Protective activity of a cell-free Klebsiella vaccine in infection in mice caused by Streptococcus pneumoniae serotypes

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial mass with hydroxylamine, for protecting mice from pneumococcal infection caused by S. pneumoniae, serotypes 3, 4 and 9N, has been studied. Klebsiella vaccine has been found to possess immunostimulating potency with respect to the S. pneumoniae serotypes under study. On day 5 this potency is manifested to a greater extent than 24 hours after immunization. The combination of Klebsiella vaccine with Proteus vulgaris, Staphylococcus aureus and Escherichia coli K-100 antigens enhances the stimulation of nonspecific resistance.     

Brugia pahangi: stage-specific antigens on microfilariae detected by serum from infected jirds or by monoclonal antibodies

Experiments were carried out to determine whether there are stage-specific antigens on microfilariae of Brugia pahangi, using sera from Mongolian jirds infected with B. pahangi and monoclonal antibodies against microfilariae of B. pahangi. These studies showed that microfilariae have both stage-specific and nonspecific antigens. The nonspecific antigens were also present on adult worms and on infective larvae. Among monoclonal antibodies, 6 out of 14 clones produced antibodies against the microfilarial stage-specific antigens, and 8 clones produced antibodies against nonspecific antigens. These monoclonal antibodies could not distinguish between adults, microfilariae, or infective larvae of B. malayi and B. pahangi.     

Studies on the role of the thymus and T cells in the in vivo suppression of delayed hypersensitivity (desensitization): radiosensitivity of the mechanism inducing nonspecific anergy.

Guinea pigs immunized with two or more antigens can have their ability to manifest delayed hypersensitivity (DH) reactions to all these antigens temporarily blocked by the administration of large (mg) amounts of one or more of these antigens 9 days after immunization. This nonspecific anergy is called desensitization. This study presents evidence for the induction of the desensitized state by an active, radiosensitive, immunoregulatory mechanism involving the peripheralization of thymocytes. Desensitization was associated with 1) a marked reduction in thymic weight; 2) an increase in mature T cells in the peripheral blood; 3) decreased responsiveness of the lymphocytes in the spleen to concanavalin-A; and 4) markedly reduced numbers of mono-nuclear cells, basophils, and polymorphs at the skin sites of specific and nonspecific desensitization. Small doses of whole-body irradiation which left DH capacity intact prevented nonspecific desensitization, but did not prevent specific desensitization, suggesting that a radiosensitive cell was involved in the production of anergy.     

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium III. Analysis of resistance induced by dialyzable factors

Dialyzable factors (DF) were prepared from ribosomal fractions of several organisms including rough mutants of Salmonella typhimurium LT2, salmonella species of different serogroups, other enteric bacteria and gram-positive organisms, and tested for their immunogenicity against S. typhimurium infection in mice. All of them conferred local resistance on mice challenged intramuscularly with S. typhimurium LT2 in the early stage of immunization before the establishment of delayed-type hypersensitivity (DTH) to salmonella antigens. Although DFs of enteric bacteria including rough mutants of S. typhimurium induced DTH to salmonella antigens, only DF of a two-heptose mutant of S. typhimurium LT2 afforded significant mouse protection but others only prolonged the mean time to death. DF of Listeria monocytogenes induced the cross-reacting immunity which afforded the low level of mouse protection as well as an increase in mean time to death without inducing DTH. Passive transfer of anti-O antibody did not enhance the mouse protection provided by each DF. Resistance conferred by DF of S. typhimurium LT2 consisted of two phases: (i) nonspecific macrophage activation resulting in reduction of organisms at the infected site, which became active in the early stage of immunization and (ii) salmonella-specific immunity capable of preventing systemic infection, which became active in the late stage of immunization.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T cell-dependent, lymphokine-mediated, activation of macrophages in response to Schistosoma mansoni antigens

Macrophages from Schistosoma mansoni-infected mice kill significant numbers of skin stage schistosomula and murine fibrosarcoma cells in vitro. In order to determine whether the macrophage tumoricidal and larvicidal activation observed in mice as a result of S. mansoni infection are mediated through T cell-dependent (lymphokine) or B cell-dependent (antibody or immune complex) mechanisms, the development of macrophage populations with cytotoxic activity against schistosome larvae or tumor cells was monitored in S. mansoni-infected nude or mu-suppressed mice. Whereas peritoneal cells from S. mansoni-infected congenitally athymic mice had no activity in either assay, cells from mu-suppressed S. mansoni-infected mice showed cytotoxic activity equivalent to that of cells from untreated S. mansoni-infected counterparts. Cells from mu-suppressed uninfected mice were not activated. The mu-suppressed animals had no detectable nonspecific IgM or specific antischistosome IgM, IgG, or IgE antibodies and showed a 90% reduction in numbers of splenic IgM+ cells upon fluorescence activated cell sorter analysis. These results indicate that antibody is not required for in vivo activation of macrophages during S. mansoni infection. Further experiments showed that lymphoid cells from S. mansoni infected mice respond in culture with various specific antigens (such as living or dead whole schistosomula or soluble adult worm antigens) by production of factors capable of activating macrophages from uninfected control mice to kill schistosomula or tumor cells in vitro. Macrophage-activating factors were produced by T cell-enriched, but not T cell-depleted or B cell-enriched, populations from spleens of schistosome-infected mice in response to schistosome antigen. Similar lymphokines may be responsible for the macrophage activation observed during chronic murine schistosomiasis. These observations emphasize the potential contribution of T cell-mediated immune mechanisms in resistance to S. mansoni infection.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. IV. Role of macrophage and its soluble factor in antigen-induced MIF production of immune T lymphocytes.

Histocompatibility-linked restriction of macrophage-T lymphocyte interaction in antigen-induced MIF production by sensitized lymphocytes was examined, by using combinations of inbred strain 2, strain 13, and JY-1 guinea pigs. The effective interaction of the antigen-bearing macrophages with the immune T lymphocytes was observed when the donor of the antigen-bearing macrophages and that of the immune lymphocytes shared Ia antigens of the major histocompatibility complex. Identities of B antigens and S antigens were not important for this cooperation. It was further demonstrated that the previously reported soluble factor derived from LPS-stimulated peritoneal adherent cells (macrophages) could help antigenic activation of the immune lymphocytes across the strain barrier provided a small number of macrophages (0.01%) from syngeneic strain were present. These results show that the presence of macrophages is absolutely required to present antigen to immune T lymphocytes in a genetically restricted manner and the soluble factor from macrophages appears to give a nonspecific effect on the lymphocyte activation in addition to or in collaboration with antigenic stimulation.     

Comparison of Streptococcal R Antigens

At least four distinct nonspecific protein R antigens were found in streptococci of groups A, B, and C by immunodiffusion in agar gel with anti-R sera.     

Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease.

Cryostat sections of various substrates were treated with carbobenzoxychloride in acetone to modify antigens. By applying specific fluorescent antibodies, it could be shown that the antigenic determinants of rabbit gamma-globulin and bovine insulin were totally masked. The antigenicity of ACTH was markedly reduced, whereas the polysaccharide antigens of Salmonella typhimurium were only partially masked. After masking, antigenicity could be restored by treatment with nonspecific protease. The reversible protection of amino groups by carbobenzoxychloride may be a way to preserve protein antigens during embedding in plastics, as such materials also bind to amino groups, blocking the antigenicity of proteins.     

Separation of Herpes Simplex Virus-Induced Antigens by Concanavalin A Affinity Chromatography

Biologically active herpes simplex virus (HSV)-induced antigens were selectively removed from extracts of infected BHK cells by affinity chromatography by utilizing an insoluble form of concanavalin A (Con A). Soluble extracts of (3)H-glucosamine-labeled, HSV-infected cells were absorbed to a Con A column. Bound material was eluted with alpha-methyl-d-mannoside (alphaMM) and NaCl. The specific activity of the eluted glycoproteins increased by 10-fold. Two broad groups of viral-induced antigens were isolated from Con A. Group I includes two antigens which bind to Con A by a specific mechanism because the antigens are dissociated by alphaMM. Group II contains three antigens which bind to Con A but apparently by a nonspecific or electrolytic mechanism. One antigen in group I was identified as the glycoprotein antigen, CP-1, described previously.     

The immunomodulating action of the surface antigens of the influenza A virus in experimental staphylococcal infection

The results of the study of influenza A virus surface antigens, hemagglutinin and neuraminidase, in the induction of nonspecific immunomodulation and protection from acute pulmonary staphylococcal infection have been studied. Protective effect, the cell composition of bronchoalveolar lavage fluid depend on the serological subtypes of surface antigens used for intranasal immunization and the infective dose of staphylococci.     

Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique.

To facilitate the immunological reaction of antibodies with antigens in fixed tissues, it is necessary to unmask or retrieve the antigens through pretreatment of the specimens. However, adjustment of heating-induced antigen retrieval is always required for different tissues and antigens. In this study, by using a low-power antigen-retrieval technique with appropriate dilution of antibodies, we successfully immunostained key antigens in pancreas such as insulin, PDX-1, glucagon, cytokeratin, and CD31, which have previously presented a particular challenge for investigators because of the rapid autodigestion and high nonspecific antibody binding in this tissue. Satisfactory results were obtained when immunohistochemistry and fluorescence in situ hybridization analysis were combined in the same slides.     

The use of paramagnetic beads for the detection of major histocompatibility complex class I and class II antigens

Specific immunoadsorbents were prepared using paramagnetic particles (Dynabeads), and their ability to immunoprecipitate major histocompatibility complex (MHC) Class I and Class II antigens compared with conventional protein A Sepharose immunoadsorbents. Lysates of lymphoblastoid cells provided the antigen source which were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Dynabeads were found to be as effective as protein A Sepharose immunoadsorbents at immunoprecipitating MHC Class I and Class II antigens, but had a much lower nonspecific binding capacity resulting in fewer interference bands and lower backgrounds.     

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund's adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund's adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.     

Formation of cells secreting antigen-dependent nonspecific immunoglobulins during immunization of mice with 2 T-independent antigens

Immunization of BALB/c and C3H/A mice with T-independent antigens (Vi-antigen of Salmonella typhi and polyvinyl pyrrolidone PVP350) induces the appearance of both antibody-forming cells (AFC) and a sharp increase in the number of cells forming nonspecific immunoglobulins (nIFC). This effect is not related to mitogenic properties of the antigens. The number of nIFC formed after simultaneous injection of both T-independent antigens does not differ from that of nIFC formed during immunization with each of these antigens alone. Thus, there was no summation of nIFC number after injection of two T-dependent or one T-dependent and one T-independent antigens. The mechanisms of nIFC formation under the influence of T-dependent and T-independent antigens are discussed.     

Partial suppression of cell mediated immunity in chromoblastomycosis

The cellular immune response of 8 patients from the Brazilian Amazon region with chromoblastomycosis was analyzed. Primary immunological responses of patients were tested by contact sensitization to 2,4-dinitro-chlorobenzene (DNCB), or rejection of first set skin allografts. 2 of 8 patients were reactive to DNCB after sensitization, and skin allograft rejection occured in an average of 14 days. Capacity of patients to mount recall immunological responses was measured by skin testing with two fungal antigens and three bacterial antigens. Delayed skin reaction to trichophytin and Candida antigens was negative in the majority of the patients. However, reactivity to mycobacterial (tuberculin), and bacterial (staphylococcal, streptococcal) antigen was high, or only slightly diminished respectively. The data suggest that patients with chromoblastomycosis have suppressed nonspecific, cell mediated immunity for some antigens (skin allografts, DNCB, fungal antigens), while reactivity to bacterial and mycobacterial antigens is not impaired.     

Polyclonal activation of lymphocytes

Literary data (48 references) on the phenomenon of polyclonal activation (PA) of lymphocytes are analysed and PA inductors of various nature are classified. A new trend in the study of PA is pointed out--the recording of nonspecific reactions to not only exogenous, but also endogenous antigens (mostly autologous erythrocytes and DNA). The problems of the mechanism of PA, the role of the normal autoimmune system, T-cells and macrophages, increased synthesis of specific and nonspecific immunoglobulins and cell proliferation are discussed. The great protective significance of PA as the quickest humoral reaction of the organism on the intrusion of antigenic products or their endogenous synthesis is emphasized. The possibility of using PA for the characterization of the function of B-cells and the normal system of autoimmunity is pointed out. In the study of PA, a standard complex of antigens should be used which should invariably include the cells of microbial autoflora of the body as well as tissue antigens for the detection of autoimmune reaction.     

The dynamics of antigenic changes in the epiblast and hypoblast of the chick during the processes of hypoblast, primitive streak and head process formation, as revealed by immunofluorescence.

Antisera were prepared to epiblast, primary hypoblast, yolk, yolk entoderm, and extraembryonic yolk sac ectoderm and were submitted to various absorption procedures. The absorbed antisera were used in the indirect immunofluorescent method to stain microscopic sections of developing chick blastoderms at different developmental stages. The antigens revealed by the staining at the periods studied were divided into groups of persistent, nonspecific, and specific antigens. The epiblast does not appear to form or include specific antigens until stage XIII (full hypoblast). The primary hypoblast is the layer which during its formation acquires specificity by the inclusion of antigenic components through a cytoplasmic segregation and probably by one or two waves of appearance of primary hypoblast specific antigens. The inductive role of the hypoblast is discussed in relation to the above antigenic manifestations. The anti-hypoblast and anti-epiblast sera after absorption with yolk were found to be suitable reagents for the detection of morphogenetic movements.     

Effect of iscoms and their adjuvant moiety (matrix) on the initial proliferation and IL-2 responses: comparison of spleen cells from mice inoculated with iscoms and/or matrix

In the iscom, antigen is attached by hydrophobic interactions to a matrix which is built up by the adjuvant Quil A and lipids. Thus, the iscom presents antigen in multiple copies on a small particle with a built-in adjuvant. By studying the specific antibody response, in vitro proliferation and IL-2 secretion by splenocytes from mice following different in vivo treatments with iscoms and/or matrix, we attempted to distinguish between nonspecific stimulatory effects, caused by the matrix or iscoms, and specific responses to the antigens incorporated into iscoms. The results strongly suggest that matrix and also iscoms exert a nonspecific adjuvant activity by a transient high spontaneous proliferation of cells collected within 2 weeks after administration of iscoms or matrix. This high rate of proliferation was preceded by suppressed proliferation, 3 days after injection with matrix or iscom. The adjuvant component included in iscoms, i.e., the matrix, does not excert a mitogenic stimulation in vitro or influence the levels of specific antibodies in serum. Specific responses to the antigens included in iscoms were recorded both as increasing levels of serum antibodies and as iscom-induced proliferation of immune spleen cells in vitro. The recruitment of IL-2 was only related to the specific stimulation induced by the antigens in iscom.     

Antigen-nonspecific and specific suppression of lymphokine production in desensitized guinea pigs

Doubly immunized guinea pigs may be desensitized with respect to delayed hypersensitivity reactions against both antigens (anergy) by injection of large doses of either one. This anergic response therefore has both a specific and nonspecific component. The specific component of desensitization persists longer than the nonspecific one. In the present study, we have explored the mechanism of both antigen-specific and antigen-nonspecific suppression during the later stages of desensitization. Guinea pigs immunized with two antigens, DNP-KLH and DNP-EA, were desensitized with DNP-EA. The lymph node cells obtained from the animals 1 day after desensitization were unable to produce MIF in the presence of either antigen. The cells obtained 3, 5, and 7 days after desensitization were able to generate MIF when stimulated with the non-specific antigen (DNP-KLH), but not with specific antigen (DNP-EA). It was shown that both T- and non-T-cell fractions obtained 1 day after desensitization had the capacity to antigen-nonspecifically suppress MIF production. In contrast, if the cells were obtained 3 or 5 days after desensitization, T cells could inhibit only the antigen-specific production of MIF, while non-T cells were still capable of suppressing antigen-specific and nonspecific MIF production. Interestingly, when these two populations were mixed back again, it was now only suppressive to the specific antigen-induced MIF production. This latter observation indicates that nonspecific suppressor non-T cells may themselves be regulated by suppressor T cells. Furthermore, antigen-specific suppressor T cells were shown to produce soluble factor(s) which inhibited the production of MIF.