Antibodies reacting with thymus and skin epithelium and antibodies to cell nuclei in immunization with streptococcal group A polysaccharide conjugated with synthetic polyelectrolytes

The antibodies to streptococcal group A polysaccharide (A-PS) have been obtained upon immunization of BALB/c mice with A-PS conjugated with synthetic polyelectrolytes (PEL). Prolonged immunization in the majority of cases revealed antibodies to cross-reactive determinant of A-PS reacting with human and mouse epithelium of the thymus and basal skin layer. These antibodies belong to autoantibodies. Later on, after the beginning of immunization some animals produced antibodies reacting with cellular nuclei. The formation of autoantibodies to nuclei is not related to crossreactions with A-PS, because A-PS do not inhibit these reactions. No antibodies reacting with the epithelial cells or with cellular nuclei have been observed upon immunization with A-PS in Freund adjuvant or with PEL alone. The production of autoantibodies to cellular nuclei is probably a result of immunoregulatory disorders associated with the damage of thymus epithelium by autoantibodies during immunization with A-PS conjugated with PEL.     

The suppression of delayed hypersensitivity to BCG antigens in BALB/c mice during the production of antibodies to the rhamnose determinants of Streptococcus group A polysaccharide]

The data obtained for the first time in our studies indicate that the production of antibodies to group A streptococcal polysaccharide (A-PS), one of the cross-reacting streptococcal antigens, may suppress delayed hypersensitivity (DH) to microbial antigens. The existence of sharply pronounced correlation between the suppression of DH and the presence of antibodies to the rhamnose area of A-PS in the blood of BALB/c mice immunized with the pepsin-treated culture of group A streptococci has been shown. The suppression of DH is absent in the immunized animals of the same group whose blood contains antibodies to the determinant, specific for A-PS. As revealed in this study, the effect of the suppression of antigen-specific cytotoxicity linked with DH to BCG antigens can be reproduced by mixing lymph node cells taken from these two groups of the animals. The data thus obtained are possibly linked with the activation of nonspecific T suppressors in the production of antibodies to the rhamnose determinants of A-PS in the animals immunized with streptococci. The mechanism of the newly discovered phenomenon is discussed.     

The effect of immunization with Streptococcus group A on the development of delayed hypersensitivity to BCG antigens in mice of different strains]

Antibodies to group A streptococcal polysaccharide (A-PS) have been shown to appear within three weeks after the injection of group A streptococcus culture, heat-killed and treated with pepsin (A-STP), in the blood of not only BALB/c mice, but also CBA mice. As revealed in this study, in BALB/c mice antibodies are mainly active against the group-specific antigenic determinant (AD) of A-PS and in CBA mice, against the rhamnose AD of A-PS, common for streptococci of different groups. This study has revealed that the appearance of antibodies to the rhamnose AD of A-PS in the blood of CBA mice inhibits antigen-specific cytotoxicity, appearing with the development of delayed hypersensitivity to BCG antigens. This effect is not linked with the immunization of the animals with high doses of streptococci. Experiments have shown that the in vitro transfer of the inhibition of antigen-specific cytotoxicity to lymph node cells of normal BCG-sensitized animals may be carried out with lymph node cells of CBA mice, immunized with A-STP and having antibodies to the rhamnose AD of A-PS, but not with the serum containing these antibodies. The mechanisms of this effect are discussed.     

Antibodies to the rhamnose determinants of the polysaccharide of streptococci group A in the sera of rheumatism patients and donors]

In the sera of patients with recurrent rheumocarditis, and especially in cases of primary rheumatism, the level of antibodies to group A streptococcal polysaccharide (A-PS) has been found, according to the results of the enzyme immunoassay, to be considerably higher than in the sera of healthy donors. The level of antibodies to rhamnose determinants (RD) of A-PS has been determined by the inhibition of the immunoenzyme reaction with A-PS under the influence of a variant of group A streptococcus and rhamnose disaccharides with the bonds alpha 1-2 and alpha 1-3. In patients with recurrent rheumocarditis the level of antibodies to A-PS has been shown to be considerably higher than in healthy donors having these antibodies. In acute primary rheumatism a high level of antibodies to A-PS has been detected only in a few cases, and at the same time the prevalence of antibodies to the specific RD of A-PS, bound with beta-N-acetylglucosamine, is observed. In the sera of patients with recurrent rheumocarditis and donors having a high content of antibodies to the rhamnose site of A-PS antibodies, seemingly active against at least two RD, have been detected. In acute primary rheumatism an insignificant amount of antibodies to the rhamnose site of A-PS may probably cause the autoimmune process accompanying rheumatism. This suggestion is substantiated by the previously established capacity of these antibodies for inducing the suppression of cytotoxic cell reactions to microbial antigens.     

Autoantibodies to different layers of epidermis during immunization of BALB/c mice with a culture of Group A Streptococcus treated with pepsin

As revealed in the indirect immunofluorescence test, antibodies to the cross-reacting group A streptococcal polysaccharide determinant (A-PS), common to the antigen of the basal cell layer of the epidermis, regularly occur at the end of the first cycle and disappear after further immunization of BALB/c mice with the pepsin-treated culture of group A streptococci. This model may be used for the study of antibodies to A-PS, cross-reacting with the cells of the basal layer of the epidermis, in the development of the autoimmune process linked++ with group A streptococcal infection.     

Level of antibodies to different determinants of Streptococcus group A polysaccharide and autoantibodies to basal layer of the skin epithelium in rheumatism]

Direct dependence was established between the presence of autoantibodies reacting with the basal layer of the skin epithelium (BLSE) and the high level of antibodies to the streptococcal group A polysaccharide (APS). By the primary active rheumatic fever (PARF) autoantibodies to the BLSE are revealed. By the recurrent active rheumatic fever (RARF) and in the control sera, autoantibodies reacting with the BLES, apparently, are directed to the rhamnose determinants of APS. These data confirm: different level of antibodies to the GS and to the rhamnose determinants of APS by PARF, RARF and in the control sera; the experiments of the autoantibody inhibition, reacting with the BLSE by the APS or the polysaccharide of streptococci A-variant, containing only the rhamnose determinants.     

Comparison of the frequency of detection of autoantibodies to epidermal antigens with the level of antibodies to polysaccharide of group A Streptococcus and the number of T-suppressors in glomerulonephritis]

By the acute glomerulonephritis (GN) of streptococcal etiology, autoantibodies (AA) reacting with the basal layer of skin epithelium (BLSE) are discovered. The presence of this AA's correlate with the high level of antibodies to the streptococcal group A polysaccharide (A-PS). In the control sera such AA's and the high level antibodies to A-PS are discovered very rarely. By the GN of non-streptococcal etiology, AA's to the BLSE apparently of other specificity are obtained in some cases, in spite of the absence of antibodies to A-PS. AA's reacting with the differentiated layers of skin epithelium are discovered in the high percent of cases by GN. The presence of these AA's do not correlate with the levels of antibodies to A-PS. The reduction of the number of T-lymphocyte suppressors is established in the blood by the presence of AA's to the BLSE by GN. This question is a subject of later investigations by the different autoimmune processes. Such data can apparently corroborate the previously expressed hypothesis, that AA's to BLSE, which as a rule react with endocrine thymus epithelium, are the cause of the beginning of immunoregulatory disorders, characteristic of autoimmune processes.     

Preparation of monoclonal antibodies to nuclear DNA of mammalian cells by immunization with group A streptococcal polysaccharide conjugated with synthetic polyelectrolytes

Monoclonal antibodies (MAb) were obtained by hybridization of spleen cells from BALB/c mice immunized with streptococcal group A polysaccharide (A-PS) conjugated with synthetic polyelectrolytes (PEL). These MAb reacted with nuclei from human and mouse cells. MAb reacting with nuclei were obtained after prolonged immunization with conjugates and were not formed by hybridization of spleen cells from non-immunized mice or by the immunization with PEL. The investigation of Mab (B1/2 and A5/2) reacting with nuclei has shown that these Mab are directed against DNA and do not react with other tissue substances. No cross-reactions of Mab with A-PS used for immunization have been revealed. Mab B1/2 and A5/2 belong to autoantibodies.     

Monoclonal autoantibodies to different epithelial structures of the thymus gland obtained by the immunization with streptococcal group A antigens

By the indirect immunofluorescence method it was shown to which epithelial thymus structures monoclonal antibodies (mAT) reacting with the different epidermal structures are directed. These mAT related to the autoantibodies were obtained earlier, as a result of lymphoid cells polyclonal activation, by the immunization of BALB/c mice with streptococcal group A nonspecific protein antigens of the cell wall. It was shown that mAT A6/1, reacting with the basal layer of the skin epithelium are directed to the epithelium of the cortical and medullar thymus zones, which is regarded as the so called endocrinal epithelium. These mAT, by the study with immunoblotting method, react with the protein of mV SOkD, B5/1 mAT to the skin epithelium, on the thymus sections react with the single cells around the Hassel bodies.     

Monoclonal antibodies to different antigens of skin epithelium obtained by immunizing mice with streptococcus group A antigens

Monoclonal antibodies (MCA) were obtained by immunization of BALB/c mice with streptococcal group A protein antigens of the cellular wall, or with whole microbial cells. In immunofluorescence test, MCA react with different skin epithelial structures (basal, suprabasal or all the epidermal layers). The majority of MCA belong to autoantibodies. The same MCA revealed no cross-reactions with streptococcal antigens in immunoenzyme and inhibition tests. MCA reacting with epithelial cells are, apparently, obtained as a result of polyclonal activation of the autoreactive clones by streptococcal antigens.     

Specificity of monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice

Antibodies reactive with left-handed Z-DNA arise spontaneously in the sera of patients with SLE and rheumatoid arthritis and in autoimmune MRL mice. However, the precise specificity of these autoantibodies has not been established. In this report, we have characterized four monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice that do not cross-react with B-DNA and can discriminate between different types of left-handed helices. Two of the monoclonal antibodies (Za and Zi) behaved similarly in that they bound to two forms of Z-DNA (Br-poly(dG-dC).poly(dG-dC) and AAF-poly(dG-dC).poly(dG-dC) but not to two other Z-form DNA (poly(dG-5BrdC).poly(dG-5BrdC) or poly(dG-5MedC).poly(dG-5MedC)). Neither antibody (Za or Zi) bound significantly to B-DNA or to denatured DNA. A third antibody (Ze) exhibited similar binding characteristics for the Z-DNA preparations, but also recognized denatured DNA. In contrast, a fourth antibody (3-7.3) bound preferentially to poly(dG-5BrC).poly(dG-5BrdC) in Z conformation. These results provide the first evidence for anti-Z-DNA autoantibodies in autoimmune mice that do not cross-react with native or denatured DNA and indicate that these antibodies exhibit considerable heterogeneity in their fine binding specificity.     

Production and characterization of monoclonal antibodies to group-specific antigenic determinant of group A streptococcal polysaccharides

Hybridomas producing monoclonal antibodies (McAb) to group A streptococcal polysaccharide (A-PS) were obtained. Of these, 3 clones were selected: 2 clones producing IgG3, precipitating McAb and 1 clone producing IgM nonprecipitating McAb. The results of the competitive inhibition in the enzyme immunoassay suggested that precipitating and nonprecipitating McAb reacted with nonidentical epitopes of A-PS, though determinants, specifically reacting with the given McAb, had a common site which included N-acetyl-D-glucosamine. On the surface of bacteria, in addition to protein M, the presence of the given determinants of A-PS was established in the direct immunofluorescence test. The newly developed method of direct immunofluorescence with the use of specially selected precipitating McAb was the basis for the development of rapid diagnosticum, permitting the identification of group A streptococci.     

Autoantibodies to the thymus and skin epithelium in NZB/N mice and (NZBxNZW)F1 hybrids

Antibodies reacting with thymus and skin epithelial cells were revealed by indirect immunofluorescence in sera of NZB/N mice and (NZB X NZW)F1 hybrids (B/W) 1-2 and 4-5 months of age. Similar antibodies were not found in sera of BALB/c mice. The inhibition experiments with DNA have shown that antibodies reacting with the thymus and skin epithelium differ from those reacting with the cellular nucleus. Positive reactions with the epithelium were obtained in all thymus and skin tissue samples of humans, guinea-pigs and NZB/N, B/W and BALB/c mice, including autologous tissues of NZB/N and B/W mice. Thus, antibodies reacting with thymus and skin epithelial tissues belong to autoantibodies. These autoantibodies are revealed during the first month of life before the onset of autoimmune processes. The role of these autoantibodies in the damage of thymus epithelium and the development of immunoregulatory disturbances, typical of autoimmune processes, needs further study.     

Precipitating monoclonal antibodies to 1 of the antigenic determinants of streptococcal group-A polysaccharide

Monoclonal antibodies (MCA) to streptococcal group A polysaccharide (A-PS) were prepared. Precipitating MCA are directed to the determinant common for A-PS and streptococcal group L polysaccharide (L-PS). The antibodies react in the immunodiffusion test and give identity reaction in A- and L-PS tests. Other MCA are non-precipitating but react with A-PS studied by immunoenzyme method. The reasons for the formation of precipitating and non-precipitating MCA to different antigenic determinants of A-PS are discussed.     

Autoantibodies to the common antigenic determinant of group A streptococcal polysaccharide and epithelial cells of mammalian thymus and skin]

A cross-reactive (CR) antigenic determinant in the polysaccharide of group A streptococus and the mammalian thymus and skin was studied. The CR antigen was found in adult humans and human embryos irrespective of the blood group, as well as in the tissues of all the animal species studied, including rabbits immunized with streptococcus and producing antibodies to A polysaccharide. Evidence was obtained that the antibodies elaborated to the CR determinant of polysaccharide A and of the tissue epithelium should be classed as autoantibodies. It is suggested that reaction of these antobodies with the CR antigen in the thymus could serve as one of the causes of autoimmune thymitis during rheumatic fever.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

Autoimmunity is triggered by cPR-3(105-201), a protein complementary to human autoantigen proteinase-3

It remains unclear how and why autoimmunity occurs. Here we show evidence for a previously unrecognized and possibly general mechanism of autoimmunity. This new finding was discovered serendipitously using material from patients with inflammatory vascular disease caused by antineutrophil cytoplasmic autoantibodies (ANCA) with specificity for proteinase-3 (PR-3). Such patients harbor not only antibodies to the autoantigen (PR-3), but also antibodies to a peptide translated from the antisense DNA strand of PR-3 (complementary PR-3, cPR-3) or to a mimic of this peptide. Immunization of mice with the middle region of cPR-3 resulted in production of antibodies not only to cPR-3, but also to the immunogen's sense peptide counterpart, PR-3. Both human and mouse antibodies to PR-3 and cPR-3 bound to each other, indicating idiotypic relationships. These findings indicate that autoimmunity can be initiated through an immune response against a peptide that is antisense or complementary to the autoantigen, which then induces anti-idiotypic antibodies (autoantibodies) that cross-react with the autoantigen.     

Analysis of the cellular interactions involved in the regulation of induced erythrocyte autoantibodies

When normal mice are immunised with rat red blood cells (RBC) they produce autoantibodies against their own red blood cells as well as antibodies against determinants on rat RBC non-cross-reacting with self-RBC. Spleen cells from mice primed with rat RBC specifically suppress the subsequent induction of autoantibody in normal mice. The response to non-cross-reacting determinants on rat RBC is not affected. The results presented here indicate that either T cells or a thymus have to be present in the recipient for suppression to be manifest. It may therefore be the case that suppression is in fact induced in the recipient by the primed T cells.     

Resistance to phagocytosis by group A streptococci: failure of deposited complement opsonins to interact with cellular receptors

The previous finding that phagocytosis-resistant M+ group A streptococci bear quantities of C3 which are sufficient for phagocytosis of their M- derivatives was investigated at two levels. It was first established that the C3 associated with M+ streptococci was not able to promote adherence to cells bearing the complement receptors CR1 and CR3 under conditions in which M- streptococci readily attached. The molecular form of C3 bound to M+ and M- streptococci was then defined by adding 125I-C3 to serum used for opsonization. C3 eluted from the bacteria by chaotropic and hydrolytic agents was analyzed by SDS-PAGE, and revealed that both cell types bound the opsonic forms of C3, C3b, and iC3b. Furthermore, approximately 80% of the C3b and iC3b associated with both cell types was covalently bound to a surface component, although most of the C3 bound to M+ streptococci was detergent-extractable, whereas greater than 50% of that bound to M- streptococci was not. These findings demonstrate that the M+ surface is interfering with the receptor binding of deposited C3b and iC3b, and that this contributes to resistance to phagocytosis by these organisms.     

Monoclonal anti-DNA IgM kappa in neuropathy binds to myelin and to a conformational epitope formed by phosphatidic acid and gangliosides

Anti-DNA antibodies that cross-react with phosphorylated epitopes of other cellular constituents may be involved in the pathogenesis of autoimmune disease. An IgM monoclonal antibody from a patient with chronic lymphocytic leukemia (CLL) and neuropathy bound to denatured DNA and immunostained myelin in peripheral nerve and spinal cord. The monoclonal IgM bound to ELISA microwells coated with a mixture of phosphatidic acid and gangliosides at serum dilutions of up to 1/100,000, but binding to phosphatidic acid alone was observed at dilutions of less than 1/100 only, and there was no binding to gangliosides alone. Incubation with micelles containing phosphatidic acid and gangliosides selectively absorbed the monoclonal IgM and inhibited its binding to denatured DNA and to myelin. These observations suggest that autoantibodies may bind to conformational epitopes formed by two separate molecules, and that autoantibodies that cross-react with phosphorylated epitopes in DNA and neural tissue could be involved in autoimmune neurologic diseases.