Advanced age contributes to clinical manifestations of many retinopathies and represents a major risk factor for age-related macular degeneration, a leading cause of visual impairment and blindness in the elderly. Rod photoreceptors are especially vulnerable to genetic defects and changes in microenvironment, and are among the first neurons to die in normal aging and in many retinal degenerative diseases. The molecular mechanisms underlying rod photoreceptor vulnerability and potential biomarkers of the aging process in this highly specialized cell type are unknown.To discover aging-associated adaptations that may influence rod function, we have generated gene expression profiles of purified rod photoreceptors from mouse retina at young adult to early stages of aging . We identified 375 genes that showed differential expression in rods from 5 and 12 month old mouse retina compared to that of 1.5 month old retina. Quantitative RT-PCR experiments validated expression change for a majority of the 25 genes that were examined. Macroanalysis of differentially expressed genes using gene class testing and protein interaction networks revealed overrepresentation of cellular pathways that are potentially photoreceptor-specific (angiogenesis and lipid/retinoid metabolism), in addition to age-related pathways previously described in several tissue types .Our study suggests a progressive shift in cellular homeostasis that may underlie aging-associated functional decline in rod photoreceptors and contribute to a more permissive state for pathological processes involved in retinal diseases. Aging is a complex biological process influenced by stochastic occurrence of molecular damage over time, but despite inherent randomness, the hallmarks of aging appear to be specific and uniform between individuals and even among species Despite a broad consensus, many changes in gene expression appear to be tissue- or even cell-type specific Age is a major risk factor for age-related macular degeneration (AMD), a leading cause of untreatable vision loss in the elderly Rod photoreceptors represent the predominant retinal cell type in most mammals and are uniquely vulnerable to the effects of age. In the human retina, about 30% of central rods are lost by the ninth decade, whereas cone density remains essentially unchanged http://research.jax.org/faculty/harrison/ger1vLifespan1.html and We had previously developed mice that express enhanced Green Fluorescent Protein (eGFP) under the control of the Neural Retina Leucine zipper promoter (Nrlp), which restricts eGFP expression in the retina to developing and mature rod photoreceptors max), sensitivity k, and a- (Vamax) and b-wave (Vbmax) amplitudes at maximum flash intensity of 1.09 log cd-s/m2. ERGs of Nrlp-eGFP mice showed a mild decline with age in rod function similar to that of age-matched C57Bl/6 controls . The dark-adapted b-wave, Vbmax, which reflects signaling downstream of the photoreceptors, declined with age in Nrlp-eGFP and C57Bl/6 mice . Rod-mediated scotopic amplitude Vmax, also declined with age but was not significantly different in Nrlp-eGFP as it was the case for C57Bl/6 mice (p<0.01). There was no change in sensitivity k with increased age in either group. For all ERG parameters, age-dependent changes in Nrlp-eGFP mice mirrored those observed in C57Bl/6 mice. Expression of eGFP in rod photoreceptors, therefore, does not appear to cause any significant difference compared to C57Bl/6 mice in retinal histology or function up to 12 months of age, though a slight decline in rod function by 12 months of age is detected in both strains.We first evaluated whether eGFP expression in rod photoreceptors had any effect on the physiology or cellular morphology by comparing Nrlp-eGFP mice to wild-type (C57Bl/6) mice at different ages. Nrlp-eGFP mice developed normally and showed no histological signs of retinal degeneration by 12 months of age . ONL thicontrols . There w6 and 9.54×105, respectively; n = 4, p = 0.846 by Student's two-tailed t test). Gene expression was compared among the three selected time points by one-way ANOVA that yielded 413 probe sets (corresponding to 375 transcribed sequences) showing >1.5-fold change at p<0.05 between at least two of the three time points ; immune system regulation - histocompatibility 2 K1, K region (H2-K1), interferon regulatory factor 6 (Irf6), and endoplasmic reticulum aminopeptidase 1 (Erap1); lipid and insulin metabolism - stearoyl coenzyme A desaturase (Scd1), oxysterol binding protein-like 1A (Osbpl1a), and insulin receptor substrate 1 (Irs1); and two genes that have not yet been characterized .Twenty-five genes with at least 2-fold change and/or potential biological relevance were selected for validation by quantitative RT-PCR (qRT-PCR) using independent rod photoreceptor RNA samples . Seventeen of the 25 genes exhibited changes in the predicted direction at both 5 months and 12 months of age relative to 1.5 months; of these, 16 showed increased expression with age, and one showed reduced expression compare . Of the Grem2, Rdh9, and Btnl9. Irf6 demonstrated a higher magnitude of change (over twofold) in retina samples than photoreceptor samples, but the remaining genes exhibited comparable fold changes with age. Expression of only one gene, C030034E14Rik, changed in an opposite direction with age in whole retinas compared to isolated rod photoreceptors.To evaluate whether gene expression changes in our analysis were characteristic of rod photoreceptors, we performed qRT-PCR analysis of the 17 validated genes using total RNA from whole retina from young (1.5 month old) and old (12 month old) Nrlp-eGFP mice . Whole rEye Morphogenesis, Electron transport chain, Motor Axon Guidance, and Antigen presentation among the top ten  enrichment analysis  top ten . Some ofRdh9) mostly increased. Both higher and lower expression changes were detected with age for genes in the tight-junction signaling and Cxcr4 signaling pathways.Ingenuity® Pathway Analysis (IPA) sorts genes into canonical pathways based on the scientific literature and indicates which are significantly overrepresented Ingenuity® network analysis revealed biological processes that were enriched among the 100 genes showing progressively higher or lower expression across the three time points see . We thenNr3c1), involved in stress response and inflammation; RhoA, an important signaling molecule associated with cytoskeletal remodeling; and Cdkn1b, which maintains cell cycle arrest. Additionally, several genes involved in overrepresented pathways (described above) were present in the direct interaction network. Overall, genes associated with stress response appeared to occupy prominent positions (hubs) in the network topology.Finally, we generated a network reconstruction using MetaCore™ from GeneGo, Inc. C. elegans have identified a genetic basis for the modulation of aging phenotypes that extends to higher organisms in vivo as it is the most accessible part of the central nervous system. Furthermore, evidence suggests shared characteristics between age-related diseases of the retina and the brain, such as AMD and Alzheimer's disease www.sph.uth.tmc.edu/Retnet/) and is uniquely amenable to non-invasive evaluation, which facilitates the study of natural history and potential therapies. However, the direct impact of aging on molecular pathways that guide photoreceptor metabolism or function has yet to be elucidated. Here, we report adaptive changes in both conserved and cell-specific age-related pathways through temporal profiling of purified rod photoreceptors. We show that a majority of these expression changes begin in young adults and continue to progress with age. Furthermore, our study reveals that single cell-type profiling provides better resolution of subtle and progressive age-related expression changes (see below), yielding a more comprehensive depiction of cellular aging.Aging-associated changes occur on multiple levels and contribute to cellular dysfunction and disease. Landmark studies in yeast and Though rod photoreceptors are highly specialized neurons involved in photoreception, we identified changes in consensus pathways of aging, including oxidative phosphorylation and stress response affecting transcription and inflammation . Rods arregulation of transcription and nucleosome assembly, and expression of genes for histone clusters 1, 2, and 3  progressively increased over time (Dnmt3a) and Suppressor of variegation 4–20 homolog 1 (SUV420h1), which was also noted to change with age in human retinas Hr) and Nuclear co-repressor 1 (Ncor1) both decreased with age, suggesting a derepression of transcription that has been associated with epigenetic changes in other aging cells Patterns of histone acetylation and DNA methylation have been shown to change with age  from yeast to humans, influencing DNA stability and gene expression patterns ver time . DNA-modIrf6 and antigen presentation genes Erap1H2-K1 suggest a similar upregulation of inflammation signals in photoreceptors. Increased expression of H2-K1 and the human homolog, HLA-C, was also reported in previous studies of aging mouse and human retina and eye tissue Fos and Nr3c1 (glucocorticoid receptor) signaling that may contribute to systemic and cognitive aging Upregulation of inflammatory response pathways has been associated with neuronal aging of rodents and humans CD59a is particularly interesting as it protects against complement attack Stress response processes, including angiogenesis and retinoid/lipid metabolism, are especially relevant for the aging retina . PhotorePpara) appear to play a role in photoreceptor aging. Altered retinoic acid metabolism has been implicated in some forms of retinal degeneration Rdh9 and Rdh18) increased dramatically with age in rod photoreceptors, perhaps representing a stress response to lipid peroxidation, another source of ROS Scd1 and Osbpl1a, and a progressive decrease in Ppara, all of which influence lipid and insulin signaling Ppara has also been observed in aging heart, liver, and kidney Ppara may represent a response to age-related changes in RA signaling or lipid peroxidation, since LCPUFAs and retinoids are endogenous ligands for PPAR-alpha Changes in retinoid and lipid metabolism via the retinoic acid receptor (RAR) pathway and the peroxisome proliferator-activated receptor alpha , and further investigations are in progress to examine their physiological relevance in rod photoreceptor aging. In addition to examining individual genes, we adopted a systems biology approach to analyze the expression data as a whole . This type of analysis keeps the focus on groups of genes and the pathways/categories/functional groups they belong to, and it is best applied to larger unbiased data sets. The strength of the observation derives from the genes in similar pathways that behave in a coordinated fashion.Our study provides an aging-associated gene expression signature for a single neuronal cell type. Studying rod photoreceptors instead of the entire retina has allowed us to identify significant age-related changes with better resolution. Both consensus and photoreceptor-specific age-related pathways, identified here, have implications for understanding the pathogenesis of AMD and other retinopathies. Notably, many expression changes are evident even at a relatively young adult age, suggesting that aging is a continuous process set in motion long before phenotypic changes are detected. Our studies reveal contributions of rod photoreceptors to retinal aging as well as potential changes in the immune-privileged status of the neuroretina with age. We cannot determine whether expression changes associated with normal aging are causative, permissive, or protective with respect to aging-associated functional decline and disease. Nonetheless, we propose that changes in gene expression represent an adaptive response of rod photoreceptors to microenvironment (such as exposure to light and oxidative stress) during the aging process. Identification of aging-associated pathways thus provides a foundation for future investigations and offers potential targets to develop treatments and therapies for age-related diseases.All mice were used in accordance with the approved Institutional Animal Care and Use Committee (IACUC) protocol.To evaluate retinal function, full field ERGs were recorded in Nrlp-eGFP mice and C57Bl/6 control mice at 4 and 12 months of age. Mice were dark-adapted overnight and were prepared under dim red illumination as described previously 2 recording system  to brief xenon white flashes from −5.8 to +1.09 log cd-s/m2/flash in steps of 0.5 log units for dark-adapted responses and from −0.91 to +1.09 log cd-s/m2/flash over a 2 log unit range in steps of 0.3 log units for light-adapted responses. Responses were amplified at 1,000 gain at 1.25 to 1000 Hz, and digitized at a rate of 2000 Hz. A notch filter was used to remove 60 Hz line noise. Dark-adapted b-wave amplitudes were fitted with a Naka-Rushton function to determine estimates for the b-wave saturated amplitude,Vmax and the response sensitivity, k. Light-adapted responses were recorded after 10 minutes of adaptation to a white 32 cd/m2 rod-suppressing background.ERGs were recorded with a Ganzfeld configuration using the Espion eRetinas from 1–2 and 10–12 months old Nrlp-eGFP and C57Bl/6 mice (n = 3 each) were dissected, fixed and processed to generate sagittal thin plastic sections encompassing the optic nerve head. The optic nerve was oriented in the section to indicate the ventral portion. ONL thickness was measured using Zeiss, AxioVision Documentation Rel.4.7 software on 40x-magnified images. Eight microscopy frames were counted ventrally and dorsally to the optic nerve head and measurements were taken in the middle of the frame. Values represent mean ± SEM.Rod photoreceptors were purified by flow-sorting, as previously described Five nanograms of total RNA from flow-sorted rod photoreceptors from Nrlp-eGFP mice at 1.5 month, 5 months and 1 year of age  were used for target generation. Ovation RNA Amplification System V2  was used for cDNA amplification according to the manufacturer's protocol. FL-Ovation cDNA Biotin Module V2 (NuGEN Technologies) was used to fragment and biotin label the amplified cDNA and for hybridization of the labeled target onto a GeneChip Mouse Genome 430 2.0 Array  with 45,000 probe sets for 34,000 well-characterized mouse genes. Washing and staining of the GeneChips was performed using the EukGE-WS2v4_450 protocol on the GeneChip Fluidic Station 450 (Affymetrix). GeneChip scanning was performed on the GCS3000-7G Scanner (Affymetrix).www.ingenuity.com, Redwood City, CA), MetaCore(TM) from GeneGO , and GO Enrichment Analysis The Robust Multichip Average (RMA) method Hprt) and ribosomal protein S26 (S26) served as internal controls. Primer pairs tested are listed in Retinas were dissected from four independent biological replicates for each of the three timepoints , enzymatically dissociated as described above, and rod photoreceptors were flow-sorted into RNAprotect (Qiagen). Whole retinas were harvested from additional mice and snap-frozen. RNA was extracted using the RNeasy Mini kit (Qiagen) and quality was evaluated as described above. Amplified photoreceptor cDNA was derived using WT-Ovation Pico System (NuGEN), and whole retina cDNA was derived using Superscript II RT-PCR system (Invitrogen). cDNA was used for real-time qRT-PCR using SYBR(R) Green PCR Mastermix  on the 7900HT Fast Real-Time PCR System (Applied Biosystems). Candidate genes were selected for analysis based on microarray predictions of fold-change >2, pattern of change, and gene function. Hypoxanthine guanine phosphoribosyl transferase Click here for additional data file.Table S225 genes evaluated by quantitative RT-PCR (photoreceptor data).(0.02 MB XLS)Click here for additional data file.


P-value < 0.05). In general, gene expressions that increased with age were associated with increased mortality. Gene expressions that decreased with age were generally associated with reduced mortality. A multivariate estimate of biological age modeled from expression data was dominated by CDC42 expression, and was a significant predictor of survival after blood draw. A multivariate model of survival as a function of gene expression was dominated by CORO1A expression. This model accounted for approximately 23% of the variation in survival among the CEU grandparents. Some expression levels were negligibly associated with age in this cross-sectional dataset, but strongly associated with inter-individual differences in survival. These observations may lead to new insights regarding the genetic contribution to exceptional longevity.We investigated the hypothesis that gene expression profiles in cultured cell lines from adults, aged 57–97 years, contain information about the biological age and potential longevity of the donors. We studied 104 unrelated grandparents from 31 Utah CEU (Centre d’Etude du Polymorphisme Humain – Utah) families, for whom lymphoblastoid cell lines were established in the 1980s. Combining publicly available gene expression data from these cell lines, and survival data from the Utah Population Database, we tested the relationship between expression of 2151 always-expressed genes, age, and survival of the donors. Approximately 16% of 2151 expression levels were associated with donor age: 10% decreased in expression with age, and 6% increased with age. Cell division cycle 42 (CDC42) and CORO1A exhibited strong associations both with age at draw and survival after draw (multiple comparisons-adjusted Monte Carlo  Caenorhabditis elegans , consists of Epstein–Barr virus-immortalized lymphoblastoid cell lines and DNA extracted there from, for 46 three-generation CEU families, each consisting of 5–15 siblings, their two parents, and two to four grandparents who were still alive at the time of the family blood draws in the early 1980s (The CEPH/Utah (CEU) family resource, maintained at the Coriell Cell Repositories in Camden, NJ, USA , we used only the 2151 always-expressed genes , to examine the relationship between individual expression levels and age at draw in the CEU grandparent cell lines. We modeled expression as a simple linear function of age at draw. Expression levels are reported on a logA more complex analysis of changes in gene expression with age over all three generations of CEU families revealed a large number of highly significant associations. However, interpretation of age-related changes in expression across three generations, ages 5–97, is difficult because senescence can be confounded with sexual or physiological maturation, as well as secular trends occurring over such a long span of donor birth years in these families. Methods and results of this analysis are described in the Supporting Information.P< 0.05. Of these, 125 increased with age and 220 decreased with age. P-value below the Bonferroni 5% threshold of 2.3 × 10−5. The strongest association of expression level with age was CDC42 (cell division cycle 42), which exhibited strongly increased expression with age, with a sex-adjusted P-value of 3.1 × 10−5, and an unadjusted P-value of 1.3 × 10−5. Among the ten most strongly age-associated expression levels  expressions were associated with age at draw in the CEU grandparents at a nominal n levels , equal nH2) for each gene listed, and the correlation of expression between spouses. Most of the genes listed in Also shown in −5, although CORO1A (coronin) comes quite close. Nine of the ten strongest associations of age-adjusted expression with mortality are negative, meaning that relative overexpression of the gene is associated with reduced mortality. Interestingly, the one exception is TERF2IP , which is thought to protect telomeric DNA from nonhomologous end-joining. Note that only one gene (CORO1A) appears in both We also tested for gene expressions most strongly associated with survival, independently of their associations with age at blood draw. P< 0.05. Of these, 48 were associated with age at blood draw at a nominal P< 0.05. A total of 167 (7.8%) expression levels were associated with survival in the CEU grandparents at a nominal The results shown in Z-scores for age effects and survival effects on (age-adjusted) expression levels, for the CEU grandparents. Using Fisher’s likelihood ratio approach (Z-scores (large red dots) to those generated by a 10% sample of 1000 random permutations of the phenotypic  data, while keeping the expression vectors constant . The dashed ellipse is drawn at the 50th largest chi-squared value observed for the 2151 always-expressed genes and 1000 permutations. The results are shown this way to approximate the fifth percentile of the null distribution, adjusted for 2151 comparisons. Three genes fall outside the threshold: CORO1A (0%), CDC42 (0.2%), and AURKB . Expression of CDC42 increases with age among the CEU grandparents, and higher age-adjusted expression is associated with higher mortality. CORO1A and AURKB represent the opposite extreme of this same pattern: expression decreases with age, and higher age-adjusted expression is associated with lower mortality.approach , we compr = 0.51; P< 2.2 × 10−16) between Z-scores for age-related expression change and age-adjusted survival in Overall there is a fairly strong positive correlation , SEPT2 (1.6), PBX3 (1.1), CIB1 (−0.91), SH3BGRL (0.77), UBE2A (−0.71), RNH1 (−0.60), PPP1R11 (0.55), QDPR (−0.48), DDX24 (0.36), GINS2 (−0.30), LPXN (0.24), and ACAA1 (0.16). Clearly, the positive association of CDC42 with age at draw dominates this model. This remains true at steps 20, 40, 60, and 80 (data not shown). Expression levels of CDC42, SH3BGRL, QDPR, and RNH1 were also among the ten most strongly associated with age at draw for CEU grandparents in the univariate analysis reported in We used the step 14 model from An alternative approach to modeling survival as a function of estimated biological age would be to model it as a function of the difference between biological and chronological age. This is equivalent to forcing both biological age and chronological age to have the same slope (with opposite signs), and is efficient only if both variables are scaled identically. We evaluated this approach by rescaling biological age to have 0 mean and unit variance, then multiplying by the standard deviation of chronological age (7.9) and adding the mean chronological age (71.5); that technique produced results (not shown here) essentially identical to our original method, reported above.The above analysis demonstrates that biological age, estimated from gene expression levels that change with age, is a significant predictor of remaining life span. An important potential weakness of this analysis is that it places too much emphasis on gene expressions that vary systematically with age in cross-sectional data. As noted above, the confounding of differential survival effects with age-related changes in expression could mask the effects of genes that regulate or contribute to survival.In an effort to circumvent this limitation, we also applied the LASSO approach to model survival as a direct multivariate function of expression levels. We computed deviance residuals from a baseline survival model adjusted for age at draw and sex, and used LASSO to identify expression levels associated with variation in the deviance residual [see Methods and reference ]. We useP-value = 4.0 × 10−8; inter-quartile relative risk (IQRR) = 2.35; median estimated survival difference = 5.5 years. Predicted mortality from the model accounts for 23% of the variation (R2 = 0.23) in survival among the CEU grandparents. Model coefficients for individual genes are converted into IQRR in P-value given in The nominal Although Inter-individual differences in gene expression profiles in the Utah CEU lymphoblastoid cell lines may reflect not only heritable genetic influences, but also environmental exposures experienced at any time prior to blood draw. Therefore, we considered the possibility that gene expressions associated with survival may simply reflect exposures (or nonexposure) to a common toxic agent, such as cigarette smoke. The data available to us do not contain information on environmental exposures; however, affiliation with the Church of Jesus Christ of Latter-day Saints (or LDS church), available from the UPDB, indirectly provides information about exposures that affect mortality risks. r2 across the entire dataset is 0.962 while the maximum r2 for 100 random pairs of grandparents was slightly smaller . Overall, then, spouse expression profiles are slightly more strongly correlated than expected by chance, although the level of correlation among all expression profiles is very high. Interestingly, however, the correlation of mortality risk between spouses  is only 0.075 , so correlations in expression are not likely to confound the survival analysis.It is apparent in It is not surprising to find that gene expression patterns vary with age, nor that variation in gene expression correlates with variation in longevity; many studies in model organisms have clearly demonstrated these associations. However, the observation that patterns of gene expression in lymphoblastoid cell lines contain information about both the age and subsequent survival of human donors is both biologically interesting and potentially of great clinical utility.et al.:While “Our findings that Cdc42 activation is associated with natural aging and that Cdc42GAP deficiency causes a DNA damage repair defect to allow cells to accumulate genomic abnormalities that leads to early senescence further suggest a functional link between Cdc42 activity and mammalian aging (The results of this study support this conclusion.CORO1A) is an actin-binding protein with potentially important functions in both T-cell mediated immunity and mitochondrial apoptosis , several single nucleotide polymorphisms near the IQGAP1 gene are strongly associated with IQGAP1 expression .Thus, the region immediately surrounding IQGAP1 may harbor genetic variants associated with variation in human lifespan.Several other genes exhibiting associations with either aging or survival are worthy of note. AURKB is a key member of the chromosomal passenger complex which is critical in the regulation and conduct of mitosis . Goring H2 = 0.09; P= 0.10 in our data) or untransformed . This may be a consequence of the cell transformation process, which activates telomerase so that cell lines may grow indefinitely in culture; possibly variable TERF2IP expression is marking some variation in telomerase activity in transformed lymphocytes that is indirectly related to longevity. A recent report links longer telomeres to increased risk of breast cancer  in lymphoblastoid cell lines is associated with increased mortality, because increased expression of TERF2IP should lead to increased telomere length , which hBecause of the large number of comparisons we have generated for a relatively small number of samples, and because the potential for measurement error is inherent in microarray data, the associations we have observed require confirmation in other settings. Associations between gene expressions and survival may be due to causal roles in regulating aging; alternatively, the expression of a gene may not reflect a regulatory role in aging, but nevertheless may serve as a reliable indicator of overall health and remaining life expectancy. Strong evidence for a regulatory role in aging would be provided by experiments in model systems showing that modulating expression extended lifespan. Within the CEU family data, we are pursuing linkage and genetic association studies to identify loci responsible for regulating expression of the heritable expression phenotypes associated with longevity. The identification of germ line variants that affect the expression of longevity-associated genes will facilitate further high-throughput genetic epidemiologic studies of human aging. However, even the nonheritable gene expression patterns we have reported may have considerable relevance to mechanisms of aging and age-related disease processes. Finally, we note that expression data are available for approximately 6000 genes not included in the ‘always-expressed’ list to which we restricted this analysis. Approximately half of these genes are expressed in many, but not all samples. The other half are rarely, if ever, expressed above background. We plan to investigate the association between longevity and expression of these genes as well.We have described some striking patterns of association of gene expression with age and mortality, based on lymphoblastoid cell lines derived from ordinary blood samples, and stored for years as a replenishable source of DNA for genetic studies. If these results can be confirmed by others, frozen cell lines may also have considerable value as sources of phenotypic information on transcription, translation, and other cellular processes helpful in predicting the future health of the donors.The CEPH/Utah family resource originated from bloods drawn from 46 three-generation families, each consisting of 5–15 siblings, their two parents, and two to four grandparents who were still alive at the time of the family blood draws in the early 1980s (http://locus.umdnj.edu/nigms/ceph/ceph.html) for a total of 247 CEU family members , including 104 of the grandparents for whom we had survival data. Expression levels for 8793 probesets were measured using Affymetrix HG-Focus arrays. The resulting expression data were deposited in the NCBI Gene Expression Omnibus database, accession numbers GSE1485 and GSE2552. We combined both datasets to maximize the number of individuals available for study.Cheung and co-workers  or ‘marginal’ (P< 0.06) in each of the grandparents’ samples. This step left us with 2151 always-expressed genes.We tested whether each gene was expressed beyond baseline in each sample using the Wilcoxon signed rank test as described in the Affymetrix Microarray Suite version 5 (gcrma) method to normalize all expression levels.We used Wu and Irizarry’s GeneChip robust multiarray averaging . For these individuals, we averaged gcrma-corrected expression levels for each probeset prior to analysis.We performed two separate analyses of expression as a function of age at draw. First, we considered only the grandparents, who were effectively unrelated to one another, although careful analysis of the records of the UPDB  has reveStrong genetic correlations in expression level should be present within each three-generation CEU family. For this reason, standard linear regression approaches are not appropriate for the study of age-related variation in expression patterns. Instead, we used linear mixed-effects models to adjust for the kinship among family members . The subr software package (http://www.r-project.org). Although some nonproportionality was detected with P-values below 0.0001, none of the genes strongly associated with survival had a P-value for nonproportionality lower than 0.28 (EMP3).Grandparents ranged in age from 57 to 97 years of age at the time of the blood draw. Median follow-up age was 84.7 years (range 65.7–100.8). Survival was measured from age at draw to age at death or follow-up. We used a proportional hazards model adjusting for sex, year of birth, and age at draw to test the association of each expression level with survival. Each proportional hazards model was tested for nonproportionality using the cox.zph function in the We computed Adjustments for multiple testing are clearly appropriate in this context, but many methods mask hidden assumptions about the dependency structure of the data or the true proportion of false null hypotheses . We therCp or by cross validation.To assess the relationship between age at draw and multiple expression levels, we employed the LASSO algorithm of Tibshirani  in our sample of 104 grandparents. We compared the resulting LOOCV curve to curves generated from 100 random permutations of the data, performed as described above. Comparing the cross-validation curve to a null distribution of cross-validation curves not only gives information on the optimal setting of the tuning parameter, but also on the probability that the result is due to chance.We set For each value of the tuning parameter, corresponding to a step in which a predictor variable can be added or dropped, the model selected was used to predict each subject’s age at draw. These ‘biological age’ estimates, together with the subjects’ actual age and sex, were used to predict age at death in a proportional hazards model.We followed the approach of


CYP1B1 is a P450 enzyme which is involved in the activation of pro-carcinogens to carcinogens as well as sex hormone metabolism. Because differences in the activity of the enzyme have been correlated with variant alleles of single nucleotide polymorphisms (SNPs), it represents an attractive candidate gene for studies into colorectal cancer susceptibility.We genotyped 597 cancer patients and 597controls for three CYP1B1 SNPs, which have previously been shown to be associated with altered enzymatic activity. Using the three SNPs, eight different haplotypes were constructed. The haplotype frequencies were estimated in cases and controls and then compared. The odds ratio for each tumour type, associated with each haplotype was estimated, with reference to the most common haplotype observed in the controls.The three SNPs rs10012, rs1056827 and rs1056836 alone did not provide any significant evidence of association with colorectal cancer risk. Haplotypes of rs1056827 and rs10012 or rs1056827 and rs1056836 revealed an association with colorectal cancer which was significantly stronger in the homozygous carriers. One haplotype was under represented in the colorectal cancer patient group compared to the control population suggesting a protective effect.Genetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations. Adverse interactions between DNA and environmental toxins may be mitigated through a range of biologic defense mechanisms, including DNA repair, cell cycle checkpoint control and xenobiotic clearance enzymes. Xenobiotic clearance is important for the removal of carcinogens and is primarily accomplished by hydroxyl conjugation, involving enzymes in the cytochrome P450 pathway . CancersAs part of a major initiative to identify major cancer susceptibility genes for colorectal cancer, we investigated three common variants in the CYP1B1 gene that individually confer a difference in the activity of the encoded protein. All three variants, codon 48G > C (rs10012), codon 119G > T rs1056827) and 432 G > C (rs1056836) have been shown to result in an increased metabolism of 16a-OH-estradiol [6827 and The Polish population is extremely homogeneous and lends itself to population based studies as there are no significant ethnic differences to take into consideration. 597 colorectal cancer patients were enrolled in the study. Enrollment occurred between 1998 and 2006 from 3 cities in Poland  and 114 bp, respectively . The resultant products were size separated on 4% agarose gels. The uncut product was 335 bp in size and the cut product was predicted to yield 4 fragments of 230,105, 91 and 14 bp in size. Only the 230,105 and 91 base pair fragments were visualized, since the 14 base pair product was too small to be readily detected using this approach  and Fisher's exact test, where appropriate. Bonferroni correction was applied to take into account multiple testing.http:// cran.r-project.org/src/contrib/Descriptions/haplo.stats.html as reported by Schaid et al. [Eight haplotypes could be generated from the three SNPs. The frequencies of each of these were estimated in the cases and controls using the genetic statistical package haplo.stats for R d et al. . The CGGEach SNP of the CYP1B1 gene generated three genotypes, including two homozygote states and one heterozygote state. For each SNP, for each site, the odds ratio and 95% confidence intervals were constructed. For these comparisons, the most common allele was considered to be the normal allele and odds ratios were constructed with reference to individuals with two common alleles. The control population for this study was used to determine the relative frequency of the CYP1B1 alleles in the underlying Polish population.For each individual SNP genotyped no significant association between it and colorectal cancer was observed (see Table When rs1056827 was investigated with rs1056836 a similar trend was forthcoming Table , where hIn order to determine if there were groups of individuals who may be greater risk by being homozygous for all three SNPs we investigated the combination of homozygote carriers against each other, where the wild type haplotypes were considered to represent population risk (see Table This study examines the potential influence of CYP1B1 genetic variants alone, and in combination, on colorectal cancer risk. Each of the three SNPs when considered individually appeared not to be associated with disease risk for colorectal cancer. When considered together, the magnitude of the colorectal cancer risk appeared to be consistent yet modest. One previous report of CYP1B1 and CRC risk provided some evidence for an association with disease but this disappeared once a correction was applied for multiple testing . The repSeveral studies have examined the relationship between individual CYP1B1 polymorphisms and cancer risk -17. The The variants in codons 119 and 432 are associated with different substrate specificities and consequently the catalytic activity of the enzyme ,17. TherFinally, in studies similar to the one reported herein it would have been improved by including the effects of environmental factors on disease risk. The current study was not intended to address environmental influences on disease risk rather to determine whether or not there is an over-representation of genetic markers in a case compared to a control population thereby providing information on the relative involvement of the genetic marker on disease risk.In summary, the CYP1B1 variants examined in this study suggest that they contribute to inter-individual differences in cancer risk and are potentially valuable in genetic risk assessment. These findings have potentially important implications for genetic risk assessment and for prevention studies, but it is important that they be confirmed in other colorectal cancer populations prior to introducing them in the clinical setting.Genetic variants within the CYP1B1 that are associated with altered function appear to influence susceptibility to a colorectal cancer in Poland. Three haplotypes were associated with altered cancer risk; one conferred protection and two were associated with an increased risk of disease. These observations should be confirmed in other populations.The authors declare that they have no competing interests.JT participated in the design and coordination of the study and carried out the molecular genetic studies, performed the statistical analysis and the manuscript. EGK participated in the collection of samples and carried out the molecular genetic studies. JS participated in the collection of samples. BM performed the statistical analysis. PSF participated in the design of the study and performed the statistical analysis. GK participated in the collection of samples. CC participated in the design of the study. BG participated in the design of the study. TH participated in the collection of samples. TB participated in the collection of samples. JG participated in the collection of samples. EZ carried out the molecular genetic studies. JK participated in the collection of samples. ZB participated in the collection of samples. RW participated in the collection of samples. EK coordinated the preparation of material for the study. JL conceived of the study and participated in its design and coordination. RJS conceived of the study and participated in its design, prepared the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/420/prepub


Pteridium aquilinum is triggered by herbivory and if so - whether it is regulated by the octadecanoid signaling pathway. Interestingly, feeding of both generalist  and specialist (Strongylogaster multifasciata) herbivores as well as application of singular and continuous mechanical wounding of fronds induced only very low levels of VOC emission. In contrast, treatment with jasmonic acid (JA) led to the emission of a blend of VOCs that was mainly comprised of terpenoids. Likewise, treatment with the JA precursor 12-oxo-phytodienoic acid (OPDA) and α-linolenic acid also induced VOC emission, albeit to a lower intesity than the JA treatment. Accumulation of endogenous JA was low in mechanically wounded fronds and these levels were unaffected by the application of oral secretions from both generalist or specialist herbivores. The emission of terpenoids upon JA treatment could be blocked with fosmidomycin and mevinolin, which are inhibitors of the MEP- and MVA pathways, respectively. These results indicate that similar to higher plants, terpenoid VOCs are produced via these pathways in bracken fern and that these pathways are JA-responsive. However, the very low amounts of terpenoids released after herbivory or mechanical damage are in stark contrast to what is known from higher plants. We speculate that S. multifasciata and S. littoralis feeding apparently did not induce the threshold levels of JA required for activating the MEP and MVA pathways and the subsequent volatile emission in bracken fern.Jasmonate-mediated regulation of VOC emission has been extensively investigated in higher plants, however, only little is known about VOC production and its regulation in ferns. Here, we investigate whether the emission of VOCs from bracken fern  Apart from this report, there is virtually no data available regarding indirect defence responses in lower plants such as ferns. In the present study, we investigate the regulatory events underlying VOC emission in the bracken fern P. aquilinum. Studying this trait, which functions in all higher plants studied so far as an indirect defence, in a phylogenetically more ancient plant like bracken might shed light on the evolutionary origin as well as the ancestral function of VOC emission.Bracken fern ?Spodoptera littoralis) and a specialist herbivore (Strongylogaster multifasciata)?Does bracken release a similar VOC blend after simple or continuous wounding and upon feeding of a generalist  involved that are also used by higher plants?Pteridium aquilinum (L.) Kuhn, Dennstaediaceae were collected from a forest about 15 km from Jena  and whole plants were brought to the greenhouse for further propagation. Experiments were done on plants vegetatively propagated from these in the greenhouse and grown at a temperature of 27–30°C and 45–50% humidity, under 16 h photoperiod in Klasmann clay substrate .Spodoptera littoralis Boisd.  was reared on artificial diet   were collected between May–June 2009 at the same field site as the plants, identified to species level P. aquilinum.The generalist herbivore, Plant volatile response is known to be regulated by members of the octadecanoid signaling pathway. We used α-linolenic acid, JA, COR and the pore-forming peptide alamethicin (ALA) to test for their capacity to induce VOC emission in bracken fern. Since ion fluxes and membrane depolarisation are elements of the early responses to herbivory −1 water . ALA was initially dissolved in methanol at 10 mg ml−1 and this stock solution was diluted in tap water to obtain the final concentration. Fronds were damaged mechanically by puncturing 2–3 rows of holes with a pattern wheel. Continuous mechanical damage was inflicted using the MecWorm system S. littoralis or S. multifasciata (both reared on fern diet) were placed on each frond, allowed to feed for a day and VOCs were collected for 24 h. S. littoralis larvae were allowed to feed on fern fronds for 1–2 days before the onset of the experiments.For elicitor treatments, JA (1 mM), OPDA (1 mM), α-linolenic acid (2 mM) and COR (100 µM) were sprayed as aqueous solution on the fern fronds of intact plants. These concentrations were chosen based on previous literature reports, in which these compounds have been shown to induce VOC emission in other plant species Spodoptera littoralis grown on P. aquilinum diet, or from field-collected Strongylogaster multifasciata larvae. To reproducibly mimic feeding of an herbivore, 20 µl of the OS was diluted 1∶1 with de-ionized water and applied to mechanically damaged (pattern wheel) intact fronds. Mechanically wounded fronds treated with water served as a control in all experiments.Oral secretions (OS) were collected from third instar larvae of JA was prepared by saponification from commercially available MeJA . COR, OPDA and the deuterated standards for phytohormone analysis were synthesized according to literature procedures Terpenoids are the main constituents of the VOC blend emitted from herbivore-induced plants. In higher plants, these VOCs are synthesized via either the MVA  or the MEP (methylerythritol phosphate) pathways. In order to understand the origin of the terpenoids produced by bracken fern, each of these pathways was specifically inhibited using certain inhibitors. Fosmidomycin was used to inhibit the DXP-reductoisomerase of the MEP pathway and mevinolin to block the HMGR-CoA reductase, the main enzyme of the MVA pathway 2H2]JA and 40 ng [13C6]JA-Ile was added to the samples and after vortexing the samples were centrifuged for 15 min at 13,200 g (4°C). The upper organic phase was transferred into a fresh tube and the leaf material was re-extracted with 0.5 ml ethylacetate. The organic phases were pooled and evaporated to dryness. The dry residue was reconstituted in 0.4 mL of 70/30 (v/v) methanol/water for analysis with an LC-ESI-MS/MS instrument . Ten µl of the sample were injected in a ProntoSIL® column  connected to a pre-column . As mobile phases 0.05% formic acid in water (solvent A) and methanol (solvent B) were used in a gradient mode with the following conditions: time/concentration (min/%) for B: 0.0/15; 2.5/15; 4.5/98; 10.5/98; 12.0/15; 15.0/15; time/flow (min/mL): 0.0/0.4; 1.5/0.2; 1.5/0.2; 10.5/0.4; 15.0/0.4. Compounds were detected in the ESI negative mode and multiple reaction monitoring (MRM) mode 2H2]JAIle.For analysis of the octadecanoids JA, JA-Ile and OPDA, 0.2 g of frozen leaf tissues were homogenized to a fine powder in liquid nitrogen. 1.0 mL of ethylacetate spiked with 200 ng [S-(OOH)-18:3 and 9(R/S)-OOH-18:3 were used as standards .As a next step, the precursors of JA biosynthesis namely the hydroperoxy and free fatty acids were analyzed in order to understand precursor availability. Hydroperoxy-fatty acids were extracted with the chloroform-methanol method as follows: 1 g of frozen leaf material was homogenized in 10 ml glass tubes containing 3.75 ml of ice-cold 2/1 (v/v) chloroform/methanol spiked with 5 ng of 15-hydroperoxy-eicosadienoic acid . After adding of 1.25 ml chloroform and 1 ml water, samples were vortexed and the phases were separated. The organic phase was collected and the water phase re-extracted with 3 ml hexane. The hexane and chloroform fractions were pooled and the solvent was evaporated under a stream of nitrogen. The samples were reconstituted in 70% (v/v) methanol/water and analyzed by liquid chromatography-(ESI)-tandem mass spectrometry (LC-MS/MS) as previously described 2SO4/water were added and after centrifugation, the organic phase was collected and the aqueous phase re-extracted with 3 ml of 3/2 (v/v) 2-propanol/hexane. The organic phases were combined, the solvent evaporated under a stream of nitrogen and the samples reconstituted in 1 ml of 1% (v/v) H2SO4/methanol. The samples were heated for 1 h at 75°C and the fatty acid methyl esters (FAMES) were extracted with hexane. FAMES were analyzed in a Varian CP-3800 GC coupled with a Varian Saturn 3800 ion trap MS in electron ionisation  mode . 1 µl of the sample was injected in splitless mode on a DB-WAX column  with helium at a constant flow of 1.0 ml min−1 as the carrier gas. The injector was at 230°C. The oven temperature program was: 130°C for 5 min, 220°C at 3.0°C/min, 5°C/min ramp to 240°C and hold for 1 min. EI spectra were recorded on Scan mode from 40 to 400 amu. Quantification was performed in the linear range of detection and based on calibration curves generated with increasing concentrations of commercial FAMES (fatty acid methyl esters) mixes  and the IS (17∶0).For fatty acid analysis, 0.5 g of leaf material was ground in liquid nitrogen, transferred into 10 ml glass tubes containing 2 ml of 2-propanol and quenched by heating for 10 min at 80°C. On cooling, 3 ml of hexane and 2 ml of 6.7% (w/v) NaBratschlauch’  that does not emit detectable volatiles by itself . The traps were eluted with 2×20 µl dichloromethane containing 200 ng µl−1 of 1-bromodecane as an internal standard. Leaves were dried for dry weight determination. VOCs samples were analysed on a Thermo Finnigan Trace GC-MS  equipped with a fused silica Alltech EC5 column  using 1.5 ml min−1 helium as carrier gas. Separation was achieved under programmed conditions . MS analysis was performed in electron impact full-scan mode at 70 eV with source temperature at 200°C and GC interface temperature at 250°C. Compounds were identified tentatively by comparison to the NIST database and subsequently collated with spectra from reference compounds . Individual compounds were quantified with respect to the peak area of the internal standard and related to the dry weight of the frond.Treated fronds were bagged individually in a PET foil ‘see e.g.  and VOCsDifferences between treatments were evaluated as a ‘general linear model’ command with ‘treatment’ as fixed and ‘plant individual’ as random factor. For multiple comparisons, LSD post hoc test tests were used in case variances were homogeneously distributed and Tamhane's T2 post hoc test if this assumption was violated. All statistical analyses were done using SPSS 17.0 .Strongylogaster multifasciata nor the generalist herbivore Spodoptera littoralis on intact fronds increased VOC emission rates in bracken . Damaging the fern fronds using a pattern wheel (singular damaging event) induced low VOC emission levels that were statistically indisting-uishable from undamaged controls. To verify whether this result was merely the consequence of the damage levels, we employed a mechanical device .Interestingly, neither feeding of the specialist herbivore  bracken . Even inP<0.02, n≥3–6 per treatment). Both coronalon (COR) and jasmonic acid (JA) induced the highest production levels of VOCs of all treatments.Next, The total amount of VOCs released by bracken upon treatment with various elicitors  was analyzed and compared to the effect of mechanical wounding and herbivore damage . The totE)-(β)-farnesene, aromadendrene epoxide, as well as a methylester of palmitic acid. Qualitative composition of the emitted VOC blends also differed strongly between different treatments (E)-β-farnesene was the most dominant compound emitted after JA or COR treatment (E)-β-farnesene was only detected after ALA-, but not after OPDA treatment. Limonene was emitted after all elicitor treatments, except after α-linolenic acid- and damage treatments. Interestingly, herbivory inflicted by a generalist herbivore  induced the emission of limonene, yet in very small amounts -β-freatment . 1-Octenreatment . ALA ind amounts .P<0.03, n = 4).To verify to which extent the observed VOC emission pattern was dose-dependent, the JA concentration applied to each intact frond was varied and the resulting emission rate of VOCs quantified. Fronds, which have been treated with different JA concentrations ranging between 0.05 mM and 1 mM, indicated that a concentration of 1 mM JA was required to significantly increase total VOC emission levels over control levels  accounted for 34% and unsaturated fatty acids  constituted 65% of the total amount of these molecules , the initial precursor for JA biosynthesis  upon wounding as a function of time . Endogenolecules . Mechaniynthesis .Spodoptera littoralis; reared on fern diet; Strongylogaster multifasciata; reared on fern diet; P>0.05, n = 5).To analyze the effect of herbivory on endogenous JA levels, oral secretions (OS) from generalist herbivores , compared to JA-treated plants, whereas the amount of monoterpenes emitted after inhibition with fosmidomycin was significantly lower compared to JA-treated plants . The observation that fosmidomycin was more effective in blocking monoterpene production than mevinolin, indicated that the MEP pathway accounted for the formation of these compounds (E)-β-farnesene was suppressed by both inhibitors by almost 90% as compared to JA treatment, which suggests that the plastid-derived MEP pathway supports the formation of both mono- and sesquiterpene synthesis in bracken fern.Since bracken produced both mono- and sesquiterpenes in response to JA treatment, we investigated which metabolic pathways were involved in the production of these compounds using specific inhibitors (fosmidomycin and mevinolin). Treatment with these inhibitors did not affect the emission rates of Compounds . On the Several fascinating functions for the emission of plant VOCs have been discovered. However, the basic questions why there is such a structural diversity of VOCs as well as how these evolved to function as an indirect defence mechanism in plants remain largely unanswered. VOC emission could have originated as a direct defence strategy against herbivores or pathogens or as a means to attract pollinators P. aquilinum is also regulated by jasmonates, ii) the same elicitors  that are known from higher plants trigger VOC emission in bracken. The impact of mechanical damage as a component of herbivore feeding was studied using a computer-controlled device (i.e. ‘Mecworm’).For many higher plants, it is well known that insect feeding induces the emission of a VOC blend that strongly resembles the one released upon exogenous application of JA Phaseolus lunatus, treatment with ALA mainly induced the emission of homoterpenes; the emitted VOC blend resembled OPDA-treated, rather than JA-treated plants. In the present study, ALA treatment elicited a VOC profile that was more similar to OPDA-treated than JA-treated plants E)-β-farnesene (E)-β-farnesene was also the most dominant VOC emitted from the fern Pteris vittataArabidopsis thalianaBotrytis cinerea, suggesting that 1-octen-3-ol may be recognized by the plant as a signal for presence of fungal pathogens When ferns were treated with COR, the VOC blend emitted resembled the one emitted from JA treated plants both qualitatively and quantitatively  and 2a. arnesene , a sesquPteridium aquilinum), elicitors like JA, COR OPDA, α-linolenic acid, and ALA induced the emission of certain VOCs, which is in line with previous studies using higher plants. Interestingly, continuous damage by the ‘Mecworm’ did not result in significantly increased VOC emission levels in the fern . Larval feeding of this herbivore, however, resulted in much lower emission levels of VOCs than were released from elicitor-treated ferns −1  and 6. TGinkgo biloba has been shown to produce increased amounts of VOCs upon JA treatment, but failed to emit any volatiles after tissue damage A. thaliana, tobacco, tomato, soybean, lima bean and maize Pteris vittata responded with an emission of terpenoids after herbivory Interestingly,


Cancer/testis (CT) antigens represent promising targets for immunotherapy. We investigated the composite expression of 13 CT antigens by RT-PCR in 79 lung cancer tissues and by immunohistochemistry in 22 lung cancer tissues. In the 79 lung cancer tissues, MAGE-3 (42%) was expressed most frequently and followed by NY-SAR-35 (33%), NY-ESO-1 (30%), MAGE-1 (27%), CT-7 (20%), MAGE-4 (19%), LAGE-1 (16%), and MAGE-10 (14%). Twenty-one tissues did not express any of the CT antigens tested, 58 (73%) expressed at least one, 36 (46%) co-expressed two, 24 (30%) co-expressed three, 17 (22%) co-expressed four, 14 (18%) co-expressed five, 8 (10%) co-expressed six, 4 (6%) co-expressed seven and 2 tissues expressed 9 of the 13 examined CT antigens. Expression of CT antigens was significantly associated with age (P<0.001), smoking history (P=0.009), and gender (P=0.001) of patients, whereas no correlation was found between the expression of CT antigens and other clinical factors, such as pT status, pN status, tumor stage, and histology history. The present results show that CT antigens are potential candidates in lung cancer patients for polyvalent immunotherapy. Lung cancer is the major cause of cancer-related mortality worldwide and is also one of the most frequent causes of death in Korea . The higAn essential condition for the development of effective immunotherapeutic strategies is the existence and identification of tumor specific antigens that are either exclusively or preferentially expressed in malignant compared to normal tissues . Human tCT antigens are immunogenic proteins expressed in normal testis and in different types of tumors ,9. CT anThe present study analyzes the frequency of expression of 13 CT antigens in 79 lung cancer tissues. The correlation between CT antigen expression patterns and pathological characteristics of lung cancer tissues was also studied.Human tumor tissues were obtained from lung cancer operations performed at the Pusan National University Hospital, Busan, Korea. Analyses were performed on tumor tissue samples of 79 patients with lung cancer confirmed pathologically. The tumor samples consisted of 33 cases of adenocarcinoma, 24 of squamous cell carcinoma, 6 of neuroendocrine carcinoma, 4 of pleomorphic carcinoma, and 12 of others . Among the 79 lung cancer patients from whom tissue samples were obtained, 68 had available records, but 11 did not. Data for the 68 tissues including gender, age, classification of TNM status, and stages were obtained from the clinical and pathological records. Tumor stage and progression were classified according to the International Staging System .Total cellular RNA was extracted from frozen tissue specimens of 79 lung cancer samples, using TRI Reagent . RNA extraction using TRIzol , a common protocol, was used. Tumor tissues removed at surgery were snap-frozen and stored at −70°C. Total-RNA was isolated from ~100 mg of each tissue sample using 1 ml TRI Reagent , extracted with chloroform, precipitated with isopropyl alcohol, washed with ethanol and re-dissolved in RNase-free water. The amount of isolated RNA was measured by a spectrophotometer  at 260 nm.The cDNA preparations used as templates in the RT-PCR reactions were prepared by using 500 ng of total-RNA in conjunction with the SuperScript First Strand Synthesis kit (Invitrogen Life Technologies).The 13 primer sets for the 13 CT antigens and the lengths of each PCR product are shown in The M3H67 monclonal antibody (to MAGE-3) was obtained from the Ludwig Institute for Cancer Research, New York Branch at the Memorial-Kettering Cancer. Immunohistochemical staining was performed according to the previously report . Briefly2 test was used to compare the correlation between disease stage, grade, and CT antigen expression. Statistical significance was accepted at P<0.05.Statistical analysis was performed with the SPSS program . Pearson χExpression of the 13 CT antigens  was assessed by RT-PCR in 79 lung cancer tissue samples. Representative RT-PCR results from lung cancer tissues are shown in The percentages of co-expressed CT antigens in lung cancer tissues are shown in The characteristics of the patients are summarized in Among the 79 lung cancer tissue specimens, 22 lung carcinoma samples from which sufficient material was available were investigated for the expression of the frequently expressed MAGE-3 protein by immunohistochemistry. Representative immunohistochemistry results of lung cancer tissues with the mAb MAGE-3 are shown in CT antigens are immunogenic proteins expressed in normal testis and in different types of tumors. Because of their tissue-restricted expression, CT antigens are ideal candidates for antigen-specific cancer immunotherapy. In general, CT antigens are expressed in 20–40% of specimens from a given tumor type . The preIn addition, 58 of 79 lung cancer tissue specimens (73%) were found to express at least one of the CT antigens. According to our results, 73% of our lung cancer patients would be eligible for specific immunotherapeutic approaches with at least one CT antigen. A possibility for the high observation frequency of CT antigen expression in lung cancer tissues could be due to the characteristics of the patients. These results indicate that expression of CT antigens was significantly associated with the age and gender of the patients, whereas no significant correlation was detected between CT antigen expression and other clinical factors such as pT status, pN status, and tumor stages . In thisThe highly homologous NY-ESO-1 and LAGE-1  are highly immunogenic, and a CTL response to an immunodominant, HLA-A2-restricted NY-ESO-1/LAGE-1 epitope can commonly be detected in cancer patients ,30. We cOf particular interest was NY-SAR-35, which represents a CT antigen as it appears to be a rare example of a cell surface antigen , which wTo confirm the existence of MAGE-3 protein expression, 22 lung carcinoma samples from which sufficient material was available were immunohistochemically stained by a specific MAGE-3 monoclonal antibody. Similarly to our previous study , we founFrom the data of our study, a high proportion  of lung cancer tissues was positive for at least one of these 13 CT antigens as targets will greatly increase the number of candidates for CT antigen-based lung cancer immunotherapy. However, our results showed no expression of CT antigens in 27% (22/79) of lung cancer tissues. For these patients, it is necessary to screen other CT antigens or tumor-specific antigens serving as immune targets for lung cancer tissue immunotherapy. In conclusion, this study demonstrated that lung cancer tissues frequently express CT antigens and a high percent express more than one CT antigen, suggesting that CT antigens are potential candidates for polyvalent immunotherapy.


Lung cancer is the most common cause of cancer-related mortality worldwide. Survival rates after non-small cell lung cancer (NSCLC) diagnosis still remain poor, despite standardization of surgery and adjuvant treatment. Cancer testis (CT) antigens as targets for immunotherapy in cancer patients have been heavily investigated. We aim to evaluate the expression of CT antigens in non-small cell lung cancer and the correlation with the clinical characteristics.The expression of MAGE-A3, MAGE-A4, MAGE-C2 and NY-ESO-1 in fresh cancer tissues from 113 patients with non-small cell lung cancer were analyzed by RT-PCR. The expression of HLA-A2 was detected by flow cytometry.49.6% non-small cell lung cancer samples were HLA-A2 positive. CT antigens were frequently expressed in non-small cell lung cancer. The expression percentage of each gene in cancer tissues samples were as follows: MAGE-A3, 70.8%; MAGE-A4, 46.9%; MAGE-C2, 61.1%; and NY-ESO-1, 15.0%. In all, Seventeen tissues did not express any of the CT antigens tested, 85.0% expressed at least one, 66.4% co-expressed two, 34.5% co-expressed three, 6.2% co-expressed four examined CT antigens. The percentage of samples co-expression HLA-A2 and CT genes were: MAGE-A3, 51.6%; MAGE-A4, 36.3%; MAGE-C2, 44.0%; and NY-ESO-1, 12.1%. MAGE-A3 was associated with male, smoking history, tumor stage, tumor differentiation and lymph node metastasis (P<0.05); MAGE-C2 was associated with tumor stage (P<0.05). There was no correlation MAGE-A4 or NY-ESO-1 expression with clinical characteristics such as gender, age, HLA-A2 positive, clinical stage, grade of differentiation and lymph node metastasis (P>0.05).CT antigens might be prognostic markers and factors related to the progress of NSCLC, and also be served as the immunotherapeutic targets of NSCLC, especially in multi-antigen vaccine preparations and tumor antigen specific lymphocytes infusion.


Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers  and 189 breast cancers, while MAGE-A proteins were present in 15% and 7–16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer. Modulation of the immune system in cancer patients has shown to efficiently generate anti-tumor immune responses, but selection of targets for effective and specific intervention remains challenging. The unique expression pattern and immunogenic properties of cancer/testis (CT) antigens make them ideal targets for different types of cancer immunotherapy, such as vaccination and adoptive transfer with recombinant T-cell receptor-transduced T cells. CT antigens are male germ cell proteins ectopically expressed in various malignancies –3. Male http://www.cta.lncc.br), but only a small number of these have been investigated for expression profiles. Although some CT antigens tend to be co-expressed in a subset of tumors, others have distinct and cancer-subtype specific expression profiles [More than 200 different CT antigens have been identified  were collected as diagnostic specimens from patients treated at the University Hospital of Odense. The ethical committee of Funen and Vejle County (VF20050069) approved the use of these tissues, without informed consent from participants. The lung (LC1502) and breast (BRC1502) carcinoma tissue microarrays were purchased from BioCat GmbH, Heidelberg, Germany. The lung carcinoma tissue microarray LC1502 contained 23 cases of lung squamous cell carcinoma, 21 lung adenocarcinoma, 5 each of lung adenosquamous carcinoma and bronchioalveolar carcinoma, 7 small cell undifferentiated lung carcinoma, 1 each undifferentiated lung carcinoma and malignant mesothelioma, 2 each of large cell lung carcinoma and carcinosarcoma, 3 neuroendocrine lung carcinoma, and 1 each of lung chronic bronchitis, lobar pneumonia and pulmonary tuberculosis, 2 normal lung tissue, duplicate cores per case (duplicated cores from the same patient were put onto upper and lower rows in the same position). The breast carcinoma tissue microarray BRC1502 contained 62 cases of ductal carcinoma, 2 lobular carcinoma and 1 each of papillary carcinoma, sarcoma, mucinous adenocarcinoma and tubular carcinoma. The estrogen receptor and HER2 status of this first cohort of breast cancers were not available. Tissue microarrays of the second cohort of breast cancers with information on receptor status were subsequently analyzed. The generation and characterization of this second cohort has previously been reported . The exp202 in Tris-buffered saline (pH 7.5) for 10 min to block endogenous peroxidase activity. Thereafter, they were rinsed in distilled H2O, demasked, processed for antigen retrieval and washed in TNT buffer . To optimize conditions for staining with antibodies raised against amino acids 122–215 of ADAM2 , amino acids 195–294 of CALR3  and amino acids 497–641 of SAGE1 , different concentrations were tested in combination with different antigen retrieval protocols using sections of human testis. The methods of antigen retrieval included microwave boiling for 15 min in 1) T-EG buffer , 2) 10 mM citate buffer, pH 6.0 or 3) Dako Target retrieval solution (Dako S1699), or subjected to proteolytic treatment using 4) 0.05% protease type XIV  in TBS, pH 7.0 for 15 min at 37°C, or 5) 0.4% pepsin  in 0.01M HCl for 20 min at 37°C. A protocol for immunohistochemical staining with mouse anti-MAGE-A  has previously been described [2O and counterstaining of sections with Mayers hematoxylin before mounting in AquaTex .Paraffin-embedded, formalin-fixed tissues were cut in 6 μm sections, deparaffinized, treated with 1.5% HTissue sections were evaluated by immunohistochemistry and considered positive if staining was observed in more than 5 cells, regardless of intensity.Breast  and lung cancer cell lines  were cultured in RPMI or DMEM  supplemented with 10% fetal bovine serum (Sigma Aldrich), penicillin (100 U/ml) and streptomycin (100 mg/ml). For MCF7 and T47D, 6–8 ng/ml insulin (Sigma Aldrich) was added to the media. When indicated, cells were grown with 5μM 5-Aza-2’-deoxycytidine (Sigma-Aldrich) for 48 hours. Cell lines were obtained from ATCC, Wesel, Germany.Total RNA was purified from cultured cells using Isol-RNA lysis reagent  and RevertAid Premium Reverse Transcriptase kit (Fermentas/Life Technologies) was used for cDNA synthesis with random hexamer primers. Relative quantification of gene expression was performed with SYBR green PCR Mastermix . PCR primers were: ADAM2 , CALR3 , SAGE1  and PUM1 .http://www.cta.lncc.br), in contrast to MAGE-A , which constitute about 85% of lung cancers ; Fig 2. We also investigated the expression of ADAM2, CALR3 and SAGE1 in 13 breast cancer cell lines  and in bThe absence of ADAM2, CALR3 and SAGE1 expression in lung and breast cancer prompted us to test their expression in a panel of melanoma cell lines ; Fig 3, Infrequent or heterogeneous expression of tumor antigens in tumors may be an obstacle to development of efficient and widely applicable cancer vaccines. Importantly, agents that inhibit the function of DNA methyltransferases have been demonstrated to induce the expression of CT antigen genes. For instance, 5-aza-2’-deoxycytidine activates MAGE-A, GAGE, CT45 and many other CT antigen genes , 26–29. We investigated the potential of three previously uncharacterized CT antigens, ADAM2, CALR3 and SAGE1, as targets for treatment of lung and breast cancer. Although we demonstrated that these CT antigens exhibit a germ cell-specific expression pattern in normal tissues, which is an important feature of antigens for cancer immunotherapy, none of these antigens were expressed in lung and breast cancer. Furthermore, the expression of these antigens could not be induced by DNMT-inhibitor treatment, in contrast to many other CT antigens. In conclusion, ADAM2, CALR3 and SAGE1 should not be considered targets for treatment of lung and breast cancer. However, our study also provides the reagents and conditions for assessing expression of these antigens in other cancer types. Furthermore, SAGE1 was detected in about 40% of melanoma cell lines, suggesting that this protein may be an interesting target. Our study represents an important step in the tedious evaluation of the long list of potential tumor antigens to identify novel therapeutic targets for different types of cancer.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


A robust and transferable algorithm is presented to objectively describe and rank robotically captured images of crystallization droplets according to their likelihood of crystalline behaviour for the efficient and accurate identification of successful crystallization. The visual inspection of crystallization experiments is an important yet time-consuming and subjective step in X-ray crystallography. Previously published studies have focused on automatically classifying crystallization droplets into distinct but ultimately arbitrary experiment outcomes; here, a method is described that instead ranks droplets by their likelihood of containing crystals or microcrystals, thereby prioritizing for visual inspection those images that are most likely to contain useful information. The use of textons is introduced to describe crystallization droplets objectively, allowing them to be scored with the posterior probability of a random forest classifier trained against droplets manually annotated for the presence or absence of crystals or microcrystals. Unlike multi-class classification, this two-class system lends itself naturally to unidirectional ranking, which is most useful for assisting sequential viewing because images can be arranged simply by using these scores: this places droplets with probable crystalline behaviour early in the viewing order. Using this approach, the top ten wells included at least one human-annotated crystal or microcrystal for 94% of the plates in a data set of 196 plates imaged with a Minstrel HT system. The algorithm is robustly transferable to at least one other imaging system: when the parameters trained from Minstrel HT images are applied to a data set imaged by the Rock Imager system, human-annotated crystals ranked in the top ten wells for 90% of the plates. Because rearranging images is fundamental to the approach, a custom viewer was written to seamlessly support such ranked viewing, along with another important output of the algorithm, namely the shape of the curve of scores, which is itself a useful overview of the behaviour of the plate; additional features with known usefulness were adopted from existing viewers. Evidence is presented that such ranked viewing of images allows faster but more accurate evaluation of drops, in particular for the identification of microcrystals. Approaches to the problem include edge detection . An image is filtered with all 38 filters in the filter bank, but only the maximum filter response for the edge and bar filters at each scale is recorded, resulting in a vector with eight  rotation-invariant filter responses per pixel. An example of the final filter response vector is shown in Fig. 2b). We chose this filter bank as it was shown by Varma and Zisserman to be the best filter bank when compared with the filter bank proposed by Leung & Malik  building a texton dictionary containing unique texture prototypes and (ii) comparing the filter response of each pixel in a new image to this dictionary to find the closest texton and label the pixel with the corresponding texton label. The conventional way of building this dictionary is by taking a fixed number of textons from each class of textures and combining them to form the final dictionary. However, precipitation behaviour yields a continuum of patterns which are difficult to classify objectively by eye, and since no labelled data set is available in the community, we took a different approach to generate this dictionary, as outlined in the following section.2.1.2.To ensure that crystal-related textons are well represented, we used separate sets of 52 arbitrarily selected crystal-containing images and 100 precipitation images to cover a wide range of patterns  was calculated using the χ2 distance between their histogram,ix and iy are the normalized bin counts of the ith feature of droplets x and y, respectively, n = 300 in this case and the distance is weighted inversely by the sum of the bin counts. The resulting 50 clusters were manually inspected and found to have visual consistency; examples of these clusters are shown in Fig. 5The precipitation patterns of 1400 images randomly selected from 28 crystallization plates were clustered to examine whether the method is able to discriminate droplets based on their patterns. Hierarchical clustering with Ward’s minimum variance method  is masked and the remaining pixels are intensity-normalized to have a mean intensity of 0 and a standard deviation of 1. The following statistical descriptors are calculated from the gradient image of the normalized image: mean, standard deviation, skewness, kurtosis and the distribution of the absolute gradients at fixed 50 bin centres ranging from 0 to 50. These 54 features are used with the area, centroid, eccentricity (ratio of difference between foci and the major axis length), major and minor axis length of the segmented droplet (described next) as inputs to a random forest classifier which predicts whether a droplet image is good or faulty. The classifier used here was trained with 11 326 images (5225 faulty and 6101 non-faulty images) and has an accuracy of over 94%. The majority of inaccurate classification occurs in experiments set up with detergent leading to the much lower contrast of the droplet boundary in such droplets .Errors owing to inaccuracies of the droplet-dispensing robots may result in empty subwells, unusually small droplets (where either the protein or reservoir solution was not added) or droplets sitting at the edge of the subwell. Errors in the imaging system may also produce images where the subwells are partially out of the field of view of the camera. To identify these failed droplets, background images were obtained for each of the subwells by taking the average of that subwell for all wells from an empty plate. This should be performed for every type of plate since it is plate-specific; in this case, we generated background images for the 3-Well Crystallization Microplate (SWISSCI), which is most commonly used at the SGC Oxford. This background image is rigidly registered to a new image by searching for the ts Fig. 7d.2.2.2.DroplIT  of the original segmented droplet. For each column (1–360°) in the polar image the droplet boundary is extended by replacing 13 pixels  outside the radius of the droplet with the median of intensities of the ten pixels closest to the droplet boundary. The median is used instead of the mean to avoid outliers from possible imperfect segmentation. The new extended polar image is then converted back into rectangular space, resulting in a padded droplet. The ring of added pixels is copied to the original droplet image. This provides a sufficient number of neighbouring pixels for the calculation of filter responses for pixels in the original edge; the filter responses for pixels in the extended region are ignored in the final histogram count. Figs. 9b), 9c) and 9d) show a comparison of texton labels with and without droplet extension.Filter responses at the edge of droplets will be dominated by the droplet boundary. To avoid the strong boundary effect, droplets are artificially extended radially and the filter response of this extended region is ignored in the final frequency count. Fig. 92.2.5.The gamma-corrected and extended image is intensity-normalized to have μ = 0 and δ = 1 for intensity invariance and is scaled by a factor of 0.25 to match the textons in the dictionary. The texton distribution is then calculated as described in §2.3.2.3.1.At the SGC, crystallization experiment droplet images are automatically captured with a Minstrel HT system (Rigaku). Experimenters label images with scores between 1 and 10, where labels 1 and 2 are for precipitates, 3–5 for microcrystals and 6–10 for mountable crystals. It should be noted that labelling is optional, and it is usually only the interesting droplets that are labelled. Two sets of training images were selected: (i) ‘interesting’ images, a random subset of 2501 images of droplets that were given labels of 3 and above captured between April 2013 and July 2013, and (ii) ‘uninteresting’ images, a random subset of 3553 images from the same period with scores <3 or with no scores recorded.2.3.2.TreeBagger function of MATLAB. The random forest algorithm was chosen for its speed and its ability to deal with the large numbers of both instances and features, unlike competing algorithms. Given a set of features from new test images, the random forest produces the posterior probability for a particular class, which is used directly as scores for the images. In this case, for the ‘interesting’ class a score of 1 indicates that the droplet is likely to contain crystals or crystalline behaviour, while a score of 0 indicates that it is ‘uninteresting’. In a typical classification exercise, a threshold is set to determine which class the data point belongs to. Tenfold cross-validation of the classifier at a cutoff of 0.5 gave an average area under the ROC curve of 0.9418 ± 0.0027, indicating good separation of scores for both classes, and an average accuracy of 0.8930 ± 0.0044. However, as the intention is to rank and not to classify droplets, no threshold was selected; instead, the scores were used to rank droplets directly.The set of 2501 ‘interesting’ droplets and 3553 ‘uninteresting’ droplets were used to train a two-class random forest classifier  with at least one recorded crystal . Of these, 101 plates were sparse-matrix screens, while the remaining 95 were optimization experiments. Each plate contained 96 wells with three subwells each, where the subwells share the same protein solution and reservoir solution at different mixing ratios . Droplets in these plates were ranked by the algorithm and the highest rank of crystals marked by crystallographers was determined either by subwell or by well, where the maximum score of the three subwells was used.c). When DroplIT failed on the original image, it was instead applied to a gamma-corrected image, as described in §For validation of the robustness of the algorithm across imaging platforms, we analysed images acquired by the Structural Biophysics group of the Novartis Institute for Biomedical Research (NIBR), Basel using a Rock Imager (Formulatrix). Images of 134 plates with at least one recorded crystal were selected. A comparison of this data set with that from the SGC is summarized in Table 12.5.A and B). Each set consisted of three sparse-matrix screens and two optimization screens of different protein targets. Two groups of five crystallographers from the SGC with varying experience were asked to evaluate each plate in a standardized time interval of 2 min, which was insufficient to view all 288 droplets in a plate and thereby simulates the end goal of the program of minimizing the time required to identify all crystals.  The first group viewed set A in a ranked order and set B in an unranked order, while the second group viewed set B in a ranked order and set A in an unranked order. The majority vote  of each group of crystallographers was used to compare against the other group for missed (micro)crystal annotations.To show that such ranked viewing of images results in drops with microcrystals being more carefully evaluated, ten plates containing (micro)crystals were randomly selected from the SGC and divided into two sets  of their probability of being ‘interesting’: this means that crystals or microcrystals are likely to be viewed first, while the ‘uninteresting’ droplets  are viewed later. In the real-world environment where time is limited and attention level decreases with time, the ranking system focuses resources on what is most likely to matter, namely the likely presence of crystallinity.The ranking score is obtained by repurposing a two-class classifier trained on images assigned manually into only two categories, namely containing crystallinity (‘interesting’) or not (‘not interesting’), and therefore containing the respective textons. When applied to new images, the classifier generates a score (0 to 1), which ordinarily would be compared against a threshold in order to assign images to either category; instead, here the score is directly employed for sorting a given set of images .e.g. ‘crystal’ over ‘microcrystal’ over ‘spherulite’ and so on); however, this ranking is algorithmically arbitrary , and moreover it is non-obvious how to rank images within classes, since the classifier scores are no longer unidirectional. On the other hand, Buchala & Wilson , which shows that for most plates the first human-annotated crystal is very high in the ranking order. We show both the results for viewing by well and subwell in Table 2Our approach appears to rank images effectively, if judged by criteria that are reasonable in routine laboratory usage: for plates where wells were viewed with our new ranking order, the first well was a crystal-containing image for 128 out of a total of 196 plates (65.31%). The number of such ‘successful’ plates increases if the criterion is relaxed to expect at least one crystal in the top 10 or top 32 ranked wells Table 2, as alsob) and Supplementary Fig. S3 illustrate how the top of the viewing order is generally enriched in images with crystals and microcrystals. Moreover, where the ranking failed to move the human-annotated crystal to the top, it was usually questionable whether the images had been correctly marked  or else the images themselves were problematic  to approximately match the resolution at the filtering stage. Table 3This result indicates that the texton approach does not need a very high level of detail in the images: SGC and Novartis images have similar field of view . A common method in vendor software is to label the images with tags and different colour schemes ; on the contrary, annotation appears to become more stringent if larger (micro)crystals have already been observed.We show here that enriching the number of crystal-containing images to inspect first does indeed result in closer attention apparently being given to interesting droplets. We tested this by comparing how effectively crystallographers could evaluate crystallization plates under extreme time pressure  both with or without the ranking system. All plates were viewed by two groups of five crystallographers, with plates viewed in rank order by one group being viewed unranked by the other, but with both groups performing both ranked and unranked viewing; the consensus score within each group was taken to reduce human error ; however, full integration with existing database and imager systems and deployment of the viewer will invariably require bespoke effort. The executable for image processing and generating ranking scores requires little effort to run once the reference background images have been generated, but it is the viewer that must be set up to fit into the infrastructure of a given laboratory; an application programming interface (API) is under development to simplify this process. We have shown that integration is possible for both the Rigaku and the Formulatrix systems, and for these particular versions of the systems it could take as little as 3 days to integrate fully for someone familiar with either system and with general database knowledge. The code and executables can be downloaded from https://github.com/thesgc/TeXRank.The software is available as a standalone package, 4.We show that textons are effective at describing crystallization experiments objectively with fast computation times, making them suitable for everyday use. These descriptors are effective for ranking droplets by their probability of containing crystals or microcrystals, and the algorithm was transferable between two widely used commercial imaging systems for sitting-drop vapour-diffusion experiments. The collection of scores across a plate forms a useful profile for a quick overview of the suitability of the protein for structural studies; the algorithm and the custom viewer can be integrated into the existing infrastructure and are freely available. We have also shown that with the system, crystallographers have been able to identify crystals and microcrystals more efficiently and accurately by prioritizing which images to view and spend more time on.10.1107/S1399004714017581/nj5198sup1.pdfSupporting Information.. DOI: