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Induced metabolic sequelae of tularemia in the rat: correlation with tissue damage.

Serum and liver zinc concentration, amino acid uptake by liver, seromucoid content, and alpha2-macrofetoprotein production were measured in vaccinated as well as nonimmune rats exposed to either virulent (SCHU S4) or attenuated (LVS) strains of Francisella tularensi. It appears that liver damage (pyogranulomatous lesions) must occur before there is any alteration in the above variables. The presence of bacteria in the liver is not of itself sufficient to lead to the onset of systemic, induced metabolic sequelae (IMS). The occurrence of zinc redistribution in all instances of increased serum protein synthesis may imply a necessary relationship between these two sequelae. Amino acid redistribution does not appear to be linked to serum protein synthesis. An endogenous mediator of systemic IMS can be detected in tularemic rats by injection of the serum of these animals into healthy recipients. The occurrence of zinc redistribution and increased serum protein synthesis in some groups of rats in the absence of amino acids uptake by liver, as well as the apparent differential dose responsiveness of these responses, are suggestive of a multiplicity of endogenous mediators.

Progress in the chemotherapy of hematologic diseases.

Progress in the chemotherapy of Hodgkin's disease and experimental therapeutic, pharmacologic, and clinical studies of the antitumor antibiotic, adriamycin, are presented in this abstract. In patients with disseminated Hodgkin's disease, the combination chemotherapy program (MOPP) produces a significant increase in the complete remission rate. This has been increased to 90% by the addition of low dose bleomycin to the MOPP program. The continuation of MOPP treatment beyond 6 months and to a total of 24 months provides improved results in patients in remission as measured either from time of onset of complete remission or from end of treatment. Finally, the pattern of relapse in patients with Hodgkin's disease provides a rationale basis for the selective use of radiotherapy in patients in complete remission. Adriamycin has a broad spectrum of antitumor activity in man. Its mechanism of action involves intercalation with DNA and inhibition of DNA function. The selective effect against tumors is not understood but may relate to membrane transport. Adriamycin is a highly important new antitumor agent for the treatment, not only of hematologic malignancies, but for a variety of solid tumors as well.

An endogenous oncornavirus of guinea pigs: its expression in leukemic cells.

Guinea pigs do not have any known infectious oncornaviruses. We have recently activated an endogenous oncornavirus (GPV) from cultured guinea pig cells after bromodeoxyuridine riboside (BUdR) treatment. GPV is noninfectious for guinea pig cells and is inducible from any guinea pig cell in culture. Although the morphogenesis of the activated virus is unique, it has the morphology and density (1.16 g/ml) of type C oncornaviruses. It contains an oncornavirus specific reverse transcriptase and a high molecular weight RNA (70S) which dissociates into 36S following denaturation. GPV contains 5 major protein components (mol. wt. 95,000 to 16,000 daltons), 2 of which are glycoproteins. The viral genome which remains repressed in normal cells is expressed in leukemic guinea pig cells. These leukemic cells contain particles which have the morphological and biochemical characteristics of BUdR activated endogenous viruses. Unlike the cells transformed by infectious oncornaviruses, the leukemic cells contain the same amount of virus specific DNA present in normal cells.

Comparison of drug effects on RNA tumor viruses and on transformed cells.

Attempts to develop in vitro methods to test inhibitors for replication of C-type RNA tumor virus are made at two levels of the life cycle of the virus: 1) Formation of the proviral DNA, and 2) transcription and processing of viral RNA. Compounds such as rifamycin derivatives were found to be effective in interfering with the first process and compounds such as cordycepin, a nucleoside analogue, blocked effectively the second process. On the other hand, a class of known anti-tumor agents, glucocorticoids, was found to stimulate (10 fold or more) the induction of C-type virus following 5-iodo-2'-deoxyuride (IDU) treatment of murine cells. This is an intriguing example of a multi-focal action of an anti-tumor agent. We are exploring the implication of this for relapse or retransformation following successful chemotherapy in leukemic mice. Attempts are being made to develop an in vitro animal cell model for quantitative cytotoxic assays for compounds active in the above system and for comparing their effects on both normal and virus transformed cells.

Consecutive cytochemical staining for the analysis of the blastic population in the acute phase of chronic myeloid leukemia.

Blastic crisis in chronic myeloid leukemia is characterized by several cytological alterations which may represent some abortive attempts to differentiate along various cell lines as a consequence of a maturation defect of the myelopoietic cells. These changes of the hematological picture are associated with alterations of the karyotype and with cytochemical abnormalities of the blast cells, possibly related to their metabolic anomalies. In this regard 14 patients with blastic crisis were investigated to achieve an evaluation of the composition of the cell population during the acute phase. A sequence of three cytochemical reactions applied consecutively on the same slide (alpha-naphthyl-acetate esterase + AS D-chloro-acetate esterase + PAS) proved to be useful for the detection of differently oriented blast cells. During the acute phase of chronic myeloid leukemia only about one half of the blast cells were expressing granuloblastic differentiation. The data may be important for some clinical and prognostic factors, since the heterogeneity of the blastic population may be associated with a particular resistance to therapy.

Anti-synaptic antibody in allergic encephalomyelitis. II. The synapse-blocking effects in tissue culture of demyelinating sera from experimental allergic encephalomyelitis.

It has been suggested that demyelination cannot account for all of the observed clinical symptoms of multiple sclerosis (MS), in particular the rapidity of onset and remission of the disease, and attention has been focussed on the role of the synapse in 'demyelinating diseases'. In the present paper we have attempted to resolve the fundamental question of the site of action of a demyelinating disease, experimental allergic encephalomyelitis (EAE), by the use of cultures of neonatal rat cerebellum. Electrophysiological and morphological development in these cultures run hand-in-hand, and in the first few days in vitro there is a 4-5 day period when synapses are both seen ultrastructurally and known to be functioning but before the onset of myelination. The serum from guinea pigs with EAE was added to these cultures at different stages during their development and the morphological and electrophysiological effects observed. An abolitionary effect on the bioelectric activity of the culture was only observed when the serum was added to mature, myelinated cultures. Also the same active sera had no effect on synaptic activity before myelination had occurred. We conclude that the synaptic blocking effect occurs only when myelin is destroyed.

The influence of complement on the neutralization of infectious bovine rhinotracheitis virus by globulins derived from early and late bovine antisera.

Calves were inoculated at four week intervals with infectious bovine rhinotracheitis virus (IBRV). Sera were obtained at eight days (early sample) and 21 days (late sample) after each inoculation. Kinetic neutralization was carried out with 7S and 19S globulins derived from these sera, both in the presence and in the absence of guinea pig complement (C). In all instances the 19S neutralizing antibody (AB) was dependent on C for neutralization of IBVR. However, C dependency was not observed with any of the 7S preparations. Neutralizing activity was readily detected in 19S globulins from the early sera after primary and subsequent inoculations but not in any of the late sera. Early sera collected after primary inoculation did not contain any 7S neutralizing AB but it was present in all the other sera tested. The 7S AB when present was always at a considerably higher concentration than 19S AB. Thus it may be possible to determine whether cattle have recently been exposed to IBRV when paired serum samples are not available by determining the presence of C-dependent 19S globulins. In addition, by comparing 19S and 7S levels, a distinction may be made between primary and secondary responses to IBRV.

Dynamic properties of axonal transport of proteins and glycoproteins: a study based on the effects of metaphase blocking drugs in the developing optic pathway of chick embryos.

Some properties of the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos and newly hatched chicks were studied by labelling retinal ganglion cells with 3H-proline or 3H-fucose. A study of the effects of colchicine (COL) and vinblastine (VLB) on embryonic axonal transport was also carried out. Marked changes in the efficiency of axonal transport were found throughout development. In particular, the fraction of retinal ganglion cell proteins which is rapidly exported toward tectal terminals increases during embryonic life but steadily decreases after hatching. Glycoprotein transport behaves similarly except that its efficiency is relatively higher at stages when critical events of synaptic maturation in the tectum are reported to occur. Embryonic axonal transport is blocked by COL and VLB at very low intravitreal concentrations. Retinal protein synthesis and the morphology of ganglion cells are profoundly altered by the drugs: in general, COL and VLB effects were much more marked in embryonic than in mature neurons. An analysis of the time course of rapid transport along embryonic optic axons was carried out by reducing the efflux of labelled proteins from the eye by giving VLB intravitreally 2 h after the pulse. It revealed some peculiar features in the retino-tectal migration of glycoproteins and confirmed their progressive accumulation within terminals as previously described by radioautography. These results suggest that axonal transport of proteins during embryonic life undergoes changes in parallel with synaptic maturation. It may thus be considered as one of the factors controlling the genesis of neuronal networks.

Immunochemical studies of organ and tumor lipids XXI. Sensitivity and specificity of the cytolipin F - sheep erythrocyte system.

The sensitivity and specificity of the sheep erythrocyte - anti-sheep erythrocyte system to inhibition by pure cytolipin F has been studied with 5 antisera, in order to compare it with the rat erythrocyte-anti-rat lymphosarcoma system and its inhibition by pure cytolipin R. The cytolipin F - sheep erythrocyte system is much more sensitive than the cytolipin R - rat erythrocyte system, inhibition of hemolysis of 6 x 10(6) sheep cells being produced by 10 ng of cytolipin F (combined with a four-fold quantity of lecithin) compared with inhibition of hemolysis of 10(6) rat cells by 50 to 100 ng of cytolipin R (also combined with lecithin). Differences in sensitivity are attributed to the larger number of available cytolipin F determinants on sheep erythrocytes compared with cytolipin R determinants on rat erythrocytes. Studies of the auxiliary lipid enhancement of cytolipin F activity by galactocerebroside, lactosyl ceramide (cytolipin H), and lecithin are also reported.

Amygdala and masseteric reflex. II Mechanism of the diphasic modifications of the reflex elicited from the "defence reaction area". Role of the spinal trigeminal nucleus (pars oralis).

It has been demonstrated (Gary Bobo and Bonvallet 1975) that long-lasting stimulation of the "amygdaloid area for the defence reaction" (basal nucleus, pars magnocellularis) elicits, after an initial facilitation, a delayed inhibition of the monosynaptic masseteric reflex (MR), while stimulation of the amygdalofugal fibers running in the ansa lenticularis provokes an immediate inhibition of the reflex. In present study, the structure which mediates these inhibitions has been identified. Using combined techniques of limited transections, localized coagulations and localized stimulation and recording, it has been demonstrated that these ingibitions are mediated by the rostral portion of the spinal trigeminal nucleus, the subnucleus oralis (NO). After localized coagulation of this nucleus, or after lesions which interrupt slectively the connections between the NO and the masticatory nucleus, long-lasting stimulation of the basal nucleus elicits only well maintained facilitation of the MR. Hence, the delayed decrease in amplitude of the reflex, observed during stimulation of the basal nucleus in the preparations with intact brain, cannot be explained by the reversal of an initial facilitatory influence to an ingibitory one. The one ingibition of the reflex is due to the superimposition, on a background of sustained facilitation of the masseteric motoneurons, of the inhibitory influence exerted on the monosynaptic masseteric circuit by a trigeminal sensory nucleus, itself activated by delayed discharges of the basal nucleus. A tentative representation of the dual control exerted on the masseteric activity by the basal nucleus is given in Fig. 9. The functional implications of this dual control during the "defence reaction" are briefly discussed.

The adaptation of cochlear and brainstem auditory evoked potentials in humans.

The adaptation of the responses from the cochlear nerve and the auditory brainstem nuclei was studied using a burst of four clicks as the stimulus. Nine experimental conditions were obtained from three stimulus levels (60, 70 and 80 dB SL) and from three intervals between click in the burst (15, 24 and 32.5 msec). Six subjects were each tested three times for each of the experimental conditions and the techniques used and results obtained are summarized. Various models of the adaptation of the brainstem responses are proposed which predict different results. The results for the adaptation of the N1 response are basically in agreement with previous work, and the more central brainstem responses showed less adaptation than the peripheral responses. These findings may be explained by postulating different adaptation mechanisms for the peripheral and central responses. The 60dB stimulus level condition gave rise to more adaptation than the 80dB level, and this is in agreement with studies of low and high threshold reception systems reported elsewhere.

Non-helical sequences of rabbit collagen. Correlation with antigenic determinants detected by rabbit antibodies in homologous regions of rat and calf collagen.

Non-helical peptide fragments were isolated from rabbit skin collagen after cleavage of alpha chains with cyanogen bromide and proteases. Determination of their amino acid sequence indicated a length of 9, 16 and 25 amino acid residues for the non-helical sequences located in the N-terminal region of alpha2 and alpha1 chain and in the C-terminal region of alpha1 chain, respectively. The C-terminal sequence Tyr-Tyr hitherto considered as the genuine end of collagen alpha1 chain is in part of rabbit collagen extended by two residues, alanine and arginine. Rabbit collagen may differ considerably in its non-helical sequences from other vertebrate collagens, particularly in the C-terminal part. Some but not all of these differences are clustered in areas occupied by antigenic determinants which are recognized in the antibody response of rabbits to rat or calf collagen. On the other hand, a high homology to rabbit collagen, e.g. in the N-terminal region of rat collagen alpha1 chain or calf collagen alpha2 chain, probably prevents immunological recognition by the rabbit. The degree of foreignness alone, however, may not necessarily determine whether a particular non-helical area is able to express immunogenic activity.

Inhibition of T- and B-lymphocyte functions by normal immunosuppressive protein.

Normal immunosuppressive protein (NIP) isolated from human plasma was studied in two well defined systems. (1) Spontaneous rosettes of sheep red blood cells with human peripheral blood lymphocytes and PHA-induced lymphocyte cytotoxicity as indicators for T-cell function. (2) Rosette formation tests of human lymphocytes with antibody-coated erythrocytes or erythrocytes coated with antibody and complement as well as antibody-induced lymphocyte-mediated cytotoxicity represented non-T-cell activity. While NIP did not inhibit the formation of any of the above mentioned rosettes, it practically prevented both PHA-induced and antibody-mediated lymphocyte cytotoxicity. Relatively small amounts of NIP inhibited PHA-induced cytotoxicity while higher doses were required for the inhibition of antibody-mediated lymphocyte cytotoxicity. Possible mechanisms of its suppressive activity are discussed. NIP was found to be heat-stable and did not show any species specificity, as NIP preparations from human plasma were immunosuppressive in human, mouse and guinea-pig systems.

Specificity of cell-mediated cytotoxicity against human melanoma lines: evidence for "non-specific" killing by activated T-cells.

The specificity of cell-mediated cytotoxicity against melanoma cells in vitro has been analyzed in a large number of studies with cells both from normal and melanoma subjects. As in a number of other, recent, similar human studies, no evidence for tumour specificity was found. Effector cells in peripheral blood responsible for the cytotoxic raction were examined by cell separation methods based on red cell rosette formation and separation through Hypaque-Ficoll mixtures. The evidence suggests that non-specificity results from killing by cells separating largely in the non-sheep red blood cell rosetting fraction and which have cytotoxic specificity directed broadly to cells with abnormal membranes. Further analysis revealed that the cells were non-phagocytic and did not bear receptors for complement. They appear to be activated into cell division and to bear surface receptors for the Fc portion of IgG. Additional evidence is presented suggesting that the cells mainly responsible are activated thymus-dependent cells present in the circulation of both tumour-bearing and normal subjects.

Relationships between mitochondrial content and glycogen distribution in porcine muscle fibres.

Serial sections of longissimus dorsi and rectus femoris muscles from 15 Yorkshire breed pigs (live weights 24-46 and 49-139 kg) were stained for glycogen (PAS) and a mitochondrial enzyme (NAD tetrazolium reductase). Muscle fibres with a low mitochondrial content in both muscles were more frequently PAS-positive than fibres with a high or intermediate mitochondrial content. However, some pigs had all their muscle fibres PAS-positive while one pig with a high post-mortem muscle pH had all rectus femoris fibres PAS-negative. Relative to lighter weight pigs, longissimus dorsi muscles of heavy pigs tended to have less fibres with a high mitochondrial content and less fibres with a positive PAS reaction. Compared to longissimus dorsi muscles, rectus femoris muscles had more fibres with a high mitochondrial content and less with a positive PAS reaction. All fibres in both muscles became PAS-negative with an accompanying decrease in pH by 24 hr post-mortem. Fibres from longissimus dorsi muscles frequently had PAS-positive sarcoplasmic cores between their myofibrils. Heavy pigs tended to have larger cores (up to a mean maximum diameter of 13.4 mum), more fibres with cores, and more cores per fibre. The pigs involved exhibited no other ante- or post-mortem muscle abnormalities.

Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum.

The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.

Histamine release from human leukocytes: modulation by cadmium ion.

The effect of cadmium on histamine release was studied in relation to the role of calcium. The antigenic histamine release from peripheral leukocytes of ragweed-sensitive patients was inhibitied in the presence of cadmium (greater than 10(-5) M). Spontaneous histamine release was not affected by cadmium except for an enhancement observed in high concentrations (greater than or equal to 10(-3)M). Cadmium did not seem to affect the first stage of histamine release but to act mainly on the calcium-dependent second stage. Increasing the level of calcium in the medium could antagonize the inhibitory effect of cadmium. The effect of cadmium could be totally abolished by deuterium oxide and partially by dibutyryl cyclic adenosine monophosphate (AMP) in certain concentrations. These results would indicate that: (1) cadmium acts as an antagonist of calcium and can be used for the study of the role of calcium in histamine release, (2) the action of calcium in the histamine release reaction seems to be related to the microtubular system, (3) cyclic AMP may potentiate histamine release when the action of calcium is inhibited.

Carrier-induced tolerance to nucleic acid antigens.

BALB/c and SJL mice were treated with nucleosides-IgG1 as a tolerogen, before either primary or secondary immunization with nucleosides-keyhole limpet hemocyanin. Nucleoside-specific responses were measured serologically by a modified Farr assay, with either 14C-labeled denatured DNA or nucleosides-131-I-labeled BSA as test antigen. Specificity of the response was tested by hapten inhition experiments. Multiple doses of nucleosides-IgG1 tolerogen given before the primary or secondary immunization effectively suppressed the secondary and tertiary anti-nucleoside responses. The tolerogen did not suppress the response to an unrelated hapten-KLH conjugate. The IgG alone did not suppress the anti-nucleoside response of BALB/c mice to nucleosides-KLH. Single doses of tolerogen before the primary or secondary immunization were less effective. Residual antibody in partially suppressed BALB/c mice showed changes in specificity as compared to controls. Suppression of the secondary response of SJL mice was measured much more readily by binding of nucleosides-131-I-BSA than by binding of denatured DNA. This reflected an altered specificity of the residual antibody; in control animals, antibodies were directed against all four nucleosides, whereas the antibodies of partially suppressed animals were directed only against guanosine. Suppression of anti-nucleic acid antibody responses may have therapeutic application in the management of systemic lupus erythematosus.

Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.

The xenogeneic effect. I. Antigen and mitogen-stimulated human lymphocytes produce a non-antigen-specific factor which reconstitutes the antibody response of T cell-deficient mouse spleen cells.

Modified Marbrook culture vessels with two chambers separated by a 0.2-mu porosity membrane have been utilized to show that antigen-stimulated human lymphocytes produce a soluble factor(s) which restores the ability of thymectomized, irradiated, and bone marrow-protected mice to mount a primary IgM plaque-forming cell response in vitro. In the initial experiments, the human lymphocytes plus antigen (sheep erythrocytes) were cultured in the lower chambers of the Marbrook vessels and the T cell-deficient mouse spleen cells plus sheep erythrocytes were cultured in the upper chambers. The response of the spleen cells was shown to be enhanced as a function of the number of human lymphocytes in the lower chambers. In subsequent experiments, the human lymphocytes were challenged with allogeneic lymphocytes or activated with a variety of T cell mitogens. Supernatants from these cultures, when placed in the lower chambers of the Marbrook vessels, were also capable of reconstituting the antibody-forming cell response of the mouse B cells. The results of the experiments are discussed in relation to a model of B cell induction which incorporates a non-antigen-specific "helpher" T cell.

Measuring ventricular extrasystoles.

Uniform ventricular extrasystoles can be divided into three types: fixed coupling, parasystole, and variable non-parasystolic coupling. Frequency distributions of coupling intervals of ventricular extrasystoles were determined from long electrocardiographic recordings of 51 patients with variable coupling. These distributions were of five types: (a) two distinct coupling intervals, (b) a preponderance of extrasystoles with short coupling intervals and less frequent extrasystoles with progressively longer coupling, (c) a preponderance of long coupling intervals and less frequent extrasystoles with progressively shorter coupling, (d) an even distribution and (3) a central distribution. Interectopic intervals were measured in these electrocardiograms (ECGs). Parasystole was found only in three recordings. It is recommended that the diagnosis of parasystole be made only if the degree of variation of the ectopic cycle (both when it is manifest and when it is concealed) is less than the variation of the coupling intervals. When the ECGs with variable non-parasystolic coupling were compared with 44 ECGs with fixed coupling, it was found that variable coupling was associated with abnormalities of the basic electrocardiographic contour, multiformity of extrasystoles and repetitive extrasystoles.

[Correlation between sensory action potentials and vibratory perception in uremic polyneuropathy (author's transl)].

1. The sensory action potentials of the tibial nerve at the medial malleolus were studied by averaging in 51 patients with chronic renal failure treated by hemodialysis. Vibratory sense was also tested quantitatively on the dorsum of the foot with a pallesthesiometer. 2. Good correlation was found between sensory tibial nerve potentials and vibration sense in subclinical as well as in clinical uremic polyneuropathy. A biphasic potential correlated with unaffected vibration sense in 18 out of 23 patients, and impaired vibratory sense with a polyphasic response in 20 of 28 patients. Maximal nerve conduction of sensory fibres was faster (mean 37.4 m/sec) in cases with normal vibratory sense, but slower (mean 31.3 m/sec), when vibratory sense was impaired. Furthermore there was a correlation between the threshold of vibratory perception and sensory nerve conduction. 3. Sensory function, tested with conventional methods, was impaired only 5 times in 28 patients with altered vibratory perception. 4. The earlier impairment, especially of the vibratory sense, may be explained by the following neurophysiological mechanisms: a) Because of the polyphasic prolonged response of the sensory potentials, no rhythmical groups of impulses reach the central nervous system, but only a continual stream of small peaks arrives, so that vibration perception does not develop. b) A multiplication of the frequency of discharges caused by alternating firing of different sensory fibres is impossible due to the reduction of the number of axons. c) The prolongation of the relatively refractory period due to demyelinization of the surviving fibres prevents the transmission of frequent impulses. 5. Alterations of the sensory action potentials of the tibial nerve, as well as of vibratory perception tested quantitatively, are earlier signs of uremic polyneuropathy than the prolonged motor nerve conduction velocity. Since not all patients give accurate information when tests of vibratory sense are performed both methods should be applied. Physiological polyphasia of sensory action potentials and diminishing vibration perception in advanced age must be taken into account.

Clinical and electrophysiological observations in patients with myotonic muscle disease and the therapeutic effect of N-propyl-ajmalin.

Six patients with congenital myotonia and 4 patients with myotonic dystrophy have been examined clinically before and after the administration of N-propyl-ajmalin, an alkaloid frequently used as a cardiac antiarrhythmic drug. All patients but one reported a good to moderate improvement of their myotonic muscle stiffness. This was verified by measuring the time the patients needed to ascend a flight of stairs and by recording the speed of opening the hand. The amplitude of the compound muscle action potential decreased during repetitive nerve stimulation in myotonic patients. This decrease was not influenced by N-propyl-ajmalin. It seems to be due to the increased after-depolarization observed in myotonic fibres which causes partial inactivation of the Na-carrying system. From one patient a muscle biopsy was taken and intracellular potentials were measured with a microelectrode. Almost all muscle cells investigated showed myotonic activity which was completely abolished by addition of 10(-5) g/ml N-propyl-ajmalin to the bathing fluid. The development and duration of "warm-up" is illustrated and a possible electrophysiological basis is discussed.

Murine intracisternal type A particles: a biochemical characterization.

Intracisternal A particle preparations from a murine neuroblastoma cell line (N18) and from a mineral oil-induced murine plasmacytoma (MOPC-104E) contain both an endogenous RNA-dependent DNA polymerase activity and high molecular-weight polyadenylic acid (poly[A])-containing RNA. The DNA polymerase activity is stimulated by oligo(dG)-poly(C) and oligo(dT)-poly(A) and to a lesser extent by oligo(dT)-poly(dA), in agreement with previous reports. The high-molecular-weight RNA is predominantly 35S and contains a poly(A) tract of approximately 220 nucleotides as judged by polyacrylamide gel electrophoresis. Small amounts of 70S RNA are also present. This RNA preparation contains RNA homologous to RNA from type-C particles, as judged by molecular hybridization experiments. However, since this RNA derives only in part from A-particles and in part from other cellular RNA, hybridization of A-particle endogenously synthesized DNA or reverse transcripts of A-particle RNA to purified type C viral 70S RNA may more accurately reflect the relationship of A-particle RNA to RNA from C-particles. None of these DNA transcripts hybridizes significantly to C-particle 70S RNA, although MOPC and N18 DNA transcripts share significant homology. Our interpretation of these results is that murine intracisternal A particles are not closely related genetically to the tested murine type C viruses, although an alternate possibility is that all the A-particle DNA transcripts are copied from only a small part of the genome, which is unrelated to C-particle RNA.

Splenectomy for complications of chronic granulocytic leukaemia.

13 patients with chronic granulocytic leukaemia (C.G.L.) which was unsatisfactorily controlled underwent splenectomy. 3 out of 4 patients with hypersplenism did well, as did 5 out of 8 patients in whom the C.G.L. had undergone metamorphosis to a refractory phase. In a case of C.G.L. complicated by severe myelofibrosis the need for transfusion was reduced but survival was short. Splenectomy should be considered when C.G.L. in its chronic phase is complicated by hypersplenism, and may be considered as a part of the treatment after C.G.L. has undergone metamorphosis to a refractory phase. However, a favourable outcome is unlikely for patients over 65 years, and in the presence of coexistent illnesses, rapidly progressive metamorphosis to an acute phase, or severe bone-marrow failure from any cause. In C.G.L., elective splenectomy early in the chronic phase must be clearly distinguished from splenectomy performed at a later stage when the disease is not well controlled.

Ischaemic heart-disease: possible genetic markers.

National death-rates from ischaemic heart-disease are significantly correlated with population frequencies of histocompatibility antigen HLA-8 and haplotype 1-8. Serum-cholesterol levels may also be correlated with population frequencies of HLA-8. Inexplicably high death-rates from ischaemic heart-disease and high levels of serum-cholesterol in Finland may be due to the combined effects of HLA-8 and W15. It is suggested that HLA-8 (and possibly W15) are linked to genes which predispose to hypercholesterolaemia and ischaemic heart-disease. These findings provide some support for i-munogenetic hypotheses for vascular disease in man.

Comparison of dopa decarboxylase inhibitor (carbidopa) combined with levodopa and levodopa alone on the cardiovascular system of patients with parkinson's disease.

The effects of carbidopa combined with levodopa (carbidopa/levodopa) and levodopa alone on the cardiovascular system of patients with Parkinson's disease were evaluated. Thirty-eight patients who had been on stable doses of levodopa underwent a complete cardiac examination, including measurement of recumbent and erect blood pressure and 24 hour ambulatory electrocardiographic monitoring. Patients were classified with respect to the presence or absence of clinically significant heart disease and ventricular arrhythmias. Nineteen of the 38 patients (50 percent) had heart disease, and 12 (32 percent) had significant ventricular arrhythmias. Eleven of the 12 with arrhythmias had underlying heart disease. The incidence of arrhythmias did not correlate with the dose of levodopa. The patients were subsequently randomly assigned to treatment groups receiving either carbidopa/levodopa or levodopa alone. There was no significant difference in the severity of ventricular arrhythmias or in the incidence of orthostatic hypotension in the group assigned to carbidopa/levodopa compared with the group receiving levodopa.

Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase).

The ability of tryptophan tRNA (tRNATrp) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNA-dependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrp and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNAs, only tRNATrp and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATrp to change the sedimentation rate of the enzyme indicates that tRNATrp is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrp aa a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme.

The effect of lipopolysaccharide on the primary immune response to the hapten NNP.

We have studied the effects of lipopolysaccharide (LPS) on the primary in vivo immune response to the hapten (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP), with special reference to the avidity and affinity of the early appearing 19S and 7S antibodies. Comparisons were made of the immune response to NNP in groups of mice given either antigen alone, LPS alone, or antigen plus LPS. The avidity of antibodies induced by LPS plus antigen were similar to that found after injection of antigen alone, in spite of the fact that the antibodies were more numerous. However, when comparing the avidity of antibodies produced in animals given only LPS with those given LPS plus antigen, the latter group was often found to have fewer low-avidity 19S-antibody-producing cells. The affinity of 7S antibodies was also similar in the two groups given antigen or antigen plus LPS. Kinetic studies of the effect of LPS on the primary immune response to NNP showed that synergy was observed only before or after the peak response in groups given antigen alone. It is concluded that LPS under synergy conditions acts preferentially on specific antigen-sensitive cells, which are distinct from those that are activated to polyclonal antibody synthesis by LPS alone. Possible mechanisms for the adjuvant effect of LPS are discussed.

Effect of the common bile acids on the fibrin/fibrinogen fragments in rheumatoid synovial fluid. A possible clue to the ameliorating effect of jaundice in rheumatoid arthritis.

Fibrin/fibrinogen degradation products observed in rheumatoid synovial fluid exhibit resistance to plasmin proteolysis. In the present study, the influence of the common bile acids on the plasmin digestion of these degradation products in 16 rheumatoid synovial fluids were quantitated immunologically by radial immunodiffusion, and qualitatively estimated by crossed immunoelectrophoresis. Addition of chenodeoxycholic acid, deoxycholic acid, and their taurine and glycine conjugates in concentrations of 3.33 mumole/ml of a mixture of rheumatoid synovial fluid and plasmin resulted in complete plasmin degradation. Cholic acid and its taurine and glycine conjugates were effective only in concentrations of 4.44 mumole/ml. A detergent, such as Triton X-100, had little or no effect on the plasmin digestion. Other proteins capable of influencing fibrinolytic activity, such as plasminogen and the inhibitors alpha1-antitrypsin and alpha2-macroglobulin, were not affected by the two detergents. The bile acids are thought to influence the fibrin/fibrinogen degradation products by unfolding the protein at a molecular level, by virtue of their properties as steroid detergents, leaving the fibrin/fibrinogen degradation products susceptible to plasmin digestion.

The soluble antigens of Rickettsia prowazeki, R. typhi and R. canada. Investigation of their interrelationship by various serological methods.

The purpose of this research is the isolation of an eventual species-specific fraction from the "soluble antigen" of Rickettsiae. The "soluble antigen" of R. prowazeki (Breinl strain), R. typhi (Wilmington strain) and R. canada were purified at 25% saturation with ammonium sulphate (PSA). Corresponding antisera were produced in rabbits. The serological methods used were the complement fixation, the micro-agglutination, the precipitation method in capillary tubes and the immuno-diffusion method carried out with complete and previously cross-absorbed antisera. Furthermore, the PSA were subjected to immuno-electrophoretic and disc electrophoretic fractionation. The PSA of R. prowazeki was found to contain at least 4 group-specific fractions. A species-specific component could be demonstrated with certainty only with the precipitation method in capillary tubes carried out with previously cross-absorbed antisera. The PSA of R. typhi contains 5 fractions of which 4 are group-specific and one is species-specific. This result was confirmed by all methods. The PSA of R. canada: the maximum of 3 components could be detected with the help of immuno-electrophoretic fractionation. A fourth antigenic determinant was made apparent by the presence of corresponding antibodies in the anti-R. canada PSA only.

Studies on topical antiperspirant control of axillary hyperhidrosis.

Axillary hyperhidrotics is reviewed from the standpoint of anatomical factors, physiological mechanisms and the history of methods of control. Anhydrous aluminum chloride and anhydrous zirconium tetrachloride are shown to be superior topical agents for partial control of axillary sweating when applied as a powder or in anhydrous nonreactive vehicles. Complete anhidrosis as demonstrated by sustained garment armpit dryness could be achieved in hyperhidrotics within 48 hours by the following trinary antiperspirant system: (1) a saturated solution of aluminum chloride hexahydrate or zirconyl chloride in absolute ethanol or isopropyl alcohol, (2) application to the dry axilla at times of sleep or other prolonged non-sweating period, (3) water vapor occlusion of area for 6 to 8 hours by means of Saran wrap. The hypothesis is presented that metallic antiperspirants act by reflux entrance into the terminal intraepidermal eccrine duct, slowly combining with the intraductal keratin, to produce a fibrillar contraction (super contraction) of keratin and hence functional closure, not histologically evident. This altered keratin is shed weeks later, with the consequent return of ductal patency and sweating.

Light microscopic differential staining of epoxy-embedded adenohypophysis.

Light microscopic histochemical methods for differential staining of adenohypophysis glandular cells were applied to Epon-embedded tissue, after prior softening of resin with saturated solution of NaOH in absolute ethanol. All investigated staining procedures, i.e. carmoisin L-orange G, PAS-orange G, PAS-range G-methyl blue, Gabe's aldehyde fuchsin-Halmi counterstain, aldehyde thionin-PAS-orange G and performic acid-alcian blue-PAS-orange G were found to give satisfactory results and tissue structure showed no signs of damage caused by the Epon-dissolving reagent. The features of light microscopic image given by plastic-embedded semithin sections: nearly 2-dimensional picture as well as the highest possible resolving power provide the opportunity for much more precise and detailed examination of adenohypophysis prepared that way, as compared with conventionally paraffin-embedded gland. Moreover, the described technique permits to establish a direct connection between light microscopic differential histochemistry of adenohypophysis and the electron microscopy by means of adjacent sections.

Quantification of the inhibitory effect of eriochrome black and sodium nitrite on non-specific immunofluorescent staining.

To evaluate non-specific staining (NSS) of animal tissues by FITC-labelled immunoglobulins a model system was developed. HeLa cells were treated with labelled antisalmonella globulins and the fluorescence intensity of the cells was determined quantitatively by means of a fibre optic probe system. This system was used to determine the optimal treatment conditions (adequate concentrations, duration of treatment) using the two NSS-reducing agents eriochrome black and sodium nitrite. Approximately the same inhibitory effects (40-50 per cent using nitrite; about 85 per cent using eriochrome black) were obtained by conjugates of different F/P ratio values. The fibre optic probe system was also used to determine the effects of the above-mentioned agents on the NSS of liver sections. In this system, the NSS was strongly reduced by eriochrome black whereas nitrite treatment did not induce any inhibitory effect. The applicability of nitrite and eriochrome black as NSS-reducing agents was further demonstrated by the fact that they had no influence on the specific fluorescence intensity of salmonella bacteria. The effect of eriochrome black was also studied in clinical specimens infected with salmonella or tularemia bacteria.

Diagnosis of Neisseria infections by defined immunofluorescence. Methodologic aspects and applications.

Defined IF techniques have been used in our laboratories for the past 15 years for diagnosing meningococcal and gonococcal infections. Conjugates against these bacteria give desired specific staining (DSS) but also undersired specific staining (USS) due to cross-reacting antigens. USS is controlled by absorption. Nonspecific staining (NSS) of protein A-containing S. aureus caused by the reaction with the Fc part of the IgG molecule is blocked by adding RB 200-labeled antiS. aureus globulins to diluted FITC conjugates against gonococci or menigococci. Nss is also avoided by using FITC-labeled F(ab')2 fragments of IgG. By optimal labeling and separation of unlabeled FITC and heavily labeled molecules on Sephadex G-25 at low ionic strength and low pH, nonspecific reactions with tissues are reduced to a minimum. Defined IF can be utilized for diagnosing pathogenic Neisseria by examination of smears prepared from clinical specimens, as confirmative tests of oxidase-positive colonies, and for demonstrating these bacteria in tissue efflorescences and tissue biopsies. It is stressed that this technique should always be used in conjunction with conventional methods.

Properties of bispecific rosette-forming cells. II. --Rosette formation by "educated" T-lymphocytes.

Normal thymus cells transferred into thymectomized and lethally irradiated syngeneic mice responded to stimulation with sheep erythrocytes by rosette formation with these erythrocytes. This response reached a peak on the 7th day and was not associated with any production of circulating antibodies. Rosettes produced by "educated" T-cells were inhibited by anti-theta serum as well as by anti-mouse Fab serum. Simultaneous stimulation of transferred thymocytes with sheep and pigeon erythrocytes provoked the appearance of a minority of cells simultaneously binding both types of erythrocytes. Depletion of B cells contaminating normal thymocyte populatins after passage through anti-mouse Ig coated columns before their transfer in thymectomized and irradiated recipients did not prevent the appearance of simple and double RFC. Moreover, when normal thymocytes or T-cells "educated" by allogeneic stimulation were incubated at 4 degrees C with anti-SRBC and anti-PRBC mouse sera a subsequent incubation at 37 degress of resulted in the dissociation of most passive rosettes formed at 4 degrees. Conversely similar incubation at 37 degrees of rosettes formed by actively immunized cells resulted in capping of about 50% simple and double rosettes. This redistribution of membrane receptors is proposed as a routine test for distinguishing active from passive rosettes.

Studies on the role of the areA gene in the regulation of nitrogen catabolism in Aspergillus nidulans.

Mutants of Apergillus nidulans with lesions in a gene, areA (formerly called amdT), have been isolated by a variety of different selection methods. The areA mutants show a range of pleiotropic growth responses to a number of compounds as sole nitrogen sources, but are normal in utilization of carbon sources. The levels of two amidase enzymes as well as urease have been investigated in the mutants and have been shown to be affected by this gene. Most of the areA mutants have much lower amidase-specific activities when grown in ammonium-containing medium, compared with mycelium incubated in medium lacking a nitrogen source. Some of the areA mutants do not show derepression of urease upon relief of ammonium repression. The dominance relationships of areA alleles have been investigated in heterozygous diploids, and these studies lend support to the proposal that areA codes for a positively acting regulatory product. One of the new areA alleles is partially dominant to areA+ and areA102. This may be a result of negative complementation or indicate that areA has an additional negative regulatory function. Investigation of various amdR; areA double mutants has led to the conclusion that amdR and areA participate in independent regulatory circuits in the control of acetamide utilization. Studies on an amdRc; areA double mutant indicate that areA is involved in derepression of acetamidase upon relief of ammonium repression.

Combination chemotherapy and radiotherapy in non-Hodgkin's lymphomata.

The results obtained with intensive chemotherapy and intensive chemotherapy plus radiotherapy in non-Hodgkin's lymphomata are reported. A quintuple drug regimen (mechloretamine, adriamycin, bleomycin, vincristine and prednisone) in histiocytic lymphomata (Stage III and IV) yielded complete remissions in 53% and complete plus partial remissions in 77%. These figures were 44% and 64% respectively in lymphocytic lymphoma. In Stage III complete responders after combination chemotherapy were subsequently irradiated (involved field irradiation). The median duration of complete remission after completion of radiotherapy was 9-5 months in histiocytic and 12-0 months in lymphocytic lymphomata. At 2 years actuarial survival in Stage III and IV was better in patients with the lymphocytic type and with nodular pattern than with histiocytic and diffuse patterns. A more recent trial compares, in Stage IV patients, cyclophosphamide, vincristine and prednisone (CVP) versus adriamycin, bleomycin and prednisone (ABP). Although the number of evaluable patients is still limited, there appears to be no difference in the response rate between CVP and ABP. In Stages I and II, 6 cycles of CVP were given as adjuvant treatment after radiotherapy. At the present moment, there is no statistical difference in the relapse rate between the group of patients treated with radiotherapy alone and that with radiotherapy plus CVP.

The endogenous reverse transcriptase activity of Gibbon ape lymphoma virus: characterization of the DNA product.

The DNA product of the endogenous reverse transcriptase reaction of Gibbon ape lymphoma virus has been analyzed and characterized. Data show that in simultaneous detection assays in which the type and/or concentration of divalent cation is varied the best yield of rapidly-sedimenting DNA was obtained from reactions containing 1.5 mM Mn2+. This yield is ten-fold better than the yield observed at the optimal Mg2+ concentration (5.0mM). Evidence is presented to show that DNA synthesized at the optimal concentration of either of these cations consists of large pieces varying in size from 4 to 12S. This DNA hybridizes efficiently to homologous viral RNA (greater than 60 percent annealing) and protects at least two-thirds of GALV 70S [32P]RNA from ribonuclease digestion. The hybrids formed with homologous viral RNA are stable as evidenced by their thermal elution patterns from hydroxylapatite columns. In contrast, DNA synthesized in reactions in which the concentration of Mn2+ or Mg2+ was greater than optimal was predominantly 4S or smaller in size and displayed a low level of hybridization (less than 10 percent) to homologous viral RNA.

Enhancing effect by metabolic inhibitors on the killing of tumor cells by antibody and complement.

Two chemically induced, antigenically distinct guinea pig hepatoma cell lines, line 1 and line 10, which are resistant to killing by rabbit anti-Forssman or specific antitumor antibody and complement, can be rendered susceptible when the cells are pretreated with metabolic inhibitors and drugs commonly used for the treatment of cancer patients. The effect appears within 7 hr after initial contact with the inhibitors and is dependent on temperature and on inhibitor concentration; the effect is reversible within 7 hr, and the process of reversion is also temperature dependent. Not all preparations of tumor cells were rendered susceptible following treatment with inhibitors. In some cases, susceptibility to killing by complement was observed with anti-Forssman antibody but not antitumor antibody. No clear correlation between known metabolic inhibitory activity of the inhibitors and conversion to the sensitive state could be made. The results suggest that properties of nucleated cells, which are under metabolic control, play an important role in the killing efficiency of antibody and complement.

Ventricular premature beats in a population sample. Frequency and associations with coronary risk characteristics.

In a large epidemiological survey, 10,880 men aged 35 to 57 years were screened for ectopic ventricular activity and estimated risk of future coronary heart disease death. A 2-minute lead I electrocardiogram (ECG) was recorded for each man, together with measurement of serum cholesterol, blood pressure, and the number of cigarettes currently smoked. Ventricular premature beats (VPB) occurred in 540 (4.96%) men; of these 459 (4.21%) were uniform, while complex forms were detected in only 7/1000 men. VPB occurring with a frequency of greater than or equal to 10 in the 2-min recording were also rare, occurring in 9/1000 men. No relation was found between the frequency of simple or complex VPB and estimated coronary risk status or between the prevalence of VPB and the individual risk factors of diastolic blood pressure, cholesterol, or cigarette smoking. There was, however, a strong positive association between VPB and the level of systolic blood pressure, and between VPB and increasing age. The lack of association between VPB and overall coronary risk status indicates that myocardial "ischemic" changes in high-risk men may not have progressed sufficiently to alter ventricular excitability or to increase the frequency of VPB.

[Excessive peripheral conversion of thyroxine (T4) to triiodothyronine (T3) in the pathogenesis of T3-hyperthyroidism (author's transl)].

In a 41-year-old woman and a 3-year-old girl, both of them with T3-thyrotoxicosis, serum levels of total and free T4 and T3 were measured serially during anti-thyroid drug treatment. Attempts to substitute thyroxine during the antithyroid treatment had to be interrupted because the patients became hyperthyroid again with excessive increases in total and free serum T3, even when concentrations of total and free T4 were brought to subnormal levels. The increased conversion of administered thyroxine ceased later on and higher amounts of oral T4 were tolerated after one year of treatment. In both patients there was an extremely low serum T4/T3 ratio, differing in this respect significantly from six other patients with T3-thyrotoxicosis and 41 patients with "conventional" T3/T4-hyperthyroidism. It is concluded that, in patients with T3-thyrotoxicosis due to excessive peripheral T4 to T3 conversion, substitution during antithyroid drug treatment should be either with very low doses of thyroxine or with triiodothyronine in divided daily doses. In such cases the level of serum T3 represents the most reliable biochemical measurement for the control of treatment, serum T4 levels being irrelevant.

Suppression of cortical epileptiform activity by generalized and localized ECoG desynchronization.

The effects of high frequency electrical stimulation of both diffusely projecting brain regions and regions of more restricted projection were studied on penicillin-induced cortical epileptiform focal activity in the cat. Results obtained were contingent on the level of focal activity present at the time of stimulation. Very active foci (spike rates above 0.5/sec) were uniformly driven by stimulation of all structures under study. Foci exhibiting weak to moderate levels of activity were, on the other hand, inhibited both during and following stimulation. Episodes of spike suppression induced through stimulation of diffusely projecting structures were typically followed by an intensified "rebound" of interictal activity. Episodes of suppression induced through stimulation of regions of limited projection were not followed by such rebounds, an effect most dramatically apparent with caudate stimulation and motor cortex foci. Results are discussed in terms of the interaction between naturally occurring brain rhythms in sleep and arousal with the epileptic process.

Isolation of parietal cells from guinea-pig gastric mucosa and the immunological characterization of their antigenic structure.

A method is described for the isolation of parietal cells from the gastric mucosa of the guinea pig by enzymatic digestion with collagenase. A suspension was obtained that contained 70-80% parietal cells. About 80% of the cells were viable immediately after incubation, but viability dropped sharply after one hour. Parietal cells were identified by their morphology on light and electron microscopy, by their uptake of neutral red, by immunofluorescent staining and by carbonic anhydrase activity. Antibodies to four distinct parietal-cell antigens were obtained from rabbits immunized with the isolated parietal cells or fractions thereof. These antibodies were directed against the microsomal fraction of the parietal-cell cytoplasm, the plasma and nuclear membranes, the soluble proteins, and Castle's intrinsic factor. The antibody against the microsomal fraction, though reacting in the same way as the antibody to parietal cell canaliculi found in the serum of patients with pernicious anaemia, showed greater species specificity.

[Methodological studies on the immunologic determination of proconvertin (factor VII)].

The influence of the different determinations of factor VII activities on the determination of the concentrations of the immunoreactive factor VII in the neutralizing test according to Good-Night were re-examined. The measurement of activity was performed with Seitz filtered cattle plasma as a complex proof of factor VII and X and with a specific artificial deficiency plasma. The examinations had the following results: In the plasma of healthy grown-up people there is a considerable, apparently physiologic range of dispersion of the immunoreactive concentration of factor VII within its normal activity which can be traced irrespective of the used measurements of activity. In plasma samples with a decreased activity of factor VII the values of measurement of the complex evidence will surpass those of the specific determination. The extent of neutralizing the activity in factor VII is not only dependent on the antibody concentration, but will mostly depend on the antibody-antigen relation. The findings obtained prove that the natural deficiency plasma of factor VII used in the original method of Goodnight can be replaced by an artificial substrate plasma.

Influence of molecular structure on the tolerogenicity of bacterial dextrans. II. The alpha1--3-linked epitope of dextran B1355.

Dextran B1355 is a branched glucose polymer containing 57 per cent alpha1--6, 35 per cent alpha1--3 and 8 per cent alpha1--2/1--4 linkages. Direct PFC responses to B1355 can be measured with sheep RBC sensitized with its O-stearoyl or palmitoyl derivative, and, as shown by inhibition analysis, are specific for an eptiope which is dependent on alpha1--3 linkages. B1355 is a potent immunogen in BALB/c mice producing peak PFC levels which approach 10(6) per spleen following an optimal dose of 1 mg. By contrast, the alpha-1--3-linked epitope of B1355 is feebly tolerogenic, for even 10 mg still induces a strong initial response. Mice given 1--10 mg sustain PFC levels 1--2 log10 above background for several months, but do not respond further to restimulation. Full recovery is attained by their spleen cells within 1 week of transfer into irradiated recipients. Deeper tolerance to this epitope was attained in vivo only when these larger doses of B1355 were injected during cyclophosphamide suppression. Two exceptions to this weak tolerogenicty were found. First, BALB/c spleen cells developed durable partial alpha1--3 tolerance following 2-hour incubation with B1355 in vitro. Second, CBA mice were fully tolerized by doses of 1 mg upwards. It is argued from these and other data in the accompanying papers that the relative resistance of BALB/c mice to induction of alpha1--3 tolerance is explicable neither as part of a more general phenomenon based on macrophage activity nor as due to an inadequate epitope density. A possible explanation based on features of the genetically determined high alpha1--3 responsiveness of BALB/c B cells is discussed.

The potentiality of antibody-producing cells. II. Evidence for two antibody molecules of different specificities secreted by micromanipulated bispecific mouse spleen cells.

Bispecific PFC appearing on the 4th day after immunization of mice with sheep erythrocytes conjugated with trinitrophenyl hapten (TNP-SRBC) were identified by their capacity to lyse native SRBC and TNP-conjugated horse erythrocytes (TNP--HoRBC) simultaneously in an open carboxymethylcellulose medium. Individual PFC thus detected, were micromanipulated into two successive media containing the indicators SRBC and TNP--HoRBC. Out of 103 transferred double cells ninety-two (89 per cent) remained double in the second medium and of the ninety-two transferred into the third medium, fifty-four (58 per cent) provoked a double lysis, eighteen (19 per cent) provoked a single lysis and twenty (23 per cent) ceased to lyse. On the other hand, when the second medium contained the indicators and the soluble specific inhibitor (TNP--BSA), out of 156 transferred double PFC, 125 (80 per cent) became single and only nineteen (12 per cent) remained double. Of these cells, 136 were transferred into the third medium (not containing inhibitor) and here again sixty-five (48 per cent) became double, forty-nine (36 per cent) remained single and twenty-two (17 per cent) ceased to provoke any lysis. Other double PFC were micromanipulated from the original revealing medium into two successive media containing only one indicator and the homologous (second medium) or the heterologous third medium) soluble inhibitor (TNP--BSA or soluble SRBC antigen), in order to see whether a soluble inhibitor suppresses only the corresponding specific lysis. Out of 186 double PFC transferred into the media containing the indicator and the corresponding inhibitor 147 (79 per cent) were specifically inhibited, whereas out of 176 double PFC transferred into the third medium (in which about 20 per cent of the cells cease to function) containing one indicator and the unrelated inhibitor, 117 (66-6 per cent) lysed the indicator in spite of the presence of the unrelated inhibitor. Since a specific inhibitor suppresses the lysis of the corresponding indicator, whereas its presence does not interfere with the lysis of the unrelated indicator in the majority of double PFC, it can be concluded that these cells secrete two types of antibody molecules possessing different specificities.

Alien histocompatibility determinants on the cell surface of sarcomas induced by methylcholanthrene. I. In vivo studies.

Groups of BALB/c mice were immunized to normal tissues (skin and/or liver plus kidney) of C3Hf, C57Bl/6, DBA/2 and AKR strains and challenged with either of two syngeneic 3-methylcholanthrene-induced immunogenic sarcomas, ST2 and TZ15, or with a "spontaneous" non-immunogenic BALB/c sarcoma, B2. It was found that anti-C3Hf and anti-DBA/2 immune mice were significantly protected against the growth of ST2, whereas anti-AKR immune mice rejected TZ15; no protection was elicited by immunizing with normal tissues of any strain against B2, which lacked individual tumor-associated transplantation antigens (TATA). The reciprocal experiment, i.e. the immunization of BALB/c mice with tumor cells and challenge with skin grafts of different strains, was also carried out with ST2 and TZ15. Accelerated rejection of all the various allogeneic skins was observed in anti-ST2 immune mice and of AKR and C3Hf skin in anti-TZ15 immune animals. In addition the Winn test demonstrated that lymph-node cells of BALB/c mice immune to C3Hf or DBA/2 tissues were specifically inhibitory for ST2, and that lymph-node cells immune to AKR tissues protected against TZ15. In a further experiment both ST2 and TZ15 tumors were left to grow in (C3Hf X BALB/c)F1, (C57Bl/6 X BALB/c)F1, (BALB/c X DBA/2)F1 and (BALB/c X AKR)F1 mice; the tumors were then excised and the "immune" mice challenged with the related tumor to measure their immune response in comparison with that elicited by the same procedure in BALB/c mice. ST2 was highly immunogenic in syngeneic BALB/c mice and in all the hybrid combinations except (C3Hf X BALB/c)F1 mice, where it completely lost its immunogenicity; TZ15 showed a certain loss of immunogenic strength in (BALB/c X AKR)F1 hybrids. It was concluded that TATA of ST2 contain antigenic determinants expressed on the normal cells of C3Hf and DBA/2 strains, and that TATA of TZ15 are likely to share antigens with AKR normal tissues.

The interaction of apurinic acid aldehyde groups with pararosaniline in the Feulgen-Schiff and related staining procedures.

The equilibrium reactions involved in the formation of the apurinic acid (APA)-Schiff chromophores in the staining phase of the Feulgen-Schiff reaction do not allow a quantitative conversion of APA to these chromophores. By modification of the sulfite and dye concentrations and the pH of the staining reagents, or by using better solvents for pararosaniline like acetic acid or dimethylsulfoxide (DMSO) a shift of these equilibria was attempted in order to obtain a higher amount of APA-bound dye. A 40% higher absorbance, when compared with the normal Schiff-staining, was obtained in model films by staining with a saturated solution of pararosaniline in a 1:1 v/v mixture of DMSO and SO2-water, followed by rinsing in SO2-water. A doubling of the absorbance resulted in the same objects when a saturated solution of pararosaniline in a 2 M acetic acid/acetate buffer of pH 4.45 was used for staining, followed by a short rinse in SO2-water. Amino groups (as found in histones) are shown to compete with the amino groups of pararosaniline for the APA aldehydes. This effect, although causing lower staining intensities, is shown not to be the explanation for the differences in stain content found between more and less compact forms of chromatin. Depending on the pH, and dye and sulfite concentrations of the staining reagents, the following components are considered as possible contributors to the mixture of chromophores (Duijndam et al., 1973 b) formed between APA and Schiff's reagent or its modifications: 1. An acid labile component with a wavelength of maximal absorbance (lambda max) near 510 nm; its structure is probably the azomethine--CH=N--; 2. A relatively acid stable component with a high value of molecular absorbance (epsilon), an lambda max near 570 nm and possibly having an enamine structure--CH=CH--NH--; 3. A component with intermediate acid stability, low epsilon, and lambda max near 540 nm, and which is probably an alkylsulfonic acid --CH(SO3H)--NH--compound. Small differences in the staining conditions in the histochemical application of the Feulgen-Schiff reaction may cause a shift in the ratio between especially components 2 and 3, resulting in variations in stain content and in lambda max.

Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue.

An 8-15S fraction of RNA isolated from hyperplastic human parathyroid tissue (primary chief-cell hyperplasia) and translated in a cell-free extract of wheat germ directs the synthesis of a protein that shares antigenic determinants and tryptic peptides with parathyroid hormone and its previously recognized immediated precursor, proparathyroid hormone. In addition, the protein contains tryptic peptides not found in proparathyroid hormone and migrates more slowly than does proparathyroid hormone on both urea-acid and urea-sodium dodecyl sulfate polyacrylamide gels, indicating that it is more acidic and larger than proparathyroid hormone. Sequential Edman degradation of the cell-free protein, radiolabeled with [35S]methionine, for 25 cycles released [35S]methionine at cycles 1, 7, 11, and 14, indicating that the NH2-terminal peptide sequence of the protein differs from that of both proparathyroid hormone and parathyroid hormone. We propose that this protein is an early biosynthetic precursor of human parathyroid hormone, pre-proparathyroid hormone, analogous to that identified recently by in vitro translation of bovine parathyroid mRNA.

The effect of alloantisera on antigen-induced T cell proliferation.

In the guinea pig, alloantisera raised by cross-immunization of strain 2 and strain 13 animals are capable of specifically inhibiting the in vitro proliferative response of (2 X 13)F1 T lymphocytes to those antigens the response to which is controlled by Ir genes linked to the genes controlling the alloantigens against which the serum is directed. However, in similar studies performed in the two parental strains, the responses to antigens not known to be under unigenic control were also markedly inhibited by the appropriate alloantisera. We have extended our studies of this "nonspecific" inhibitory effect of alloantisera on T cell proliferation and have demonstrated that the proliferative response of strain 2 and strain 13 T cells to a large number of antigens is markedly inhibited by anti-2 and anti-13 sera, respectively. Antisera to the B alloantigen, the product of a linked but distinct histocompatibility locus, which is present in both strain 2 and strain 13 animals, also produced a marked inhibition of T-lymphocyte proliferation. A number of possible explanations for the generalized inhibitory effect of alloantisera on T cell proliferation are discussed.

The major histocompatibility complex of the guinea pig. I. Serologic and genetic studies.

Serologic and genetic studies of the antigens which comprise the guinea pig MHC have demonstrated three distinct but linked genetic regions. Antisera to the B region were raised by cross-immunization of random-bred animals; this region controls antigens B.1, B.2, B.3, and B.4 which behave as alleles at a single locus and which resemble the products of the murine D or K region genes in their tissue distribution and molecular characteristics. Cross-immunization of inbred strain 2 and strain 13 animals, both of which bear the B.1 antigen, leads to sera which identify antigens which resemble the products of the I region of the murine MHC. Specific absorption experiments have demonstrated four distinct I region antigens. In addition to the B and I regions, inbred strain 2, strain 13, and some outbred animals bear an antigen (S.1) which is the product of a third genetic region and which also resembles the murine D or K region gene products in molecular size. The results of these studies should facilitate the use of the guinea pig as an experimental model for studies of genetic control of the immune response and the function of the histocompatibility-linked Ir genes.

Bifunctional major histocompatibility-linked genetic regulation of cell-mediated lympholysis to trinitrophenyl-modified autologous lymphocytes.

Murine thymus-derived lymphocytes can be sensitized in vitro to trinitrophenyl (TNP)-modified autologous spleen cells (1, 2). Cytotoxic effector cells were generated which were specific for TNP-modified target cells expressing the same H-2K and H-2D serological regions as the modified stimulator cells (3, 7). Spleen cells from two C57BL/10 congenic strains of mice sharing common I-C, S, and D regions, but differing at K, I-A, and I-B regions, generated different levels of lytic responses to the shared modified H-2Dd products upon sensitization with auto logous TNP-modified cells. Lymphocytes from an F1 between responder and nonresponder strain generated a level of cytolysis toward the H-2Dd modified specificity which was of the same order of magnitude as that obtained with the high responder, irrespective of whether F 1 or either parental strain of modified stimulator cell was used. These results suggest that the modification of H-2Dd products resulted in formation of new antigenic determinants in both parental strains. However, the difference observed in responsiveness appeared to be due to a gene or genes mapping in the K, I-A, or I-B region which influenced the ability of the responding lymphocytes to react to these modified H-2Dd products. Responsiveness was expressed as a dominant trait in the F1.

A quantitative analysis of isotope concentration profiles and rapid transport velocities in the C-fibers of the garfish olfactory nerve.

In the olfactory nerve of the long-nosed garfish (Lepisosteus osseus), unusually well-defined isotope concentration distributions can be established with the rapid transport process. Transport velocities of two profile loci can be accurately described and a quantitative profile analysis is possible after profile normalization. Results from such studies indicate that: (1) peak amplitudes decrease exponentially as a function of distance from the olfactory mucosa according to the equation p = 2130 exp (-0.109chi); (2) the wavefront base and the peak apex loci move at rates of 221 +/- 2 and 201 +/- 4 mm/day, respectively (at 23 degrees C), revealing a peak dispersion or broadening during transport; (3) the broadening is asymmetric with material shifting to the rear of the peak; (4) plateau regions are established behind the peak with material deposited by the peak; (5) only 20% of the total radioactivity in a cut nerve reaches the nerve terminals in the rapid transport peak while 80% is deposited along the axon; (6) profile areas from cut nerves decrease and lose 15% of their activity in 20 hr, while intact nerve profiles increase 10% in 16 hr due to continued somal contribution to the profile; (7) the displacement of the wavefront base (WFB) and peak apex (PA) profile loci can be described by the functions s(WFB) = (0.055T - 0.345)t - 1.43 s(PA) = (0.053T - 0.391)t - 2.71 (8) transport velocities are linear functions of temperature between 10 and 25 degrees C and increase 370% in that range. A linear extrapolation of the WFB and PA functions to 37 degrees C yields 410 and 377 mm/day, respectively.

[Blood flow and oxidative metabolism of the brain in patients with dementia (author's transl)].

The purpose of this retrospective study was to investigate how the blood flow and oxidative metabolism of the brain was changed in dementia and the influence of the age factor. Cerebral blood flow (CBF) was measured in 115 patients aged from 40 to 83 years by means of the Kety-Schmidt technique with the modification of Bernsmeier and Siemons. The cerebral metabolic rates of oxygen and CO2 were determined by the van Slyke method and by gaschromatography respectively and of glucose and lactate by standard enzymatic methods. All cases of dementia due to head injuries, cerebral infections, cerebral infarctions, exogenous or endogenous intoxications or circulatory diseases were excluded from this study, but no classification of the dementias was made. Statistical calculations were carried out by means of the analysis of variance for a two-way design. Cerebral blood flow did not show a normal distribution curve but was at least triphasic; CBF in demented patients was either lower than normal, normal or higher than normal. The distribution curves showed further that a low cerebral blood flow of mean 32.5 ml/100 g min coincided with a low CMR oxygen of 2.50 ml/100 g min; however, CMR glucose was either low (2.50 mg/100 g min), or nearly normal (4.50 mg/100 g min) or elevated (7.50 mg/100 g min). A normal (45.0 ml/100 g min) or enhanced (62.5 ml/100 g min) CBF correlated with a CMR oxygen which was either decreased to 2.75 ml/100 g min or increased to 4.75 ml/100 g min; CMR glucose was either decreased to 1.50 mg/100 g min, or nearly normal (4.50 mg/100 g min), or was elevated to 6.50 and 10.50 mg/100 g min with respect to the peaks of the distribution curves. It is assumed that the variability of the findings with respect to the blood flow and oxidative metabolism of the brain in dementia is due to different pathophysiological and pathobiochemical disturbances in the brain. A significant influence of age on CBF and metabolism in patients with dementia was not found.

Chemical and biologic properties of isolated radiolabeled bleomycin preparations.

Cobalt-57-bleomycin is a diagnostically useful radiopharmaceutical, but little is known about the nature of its individual fractions in regard to their metal-binding capacity and their in vivo distribution. Bleomycin was separated by high performance liquid chromatography (HPLC) into four major components. These were labeled and the distribution studied in tumor-bearing rats at 2 and 24 hr. In vivo radiochemical purity was also determined. Of the nine HPLC systems studied, Porasil A eluted with a mobile phase of 0.3% ammonium formate in methanol gave the best separation of the fractions. These fractions were copper free and retained their biologic activity and purity. An in vitro competitive binding study of 57Co-bleomycin with either 57Co-human serum albumin (HSA) or 57Co-ethylenediaminetetraacetic acid (EDTA) showed the labeled bleomycin to be a strong chelate. The biologic distribution in tumor-bearing rats showed significantly higher concentration in tumors at 2 hr for fractions A2 and B2 as compared to the bleomycin mixture. The other fractions, A1 and demethylA2, gave lower tumor concentrations than the bleomycin mixture. The tumor-to-blood ratios for A2 and B2 were not significantly different from the bleomycin mixture, suggesting that the concentration of the bleomycin in the tumor was related to blood concentration. Tumor-to-blood ratios of greater than 10:1 at 2 hr were achieved for A2, B2, and the mixture; ratios of greater than 31:1 were achieved at 24 hr for all three. From these data it appears that the major components A2 and B2 are the most useful for diagnostic tumor imaging.

Oncofetal protein accompanying irradiation-induced small-bowel adenocarcinoma in the rat.

A tumor-associated protein was found in tissue derived from an X-irradiation-induced adenocarcinoma in the small bowel of the rat. The protein was associated with the cell membranes of the tumor tissue. It shared common antigenic determinants both with a rat fetal protein and a perchloric acid-soluble protein isolated from the serum of the tumor-bearing rat.

In vitro transcription of DNA from the 70S RNA of Rous sarcoma virus: identification and characterization of various size classes of DNA transcripts.

DNA transcripts, ranging from 1,500 to 4,500 nucleotides in length, can be identified in the DNA product synthesized in vitro by the Rous sarcoma virus-associated RNA-directed DNA polymerase. Although these DNA transcripts are considerably larger than the size classes of the DNA product previously reported, they only represent a minor proportion (less than 5%) of the total DNA synthesized in standard reaction mixtures containing rate-limiting concentration of one of the four deoxynucleoside triphosphates. However, the proportion of these larger transcripts relative to the total DNA product increases substantially when the enzymatic synthesis of DNA is performed in the presence of equimolar concentrations of deoxynucleoside triphosphate precursors. Both rate-zonal sedimentation in alkaline sucrose and nucleic acid hybridization techniques have confirmed the length and genetic complexity of these larger DNA transcripts. The identification of large DNA chains in the DNA product synthesized in vitro by the avian oncornavirus RNA-directed DNA polymerase provides an explanation for the paradox that exists between the limited number of primer sites per 70S RNA genome, the small size of the bulk of the DNA product, and the extent of the Rous sarcoma virus genome represented by the DNA product.

Hypercalcemia and neoplasia. Biologic, biochemical, and ultrastructural studies of a hypercalcemia-producing Leydig cell tumor of the rat.

A localized, transplantable testicular tumor of the Fischer rat regularly produces hypercalcemia and increased phosphorus clearance in host animals. Light and electron microscopic examinations of the tumor indicate that it is of Leydig origin. There is no evidence that the tumor secretes any biologically active sex steroids, judges by weights of target tissues, when the tumor is grown in castrated or spayed rats. No radioactive steroid hormone formation in vitro was detected using 1-14C-acetate as a precursor although 14C was incorporated into the "C27" sterol fraction. Mass (micrograms) amounts of sex steroids were not detected after purifying large amounts of tumor extracts. The phytosterols, beta-sitosterol, stigmasterol, campesterol, were tentatively identified in tumor extracts but were also found in other tissues and in tumors not associated with hypercalcemia. Administered in vivo, human chorionic gonadotropin caused an acute rise in serum calcium in 3 to 5 hours in tumor-bearing hypercalcemic rats. Only trophic hormones with luteinizing hormone activity were found to compete with 125I-human chorionic gonadotropin for binding to the tumor homogenate in vitro indicating the tumor possessed luteinizing hormone receptors. When the tumor was transplanted intrasplenically, hypercalcemia did not occur unless adhesions formed, suggesting that the tumor hormone was rapidly metabolized by the liver and was probably of small molecular weight. Secretory granules, usually thought to be associated with peptide hormone secretion, were not detected at the ultrastructure level. Cortisol, conjugated estrogen, and an inhibitor of sterol biosynthesis (AY-9944) were effective in lowering the elevated serum calcium. Definitive identification of the agent causing lethal hypercalcemia has not been accomplished. The available data suggest it is not parathyroid hormone or vitamin D. The Leydig cell origin of the tumor, its response to human chorionic gonadotropin in vivo, the lack of secretory granules at the ultrastructural level, and biologic characteristics, all lead to the speculation that the secretory product of the tumor is a new hormonal substance, possibly a steroid precursor or related substance not previously described or is a known substance of small molecular weight whose calcium-mobilizing properties have not been fully characterized. This transplantable tumor may represent a model for one form of neoplastic hypercalcemia occurring in man and may have important implications in the general area of calcium and phosphorus homeostasis.

A new type of familial hypercholesterolaemia.

Two members of a family (proband and daughter) with hypercholesterolaemia have an abnormal low-density lipoprotein which fails to suppress the activity of a rate-determining enzyme for cholesterol biosynthesis (3-hydroxy-3-methyl glutaryl-CoA reductase) in leucocytes of the patients and controls. However, the proband's leucocytes are inhibited by lipoproteins from other sources demonstrating that the mechanism for cellular regulation of the enzyme is intact. This mutant lipoprotein may have a role in the production of hypercholesterolaemia.

Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange.

A staining technique that detects sister chromatid exchanges (SCEs) has been used to examine the response of chromosomes in cultured Chinese hamster cells to a wide variety of mutagens-carcinogens. The test gives a very sensitive and rapid method for detecting chromosome mutagenicity of chemical agents and provides a powerful new method for detecting environmental mutagens.

The release of membrane antigens into culture by adult Schistosoma mansoni.

Antigens sharing determinants with surface membranes and soluble proteins of adult Schistosoma mansoni have been detected in culture media after incubation of radioactively labelled worms. The relative quantities of these antigens were measured with specific antisera raised in rabbits and with serum from an immune rhesus monkey. It was found that 12-16% of TCA-precipitable radioactivity in the culture medium consisted of membrane antigens and 6-8% consisted of antigens sharing determinants with proteins found in the soluble fraction of adult worms. Over half the membrane antigens were present in particulate form, while other antigens were present in solution. Surface labelling the adult worms with [125I]confirmed that some of the particles in the culture medium were derived from the surface membrane of the adult worm and electron microscope examination of such particles showed that large membrane fragments were present. These results support the hypothesis that antibodies against schistosome membrane antigens are induced by particulate membrane antigens released by the parasite.

Isolation of "speckled" nuclear antigen reactive with autoantibodies in patients with cancer and autoimmune diseases.

An antigenic substance reactive with autoantibodies found in patients with cancer and autoimmune diseases was isolated from calf thymus. The purification procedure included extraction of the tissues with acetone powder, batch and column chromatography on DEAE-resins, ammonium sulfate precipitation, gel filtration on Sephadex G-200, and affinity chromatography on antibody-Sepharose 4B. Indirect immunofluorescence examination of cultured human embryo cells, using the serum of patients with nasopharyngeal cancer, showed a speckled nuclear pattern. The antigenic factor was a soluble acidic protein with a pI of 5.0 and a molecular weight of 250,000. The antigenic activities of this purified substance from calf thymus, and of the material on the cultured human embryo cells, were destroyed by proteases, ribonuclease, and alkaline phosphatase. The determinants were also sensitive to periodate oxidation. Thermal stability to 60 degree C and pH stability between 2.6 and 8.5 were demonstrated. Cross-reactivity of the antigenic substance with antibodies isolated from individuals with cancer and autoimmune diseases was shown by immunofluorescence, with appropriate blocking and absorption controls.

HL-A27 and anterior uveitis.

HL-A types were determined in 90 successive patients with non-granulomatous uveitis. Fifty-one were HL-A27 positive (55.7%) compared to 8.2% of controls. Of 16 patients with ankylosing spondylitis, 13 were HL-A27 positive, as were two patients with a history of Reiter's syndrome. Twenty-eight patients were HL-A27 positive but had no evidence of rheumatic disease. The findings are discussed in relation to the possible pathogenesis of uveitis.

Prostatitis: Man's hidden infection.

Prostatitis exists when inflammation of prostatic glands and tissues results from infection or allergy. Gram-positive and negative bacteria cause most prostatic infections, but infections may also be caused by fungi, mycoplasma, viruses, and other nonbacterial infecting agents. Precise diagnostic localization of infection to prostatic glands is accomplished by obtaining divided urinary specimens and prostatic fluid and observing numbers of bacteria (or other infecting agents) present in each specimen. Treatment of prostatitis remains difficult because of several factors: patients may lack the normal prostatic antibacterial factors and only a few commonly used antibiotics pass the plasma-prostate barrier and enter prostatic fluid. For proper therapy, one must select antibiotics known to penetrate into prostate tissue and fluid and to which the infecting organism is demonstrated to be sensitive. Even so, optimal cure rates following antibiotic therapy seem no better than 33 per cent. Nonspecific methods of treatment such as surgical excision of prostatic tissue, prostatic massage, sitz baths, relaxants, and supportive psychologic therapy contribute to the rehabilitation of patients with prostatitis. Relapse or recurrence of prostatitis is frequent. Longterm (in excess of six months) follow-up is required to ascertain cure.

Embryology of the epidermis: ultrastructural aspects. II. Period of differentiation in the mouse with mammalian comparisons.

A detailed light and electron microscopic study of the cellular morphology of the epidermis in the 13 through 16 day mouse fetus reveals that an occasional intermediate cell is interposed between the basal and peridermal layers on day 13. All layers are mitotically active. Tonofilaments, unassociated with desmosomes, are present within the basal cell cytoplasm and the mitotic axis of the basal cells has changed from a parallel to a perpendicular plane with respect to the epidermal surface. At 14 days, a complete stratum intermedium, composed of one or two cell layers, is present. Rarely, developing hemidesmosomes are observed. Pools of glycogen are present in all cells below the periderm. The periderm is dense and no longer mitotically active. The skein of filaments, present in the inferior cytoplasmic region of the basal cells on days 12 and 13, is now absent. In the 15 day fetus, numerous developing hemidesmosomes are present. The stratum intermedium contains three to four layers of cells, and filaments are located deep within the cytoplasm of these intermediate cells. Rarely, a few developing keratohyalin granules and keratinosomes are present. A stratum intermedium is no longer present in the 16 day fetus. This region is now composed of a stratum spinosum and a stratum granulosum. Numerous keratinosomes are located in the upper stratum spinosum and lower stratum granulosum. The cells in the stratum granulosum are nucleated and the uppermost cells contain large keratohyalin granules. Three heterogeneous and one homogeneous type of keratohyalin granule is described. Dense bodies are present within mitochondria, nuclei, glycogen pools and the peridermal cytoplasm. The periderm is no longer dense and glycogen and keratohyalin granules are not observed in this layer.

Selective staining of animal chromosomes with synthetic dyes following iodine-dye-procedure.

The paper embodies results of the use of 51 synthetic dyes, belonging to different chemical groups for staining of animal chromosomes following iodine-dye procedure. It has been found that some of these dyes can replace gentian violet, crystal violet and safranin when used after this procedure. It has further been found that the fluorescent dyes, acriflavine and acridine yellow can also be used to stain animal chromosomes and that some of the dyes belonging to one chemical group can be successfully used whereas others of the same group are of no use. Dyes of the monoazo group are absolutely useless. Amongst the dyes successfully used, the preparations remain stable when stained with most of them except methyl green, malachite green, brillant green, iodine green and cresyl violet and amongst acid dyes, acid fuchsin. Cytochemical studies presented herein indicate that the components of the animal chromosomes stainable with crystal violet are the nucleic acids and that these substances should be highly polymerised and should not be even in a semi-degraded state. Removal of any one of these nucleic acids makes the chromosomes unstainable with iodine-crystal violet.

Serotypes of hepatitis B antigen (HBs Ag): the problem of "new" determinants, as exemplified by "t".

The principal antigenic determinants of hepatitis-B surface antigen (HBs Ag) are specified by distinct genotypes of hepatitis-B virus (HBV). This hypothesis still stands undisproven. These specificities include the common antigen(s), called a, and two pairs of subdeterminants, d/y and w/r. The members of each pair are in general mutually exclusive, resulting in four "primary" phentoypes of HBs Ag: adw, adr, ayw, and ayr. The newer "a(w) subcategories" probably also reflect differences in viral genotype; if so, the number of "subtypes" rises to eight. Further subdivision into independent strains of HBV may eventually become feasible on the basis of one or more of the following "new" HBs Ag reactivities, once these have been shown to be virus-coded: g, n, q, x, and t. The requirements for accepting new reactivities as HBV-specific, and for comparing them among themselves, are discussed, using t as an example. A peculiar feature of t is its variable physical behaviour: "overt" t reactivity is correlated with the adw phenotype; "cryptic" [t], with ayw; while adr and ayr have so far been t-negative. Pending the indispensable tests of experimental transmission, this uneven distribution would seem to suggest that expression of t is regulated by the HBV genome.

Relationship of hepatitis A antigen to viral hepatitis.

Progress in research on hepatitis type A has begun to accelerate because of the recent discovery of an antigen associated specifically with hepatitis type A infection and the development of tests for antibody to the antigen. Hepatitis A antigen is associated with 27 nm virus-like particles found in the liver and stool of animals experimentally infected with hepatitis type A and in the stool of humans experimentally or naturally infected with the virus. The density of the particulate antigen when isolated from the liver is 1.34, but antigen particles with densities ranging from 1.32 to 1.40 have been detected in stool. However, antigens from the liver and from the stool appear to be antigenically related. Using immune electron microscopy as a serologic tool for detecting antibody to hepatitis A antigen, we detected antibody in convalescent sera from 100 per cent of patients experimentally or naturally infected with hepatitis type A. In contrast, patients with hepatitis type B or non-B hepatitis not epidemiologically compatible with a diagnosis of hepatitis type A did not have a serologic response to hepatitis A antigen. Antibody was found in approximately 50 per cent of normal individuals tested; the frequency was directly related to age. By the use of immune electron microscopy for the detection of hepatitis A antigen and antibody, the temporal relationship of antigen, antibody and liver damage was determined in experimentally infected humans and chimpanzees. On the basis of serologic comparisons, hepatitis type A does not appear to be related to experimental hepatitis caused by the GB agent of Deinhardt, nor is the hepatitis A antigen serologically related to the fecal antigen of Cross.

Development and utilization of complement-fixation and immune adherence tests for human hepatitis A virus and antibody.

The reliable propagation of CR326 strain of human hepatitis A virus in Saguinus mystax marmosets has permitted the development of specific serum neutralization, complement-fixation (CF), and immune adherence (IA) assays for hepatitis A antigen and antibody. The CF and IA assay were made possible by the use of livers of CR326-infected marmosets as a source of hepatitis A antigen. All assays were shown to be specific for hepatitis A. Patients with hepatitis B did not show development of hepatitis A antibody. Hepatitis A antibody appeared following onset of illness, and, in the longest time period studied, has persisted for seven years. Epidemiologic studies have been performed on several Costa Rican families with outbreaks of hepatitis, with the IA and CF assays. Also, several populations in the U.S.A. were studied. These indicated a high incidence of hepatitis A at an early age in Costa Rica and a relatively low incidence of hepatitis A antibody among adults in the U.S.A. It was shown that human immune globulin can be standardized for hepatitis A antibody content by the IA assay. Finally, the IA assay indicated probable hepatitis A antibody in uninoculated chimpanzees, grivet monkeys, and rhesus monkeys.

[Effect of chlormandinone acetate upon seizures in epileptic children with latent signs of hyperandrogenism].

The effect of chlormadinone acetate (24 mg/day) upon the plasma levels of pituitary gonadotropins and gonadal hormones on the number of generalized convulsions and spike EEG density was investigated in a group of epileptic children with intractable seizures and with clinical signs suggestive of hyperandrogenism. In each case, the effect of chlormadinone was evaluated in relation to hormonal levels and seizures observed during a control period and under the effect of placebo as follows: Control (PC)-Chlormadinone acetate (PCL1)-Placebo (PP)-Chlormadinone acetate (PCL2). In a male child (4MS), the number of convulsive attacks observed in the control period (26/month) was reduced during PCL1 (2/month) increased during PP (12/month) and was reduced again during PCL2 (0/month). Spike EEG density showed a parallel course to the clinical attacks. In this case, control levels of testosterone were markedly elevated (40 ng/ml) and were decreased during PCL1 to 4.0 increased again during PP to 34.0 and decreased again during PCL2 to 1.2 ng/ml. Plasma levels of pituitary gonadotropins were unchanged throughout the entire period of study. In other cases, neither the number of epileptic attacks nor spike EEG density were apparently affected by this regime and plasma levels of pituitary gonadotropins and gonadal hormones were also unmodified. These results suggest that a latent state of hyperandrogenism may be detected in some epileptic patients with intractable seizures and that chlormadinone may reduce convulsive attacks in these patients, probably by decreasing testosterone plasma levels.

Nitroglycerin and premature ventricular complexes in myocardial infarction.

Because of clinical observations suggesting that nitroglycerin may suppress premature ventricular complexes during acute ischaemia, a study was undertaken to assess the effect of nitroglycerin on the incidence of premature ventricular complexes in patients with acute myocardial infarction. Forty patients with acute myocardial infarction were studied. Twenty-six patients received 0.4 mg nitroglycerin sublingually every 4 hours for the first 24 hours after admission to the coronary care unit. The total premature ventricular complex count for the 26 patients for 15 minutes before nitroglycerin was 592, and 276 for the 15 minutes after the drug (P less than 0.005). In the remaining 14 patients on the same nitroglycerin schedule, a single electrocardiographic lead was continuously recorded on tape. During the first hour after nitroglycerin, the premature ventricular complex count decreased by 58 per cent, and the second and third hours showed a decrease from control count of 71 and 65 per cent respectively. By the end of 4 hours the ectopic count was back to control level. The data indicate that nitroglycerin may decrease the number of premature ventricular complexes for up to 3 hours in patients with acute myocardial infarction. The mechanism of action of nitroglycerin is not elucidated by this study, but the observation may be of value in further studies of specific antiarrhythmic therapy and prevention of arrhythmias in patients with coronary artery disease.

On the interaction of 3,4,5,6-tetrahydrouridine with human liver cytidine deaminase.

In contrast to the rapid inhibition of bacterial cytidine deaminase by 3,4,5,6-tetrahydrouridine, the onset of inhibition of the enzyme from human liver was found to be relatively slow. Inhibition was found to be reversible, and the corrected rate constants for binding (kon = 2.4 x 10(4) M-1 sec-1) and release (koff = 5.6 x 10(-4) sec-1) were in reasonable agreement with a Ki value (2.9 x 10(-8) M) measured separately under steady-state conditions, which was several orders of magnitude lower than estimates previously reported in the literature. Rates of binding and release of this potential transition state analogue were not appreciably affected by the substitution of deuterium oxide for solvent water. The slow onset of inhibition, which was also observed for cytidine deaminase from HeLa cells, suggests that structural reorganization precedes the formation of a stable enzyme-inhibitor complex. 6-Azacytidine, which favors a "high-anti" configuration at the glycosidic bond, was found to be active as a substrate for cytidine deaminase, with a turnover number exceeding that of cytidine. 2,2'-Anhydro-1-beta-D-arabinofuranosylcytosine, which is restricted to the "syn" configuration, was found to be without activity as a substrate or an inhibitor.

Is a 'second look operation' justified in suspected recurrences after abdominal cancer surgery?

Seventy-three patients have been submitted to 74 further laparotomies for suspected recurrent malignant abdominal disease over a period of 13 months. The original tumour was situated in the large bowel in 42, oesophagus or stomach in 24, ovary in 3, small intestine in 2 and pancreas and retroperitoneum in 1 instance each. There were 10 examples of benign lesions, 16 of further primary cancer and 24 of resectable local recurrences or metastases. Seventeen patients underwent some palliative procedure, and only 7 were beyond any surgical help.

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons of the rat. II. Action of vincristine on axonal flow and neurotubules in the paraventricular and supraoptic nuclei.

Intrathecal administration of 20 mug of vincristine sulphate in the rat induced in vivo the formation of paracrystalline inclusions mainly in axonal processes. This is associated with an impairment in the migration of neurosecretory granules as shown by their accumulation in the perikarya of the magnocellular neurons. The granules are intermixed with numerous dense bodies of various shape, sometimes with a fibrillar content, and probably of lysosomal origin. In addition to the impairment of the flow of neurosecretory granules, there is also a striking accumulation of mitochondria and synaptic vesicles, and an apparent proliferation of the smooth endoplasmic reticulum. In the posterior lobe, the axonal endings contain a large number of neurosecretory granules, intermingled with bodies of varying shapes and electron density. Occasionally, a dense membrane surrounding a group of elementary granules is observed, reacting positively for acid phosphatase. This suggests an attempted crinophagia.

Basophilic leucocytes: structure, function and role in disease.

We have attempted to review the current state of our knowledge concerning the human basophilic leucocyte, drawing on experimental data derived from animals when necessary. Long neglected, a great deal has been learned about these cells in recent years, about their morphology, their biochemical constitutents and their ability to synthesize certain of these constitutents, their interactions with homocytotropic antibodies, their release of mediators in anaphylaxis, their response to chemotatic stimuli, their participation and progressive degranulation in cell-mediated hypersensitivity reactions, and their capacity for ingesting and releasing certain exogenous tracers. Despite this vast accumulation of new information, much more must be learned before we can confidently describe the role of basophils, or of the closely related mast cells, in health or disease. It seems most unlikely that either cell exists for the purpose of destroying the organism in anaphylactic shock. Nonetheless, it is highly probably that basophil/mast cell function is closely related to the potent chemicals stored within their cytoplasmic granules. One likely possibility holds that small amounts of these chemicals are required for homeostasis (e.g., for regulation of the tone of the microvasculature) and that these cells function by releasing such substances continuously, as they are needed, in small aliquots rather than by explosive discharge. This hypothesis requires that basophils be capable of releasing their contents in piecemeal fashion. Such gradual release apparently occurs in delayed-type hypersensitivity reactions, but the mechanisms responsible for this form of degranulation have not yet been identified. This hypothesis also requires that physiological, rather than pharmacological, roles be found for histamine, heparin and possibly for other components of the basophils/mast cell granules. Progress in this direction has been extremely slow.

[Maturation of interhippocampal responses. Report and localization of inhibitory and excitatory synapses in the horn of Ammon of the young rabbit].

Extracellular recording from hippocampal areas of new-born rabbits up to 3 months of age was carried out to examine the responses evoked by stimulation of the contralateral alveus of CA1. In CA1, the extracellular potential fields show a positive wave whose amplitude is maximal at the somata of the pyramidal cells at any age. This positive wave has the same latency as an inhibitory postsynaptic potential induced in the same conditions and presumably represents inhibition at this level. In stratum oriens and at the top of stratum pyramidale, this positive wave is accompanied by ripples (at a frequency of about 200 c/sec) that are produced by basket cells. These ripples exhibit a very small amplitude up to 10 days of age. This positivity decreases in the depth towards the apical dendrite, reverses at the beginning of stratum radiatum and becomes rapidly negative. A second positive wave with ripples could be recorded from the layer of deep pyramidal cell somata belonging to the underlying CA3-CA4, only after 7 days. It is concluded that, in both the new-born and the adult rabbit, inhibitory synaptic action from contralateral stimulation is present on or close to the pyramidal cell somata, whereas excitatory action is located in the apical dendrites near the main branching.

Interaction between the activity of an epileptic focus and discrete skilled movements in rats.

Sixteen male hooded rats were trained to reach into a narrow feeding tube for small food pellets. The paw movements were photoelectrically detected. An epileptic focus established by local application of 1% picrotoxin on the exposed motor cortex increased the frequency of reaching with the ipsilateral paw and impaired reaching with the contralateral paw. Interictal discharge rate of all ipsilateral foci was increased by reaching in the same way as the slow activity (less than 0.5/sec) of contralateral foci. On the other hand, fast activity (greater than 0.5/sec) of contralateral foci was decreased by reaching. Computer analysis of interictal discharge indicence during 512 msec before and after reaching onset showed that the brief facilitation of discharge (50 msec) during the actual movement was often preceded and followed by more prolonged inhibition (200 msec). The inhibition was better expressed in the contralateral hemisphere. The results are interpreted as due to changes of cortical excitability associated with reaching and to interference of the epileptic focus with the cortical elaboration of the skilled movement.

[Investigations on the influence of Rh immunoglobulin prophylaxis on the immune response to postpartum rubella vaccination (author's transl)].

A preliminary investigation showed that patients with rubella HHT antibody titres of 1:8 or greater did not show a significant rise in the antibody titre following rubella vaccination. The rubella antibody titre was determined in 651 obstetric patients. Of these, 43 (6.6%) had no significant antibodies to rubella (HHT less than 1:8) and were included in the present investigation. Patients in Group A received 0.5 ml. of the rubella vaccine Meruvax on the fifth postpartum day. Patients of Group B(Rh negative and Rh positive) received 250 mug anti-D in a 16% gammaglobulin solution intra-muscularly 48 hours postpartum and the rubella vaccination 3 days later. Three weeks following the rubella vaccination the mean geometric rubella antibody titre had risen +/- 1 Standard deviation to 19.6 +/- 7.7 in Group A (17 patients) and to 18.0 +/- 6.3 in Group B (12 patients). Six weeks following the rubella vaccinations Group A (19 patients) showed titres of 61.7 +/- 2.9 and Group B (14 patients) showed titres of 70.0 +/- 2.6. There was no statistically significant difference (greater than 0.5). The conversion rate in both groups was 100%. Patients can therefore be vaccinated against rubella in the postpartum period even though they will receive a concomitant prophylaxis with Rh immunoglobulin.

Common antigenic structures of HL-A antigens. VI. Common antigenic determinants located on the 33,000 Dalton alloantigenic fragment portion of papain-solubilized HL-A molecules.

HL-A 33,000 Dalton fragments were isolated from three papain-solubilized HL-A preparations carrying different HL-A allospecificity by the dissociation induced in the reaction with rabbit antibodies to HL-A 11,000-Dalton fragment (i.e. human beta2-microglobulin). By assaying the binding activity with rabbit antisera against papain-solubilized HL-A molecules and against each of the HL-A component fragments and also with a battery of HL-A alloantisera, the 'antibody-dissociated' 33,000-Dalton fragments were shown to retain not only the HL-A alloantigenic determinants but also the HL-A common antigenic determinants that are native to the 33,000-Dalton fragment portion of undissociated HL-A molecules and to be devoid of the cryptic HL-A common antigenic determinants that are found on HL-A 33,000-Dalton fragments obtained by dissociation with chemical reagents including acid. However, on exposure to acid, the antibody-dissociated 33,000-Dalton fragments were found to lose their HL-A alloantigenic and common antigenic characteristics and gain the cryptic HL-A common antigenic determinants. The greater part of the HL-A common antigenic determinants found here appears to be primate cross-reacting determinants.

Secondary specific immune response in vitro to MSV tumor cells.

The interactions which occur between antigenic tumor cells and normal or immune lymphoid cells in a 3-day in vitro culture, have been studied with a murine sarcoma virus (MSV)-induced tumor. The 3H-thymidine incorporation of lymphoma cells growing in suspension, and the radioactive-chromium release of freshly sampled lymphoma cells regularly added to the culture, have been compared to determine the part played by immune lymphoid cells in cytolysis and cytostasis of the tumor-cell population. The cytolytic activity increases in the culture from day 0 to day 3. It is due, predominantly, to T-cells, and remains specific to antigens shared by MSV tumors and related lymphomas. This activity would be difficult to detect unless freshly sampled ascitic cells were used as targets, since the lymphoma cells spontaneously lose a part of their sensitivity to immune cytolysis during in vitro culture. The method used in the present experiments is a secondary chromium release test (SCRT), which measures the invitro secondary stimulation of cytotoxic T-lymphocytes (CTL) by tumor cells. In the absence of stimulatory cells, the CTL activity would have rapidly fallen in vitro. The cytostatic activity also increases during the 3 days in vitro, in parallel to the cytolytic activity: it is due to non-T-cells and remains mainly non-specific. The significance of these data for the interpretation of invitro demonstrated cell-mediated anti-tumor immune reactions is briefly discussed, as well as their relevance in the in vivo role of immune CTL.

Myoepithelial cells in human thymus: staining, polarization and fluorescence microscopic studies.

Myoid cells in human thymus were studied around the turn of the century, and alterations in patients with cardiovascular disease were reported. It was therefore deemed of interest to reinvestigate these long forgotten cells. The configurational staining, polarization and fluorescence microscopic properties of smooth myofibrils in thymic epithelial cells were identical with those of classical myoepithelial cells, smooth muscle, and A bands of striated muscle. Cross-striated myoid cells could not be found in thymus of children. Myoepithelial cells formed a layer at the surface of thymic lobules; others were scattered throughout the cortex and medulla. In addition, the medulla contained seemingly hypertrophic myoepithelial cells. Hassall's corpuscles consisted of layers of myoepithelial cells. Hammar (1905) regarded epithelial cells with smooth myofibrils in human thymus as equivalents of the cross-striated myoid cells in lower vertebrates. The myoepithelial cells observed in this study are apparently identical with the smooth myoid cells of early anatomists; the hypertrophic myoepithelial cells correspond to the unicellular Hassall's corpuscles. The functions of these cells are not yet clear; the wide variations from case to case in the same age group indicate that the myoepithelial cells are affected by a variety of diseases.

Morphological heterogeneity of HeLa cell mitochondria visualized by a modified diaminobenzidine staining technique.

The diaminobenzidine (DAB) technique for the ultrastructural localization of sites of cytochrome c oxidase activity in animal tissues has been adapted to the visualization of mitochondria in animal cells growing in culture. The modified technique allows the staining of mitochondria in all cells in coverslip preparatins for light microscopy. Electron microscopy of thin sections of material treated by this method has revealed that all mitochondrial profiles within a cell (and only these) are stained and they exhibit a well preserved size and internal structure. Coverslip cultures of synchronized and unsynchronized HeLa (F-315) cells stained with the DAB reaction were examined under oil immersion. In the majority of the cells, mitochondria were recognized as discrete bodies in the thinner peripheral portion of the cytoplasm. This observation indicates that in a large proportion of HeLa F-315 cells, at least under the growth conditions used here, the mitochondrial complement is dividied into distinct organelles. This examination also revealed a considerable morphological heterogeneity of mitochondria, which exhibited an ovoid or short rod-like or, less frequently, long filamentous shape, with some evidence of branching. The variability in mitochondrial morphology appeared to be far more prounced between different cells than within individual cells; this cellular heterogeneity was not related in any obvious way to cell-cycle-dependent changes.

Mustard meal in dairy rations.

Consumption of 0% mustard meal and 15% soybean meal, 7.5% mustard meal and 7.5% soybean meal, or 15% mustard meal and 0% soybean meal rations did not differ in palatability studies with 10 group-fed lactating cows when the mustard meal was treated with 3% caustic soda. Order of preference was for 0, 7.5, and 15% mustard meal rations when mustard meal was untreated. Twelve lactating cows were in each of two lactation trials to compare the three rations of untreated mustard meal. Milk, milk fat, and solids-not-fat, and milk protein did not differ for either trial. Protein-bound iodine of plasma for all cows were within the normal range. Three cows were placed on each of the three rations and received a minimum of 9 kg per day for 6 mo preparturition to determine goitrogenic effects. All cows gave birth to normal, vigorous calves. Limited organoleptic evaluations of milk indicated that untreated mustard meal may impart a detrimental flavor to milk, but a taste panel could not differentiate between milk from cows on the three rations of treated mustard meal. Twenty-one male and 43 female Holstein claves received either 0, 10, or 20% mustard meal starter rations from birth to 3 mo of age. Growth, feed consumption, or plasma protein-bound iodine did not differ.

Atomic absorption spectrophotometry applied to photographic densitometry.

For this study of photographic densitometry, sections of cartilage stained with Alcian Blue, safranin O and high iron diamine were photographed at x40 with Nikon photomicrography equipment on Kodak Panatomic X film with appropriate filters to enhance contrast. Portions of the developed negative films were selected from intercellular matrix regions, and circles of film equivalent in diameter to a 30-mu circle of tissue were obtained with a hand-held paper punch. Silver was eluted from the circles of film with 35% nitric acid, and the quantity of silver deposited on the film was determined by atomic absorption spectrophotometry as a measure of stain intensity. The intensity determined by this analytic procedure compared favorably with results obtained previously from the same tissue with microspectrophotometry. This method of silver analysis has advantages over earlier studies which used silver elution to determine photographic densitometry in its technical ease, accuracy and sensitivity. Furthermore, this method compares well with microspectrophotometry in its results and has the advantages of relative inexpensiveness and availability of equipment.

Recognitive specificity of human cytotoxic T lymphocytes. I. Antigen-specific inhibition of human cell-mediated lympholysis.

The specificity of antigen recognition by in vitro sensitized human cytotoxic T lymphocytes (CTLs) has been studied using a sensitive cell-mediated lympholysis (CML) assay. Frequently, high levels of cytotoxicity are observed on third-party targets unrelated to sensitizing or responding cells; however, no cytotoxicity differing significantly from zero has been observed on targets autologous to the responding CTLs. This "cross-killing" of third-party target cells has been observed when stimulating and third-party cells bear no cross-reacting serologically defined (SD) antigens, thought to be the target antigens recognized by CTLs. CML-blocking studies, using unlabeled normal human lymphocytes to inhibit 51Cr release from radiolabeled target cells, have shown that cross-killing, even in the absence of shared SD determinants, results from CTLs recognizing antigens shared by the third-party targets and the initial stimulating population. Furthermore, these antigens have been mapped to the major histocompatibility complex (MHC). The ability of human CTLs to specifically recognize MHC-controlled antigens not detected serologically suggests that SD antigens may be recognized differently by alloantisera and CTLs, or that MHC antigens other than SD may be the targets of CTLs in CML.

Tumor imaging after administration of 99mTc-labeled bleomycin.

Bleomycin, an anticancer drug, was labeled with 99mTc using stannous chloride and ascorbic acid and specific activities of 1-3 mCi/mg-eq with labeling efficiencies of 50-75% were achieved. Very rapid excretion of 99mTc-bleomycin through the kidney and concomitant rapid decrease of radioactivity in blood, various tissues and organs, and whole body were observed after intravenous administration of the radiopharmaceutical into tumor-bearing mice. In such animals, approximately 1% of the label was found in a transplanted fibrosarcoma within 30 min while 0.58% was recovered in such lesions even after 24 hr. In patients positive tumor images were obtained by scintigraphy as early as 1 hr after intravenous administration of 3-5 mCi of 99mTc-bleomycin. A total of 142 cases were examined by scintigraphy after administration of 99mIc-bleomycin and/or 67Ga-citrate. In 93 cases with various malignant tumors, tumor was detected in 80% using 99mTc-bleomycin and in 63% using 67Ga-citrate. Technetium-99m-bleomycin scintigraphy successfully detected tumors of the thyroid, lung, face, breast, extremity, and digestive tract and was also useful in finding metastatic lesions and brain tumors. However, 67Ga scintigraphy gave superior results in detecting lesions in patients with malignant lymphomas. In patients with inflammatory diseases, accumulation in lesions was detected in 13% using 99mTc-bleomycin and in 48% using 67Ga-citrate. The further use of 99mTc-bleomycin scintigraphy for tumor detection in patients appears to be warranted.

A transmission electron microscope study of the effects of ion etching on cells.

The effects of ion etching on blood cells have previously been studied by scanning electron microscopy. This present study by transmission electron microscopy was undertaken to evaluate the effects of the etching process on the cells. Critical point dried preparations were made, etched and subsequently processed and embedded in Araldite. Examination of thin sections of erythrocytes revealed disintegration of the plasma membrane; the residual membrane destruction products formed the tips of cones produced by long etching times. The effect of etching varied in erythrocytes in the same preparation. Nucleated cells showed a similar disintegration of the plasma membrane, but membranes of mitochondria, granules, vesicles and vacuoles did not exhibit effects of etching comparable to those of the plasma membranes. After treatment with a number of different fixatives, erythrocytes on carbon-coated copper grids were also etched and examined directly in a high voltage electron microscope at 1 MV. The effects were comparable to those seen in thin sections. To study the etch rates of biological materials, the resonant frequencies of quartz crystals were measured after application of thin films of albumen and cholesterol and again after these had been etched. the ratio of the frequency changes indicated that the etch rate of albumen was approximately 2-5 times that of cholesterol. The results are discussed in the light of theories of the mechanisms involved in ion etching.

Controlled trial of bromocriptine, quinoestrol, and placebo in suppression of puerperal lactation.

2-bromo-alpha-ergocryptine (bromocriptine) in a dosage of 2-5 mg twice daily caused a rapid fall in plasma prolactin. It was more effective than either a single dose of 4 mg quinoestrol or a placebo in suppressing puerperal lactation, as judged by milk flow and the relief of breast pain and congestion. Patients who received quinoestrol were more comfortable than those who received placebo.

Early syphilitic hepatitis.

17 out of 175 cases of early syphilis had clinical, biochemical, and immunological evidence of liver damage. Before penicillin therapy the histological appearance of the liver was abnormal in 14 of the 15 patients from whom biopsy specimens were obtained. In 7 cases, treponemes were seen in the liver. After two months' penicillin therapy the extent and severity of the histological abnormality was reduced. In the repeat liver-biopsy specimens obtained after penicillin treatment no treponemes could be demonstrated. It is suggested that the hepatitis found in these 17 cases of early syphilis was produced by treponemes.

Coagulation changes during second-trimester abortion induced by intra-amniotic prostaglandin E2 and hypertonic solutions.

The coagulation system was studied in twenty-seven patients undergoing second-trimester abortion induced by intra-amniotic prostaglandin (P.G.) E2 alone and in combination with a hypertonic solution of urea or glucose. Changes consistent with intravascular coagulation, namely a rise in fibrin degradation products and a fall in plasma-fibrinogen and platelet-count, were observed in those patients treated using P.G.E2 with hypertonic urea. Similar but less pronounced changes were found in the group treated using P.G.E2 with hypertonic glucose. In patients treated with P.G.E2 alone no changes suggestive of intravascular coagulation were detected. One patient treated using P.G.E2 with hypertonic urea who did not abort for 26 hours demonstrated changes indicative of a pronounced degree of disseminated intravascular coagulation. These findings show that when abortion is induced using P.G.E2 and a hypertonic solution, particularly hypertonic urea, disseminated intravascular coagulation can occur as a result of a hypertonic agent being used.

Bromocriptine therapy in acromegaly.

Bromocriptine (CB-154, Sandoz) has been given to 21 acromegalic patients (11 female, 10 male) for a period of 6-10 months. The mean serum growth-hormone (G.H.) levels ranged from 10 mug/1 to 512 mug/1 before therapy. Bromocriptine suppressed G.H. values to 5 mug/1 or less in 4 patients and to less than 10 mug/1 in a further 8 patients, but in 2 patients G.H. levels did not show any significant reduction. Bromocriptine did not block stress-induced G.H. secretion. It did not distrub pituitary function other than secretion of prolactin and had negligible side-effects. Its effect on tumour size is uncertain and it is therefore unsuitable for patients with suprasellar extension of the tumour. Otherwise it seems reasonable to offer a trial of bromocriptine to all patients with acromegaly where therapy is deemed necessary. In those who show a full response of G.H. levels with a dose of 20-40 mg of bromocriptine per day, external radiation to the pituitary can be used to prevent tumour expansion and bromocriptine withdrawn at intervals to assess the effect of the radiation. In patients with a partial response to bromocriptine, the decision to offer alternative therapy depends on the extent of the response and on the age and medical condition of the patient. In patients who fail to respond to bromocriptine, particularly those younger patients with active disease, more definitive local treatment (e.g., trans-sphenoidal removal of the tumour or yttrium-90 implantation) would be indicated. Bromocriptine may also be used with benefit in the large number of patients who have shown a partial response to other forms of therapy.

Mechanisms of reflex cardiac arrest in tetraplegic patients.

Four patients with physiologically complete high cervical spinal-cord lesions, sustained within the previous 6 weeks, were observed. All needed intermittent positive-pressure ventilation. In the stage of spinal shock, stimuli to the trachea induced bradycardia, and in two patients cardiac arrest resulted. The bradycardia occurred when the patients were hypoxic, and seemed to be due to a vaso-vagal reflex. Normally this reflex is opposed by sympathetic activity, and during hypoxia by increased pulmonary (inflation) vagal reflex activity due to increased breathing. In these patients, however, compensatory sympathetic activity was prevented by the cervical cord lesion, and increased pulmonary vagal reflex activity by the fact that the breathing was artificial and therefore did not increase with hypoxia. Treatment in emergency includes the administration of atropine. Adequate oxygenation and, if this cannot be achieved, maintenance atropin should prevent the bradycardia and cardiac arrest associated with stimulation of the trachea in artificially ventilated tetraplegic patients.

Natural course of symptomless autoimmune thyroiditis.

Out of 18 subjects with symptomless autoimmune thyroiditis (S.A.T.) 5 (28%) became hypothyroid within 4 to 39 months of observation, whereas 13 (72%) remained euthyroid up to atleast 28 to 50 months. In all those who developed hypothyroidism the basal serum level of thyroid-stimulating hormone (T.S.H.) was already initially above normal (normal range 1-6--6-9 muU/ml) and 4 had markedly elevated concentrations (less than 19 muU/ml). All those subjects developing hypothyroidism also had initially an exaggerated response to thyrotropin-releasing hormone (T.R.H.) (upper normal limit, delta T.S.H. 30 muU/ml), and in 4 the response was much exaggerated (delta T.S.H. less than 70 muU/ml). In 3 of these subjects the basal T.S.H. and the response to T.R.H. were reassessed before starting the substitution therapy and in all there was a further increase in both values. The basal serum T.S.H. was initially also slightly increased in 2 and the response to T.R.H. slightly above normal in 4 subjects who remained euthyroid. The basal T.S.H. level became normal in both cases with elevated values; and the response to T.R.H. declined to a normal level in 3 of the latter 4 subjects, but showed a further increase in 1. The thyroglobulin antibody (TgA) titres were initially significantly elevated in 15 subjects and the thyroid microsomal antibodies (MsA) in 1. The TgA titres decreased during the observation period in all but 1 subject and a similar trend was observed as regards the MsA titres. It is concluded that within a few years of observation a substantial number of subjects with S.A.T. will be hypothyroid. A definitively increased basal serum T.S.H. level and a markedly exaggerated response to T.R.H. in the symptomless stage of the disease is connected with a high risk of late hypothyroidism.

[Pollution of human milk in France by organochlorine insecticide residues].

The monthly analysis of milk mixtures from ten French human milk banks, over a period of two years, showed that there was marked pollution by organochlorine insecticide residues. The following averages were noted: hexachlorocyclohexane + hexachlorobenzene 2,75 mg/kg of pure fat, DDT and derivatives 3,24 mg/kg fat, heptachlor epoxide 0,28, dieldrin 0,23. The main insecticides were, with average values, beta hexachlorocyclohexane (1,67), hexachlorobenzene (0,98), and dichlorodiphenyldichloroethylene (2,40). All these milks were invariably very polluted throughout the year, but there were however noted some qualitative differences according to the geographic position of the milk banks: South of the Loire river, the main polluting factor was hexachlorocyclohexane, whilst North of the Seine, fairly high values were found for heptachlor epoxide and dieldrin. The special study of human milk from Lille showed a 50% reduction in pollution between 1970 and 1973. The probable routes of contamination are human food and the ill-advised domestic use of pesticides.

Structural features of double-stranded polyribonucleotides required for immunological specificity and interferon induction.

Purified antibody to poly(adenylic acid)-poly(uridylic acid) was used in quantitative microcomplement fixation assays to detect conformational variations among several double-helical polyribonucleotide analogs of poly(adenylic acid)-poly(uridylic acid) or poly(inosinic acid)-poly(cytidylic acid) that had been previously evaluated for their ability to induce interferon. Modification at the furanose 2'-position of one or both strands resulted in a dramatic decrease in serological reactivity. Most modifications of the bases caused smaller serological changes, and no base modification caused complete loss of reactivity. The reaction patterns support the conclusion that the structure of the furanose and the overall conformation of the helix are critical in the formation of antigenic determinants. The backbones of both strands appear to be involved in forming a single antigenic site, and base modifications may alter the steric relationship between the backbones. In addition, the same structural changes that substantially alter recognition by antibody also lead to large changes in the interferon-inducing ability of the nucleic acid.

Endogenous immune complex nephropathy associated with malignancy I. Studies on the nature and immunopathogenic significance of glomerular bound antigen and antibody, isolation and characterization of tumor specific antigen and antibody and circulating immune complexes.

Three patients with clear cell renal carcinoma and one with another intrarenal malignancy were studied for the presence of glomerular localized immunoglobulins, complement components and tumor specific antigen and antibody by immunofluorescence. To determine the association and elucidate the pathogenic mechanisms involved in the relationship between tumors and glomerular deposits, antibody eluted from tumor tissue and renal glomeruli, cryoproteins, serum antibodies and rabbit antisera to tumor tissue were tested for specificity to antigen. The relationship between tumor antigens and the lipoprotein antigen localized in normal proximal tubular brush border (RTE) and the small bowel mucosa, was studied by immunofluorescence, absorption and blocking studies as well as complement fixation. Immunoglobulins and complement components were localized in the glomeruli and tumor membrane of all patients. Sera and glomerular fixed antibody from three patients with renal cell carcinoma localized to normal proximal tubular brush border and jejunal mucosa as well as to tumor membrane and the glomeruli and proximal tubules of all of these three patients. Anti RTE activity was also detected by complement fixation. Immunologic similarity between RTE and renal cell carcinoma antigen was confirmed by absorption studies. Furthermore, cryoprecipitable complexes of tumor antigen and specific antibody were isolated from the serum. The tumor antibody was immunologically similar to RTE. In the other case the rabbit anti-tumor antibody and the patient's serum fixed to the tumor membrane and kidney of the patient but did not show cross reactivity with the renal cell carcinoma or RTE. These studies suggest that the tumor antigen in renal cell carcinoma is similar to RTE and the glomerular deposits represent tumor antigen and antibody complexes. In addition these investigations support the hypothesis that tumor immune complexes are significant in the glomerular lesions, and that the origin of renal cell carcinoma is in the proximal tubule. The investigations also show that tumor antibodies are specific for tumors of the same morphological type but not for other tumors in the same tissue. Moreover, the renal glomerulus appears to be a chosen anatomic site for deposition of tumor antigens and antibodies and studies of the kidney may provide insight into the nature of tumor antigens and antibodies. Cryoprecipitation appears to be a valuable method in isolation of tumor complexes and characterization of tumor specific antigen and antibody.

Long-term prognosis of 66 permanent anterior teeth with root fracture.

A longitudinal clinical and radiographic follow-up study was made of all permanent teeth with root fractures referred to the Oslo University Department of Pedodontics between 1953 and 1972 (n = 66). The material included 51 patients aged 6-21 years. The mean observation period was 5.2 years, ranging from 1 to 19 years. The present report documents background data and the long-term results. Two teeth with exarticulation of the coronal fragments (3%) were immediately extracted. Repair of the fracture area occurred in 51 teeth (77%). Pulp necrosis was found in 13 teeth (20%), nine of which were successfully treated endodontically; only four teeth had to be extracted. Several factors were found to influence the prognosis, most notably the degree of dislocation of the coronal fragment. The localization of the fracture influenced repair only slightly. Despite somewhat increased mobility in some cases, the longevity of teeth with fractures even in the coronal third of the root was not significantly shortened. It is concluded that when optimally treated by repositioning, fixation and relief of occlusion, anterior teeth with root fracture have a favorable prognosis. Even when pulp necrosis occurs, the long-term prognosis is good.

Identification of tissue-specific antigens and concanavalin A receptors on rat epidermal cells using a radio-immunoprecipitation method.

Iodination with lactoperoxidase - 125I- - H2O2 was used to label surface components of rat epidermal cells. Lysis of the cells in non-idet P40 resulted in the solubilization of tissue-specific antigens and of concanavalin A receptors. These specificities were demonstrated using a radio-immunoprecipitation method. The tissue-specific antigens were recognized using absorbed rabbit anti-rat epidermal cell sera (Lloyd & Darnule 1974); they reacted with two low molecular weight components in the lysate (9,000 and 12,000 daltons). Concanavalin A reacted with three major components. Two had high molecular weights (75,000 and 95,000 daltons). The possibility that one of these components was radioiodinated lactooperoxidase, which would have reacted with concanavalin A, was disproved. Another component (which occasionally appeared as two peaks) was similar in size to the species detected by the tissue-specific antisera. Their non-identity was, however, demonstrated by the finding that the two specificities could be precipitated independently of each other.

Harvesting of granulocytes using a hydroxyethyl starch solution.

The need for sophisticated ocmponent therapy has resulted in improved techniques for obtaining concentrates of platelets and granulocytes. The use of single donors as a source for these products is advisable to avoid multiple sensitizations. Obtaining concentrated granulocytes represents a problem because of the difficulty in separating granulocytes from red blood cells by differential centrifugation or sedimentation since the specific gravities are similar. Hydroxyethyl starch (HES) makes the separation more effective. A solution made of 250 ml of 6 per cent HES, 250 ml of distilled water, and 15 g of sodium citrate in 30 ml distilled water provided a satisfactory anticoagulant solution for this purpose. The granulocytes collected averaged 49 per cent of the total available in the processed blood; the platelets averages 82 per cent. A satisfactory yield could thus be obtained from a single donor, and this could be repeated several times in a month. The value of ABO, Rh, and HL-A compatibility between donor and recipient probably increases the viability and safety of this procedure. The Haemonetic No. 30 Cell Separator provided an easy and rapid method for this procedure.