Showing papers in "Journal of Experimental Medicine in 1963"

The particulate hydrolases of macrophages : i. comparative enzymology, isolation, and properties

Abstract: The contents of selected hydrolytic enzymes of oil-induced peritoneal, normal alveolar, and BCG-induced alveolar macrophages have been studied. On a per cell or nitrogen basis the normal alveolar cells contained considerably more acid phosphatase, cathepsin, acid ribonuclease, lysozyme, and lipase than peritoneal cells. The BCG-induced alveolar macrophage exhibited increased levels of acid phosphatase, lysozyme, and lipase as compared to alveolar macrophages from unstimulated rabbits. The morphological differences between these cells was discussed and electron micrographs of the BCG-induced macrophage presented. Fractionation of the BCG-induced macrophage by differential centrifugation showed that 60 to 80 per cent of the total cell content of acid phosphatase, cathepsin, beta glucuronidase, acid ribonuclease, acid deoxyribonuclease, aryl sulfatase, lysozyme, and lipase were localized in a postnuclear fraction which sedimented at 15,000 g. This fraction also contained the majority of the mitochondria as evidenced by its content of cytochrome oxidase. Non-specific esterase was not localized to this fraction. A separation of the hydrolase-containing particles and mitochondria was achieved by isopycnic sucrose gradient centrifugation. Under the conditions employed, the mitochondria distributed at densities of 1.19 to 1.20, whereas the hydrolase particles sedimented to a density of 1.26 to 1.27. Each of the hydrolases including acid phosphatase, beta glucuronidase, cathepsin, lysozyme, and acid ribonuclease exhibited maximum activities in the same gradient fraction. The isolated granules exhibited enzymatic latency, and activation could be achieved by cycles of freezing and thawing or surface active agents. The majority of each of the hydrolytic enzymes could be liberated in a non-particulate form by mechanical trauma. Macrophages which had been stained supravitally with neutral red were fractionated by differential and gradient centrifugation. More than 70 per cent of the dye could be recovered in the particulate hydrolase fraction. The isolated, stained granules resembled those seen in the intact cell.

Antibody formation initiated in vitro. II. Antibody synthesis in x-irradiated recipients of diffusion chambers containing nucleic acid derived from macrophages incubated with antigen.

Abstract: The diffusion chamber technique permitted the demonstration of specific antibody formation in x-irradiated recipients of such chambers filled with normal lymph node cells and a cell-free homogenate of macrophages which had been incubated in intro with T2 bacteriophage. The activity of the cell-free homogenate was retained in its RNA fraction isolated by means of the phenol method. No antibody formation occurred if such RNA was treated with RNAase. On sucrose gradients (5 to 20 per cent), the active RNA was found to be present in the top third layer. The question of the possible presence of antigen complexed to the RNA is discussed.

Chromatographic resolution of the first component of human complement into three activities.

Abstract: A euglobulin fraction of human C'1 has been chromatographically resolved into three distinct activities, designated C'1q, C'1r, and C'1s, in the order of their elution from DEAE cellulose. All three of these activities have been shown to participate in various hemolytic reactions requiring C'1, including the cold phase of the Donath-Landsteiner reaction, and to be necessary for generation of C'1 esterase. C'1q was identical with a previously described serum protein implicated in a very early step of complement action and designated the 11S component on the basis of its sedimentation constant. C'1r could not be related to a known complement activity and has been presented as a new component. C'1s, on the basis of chromatographic evidence, was identified with C'1 proesterase. Methods of assay of these components of C'1 have been presented. The significance of C'1q, C'1r, and C'1s in generation of C'1 esterase and the central role of this enzyme in reactions involving C'1, C'4, and C'2 have been discussed.

Studies on antibody production viii. the inhibitory effect of chloramphenicol on the synthesis of antibody in tissue culture

Abstract: When lymph node fragments from previously immunized rabbits were stimulated in vitro to produce a secondary response, the continuous presence of 50 µg/ml (0.15 mM) of chloramphenicol in the medium during the entire incubation period of 15 to 21 days produced nearly complete suppression of the response. Concentrations as low as 5 µg/ml (0.015 mM) produced approximately 80 per cent suppression of the response. When 50 µg/ml of chloramphenicol was present during only the first 6 days of culture, the secondary response was reduced 90 per cent. When it was absent for the first 6 days but present for the next 9 to 15 days, the response was reduced only 40 per cent. Since over 95 per cent of the antibody of the secondary response in most experiments appeared in the medium after the 6th day, chloramphenicol apparently inhibits antibody production by interfering with some early phase of the response. It is suggested that this interference involves messenger RNA and that animal cells have appeared resistant to this drug only because their complement of messenger RNA present when the drug has been added is stable over the short periods during which protein synthesis has usually been studied.

Evidence that the l-asparaginase of guinea pig serum is responsible for its antilymphoma effects : i. properties of the l-asparaginase of guinea pig serum in relation to those of the antilymphoma substance

Abstract: A number of the properties of the L-asparaginase present in guinea pig serum have been examined and shown to be indistinguishable from those of the agent responsible for inhibiting cells of lymphoma 6C3HED in vivo. The patterns of instability of the enzyme to changes in temperature and pH were found to parallel closely those of the antilymphoma agent. L-Asparaginase activity was essentially absent from the serum of newborn guinea pigs and this failed to inhibit 6C3HED cells. On separating guinea pig serum proteins by salt precipitation, electrophoresis, and chromatography on DEAE cellulose, antilymphoma activity was found only in fractions which contained L-asparaginase.

Properties of guinea pig 7S antibodies. II. Identification of antibodies involved in passive cutaneous and systemic anaphylaxis.

Abstract: Guinea pig 7Sγ1 antibodies were demonstrated to mediate passive systemic or cutaneous anaphylaxis; guinea pig 7Sγ2 antibodies were unable to mediate these reactions. Gamma-2 antibodies specifically inhibited passive cutaneous anaphylactic reactions provoked by gamma-1 antibodies by competing for antigen. However, gamma-2 antibodies were unable to inhibit passive cutaneous sensitization of guinea pigs by a heterologous antibody system. Guinea pig 7Sγ2 antibodies appear to lack receptors for fixation to guinea pig tissues and do not compete with sensitizing antibody for receptor sites.

The particulate hydrolases of macrophages ii. biochemical and morphological response to particle ingestion

Abstract: The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme. With more prolonged incubation the majority of the soluble intracellular pool of acid ribonuclease and lysozyme was lost to the extracellular medium. No change in the total content of any of the hydrolases was noted during 180 minutes of incubation in vitro. The morphological fate of the granules was studied by a histochemical method for acid phosphatase. After the phagocytosis of yeast cell walls there was a disappearance of acid phosphatase-positive granules and an accumulation of reaction product about the ingested particle. Experiments employing macrophages which were supravitally stained with neutral red also demonstrated the loss of neutral red-positive granules and the accumulation of the dye about the yeast cell walls. These results strongly suggest that lysis of macrophage granules occurs following phagocytosis and that a portion of the granule contents are then resegregated within the newly formed phagocytic vacuole.

Antibody formation. IV. Formation of rapidly and slowly sedimenting antibodies and immunological memory to bacteriophage phi-X 174.

Abstract: Injection of a sufficient dose of bacteriophage phiX 174 into guinea pigs results in the formation of rapidly sedimenting antibody molecules (19S), and later, slowly sedimenting molecules (7S). Above a threshold dose of antigen, the relative rate of 19S formation is maximal and dose-independent; below this dose, slower relative rates are obtained. The time for doubling the serum 19S level is as short as 6 to 8 hours, suggesting that the absolute rate of antibody formation per cell is increasing in addition to proliferation of antibody-producing cells. Synthesis of 19S after injection of 10(10) phiX virtually ceases at 10 days after which 19S antibody activity disappears from the circulation with a half-life of approximately 24 hours. A second injection of phiX on day 5 or 9 prolongs 19S synthesis, indicating that antigen not only can regulate the relative rate, but also is essential for continued synthesis of 19S. 19S synthesis is also prolonged in guinea pigs by injection of phiX with endotoxin or by 400 r whole body x-irradiation 24 hours after injection of phage into rabbits. The primary 7S response is not detected until approximately 1 week after immunization and relative rates are antigen-dependent. Primary 7S synthesis can continue for many months and leads to preparation for a secondary antibody response (immunological memory) during which only 7S is detected. In contrast, in animals that form precipitating 19S without detectable 7S, a second injection of phage 1 month later results in a second 19S response which closely resembles the first. These findings have led to the suggestion that formation of 19S does not lead to persisting immunological memory.

Fetal response to antigenic stimulus. II. Antibody production by the fetal lamb.

Abstract: The fetal lamb in utero is able to form large amounts of specific antibody in response to antigenic stimulus as early as the 66th to 70th day of the 150 day gestation period. Among the several antigens employed, the fetal lamb responded earliest, and with the highest titers, to bacteriophage φX. Slightly less effective as an antigen was horse ferritin, while ovalbumin proved to be a weak antigen, especially in younger fetuses. Ineffective in stimulating an antibody response at any time during fetal or early neonatal life were diphtheria toxoid, Salmonella typhosa , and BCG. Thus, it may not be feasible to fix precisely the time of onset of immunologic responsiveness in a species, inasmuch as it appears to differ so greatly from one antigen to another. The quantity of antibody found 10 days after φX immunization was not significantly different in fetuses injected at 60 to 120 days of gestation. The earliest anti-phage antibody produced by the lamb fetus is a macroglobulin sensitive to the action of 2-mercaptoethanol. Only in older fetuses with longer lasting stimuli were appreciable amounts of 7S γ-globulin antibodies formed. The conformity of these observations to theories on the ontogenesis of the immune response is discussed.

The antibody response of rats depleted of lymphocytes by chronic drainage from the thoracic duct

Abstract: The chronic drainage of lymph and cells from a thoracic duct fistula in rats results in a reduction in the weight of all the lymph nodes and of their content of small lymphocytes. The primary immune response to tetanus toxoid or sheep erythrocytes is severely depressed or abolished in such animals. This unresponsive state is related to the loss of lymphocytes from the thoracic duct fistula and, not to stress factors ensuing from the trauma of operation and restraint; it can be reversed by injecting inocula which contain almost exclusively small lymphocytes. In contrast to the severe impairment of the primary immune response in lymphocyte-depleted rats, such animals show a normal response to a second injection of tetanus toxoid. The mechanism by which small lymphocytes mediate the primary immune response is discussed.

Evidence that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects. II. Lymphoma 6C3HED cells cultured in a medium devoid of L-asparagine lose their susceptibility to the effects of guinea pig serum in vivo.

Abstract: Cells of the original line of lymphoma 6C3HED, which regularly prove susceptible to the effects of guinea pig serum in vivo, were cultured in Eagle's medium devoid of L-asparagine; after a latent period of 2 or more weeks, during which time the cell population declined markedly, some of the cells began to proliferate, and thereafter continued vigorous growth. On implantation into mice the proliferating cells were found, however, to have completely and permanently lost their susceptibility to the effects of guinea pig serum. By contrast, when cultures of the original line of 6C3HED cells were prepared in Eagle's medium to which L-asparagine was added in a concentration of 20.0 mg/liter or more, they proliferated vigorously from the beginning; after long periods of growth in the enriched medium in vitro they remained susceptible to the effects of guinea pig serum upon test in vivo. Other amino acids, purines, and pyrimidines were unable to substitute for L-asparagine in this relation. Furthermore, a variant subline of 6C3HED cells which had become insensitive to guinea pig serum under in vivo conditions did not require L-asparagine for growth in tissue culture. It seems plain from the findings as a whole, that in 6C3HED cells, L-asparagine dependence in vitro is associated with the in vivo character of guinea pig serum sensitivity, and conversely L-asparagine independent variants are insusceptible to the effects of guinea pig serum. The implications of the findings complement those of a companion paper in which direct evidence is provided that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects.

Studies on artificial antigens : iii. the genetic control of the immune response to hapten-poly-l-lysine conjugates in guinea pigs

Abstract: The genetic transmission of the capacity to develop an immune response to hapten-polylysine conjugates was studied in guinea pigs. 82 per cent of the 22 offspring of 8 pairs of responder (guinea pigs which are capable of an immune response) parents were also responders, whereas, none of the 26 offspring of 9 pairs of non-responder parents were responders. None of 11 strain 13 guinea pigs and 100 per cent of 40 strain 2 guinea pigs were responders. These findings are consistent with the view that the capacity to respond immunologically to hapten-polylysine conjugates is genetically transmitted as a unigenic Mendelian dominant.

In vitro studies of ulcerative colitis. II. Cytotoxic action of white blood cells from patients on human fetal colon cells.

Abstract: Freshly isolated fetal human colon cells were labeled with (32)P-orthophosphate or (14)C-amino acids and exposed to white blood cells from children with ulcerative colitis or from healthy controls. Exposure of the colon cells to patients' white cells led to a rapid isotope release, significantly higher than that obtained with normal white cells. After 150 minutes of incubation, 75 per cent of the total isotope present was found in the media of the colitis samples but only 40 per cent in those of the controls. Consistent results were obtained with white blood cells from 14 patients and 18 healthy individuals. Similar results were obtained with either fresh white cells or with white cells aged for 12 to 18 hours and consisting to 60 to 70 per cent of lymphocytes and to 20 to 30 per cent of large mononuclear cells. No specific cytotoxic activity could be conferred onto normal white cells by pretreating them with patients' serum containing antibodies against colon antigen. The cytotoxic action of the patients' white cells was immunologically specific, since no difference from the controls was found in the isotope release when cells from other organs or animals were similarly treated. Preliminary experiments suggested that the patients' white cells could be desensitized by pretreating them with colon extract. For obtaining a significant cytotoxic effect of the patients' white cells, the presence of 10 to 20 per cent of fresh guinea pig or human serum in the incubation medium was required.

Properties of guinea pig 7s antibodies iii. identification of antibodies involved in complement fixation and hemolysis

Abstract: Guinea pig 7Sgamma(2) antibodies were demonstrated to fix complement in the presence of antigen and to sensitize antigen-coated, tanned erythrocytes for lysis in the presence of complement; guinea pig 7Sgamma(1) antibodies did not participate in these reactions. Gamma-2 antibodies were more efficient in provoking hemorrhagic necrosis in reverse passive Arthus reactions than equal amounts of non-complement-fixing gamma-1 antibodies. Unlike anaphylaxis in the guinea pig, both guinea pig 7Sgamma(1) and 7Sgamma(2) antibodies provoked passive cutaneous anaphylactic reactions in the rat. Efficient hemolytic activity attributable to 7S guinea pig anti-sheep erythrocyte antibodies migrated faster than the peak of complement-fixing activity, but slower than the peak of PCA activity in starch block electrophoresis. It is uncertain whether this activity is a function of a third type of antibody produced in response to the particulate property of the antigen or whether it is due to the antigenic heterogeneity of the erythrocyte cell membrane.

Properties of guinea pig 7S antibodies. I. Electrophoretic separation of two types of guinea pig 7S antibodies.

Abstract: Guinea pigs hyperimmunized with single protein antigens or hapten conjugates emulsified in complete adjuvants produced two types of precipitating antibodies with different electrophoretic mobilities. "Slow" migrating antibody generally appeared earlier and "fast" migrating antibody later in the course of immunization. Animals initially immunized by the intraperitoneal route with hapten conjugates without adjuvants produced primarily fast migrating antibody. Purified guinea pig antibodies were also separable into slow and fast migrating components by electrophoresis in supporting media. Using suitable antisera prepared in rabbits hyperimmunized with guinea pig serum, it was demonstrated that slow and fast antibodies have both common and distinct antigenic determinants. Analytical ultracentrifugation disclosed that both antibodies have sedimentation coefficients of approximately 7S. These antibodies have been designated guinea pig 7Sγ1 and 7Sγ2.

Isolation and description of the fourth component of human complement.

Abstract: Purification of the activity of the fourth component of human complement resulted in the isolation of a highly homogeneous serum protein Since this protein has not been recorded previously it was called s1E-globulin on the basis of its immunoelectrophoretic behavior C'4 activity and s1E-globulin were found to have highly similar, if not identical physicochemical characteristics Moreover, s1E-globulin was shown to exhibit the specific behavior of C'4 activity in that it is taken up only by cells which contain activated C'1 DFP-inactivated C'1 failed to catalyze uptake of the protein Treatment with hydrazine which is known to destroy C'4 activity, led to changes in the physicochemical properties of s1E-globulin and rendered the molecule incapable to combine with C'1-containing cells The evidence indicates that s1E-globulin represents the fourth component of human complement

Isolation of granules from eosinophil leucocytes and study of their enzyme content.

Abstract: Eosinophils were separated from other types of cells in horse blood or rat peritoneal fluid by centrifugation in concentrated albumin solutions. Eosinophils did not appear to be damaged by this separation procedure. A technique was also devised for isolation of cytoplasmic granules from eosinophils, thus allowing studies on enzyme content of the granules. Granules from both horse and rat eosinophils contained a number of hydrolytic enzymes, similar in variety and in concentration to those previously found in granules of rabbit polymorphonuclear leucocytes. Eosinophil granules differed from those of the rabbit granulocyte in their high content of peroxidase and the absence of lysozyme and phagocytin. On disruption of eosinophil granules by repeated freezing and thawing in saline, cathepsin, ribonuclease, arylsulfatase and beta glucuronidase were released into solution, but phosphatases were partially and peroxidase completely bound to the insoluble granule residue. Peroxidase could be extracted from the granule residue with weak acid. Eosinophil granules thus are lysosome-like structures.

Studies on artificial antigens i. antigenicity of dnp-polylysine and dnp copolymer of lysine and glutamic acid in guinea pigs

Abstract: Dinitrophenyl conjugates of poly-L-lysine, varying in percentage conjugation and molecular weight have been found to induce skin reactivity and precipitating antibodies in guinea pigs. At best, 40 per cent of immunized animals developed delayed and immediate responses to DNP-polylysine, which is believed to reflect constitutional differences among the animals assayed. Only those animals capable of responding to DNP-polylysine, responded to an immunologically distinct poly-α-amino acid consisting of glutamyl and lysyl residues ("copolymer glu-lys"). The percentage of animals responding to the DNP-polylysine antigen decreased as the degree of DNP conjugation increased.

The fate of bacteria within phagocytic cells i. the degradation of isotopically labeled bacteria by polymorphonuclear leucocytes and macrophages

Abstract: The intraleucocytic fate of a variety of P32- and C14-labeled bacteria has been studied in both polymorphonuclear leucocytes and macrophages. Both cell types brought about extensive degradation of bacterial lipids, nucleic acids, and proteins. Intracellular breakdown was primarily dependant upon the composition of the ingested particle rather than on the type or source of the phagocyte. Evidence is presented for the reincorporation of bacterial constituents into leucocyte lipid. More than 50 per cent of the acid-soluble degradation products of P32-labeled bacteria appear as inorganic phosphate. Bacterial RNA is degraded more readily than DNA. Following phagocytosis, labeled bacteria lose their pool of small molecular weight intermediates. This is followed by the degradation of acid-insoluble constituents. The majority of bacterial breakdown products are then excreted by the leucocyte and appear in the medium. Heat-killed bacteria were more readily broken down than viable organisms. Only small amounts of C14-labeled bacteria were completely oxidized by leucocytic enzymes to C14O2. Acid extracts of polymorphonuclear leucocyte granules, which were highly bactericidal, liberated the acid-soluble constituents of labeled bacteria but did not significantly degrade bacterial macromolecules.

THE CELLULAR ORIGIN OF HUMAN IMMUNOGLOBULINS (γ2, γ1M, γ1A)

Abstract: A study was made of the cellular origin of human immunoglobulins (γ2, γ1M, γ1A). The results indicated that two closely related families of cells form immunoglobulins in human lymphoid tissue: germinal (reticular) centers and plasma cells. Thus their cellular origin in addition to their known antigenic relations further justifies placing the immunoglobulins in one family of proteins. Immunoglobulins were also formed to a small extent in primitive reticular cells which resembled those of germinal centers but were separated from them. Possibly such cells were undergoing transition to the much more numerous plasma cells with which they were commonly associated. The mantles of small lymphocytes which surrounded germinal centers did not contain detectable quantities of immunoglobulins. While in general only one type of immunoglobulin was present in an individual cell or germinal center, γ2- and γ1M-globulin were identified on occasion in the same plasma cell and germinal center. A peculiarity of the fetal thymus gland was the presence of immunoglobulin, mainly γ1M, in a small number of cells of small and intermediate size and primitive reticular appearance and in Hassall's corpuscles.

Mechanisms of endotoxin tolerance. I. Relationship between tolerance and reticuloendothelial system phagocytic activity in the rabbit.

Abstract: Pyrogenic tolerance following 7 daily intravenous injections of 2.0 µg/kg E. coli endotoxin in albino rabbits was associated with significant increases in RES phagocytic activity as measured with colloidal carbon. Nevertheless, 4 hours after RES blockade with thorotrast (3 ml/kg), the tolerant rabbits exhibited significantly lower fever indices following intravenous endotoxin challenge than did non-tolerant control animals despite comparably depressed capacities to clear carbon from the blood. Moreover, plasma from rabbits tolerant to endotoxin induced significant tolerance in normal rabbits prepared by thorotrast blockade without enhancing the depressed carbon clearance. This passive protection extended to heterologous endotoxins. Analysis of the data indicates that RES blockade does not abolish tolerance; rather blockade resets the reactivity to endotoxin in the normal and tolerant animal, rendering both exquisitely reactive, but permitting retention of the major portion of tolerance. Apparently the tolerant animal possesses a dual endotoxin defense system. One system is abolished by thorotrast; the other is in part humoral, accounts for the greater portion of tolerance, and is thorotrast-resistant. The nature of the humoral component is not defined but is consistent with that of an opsonin with high endotoxin specificity.

Factors controlling serum gamma-globulin concentration.

Abstract: Both synthetic and catabolic processes determine the serum gamma-globulin level. The rate of gamma-globulin synthesis appears to be the primary factor determining the amount of serum gamma-globulin. Increase of gamma-globulin synthesis (as may occur following immunization or development of plasma cell tumor) elevates the serum gamma-globulin level. This, in turn, accelerates the fractional rate of gamma-globulin catabolism. The change in catabolic rate reduces the dimensions of the serum change from that which would occur if synthesis alone determined the serum gamma-globulin level. The present studies indicate the existence of a homeostatic mechanism controlling the rate of gamma-globulin catabolism. The mechanisms of gamma-globulin catabolism are specific and selective. Marked serum increase of other immunoglobulin components (beta(2A)-globulins and gamma(1)-macroglobulins) do not accelerate gamma-globulin catabolism. Similarly, serum albumin increases do not influence gamma-globulin catabolism. The site determining gamma-globulin catabolism is restricted to a part of the gamma-globulin molecule; i.e., on the F piece obtained by papain digestion and, by inference, on the H chains obtained by reduction and alkylation of gamma-globulin molecules.

SEQUENCES OF SYNTHESIS OF γ-1 MACROGLOBULIN AND γ-2 GLOBULIN ANTIBODIES DURING PRIMARY AND SECONDARY RESPONSES TO PROTEINS, SALMONELLA ANTIGENS, AND PHAGE

Abstract: The nature of the antibodies produced by the rabbit during the primary and secondary responses to T2 phage, proteins, and the O and H antigens of Salmonella typhosa has been determined. Immune sera have been fractionated by zone electrophoresis, sucrose density ultracentrifugation, and anion exchange chromatography. The resulting fractions have been assayed by phage neutralization or hemagglutination (antisera to proteins) or bacterial agglutination. In confirmation and extension of earlier work from this laboratory, the primary response to these antigens, with the exception of the O antigen of the Salmonella , included the early synthesis of 19S, γ-1 globulin antibody, and the later synthesis of 7S, γ-2 globulin antibody. The primary response to the O antigen consisted of the synthesis of only a macroglobulin agglutinin. The secondary response to the proteins, including the H antigen of the Salmonella , comprised the early synthesis of large amounts of the 7S γ-2 globulin antibody to the same level attained during the primary response. The secondary response to the phage consisted in the synthesis of 7S, γ-2 globulin antibody alone. Treatment of the macroglobulin phage-neutralizing antibody with mercaptoethanol resulted in complete loss of its neutralizing activity. A working hypothesis to explain these observations was presented. A salient feature of this hypothesis was the suggestion that different cells synthesized the two distinct molecular forms of antibody. The significance of the sequential synthesis of the two forms of antibody is not known. It was proposed that the system for synthesis of macroglobulin antibody is an auxiliary system for antibody synthesis, perhaps the first to develop phylogenetically and ontogenetically. It is felt that the present observations indicate a clear-cut qualitative distinction between the primary and secondary responses to immunization whereby these responses might be identified in various experimental situations. It is also felt that these findings with the primary and secondary responses to various antigens in the rabbit may be of widespread occurrence in nature among a variety of species.

Separation, characterization, and biological significance of a common antigen in enterobacteriaceae

Abstract: An antigen common to Enterobacteriaceae and closely associated with endotoxin fractions has been separated by chromatography on DEAE cellulose employing elution with a NaCl gradient. The purified common antigen fails to coat erythrocytes, is poorly, if at all antigenic, it is non-dialyzable and excluded from sephadex G-100 gel. It is composed of polysaccharide and polypeptide. The most important property of this antigen thus far determined appears to be its interference with the specificity of the hemagglutination test commonly employed to measure antibody to O antigen of Enterobacteriaceae. It may also have taxonomic significance in classification of this family of bacteria.

Experimental transmission of influenza virus infection in mice. ii. some factors affecting the incidence of transmitted infection.

Abstract: Evidence has been presented that with the experimental model described, infected mice vary in their ability to transmit influenza virus infection. This variation is not explained by differences in titers of influenza virus in the nose, throat, trachea, or lungs of good transmitters. Older mice acquire transmitted influenza virus infection more readily than younger mice. Seasonal variations in the incidence of transmitted influenza virus infection occur.

The histogenesis of basement membranes

Abstract: A parietal yolk sac carcinoma of the mouse that secretes large quantities of basement membrane-like material has been used to study the formation of basement membranes. Suitably characterized fluorescein-labeled antibodies against this material stained basement membranes of epithelial structures and vessels, as well as reticulin. When absorbed with reticulin and vascular basement membranes of the spleen until these structures no longer fluoresced, the antibody still stained the basement membrane-like material of the tumor, its normal embryonic counterpart (Reichert's membrane), and the basement membranes at the bases of epithelial cells. The observation made previously that parietal yolk sac cells secreted, in the absence of connective tissue and reticulin, the basement membrane (Reichert's membrane) upon which they rested has been confirmed through the localization of ferritin-labeled antibody to the endoplasmic reticulin of the secreting cells. Since a basement membrane proven to be an epithelial secretion is antigenically similar to basement membranes at the bases of all epithelial cells studied but antigenically different from connective tissue elements, it is postulated that the basement membranes at the bases of epithelial cells in general are an epithelial secretion, and are not a condensation of ground substance as is commonly believed.

Components of guinea pig complement. i. separation of a serum fraction essential for immune hemolysis and immune adherence.

Abstract: Employing sheep erythrocytes sensitized by antibody and the first and fourth components of complement (EAC'1,4), in such a manner as to prevent the development of immune adherence (I-A) reactivity during preparation, four separate substances required for the conversion of EAC'1,4,2 to the final damaged state (E*) were identified in whole guinea pig serum by cellulose chromatography, and tentatively termed C'3c, C'3b, C'3a, and C'3d. I-A reactivity was induced in EAC'1,4,2 after interaction with only one of these four substances, C'3c. A detailed comparison of the effects of heat, hydrazine, low pH, freezing, absorption by immune complexes, and elution from cellulose columns indicated that this same substance which was capable of imparting I-A reactivity to EAC'1,4,2 was also essential for immune hemolysis. Other experiments showed that I-A-reactive cells prepared either by treating EA with different concentrations of whole C' at 0°C, or by treating EAC'1,4,2 with C'3c, underwent lysis by C'2 + C'3b + a + d in proportion to the amount of whole C' or of C'3c used to make the cells reactive in I-A. These data provide strong evidence that a single factor, C'3c, is required both for the conversion of EAC'1,4,2 to an I-A-reactive complex (EAC'1,4,2,3c) and for the lysis of EAC'1,4,2 by C'3b + a + d. C'3c is the only one of the components studied which can induce I-A reactivity, and is the first to react with EAC'1,4,2. Formation of EAC'1,4,2,3c proceeds even at 0°C, but is much more rapid at elevated temperatures, showing a maximum in from 5 to 15 minutes at 37° or 30°C respectively. Prolonged incubation at these temperatures results in a decline in hemolytic reactivity without a noticeable effect on I-A. This loss was resolved into three phenomena: (a) a rapid loss of ability of SAC'1,4,2,3c to react with C'3b, presumably as a result of decay of the C'2 moiety in the complex, which is readily reversed by addition of fresh C'2; (b) a slow, irreversible spontaneous inactivation of SAC'1,4,2,3c; (c) a moderately rapid, irreversible inactivation of SAC'1,4,2,3c by some factor present in C'3c preparations.

Activation of lysosomal enzymes in virus-infected cells and its possible relationship to cytopathic effects

Abstract: Activities of the enzymes s-glucuronidase, acid phosphatase, acid DNAase, acid RNAase, and acid protease have been measured in the lysosomal and supernatant fractions of mouse liver cells and monkey kidney cells before and after infection with mouse hepatitis virus and vaccinia virus, respectively. In the infected cells there was easily measurable release of lysosomal enzymes into the supernatant fraction. Evidence was presented that this is not an artefact of homogenization and precedes cell degeneration demonstrable histologically. It is suggested that release of lysosomal enzymes may explain some of the biochemical changes found in infected cells and may contribute to the cytopathic effects of some viruses.

THE ASSOCIATION OF SKIN-SENSITIZING ANTIBODY WITH THE ß2A-GLOBULINS IN SERA FROM RAGWEED-SENSITIVE PATIENTS

Abstract: 1. The removal of the beta(2)A-globulins from three sera from treated ragweed-sensitive individuals by immune absorption was associated with the loss of all detectable skin-sensitizing antibody activity as demonstrated by Prausnitz-Kustner testing. 2. Gel filtration studies, with sephadex G-200, indicated that the fractions containing only macroglobulins were devoid of all detectable skin-sensitizing antibody activity. 3. The immune absorption of the gamma-globulins from a serum fraction containing beta(2)A-globulins, gamma-globulins, and a trace of beta(2)M-globulins had no detectable influence on the skin-sensitizing antibody activity. 4. The removal of a portion of the albumin from the allergic sera by immune absorption, with retention of the skin-sensitizing activity, indicated that the loss of skin-sensitizing antibody was not due to non-specific absorption on an antigen-antibody precipitate. 5. No inhibition of the Prausnitz-Kustner reaction was observed when sheep serum, normal human serum, or normal human gamma-globulins were tested in concentrations described. 6. We conclude that the skin-sensitizing antibody activities in the three sera from treated ragweed-sensitive individuals studied were associated with the beta(2)A-globulins.

Experimental glomerulonephritis : ii. immunologic events in the pathogenesis of nephrotoxic serum nephritis in the rat

Abstract: The primary phase of nephrotoxic serum nephritis produced by rabbit nephrotoxic serum appears to be dependent to a great extent, but not completely, upon the participation of serum complement. On the other hand, duck nephrotoxic serum produces its primary renal injury without detectable utilization of or dependence upon serum complement. The secondary phase of nephrotoxic serum nephritis appears to be largely or entirely dependent upon the host's antibody response to the heterologous gamma globulin fixed in the glomeruli. No evidence could be obtained for the existence of an autoimmune antikidney response by the host in this experimental model.