Unimmunized Animals Research Articles

Enzymatic modification of lymphocyte receptors for antigen. III. Resistance of receptors to trypsin at the peak of the immune response.

Using sheep erythrocytes (SRBC) as the antigen, two subpopulations of spleen antigen-binding lymphocytes could be distinguished by a marked difference in the susceptibility of their receptors to trypsin. In unimmunized animals, 30% of the antigen-binding cells were trypsin-resistant, whereas at 5 days after immunization, 80-90% were trypsin-resistant, indicating an increase of about 50-fold in trypsin-resistant antigen-binding cells per spleen. In contrast, trypsin-sensitive cells per spleen were only 4-fold higher on day 5 than before immunization. The rise in % trypsin sensitivity preceded the increase in rosettes per spleen, implying that immunization produced a preferential increase in trypsin-resistant antigen binding cells partly by converting sensitive cells to resistant cells. After the 5th day, the trypsin sensitivity of antigen-binding cells slowly returned toward the unimmunized level, but a booster injection of SRBC restored trypsin resistance. Trypsin resistance was not lost in the presence of sodium azide or protein synthesis inhibitors. But a slightly increased trypsin susceptibility was conferred by 2-deoxyglucose, implying that glycolysis or the glycosylation of protein may be involved in maintaining trypsin resistance.

Structural Studies on Induced Antibodies with Defined Idiotypic Specificities

Abstract Amino acid sequence analysis has been performed on three groups of heavy (H) chains of A/J mice. H chains derived from unimmunized animals were compared to anti-p-azophenylarsonate (anti-Ar) antibodies which were further subdivided into those possessing and those depleted of a cross-reacting idiotype (CRI). It was found that anti-Ar antibodies bearing the CRI are homogeneous through the first hypervariable region of the H chain. The same sequence was obtained for pooled antibody isolated from the ascites fluid of 18 A/J mice or from a single mouse. The H chains appear to belong to a minor VH subgroup. In the first 30 positions Anti-Ar antibodies depleted of the CRI had the same sequence as those containing the CRI (with small amounts of heterogeneity at some positions), but contained a mixture of sequences in the first hypervariable region of the H chain. These studies indicate that antibodies with similar specificity and with identical framework sequences, but which differ in their hypervariable regions, contain different idiotypic determinants, and support the concept that the idiotypic determinants reside primarily within hypervariable regions.

Interaction of toxin of Corynebacterium diphtheriae with phagocytes from susceptible and resistant species.

The interaction of the toxin of Corynebacterium diphtheriae with leukocytes from sensitive and resistant animal species was examined by determining the ability of toxin to inhibit protein synthesis by several types of phagocytic cell. Small amounts of toxin (25 minimal lethal doses) impaired protein synthesis in both polymorphonuclear leukocytes and mononuclear cells from humans and guinea pigs, whereas large amounts (2,000 minimal lethal doses) were required for minimal inhibition of mouse phagocytes. Peritoneal macrophages from hyperimmunized guinea pigs exhibited the same high degree of sensitivity to diphtheria toxin as did those from unimmunized animals. Prolonged incubation with toxin resulted in a 75% reduction in phagocytosis of polystyrene latex spheres but had no effect on transport of the glucose analogue 2-deoxy-D-glucose by guinea pig macrophages. Thus phagocytic cells, although they are endowed with a high level of phagocytic cell appear to reflect the native resistance or sensitivity of the host species of origin.

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals. When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.

Differentiation of Lymphoid Cells: Enhancement of the Induction of Immunoglobulin Production by Thymus Cells and a Product Secreted by Thymus Cells

Abstract The induction of immunoglobulin M production, as a consequence of the incubation of rabbit spleen cells for 72 hr in cell culture in the absence of added antigen, was markedly enhanced when thymus cells were cultured along with spleen cells. In contrast, immunoglobulin production by lymph node cells was only slightly stimulated by the presence of thymus cells during culture. Immunoglobulin production by spleen cells was equally enhanced, regardless of whether the thymus cells were derived from unimmunized animals or from animals immunized with a dinitrophenyl-antigen. Thymus cells added to cultures of spleen cells at 48 hr were still markedly stimulatory even though the induction process in the absence of thymus cells appears to be largely complete at 48 hr. Ficoll density gradient separation of spleen cells revealed some fractions that were slightly enhanced and others that were markedly enhanced by thymus cells. Finally, a cellfree mediator secreted into the culture medium by thymus cells also enhanced immunoglobulin production to about the same extent as thymus cells.

The effect of hydrocortisone on the sheep red cell response in adult Xenopus laevis, the South African clawed toad.

AbstractThe effect of hydrocortisone on the anti‐erythrocyte (SRBC) response of the South African clawed toad, Xenopus laevis, was studied. Hydrocortisone was administered two days prior to antigen injection and was found to reduce the eight‐day antigen‐binding response of sensitized spleen cells to 17% of its normal peak value. Control animals received injections of the vehicle solution the hormone is normally suspended in. Hemagglutination assays were also performed to ascertain whether the hydrocortisone treatment merely altered the ability of the spleen cells to bind antigen or suppressed their ability to produce antibody. Serum antibody titers were found to be depressed to 9% of the peak value by hydrocortisone treatment. Both thymus and spleen cells from the sensitized, hydrocortisone‐treated and control adult Xenopus were tested by immunocytoadherence. Unimmunized animals were also used to establish background levels. It was found that while the antigen‐binding response by immunized spleen cells was greatly decreased by hydrocortisone, thymocytes were particularly effected as their antigen‐binding capacities were reduced to background levels. It was concluded that these data support the view that the toad immune system is comparable to the mammalian with regard to hydrocortisone sensitivity of the anti‐red cell response.

Cellular immunity to herpes simplex virus mediated by interferon.

Rabbit kidney cell monolayers infected with herpes simplex virus (HSV) were incubated with leukocytes from rabbits immunized with complete Freund's adjuvant. When the leukocytes were exposed to tuberculin purified protein derivative (PPD), viral replication and plaque formation were markedly inhibited. Similarly, when leukocytes from animals immunized with HSV were exposed to UV-inactivated HSV, viral replication was markedly inhibited. Exposure of leukocytes from unimmunized animals or animals immunized with incomplete Freund's adjuvant to UV-inactivated virus or PPD produced relatively little inhibition of viral replication. Examination of supernatant fluids from stimulated cultures revealed a soluble mediator that had the properties of interferon. Interferon production was detected within several hours after exposure of sensitized leukocytes to antigen. Supernatant fluids from as few as one sensitized leukocyte per 200 rabbit kidney cells inhibited HSV replication by over 90%. These findings support the concept that the cellular immune response to HSV consists of two phases: an immunologically specific antigen recognition phase, and a nonspecific effector phase that stops HSV spread by generating interferon.

Protective immunity to amebic infection demonstrated in guinea pigs.

Guinea pigs were given a series of intramuscular injections of Entamoeba histolytica antigens mixed with Freund's complete adjuvant. The antigens were sonicated whole amebae and fractions obtained from amebae separated by column chromatography and concentrated by passage through ultrafiltration membranes. After three immunizing doses the animals were challenged with intracecal injection of amebae and were killed 6 days later. Gross and microscopic observations were made, the degree of protection was noted and the antibody response was studied. Fraction I had the largest molecular weight and complete protection was obtained. Partial protection was noted with the other antigens; 70% with whole amebae, 64% with fraction III and 43% with fraction II. Ninety-two percent of unimmunized animals were infected. Unusual lesions were observed in the liver of the immunized animals and their significance is discussed.

Neutralization of Friend leukaemia by sera of unimmunized animals.

SUMMARY The sera of several animal species neutralize Friend leukaemia virus. Among 54 serum samples taken from 16 different species only those of mouse, rat and sheep were found to be devoid of this property. The neutralizing activity is partially heat-labile and appears to require the presence of complement. Its biological significance might be considerable.

Dissociation of in vitro lymphocyte transformation from production of a mononuclear leucocyte chemotactic factor in agammaglobulinemic chickens

Cultures of splenic and peripheral lymphocytes from normal chickens immunized intravenously with Brucella abortus organisms were stimulated by this antigen to incorporate significantly greater amounts of 3H-thymidine and 14C-leucine than lymphocytes from unimmunized animals. Lymphocytes from immunized agammaglobulinemic chickens were unresponsive to Brucella. This defect could not be corrected by the addition of either normal nonimmune or irradiated normal immune spleen cells to cultures of ag chicken lymphocytes which suggests that normally B cells transform in vitro in response to this antigen. In contrast, cultured peripheral blood leucocytes from both immunized normal and agammaglobulinemic chickens produce significantly more monocyte chemotactic factor in response to Brucella than leucocytes from nonimmune chickens. This indicates that the production of this mediator is a B cell independent function and suggests that T cells are the producers of this lymphokine.

BCG immunization in marmosets.

Clinical and immune responses of marmosets to BCG immunization were studied. Ten of 13 animals responded to BCG immunization by developing lymphocyte reactivity to purified protein derivative (PPD), while the lymphocytes of only 3 of 64 unimmunized animals showed reactivity to PPD.

Intracellular response by Tetrahymena pyriformis to fluids from unimmunized animals.

Dans leTetrahymena pyriformis, les sérums des lapins et humains et les ascites fluides des souris ont une apparence bipolaire. Les petit «corps» s'accumulent antérieurement, les grands «corps» postérieurement. On peut présumer que la réduction de la tension du O2 et/ou l'augmentation de la tension du CO2 sont requis, puisque l'état bipolaire ne s'est produit que dans des conditions limitées d'exposition à l'air. La bipolarisation est reversible. Les animaux bipolaires qui ont été exposés brièvement à l'air ressemblent aux animaux normaux.

Shared antigens between heterologous bacterial species.

Sera from normal rabbits were shown to have antibodies that bound radiolabeled test antigens derived from many taxonomically unrelated bacteria. Sera from rabbits that had been immunized with sonically treated material of 12 different bacteria, including M. bovis strain BCG, had antibodies that bound not only radiolabeled homologous test antigens but also radiolabeled antigens from many unrelated bacteria. Binding by normal and immunized sera to radiolabeled test antigens was inhibited by homologous unlabeled test antigens but not by substances such as bovine serum albumin, polyvinylpyrrolidone, sheep erythrocytes, and endotoxin. The broad range of shared or cross-reactivity among antigens in bacteria may explain the presence of antibodies to many bacteria in sera from normal humans and previously unimmunized experimental animals. The presence of these antibodies raises the question whether resistance to many bacterial infections may be partly due to immune mechanisms, whether cellular or humoral, that have been stimulated by unrelated bacteria.

Release of intracellular constituents from rabbit polymorphonuclear leukocytes exposed to soluble and insoluble immune complexes.

Polymorphonuclear leukocytes (PMNL) obtained from peritoneal exudates of unimmunized rabbits consistently released several intracellular constituents into the suspending medium when incubated with equivalence precipitates, which were prepared with rabbit antiserum to bovine serum albumin (BSA). Identical concentrations (AbN) of soluble immune complexes in antigen excess were neither as potent nor as predictable in triggering these phenomena. Precipitates prepared from antisera at 4 weeks after immunization were slightly more potent than those prepared from 6, 8 or 10-week antisera. On the other hand, soluble complexes prepared from 10-week antisera proved more potent than those derived from antisera of earlier bleedings. When PMNL were harvested from rabbits immunized with BSA and then exposed to immune complexes containing autologous antibodies, the ‘immune’ PMNL appeared to be less responsive to these stimuli when compared to PMNL obtained from unimmunized animals.

The macrophage aggregation assay for delayed hypersensitivity: Development of the response, role of the macrophage, and the independence of humoral antibody

In vitro aggregation of guinea pig peritoneal exudate cell (PEC) suspensions in the presence of antigen was found to correlate with cutaneous delayed hypersensitivity. In contrast, PEC from animals with humoral antibody and negative delayed skin tests did not aggregate when antigen was added. Separation of lymphocytes and macrophages in the PEC suspensions demonstrated that the macrophages were the aggregating cells which could be derived from either immunized or unimmunized animals. Macrophage aggregation took place in the presence of sensitive lymphocytes plus antigen or upon incubation of macrophages with supernatant fluids from cultures of sensitive lymphocytes with antigen. Following immunization with Bacillus Calmette-Guerin the macrophage aggregation assay paralleled the conversion of skin tests.

The fate of circulating methylcholanthrene tumor cells in mice with tumor-specific immunity.

Syngeneic mice were immunized to a methylcholanthrene-induced tumor, and immunity was demonstrated by the failure of cell suspensions of the tumor to grow when inoculated intramuscularly. Identical cell suspensions inoculated intravenously, however, produced pulmonary tumors in all immunized animals. Intravenous inoculations of blood containing circulating tumor cells obtained from donor mice bearing the same tumor produced identical numbers of pulmonary tumors in immunized and unimmunized animals. Thus, in this well-studied experimental model, specific immune inhibition of tumor growth as conventionally assessed was not reflected in destruction of cell suspensions of the tumor or circulating tumor cells shed from the tumor when these cells are injected into the circulatory system.

Antigen in feed as cause of antibody in unimmunized animals.

Lymphoid cells which reacted with BSA labelled with fluorescein or <sup>125</sup>I were found in mesenteric and popliteal lymph nodes, spleen and lamina propria of the small intestine from unimmunized Syrian golden hamsters. Hamster and rabbit feed contain BSA and this probably is the antigenic stimulus for the cells containing antibody to BSA. Unimmunized hamsters had no serum antibody to BSA as judged by radioimmunoelectrophoresis and PGA in the guinea pig.

A Cytolytic System for the in Vitro Detection of Cell-Mediated Immunity to Soluble Antigen

Abstract An in vitro cytotoxic test is described for the demonstration of cell-mediated immunity to soluble antigen. This system has been used to study the development of a cytotoxic splenic lymphocyte population in the guinea pig after immunization with dinitrophenylated human IgG (DNP-HGG). As “target” cells, 51Cr-labeled mouse mastocytoma cells were used, and were coupled to DNP-HGG with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. “Target” cell destruction was assessed by comparing the rates of 51Cr release from these cells in the presence of splenic lymphocytes from specifically sensitized animals with that released in the presence of the same number of lymphocytes from unimmunized animals. The mastocytoma cells were found to be preferable to erythrocytes as carriers of antigen, in that they were more sensitive to membrane permeability changes. Splenic lymphocytes from guinea pigs immunized with DNP-HGG caused specific lysis of DNP-HGG-coated mastocytoma cells. Cells coated with DNP-BSA and HGG were lysed to a lesser extent, while cells coated with antigens immunochemically unrelated to the antigen used for sensitization were not lysed to any greater extent than that observed with lymphocytes from unimmunized animals. Several factors distinguished cytolysis by “sensitized” lymphocytes from that mediated by serum antibody in the presence of complement. Cytolysis by lymphocytes was not inhibited by anti-guinea pig immunoglobulin antisera. The total cytolytic activity of splenic lymphocytes did not parallel measured serum antibody. The specificity of cytolysis by splenic lymphocytes from animals immunized with DNP-HGG was markedly carrier-dependent.

Specific inhibition of tumor cell DNA synthesis in vitro by lymphocytes from peritoneal exudate of immunized syngeneic guinea pigs.

Tumor immunity to a transplantable diethylnitrosamine-induced hepatoma in inbred guinea pigs has been found to be immunologically specific and cell mediated. We have investigated this cellular immunity using a quantitative, reproducible, and simple assay based on the ability of leucocytes to inhibit the incorporation of tritiated thymidine (TdR(3)H) by tumor cells. Tumor cell suspensions were obtained from the ascites form or tissue culture monolayers of the hepatoma. Cells from the peritoneal exudate of immunized guinea pigs inhibited tritiated thymidine uptake by tumor target cells to a significantly greater degree than cells from the peritoneal exudate of unimmunized animals. Immune lymph node, peripheral blood, and spleen leucocytes were not inhibitory. The assay was sufficiently sensitive to detect relatively weak tumor immunity. The in vitro inhibition was correlated directly with the presence of delayed hypersensitivity and/or inhibition of tumor cell growth in local passive transfer studies. Irradiation of peritoneal exudate cells (3000 R) blocked their inhibitory effects on tumor cells. Fractionation of the peritoneal exudate cells by centrifugation in zones of bovine serum albumin of different density also revealed the lymphocytes to be responsible for the specific inhibitory effects whereas macrophages inhibited in an immunologically nonspecific fashion.

Effects of immobilizing antiserum and normal serum on Paramecium surface antigen synthesis.

SYNOPSIS. Rabbit antiserum can transform paramecia from one scrotype to a 2nd antigenic type. Altho homologous antisera made against antigens sharing the same specificity as that of a treated cell are most effective, other sera lacking crossreacting antibodies also can induce transformation. Furthermore, normal serum from unimmunized animals will often have the same effect. A 0‐40% ammonium sulfate fraction of serum removes a lethal factor, leaving behind an efficient agent for transformation. The activily of this agent will vary from rabbit to rabbit, and apparently is characteristic of a particular rabbit.