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Showing papers in "Journal of Experimental Medicine in 1968"


Journal ArticleDOI
TL;DR: It was concluded that the rapidly dividing mononuclear phagocytes of the bone marrow, called promonocytes, are the progenitor cells of the monocytes and give rise to peritoneal macrophages.
Abstract: The origin and turnover of efferent populations of mouse mononuclear phagocytes has been described. Mononuclear phagocytes were defined as mononuclear cells which are able to adhere to glass and phagocytize. In vitro labeling studies with thymidine-3H showed that monocytes in the peripheral blood and peritoneal macrophages do not multiply and can be considered end cells in a normal, steady state situation. However, the mononuclear phagocytes of the bone marrow appear to be rapidly dividing cells. This conclusion was supported by in vivo labeling experiments. A peak of labeled mononuclear phagocytes of the bone marrow was found 24 hr after a pulse of thymidine-3H. This was followed, 24 hr later, by a peak of labeled monocytes in the peripheral blood. From these experiments it was concluded that the rapidly dividing mononuclear phagocytes of the bone marrow, called promonocytes, are the progenitor cells of the monocytes. Labeling studies after splenectomy and after X-irradiation excluded other organs as a major source of the monocytes. Peak labeling of both the blood monocyte and peritoneal macrophages occurred at the same time. A rapid entry of monocytes from the blood into the peritoneal cavity was observed, after a sterile inflammation was evoked by an injection of newborn calf serum. These data have led to the conclusion that monocytes give rise to peritoneal macrophages. No indications have been obtained that mononuclear phagocytes originate from lymphocytes. In the normal steady state the monocytes leave the circulation by a random process, with a half-time of 22 hr. The average blood transit time of the monocytes has been calculated to be 32 hr. The turnover rate of peritoneal macrophages was low and estimated at about 0.1% per hour. On the basis of these studies the life history of mouse mononuclear phagocytes was formulated to be: promonocytes in the bone marrow, → monocytes in the peripheral blood, → macrophages in the tissue.

1,413 citations


Journal ArticleDOI
TL;DR: When C' factors from mouse serum are added to the antigen-19S antibody complex (EAC'), rosettes are formed on most mouse peritoneal macrophages and PMN and on a few monocytes, and EAC' also adheres to 10–25% of lymph node lymphocytes but not to thymus lymphocytes.
Abstract: Sheep red blood cells sensitized by 7S, but not by 19S rabbit anti-Forssman antibodies, adhere and form rosettes on mouse macrophages and on a few monocytes and polymorphonuclear cells (PMN). When, however, C' factors from mouse serum are added to the antigen-19S antibody complex (EAC'), rosettes are formed on most mouse peritoneal macrophages and PMN and on a few monocytes. In addition EAC' also adheres to 10-25% of lymph node lymphocytes but not to thymus lymphocytes. EAC' prepared with 7S anti-Forssman antibodies has identical properties. The adherence of red cells induces an increase in the membrane activity of the leukocytes and causes injury to the red cells which rapidly become deformed and fragmented. Adherence of EAC' occurs at 37 degrees C and is minimal at 4 degrees C. Probably only the first four C' components are involved in this phenomenon as mouse serum deficient in C'5 or rabbit serum, deficient in C'6 can be used as a source of C' components. Treatment of EAC' with EDTA does not modify its leukocyte-adherence properties. The adherence of EAC' to the leukocytes is not inhibited in the presence of serum. The receptors for C' on macrophages, PMN, and monocytes differ from those found on lymphocytes. Rosette formation by EAC' on macrophages, PMN, and monocytes depends on divalent cations (Mg(++)) and can be reversed by Na(3)H EDTA, while adherence to lymphocytes is independent of these ions and occurs in the presence of 0.01 M Na(3)H EDTA. Both types of receptors for C' components are destroyed by trypsin treatment of the leukocytes, in contrast with the receptors for 7S antibodies on the same cells which persist after enzyme treatment.

610 citations


Journal ArticleDOI
TL;DR: The results were considered to support the concept that memory resides in the T cell population and that collaboration between T and B cells is necessary for an optimal secondary antibody response.
Abstract: The number of discrete hemolytic foci and of hemolysin-forming cells arising in the spleens of heavily irradiated mice given sheep erythrocytes and either syngeneic thymus or bone marrow was not significantly greater than that detected in controls given antigen alone. Thoracic duct cells injected with sheep erythrocytes significantly increased the number of hemolytic foci and 10 million cells gave rise to over 1000 hemolysin-forming cells per spleen. A synergistic effect was observed when syngeneic thoracic duct cells were mixed with syngeneic marrow cells: the number of hemolysin-forming cells produced in this case was far greater than could be accounted for by summating the activities of either cell population given alone. The number of hemolytic foci produced by the mixed population was not however greater than that produced by an equivalent number of thoracic duct cells given without bone marrow. Thymus cells given together with syngeneic bone marrow enabled irradiated mice to produce hemolysin-forming cells but were much less effective than the same number of thoracic duct cells. Likewise syngeneic thymus cells were not as effective as thoracic duct cells in enabling thymectomized irradiated bone marrow-protected hosts to produce hemolysin-forming cells in response to sheep erythrocytes. Irradiated recipients of semiallogeneic thoracic duct cells produced hemolysin-forming cells of donor-type as shown by the use of anti-H2 sera. The identity of the hemolysin-forming cells in the spleens of irradiated mice receiving a mixed inoculum of semiallogeneic thoracic duct cells and syngeneic marrow was not determined because no synergistic effect was obtained in these recipients in contrast to the results in the syngeneic situation. Thymectomized irradiated mice protected with bone marrow for a period of 2 wk and injected with semiallogeneic thoracic duct cells together with sheep erythrocytes did however produce a far greater number of hemolysin-forming cells than irradiated mice receiving the same number of thoracic duct cells without bone marrow. Anti-H2 sera revealed that the antibody-forming cells arising in the spleens of these thymectomized irradiated hosts were derived, not from the injected thoracic duct cells, but from bone marrow. It is concluded that thoracic duct lymph contains a mixture of cell types: some are hemolysin-forming cell precursors and others are antigen-reactive cells which can interact with antigen and initiate the differentiation of hemolysin-forming cell precursors to antibody-forming cells. Bone marrow contains only precursors of hemolysin-forming cells and thymus contains only antigen-reactive cells but in a proportion that is far less than in thoracic duct lymph.

565 citations


Journal ArticleDOI
TL;DR: It is concluded that thoracic duct lymph contains a mixture of cell types: some are hemolysin-forming cell precursors and others are antigen-reactive cells which can interact with antigen and initiate the differentiation of hemoly sin-forming Cell precursor to antibody-forming cells.
Abstract: An injection of viable thymus or thoracic duct lymphocytes was absolutely essential to enable a normal or near-normal 19S liemolysin-forming cell response in the spleens of neonatally thymectomized mice challenged with sheep erythrocytes. Syngeneic thymus lymphocytes were as effective as thoracic duct lymphocytes in this system and allogeneic or semiallogeneic cells could also reconstitute their hosts. No significant elevation of the response was achieved by giving either bone marrow cells, irradiated thymus or thoracic duct cells, thymus extracts or yeast. Spleen cells from reconstituted mice were exposed to anti-H2 sera directed against either the donor of the thymus or thoracic duct cells, or against the neonatally thymectomized host. Only isoantisera directed against the host could significantly reduce the number of hemolysin-forming cells present in the spleen cell suspensions. It is concluded that these antibody-forming cells are derived, not from the inoculated thymus or thoracic duct lymphocytes, but from the host. Thoracic duct cells from donors specifically immunologically tolerant of sheep erythrocytes had a markedly reduced restorative capacity in neonatally thymectomized recipients challenged with sheep erythrocytes. These results have suggested that there are cell types, in thymus or thoracic duct lymph, with capacities to react specifically with antigen and to induce the differentiation, to antibody-forming cells, of hemolysin-forming cell precursors derived from a separate cell line present in the neonatally thymectomized hosts.

564 citations


Journal ArticleDOI
TL;DR: Enhancement of the antinuclear antibody response by active immunization of young NZB/W mice with DNA-methylated BSA hastens the development and increases the severity of the glomerulonephritis.
Abstract: The development of glomerulonephritis in NZB/W mice is closely related to the formation of antinuclear, particularly anti-DNA, antibodies. The developing inflammatory glomerular lesions are characterized by the deposition of gammaG- and beta(1C)-globulins plus DNA and possibly other nuclear antigens, presumably as complexes, in a granular to lumpy pattern along the capillary walls and in the mesangia. Elution studies revealed the gammaG-globulin in the glomeruli to be largely gammaG(2A)-type antibody to soluble nuclear antigens. Enhancement of the antinuclear antibody response by active immunization of young NZB/W mice with DNA-methylated BSA hastens the development and increases the severity of the glomerulonephritis. Similarly, injections of soluble DNA into NZB/W mice with circulating anti-DNA antibodies but with as yet little nephritis causes rapid progression of nephritis.

517 citations


Journal ArticleDOI
TL;DR: The tentative conclusion is reached that follicular antigen in a primary follicle encounters natural antibody on the surface of certain antigen-reactive lymphocytes, which causes blast cell transformation and eventually the genesis of a germinal center.
Abstract: This paper describes the trapping of antigen in lymphoid follicles of rat popliteal lymph nodes as revealed by electron microscopic radioautographs following injection of (125)I-labeled Salmonella adelaide flagella and other materials. The antigen was taken up vigorously, and to an approximately equal extent, by both primary and secondary follicles. The rate of uptake was faster in preimmunized than in virgin adult rats. The bulk of the antigen in follicles was extracellular, and persisted in this location for at least 3 wk. Label was most frequently found at or near the surface of fine cell processes. Many of these were branches of dendritic follicular reticular cells. Such processes interdigitated with equally fine processes of lymphocytes, creating an elaborate meshwork. In some cases, antigen was found between lymphocytes which appeared to be in close apposition. Occasionally, a few grains appeared over lymphocyte nuclei and study of serial sections suggested that this probably represented true entry of small amounts of antigen into lymphocytes. The characteristic "tingible body" macrophages (TBM) of germinal centers appeared to play only a secondary role in follicular antigen retention. They showed degrees of labeling over their phagocytic inclusions varying from negligible to moderately heavy. Moreover, follicles lacking or poor in TBM retained antigen just as effectively as those containing numerous TBM. The hypothesis is advanced that TBM may be derived from monocytes that migrate down from the circular sinus. Follicular localization of three other materials was also studied, though not in such detail. These were (125)I-HSA complexed to anti-HSA: (125)I-labeled autologous IgG; and (125)I-monomeric flagellin. All of these showed the basic features of intercellular, membrane-associated deposition noted with (125)I-flagella. The role of follicular antigen depots in immune induction is discussed. The tentative conclusion is reached that follicular antigen in a primary follicle encounters natural antibody on the surface of certain antigen-reactive lymphocytes. The resultant reaction causes blast cell transformation and eventually the genesis of a germinal center.

463 citations


Journal ArticleDOI
TL;DR: Property of the human leukocyte elastase differ sufficiently from those of pancreatic elastases of different species as to suggest that the former enzyme is a distinct and separate entity, and that elastica staining of human arterial vessels is reduced by incubation of tissues with humanLeukocyte granule extracts in vitro.
Abstract: The present study demonstrates that a granule fraction derived from human polymorphonuclear leukocytes possesses elastinolytic activity, and that the latter can be separated from the collagenase present in these cells. Properties of the human leukocyte elastase differ sufficiently from those of pancreatic elastases of different species as to suggest that the former enzyme is a distinct and separate entity. Thus, soybean trypsin inhibitor and salivary kallikrein inhibitor (Trasylol) fail to inhibit elastolysis by the pancreatic enzyme, but do inhibit the leukocyte elastinolytic agent. Elastolysis by human leukocyte granule extract does not show significant salt inhibition, whereas that catalyzed by pancreatic elastase is markedly reduced when ionic strength is increased to physiological levels. The leukocyte granule extract is at least 10 times more resistant to serum elastase inhibitor than is the purified pancreatic enzyme. Both enzymes show optimal elastolysis above pH 8.5, but the leukocyte factor still retains 50% of its maximal elastolytic activity at pH 6–7; whereas the activity of the pancreatic enzyme falls to 10% or less of its maximal value under the same conditions. The foregoing characteristics of the human leukocyte elastase suggest that it, rather than pancreatic (serum) elastase, may mediate pathological elastolysis during acute arteritis in man. In keeping with this suggestion, the present experiments also show that elastica staining of human arterial vessels is reduced by incubation of tissues with human leukocyte granule extracts in vitro.

440 citations


Journal ArticleDOI
TL;DR: It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells, and that nodes containing such cells can participate in an immunological response against sheep red cells.
Abstract: The relationship between hematopoietic colony-forming stem cells and cells in the thymus and lymph nodes of unirradiated mice has been investigated using a chromosome-marker technique. It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells. It was also found that cells belonging to the same clone as colony-forming cells may reach the lymph nodes, and that nodes containing such cells can participate in an immunological response against sheep red cells. Either the precursors of cells in thymus and lymph node are identical with hematopoietic colony-forming cells, or they are both descendants of a common precursor which has not yet been identified. The results are compatible with the view that cells of the hematopoietic system and the immune system may be derived from the same stem cell.

429 citations


Journal ArticleDOI
TL;DR: The C'5 anaphylatoxin failed to cross-desensitize Guinea pig ileum to the contracting capacities of C'3 and guinea pig anaphlyatoxin and vice versa.
Abstract: Anaphylatoxin activity was derived from both human C'5 and C'3 molecules. This was achieved in the case of C'5 by interaction with trypsin or with EAC'4, oxy2a, 3. The smooth muscle-contracting material obtained from the treated C'5 was found to be a fragment of approximately 9,000–11,000 molecular weight. Its action was inhibited with antihistamine. The trypsinized C'5 also increased vascular permeability in guinea pig skin. When human C'3 was incubated with C'3 inactivator complex, which consists of a cobra venom protein and a s-globulin of human serum, anaphylatoxin activity was observed. The activity was associated with a fragment cleaved from the C'3 molecule, having a molecular weight of between 6,000 and 15,000 as determined by gel filtration techniques. Similar activity was derived from C'3 by the C'3-converting enzyme in free or in cell-bound form. The C'5 anaphylatoxin failed to cross-desensitize guinea pig ileum to the contracting capacities of C'3 and guinea pig anaphylatoxin and vice versa. Anaphylatoxin prepared from C'3 by all methods mentioned above caused cross-desensitization to the other C'3 derivatives, but failed to desensitize to guinea pig anaphylatoxin.

418 citations


Journal ArticleDOI
TL;DR: The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types, and one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of γG2-heavy chains and kappa light chains.
Abstract: The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types Certain antibodies such as anti-tetanus toxoid and anti-A isoagglutinins were predominantly of the major γG1-type However, a high preponderance of molecules of the minor γG2-subgroup was found for antibodies to dextran, levan, and teichoic acid These findings explain some unusual features previously noted for anti-dextrans such as weak PCA reactions and lack of Gm antigens Studies of several isolated antibodies from single heterozygous individuals showed a selective absence of genetic markers in certain antibodies and their presence in others The "allelic exclusion" principle was clearly evident in the isolated antibodies of two different individuals Large differences in the ratio of kappa to lambda light chains were observed for the same type of antibody from different individuals Subfractionation of dextran antibodies by affinity for specific glycosidic linkage or combining site size produced marked changes in the ratios The isomaltohexaose eluates of the dextran antibodies from two subjects were primarily kappa and the isomaltotriose eluates were predominantly lambda The one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of γG2-heavy chains and kappa light chains By these criteria as well as others, it closely resembled myeloma proteins

410 citations


Journal ArticleDOI
TL;DR: The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora.
Abstract: Colonization of the gastrointestinal tract by bacteria of the normal flora was followed by bacteriological and special histological techniques in mice from several colonies These histological techniques were designed to preserve the intimate associations that become established between particular strains of microorganisms and the epithelium of the mucosa of certain areas of the gut The findings were as follows: 1 The various strains of bacteria of the normal flora became established in the different areas of the guts of infant mice according to a definite time sequence 2 The first types of bacteria that could be cultured from the gut were lactobacilli and Group N streptococci Within the first day after birth, these bacteria colonized the entire digestive tract and formed layers on the stratified squamous epithelium of the nonsecreting portion of the stomach and of the distal esophagus 3 The bacterial types that appeared next were coliforms and enterococci From about the 9th to the 18th day after birth, these bacteria could be cultured in extremely high numbers from the cecum and the colon Histological sections of those organs taken during the first 2 or 3 days of that interval revealed microcolonies of Gram-positive cocci in pairs and tiny Gram-negative rods embedded in the mucous layer of the epithelium The microcolonies were well separated from the mixture of digesta and bacteria that occupied the center of the lumen; they may have consisted of the coliforms and enterococci mentioned above; but this possibility remains to be proved 4 Histological sections also revealed that, at about the 12th day after birth, long, thin Gram-variable rods with tapering ends were present, side by side, with the small Gram-negative rods and Gram-positive cocci in the mucous layer By the 15th day after birth, the fusiform bacteria formed thick layers in the mucus, and seemed to be the only bacteria remaining in that location It has not yet been possible to enumerate these tapered rods by culture methods, but as judged by visual appearances in the histological sections, they seemed to outnumber all other bacteria in the cecum and the colon by a factor of as much as 1000 It must be stressed that these bacterial layers are readily disrupted and even washed away by conventional histological techniques; their discovery was largely due to the use of the special histological techniques described in the text The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora The constant occurrence of several distinct associations of bacteria with the special histological structures of the animal host renders obsolete the notion that the intestine constitutes a chemostat in which the bacterial populations are randomly mixed For a full understanding of the ecology of the normal microflora, it is necessary to think of body surfaces as distinct microenvironments in which virtually pure cultures of a few species of microorganisms interact with their host and the adjacent microbial populations Experiments based on this hypothesis are admittedly difficult to design, but on the other hand studies based on the assumption that microorganisms exist as mixtures in the gastrointestinal tract will be only of limited value and may often be misleading

Journal ArticleDOI
TL;DR: In heavily irradiated mice, bone marrow regeneration of either endogenous or exogenous origin was shown to occur in discrete foci comparable to the more intensively studied spleen colonies, with a small percentage of "mixed" colonies.
Abstract: In heavily irradiated mice, bone marrow regeneration of either endogenous or exogenous origin was shown to occur in discrete foci comparable to the more intensively studied spleen colonies. The number of endogenous bone marrow colonies was inversely related to dose of whole body X-irradiation. Endogenous marrow colonies were found after higher doses of irradiation than were endogenous spleen colonies. Most of them were granulocytic in nature. Exogenous bone marrow colonies in lethally irradiated mice injected with bone marrow cells were proportional in number to the dose of cells injected, appeared at a time comparable to spleen colonies like which, at 7 or 8 days, they were of single differentiated cell line, either granuloid or erythroid or megakaryocytic, with a small percentage of "mixed" colonies. Whereas erythroid colonies outnumber granuloid colonies in spleen, either in situ or subcutaneously transplanted (E:G colony ratio of about 3.5), granuloid colonies outnumber erythroid in bone marrow (E:G colony ratio of about 0.7). The characteristic E:G colony ratios of spleen and marrow appear more likely to be the result of a hemopoietic organ stromal influence on pluripotent colony forming units (CFU's) than of selective lodgment of committed (unipotent) granuloid and erythroid CFU's in bone marrow and spleen, respectively, as indicated by the following. Bone marrow stem cells (CFU) which had reseeded the marrow cavity of irradiated primary recipients 18-24 hr earlier, were reharvested and retransplanted intravenously into irradiated secondary hosts. The E:G colony ratio of the colonies formed in the spleen of the secondary hosts was typical of primary spleen colonies (2.8), that of the colonies formed in the marrow cavity was typical of bone marrow colonies (0.6). Pieces of marrow stroma containing reseeded CPU's from the contralateral femur of these same primary recipients were implanted by trocar directly into the spleens of other irradiated secondary recipients. Those CPU's that developed in the intrasplenic-implanted marrow stroma yielded an. E:G colony ratio of 0.1. Those that migrated into the contiguous and remote portions of the spleen gave E:G colony ratios of 2.9 and 2.4, respectively. Irradiated marrow stroma and normal spleen CPU's (a 1 mm cube of spleen) were loaded into the same trocar and implanted directly into the spleens of irradiated mice. The spleen CFU's that migrated into the implanted marrow stroma yielded five granuloid and two mixed colonies. The larger number that developed in the host spleen yielded an E:G colony ratio of 2.9 or higher. Of those 19 mixed colonies that bridged the junction of spleen and implanted marrow stroma in each of the above two experiments, in every case, the erythroid portion of the colony was in the splenic stroma, the granuloid portion was in the marrow stroma.

Journal ArticleDOI
TL;DR: The kinetics of the appearance of PFC in the mouse spleen after injection of SRC suggest that the depressive effect of 7S antibody simulates a reduction in SRC dose, whereas the enhancing effect of 19S antibody appears as a temporary increase in the rate at which PFC appear.
Abstract: Prior to sheep red cells (SRC) mice were given 7S or 19S anti-SRC antibodies or mixtures of both. All 7S preparations suppressed the immune response. All 19S preparations enhanced the primary response, as measured by an up to 15-fold increase in the number of PFC per spleen. Results obtained with mixtures showed that 7S and 19S antibodies are competitive in their effect. The kinetics of the appearance of PFC in the mouse spleen after injection of SRC suggest that the depressive effect of 7S antibody simulates a reduction in SRC dose, whereas the enhancing effect of 19S antibody appears as a temporary increase in the rate at which PFC appear. Antibodies from one animal species are quite effective in another species.

Journal ArticleDOI
TL;DR: Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro and restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions.
Abstract: Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro An ascites leukemia, phenotype TL1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C Thus modulation apparently is an active cellular process Antigens TL 1,2, and 3 are all modulated by anti-TL1,3 serum and by anti-TL3 serum This modulation affects all three TL components together, even when antibody to one or two of them is lacking aAnti-TL2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions

Journal ArticleDOI
TL;DR: These experiments support the hypothesis that certain biological effects induced by endotoxins may be mediated via the C' system, and may account for some of the known similarity in the reactivities evoked by endotoxic lipopolysaccharide, zymosan, or preformed immune complexes in vivo.
Abstract: Large amounts of each C'3, C'5, C'6, C'7, C'8, and C'9 were consumed when guinea pig serum was incubated with endotoxic lipopolysaccharide, zymosan, or preformed immune complexes. Since these C' components subserve several of the biological activities which follow the injection of endotoxins into experimental animals, these experiments support the hypothesis that certain biological effects induced by endotoxins may be mediated via the C' system, and may account for some of the known similarity in the reactivities evoked by endotoxins and immune complexes in vivo.

Journal ArticleDOI
TL;DR: The cytopathic effect of lymph node cells from tuberculin-sensitized rats on rat embryo fibroblasts in the presence of PPD was not enhanced by admixture of normal (nonsensitized) lymph nodes, and preincubation studies showed that this in vitro response is initiated by the reaction of lymphocytes with specific antigen.
Abstract: The cytopathic effect of lymph node cells from tuberculin-sensitized rats on rat embryo fibroblasts in the presence of PPD was not enhanced by admixture of normal (nonsensitized) lymph node cells. Preincubation studies showed that this in vitro response is initiated by the reaction of lymphocytes with specific antigen, beginning within 30 min, rather than uptake of antigen by the fibroblasts. The supernatant fluids from suspensions of sensitized cells incubated with PPD for 17 hr or more possessed cytotoxic activity. The target fibroblasts showed a marked increase in acid phosphatase content within 48 hr after the addition of sensitized lymph node cells and antigen.

Journal ArticleDOI
TL;DR: The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.
Abstract: The nephritogenic antigen, responsible for the immunogenic stimulus in experimental allergic glomerulonephritis induced with tubular antigen, has been identified as a renal tubular epithelial (RTE)-specific antigen and has been isolated in a relatively purified form. This antigen, RTE-α5, is a distinct and antigenically specific lipoprotein of high density which is derived primarily from the brush border of proximal convoluted tubular epthelium of the rat kidney. It has been suggested that this molecule may be a plasma membrane subunit. Immunization of rats with as little as 3 µg N of RTE-α5 in complete Freund's adjuvant has effectively induced this form of membranous glomerulonephritis. RTE-α5 is not a constituent of normal rat glomeruli; however, with the onset of autologous immune complex nephritis it is deposited in a granular fashion along glomerular capillary walls indistinguishable from the deposits of γ-globulin and complement. The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.

Journal ArticleDOI
TL;DR: The administration of ASA to rabbits (in doses which inhibited collagen-induced platelet aggregation) impaired hemostasis, prolonged platelet survival, and diminished the amount of deposit formed in an extracorporeal shunt.
Abstract: Acetylsalicylic acid (ASA, aspirin) and sodium salicylate inhibit platelet aggregation induced by collagen, antigen-antibody complexes, gamma globulin-coated particles or thrombin. These compounds suppress the release of platelet constituents, such as adenosine diphosphate (ADP) and serotonin, induced by such stimuli. Since ASA and sodium salicylate do not inhibit ADP-induced platelet aggregation, it appears that their effect on the action of the other stimuli is due to a decrease in the amount of ADP released. The administration of ASA to rabbits (in doses which inhibited collagen-induced platelet aggregation) impaired hemostasis, prolonged platelet survival, and diminished the amount of deposit formed in an extracorporeal shunt.

Journal ArticleDOI
TL;DR: The observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.
Abstract: A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.

Journal ArticleDOI
TL;DR: The results of these experiments strongly support the premise that the proliferative response in mixed cultures of lymphocytes represents a de novo immunologic reaction on the cellular level against histocompatibility antigens.
Abstract: The proliferative interaction of cultured rat lymphocytes of immunogenetically disparate origin—the mixed lymphocyte interaction—was employed as an experimental model to examine the initial stages of the immune response mechanism Using mixed cultures of cells derived from parental strain and F1 hybrid rats, in which only the parental lymphocytes respond, the following observations were made on the magnitude and kinetics of the reaction After initiation of the cultures, there was a latent period of approximately 40 hours during which time no mitotic activity was detected This inactive phase was followed by a period of proliferation in which previously nondividing cells entered the mitotic cycle for the first time Activity in the cultures, as detected by incorporation of radioactive thymidine and measured by radioautography or scintillation spectrometry, increased exponentially with a doubling time (T2) of 9–10 hr In this exponential proliferative phase, lasting approximately 100 hr, the dividing cells underwent a series of rapid sequential divisions with a generation time (Tc) of 8 hr, and few, if any, dropped out of the mitotic cycle In addition to the cells which first entered mitosis at the beginning of the proliferative phase and then proceeded through multiple divisions, significant numbers of new, previously nondividing cells continued to enter the mitotic cycle during the entire exponential growth phase The total number of these newly responsive, first division cells throughout the total culture period amounted to 1–3% of the original parental cell inoculum This is a surprisingly large proportion of peripheral blood lymphocytes with demonstrable reactivity to a particular antigen system, if it is assumed that these first division cells in vitro are functionally related to the hypothetical antigen-sensitive cells which proliferate and differentiate into immunological effector cells At present there is no entirely satisfactory explanation for this large number of reactive cells in the mixed lymphocyte interaction

Journal ArticleDOI
TL;DR: Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits by coupling it with its antigen and revealing the sites of peroxidase activity cytochemically.
Abstract: Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

Journal ArticleDOI
TL;DR: It is concluded that the chemotactic factor is able to deactivate or induce chemotaxis depending upon experimental conditions, and the involvement of the activatable esterase of the rabbit polymorphonuclear leukocyte is a serine ester enzyme with a special affinity for aromatic amino acid derivatives.
Abstract: As shown previously, immune complexes engender in rabbit serum a factor capable of inducing chemotaxis of rabbit polymorphonuclear leukocytes. This chemotactic factor consists of a complex of the fifth, sixth, and seventh components of complement. As demonstrated here, the polymorphonuclear leukocytes incubated with such treated rabbit serum lose their ability to respond chemotactically to the chemotactic factor. They are "deactivated." The process of "deactivation" is a function of the duration of contact of the cells with, and the concentration of, the treated serum. There is a parallelism between the time course of deactivation and of chemotaxis, as well as the dose-response curves for the two processes. Chemotactic factor purified by isoelectric precipitation and ion-exchange chromatography produces deactivation in the same manner as the treated serum. The deactivating activity requires, as does the chemotactic factor, the sixth component of complement; like the chemotactic factor, it is heat-stable and nondialyzable. Deactivation is prevented by the same phosphonate esters shown previously to prevent chemotaxis by the complement-associated chemotactic factor. The profiles of the phosphonates in protecting against deactivation are the same as the profiles for the chemotactic factor-dependent inhibition of chemotaxis. Aromatic amino acid derivatives prevent both chemotaxis and deactivation. We conclude from this evidence that the chemotactic factor is able to deactivate or induce chemotaxis depending upon experimental conditions. The fact that the profiles given by the phosphonates for protection against chemotactic factor-dependent deactivation and for chemotactic factor-dependent inhibition of chemotaxis are the same indicates that the "activatable esterase" is involved in both processes. Acetate esters such as ethyl acetate and others shown previously to prevent chemotaxis by inhibiting the "activated esterase" do not prevent deactivation. This indicates that deactivation can occur without participation of the latter enzyme, implying that deactivation involves only a part of the biochemical mechanism of chemotaxis. The protection against deactivation afforded by aromatic amino acid derivatives is specific, insofar as nonaromatic amino compounds and simple acetate esters have no effect. In addition, as stated, the aromatic amino acid derivatives inhibit deactivation and chemotaxis by the chemotactic factor. This latter finding, together with the demonstration of the involvement of the activatable esterase in both deactivation and chemotaxis, suggests that the activatable esterase of the rabbit polymorphonuclear leukocyte is a serine esterase with a special affinity for aromatic amino acid derivatives.

Journal ArticleDOI
TL;DR: In serum sickness, morphologic observations showed an increase in vascular permeability during which large molecules were capable of being trapped by a filtering membrane in the vessel wall, and rabbits were treated with antagonists of histamine and serotonin markedly inhibited the localization of immune complexes in glomeruli, the development of proteinuria, and glomerular endothelial proliferation.
Abstract: In serum sickness, mechanisms by which circulating immune complexes become localized in the walls of vessels and glomeruli have been studied. In affected arteries, morphologic observations showed that circulating marker particles of carbon would rapidly deposit along the luminal surface of the internal elastic lamina. This, as in previous studies, suggested an increase in vascular permeability during which large molecules were capable of being trapped by a filtering membrane in the vessel wall. In attempts to prevent the increase in vascular permeability, rabbits were treated with antagonists of histamine and serotonin. Such treatment markedly inhibited the localization of immune complexes in glomeruli, the development of proteinuria, and glomerular endothelial proliferation. Cardiovascular lesions also were largely prevented from developing. Depletion of platelets, the principal reservoir of vasoactive amines, had a similar though less pronounced effect. While the deposition of immune complexes was inhibited, allergic inflammation in general was not, since normal rabbits treated as above were found capable of developing full Arthus reactions and acute nephrotoxic nephritis. Hydrodynamic factors were noted to be important in determining the location of arterial lesions. Studies of aortas from unmodified rabbits and from those with surgically induced coarctations of the abdominal aorta revealed intimal lesions concentrated at areas of high turbulence, such as at branches, bifurcations, outflows and zones of configurational change. Lesions in these areas were also largely inhibitable by depletion of platelets or by antagonists of histamine and serotonin.

Journal ArticleDOI
TL;DR: The evidence is reviewed that a single genetic system, the major histocompatibility locus in man, HL-A, determines most of the antigens measured by presently available leukocyte isoantisera, and also controls reactivity in one-way mixed leucocyte culture tests.
Abstract: The evidence is reviewed that a single genetic system, the major histocompatibility locus in man, HL-A, determines most of the antigens measured by presently available leukocyte isoantisera, and also controls reactivity in one-way mixed leucocyte culture tests. Studies in 12 families are presented to support this conclusion. Some interesting exceptions to the general typing-MLC tests correlation are presented and discussed.

Journal ArticleDOI
TL;DR: It is demonstrated that generation of chemotactic factor by endotoxin in serum is dependent upon C' system activation involving at least C'5, and the relatively low molecular weight of this factor suggests that it might be derived from activation of a single complement component rather than from complexing of multiple complement components.
Abstract: Endotoxic lipopolysaccharide has recently been shown to fix large amounts of the complement components related to the biologic activities mediated by that system. The present study sought to determine whether the generation of chemotactic factor by endotoxin in serum was dependent upon complement system activation. Preheating serum, incubating at 0°C, or incubating in the presence of EDTA, all prevented chemotactic factor generation as well as complement fixation by endotoxin. "Endotoxoids" deficient in complement-firing activity were also deficient in chemotactic factor generation. Chemotactic factor could not be generated by endotoxin in sera of mice congenitally deficient in the C'S component of complement, while chemotactic factor was generated by endotoxin in the sera of coisogenic mice with normal complement levels for that species. The chemotactic factor induced by endotoxin was heat stable and nondialyzable. Molecular sieve chromatography and sucrose density gradient ultracentrifugation demonstrated that the chemotactic factor was a relatively low molecular weight product (15,000–30,000) and as such different from previously scribed C' system-derived chemotactic factors. These experiments demonstrate that generation of chemotactic factor by endotoxin in serum is dependent upon C' system activation involving at least C'5. Furthermore, the relatively low molecular weight of this factor suggests that it might be derived from activation of a single complement component rather than from complexing of multiple complement components.

Journal ArticleDOI
TL;DR: The transfer of spleen cells from (C3H x C57Bl/6) F1 mice into irradiated C3H parental recipients, normally incapable of responding to (T,G)-A--L, transfers the ability to make either a primary or secondary immune response to this synthetic polypeptide antigen, indicating that the genetic control is exerted upon a process directly related to antibody formation.
Abstract: The transfer of spleen cells from (C3H x C57Bl/6) F(1) mice, capable of responding to (T,G)-A--L, into irradiated C3H parental recipients, normally incapable of responding to (T,G)-A--L, transfers the ability to make either a primary or secondary immune response to this synthetic polypeptide antigen. This localizes the genetic control of the ability to respond to the spleen cell population and indicates that the genetic control is exerted upon a process directly related to antibody formation. Studies with congenic strains of mice and linkage studies in segregating backcross populations show that the ability to respond to (T,G)-A--L and (H,G)-A--L is linked to the H-2 locus and can thus be localized to the IXth mouse linkage group. Note Added in Proof: Of the three possible recombinant animals noted in Tables IV and V, two were infertile. The third animal was not a recombinant, since progeny testing and reimmunization showed that this animal was an H-2(2)/H-2(k) heterozygote capable of responding well to (T,G)-A--L.

Journal ArticleDOI
TL;DR: In the presence of specific antigen, lymph node cells from inbred rats with delayed hypersensitivity to tuberculoprotein, bovine gammaglobulin, and egg albumin produced progressive destruction of monolayers of rat embryo fibroblasts in tissue culture.
Abstract: In the presence of specific antigen, lymph node cells from inbred rats with delayed hypersensitivity to tuberculoprotein, bovine gammaglobulin, and egg albumin produced progressive destruction of monolayers of rat embryo fibroblasts in tissue culture, first apparent at 48 hr and maximal at 72 hr. The effect was specific and did not depend on a genetic difference between the lymph node cells and target cells. It required antigen concentrations equal to or greater than 1.25 µg/ml and lymphocyte: target cell ratios of approximately 10 or 20:1. It could be evaluated both by a plaquing technique and by cell enumeration with an electronic particle counter.

Journal ArticleDOI
J. D. Broome1
TL;DR: The finding that resistant cells have not only a higher asparagine synthetic capacity than sensitive cells but appear able to utilize endogenous asParagine preferentially for protein synthesis is provided.
Abstract: L-asparaginases of agouti serum and Escherichia coli cause a profound lowering in the level of free asparagine in the blood of treated mice and also in the tissues. During treatment, normal tissues and resistant 6C3HED lymphomas survive unharmed with intracellular asparagine levels which are critically low for sensitive lymphomas. An explanation for this contrast between the two types of lymphoma is provided by the finding that resistant cells have not only a higher asparagine synthetic capacity than sensitive cells but appear able to utilize endogenous asparagine preferentially for protein synthesis. Cell-free extracts of resistant cells contain an asparaginase synthetase, but this is not found in preparations from sensitive cells.

Journal ArticleDOI
TL;DR: It has been found that a graft must be in residence for a minimum period of 4 days to evoke the development of a detectable level of sensitivity in the host, and evidence has been obtained suggesting that reestablishment of a functional lymphatic system in a free skin graft may take as long as 9 days.
Abstract: Experiments have been carried out on guinea pigs of two isogenic strains to elucidate the role of afferent lymphatic vessels in the rejection of orthotopic skin homografts. Graft beds were prepared in partially isolated skin flaps with an intact sustaining vascular "umbilical cord" in which a lymphatic connection with the host could be retained or abolished at will. In the absence of demonstrable lymphatic connections between flap and host, intra-flap homografts long outlived similar grafts transplanted to conventional sites in intact skin and, rather than being specifically rejected, died as a consequence of ischemic necrosis of the flap. When lymphatic drainage was retained, intra-flap homografts were rejected in the usual manner. Hosts of long-term intra-flap homografts did not develop sensitivity, as evidenced by the "first set" type rejection of subsequent test grafts, or by the long-term survival of a second skin graft transplanted to a new flap raised on the opposite side of the host's body. Intra-flap skin homografts were rejected if (a) the hosts had been presensitized, (b) they were grafted concomitantly with a skin homograft placed in a conventional site, or inoculated with a suspension of donor lymphoid cells, or (c) if the lymphatic drainage was restored by reimplantation of the hitherto partially isolated flap to an appropriate vascular bed. These findings and others indicate that an intact lymphatic drainage in its bed is essential for an orthotopic skin homograft to sensitize its host. Various experiments were carried out in which intra-flap homografts were used as "indicators" for the acquisition of specific active or adoptive immunity by their hosts. By transplanting skin homografts to conventional beds concomitantly with intra-flap grafts and then excising the former at various intervals, it has been found that a graft must be in residence for a minimum period of 4 days to evoke the development of a detectable level of sensitivity in the host. Furthermore, by replacing either freshly prepared or long-term skin flaps bearing skin homografts in vascular beds on the trunk and determining the subsequent survival times of the homografts, evidence has been obtained suggesting that reestablishment of a functional lymphatic system in a free skin graft may take as long as 9 days. Using intra-flap homografts as indicators of adoptive immunization of the host, we found that as few as 50 x 106 isologous peripheral blood leukocytes from a specifically sensitized animal will transfer an effective level of sensitivity. We also found that hyperimmune serum, in relatively large amount, exerts a weak but definite adverse effect upon either freshly or recently transplanted intra-flap grafts.

Journal ArticleDOI
TL;DR: Quantitative analyses indicated higher levels of γA2-proteins in external secretions and the occurrence of two different antigenic types in all normal sera as well as saliva and colostrum that showed the unique interchain disulfide linkage.
Abstract: The gammaA2-subgroup of gammaA-globulins, previously delineated by antigenic studies, was found to differ strikingly from other immunoglobulins in the manner in which the polypeptide chains are bound together. The heavy and light chains were not linked to each other by disulfide bonds. Instead the light chains were disulfide linked to one another, and were present in the gammaA2-molecule as disulfide bridged L-L dimers. Antisera specific for gammaA2-proteins indicated the occurrence of two different antigenic types in all normal sera as well as saliva and colostrum. Both of these showed the unique interchain disulfide linkage. Quantitative analyses indicated higher levels of gammaA2-proteins in external secretions.