all three icus .
the introduction of simple rules on gc had the largest effect in icu - a , since gc became more stable with less variation .
subsequent changes in the guidelines did not have effects as large as the introduction of the guideline itself .
mean bgl in icu - b was reduced by the introduction of the guideline but the mean bgl still remained higher and less stable from quarter to quarter than in the other two icus .
similar to icu - a , in icu - c the implementation of the gc guideline had a large effect on bgl , but with the introduction of the computerized decision support system ( cdss ) the mean bgl decreased further and gc stabilized.fig .
1control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) .
an asterisk means that the indicator was not only influenced by performance but also by definition of targets , and that because of the latter sharp changes over time could be recognized . when the data points are , without any special - cause variation , within the process control limits then the process is said to be in control and stable .
common rules for distinguishing a special - cause variation ( i.e. , a structural change ) : one or more points above or below the process control limit , a run of eight ( or seven ) or more points on one side of the center line , two out of three consecutive points appearing beyond 2 sigmas on the same side of the center line , a run of eight ( or seven ) or more points all trending up or down . because the time of intervention ( major changes ) is known and because the process was stable ( i.e. , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits are recalculated in the intervention period control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) .
an asterisk means that the indicator was not only influenced by performance but also by definition of targets , and that because of the latter sharp changes over time could be recognized .
when the data points are , without any special - cause variation , within the process control limits then the process is said to be in control and stable .
common rules for distinguishing a special - cause variation ( i.e. , a structural change ) : one or more points above or below the process control limit , a run of eight ( or seven ) or more points on one side of the center line , two out of three consecutive points appearing beyond 2 sigmas on the same side of the center line , a run of eight ( or seven ) or more points all trending up or down . because the time of intervention ( major changes ) is known and because the process was stable ( i.e. , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits are recalculated in the intervention period the percentage of bgls within locally defined targets increased after introducing gc guidelines in all three icus . in icu - a , the subsequent revisions in the protocol did not change this percentage . as with mean bgls ,
the percentage of bgls within target in icu - b changed significantly with the two changes in the protocol . in icu - c , both the introduction of gc as well as the introduction of the cdss increased the percentage of bgls within targets .
time to reach targets decreased after introducing the first gc guideline in icu - a and icu - c .
the means of this indicator in icu - a were better than in icu - c before introducing the protocol .
but in icu - c after protocol introduction , and especially with cdss implementation , the mean of this indicator rapidly decreased and eventually it was half of that in icu - a .
after protocol implementation in icu - b , the mean time to reach target ranges decreased stepwise with changes in the protocol , but was comparable to the other two icus with less stability and higher mean .
process control charts of safety - related quality indicators are shown in figs . 2 and 3 .
as soon as the guidelines were introduced , in all three icus the percentage of bgl measurements 40 mg / dl increased . in icu - a , the first revision of the guideline decreased both severe and nonsevere hypoglycemia events . in icu - b , with all changes in the protocol , the incidence of hypoglycemia increased . in icu - c , these indicators increased after introducing gc and further increased after cdss implementation . compared to the other two icus , the percentage of bgl measurements 40 mg / dl in icu - b was less stable with more variation from quarter to quarter before the third revision in the gc guideline.fig .
2control charts of percentage of patients with at least one bgl 40 mg / dl and percentage of bgl 40 mg / dl ( safety - related indicators)fig
. 3control charts of mean hyperglycemia index and percentage of bgl > 150 mg / dl ( safety - related indicators ) control charts of percentage of patients with at least one bgl 40 mg / dl and percentage of bgl 40 mg / dl ( safety - related indicators ) control charts of mean hyperglycemia index and percentage of bgl > 150 mg / dl ( safety - related indicators ) mean interval between bgl measurements decreased after introducing the gc guidelines in icu - a and icu - c ( fig . 4 ) .
in icu - a this interval was smaller than in icu - c before introducing the protocol .
but after introduction of the guideline in icu - c , especially with the cdss implementation , this mean rapidly decreased and was even half of that in icu - a . with the introduction and subsequent changes of the guideline in icu - b ,
the mean interval between bgl measurements decreased , but compared to the other two icus , with less stability and with higher means especially before the third revision .
4control chart of mean bgl sampling intervals ( protocol - related indicator ) control chart of mean bgl sampling intervals ( protocol - related indicator ) the results of nonparametric and chi - squared tests on the effect of introducing gc and related changes on these indicators were concordant with the spc results ( data not shown ) .
we used all data from 31 september 2005 till 31 september 2007 because all icus declared there were no other interventions that could have affected gc in this period .
the spc charts also showed the processes were stable for all indicators in all three icus in this period .
median bgl in icu - c and icu - b was significantly lower than icu - a .
the percentage of patients with at least one hypoglycemia event and overall percentage of hypoglycemia and severe hypoglycemia events were significantly higher in icu - c and icu - b .
other statistically ( and seemingly clinically ) significant differences were in bgl measurement interval , and time to reach target , which were lower in icu - c ( table s3 in the esm ) .
in total 9,392 , 2,968 , and 4,751 admissions from icu - a and icu - b ( from 1 january 1999 to 31 september 2007 ) and icu - c ( from 1 january 2001 to 31 september 2007 ) , respectively , were extracted and analyzed .
table s2 in the esm shows the patient baseline characteristics including age , gender , apache iii score , admission type , icu mortality , and length of stay in each year of study .
spc showed that they were stable and did not change significantly during the study period .
figure 1 shows the quality process control charts for the effectiveness / efficiency - related indicators of gc in the three icus .
( i.e. , a change was detected ) after implementing the gc guideline in all three icus .
the introduction of simple rules on gc had the largest effect in icu - a , since gc became more stable with less variation .
subsequent changes in the guidelines did not have effects as large as the introduction of the guideline itself .
mean bgl in icu - b was reduced by the introduction of the guideline but the mean bgl still remained higher and less stable from quarter to quarter than in the other two icus .
similar to icu - a , in icu - c the implementation of the gc guideline had a large effect on bgl , but with the introduction of the computerized decision support system ( cdss ) the mean bgl decreased further and gc stabilized.fig .
1control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) .
an asterisk means that the indicator was not only influenced by performance but also by definition of targets , and that because of the latter sharp changes over time could be recognized .
when the data points are , without any special - cause variation , within the process control limits then the process is said to be in control and stable .
common rules for distinguishing a special - cause variation ( i.e. , a structural change ) : one or more points above or below the process control limit , a run of eight ( or seven ) or more points on one side of the center line , two out of three consecutive points appearing beyond 2 sigmas on the same side of the center line , a run of eight ( or seven ) or more points all trending up or down . because the time of intervention ( major changes ) is known and because the process was stable ( i.e. , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits are recalculated in the intervention period control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) .
an asterisk means that the indicator was not only influenced by performance but also by definition of targets , and that because of the latter sharp changes over time could be recognized .
when the data points are , without any special - cause variation , within the process control limits then the process is said to be in control and stable .
common rules for distinguishing a special - cause variation ( i.e. , a structural change ) : one or more points above or below the process control limit , a run of eight ( or seven ) or more points on one side of the center line , two out of three consecutive points appearing beyond 2 sigmas on the same side of the center line , a run of eight ( or seven ) or more points all trending up or down . because the time of intervention ( major changes ) is known and because the process was stable ( i.e. , not out of control
according to the spc rules ) before and after the intervention , the mean and process control limits are recalculated in the intervention period the percentage of bgls within locally defined targets increased after introducing gc guidelines in all three icus . in icu - a , the subsequent revisions in the protocol did not change this percentage . as with mean bgls ,
the percentage of bgls within target in icu - b changed significantly with the two changes in the protocol . in icu - c , both the introduction of gc as well as the introduction of the cdss increased the percentage of bgls within targets .
time to reach targets decreased after introducing the first gc guideline in icu - a and icu - c .
the means of this indicator in icu - a were better than in icu - c before introducing the protocol .
but in icu - c after protocol introduction , and especially with cdss implementation , the mean of this indicator rapidly decreased and eventually it was half of that in icu - a .
after protocol implementation in icu - b , the mean time to reach target ranges decreased stepwise with changes in the protocol , but was comparable to the other two icus with less stability and higher mean .
process control charts of safety - related quality indicators are shown in figs . 2 and 3 .
as soon as the guidelines were introduced , in all three icus the percentage of bgl measurements 40 mg / dl increased . in icu - a , the first revision of the guideline decreased both severe and nonsevere hypoglycemia events . in icu - b , with all changes in the protocol , the incidence of hypoglycemia increased . in icu - c , these indicators increased after introducing gc and further increased after cdss implementation . compared to the other two icus , the percentage of bgl measurements 40 mg / dl in icu - b was less stable with more variation from quarter to quarter before the third revision in the gc guideline.fig .
2control charts of percentage of patients with at least one bgl 40 mg / dl and percentage of bgl 40 mg / dl ( safety - related indicators)fig .
3control charts of mean hyperglycemia index and percentage of bgl > 150 mg / dl ( safety - related indicators ) control charts of percentage of patients with at least one bgl 40 mg / dl and percentage of bgl 40 mg / dl ( safety - related indicators ) control charts of mean hyperglycemia index and percentage of bgl > 150 mg / dl ( safety - related indicators )
mean interval between bgl measurements decreased after introducing the gc guidelines in icu - a and icu - c ( fig .
- a this interval was smaller than in icu - c before introducing the protocol .
but after introduction of the guideline in icu - c , especially with the cdss implementation , this mean rapidly decreased and was even half of that in icu - a . with the introduction and subsequent changes of the guideline in icu - b ,
the mean interval between bgl measurements decreased , but compared to the other two icus , with less stability and with higher means especially before the third revision .
4control chart of mean bgl sampling intervals ( protocol - related indicator ) control chart of mean bgl sampling intervals ( protocol - related indicator ) the results of nonparametric and chi - squared tests on the effect of introducing gc and related changes on these indicators were concordant with the spc results ( data not shown ) .
we used all data from 31 september 2005 till 31 september 2007 because all icus declared there were no other interventions that could have affected gc in this period .
the spc charts also showed the processes were stable for all indicators in all three icus in this period .
median bgl in icu - c and icu - b was significantly lower than icu - a .
the percentage of patients with at least one hypoglycemia event and overall percentage of hypoglycemia and severe hypoglycemia events were significantly higher in icu - c and icu - b .
other statistically ( and seemingly clinically ) significant differences were in bgl measurement interval , and time to reach target , which were lower in icu - c ( table s3 in the esm ) .
the effect of introducing gc guidelines and the various implementations on the quality of gc , especially for an extended period of time , is unknown .
the implementation strategies of gc ranged from raising awareness to employing a computerized decision support system . in all three icus ,
regardless of strategy , there was a continuous and significant improvement in the effectiveness and efficiency indicators , an increase in hypoglycemic - related indicators , and an increase in bgl measurements .
however , the speed of change and the final outcomes differed significantly among the three icus . with spc ,
data are plotted and interpreted in a time series rather than merely comparing before and after measures . with this method
process control charts showed which parts of the processes were more stable and also showed the duration of change after each intervention .
1 , 2 , 3 and 4 showed the diversity in results for implementing similar protocols in three different centers and reflected a range of error
, all else being equal , in implementing a protocol with differing interpretations and implementation details .
those wishing to use spc as a tool for longitudinal self - examination of performance are referred to .
first , to our knowledge this is the first report on effects of gc in routine daily clinical practice over an extended period of time .
second , to visualize and make inferences on the longitudinal development of quality indicators , we used the powerful instrument of process control charts from the field of spc .
first , we did not perform subgroup analysis ( such as surgical vs. medical patients ) .
second , we did not investigate the influence of gc on clinically relevant endpoints such as survival .
however , as gc has been shown to be an evidence - based strategy that decreases morbidity and mortality [ 5 , 6 ] , adherence to this strategy is commonly advocated .
however , the fact that the quality indicators were significantly influenced implies that the protocols are being increasingly followed . finally , because nutrition data were not available for the whole study period , we could not investigate the possible effect of nutrition on the quality of gc . however , nutrition input is not likely to have changed over the study period within each hospital .
in addition , the protocol should be robust to reasonable fluctuations in the given nutritional carbohydrate levels .
usually routine clinical practice characteristics and limitations are not considered in randomized control trial ( rcts ) and guidelines .
this is one explanation for the underuse of treatments in routine practice that were beneficial in trials and that are recommended in guidelines . among tight glycemic control ( tgc )
studies , some have explicitly considered routine practice issues in their design , e.g. , [ 14 , 15 , 17 ] . however , we in addition monitored performance during actual use of the protocol over time with spc .
relaxing the gc range to a wider , locally workable target can perhaps be explained by an associated fear of hypoglycemia .
. showed that the rate of patients with at least one episode of severe hypoglycemia ( 40 mg / dl ) was higher in the study group than in the control group ( 17.0 vs. 4.1% , p<0.001 ) .
although they reported that no serious adverse events were found , the trial was nonetheless stopped due to the hypoglycemic episodes .
interestingly our results showed that in routine practice , and in contrast to the clinical trials , gc was not stopped although the rate of patients with at least one hypoglycemia event were relatively high even with less tight target ranges ( 9% in the icu - b and icu - c after the last guideline revision ) .
although concern about increasing the percentage of hypoglycemic patients resulted in terminating the use of the more detailed protocol in icu - b , thereafter an even tighter target range was used in this icu , and the percentage of hypoglycemic patients increased even after these revisions . protocol failure and/or lack of compliance could partly explain this result , but we do not have data to test these hypotheses .
vriesendorp et al . and chase et al . showed that tgc - induced hypoglycemia was not associated with worse outcome [ 17 , 33 ] , although recent studies have reported such an association [ 22 , 34 ] .
fear of hypoglycemia and its reputation as being more dangerous than hyperglycemia in the critically ill may well be based on deeply rooted emotional beliefs rather than on evidence .
our study demonstrates that as long as continuous measurement tools are not available , hypoglycemia events are considered acceptable and can be consequently managed .
our results also showed that any decrease in mean bgl resulted in an increase in the percentage of hypoglycemia events .
icu - c , which eventually had the lowest mean bgl , also had the highest percentage of hypoglycemia events .
the complex guidelines and the use of the cdss , resulting in a mean measurement interval in icu - c of 120 min , may have led to more early ( and possibly more frequent ) detection of the hypoglycemia .
also minimum bgl in icu - a and icu - b was associated with maximum percentage of hypoglycemia events . revising the protocol in icu -
a decreased the hypoglycemia events but could not return the level to what it was before implementing gc .
shortly after the first presentation on tgc in november 2001 , there were either no written guidelines in the participating icus or the guidelines were very simple and in a development phase .
spc charts showed that awareness of tgc , regardless of the guidelines in place , brought about the maximum changes in the related indicators .
after this stage , stronger interventions , such as more complex protocols and the use of a cdss , were called upon to bring about change , but the level of change was smaller .
all have managed to reach an acceptable control but with different speeds of change and different variability over time . both icus with detailed written protocols ( icu - a and icu - c ) had better results and more stable processes with lower variability from quarter to quarter than icu - b . using a detailed written protocol with clearly defined steps compared to simple rules ( icu - b ) seems to help the nurses to make better decisions to control blood glucose .
our results show that a complex protocol with more steps and detail in icu - c had improved effects but only after including the cdss as an intervention . in icu - c the mean bgl measurement interval also became smaller than in icu - a after the cdss .
we can conclude that the cdss influenced the nurses behavior by reminding them about the time of the next bgl test .
probably the shorter time between measurements is based on differences between protocols , not the effect of cdss .
however it is unclear whether the beneficial effects of the cdss are mainly due to the more frequent measurements or due to the complex protocol rules . with the current data we can not answer this question . in addition , to achieve such a low mean bgl , higher costs ( cdss and more nursing and laboratory utilization ) were incurred and patients experienced more hypoglycemia events . cost - benefit analysis merits more research when aiming at lower mean bgl .
frequent bgl measurement is a key element in tgc , in order to steer the process in a timely manner [ 14 , 17 , 36 ] . in the three studied icus , better results were associated with more frequent measurements . in icu - a , there were many bgl measurements before introducing gc , since bgl measurements were included with blood gas analysis , even when there was no intention to measure it .
this might explain some of the fast improvement in glucose regulation in icu - a after introducing the guideline and the very small variation in sample interval .
there are different successful strategies to realize gc in routine clinical practice but they have various speeds and implementations .
more intensive implementation strategies resulted in better control of the process but at the cost of more icu resources , including the use of a decision support system .
spc is a useful tool for monitoring phenomena over time and allows for capturing within - institution changes . within quality measurement and/or improvement efforts
, spc can show where the special variations are and where opportunities lie for improving ( adherence to ) protocols .
below is the link to the electronic supplementary material .
supplementary material ( doc 80 kb ) | backgroundglucose control ( gc ) with insulin decreases morbidity and mortality of critically ill patients . in this study
we investigated gc performance over time during implementation of gc strategies within three intensive care units ( icus ) and in routine clinical practice.methodsall adult critically ill patients who stayed for > 24 h between 1999 and 2007 were included .
effects of implementing local gc guidelines and guideline revisions on effectiveness / efficiency - related indicators , safety - related indicators , and protocol - related indicators were measured.resultsdata of 17,111 patient admissions were evaluated , with 714,141 available blood glucose levels ( bgl ) measurements .
mean bgl , time to reach target , hyperglycemia index , sampling frequency , percentage of hyperglycemia events , and in - range measurements statistically changed after introducing gc in all icus .
the introduction of simple rules on gc had the largest effect .
subsequent changes in the protocol had a smaller effect than the introduction of the protocol itself .
as soon as the protocol was introduced , in all icus the percentage of hypoglycemia events increased .
various revisions were implemented to reduce hypoglycemia events , but levels never returned to those from pre - implementation .
more intensive implementation strategies including the use of a decision support system resulted in better control of the process.conclusionthere are various strategies to achieve gc in routine clinical practice but with variable success .
all of them were associated with an increase in hypoglycemia events , but gc was never stopped . instead , these events have been accepted and managed .
statistical process control is a useful tool for monitoring phenomena over time and captures within - institution changes.electronic supplementary materialthe online version of this article ( doi:10.1007/s00134 - 010 - 1924 - 3 ) contains supplementary material , which is available to authorized users . | Electronic supplementary material
Introduction
Methods
Study locations
Local glucose control guidelines
Patients
Performance indicators and definitions
Statistical analysis
Results
Patients
Effectiveness and efficiency of glucose control
Safety of glucose control
Protocol-related indicator
Discussion
Conclusions
Electronic supplementary material | the online version of this article ( doi:10.1007/s00134 - 010 - 1924 - 3 ) contains supplementary material , which is available to authorized users . , blood glucose level ( bgl ) of 80110 mg / dl , frequently referred to as tight glycemic control ] decreased morbidity and mortality of critically ill patients in two randomized controlled trials [ 5 , 6 ] . recent published meta - analysis [ 7 , 8 ] could not confirm the benefits of gc , but there are some important methodological concerns about this study [ 9 , 10 ] and many intensive care units ( icus ) still use gc in their routine clinical practice . in icu - c ,
the term sliding scale here refers to a dynamic protocol for intravenous insulin infusion icu intensive care unit , bgl blood glucose level , no advice given in the protocol description of guidelines revisions in three studied icus all adult critically ill patients who stayed for > 24 h between 1999 and 2007 were included . the indicators , described in table s1 in the electronic supplementary material ( esm ) , were categorized in three groups : effectiveness / efficiency - related indicators ( mean bgl , bgl within predefined targets , and time to reach target ) , safety - related indicators ( severe hypoglycemia , hyperglycemia , and hyperglycemia index ) , and protocol - related indicators ( sampling frequency ) . in icu - c ,
the term sliding scale here refers to a dynamic protocol for intravenous insulin infusion icu intensive care unit , bgl blood glucose level , no advice given in the protocol description of guidelines revisions in three studied icus
all adult critically ill patients who stayed for > 24 h between 1999 and 2007 were included . the indicators , described in table s1 in the electronic supplementary material ( esm ) , were categorized in three groups : effectiveness / efficiency - related indicators ( mean bgl , bgl within predefined targets , and time to reach target ) , safety - related indicators ( severe hypoglycemia , hyperglycemia , and hyperglycemia index ) , and protocol - related indicators ( sampling frequency ) . figure 1 shows the quality process control charts for the effectiveness / efficiency - related indicators of gc in the three icus . the introduction of simple rules on gc had the largest effect in icu - a , since gc became more stable with less variation . similar to icu - a , in icu - c the implementation of the gc guideline had a large effect on bgl , but with the introduction of the computerized decision support system ( cdss ) the mean bgl decreased further and gc stabilized.fig . 1control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) . , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits are recalculated in the intervention period control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) . figure 1 shows the quality process control charts for the effectiveness / efficiency - related indicators of gc in the three icus . the introduction of simple rules on gc had the largest effect in icu - a , since gc became more stable with less variation . similar to icu - a , in icu - c the implementation of the gc guideline had a large effect on bgl , but with the introduction of the computerized decision support system ( cdss ) the mean bgl decreased further and gc stabilized.fig . 1control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) . , not out of control according to the spc rules ) before and after the intervention , the mean and process control limits are recalculated in the intervention period control charts of mean bgl , time to reach target range , percentage of bgls in range predefined in the protocols , and percentage of bgls between 63 and 150 mg / dl ( efficiency - related indicators ) . as soon as the guidelines were introduced , in all three icus the percentage of bgl measurements 40 mg / dl increased . our results also showed that any decrease in mean bgl resulted in an increase in the percentage of hypoglycemia events . more intensive implementation strategies resulted in better control of the process but at the cost of more icu resources , including the use of a decision support system . spc is a useful tool for monitoring phenomena over time and allows for capturing within - institution changes . | [
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the rational prescribing of drugs is an essential skill that requires sound knowledge of clinical pharmacology and therapeutics .
many medical graduates are insufficiently prepared to assume prescription responsibilities after graduation and are likely to cause prescribing errors .
in addition , during internship , a period of medical apprenticeship , the intern is expected to demonstrate the knowledge and skills of selecting right drug for right patient , identify and report adverse drug reaction , and should have the sound knowledge and skills of rational pharmacotherapeutics and essential medicines .
however , it has been reported that medical students and interns lack the confidence of prescribing and their training in pharmacology is inadequate .
this is due to the general perception that pharmacology course in medical schools has failed to keep pace with the rapid changes and requirements of clinical practice .
traditional pharmacology teaching which focuses more on factual information with little emphasis on clinical and rational therapeutics has been considered to be responsible . in india ,
undergraduate ( ug ) medical course consists of 4 years ( 9 semesters ) followed by 1 year of compulsory rotating internship .
the medical council of india proposes graduate medical education regulations for each discipline and regularly updates the recommendations .
these regulations describe ug pharmacology curriculum with a clear emphasis to teach essential skills that will help the students select the medicines safely and effectively throughout their professional life .
an attempt was made to revise pharmacology curriculum with special emphasis on the who guidelines to good prescribing that help the students to select the medicines safely and effectively .
in addition , other clinical pharmacology exercises such as evaluation of fixed - dose combinations ( fdcs ) , sources of drug information , dosage calculation , evaluation of drug promotional literature , and adverse drug reaction ( adr ) reporting for rational prescribing were also introduced as pharmacology practical exercises .
while these exercises sensitize the students for a rational use of drugs , it is not known to what extent they improve the prescribing skill .
unfortunately , these skills are not reinforced during clinical terms and so their practical application remains incomplete .
the present educational research was undertaken to find out the impact of teaching clinical pharmacology and rational therapeutics ( cpt ) with respect to the knowledge and attitude of ug medical students and interns and to determine whether teaching cpt has prepared fresh medical graduates ( interns ) to prescribe safely and rationally .
a prospective , cross - sectional study was conducted on medical ugs and interns at b. j. medical college , ahmedabad .
ugs 2 year mbbs iii term ( ii / iii ) , 3 year mbbs part i ( iii / i ) and part ii ( iii / ii ) and interns who had completed clinical rotation in major subjects and learnt cpt by lectures , practicals in ii mbbs were included in the study .
the study was approved by the institutional ethics committee and prior informed consent was obtained ( reference no .
the knowledge component was assessed by written clinical case scenario of malaria , drug drug interaction ( ddi ) by case scenario of tuberculosis , identifying rational / irrational fdcs .
their attitude was assessed by their perceptions , characteristics , and advantages of various clinical pharmacology exercises and confidence in prescribing selected drugs from the given list . for ugs
, the practice element was evaluated by questions related to sources of drug information used and reporting of adrs . whereas the intern s questionnaire had questions related to prescribing practices , especially their confidence in prescribing selected drugs , prescribing in special patient population , specific problems encountered while prescribing , commonly prescribed fdcs , adr observed and reported , etc .
data were entered into microsoft excel and analyzed using anova test and chi - square test .
out of 379 ugs , 121 were from the second mbbs iii term ( ii / iii ) , 124 iii mbbs part i ( iii / i ) , and 134 iii mbbs part ii ( iii / ii ) students .
the majority of ugs ( 373 , 98% ) accepted having received cpt training in ii mbbs .
however , only 209 ( 55.1% ) ugs rated their cpt knowledge as good and 116 ( 31% ) as average .
of 96 interns , the majority ( 93 , 96% ) accepted having received cpt training in ii mbbs and rated their cpt knowledge as good ( 51 , 53% ) and average ( 31 , 32% ) .
of 121 2 year students , 21 ( 17.3% ) correctly answered written case scenario of plasmodium vivax malaria .
however , the majority of students ( 72 , 59.5% ) could not identify ddi .
second , only 43 ( 35.5% ) could identify two out of four irrational fdcs and 34 ( 28% ) could enlist three rational fdcs [ table 1 ] .
surprisingly , the majority of the students correctly answered questions related to p - drug ( 69 , 57% ) and drugs banned due to adrs ( 60 , 49.5% ) .
assessment of clinical pharmacology and rational therapeutics knowledge among undergraduates students ( n=379 ) out of six clinical pharmacology exercises , p - drug ( 80 , 66.1% ) was considered to be most useful followed by adr reporting ( 39 , 32.2% ) among ii mbbs students [ figure 1 ] .
secondly , majority ( 52 , 42.9% ) believed they could comfortably prescribe analgesics without supervision [ figure 2 ] .
in addition , majority of students considered efficacy 64 ( 52.8% ) as an important characteristic in choosing p - drug and availability of drugs will be an advantage of prescribing from the essential medicines list ( eml ) ( 71 , 58.6% ) .
interestingly , majority of students 117 ( 96.6% ) considered adr reporting is important ; however , 70 ( 57.8% ) stated only serious adr should be reported .
attitude of undergraduates towards various clinical pharmacology exercises perception of undergraduates and interns for comfortable prescribing without supervision majority of students 106 ( 87.6% ) used textbooks followed by the internet ( 43 , 35.5% ) as a source of drug information . while none of the students witnessed any adrs during the clinical postings . of 124 iii / i and 134 iii / ii students , only 2 ( 1.6% ) and 45 ( 33.5% )
however , majority of iii / i ( 66 , 53.2% ) and iii / ii ( 84 , 62.6% ) could not identify ddi and only 46 ( 37% ) iii / i and 50 ( 37.3% ) iii / ii students could identify two out of four irrational fdcs . surprisingly , 42 ( 33.8% ) iii / i ugs could mention all three rational fdcs which was even less ( 39 , 29% ) in iii / ii students . moreover , majority 98 ( 79% ) iii / i and 95 ( 70% ) iii / ii correctly answered p - drug concept . while 51 ( 41.1% ) iii / i and 37 ( 27.6% ) iii / ii students listed two drugs banned due to adrs .
interestingly , both the groups mentioned p - drug ( 73 , 58.8% and 78 , 58.2% ) as the most useful clinical pharmacology exercise followed by evaluation of fdcs by iii / i students ( 56 , 45.1% ) and adr by iii / ii students ( 64 , 47.7% ) [ figure 1 ] .
majority of the students in both the groups affirmed comfortably prescribing nonsteroidal anti - inflammatory drugs without supervision ( 91 , 73% ; 106 , 79% ) [ figure 2 ] .
similarly , majority of the students in both the group labeled efficacy as the most important characteristic of the drug for choosing p - drug ( 65 , 52% ; 79 , 58.9% ) .
moreover , both the groups confirmed that the availability of drugs would be the advantage while prescribing drugs from eml ( 75 , 60% ; 92 , 68.6% ) .
further , majority of students ( 122 , 98.3% , 129 , 96.6% ) considered adr reporting is important ; however , 85 ( 68.5% ) and 80 ( 59.7% ) stated only serious adr should be reported .
majority of iii / i students ( 110 , 88.7% ) and iii / ii ( 114 , 85% ) refer textbooks followed by the internet ( 70 , 56.4% ; 78 , 58.2% ) as sources of drug information . while none of the students witnessed any adrs during their clinical postings .
it was observed that 13 ( 13.5% ) interns correctly answered written case scenario of p. vivax malaria .
however , majority of interns 37 ( 38.5% ) could not identify ddi and only 39 ( 40.6% ) interns could mention two rational fdcs . surprisingly , almost half of the interns 49 ( 51% ) could not answer question related to p - drug . while 45 ( 46.8% ) could answer two drugs banned due to adrs .
a significant reduction in knowledge score was observed ( 4.73 2.3 , p < 0.0001 ) as compared to ugs [ table 1 and figure 3 ] .
comparison of knowledge score among undergraduates and interns majority interns ( 95 , 98.9% ) acknowledged comfortable prescribing of analgesics followed by antacids ( 78 , 81.2% ) without supervision .
similarly , majority of the interns believed efficacy as the most important characteristic of the drug for choosing p - drug ( 47 , 48.9% ) and availability of drugs would be the an advantage while prescribing drugs from edl ( 42 , 43.7% ) . moreover ,
majority of interns ( 86 , 89.5% ) considered adr reporting is important ; however , 59 ( 61.45% ) interns stated only serious adr should be reported while 20 ( 20.8% ) interns believed that adrs related to the new drug should be reported .
surprisingly , the internet significantly ( 72 , 75% ) topped the list for seeking drug information followed by textbooks ( 70 , 72.9% ) by interns as compared to ugs ( p < 0.05 ) [ figure 3 ] .
in addition , 40 ( 41.6% ) interns seek drug information from senior colleagues , and 13 ( 13.5% ) relied on medical representatives ( mrs ) .
interestingly , majority of interns ( 61 , 63.5% ) confessed prescribing problems in special group of patients ( 34 , 35.4% ) and selection of drug ( 32 , 33.3% ) .
although majority agreed to be confident in prescribing for adult patients ( 86 , 89.5% ) , they were hesitant to prescribe opioids ( 74 , 77% ) , steroids ( 73 , 76% ) , anti - hypertensives ( 60 , 62.5% ) .
moreover , the interns referred source of information before prescribing ( 72 , 75% ) for details of drug administration ( 32 , 33.3% ) .
majority of interns had prescribed multivitamins ( 81 , 84.3% ) , co - amoxiclav ( 80 , 83.3% ) , cough mixtures ( 70 , 72.9% ) during internship [ table 2 ] .
however , majority had not applied p - drug concept ( 64 , 66.6% ) , or reported adr ( 90 , 93.7% ) during internship .
prescribing practices of fixed dose combination among interns ( n=96 ) majority of ug students ( 75% ) and interns emphasized teaching on drug selection ( 49% ) followed by dosage schedule ( 45% ) and drug interaction ( 38.25% ) , while 61% of interns emphasized teaching on dosage schedule followed by prescribing in special patient population ( 51% ) and drug selection ( 47% ) .
of 121 2 year students , 21 ( 17.3% ) correctly answered written case scenario of plasmodium vivax malaria .
however , the majority of students ( 72 , 59.5% ) could not identify ddi .
second , only 43 ( 35.5% ) could identify two out of four irrational fdcs and 34 ( 28% ) could enlist three rational fdcs [ table 1 ] .
surprisingly , the majority of the students correctly answered questions related to p - drug ( 69 , 57% ) and drugs banned due to adrs ( 60 , 49.5% ) .
assessment of clinical pharmacology and rational therapeutics knowledge among undergraduates students ( n=379 ) out of six clinical pharmacology exercises , p - drug ( 80 , 66.1% ) was considered to be most useful followed by adr reporting ( 39 , 32.2% ) among ii mbbs students [ figure 1 ] .
secondly , majority ( 52 , 42.9% ) believed they could comfortably prescribe analgesics without supervision [ figure 2 ] .
in addition , majority of students considered efficacy 64 ( 52.8% ) as an important characteristic in choosing p - drug and availability of drugs will be an advantage of prescribing from the essential medicines list ( eml ) ( 71 , 58.6% ) .
interestingly , majority of students 117 ( 96.6% ) considered adr reporting is important ; however , 70 ( 57.8% ) stated only serious adr should be reported .
attitude of undergraduates towards various clinical pharmacology exercises perception of undergraduates and interns for comfortable prescribing without supervision majority of students 106 ( 87.6% ) used textbooks followed by the internet ( 43 , 35.5% ) as a source of drug information . while none of the students witnessed any adrs during the clinical postings . of 124 iii / i and 134 iii / ii students , only 2 ( 1.6% ) and 45 ( 33.5% )
however , majority of iii / i ( 66 , 53.2% ) and iii / ii ( 84 , 62.6% ) could not identify ddi and only 46 ( 37% ) iii / i and 50 ( 37.3% ) iii / ii students could identify two out of four irrational fdcs .
surprisingly , 42 ( 33.8% ) iii / i ugs could mention all three rational fdcs which was even less ( 39 , 29% ) in iii / ii students . moreover , majority 98 ( 79% ) iii / i and 95 ( 70% ) iii / ii correctly answered p - drug concept . while 51 ( 41.1% ) iii / i and 37 ( 27.6% ) iii / ii students listed two drugs banned due to adrs .
interestingly , both the groups mentioned p - drug ( 73 , 58.8% and 78 , 58.2% ) as the most useful clinical pharmacology exercise followed by evaluation of fdcs by iii / i students ( 56 , 45.1% ) and adr by iii / ii students ( 64 , 47.7% ) [ figure 1 ] .
majority of the students in both the groups affirmed comfortably prescribing nonsteroidal anti - inflammatory drugs without supervision ( 91 , 73% ; 106 , 79% ) [ figure 2 ] . similarly , majority of the students in both the group labeled efficacy as the most important characteristic of the drug for choosing p - drug ( 65 , 52% ; 79 , 58.9% ) .
moreover , both the groups confirmed that the availability of drugs would be the advantage while prescribing drugs from eml ( 75 , 60% ; 92 , 68.6% ) .
further , majority of students ( 122 , 98.3% , 129 , 96.6% ) considered adr reporting is important ; however , 85 ( 68.5% ) and 80 ( 59.7% ) stated only serious adr should be reported .
majority of iii / i students ( 110 , 88.7% ) and iii / ii ( 114 , 85% ) refer textbooks followed by the internet ( 70 , 56.4% ; 78 , 58.2% ) as sources of drug information . while none of the students witnessed any adrs during their clinical postings .
of 121 2 year students , 21 ( 17.3% ) correctly answered written case scenario of plasmodium vivax malaria .
however , the majority of students ( 72 , 59.5% ) could not identify ddi .
second , only 43 ( 35.5% ) could identify two out of four irrational fdcs and 34 ( 28% ) could enlist three rational fdcs [ table 1 ] .
surprisingly , the majority of the students correctly answered questions related to p - drug ( 69 , 57% ) and drugs banned due to adrs ( 60 , 49.5% ) .
assessment of clinical pharmacology and rational therapeutics knowledge among undergraduates students ( n=379 ) out of six clinical pharmacology exercises , p - drug ( 80 , 66.1% ) was considered to be most useful followed by adr reporting ( 39 , 32.2% ) among ii mbbs students [ figure 1 ] .
secondly , majority ( 52 , 42.9% ) believed they could comfortably prescribe analgesics without supervision [ figure 2 ] .
in addition , majority of students considered efficacy 64 ( 52.8% ) as an important characteristic in choosing p - drug and availability of drugs will be an advantage of prescribing from the essential medicines list ( eml ) ( 71 , 58.6% ) .
interestingly , majority of students 117 ( 96.6% ) considered adr reporting is important ; however , 70 ( 57.8% ) stated only serious adr should be reported .
attitude of undergraduates towards various clinical pharmacology exercises perception of undergraduates and interns for comfortable prescribing without supervision majority of students 106 ( 87.6% ) used textbooks followed by the internet ( 43 , 35.5% ) as a source of drug information . while none of the students witnessed any adrs during the clinical postings .
of 124 iii / i and 134 iii / ii students , only 2 ( 1.6% ) and 45 ( 33.5% ) could correctly answered written case scenario of p. vivax malaria , respectively .
however , majority of iii / i ( 66 , 53.2% ) and iii / ii ( 84 , 62.6% ) could not identify ddi and only 46 ( 37% ) iii / i and 50 ( 37.3% ) iii / ii students could identify two out of four irrational fdcs .
surprisingly , 42 ( 33.8% ) iii / i ugs could mention all three rational fdcs which was even less ( 39 , 29% ) in iii / ii students . moreover , majority 98 ( 79% ) iii / i and 95 ( 70% ) iii / ii correctly answered p - drug concept . while 51 ( 41.1% ) iii / i and 37 ( 27.6% ) iii / ii students listed two drugs banned due to adrs .
interestingly , both the groups mentioned p - drug ( 73 , 58.8% and 78 , 58.2% ) as the most useful clinical pharmacology exercise followed by evaluation of fdcs by iii / i students ( 56 , 45.1% ) and adr by iii / ii students ( 64 , 47.7% ) [ figure 1 ] .
majority of the students in both the groups affirmed comfortably prescribing nonsteroidal anti - inflammatory drugs without supervision ( 91 , 73% ; 106 , 79% ) [ figure 2 ] .
similarly , majority of the students in both the group labeled efficacy as the most important characteristic of the drug for choosing p - drug ( 65 , 52% ; 79 , 58.9% ) .
moreover , both the groups confirmed that the availability of drugs would be the advantage while prescribing drugs from eml ( 75 , 60% ; 92 , 68.6% ) .
further , majority of students ( 122 , 98.3% , 129 , 96.6% ) considered adr reporting is important ; however , 85 ( 68.5% ) and 80 ( 59.7% ) stated only serious adr should be reported . majority of iii / i students ( 110 , 88.7% ) and iii / ii ( 114 , 85% ) refer textbooks followed by the internet ( 70 , 56.4% ; 78 , 58.2% ) as sources of drug information . while none of the students witnessed any adrs during their clinical postings .
it was observed that 13 ( 13.5% ) interns correctly answered written case scenario of p. vivax malaria .
however , majority of interns 37 ( 38.5% ) could not identify ddi and only 39 ( 40.6% ) interns could mention two rational fdcs . surprisingly , almost half of the interns 49 ( 51% ) could not answer question related to p - drug . while 45 ( 46.8% ) could answer two drugs banned due to adrs .
a significant reduction in knowledge score was observed ( 4.73 2.3 , p < 0.0001 ) as compared to ugs [ table 1 and figure 3 ] . comparison of knowledge score among undergraduates and interns majority interns ( 95 , 98.9% ) acknowledged comfortable prescribing of analgesics followed by antacids ( 78 , 81.2% ) without supervision .
similarly , majority of the interns believed efficacy as the most important characteristic of the drug for choosing p - drug ( 47 , 48.9% ) and availability of drugs would be the an advantage while prescribing drugs from edl ( 42 , 43.7% ) .
moreover , majority of interns ( 86 , 89.5% ) considered adr reporting is important ; however , 59 ( 61.45% ) interns stated only serious adr should be reported while 20 ( 20.8% ) interns believed that adrs related to the new drug should be reported .
surprisingly , the internet significantly ( 72 , 75% ) topped the list for seeking drug information followed by textbooks ( 70 , 72.9% ) by interns as compared to ugs ( p < 0.05 ) [ figure 3 ] .
in addition , 40 ( 41.6% ) interns seek drug information from senior colleagues , and 13 ( 13.5% ) relied on medical representatives ( mrs ) .
interestingly , majority of interns ( 61 , 63.5% ) confessed prescribing problems in special group of patients ( 34 , 35.4% ) and selection of drug ( 32 , 33.3% ) .
although majority agreed to be confident in prescribing for adult patients ( 86 , 89.5% ) , they were hesitant to prescribe opioids ( 74 , 77% ) , steroids ( 73 , 76% ) , anti - hypertensives ( 60 , 62.5% ) .
moreover , the interns referred source of information before prescribing ( 72 , 75% ) for details of drug administration ( 32 , 33.3% ) . majority of interns had prescribed multivitamins ( 81 , 84.3% ) , co - amoxiclav ( 80 , 83.3% ) , cough mixtures ( 70 , 72.9% ) during internship [ table 2 ] . however , majority had not applied p - drug concept ( 64 , 66.6% ) , or reported adr ( 90 , 93.7% ) during internship . prescribing practices of fixed dose combination among interns ( n=96 )
majority of ug students ( 75% ) and interns emphasized teaching on drug selection ( 49% ) followed by dosage schedule ( 45% ) and drug interaction ( 38.25% ) , while 61% of interns emphasized teaching on dosage schedule followed by prescribing in special patient population ( 51% ) and drug selection ( 47% ) .
effective teaching and training in cpt is the backbone to inculcate a rational and scientific basis of prescribing .
who-6 step method has been proved to be effective for drug selection in medical students . in view of the above
, this cross - sectional study was conducted among ug students and interns who learnt cpt based on who model along with other clinical pharmacology exercises in 2 year mbbs .
the objective of this study was to find out the impact of teaching and its retention in fresh medical graduates .
this study observed that cpt teaching is effective in the transfer of knowledge to ugs , unfortunately , its retention was poor after graduation , i.e. , in internship as evidenced by a significant reduction in knowledge score .
in addition , the application of principles of cpt while prescribing such as the process of selection of drugs , identification of rational / irrational fdcs , and adr reporting were not followed .
a significant proportion of ugs and interns agreed being taught the rational use of drugs and cpt in 2 year mbbs . despite this , half of them rated their knowledge as good and half as average and poor .
moreover , the ug knowledge was significantly reduced by the time the student completes internship .
this finding indicates that knowledge was not retained until internship and in fact , it was significantly reduced as compared to ugs .
the knowledge questions were pertaining to common clinical problems such as malaria and tuberculosis , identifying rational / irrational fdcs , etc .
, this observation is alarming and indicates that classroom teaching of clinical pharmacology and therapeutics is not sufficient but needs to be aligned with clinical teaching during mbbs and reinforced during internship .
although p - drug along with evaluation of fdcs and adr reporting was considered to be most useful exercises among ugs , it was seldom followed in clinical practice .
probably , this exercise involved the active participation of students in practical class and brought them close to real life situation .
however , application and implementation of these p - drug concept and rational fdc in internship was lacking .
exercise on the selection of p - drug improves the performance of the students in clinical pharmacology case report .
evaluations of fdcs are important in country like india where a good number of irrational fdcs are aggressively promoted and freely available in the indian market . moreover , substantial number of ugs and interns believed that adr reporting is important , but it was not reported or witnessed in internship .
conversely to our observation , a higher rate of adr reporting among interns has been observed by tobaiqy et al .
this can be attributed to the lack of spontaneous adr reporting by clinicians and lack of awareness in spite of increase efforts by pharmacovigilance programme of india .
if adr reporting is taught in cpt , but not preached by doctors , the students will not form a habit and the impact will be is less or negligible .
this indicates need to increase awareness of adr reporting and surveillance among the health - care professionals .
comparison of practices of clinical pharmacology and rational therapeutics among interns with other studies interestingly , textbook and internet were the most common source of drug information .
this is similar to bangladesh study where interns rely on text books ( 72% vs. 43% ) .
interestingly , oshikoya et al . showed that interns use national formulary as most common source for drug information [ table 3 ] .
this is an important step to reduce prescribing errors and adrs , while this study showed that interns also relied on senior colleagues and mr for source of drug information .
this is alarming and can be attributed to their clinical exposure , observation of day - to - day practices followed by health - care professionals in hospital setup and influence of external environment on their practice .
a substantial number of interns perceived themselves sufficiently prepared to prescribe a variety of drugs unsupervised .
however , these findings did not correlate with their cpt knowledge which was reduced to a great extent .
it seems that the selection of drugs reflects their observation of most commonly prescribed drugs during clinical rotation rather than their actual knowledge of drug safety .
, who also found that interns were confident in prescribing laxatives and analgesics including opioids and nonopioids , and antacids .
islam et al . have reported that interns were confident in prescribing vitamins , drugs for peptic ulcer , and anti - histaminics .
showed that interns were confident in prescribing anti - malarials , vitamins , and antibiotics [ table 3 ] .
these findings show the fresh medical graduates develops the general perception about safe drugs from day - to - day prescribing practices and over prescribing rather than a true appreciation of risk - benefit ratio .
however in this study , interns were hesitant to prescribe opioids , corticosteroids , anti - hypertensives , and oral contraceptive pills .
probably this was due to high prevalence adrs and abuse liability associated with these drugs .
around 63% of interns found problem in prescribing like drug interaction , and selection of drug . they were confident in prescribing in adult and elderly but not in patient with liver disease and renal disease .
this indicates that interns are not confident prescribing in special conditions , especially where the selection of drug and dose is difficult and requires sound knowledge .
it can be stated that student must be taught clinical pharmacology by various case base scenarios which focus on this special condition .
for example , it was a single center study ; the prescribing skills were assessed using written case scenario and not actually observing interns in simulated cases or clinical setup . in addition , the confidence of unsupervised prescribing was self - rated and may not necessarily be translated into actual rational practical prescribing .
the variation in the background knowledge , clinical exposure , and activities of different ugs and interns are also likely to affect their perceptions of cpt teaching . however , the data generated lead to some important conclusion and useful information for undertaking corrective measures for improving prescribing skills .
the class room and sequential cpt teaching , where the learning and the application of knowledge are separate is not sufficient to make ugs and interns rational prescribers .
gaining knowledge and simultaneously allowing students to put into practice with real patients under appropriate supervision is essential for learning complex task of prescribing skill .
this would substantially allow students to work in context and carry out prescribing skills and prepare better for future practice .
it can be concluded that theoretical and sequential cpt teaching do not adequately prepare ugs and interns to prescribe safe and rational drugs .
there is a need to strength cpt teaching by providing opportunities to practice practical skills during training and reinforce principles of cpt in internship . | objectives : to find out the impact of teaching clinical pharmacology and rational therapeutics ( cpt ) to medical undergraduates ( ugs ) and interns.materials and methods : this cross - sectional , prospective study was conducted on three ugs batches and interns using two pretested validated structured questionnaires , modified from the work of tobaiqy et al .
the study was approved by the institutional ethics committee .
anova and chi - square test were used for statistical analysis . the value of p < 0.05 was considered statistically significant.results:a total of 379 ugs and 96 interns participated in this study .
mean knowledge score of interns was significantly reduced as compared to ugs ( p < 0.0001 ) . a significant increase in confidence for unsupervised prescribing of nonsteroidal anti - inflammatory drugs ( 99% ) , oral rehydration salt ,
iron salts was perceived among interns as compared to ugs ( p < 0.05 ) . however , 63.5% confessed problems in selection of drugs , drug drug interactions , prescribing in special patient population .
although they were confident prescribing fixed dose combination for adult patients ( 89.5% ) , majority were hesitant to prescribe opioids ( 77% ) , steroids ( 76% ) , vaccines ( 75% ) , and antihypertensives ( 62%).conclusion : the theoretical cpt teaching transfers knowledge to ugs ; however , it is not retained in internship and does not adequately prepare interns to prescribe safe and rational drugs . | I
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Undergraduates
Second year MBBS III term (II/III, 5
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Conflicts of interest | the present educational research was undertaken to find out the impact of teaching clinical pharmacology and rational therapeutics ( cpt ) with respect to the knowledge and attitude of ug medical students and interns and to determine whether teaching cpt has prepared fresh medical graduates ( interns ) to prescribe safely and rationally . a prospective , cross - sectional study was conducted on medical ugs and interns at b. j. medical college , ahmedabad . the study was approved by the institutional ethics committee and prior informed consent was obtained ( reference no . whereas the intern s questionnaire had questions related to prescribing practices , especially their confidence in prescribing selected drugs , prescribing in special patient population , specific problems encountered while prescribing , commonly prescribed fdcs , adr observed and reported , etc . of 124 iii / i and 134 iii / ii students , only 2 ( 1.6% ) and 45 ( 33.5% )
however , majority of iii / i ( 66 , 53.2% ) and iii / ii ( 84 , 62.6% ) could not identify ddi and only 46 ( 37% ) iii / i and 50 ( 37.3% ) iii / ii students could identify two out of four irrational fdcs . a significant reduction in knowledge score was observed ( 4.73 2.3 , p < 0.0001 ) as compared to ugs [ table 1 and figure 3 ] . moreover ,
majority of interns ( 86 , 89.5% ) considered adr reporting is important ; however , 59 ( 61.45% ) interns stated only serious adr should be reported while 20 ( 20.8% ) interns believed that adrs related to the new drug should be reported . surprisingly , the internet significantly ( 72 , 75% ) topped the list for seeking drug information followed by textbooks ( 70 , 72.9% ) by interns as compared to ugs ( p < 0.05 ) [ figure 3 ] . interestingly , majority of interns ( 61 , 63.5% ) confessed prescribing problems in special group of patients ( 34 , 35.4% ) and selection of drug ( 32 , 33.3% ) . although majority agreed to be confident in prescribing for adult patients ( 86 , 89.5% ) , they were hesitant to prescribe opioids ( 74 , 77% ) , steroids ( 73 , 76% ) , anti - hypertensives ( 60 , 62.5% ) . prescribing practices of fixed dose combination among interns ( n=96 ) majority of ug students ( 75% ) and interns emphasized teaching on drug selection ( 49% ) followed by dosage schedule ( 45% ) and drug interaction ( 38.25% ) , while 61% of interns emphasized teaching on dosage schedule followed by prescribing in special patient population ( 51% ) and drug selection ( 47% ) . assessment of clinical pharmacology and rational therapeutics knowledge among undergraduates students ( n=379 ) out of six clinical pharmacology exercises , p - drug ( 80 , 66.1% ) was considered to be most useful followed by adr reporting ( 39 , 32.2% ) among ii mbbs students [ figure 1 ] . a significant reduction in knowledge score was observed ( 4.73 2.3 , p < 0.0001 ) as compared to ugs [ table 1 and figure 3 ] . moreover , majority of interns ( 86 , 89.5% ) considered adr reporting is important ; however , 59 ( 61.45% ) interns stated only serious adr should be reported while 20 ( 20.8% ) interns believed that adrs related to the new drug should be reported . surprisingly , the internet significantly ( 72 , 75% ) topped the list for seeking drug information followed by textbooks ( 70 , 72.9% ) by interns as compared to ugs ( p < 0.05 ) [ figure 3 ] . interestingly , majority of interns ( 61 , 63.5% ) confessed prescribing problems in special group of patients ( 34 , 35.4% ) and selection of drug ( 32 , 33.3% ) . although majority agreed to be confident in prescribing for adult patients ( 86 , 89.5% ) , they were hesitant to prescribe opioids ( 74 , 77% ) , steroids ( 73 , 76% ) , anti - hypertensives ( 60 , 62.5% ) . prescribing practices of fixed dose combination among interns ( n=96 )
majority of ug students ( 75% ) and interns emphasized teaching on drug selection ( 49% ) followed by dosage schedule ( 45% ) and drug interaction ( 38.25% ) , while 61% of interns emphasized teaching on dosage schedule followed by prescribing in special patient population ( 51% ) and drug selection ( 47% ) . in view of the above
, this cross - sectional study was conducted among ug students and interns who learnt cpt based on who model along with other clinical pharmacology exercises in 2 year mbbs . the objective of this study was to find out the impact of teaching and its retention in fresh medical graduates . this finding indicates that knowledge was not retained until internship and in fact , it was significantly reduced as compared to ugs . however in this study , interns were hesitant to prescribe opioids , corticosteroids , anti - hypertensives , and oral contraceptive pills . it can be concluded that theoretical and sequential cpt teaching do not adequately prepare ugs and interns to prescribe safe and rational drugs . | [
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the japanese traditional herbal medicine , kampo , is a form of pharmacological therapy that originated in medieval china and was developed further in japan .
treatment with kampo means a possibility for combination therapy with modern western and traditional asian medical practices .
most such traditional medicines include various components , and they are often prescribed for patients with medically unexplained physical symptoms .
however , the detailed mechanism(s ) of the pharmacological action of kampo medicines is yet unknown .
consequently , a standardized educational system of these medicines has not been fully established until recently for medical students or trainees . in other words ,
the japanese ministry of health and welfare has approved the use of many kampo medicines as ethical medicines based on the symptoms of the respective patients .
therefore , therapeutic strategies that use kampo medicines based on proven evidence have yet to be established . hence , kampo therapy is referred to as experience - based medicine and not evidence - based medicine , even though these medicines are prescribed in most clinical fields in japan
. however , extensive evidence for the clinical efficacies or pharmacological mechanism(s ) of kampo medicines has gradually accumulated over past decades .
this review focuses on the basic and clinical evidence for efficacies of kampo medicines in treating various disorders of the gastrointestinal tract ( gi ) tract and introduces the current novel insights in this field ( figs . 1 , 2).fig .
1scheme of the pharmacological actions and functional site of kampo medicines for the upper gi tractfig .
2scheme of the pharmacological actions and functional sites of kampo medicines for the middle and lower gi tracts scheme of the pharmacological actions and functional site of kampo medicines for the upper gi tract scheme of the pharmacological actions and functional sites of kampo medicines for the middle and lower gi tracts
recently , an increase in the prevalence of gerd has been reported in japan and in western countries owing to factors such as obesity associated with the intake of western food , a decrease in the helicobacter pylori infection rate , and increased prevalence of h. pylori eradication therapy [ 14 ] .
the primary pathophysiology of gerd is the reflux of gastric contents , including gastric acid .
esophageal motor dysfunctions such as an increase in transient lower esophageal sphincter relaxation ( tlesr ) are also associated with the occurrence of gerd .
other factors contributing to the pathophysiology of gerd include esophageal peristalsis , mucosal hypersensitivity at the bottom of the esophagus to reflux contents such as gastric acid or bile acid , or disorders of gastric motility ( gastric reservoir function or emptying ) .
hence , the inhibition of acid secretion by using proton pump inhibitors ( ppis ) has been suggested as the most effective therapeutic strategy for gerd .
however , certain agents that regulate the physiological functions of the esophagus , e.g. , its motor functions , may also prove useful in gerd treatment
, glycyrrhizae radix ( 4.7 % ) , zingiberis rhizoma ( 2.3 % ) , atractylodislanceae rhizoma ( 18.6 % ) , zizyphi fructus ( 9.3 % ) , aurantiinobilis pericarpium ( 9.3 % ) , ginseng radix ( 18.6 % ) , pinelliae tuber ( 18.6 % ) , and hoelen ( 18.6 % ) , and is widely prescribed for patients with upper gi symptoms .
basic studies show that rikkunshito exhibits a pharmacological action similar to that of prokinetic agents , as shown by its attenuation of gastric dysmotility induced by a nitric oxide - synthesizing enzyme inhibitor [ 9 , 10 ] .
in addition , rikkunshito improves the delay of gastric emptying mediated by serotonin ( 5-ht ) type 3 receptor but not dopamine receptors .
conversely , rikkunshito increases plasma ghrelin levels , and regulates motility at the esophagogastric junction [ 12 , 13 ] .
ghrelin is known to have a strong orexigenic effect [ 14 , 15 ] and enhances gi motility [ 1618 ] .
nahata et al . showed impairment of gi motility in a rat gerd model via dysfunction of ghrelin signaling and that rikkunshito restored gi motility by improving the ghrelin response .
these data indicate the possible efficacy of rikkunshito in the treatment of gerd owing to its pharmacological action on motor functions throughout the upper gi tract .
reported that rikkunshito improved nausea symptoms in children with gerd by increasing esophageal clearance and decreasing esophageal acid exposure time for the esophagus from a 24-h ph monitoring study .
rikkunshito is also effective for the relief of reflux symptoms such as heartburn and acid regurgitation .
acid secretion inhibitors such as ppis are the first - choice agents for gerd treatment ; however , we often encounter ppi - refractory gerd adult patients .
thus , antacid agents may not always be effective for gerd , especially in the case of a patient with no erosions on the esophageal mucosa ( non - erosive reflux disease : [ nerd ] ) . in our recent report of a randomized , parallel comparative study of ppi - refractory gerd patients
, we stated that rikkunshito combined with standard - dose rabeprazole therapy shows clinical efficacy similar to that of double - dose ppi . in subgroup analyses of this study ,
the effectiveness of rikkunshito was more pronounced in male nerd patients , particularly leptosomatic nerd patients . by using a rat reflux esophagitis model , miwa et al
. showed that rikkunshito improves acid regurgitation - associated circumstances by promoting tight junction protein formation and suppressing the dilation of intercellular spaces in the epithelial mucosa .
in addition to gastric acid reflux , bile acid reflux to the bottom part of the esophagus may also be an important factor in the pathogenesis of mucosal hypersensitivity leading to ppi - refractory gerd .
recently , araki et al . showed that rikkunshito exhibits potent and differential absorption of bile salts .
correctively , these findings suggest that rikkunshito may have the potential for improving hypersensitivity to gastric acid or bile acid at the bottom part of the esophagus .
these pharmacological functions of rikkunshito different from motor function may suggest other explanations why it relieves the symptoms of ppi - refractory gerd patients , especially for nerd .
currently , double - dose ppi and long - term maintenance therapy is the standard therapeutic strategy for such ppi - refractory gerd patients ; however , a new regimen that includes rikkunshito should be considered for gerd patients including ppi - refractory cases .
acute inflammation of the stomach is often caused by factors such as non - steroidal anti - inflammatory drugs ( nsaids ) , ethanol , systemic stress , or h. pylori infection , all of which may cause mucosal injuries [ 2628 ] .
acid secretion inhibitors , such as ppis and histamine type 2 receptor antagonists ( h2ras ) , or mucoprotective agents , such as rebamipide and teprenone , are effective for treating mucosal injuries [ 29 , 30 ] . aside from these drugs ,
reported that hangeshashinto and shosaikoto inhibited ethanol - induced gastric mucosal injuries in a dose - dependent manner by eliminating oxygen radicals .
hangeshashinto is also reported to elevate secretion of mucin , which protects against acute gastric mucosal injuries .
hangeshashinto improves mucosal injuries caused by water - immersion stress and an acute mucosal injury model ; however , shosaikoto did not show this property .
this disparity in their effects may be due to differences in the respective components of these medicines .
the effect of rikkunshito is mediated by the inhibition of decrease in mucin content of the gastric mucosa , and this effect is in part mediated by nitric oxide ( no ) but not prostaglandins or sulfhydryls .
thus , these findings suggest that both hangeshashinto and rikkunshito exert protective effects against acute mucosal injuries in the stomach of rats
. chronic gastritis , which is characterized by chronic infiltration of inflammatory cells , hypertrophy or atrophy of the gastric glands , or intestinal metaplasia , has been proved to be caused by persistent h. pylori infection .
therefore , chronic gastritis is also referred to as histological gastritis , the standard treatment for which includes triple antimicrobial therapy .
however , some traditional herbal medicines have shown evidence of antimicrobial efficacy against h. pylori .
ishii reported that 104 of 109 kinds of medicinal plants and 28 types of spices had anti - h .
gosyuyuto has four constituents ; evodia fructus , zizyphi fructus , zingiberis rhizoma , and ginseng radix . in a clinical study report , higuchi et al .
reported that gosyuyuto combined with omeprazole and amoxicillin yields a higher eradication rate ( 84 % ) than dual therapy ( 59 % ) without causing any significant adverse effect .
moreover , hamasaki et al . analyzed the components of gosyuyuto and reported that two quinolone compounds ( am quinolones ) , 1-methyl-2-[(z)-8-tridecenyl]-4-(1h)-quinolone and 1-methyl-2-[(z)-7-tridecenyl]-4-(1h)-quinolone , which were purified from the evodia fructus have strong and selective antibacterial activity against the reference strains and isolated strains of h. pylori from clinical patients .
further , in vitro studies revealed that the antibacterial effect of these two am quinolones against h. pylori is mediated by the inhibition of respiration and not dna synthesis , and in vivo studies revealed that they significantly decreased the number of viable h. pylori in the stomachs of h. pylori - infected mongolian gerbils and reduced neutrophil infiltration without causing harmful adverse events , including animal mortality .
the agents with a unique antimicrobial mechanism(s ) mediated by the inhibition of bacterial respiration , i.e. , mechanisms different from that of antibiotics such as amoxicillin , clarithromycin , or metronidazole , may be beneficial for the improvement of h. pylori - associated gastric diseases , whether they are used alone or in combination with the above mentioned antibiotics and ppis .
thus , there are some candidates among traditional medicines to treat or protect against gastric mucosal injuries caused by the above various mechanism(s ) , including h. pylori infection .
another form of chronic gastritis in japan is a symptomatic gastritis called functional dyspepsia ( fd ) , and it is classified as a functional gi disorder ( fgid ) . according to the rome iii criteria published in 2006 , the diagnosis of fgids is based on the following conditions : patients exhibit symptoms originating from the gi tract without evident organic disease , and these bothersome symptoms persist even after various treatments , thus decreasing their quality of life ( qol ) .
the pathophysiology of fd is characterized by multifunctional disorders of the upper gi tract , e.g. , disorders of gi motility , abnormal acid secretion , visceral hypersensitivity , h. pylori infection , psychological factors [ 44 , 45 ] , and imbalance of the autonomic nervous system ( ans ) . of these factors ,
most studies mainly focus on the disorders of gi motility , because they are closely related to the pathogenesis of fd . in general ,
while previous reports have mainly focused on the delay in gastric emptying , it is necessary to consider the total coordination of these phases for the better understanding of fd .
gastric accommodation , a reservoir function , originates from the proximal stomach and is important for the physiological function of the stomach .
neurological transmitters such as no that are essential for this function have already been elucidated [ 48 , 49 ] .
impairment of gastric accommodation has also been reported to cause clinical symptoms such as epigastric discomfort , early satiety , and bloating of fd patients .
furthermore , we have previously reported that the impairment of reservoir function is associated with delay in gastric emptying , which results in dyspeptic symptoms .
thus , drugs that can improve the impairment of reservoir function of the stomach are desirable . with regard to traditional medicines ,
hayakawa et al . reported that , in isolated guinea pig stomachs , rikkunshito promotes adaptive relaxation , which means gastric accommodation .
rikkunshito also induces the relaxation of gastric fundus smooth muscles isolated from diabetic neuropathic rats with gastric dysmotility .
in addition , rikkunshito can improve gastric dysmotility mediated by no or 5-ht3 receptor pathways [ 10 , 11 ] . considering this basic evidence
, rikkunshito may be a promising drug for the treatment of fd in the clinical field .
unfortunately , an appropriate treatment regimen for fd has not yet been established , even though a variety of clinical trials considering the above pathogenesis have been conducted in japan . therefore , treatment may be empirically adopted for fd patients based on the predominantly suspected pathogenesis related to respective symptoms of fd .
there is some evidence for the effectiveness of kampo medicines in the treatment of fd under such conditions . when fd was still called non - ulcer dyspepsia ( nud ) ,
illustrated the efficacy of rikkunshito on gastric emptying in patients with nud , which supports the data shown in the above basic studies .
interestingly , in addition to this effect on gastric motility , rikkunshito also improved gi symptoms in patients with nud . to our knowledge , this is the first report in the english literature that describes rikkunshito as a promising drug for the improvement of symptoms of fd patients .
kusunoki et al . showed that rikkunshito may be beneficial for the treatment of fd patients with impaired gastric accommodation and gastric motility evaluated by the extracorporeal ultrasonography method . on the other hand
, physical and psychological stress often causes gastric hypersensitivity to stimulation by mechanical balloon distension , which is generally well known as a pathogenesis of fd .
shiratori et al . showed that rikkunshito may improve stress - induced gastric hypersensitivity and/or changes in gastric wall tone by using gastric barostat method .
one report assessed the efficacy of some herbal medicines under the condition of venipuncture stress .
rikkunshito and hangeshashinto but not hangekobokuto or ninjinto reversed the increase in plasma levels of neuropeptide y , a representative neurotransmitter of the sympathetic nervous system of ans , to the control levels .
in addition , administration of rikkunshito decreases the afferent activity of the gastric vagal nerve , but increased the efferent activities of the gastric branches of the vagal nerve . from these findings with the fact that activity of sympathetic nervous system in the ans of fd patient was relatively higher compared to that of the parasympathetic nervous system
, the above might be another pharmacological action of rikkunshito for the improvement of dyspeptic symptoms . according to another comparative human study , rikkunshito but
not domperidone also improves symptoms of patients with fd in parallel with plasma ghrelin levels .
rikkunshito also has an impact on delayed gastric emptying in severely handicapped patients and on the coordination of the gastric myoelectric activity in post - operative dyspeptic children after gi surgery .
these findings suggest that rikkunshito improves dyspeptic symptoms mediated by regulating gastric sensorimotor function or some inflammatory and/or neuroendocrinal mediators .
this efficacy is mediated by a reduction of gas volume in parallel with the improvement of symptoms of abdominal pain , indigestion , and constipation
. thus , there are some candidates among various japanese traditional medicines for the treatment of both histological and symptomatic gastritis .
the physiological function of the small intestine mainly comprises ingestion and absorption of foods throughout gi transit .
therefore , a regulated intestinal motility is important to maintain its integrity and achieve these functions .
however , disorders of intestinal motility often occur under some pathological conditions due to surgical operation and abdominal irradiation for treating some neoplasms , the unfavorable conditions of which lead to serious symptoms .
therefore , some effective drugs including japanese traditional medicines that regulate intestinal motility are needed in the clinical field .
apart from rikkunshito , a variety of reports have documented the effectiveness of daikenchuto in such cases .
daikenchuto is composed of zanthoxyli fructus , ginseng radix , zingiberis siccatum rhizoma , and maltose syrup powder and generally accelerates intestinal motility in various animal models of intestinal dysmotility , viz . , mouse [ 62 , 63 ] , rat [ 6466 ] , guinea pig [ 67 , 68 ] , rabbit , or dog [ 70 , 71 ] .
the pharmacological functions of this drug are usually mediated by an increase in acetylcholine release through the vagal nerve , cholinergic neuron , or 5-ht3 and/or 5-ht4 receptors [ 6467 , 7072 ] .
another report showed that a possible role of daikenchuto in postoperative adhesive obstruction via transient receptor potential vanilloid type 1 channel .
in addition , daikenchuto exhibit anti - inflammatory activity in rat intestinal mucosal injuries , mediated by decreased expressions of interleukin-1 and interferon ( ifn)- and in bacterial translocation models , mediated by reducing inflammatory reaction and maintaining intestinal integrity .
further examinations showed that the main component exerting this pharmacological function is hydroxy sanshool , one of the chief constituents of zanthosyli fructus included in daikenchuto [ 66 , 6971 , 76 ] .
these findings from various basic studies correctively suggest that daikenchuto may be useful for the treatment of post - operative adhesive obstructions or ileus . in clinical studies ,
. showed that daikenchuto led to an increase in intestinal motility in a randomized placebo controlled study in healthy humans .
another study with human subjects suggested that the effect of daikenchuto on intestinal transit may be mediated by the releases of motilin and vasoactive intestinal peptide ( vip ) into the plasma through muscarinic receptor 1 .
daikenchuto is reported to improve the stasis of patients with total gastrectomy and jejunal pouch interposition , decrease the prevalence rate of re - operation associated with ileus after abdominal surgery , and attenuate radiation - induced enteritis .
thus , it was concluded that the pharmacological action of this drug is mainly mediated by improvement of intestinal motility .
interestingly , daikenchuto is also reported to increase human portal blood flow mediated by an increase in the intestinal blood flow without an increase in the cardiac output [ 82 , 83 ] .
it is also confirmed that an increase in intestinal blood flow by daikenchuto is mainly mediated by calcitonin gene - related peptide ( cgrp ) .
thus , japanese traditional medicines could regulate the physiological function of the small intestine , particularly , intestinal motility , although definitive evidence is limited only to daikenchuto at present .
colonic transit in cooperation with mucus secretions plays an important role in the regulation of fecal conditions , such as constipation and diarrhea , after ingestion of food intake .
constipation and diarrhea are usually recognized as the common complaints but are not associated with severe life - threatening diseases in the general clinical field .
it is also well known that there seems to be a high prevalence of patients with these symptoms , including those individual who have never visited any medical institutions despite having these unfavorable conditions .
in addition to the efficacy of japanese traditional medicines for intestinal contraction , some of these medicines can also regulate colonic motility and improve fecal conditions and their related symptoms . in basic studies ,
daikenchuto has been found to exhibit prokinetic effects on the total gi tract , including the stomach , duodenum , jejunum , ileum , and colon during the fasting state .
however , this effect was attenuated during the fed state when daikenchuto was administered orally .
conversely , intracolonic administration of daikenchuto induced a colonic contraction similar to that during both the fasting and the fed states , and the related defecation occurred despite the differences in administration site and timing . based on these findings
, the focal administration of this drug may be optimal for the treatment of intestinal obstruction , such as postoperative ileus .
moreover , daikenchuto improves the opioid - induced disorders of the colonic motility , which is similar to that seen by naloxone . as a result , wood et al
. concluded that daikenchuto may have a beneficial effect on the restoration of colonic motility in postoperative patients with opiate analgesic therapy .
human studies revealed that the prokinetic effect of daikenchuto on colonic motility was mediated by increase in the levels of plasma motilin , vip , and 5-ht [ 87 , 88 ] . on the other hand , kono et al .
showed that , in rats , daikenchuto can also increase blood flow in the colon via cgrp and receptor - activity modifying protein 1 . in another human study ,
increase in the blood flow by daikenchuto was mediated by increase in the levels of plasma cgrp and substance p . in a clinical study with healthy volunteers , occurrence of colonic motility
was immediately confirmed by extrasomatic ultrasonography for 30 min after administration of daikenchuto into the ascending colon .
however , no colonic contractions were observed in the right colon after administration of saline solution alone . in patients hepatectomized by laparotomy
, paralytic ileus occasionally occurs as a complication during the early postoperative period , which often causes post - resection liver failure due to hyperammonemia .
daikenchuto can prevent such unfavorable condition by improvement of delayed flatulence and hyperammonemia , and also accelerate the times to initial flatus and tolerance of regular diet by increasing bowel movement in patients after colorectal surgery .
thereafter , it has been confirmed that daikenchuto improves some disorders of the colon such as obstructive bowel disease or anorectal dysfunction with severe constipation in children [ 95 , 96 ] , and constipation in patients with parkinson s disease .
based on these findings , daikenchuto has been recognized as a prokinetic agent that is effective in the treatment of constipation and postoperative motility disorders . furthermore , daikenchuto exhibits anti - inflammatory effects . in a representative experimental colitis model induced by trinitrobenzene sulfonic acid ( tnbs ) ,
furthermore , daikenchuto inhibits mucosal injuries and adhesion of the colonic serosa caused by focal inflammation and decreases cytokine levels of tumor necrosis factor and ifn- via the same mechanism . as the pathophysiology of irritable bowel syndrome ( ibs ) , low grade inflammation of the intestine
therefore , both regulation of colonic motility and inflammation are important for the therapeutic strategy .
intestinal muscle hypercontractility associated with inflammation in a t cell - mediated model of enteropathy was ameliorated by daikenchuto , which indicated that this pharmacological function was mediated by decrease in pro- and anti - inflammatory cytokines .
therefore , the possibility of daikenchuto for treating patients with ibs was suggested . as another function of daikenchuto , inoue et al . reported the regulation of intestinal fibrosis associated with decreasing expression of heat shock protein 47 and collagen content in this model .
this disease often impairs the qol of the affected individual . unfortunately , there is no definite therapeutic rationale for this disease .
however , one case report states that daikenchuto has the potential to manage radiation - induced enteritis perhaps owing to its anti - inflammatory action . in a retrospective observational clinical study ,
daikenchuto reduced the duration of long - tube decompression and saved costs by prevention of postoperative adhesive small bowel obstruction , which is a major complication of abdominal surgery .
the comparative study showed that daikenchuto lowered the inflammatory responses associated with laparoscopic colorectal resection and shortened the time to first flatus .
thus , daikenchuto may be useful as a prokinetic and anti - inflammatory drug under various pathological conditions .
further , hangeshashinto has a potent and special effect on the lower intestinal tract . through a series of pharmacological studies of hangeshashinto for colonic functions , kase et al .
firstly , they showed the dose - dependent effects of this drug via an increase in water absorption in the colon but not inhibition of colonic transit in a castor oil - induced diarrhea model [ 104 , 105 ] .
the other mechanism(s ) of anti - diarrheal effects of hangeshashinto was proved by increases in serum corticosterone levels and fluid secretion .
it was also confirmed that a decrease in prostaglandin e2 contents associated with anti - diarrheal effects was caused by an inhibition of cyclooxygenase type 2 .
in addition , there are some reports on the association between hangeshashinto and gi motility regulatory peptides ( somatostatin , motilin , gastrin , and vip ) or neuropeptides ( 5-ht , cgrp , and substance p ) .
hangeshashinto increases the plasma levels of somatostatin , motilin , gastrin , cgrp , and substance p , but did not have an effect on the levels of 5-ht , mineral balance , or aldosterone [ 105110 ] .
hangeshashinto also improved macroscopic injuries and body weight loss in a tnbs - induced colitis model .
unfortunately , thus far , pharmaceutical evidence for hangeshashinto might be derived only from these complicated interactions .
however , hangeshashinto also has the potential to maintain the integrity of the gi tract , including the improvement of clinical colitis .
in the clinical field , most clinicians often encounter various patients with general malaise and have trouble treating these patients in coordination with the severity of their symptoms .
general malaise includes anorexia , headache , dizziness , nausea , emesis , discomfort , and diarrhea .
furthermore , because the qol of these patients lowers drastically with the severity of their malaise , clinicians need to improve their symptoms as soon as possible . in such cases ,
kampo medicines are used more frequently than the usual common drugs as comprehensively alternative medicines , because it is well known that kampo medicines can affect the physiological as well as the mental conditions .
therefore , kampo therapy may be the logically correct strategy for treatment in patients with numerous general malaise .
however , it is difficult to establish the basic and clinical evidence for kampo medicines in the treatment of general malaise . among various pathological conditions
, it is well known that systemic chemotherapy using anti - cancer drugs causes symptoms similar to general malaise including anorexia , emesis , or diarrhea , which sometimes lead to serious life - threatening conditions .
for instance , irinotecan hydrochloride ( cpt-11 ) is often effectively used to treat cancers of the lung , colon , or ovarian cancers but can cause severe diarrhea as an adverse event .
hangeshashinto prevented cpt-11-induced symptoms such as diarrhea , anorexia , and body weight loss in rats , without a loss of its antitumor effects [ 112 , 113 ] .
in the same model , hangeshashinto led to an increase in water absorption in the colon , mediated by an inhibition of prostaglandin e2 synthesis .
another clinical study confirmed the preventive effect of hangeshashinto toward cpt-11-induced diarrhea in patients with advanced non - small - cell lung cancer .
we believe that , in the current literature , there is some evidence supporting the efficacy of this agent in patients with gastric or colon cancers . apart from diarrhea
however , only a limited number of agents that can effectively increase food intake have been established .
some reports show that rikkunshito can regulate some humoral factors , including gut regulatory peptides ( somatostatin , gastrin , vip , or cgrp ) [ 110 , 116 ] , and improve stress - associated diseases by decreasing the levels of adrenocorticotropic hormone or cortisol . considering these effects of rikkunshito and its efficacy in patients with fgids
recently , it has been proved that rikkunshito can increase food intake and improve anorexia under unfavorable condition by increasing in plasma ghrelin levels .
first , takeda et al . showed that rikkunshito suppresses cisplatin - induced anorexia in rats via 5-ht2 receptor antagonism .
thereafter , yakabi et al . [ 13 , 118 ] showed that rikkunshito led to improvement for the reduced secretion of ghrelin in the hypothalamus and resulted in the improvement of cisplatin - induced anorexia .
another report states that a component of rikkunshito ( 10-gingerol ) improves cisplatin - induced anorexia by inhibiting acylated ghrelin degradation .
basic research shows that rikkunshito suppresses anorexia , emesis , or decrease in qol under the condition of systemic chemotherapy with cisplatin etc . by increasing the secretion of plasma ghrelin [ 120 , 121 ] .
therefore , some kampo medicines seem to be more and more useful for treatment for general malaises in the future .
the japanese medical treatment kampo is frequently prescribed for patients with numerous gi tract disorders associated with various symptoms , including general malaise .
this treatment should be recommended theoretically , because it combines modern western and traditional asian medical practices .
however , kampo medicines are not used widely worldwide and are considered complementary medicines and not mainstream .
this is because the mechanism of their pharmacological actions is not so clear and because of the lack of established kampo educational system in japan .
it is suggested that kampo medicines must be developed to the evidenced - based medicine from the empirical - based medicine in the near future by making known their much more benefits to the whole world . | treatment with kampo , the japanese traditional medicine , is a form of pharmacological therapy that combines modern western and traditional asian medical practices . in japan ,
various traditional medicines are often combined with western medicines and prescribed for patients with diseases such as gastroesophageal reflux disease , functional dyspepsia , chronic gastritis , irritable bowel syndrome , and post - operative ileus .
based on numerous past observations , japanese traditional medicines are thought to be particularly useful in the treatment of medically unexplained physical symptoms such as nausea , abdominal discomfort , and anorexia . however , the detailed mechanism by which they mediate their pharmacological action is yet unknown .
in addition , the clinical evidence to support their use is insufficient .
this review focuses on the basic evidence of the pharmacological action and the clinical efficacies of kampo medicines accumulated over several past decades .
in addition , we introduce both the current novel insights into kampo medicines and the therapeutic approach employed when they are used to treat various disorders of the gastrointestinal tract . | Introduction
Gastroesophageal reflux disease (GERD) associated with esophageal motor function and visceral hypersensitivity
Mucosal injuries and histological gastritis (
Functional dyspepsia (FD)
Motility disorders and inflammations of the small intestine
Colonic motility, secretions, and inflammation-colitis and irritable bowel syndrome
Miscellaneous: supportive care during systemic chemotherapy
Concluding remarks | the japanese traditional herbal medicine , kampo , is a form of pharmacological therapy that originated in medieval china and was developed further in japan . treatment with kampo means a possibility for combination therapy with modern western and traditional asian medical practices . most such traditional medicines include various components , and they are often prescribed for patients with medically unexplained physical symptoms . however , the detailed mechanism(s ) of the pharmacological action of kampo medicines is yet unknown . in other words ,
the japanese ministry of health and welfare has approved the use of many kampo medicines as ethical medicines based on the symptoms of the respective patients . however , extensive evidence for the clinical efficacies or pharmacological mechanism(s ) of kampo medicines has gradually accumulated over past decades . this review focuses on the basic and clinical evidence for efficacies of kampo medicines in treating various disorders of the gastrointestinal tract ( gi ) tract and introduces the current novel insights in this field ( figs . 1scheme of the pharmacological actions and functional site of kampo medicines for the upper gi tractfig . 2scheme of the pharmacological actions and functional sites of kampo medicines for the middle and lower gi tracts scheme of the pharmacological actions and functional site of kampo medicines for the upper gi tract scheme of the pharmacological actions and functional sites of kampo medicines for the middle and lower gi tracts
recently , an increase in the prevalence of gerd has been reported in japan and in western countries owing to factors such as obesity associated with the intake of western food , a decrease in the helicobacter pylori infection rate , and increased prevalence of h. pylori eradication therapy [ 14 ] . , its motor functions , may also prove useful in gerd treatment
, glycyrrhizae radix ( 4.7 % ) , zingiberis rhizoma ( 2.3 % ) , atractylodislanceae rhizoma ( 18.6 % ) , zizyphi fructus ( 9.3 % ) , aurantiinobilis pericarpium ( 9.3 % ) , ginseng radix ( 18.6 % ) , pinelliae tuber ( 18.6 % ) , and hoelen ( 18.6 % ) , and is widely prescribed for patients with upper gi symptoms . chronic gastritis , which is characterized by chronic infiltration of inflammatory cells , hypertrophy or atrophy of the gastric glands , or intestinal metaplasia , has been proved to be caused by persistent h. pylori infection . another form of chronic gastritis in japan is a symptomatic gastritis called functional dyspepsia ( fd ) , and it is classified as a functional gi disorder ( fgid ) . according to the rome iii criteria published in 2006 , the diagnosis of fgids is based on the following conditions : patients exhibit symptoms originating from the gi tract without evident organic disease , and these bothersome symptoms persist even after various treatments , thus decreasing their quality of life ( qol ) . impairment of gastric accommodation has also been reported to cause clinical symptoms such as epigastric discomfort , early satiety , and bloating of fd patients . considering this basic evidence
, rikkunshito may be a promising drug for the treatment of fd in the clinical field . there is some evidence for the effectiveness of kampo medicines in the treatment of fd under such conditions . therefore , some effective drugs including japanese traditional medicines that regulate intestinal motility are needed in the clinical field . thus , japanese traditional medicines could regulate the physiological function of the small intestine , particularly , intestinal motility , although definitive evidence is limited only to daikenchuto at present . based on these findings
, the focal administration of this drug may be optimal for the treatment of intestinal obstruction , such as postoperative ileus . thereafter , it has been confirmed that daikenchuto improves some disorders of the colon such as obstructive bowel disease or anorectal dysfunction with severe constipation in children [ 95 , 96 ] , and constipation in patients with parkinson s disease . as the pathophysiology of irritable bowel syndrome ( ibs ) , low grade inflammation of the intestine
therefore , both regulation of colonic motility and inflammation are important for the therapeutic strategy . however , it is difficult to establish the basic and clinical evidence for kampo medicines in the treatment of general malaise . the japanese medical treatment kampo is frequently prescribed for patients with numerous gi tract disorders associated with various symptoms , including general malaise . this treatment should be recommended theoretically , because it combines modern western and traditional asian medical practices . however , kampo medicines are not used widely worldwide and are considered complementary medicines and not mainstream . | [
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] |
an optical biopsy ( ob ) is the examination of tissue using a nonintrusive diagnostic method .
it is performed with one of the following techniques : laser , optical coherence tomography ( oct ) , infrared , fluorescence , spectroscopy , and so forth .
tissue is accessed through the skin or by endoscopy . in pathology the gold standard
is the histology of the fixed tissue ( dead tissue ) . in ob techniques ,
images are obtained in real time together with complementary information , which allows evaluation of the disease in vivo , but gold standards are still lacking . from the technical point of view
, the methods can be divided in two major groups : those based on morphological images ( figure 1 ) and those not associated with images .
the term optical biopsy was established for the latter group , but if we use the definition use of optical energy to obtain information regarding structure and function of the tissue , without disrupting it , then this includes any of the noninvasive techniques .
this definition is closer to pathology competencies by training because the diagnosis is based either on normal tissue or on changes in cell or tissue morphology .
in general , ob images have higher resolution than the rayleigh law 0.61*/na which considers that two points can be resolved when the centre of the point spread function ( psf ) is inside the first zero of the psf centred in the second point , provided that contrast is higher than 26% , because lateral resolution depends on object luminance and contrast ( figure 2 ) . the superesolution formula ( n )
is such that /2 n na is the limit of resolution with n*resolution being equal to /2 na .
the optical resolution is limited by sampling in ccd cameras . according to nyquist sampling theory , to distinguish an object this
should be separated at least by 2.3 to 3 times the microscopic power ( m ) per the optical resolution ( d ) divided by the size of the camera pixel ( x ) furthermore , pixel size ( the smallest is 2.7 m in 2/3 chips ) , as well as the degree of integration of sensitive rgb pixels in single chip colour cameras ( see figure 3 ) , also modifies the resolution . super resolution can be achieved using information theory tools , such as image analysis and pattern recognition .
starting with noise control of the image capture device by averaging , followed by adaptive contrast filters , which detect borders and operate inside the region , up to the trough focus scanning optical microscope ( tsom ) technique that achieves 3 nm of resolution .
numerous microscopic techniques based on structured illumination , interferometry , and holography also achieve super resolution ; while spectroscopic techniques that detect physical and chemical specimen properties ( spectral pathology ) can also be at the super resolution limit . finally , optic nonlinear techniques use excitation coherent light whose tissue scattering is not related to the incident light ( double frequency ) providing information about structure and molecular interactions . these systems are called chemical microscopes because they detect molecular changes or molecular arrangement and distribution .
all of them can be miniaturized using new materials and micro - electronic machines ( mems ) and incorporated into endoscopic devices to obtain chemical or imaging micro - endoscopies ; these are usually managed by endoscopists not trained in the pathology domain .
this enhances not only the push endoscopy ( pe ) but double balloon endoscopy ( dbe ) that allows visualising the whole small intestine up to the more recent video - endoscopy capsule ( vce ) , a wireless endoscopic capsule ( wce : wireless endoscopic capsule ) . any of them can carry optical biopsy systems .
confocal microscopy uses a light beam at the limit of optical diffraction , and other technique such as two photon imaging or third harmonics use smaller light beams .
finally the most sophisticated applications correlate captured and expected images as in holographic techniques and image processing ; when both images are similar , differences reach super resolution .
if emitted photons are of similar energy and wavelength as incident light we get a rayleigh scatter .
fraction emitting photons different from initial excitation produce a raman scatter : predominantly of higher energy producing the stokes effect , but also of lower energy in the antistokes effect .
fluorescence spectroscopy studies properties of tissue taking into consideration the intensity and wavelength emitted by fluorescent or autofluorescent objects excited by uv , near infrared - nir ( 640850 nm ) or compact/ tuned laser induced fluorescence ( lif ) .
there are two methods : light scattering spectroscopy ( lss ) for macroscopic areas and fast excitation emission matrix ( fasteem ) for microscopic areas using optical fibers .
three types of information can be studied , together known as tri - model spectroscopy ( tms ) : fluorescence , reflectance and elastic dispersion of light .
this is the case with visual enhanced lesion scope ( velscope ) from led dental inc . ,
( pmti ) photonic molecular tissue imager from mediscience technologies uses uv-320 nm and yellow light at 580 nm in cancer detection ( cd - ratiometer ) and human papilloma virus ( hpv ) .
, acquired by sprectrascience uses uv and visible light between 360 and 720 nm .
hyperspectral diagnostic imaging system ( hsdi ) from sti medical systems , hawaii , registered the term virtual biopsy as capture and automatic analysis by cad ( computer - aid - diagnosis ) of direct , fluorescent , and scattered white light , to localize malignancy risk areas .
simpler is dynamic spectral imaging system ( dysis ) from forth - photonics showing pseudocolor after cleaning the colposcopic area with dilute acetic acid .finally light - induced fluorescence endoscope ( oncolife ) from xillix uses blue light ( 425455 nm ) with a xenon lamp .
fluorescence is collected with a ccd and analyzed with green ( 480580 nm ) and red filter ( 620720 nm ) .
this technique has been incorporated into gastrointestinal tract analysis with fujii intelligent chromoendoscopy ( fice ) technology from fujinon .
this is the case with visual enhanced lesion scope ( velscope ) from led dental inc .
( pmti ) photonic molecular tissue imager from mediscience technologies uses uv-320 nm and yellow light at 580 nm in cancer detection ( cd - ratiometer ) and human papilloma virus ( hpv ) .
, acquired by sprectrascience uses uv and visible light between 360 and 720 nm .
hyperspectral diagnostic imaging system ( hsdi ) from sti medical systems , hawaii , registered the term virtual biopsy as capture and automatic analysis by cad ( computer - aid - diagnosis ) of direct , fluorescent , and scattered white light , to localize malignancy risk areas .
simpler is dynamic spectral imaging system ( dysis ) from forth - photonics showing pseudocolor after cleaning the colposcopic area with dilute acetic acid .
finally light - induced fluorescence endoscope ( oncolife ) from xillix uses blue light ( 425455 nm ) with a xenon lamp .
fluorescence is collected with a ccd and analyzed with green ( 480580 nm ) and red filter ( 620720 nm ) .
this technique has been incorporated into gastrointestinal tract analysis with fujii intelligent chromoendoscopy ( fice ) technology from fujinon .
( b ) fasteem [ see ] this method has structured illumination : fluorescence is excited by a pulse laser ( 308480 nm ) and two pulses of white light through an optical fibre probe ( 270800 nm ) .
a spectrograph analyzes the first - order diffraction and eliminates the second order ( > 540 nm ) with a high - pass filter .
the optical fibre lif probe can be integrated in an mis - colposcope ( multispectral imaging system ) or in an endoscope through the biopsy channel .
cervicograms - tissuegrams , which means that mis - colposcopy and tissue images that can be stored and consulted at distance .
in fact it is a virtual biopsy if the term had not been registered by sti medical systems .
this method has structured illumination : fluorescence is excited by a pulse laser ( 308480 nm ) and two pulses of white light through an optical fibre probe ( 270800 nm ) .
a spectrograph analyzes the first - order diffraction and eliminates the second order ( > 540 nm ) with a high - pass filter .
the optical fibre lif probe can be integrated in an mis - colposcope ( multispectral imaging system ) or in an endoscope through the biopsy channel .
all systems allow cervicograms - tissuegrams , which means that mis - colposcopy and tissue images that can be stored and consulted at distance .
in fact it is a virtual biopsy if the term had not been registered by sti medical systems .
fourier transform infrared spectroscopy ( ftir ) similarly to any ir system is a process of absorption but in this case analyzed in the frequency domain .
fluorescence and the ftir are the most sensitive and popular methods of optical biopsy . in cancer of the uterine cervix , the ftir has a sensitivity of 98.6% and a specificity of 98.8% , with positive and negative predictive values of 99.5% and 96.5% , respectively , while pap - cytology achieves 95.9% and 72.3% .
raman spectroscopy includes : resonance , surface - enhanced raman scattering ( sers ) , and microspectroscopy .
raman phenomena occur with any type of incident light , but probes have difficulties detecting spatial distribution of the tissue , because the signal is very low . in medicine
, raman spectroscopy in near infrared ( raman - nir ) is used to detect preneoplasic skin lesions , gastrointestinal lesions by endoscopy ( see below vce ) , changes linked to atheromatosis , as well as a therapeutic aid for detecting tumour margins in surgery .
a structured illumination is obtained with the raman - hollow optical fiber ( hof ) whose optical fibre does not produce noise because the light is limited to the centre .
the probes do not require filters , have several wavelengths , and one fibre can conduct the excitation light and collect scatter light ; the resulting raman scatter has a high signal to noise ratio ( snr ) and does not require background correction .
raman spectroscopy has been integrated into regular microscopy as well as confocal microscopy . in the latter , 3d images of high resolution
are obtained to study tissue properties at a subcellular level , being of great value in chemical microscopy .
based on structured illumination , it uses two small light apertures ( pinholes ) in conjugated planes ( confocal ) ; one in front of the excitation light and the other in front of the detector .
thus , fluorescence out of focus is avoided since the confocal aperture only allows light from the focal plane , improving lateral , and axial resolution .
axial resolution allows optical sectioning , focussing one plane at a time and reconstructing images electronically .
they use ultrashort pulsed lasers with rod grin microlenses ( gradient refractile index ) to control chromatic dispersion and phase modulation .
initially these were of 0.9 mm in diameter , with 30 kfibers directly connected to a grin ( 1 mm .
diameter ) of 4.8 magnification ( low na = 0.2 ) , with a total diameter of 1.2 mm ; the space between fibers was 3 m limiting the lateral resolution of sampled area to 0.6 m .
the most widely used system is optiscan ( optiscan , australia ) integrated on the tip of a conventional colonoscope ( pentax ec3870k ; pentax , tokyo , japan ) , which allows an optical biopsy of the epithelial surface and lamina propia during endoscopy ( figure 4 ) .
contrast enhanced with sodium fluoresceine or methylene blue it gives rise to multispectral confocal microendoscopy ( mis ) capable of detecting immune markers such as anti - cd44v6 for aberrant crypts and producing an immune - microendoscopy .
mauna kea technologies has built cellvizio , a family of confocal - microscopy probes on miniaturized objectives compatible with any colonoscope , gastroscope , or bronchoscope that allows reaching very small conducts obtaining cholangioscopies and alveoloscopies .
their lateral resolution is 1 m and the axial resolution is 150 m . their diameters are from 0.93 to 2.5 mm and they are up to 4 meters long .
video - endoscopy capsule ( vce ) allows direct examination of the small intestine in a noninvasive , secure and well - tolerated manner , although stenosis and diverticulus are contraindicated .
for that reason the capsules are now marked with rfid to easily locate and extract them .
wec or wireless endoscopic capsule m2a ( pillcam sb from given imaging ) is of 3.7 g , and measures 11 26 mm , with a cmos camera and 6 leds , equipped with uhf transmission , sending 2 frames per second during 7 hours .
the ec type 1 is from olympus with a high resolution ccd - camera and a real - time viewer .
the cpe compact photonic explorer , from mediscience technology , contains spectroscopic fluorescence able to detect malignant lesions even at the molecular level .
the third generation of capsules is the norika 3 rf endoscopic robot capsule in 2001 .
it does not have batteries because it is charged with an external electromagnetic field that also powers rotation .
it measures 9 23 mm and 40% is empty and available for surgical or therapeutic gadgets such as sprays , lasers , and ph detectors .
new releases are of 5.8 15 mm which can be used in children .
sayaka - ce from rf system lab , japan , sends 30 fps , does not use batteries , and also builds mosaics ( figure 5 ) .
this is the case with versatile ( endoscopic capsule for gastrointestinal tumor recognition ) that also includes nir - spectroscopy .
resolution improvement with differential interference contrast ( dic ) , the nomarski technique , has been known for many years .
today , the interferometric techniques with diffraction gate can be compared to confocal microscopy but with better tissue penetration ( figure 1 ) ; axial resolution is linked to the coherence of light and transversal or lateral resolution to the size of the light beam .
there are three modalities with low coherence light or low coherence interferometry ( lci ) ; coherence probe microscopy ( cpm ) , optical coherence tomography ( oct ) , and optical coherence microscopy ( ocm ) .
the first uses halogen light and the other two super - leds or ( sdl ) super - luminescence laser diodes .
the applications with halogen light are known as full field ( ff - oct and ff - ocm ) .
iim can be adapted to existing microscopes without entering the pupil plane of the objective .
it uses structured light obtained through a gate of less than wavelength ( ) , at the limit of the na of the optical system , which produces a shift in spatial frequencies , recording and combining several images in amplitude and phase to improve the resolution .
iim works at lower power with low aperture objectives ( na < 0.5 ) but obtains twice the resolution of high aperture objectives ( na = 1.4 ) .
the depth of field ( dof ) , vision area and working distance , is that of a low power objective , although the final integrated image has a resolution at the linear limit of the transmission media ( refraction index n ) /4n . by shifting the objective plane and transforming the observed spatial frequencies ( reference image ) into spatial frequencies of the real image
molecular interferometry is a step forward ; mi2 or molecular interferometric imaging allows the detection of biomolecules .
endoscopes with high quality image are large because the sensors are also large ( 1 cm or more in diameter ) .
standard microendoscopes ( 2.4 to 1.2 mm in diameter ) use a limited bundle of optical fibres , improving flexibility but losing image quality .
single - fibre microendoscopes , with the thickness of a hair ( 80 nm ) in 2006 , have hof effect , and are made of photonic crystal fibers ( pcf ) .
see or spectrally encoded endoscopy uses multicolour light projected into the tissues through a single fibre of the thickness of a hair . the scattering is collected and the various wavelengths are analyzed by spectroscopy .
simultaneously , an interferometer computes the structural information based on two light sources giving a 3d image .
oct is a noninvasive technique that uses low coherence light sources such as sld , some of them in the nir - region ( niris oct of imalux ) , to produce tomography by interferometry tdoct - time - domain oct or interferometry sdoct - spectral - domain oct . by definition it works with low numerical apertures ( na < 0.5 ) and
the axial and lateral resolution using slds was of 30 m ; in 2001 , using broadband ( range > 100 nm ) pulsed - lasers it was of 10 m ( h - oct or high resolution - oct ) , and today using ultra - high resolution ( uh - oct ) it reaches 0.5 m ( figure 6 ) . oct is widely used in ophthalmology , and is extending into endoscopy ( i.e. , bladder tumor detection with a sensitivity of 92% and a specificity of 85% ) , colonoscopy and other imagining techniques .
there are on the market oct - endoscopes of 1 mm in diameter and oct - fiberscopes of 400 m in diameter with grin or drum optics [ 1619 ] . in ophthalmology , adaptive optics ( aos )
is required to compensate for irregularities of the ocular system , which makes the devices much more expensive [ 20 , 21 ] .
although ocm is used when the numeric aperture is high ( na > 0.5 ) the fact is that there are grins optics with na = 1.2 , achieving important optical detail .
furthermore , on a classical oct a synthetic aperture can be built to yield an interferometric synthetic aperture microscopy ( isam ) , whose advantages are used in optical frequency domain imaging ( ofdi ) .
it is well known that the depth of field ( dof ) and resolution are inversely related , because one improves when the other decreases and vice versa . with the isam method
there is no such limitation because it captures stable phase data including reference phase ( i.e. , coverslip ) in the object .
the macroscopic oct is the ffoct or full - field optical coherence tomography that uses simple halogen lamps and ccd cameras as detectors , achieving an snr of 90100 db .
similarly , ff - ocm or full - field optical coherence microscopy uses broadband thermal light from a tungsten lamp to produce ocm spectroscopy .
this technique , with low power water immersion , objectives 10x of low numeric aperture na = 0.3 , captures spectral information with colour cameras , and produces high resolution images ( axial 0.8 m and lateral 1.4 m ) by means of image processing .
microscopic detail provides a pseudohistological stain in the hue - saturation - luminescence ( hsl ) color space , where h represents variation , s temporal shift , and l is constant . in other words , hue is the predominant wavelength , saturating the spectral purity and luminancing the colour intensity . in the h - channel ,
changes in the wavelength of the reflected light are codified in red , green , or yellow depending on whether they increase , decrease , or are maintained . in the s - channel of each wavelength ,
ofdi is an improvement on the oct , because instead of examining point by point , 1000 points are analyzed simultaneously .
the tip of the probe of the optical fibre is permanently rotating and emits a laser light that changes the wavelength from 1264 to 1376 nm . an image is created measuring the temporal delay of the echo as well as the spectral interference between the tissue and the reference in each wavelength , whose fourier transformation ( optical frequency domain ) gives a microscopic view .
this uses electromagnetic high wavelength ( thz ) , localized between microwaves ( mw ) and infrared ( ir ) light , with a frequency of 10 hz .
these waves propagate in metals and nonpolar material and are therefore highly appreciated as metal detectors for weapons and so forth .
they are highly sensitive to molecular resonance and therefore used in interferometric image sensors and detectors .
the thz generators are classified in three categories : thermal incoherent , broadband pulsed , and continuous wave narrow band .
the majority use tdt or pulsed spectroscopic imaging based on time - domain terahertz spectroscopy .
it allows one to obtain any type of information regarding spectral absorption , depth , and type of objects .
the disadvantage is that the optical components are large , expensive , and with low potential .
in contrast , cw or continuous - wave thz imaging system is simple , of low cost , and does not require expensive devices or complex optics . for a multifunction broadband system we can use multiple discrete frequencies or tuned lasers .
it is a matter of time before we obtain a quicker detector to improve endoscopic molecular imaging . in holography ,
the dispersion of light from an object is recorded in a medium while it is illuminated with a second point of light ( reference light ) .
the light field obtained from the interference of both light sources is the hologram of the object .
holographic gratings are used in numerous noninvasive applications of uv or medium - ir . only those of industrial quality
they consist of a miniaturized holographic system capable of recording reflection holograms , with 3 parts : the cartridge , the diagram , and a single mode fiber of 4 m in diameter .
contrast methods ( fluorescence , staining ) are often used before recording , as in gynaecology or enteroscopy . with the new techniques in holography
the result is an electronic object in 3d that can be seen , for example , in scanning tunnelling microscopes . in the same hologram we can record several images in several wavelengths that can be read as a book just by changing the wavelength .
directly related with laser technology , it uses ultrashort pulses ( high peak and low potency ) on optical fibers with high aperture lenses .
specimen response is nonlinear to the incident light , but parametric since there is no energy transfer .
two phenomena are included : harmonic generation and surface enhanced raman scattering ( sers ) .
nonlinear single fiber optical endoscopes of low numeric aperture use thick optical fibers with limited sensitivity . to improve them ,
long and thin fibers are used instead , very efficient in both senses ( excitation collection and emission ) emitting light at the limit of optical diffraction and capable of supporting laser ultrashort pulses .
these are the pcfs photonic crystal fibers that together with mems allow building miniaturized single - fiber endoscopes .
these techniques will only be covered superficially since they integrate chemical microscopes with nanometric resolution far removed from surgical pathology .
cars microscope consists of two lasers ( pump and antistoke ) synchronized in time , and whose frequency depends on the particle under study .
the first is proportional to the square of raman scatter , and the resulting signal is the cube of the initial one .
it is very sensitive to carbon - hydrogen vibration and for that reason is used to detect lipids .
cars has been integrated with confocal microendoscopy in order to obtain high resolution images of vibrating particles .
it uses the principles of cars and ocm to obtain a transversal section of the particular molecules inside the specimen .
two cars signals are generated , one from the reference molecules and the other from the molecule we wish to study .
the two coherent light signals mutually interfere ; the envelope or combined signal measures the concentration and the interference fringes give information regarding the phase and allow us to determine the vibrational characteristics of the molecules .
duv microscopy on thick tissues extends regular optical microscopy to the region of the duv in order to detect endogenous signals from nonmarked materials .
duv wavelength is between 230 nm and 350 nm , although it is not completely quantified , because it can not be transmitted through glass and therefore can not be observed with usual methodology .
we can use a single photon one - photon excitation ( ope ) or two photons tpe ( two - photon excitation microscopy ) , the latter with ir at 730 nm .
the ir photons improve tissue penetration due to the fact that , in contrast to uv , the ir has less absorption coefficient and a limited dispersion coefficient .
finally , multiphoton excitation mpe uses the same principle but with a pulsed laser of 100 fs at 80 mhz and wavelength from 700 to 1050 nm .
since fluorescence only appears in the focused volume there is no excitation out of this and the 3d resolution is intrinsic .
photon excitation endoscopes had been miniaturized over tpe with mems scanners and ultrashort pulsed - lasers on hollow - core photonic - bandgap fiber ( hof ) that virtually eliminates pulse distorsion , which is currently applied in neurobiology .
shg microscopes detect the second harmonic which is a property of noncentrosymetric structures such as collagen and has been applied with success for the study of the cornea , and lamina cribosa of the sclera .
the nsom scans the object with a wave of light under the wavelength a . the emission point is located close to the object ( distance < a ) , obtaining a resolution closer to a and an n = /2a 10 times superior .
the optical fiber is usually tapped and the light reflects on the tapper , with only a very small wavelength leaving the fiber .
it is often combined with other photonic excitation techniques such as the trifunctional microscope in which one can move from cm or confocal to snom and to afm ( atomic force microscope ) , moving the microscopic tower .
another nanometric solution is the s - scattering - type scanning near - field optical microscopy ( snom ) .
this allows 20 nm resolution , breaking the diffraction barrier with visible light , ir and thz .
the sensitive point is located at the end of the cantilever and can work touching the surface of the specimen or not .
this is a hybrid method since it includes three techniques : optic irradiation , ultrasound detection , and image production .
illumination is performed with 804 nm wavelength that produces a deformation in the tissue which is captured by a 5 mhz ultrasound probe , providing high resolution images ( axial resolution of 15 m and lateral resolution of 45 m ) with high penetration in the tissue ( up to 3 cm ) [ 28 , 29 ] .
regular microscopes can not normally be used for nanometric detection ( < 100 nm ) because of optical resolution limitations due to the wavelength used .
the method uses noncoherent , nonstructured illumination processed by image analysis of images taken by a regular camera through a regular optical microscope equipped with automatic staging and established z - focus movements .
this method extends the range of the regular optical microscope to the nanoscale , detecting differences as small as 3 nm . out of focus images at a given z - distance are stored and piled up .
an image analysis algorithm detects differences between the congruent parts to finally produce in - focus images of very small particles at the level of nanoparticles , ten times smaller than the wavelength used .
the so - called photon - migration imaging is used to achieve a tissue penetration which exceeds oct limits .
spectral window from 700 to 900 nm where light absorption due to chromophores , such as blood and water , is low . in this window
nir ( near - ir ) allows clear visualization of the oxygenation of haemoglobin and the quantity of circulating blood in muscle , cerebrum , or breast , for example .
other types of element detection require specific molecular beacons such as tricarbocianine , which is fluorescent to nir , linked to a specific tumour protein , therefore showing the location of the tumour .
it has also been used with other biomarkers such as amyloidal - b in alzheimer 's disease .
some years ago pathology departments were actively incorporating clinical specialists for specific organ biopsies ( hepatic , renal , gynaecological , ophthalmologic , dermatologic , etc . ) .
now clinical departments will carry out morphological diagnoses based on noninvasive biopsy techniques . and pathologists will need to update their knowledge to cooperate at a distance ( telediagnosis ) otherwise laboratories will be empty of classical surgical diagnostic contents and full of advanced subspecialties according to new technology and technological advances . we can conclude that nonstructured and structured illumination and interferometry , including holography and hybrid methods , produce high quality images comparable and sometimes better than optical microscopy and should be interpreted by a pathologist .
this should be the optical biopsy section in pathology departments where optical biopsies are received in teleconsultation .
since obs are taken by microendoscopic techniques , it is not feasible for pathologists always to be present , but their expertise is required to make an exact diagnosis . from the methods reviewed here , confocal endoscopy , pam , and
uh - oct with or without contrast provide enough histological quality to be incorporated in diagnostic procedures , in collaboration with the pathology department , but for this , gold standards have to be established . pathology knowledge and the gold standards of surgical pathology are built on the following premises : ( 1 ) experience is better than evidence ; ( 2 ) knowledge based on the literature ; ( 3 ) scientific relevance or eminence ; ( 4 ) interpretation ; ( 5 ) personal impression .
it seems obvious that the sooner we gain sufficient experience and images available and accessed by all surgical pathologists , the better and sooner the gold standard for optical biopsy will be established .
obs , particularly morphologically based , will only be incorporated into routine diagnostic procedures when its gold standards have been established .
this is the reason why an image bank of optical biopsies accessible by the scientific community in a grid environment is urgently needed , something similar to the protein atlas initiative .
furthermore , we can not forget the extreme efficiency of these techniques in medical support for underserved and isolated areas .
we have seen the high microscopic and immunologic quality of confocal endomicroscopy , whose drawback is its limited tissue penetration ( see figure 1 ) , which is why it is widely implemented in intestinal endoscopy .
alternative techniques such as pam or uh - oct with better penetration should progressively be introduced to widen the optical biopsy area .
furthermore the hof technique can provide superior microscopic quality as compared to regular optical microscopy , and improvement of molecular techniques will extend immune - endoscopy towards pharmacy - pathology and personalized therapies as mentioned elsewhere . | the ability to obtain information about the structure of tissue without taking a sample for pathology has opened the way for new diagnostic techniques .
the present paper reviews all currently available techniques capable of producing an optical biopsy , with or without morphological images .
most of these techniques are carried out by physicians who are not specialized in pathology and therefore not trained to interpret the results as a pathologist would . in these cases , the use of telepathology or distant consultation techniques is essential . | 1. Introduction
2. Super Resolution
3. Techniques
4. Discussion | an optical biopsy ( ob ) is the examination of tissue using a nonintrusive diagnostic method . in pathology the gold standard
is the histology of the fixed tissue ( dead tissue ) . from the technical point of view
, the methods can be divided in two major groups : those based on morphological images ( figure 1 ) and those not associated with images . the term optical biopsy was established for the latter group , but if we use the definition use of optical energy to obtain information regarding structure and function of the tissue , without disrupting it , then this includes any of the noninvasive techniques . all of them can be miniaturized using new materials and micro - electronic machines ( mems ) and incorporated into endoscopic devices to obtain chemical or imaging micro - endoscopies ; these are usually managed by endoscopists not trained in the pathology domain . any of them can carry optical biopsy systems . in cancer of the uterine cervix , the ftir has a sensitivity of 98.6% and a specificity of 98.8% , with positive and negative predictive values of 99.5% and 96.5% , respectively , while pap - cytology achieves 95.9% and 72.3% . initially these were of 0.9 mm in diameter , with 30 kfibers directly connected to a grin ( 1 mm . diameter ) of 4.8 magnification ( low na = 0.2 ) , with a total diameter of 1.2 mm ; the space between fibers was 3 m limiting the lateral resolution of sampled area to 0.6 m . the most widely used system is optiscan ( optiscan , australia ) integrated on the tip of a conventional colonoscope ( pentax ec3870k ; pentax , tokyo , japan ) , which allows an optical biopsy of the epithelial surface and lamina propia during endoscopy ( figure 4 ) . contrast enhanced with sodium fluoresceine or methylene blue it gives rise to multispectral confocal microendoscopy ( mis ) capable of detecting immune markers such as anti - cd44v6 for aberrant crypts and producing an immune - microendoscopy . wec or wireless endoscopic capsule m2a ( pillcam sb from given imaging ) is of 3.7 g , and measures 11 26 mm , with a cmos camera and 6 leds , equipped with uhf transmission , sending 2 frames per second during 7 hours . single - fibre microendoscopes , with the thickness of a hair ( 80 nm ) in 2006 , have hof effect , and are made of photonic crystal fibers ( pcf ) . this technique , with low power water immersion , objectives 10x of low numeric aperture na = 0.3 , captures spectral information with colour cameras , and produces high resolution images ( axial 0.8 m and lateral 1.4 m ) by means of image processing . this uses electromagnetic high wavelength ( thz ) , localized between microwaves ( mw ) and infrared ( ir ) light , with a frequency of 10 hz . it allows one to obtain any type of information regarding spectral absorption , depth , and type of objects . in holography ,
the dispersion of light from an object is recorded in a medium while it is illuminated with a second point of light ( reference light ) . only those of industrial quality
they consist of a miniaturized holographic system capable of recording reflection holograms , with 3 parts : the cartridge , the diagram , and a single mode fiber of 4 m in diameter . to improve them ,
long and thin fibers are used instead , very efficient in both senses ( excitation collection and emission ) emitting light at the limit of optical diffraction and capable of supporting laser ultrashort pulses . these techniques will only be covered superficially since they integrate chemical microscopes with nanometric resolution far removed from surgical pathology . cars has been integrated with confocal microendoscopy in order to obtain high resolution images of vibrating particles . the ir photons improve tissue penetration due to the fact that , in contrast to uv , the ir has less absorption coefficient and a limited dispersion coefficient . the optical fiber is usually tapped and the light reflects on the tapper , with only a very small wavelength leaving the fiber . this should be the optical biopsy section in pathology departments where optical biopsies are received in teleconsultation . from the methods reviewed here , confocal endoscopy , pam , and
uh - oct with or without contrast provide enough histological quality to be incorporated in diagnostic procedures , in collaboration with the pathology department , but for this , gold standards have to be established . it seems obvious that the sooner we gain sufficient experience and images available and accessed by all surgical pathologists , the better and sooner the gold standard for optical biopsy will be established . furthermore , we can not forget the extreme efficiency of these techniques in medical support for underserved and isolated areas . | [
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