Showing papers in "Journal of Experimental Medicine in 1965"

Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques

Abstract: Two methods were used to demonstrate the presence of tumor-specific antigens in adenocarcinomata of the human colon: (a) rabbits were immunized with extracts of pooled colonic carcinomata, and the antitumor antisera thus produced were absorbed with a pooled extract of normal human colon and with human blood components; (b) newborn rabbits were made immunologically tolerant to normal colonic tissue at birth, and were then immunized with pooled tumor material in adult life. Normal and tumor tissues were obtained from the same human donors in order to avoid misinterpretation of results due to individual-specific antigenic differences. The antisera prepared by both methods were tested against normal and tumor antigens by the techniques of agar gel diffusion, immunoelectrophoresis, hemagglutination, PCA, and immunofluorescence. Distinct antibody activity directed against at least two qualitatively tumor-specific antigens, or antigenic determinants, was detected in the antisera prepared by both methods and at least two additional tumor antigens were detected exclusively in antisera prepared by the tolerance technique. Whether these additional antigens were qualitatively different from normal tissue antigens, or merely present in tumor tissue in higher concentrations than in normal tissue has not as yet been determined. Furthermore, it was shown that the tumor-specific antibodies were not directed against bacterial contaminants or against the unusually high concentrations of fibrin found in many neoplastic tissues. It was concluded from these results that the pooled tumor extracts contained tumor-specific antigens not present in normal colonic tissue. Identical tumor-specific antigens were also demonstrated in a number of individual colonic carcinomata obtained from different human donors.

Specific carcinoembryonic antigens of the human digestive system

Abstract: A wide variety of human adult and fetal tissues were studied by immune-diffusion techniques in agar gel to determine whether they contained the tumor-specific antigen(s) previously found in coionic cancers. In the adult tissues it was demonstrated that identical antigens were present in all tested specimens of malignant tumors of the entodermally derived epithelium of the gastrointestinal tract and pancreas, but were absent from all other tested adult tissues. The common antigenic constituents, therefore, represent system-specific cancer antigens of the human digestive system. System-specific cancer antigens have not previously been demonstrated in humans. Experiments with fetal tissues demonstrated that identical antigens were also present in fetal gut, liver, and pancreas between 2 and 6 months of gestation. These components were named "carcinoembryonic" antigens of the human digestive system. On the basis of the present findings and the recent work regarding control of the expression of genetic potentialities in various types of cells, it was concluded that the carcinoembryonic antigens represent cellular constituents which are repressed during the course of differentiation of the normal digestive system epithelium and reappear in the corresponding malignant cells by a process of derepressive-dedifferentiation.

Characteristics of an immune system common to certain external secretions

Abstract: The γ1A present in saliva and colostrum exists largely in the form of higher polymers, the major component of which has a sedimentation coefficient of 11S. The 11S γ1A in these fluids differs from the polymers found in normal and myeloma sera both immunologically and by the fact that their sedimentation coefficients are unaffected by disulfide bond reduction in the absence of urea. However, like other γ-globulins the 11S γ1A molecules consist of multiple polypeptide chains linked by disulfide bonds. Local synthesis of γ1A in the salivary gland has been shown by fluorescent and autoradiographic studies, although the fraction of the total salivary γ1A which is derived from local production is uncertain. No evidence of transport of intravenously administered I131-labeled 7S γ1A from serum to saliva was obtained. Immunological specificity has been demonstrated in the salivary and colostral γ1A. Whether that portion of the γ1A which is immunologically specific is a piece incorporated during the local synthesis of γ1A in the gland or is added by the epithelial cell in the process of transport remains to be determined. Antibody activity (isohemagglutinins) have been demonstrated in saliva and colostrum and have been shown to be of the γ1A-type. In both of these fluids activity is associated primarily with γ1A-polymers of 11S and 18S sizes. There appears to be an immunological system which is characteristic of certain external secretions. Its properties including the local production of a distinctive type of antibody separate it from the "systemic" system responsible for the production of circulating antibody. This system may play a significant role in the body's defense mechanisms against allergens and microorganisms.

The differentiation of mononuclear phagocytes morphology, cytochemistry, and biochemistry

Abstract: The in vitro differentiation of homogeneous populations of monocyte-like cells from the unstimulated mouse peritoneal cavity is described. Under the conditions employed, a progressive increase in cell size occurs without significant cell division. This process is characterized morphologically by the accumulation of phase-dense and neutral red-positive granules, mitochondria, and lipid droplets. The phase-dense granules react strongly for acid phosphatase. Biochemical determinations indicate marked increases in the total content and specific activity of acid phosphatase, cathepsin, and beta-glucuronidase. The production of acid phosphatase is more rapid and extensive than that of the other two hydrolases. From these data it appears that the conversion of a monocyte-like cell to a mature macrophage is accompanied by the formation of increased numbers of lysosome-like cytoplasmic organelles. Mouse peritoneal phagocytes stimulated in vivo with a bacterial lipopolysaccharide undergo a similar series of morphological and biochemical events.

Cellular localization of immunoglobulins with different allotypic specificities in rabbit lymphoid tissues

Abstract: The cellular localization of allotypes in rabbit lymphoid tissues has been studied by immunofluorescence. In heterozygous animals the double staining for two allotypes controlled by allelic genes (A1 and A2; A4 and A5; A4 and A6) has shown the existence of two populations of plasma cells, one containing one allotype and the other the alternative one. The localization in different cells of immunoglobulins marked by allelic allotypic specificities has been confirmed by microspectrography of single cells. An exception to this rule was given by the presence in the germinal centers of lymphoid follicles of apparently uniform mixtures of products of the two allelic genes. Double staining for two allotypes controlled by genes at different loci showed, instead, the presence of many cells containing both allotypes; the number of these cells was highest in doubly homozygotes, in the other it was consistent with random association of non-allelic specificities. In addition double staining for one allotype and gamma G globulins in the lymphoid tissues of rabbits homozygous at the a or at the b locus, has shown the presence of cells containing immunoglobulins that lack one allotype.

A role of polymorphonuclear leukocytes and complement in nephrotoxic nephritis.

Abstract: In acute nephrotoxic nephritis, polymorphonuclear leukocytes (polymorphs) accumulated in large numbers in the glomeruli in the first 12 hours. The endothelial cells were dislodged by the polymorphs which then came to lie immediately adjacent to the glomerular basement membranes. Ultrastructural changes in neither polymorphs nor basement membranes were observed. Depletion of polymorphs in both rats and rabbits prevented the development of proteinuria. This occurred when doses of nephrotoxic globulin were employed that produced proteinurias of as much as 1800 mg/kg/24 hours in intact rabbits, or enough to yield near maximal immediate proteinuria in intact rats. In addition, measurable glomerular damage was frequently averted until the onset of the secondary stage of NTN. Controls indicated that the polymorph depleted animals exhibited minimal non-specific changes in the blood, that the ability of their vascular beds to react to stimuli was not affected, and that deposition of nephrotoxic antibody and C' in the glomeruli was not inhibited. Elimination of polymorphs from the circulation was only partially effective in preventing glomerular damage when large doses of nephrotoxic globulin were used. This indicated that under these circumstances, a polymorph independent glomerular injury may also take place in first stage nephrotoxic nephritis. An indirect role of C', i.e ., the accumulation of polymorphs, in bringing about glomerular injury in first stage nephrotoxic nephritis was apparent. When rabbit nephrotoxic globulin was injected into rats depleted of C', or when duck nephrotoxic globulin that fixed C' poorly was injected into normal rats, C' failed to bind with the antibody along glomerular basement membranes and polymorphs did not accumulate.

ISOLATION OF ß1F-GLOBULIN FROM HUMAN SERUM AND ITS CHARACTERIZATION AS THE FIFTH COMPONENT OF COMPLEMENT

Abstract: At least 3 complement factors were found necessary for the conversion of the thermolabile intermediate complex EAC'1a,4,2a to a thermostable state. One of these factors is the earlier described β1C-globulin. The second, a heretofore unrecorded serum protein, β1F-globulin. The third factor has not yet been defined as a discrete serum protein entity. Kinetic experiments indicated that β1C reacted prior to β1F, which in turn seemed to precede the third factor in the reaction sequence. Therefore, the 3 components were tentatively designated the third (C'3), the fifth (C'5), and the sixth (C'6) components of complement, respectively. A procedure was developed allowing the isolation of highly purified β1C-(C'3) and β1F-globulin (C'5) and of partially purified C'6. With respect to its function in immune hemolysis, β1F-globulin or C'5 was found to be closely dependent on the simultaneous presence of C'6. The hypothesis that C'5 and C'6 form a functional unit was supported by the finding that both components interact with each other in solution resulting in the formation of a complex. A similar complex was also found in fresh human serum.

Genetic control of the antibody response. I. Demonstration of determinant-specific differences in response to synthetic polypeptide antigens in two strains of inbred mice.

Abstract: Immunization of CBA and C57 mice with a branched, multichain synthetic polypeptide, poly (tyr,glu)-poly DL-ala--poly lys, ((T,G)-A--L), in Freund's complete adjuvant results in a tenfold or more difference in the antigen-binding capacity of sera from the two strains, although they respond equally to bovine serum albumin. Immunization of CBA x C57 F1, F1 x CBA, and F1 x C57 mice reveals definite genetic control of the response to (T,G)-A--L, which appears to be due to a single major genetic factor, with perhaps one or more modifying factors. Immunization of CBA and C57 mice with (H,G)-A--L, a synthetic polypeptide in which histidine replaces tyrosine, gives the opposite result, CBA's respond and C57's do not. From this, it appears that the genetic control of the response to (T,G)-A--L is specific for the antigenic determinant. The implications of these results are discussed.

Studies on rabbit lymphocytes in vitro. i. stimulation of blast transformation with an antiallotype serum

Abstract: Rabbit lymphocytes may be stimulated in vitro with specific antiallotype sera to transform into "blast" cells and to synthesize DNA. This transformation only occurs when the donor cells are obtained from a rabbit having a given gamma-globulin allotype (As4) and these cells are cultured in the presence of an antiserum prepared against the given allotype (As4). Heterologous (sheep, goat, and guinea pig) anti-rabbit gamma-globulin sera also induce significant blast transformation and DNA synthesis in rabbit lymphocytes. Allotypic transformation and DNA synthesis are due to 7S antiallotype antibodies and do not require complement. The degree of transformation and rate of DNA synthesis is related to the concentration of antibody. Incubation of the appropriate cells with the antiallotype antibody for as short a time as 15 minutes results in a significant degree of "blast" transformation, indicating that the recognition of the antiallotype specificity in the cells and stimulation of the cellular changes leading to eventual transformation is rapid. The activity of the antiallotype sera as measured by transforming or haemagglutinating capacity, may be absorbed by lymphocytes of the appropriate allotype, but is not absorbed by lymphocytes from a donor rabbit not having the allotype to which the antiserum is directed. Transformation does not occur with mixtures of lymphocytes from different rabbits even if 1 donor is immunized against an allotype present in the other donor. Peripheral rabbit lymphocytes can also be induced to undergo "blast transformation" in vitro by phytohaemagglutinin and staphylococcal filtrate. The lack of demonstrable leucoagglutinins in staphylococcal filtrate and antiallotype serum indicates that agglutination is not a necessary prerequisite to the induction of blast transformation.

The role of serum complement in chemotaxis of leukocytes in vitro

Abstract: By the use of chambers containing two compartments with an interposed micropore filter, chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro was studied employing various agents that fixed serum complement (C'). Antigen-antibody complexes, zymosan, and aggregated human gamma globulin, in the presence of fresh rabbit, guinea pig, or mouse serum resulted in the migration of PMN's through the micropore filter. Pepsin-degraded rabbit antibody or unaltered duck serum containing antibody did not exhibit such activity after addition of antigen. Heating of the serum before treatment or the presence of EDTA prevented the generation of the chemotactic factor. The chemotactic factor could not be generated in whole serum from rabbits genetically deficient in C'. However, the defect in this rabbit serum could be corrected by addition of rabbit or human C'6. Serum of B10.D2 mice deficient in hemolytic C' also yielded poor chemotactic activity. Interaction of the first four reacting components of guinea pig C' did not result in significant chemotactic activity unless guinea pig euglobulin with heat labile components was also present. In rabbit serum, C'5 and C'6, when "activated" by interaction with the first four reacting components, behaved like a protein-protein complex and exhibited marked chemotactic activity. By employing conditions favoring dissociation of the complex, the individual components were isolated and shown to be chemotactically inactive. Upon recombination of the two components, however, activity reappeared. Using another approach, the C'5-C'6 complex was isolated intact, and shown to be chemotactically active while other fractions not containing these components were not active. It is postulated that the C'5-C'6 complex is the active chemotactic factor generated in serum after the addition of C'-fixing agents.

A new class of human immunoglobulins. i. a unique myeloma protein.

Abstract: The unique myeloma protein from S. J., a patient with multiple myeloma, was isolated and characterized. It resembled other myeloma proteins in many respects. The S. J. myeloma protein migrated in a distinct peak in the slow β-globulin region on zone electrophoresis, appeared as a single band on starch gel electrophoresis, and sedimented at 7.04S in the ultracentrifuge. Papain and cysteine treatment produced Fc (fast) and Fab (slow) fragments. Reduction and alkylation of the myeloma protein produced heavy and light chains in a ratio of approximately 3:1. The S. J. myeloma protein had type L (type II) light chains. These were antigenically similar to the Bence Jones protein also found in this patient. The S. J. myeloma protein was unique in the properties of its heavy chains. The myeloma protein (and its heavy chains and Fc pieces) did not contain antigenic determinants specific for IgG, IgA, or IgM. The myeloma protein (and its heavy chains), however, did contain antigenic determinants which are characteristic of a new class of immunoglobulin. The S. J. myeloma protein was unusual also in its effect on the metabolism of normal IgG and in the electrophoretic mobility of the Fc fragment produced by papain digestion. No evidence was obtained to indicate that the entire heavy polypeptide of the S. J. protein was a grossly abnormal product of malignant cell metabolism. The unique properties of the S. J. myeloma protein (and its heavy chains) are believed to represent, in large measure, properties to be found in a small part of the normal immunoglobulin population.

The development of the bacterial flora in the gastrointestinal tract of mice

Abstract: Selective culture media, and equipment for anaerobic incubation of large numbers of specimens, have been developed to facilitate the quantitative enumeration of the various aerobic and anaerobic bacterial species present in the gastrointestinal tract. The evolution of this flora has been followed in young mice from several colonies by cultivating homogenates of the different parts of the gastrointestinal tract at daily intervals from the time of birth to the time of weaning. It has been found that the lactobacilli and anaerobic streptococci become established immediately after birth and persist in large numbers, not only in the large intestine but also in the stomach and in the small intestine. In contrast, the anaerobic bacilli of the bacteroides group become established only after the 16th day; they multiply only in the large intestine but persist in this organ in very large numbers. Other bacterial species become established at different periods of time after birth, exhibit characteristic anatomic localizations, and greatly fluctuate in numbers. In general, the populations of enterobacilli and enterococci decrease precipitously after having reached a maximum level shortly after the beginning of colonization.

Morphological and biological studies on a virus in cultured lymphoblasts from burkitt's lymphoma.

Abstract: Lymphoblasts of two tissue culture strains (EB1 and EB2) from different biopsy specimens of Burkitt's lymphoma have been examined in thin sections by electron microscopy, and have each been found to carry a morphologically identical virus. The virus was observed in samples taken over many months, being present in about 1 to 2 per cent of the cells in two forms: Immature particles about 75 mµ in diameter which were seen in both the nucleus and cytoplasm; and larger mature particles with a diameter of 110 to 115 mµ, which were either within membrane-bounded cytoplasmic spaces or at the cell surface. There was some indication that the particles matured by budding through the cytoplasmic membranes. Both types of particle occurred in dead degenerating cells or, less frequently, in intact altered cells. The characteristic alterations of the latter included margination of the chromatin, fragmentation of the nuclear envelope, beaded opaque material in the mitochondria, and, with one of the cell strains (EB1), sheaves of altered spindle tubules. All attempts to isolate and identify the virus carried by the two strains of lymphoblasts failed. No pathological effects were caused in 8-day chick embryos inoculated either with whole lymphoblasts or extracts of disrupted lymphoblasts, using the intraallantoic, amniotic, and chorioallantoic routes, and the extraembryonic fluids of such chicks were without haemagglutinating activity for human, chicken, guinea pig, or monkey erythrocytes. Whole lymphoblasts or lymphoblast extracts were likewise without effect when inoculated intraperitoneally into newborn hamsters or two strains of newborn mice. Similar lymphoblast inocula did not cause detectable changes in 9 different test tissue culture systems even after 8 blind passages. The nature of the unknown, unidentified virus in the cultured lymphoblasts from Burkitt's lymphomas is considered and its possible relationship to the cells discussed.

Indigenous, normal, and autochthonous flora of the gastrointestinal tract.

Abstract: The bacterial flora of the gastrointestinal tract differs qualitatively and quantitatively from one colony of mice to another. Certain components of this flora, however, are always present in large and approximately constant numbers in healthy adult mice, irrespective of the colony from which the animals are derived. Lactobacilli and anaerobic streptococci are extremely numerous in the stomach, the small intestine, and the large intestine. In contrast, organisms of the bacteroides group proliferate only in the large intestine. These three bacterial species persist at approximately constant levels in their characteristic localization throughout the life span of healthy animals. They are closely associated with the walls of the digestive organs, and are probably concentrated in the mucous layer. A few experiments carried out with rats and young swine indicate that lactobacilli are also present in large numbers in the stomach of these animal species. It is suggested that some of the components of the gastrointestinal flora have become symbiotic with their hosts in the course of evolutionary development and thus constitute a true autochthonous flora. The other components of the indigenous flora are acquired early in life either through accidental contact or because they are ubiquitous in the environment. The "normal" flora is that which is always present in the environment of the animal colony under consideration.

A new class of human immunoglobulins. ii. normal serum igd

Abstract: A new class of immunoglobulin, IgD, was identified in normal human serum by immunochemical technics. Antiserums prepared against the unique S.J. myeloma protein facilitated recognition of the related normal protein. IgD was shown to possess type K (I) and type L (II) light chain determinants, similar to those present in other classes of immunoglobulins. IgD does not possess determinants which are specific to IgG, IgA, or IgM. The IgD proteins possess their own specific antigenic determinants. IgD migrates in the fast gamma-region on immunoelectrophoresis. The properties on sephadex gel filtration and DEAE cellulose chromatography are described. IgD was found to have a median level of 0.03 mg/ml in 100 normal serums. The range of concentrations found in individual normal serums is much wider, however, than that of other classes of immunoglobulins. IgD, on the average, accounts for less than 1 per cent of the normal serum immunoglobulins.

The catabolism of homologous and heterologous 7s gamma globulin fragments.

Abstract: The catabolism of homologous and heterologous 7S gamma globulin fragments obtained by pepsin and papain digestion was studied in rabbits, guinea pigs, and mice. The elimination from the circulation of I* labeled gamma globulin fragments was followed and the urinary excretion of the total and protein-bound I* activity determined. Evidence is presented that the molecular structure responsible for the catabolism of 7S gamma globulin is located in papain fragment III. The elimination of papain fragment III was slow and closely related to the intact gamma globulin, whereas the pepsin fragment and papain fragments I and II were rapidly eliminated and catabolized in all species examined. Prolonged incubation with cysteine altered papain fragment III as shown by a rapid catabolism of a large portion of incubated fragment III within 24 hours after injection. Small amounts of intact RGG and RGG papain fragment III were excreted as protein-bound I* activity in the urine. On the other hand, large amounts of the pepsin fragment and papain fragments I and II of RGG were excreted as protein-bound I* activity in the urine. The possibility of a molecular structure present in papain fragment III, which may be responsible for tubular reabsorption in the kidney, is discussed. The rate of urinary excretion of fragments obtained from RGG was different from that of fragments obtained from gamma globulin of several other species. In general, small amounts of the pepsin fragment and papain fragment III obtained from gamma globulin other than RGG were excreted as protein-bound I* activity. The amounts of fragment I* excreted as protein-bound I* activity depended on the species in which it was injected, as well as the source of the gamma globulin. The rapid catabolism of the pepsin fragment and papain fragments I or II which bear antibody-combining sites suggest that their use for the prophylactic treatment of tetanus and diphtheria in man is limited.

The immunological development of the human fetus.

Abstract: The immunogenesis of the human fetus has been investigated by means of the formation of immunoglobulins in vitro, immunofluorescence, morphological studies, and analysis of the immunoglobulins in the serum. Twenty fetuses which were born alive but died soon after delivery, were studied; their ages ranged from 13 to 31 weeks. The results of the spleen cultures demonstrated the synthesis of IgG and IgM, which starts at about the twentieth week of gestation. In the serum, IgM could be detected at about the same period. The immunofluorescent staining of the spleen tissue showed that medium sized and large lymphoid cells as well as plasma cells, even with Russell bodies, were positive for either IgG or IgM. The peripheral blood was also found to contain a small number of medium sized IgG and IgM-positive cells. Both the spleen and the peripheral blood showed a considerable number of fluorescent small lymphocytes which exclusively contained IgM. The relatively high ratio of IgM to IgG production prenatally as compared to the postnatal situation, agrees with a predominantly primary antibody response in fetal life. In general, the fetal thymus did not synthesize immunoglobulins. No indications for the synthesis of IgA and IgD during fetal life were found.

Association of germfree mice with bacteria isolated from normal mice

Abstract: Germfree mice were given food contaminated with pure cultures of various bacterial species isolated from ordinary healthy mice. The cultures were given singly, or in association, or consecutively at weekly intervals. Whatever the technique of administration, the lactobacilli and anaerobic streptococci immediately established themselves throughout the gastrointestinal tract, and became closely associated with the walls of the organs. In contrast, the organisms of the bacteroides group were found in large numbers only in the large intestine. Within a week after exposure, the populations of these three bacterial species reached levels similar to those found in ordinary mice. They remained at these characteristic levels throughout the period of observation (several months). Their presence resulted in a progressive decrease in the size of the cecum which eventually became normal in gross appearance. Coliform bacilli multiplied extensively and persisted at high levels in all parts of the gastrointestinal tract of germfree mice, even after these had become colonized with lactobacilli, anaerobic streptococci and bacteroides. However, the coliform population fell precipitously within a few days after the animals were fed the intestinal contents of healthy pathogen-free mice.

The in vitro differentiation of mononuclear phagocytes. ii. the influence of serum on granule formation, hydrolase production, and pinocytosis.

Abstract: The concentration of newborn calf serum in the medium has marked effects on the morphological and biochemical properties of mouse mononuclear phagocytes. At a low serum concentration, the cells developed small numbers of tiny cytoplasmic granules and little or no increase in acid phosphatase, cathepsin, and β-glucuronidase. As the serum concentration was raised, granules were formed at a more rapid rate and were larger in size. The rate of production and total amount of three hydrolytic enzymes was increased at higher levels of serum. Observations on living cells indicated that the phase-dense granules which accumulated in the perinuclear region were derived from pinocytic vesicles. These clear vesicles fused and migrated to the centrosphere where they underwent a gradual increase in phase density and reacted positively for acid phosphatase. A microscopic technique was described for the evaluation of the pinocytic process. When this method was employed, the rate of pinocytosis increased curvilinearly with elevations in the calf serum concentration of the medium. The comparative influence of bovine, horse, and rabbit serum on mouse cells was evaluated. It is suggested that pinocytosis is a major regulator of granule formation and hydrolytic enzyme production by the mouse macrophage.

The origin of the cells in the efferent lymph from a single lymph node

Abstract: The popliteal nodes of sheep were perfused continuously for over 100 hours with a solution containing 3H-thymidine. The labelling pattern of the lymphocytes in the efferent lymph from the perfused nodes showed that under normal conditions not more than 4 per cent of these lymphocytes were actually produced in the node. The transitional cells, large lymphocytes and cells of the plasma cell series which appeared in the lymph following antigenic stimulation of the node were all produced in the node.

Bound complement and immunologic injury of blood vessels

Abstract: Rats and guinea pigs were depleted of complement (C') by treatment with heat aggregated human γ-globulin (agg HGG), zymosan, anti-s1C globulin, and carrageenan. Although antigen and antibody were bound to vascular structures, Arthus reactions were inhibited. This inhibition was characterized by the lack of C' binding to walls of vessels, the lack of polymorphonuclear (PMN's) cellular infiltrates, and the lack of significant vascular damage. When the same animals were followed for several hours thereafter, levels of serum C' began to rise, C' was bound in tissues, PMN infiltrates appeared, and immunologic vasculitis developed. Blood counts, chemotaxis of PMN's induced by lysates of PMN granules, together with studies on motility and phagocytosis by PMN's obtained from C' depleted rats, failed to establish any abnormality in these cells which would account for inhibition of Arthus reactions. The specificity of C' depletion in terms of effects in the first four reacting components of guinea pig C' was studied. Treatment with agg HGG led to loss of activity in all components, whereas zymosan and anti-s1C globulin predominately affected the third component (C'3c). Carrageenan mainly affected the first two reacting components of C'. Thus, the availability of the 3c component, or a subsequently reacting component, correlated with the attraction of PMN's to immune reactants in vivo . Various antibodies with different C' fixing capacities in vitro were tested for their ability to induce immunologic vasculitis in normal animals. In rats, only those antibodies which fixed C' in vitro possessed biological activity, whereas in guinea pigs, all antibodies tested, regardless of C' fixation in vitro , induced Arthus reactions. For a given antibody in rats the vasculitis-inducing property was reflected in its ability to bind C' in vascular structures. Rats depleted of circulating PMN's by specific antibody were tested for Arthus activity. Although concentrations of immune reactants and C' were readily detected in vascular structures, no PMN infiltration occurred and significant vascular damage was averted.

Antibody synthesis at the cellular level. antibody-induced suppression of 19s and 7s antibody response.

Abstract: The suppressing activity of passively transferred antibodies on antibody synthesis against sheep red cells was investigated at the cellular level by the agar-plaque technique developed by Jerne. Humoral antibodies injected prior to the antigen suppressed the appearance of plaque-forming spleen cells producing 19S antibodies completely. Antibodies given during the first 4 days after antigen injection also showed such action, but only after a latency period of 40 hours. The inhibiting efficiency of 7S antibodies was about 100 to 200 times greater than that of 19S antibodies. The results support the conclusion that humoral antibodies inhibit the immune response by removing the stimulus for the proliferation of the antibody producing cells and not by directly depressing antibody synthesis in already committed cells. Passively transferred antibodies inhibited the 7S response if given prior to, or 24 hours after the antigen injection, in analogy with previous results concerning 19S response. In contrast to these previous results on 19S synthesis, antibody transfer had no detectable effect during the early exponential phase of 7S production (5 to 7 days after antigen injection). Only limited inhibition was observed 3 days after the antigen. One possible explanation of this difference is that 7S-producing cells do not divide, or divide at a slow rate. Antigen injection would stimulate the proliferation of 19S-producing cells. Subsequently these would switch to the synthesis of 7S antibodies. These would inhibit the initiation of new 19S-producing cells by combining with the antigen. They would thus suppress the recruitment of their own precursors. A steady state of 7S antibody production by cells with a long lifetime would be the result. This hypothesis ascribes an important regulatory function to 7S antibodies. They would be parts of a feed-back system preventing excessive cell multiplication in response to a single antigen.

Phylogenetic origins of antibody structure. I. Multichain structure of immunoglobulins in the smooth dogfish (Mustelus canis).

Abstract: The elasmobranch Mustelus canis has been shown to produce antibodies to Limulus hemocyanin The serum of both normal and immunized M canis contains immunoglobulins having sedimentation coefficients of approximately 7S and 17S Antibody activity was found in the 17S immunoglobulin which may be dissociated to 7S components with concomitant loss of activity Both 17S and 7S serum, immunoglobulins were antigenically identical They consisted of light and heavy chains present in amounts comparable to those of higher vertebrates Peptide maps indicated that the light chains had an entirely different primary structure than the heavy chains, but that the corresponding chains of 7S and 17S dogfish serum immunoglobulins were similar in primary structure The heavy chains appeared to resemble the n chains of immunoglobulins of higher vertebrates in their starch gel electrophoretic behavior It is suggested that the elasmobranch M canis may have only one major class of immunoglobulins resembling that of macroglobulins (γM-immunoglobulins) seen in higher vertebrates The results indicate that the multichain structure of antibodies is an ancient evolutionary development

The isolation and biological activities of rabbit gamma m- and gamma g-anti-salmonella typhimurium antibodies.

Abstract: Rabbit γM- and γG-anti-S. typhimurium antibodies were isolated by combined immune specific and physical methods and some of their properties in immunological systems were measured. γM-Antibody was detectable at lower concentrations and revealed a higher specific activity than the γG-globulins. Indirect studies indicated that the γG-globulin was the more "avid" immunoglobulin. Treatment of the γM-globulin with the reducing agent 2-ME decreased but did not destroy the immune activity of the subunits. The results confirm the necessity of analysis of purified immunoglobulin antibodies to evaluate the significance of their biological properties secondary to their interaction with antigens.

Quantitative studies on the behavior of sensitized lymphocytes in vitro : i. relationship of the degree of destruction of homologous target cells to the number of lymphocytes and to the time of contact in culture and consideration of the effects of isoimmune serum.

Abstract: When lymphoid cells, derived from rats immunized with respect to homologous skin, were cultured with target cells originally of donor origin, cytocidal and cytostatic activities of the attacking lymphocytes became evident. By application of a sensitive and reproducible quantitative assay system, various aspects of the mechanism of this destructive interaction between target cells and lymphocytes were examined with the following results. 1. The degree of survival of target cells was inversely related to the number of sensitized lymphocytes. Graphic plots of the data indicated that this relationship was an exponential one similar to "single-hit" inactivation phenomena. One interpretation which could be placed on these results is that a single lymphocyte, if immunologically active, was sufficient to destroy or at least have a detectably adverse effect on one target cell. Furthermore, from such a model it could be computed that, of the lymphocytes derived from an immunized animal, approximately 1 to 2 per cent of the cells were immunologically active; i.e., capable of demonstrable destructive activities against homologous target cells in vitro. 2. Morphological studies on the effect of sensitized lymphoid cells on homologous target cells, aftervarious lengths of time in culture, showed that by 7 hours of incubation, the attacking lymphocytes firmly adhered to the target cells. The cytotoxic effect of these lymphocytes generally occurred after the 20th hour. Quantitative studies supported this conclusion; the latent period, i.e., the time required for detectable degrees of target cell destruction to occur, was approximately 20 hours. 3. A consequence of the incubation of target cells with normal lymphoid cells or even with small numbers of sensitized lymphoid cells was an increase in the rate of division of the target cells. As might be expected, this was reflected in a shorter doubling time of these cells. 4. Extracts prepared from sonically disrupted sensitized lymphocytes proved to be no more deleterious to target cells than similar preparations from normal lymphoid cells. Furthermore, no evidence could be obtained that sensitized lymphoid cells, separated from target cells by a Millipore membrane, were cytocidally effective. These data indicated that if a cell-bound substance is involved in the destruction of homologous cells, either it is not toxic by itself, or it cannot be detached from the sensitized cells. In any case, close apposition of the lymphocytes to the target cells is apparently required for the destruction of the latter in vitro. 5. Serum obtained from immunized animals, if heat-inactivated, did not adversely affect homologous target cells; if employed fresh, slight degrees of toxicity resulted. Specific isoimmune sera did not impart any detectable degrees of immunological reactivity upon otherwise normal lymphoid cells. Immune sera, even in high concentrations, did not augment the effect of sensitized lymphoid cells upon homologous target cells; rather a slight inhibitory effect of these sera was detected. 6. Attempts to detect the presence of complement activity, which might have been provided by the lymphoid cells in culture, were unsuccessful. On the basis of these results, it was suggested that the destruction of homologous target cells by sensitized lymphoid cells occurs as a two step process. First, the attacking lymphocytes attach to their targets via a non-toxic cell-bound substance having an immunologic specificity, and then, destruction of the target cells follows the result of some process dependent on the metabolic activity of the attacking lymphoid cells.

Histochemical Studies of Lysosomes and Lysosomal Enzymes in Virus-Infected Cell Cultures.

Abstract: The appearance of lysosomes and the distribution of lysosomal enzymes have been studied in a number of cell cultures exposed to viruses. Lysosomes were shown by fluorescence microscopy after vital staining with aminoacridines and light microscopy after vital staining with neutral red. The lysosomal enzymes studied histochemically in unfixed and fixed cells were acid phosphatase and 5-bromo-4-chloro-indoxyl acetate esterase. Activation of lysosomal enzymes was found to take place in three stages. The first is characterized by permeability of lysosomal membranes without release of enzymes. This is demonstrable by staining of lysosomal enzymes in unfixed cells and by increased uptake of aminoacridine fluorochromes and neutral red into lysosomes. In cell sheets initially stained with neutral red this gives rise to red plaques. This stage can be fully reversible; cells infected with, and yielding, the red-plaque strain of NDV, recover fully afterwards. In the second stage lysosomal enzymes are released into the cytoplasm, the cells round up and there is decreased uptake of aminoacridines and neutral red into lysosomes. In cell monolayers this results in the formation of white plaques. In the third stage, not usually seen in cell cultures, lysosomal enzymes are released from or inactivated in the cells and are not seen in either fixed or unfixed preparations. The possible roles of lysosomal enzymes in production of cytopathic effects, polykaryocytosis and malignant cell transformation are discussed.

The immunoglobulins of mice. v. the metabolic (catabolic) properties of five immunoglobulin classes.

Abstract: The metabolic properties of immunoglobulin were investigated by comparing five classes of mouse immunoglobulin. Three forms of 7S immunoglobulin had different rates of catabolism. The fractional rates of catabolism were found to be about 13 per cent per day for 7S γ2a-globulin; 25 per cent for 7S γ2b-globulin; and 17 per cent for 7S γ1-globulin. Catabolism of the three classes of 7S γ-globulin (γ2a, γ2b, and γ1) were prolonged at low serum 7S γ-globulin levels and accelerated at high serum 7S γ-globulin levels. Each of the 7S γ-globulin components was influenced by the serum level of the other mouse 7S γ-globulin components and by exogenously administered human 7S γ-globulin. They were not appreciably altered, however, by the serum level of IgA (γ1A-, β2A-globulin). The progressively changing (longer) half-times observed in turnover studies of normal IgG (7S γ-globulin) may be caused by catabolic heterogeneity of normal 7S immunoglobulins which are immunochemically and catabolically related to γ2a-, γ2b-, and 7S γ1-myeloma proteins. These studies indicate that the 7S γ2a-, 7S γ2b-, and 7S γ1-globulins share a common catabolic control mechanism. This mechanism is influenced by the serum level of each of these components, but is independent of the serum level of IgA (γ1A-globulin) and probably is independent of IgM (γ1M-globulin). Catabolism of IgA (γ1A-, β2A-globulin) and IgM (γ1M-globulin) was much more rapid than the catabolism of the 7S γ-globulins. The halftimes of the IgA and IgM were approximately 1.2 and 0.5 days respectively. The fractional rate of catabolism of IgA and IgM seemed to be independent of their serum concentration. The rate of catabolism, as well as the rate of synthesis, was shown to play a major role in determining the serum level of each class of immunoglobulin.

Pathogenic factors in vascular lesions of experimental serum sickness

Abstract: The present studies suggest that polymorphonuclear leukocytes (PMN's) are essential for the development of cardiovascular lesions in serum sickness. In the absence of PMN's, necrotic vascular lesions were never seen and endothelial proliferation in arteries was inhibited. Zones of fibrinoid deposits did not occur. By contrast, at least two-thirds of the control animals exhibited endothelial proliferation, and half had necrosis of arterial walls, usually with fibrinoid deposits. In arterial lesions that involved the intima and media, the internal elastic lamina was disrupted. This was associated with accumulations of PMN's and was prevented when PMN's were depleted. The observations suggested that the elastic lamina acts as a barrier to the outward spread of inflammation in arteries and that it is an important substrate of PMN action. Although glomerulitis and proteinuria developed in PMN-depleted animals, no conclusions could be drawn concerning the pathogenic role of PMN's in renal lesions, since PMN depletion could not be effected before the onset of immune elimination without influencing the immune response itself. Host complement (s1C-globulin) was localized along with the antigen and rabbit gamma globulin in glomeruli and arteries showing lesions. In the glomeruli these deposits formed a granular lining along the area of the basement membrane. In arteries the fluorescent amorphous particles were in the intima and media of inflamed vessels. The immune response to BSA and the incidence and severity of cardiovascular and renal lesions were enhanced by the intravenous administration of pooled rabbit antiserum to BSA given 18 hours before BSA antigen and by injecting endotoxin along with the BSA. These additions to the usual procedure of inducing serum sickness did not appear to change the quality of the disease. In normal rabbits, the peak incidence of cardiovascular lesions was early in immune elimination of antigen, at a time when levels of circulating complexes was maximal. Conversely, the severest renal injury was noted several days later, at the completion of immune elimination.

Fibrinogen precipitation by streptococcal m protein. i. identity of the reactants, and stoichiometry of the reaction.

Abstract: Evidence confirming the identity of fibrinogen-precipitating factor and streptococcal M protein is provided by the demonstration of bactericidal, mouse protective, and long chain-producing antibodies in the sera of rabbits immunized with washed M-fibrinogen precipitates. Two precipitin lines were observed in immunoelectrophoresis of human plasma vs. rabbit anti-M-fibrinogen antiserum; no precipitin reactions were observed between human serum and rabbit anti-M-fibrinogen antiserum. The reaction between M protein and fibrinogen was stoichiometric; a constant equimolar ratio of reactants was observed in precipitates formed with a wide range of M protein concentrations.

Studies on rabbit lymphocytes in vitro : iii. protein, rna, and dna synthesis by lymphocyte cultures after stimulation with phytohaemagglutinin, with staphylococcal filtrate, with antiallotype serum, and with heterologous antiserum to rabbit whole serum

Abstract: In vitro cultures of the peripheral blood lymphocytes of rabbits may be stimulated with phytohaemagglutinin, staphylococcal filtrate, antiallotype serum, or sheep anti-rabbit whole serum to synthesize protein, RNA and DNA as indicated by the incorporation of radiolabelled precursor substances into these products. A sequence of events found in all stimulated cultures characteristically shows protein synthesis followed by RNA synthesis, histologic blast transformation, DNA synthesis, and mitosis, with the complete sequence requiring 48 hours. All four stimulants induce essentially identical metabolic changes. Characterization of the proteins synthesized by lymphocytes in vitro has failed to demonstrate immunoglobulin synthesis by stimulated or non-stimulated cultures. It is concluded that the majority of proteins produced by peripheral lymphocytes stimulated in vitro are most likely cellular proteins related to the metabolic alterations necessary for mitosis. Absorption of sheep antisera to whole rabbit serum with rabbit IgG does not always remove the transforming capacity of the sheep antisera. Thus, it is likely that antibodies to proteins other than IgG present in the small lymphocyte may also be able to stimulate transformation. A possible common mechanism for the induction of lymphoblast transformation may be the ability of both specific and non-specific stimulants to react with protein constituents of the lymphocyte which may also be present in serum.