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.     

Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus.

A hamster monoclonal antibody (MAb), designated 8.1.1, was raised against murine thymic stromal cell lines and was found to react with cell surface molecules expressed by a morphologically distinct population of epithelial cells of the murine thymus comprising the subcapsular environment, cells investing vascular structures throughout the thymus, and some of the cellular elements in the medulla. The epithelial nature of the labeled cells was confirmed with immunoelectron microscopy. Reactivity with MAb 8.1.1 was associated with thymic epithelial cells in contact with basal laminae. Ontological studies of thymic tissue demonstrated that the epitope recognized by this MAb was expressed before Day 14 of gestation, although the restricted subcapsular and medullar expression of 8.1.1 was not apparent until sometime after birth. MAb 8.1.1 also reacted with a number of extra-thymic tissues, including lamina propria of gut, glomeruli and tubules in the kidney, mesothelia covering a number of organs, and the dermis and epidermis of skin. Within the epidermis, reactivity of MAb 8.1.1 was largely restricted to basal epithelial cells. Immunochemical analysis of 8.1.1 reactivity with detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that detergent-soluble extracts of thymic stromal cell lines and thymus tissue indicated that the epitope recognized by this MAb was associated with a glycoprotein bearing terminal N-acetylglucosamine residues and possessing an Mr of approximately 36-38 KD under reducing or non-reducing conditions.     

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.     

Antibodies to an antigen of human thymus epithelial tissue common to the epidermis of the skin in malignant myasthenia]

It has been demonstrated by the indirect immunofluorecent technique that many of the sera of patients with myasthenia gravis react with the anticells of the human thymus epithelial tissue. Sorption of the sera with the suspension of the epidermis cells and the homogenates of the tissues of other human organs showed that the epithelial cell antigen with which the sera of patients with myasthenia reacted were epidermal heteroorganic thymus antigens, i.e. common for the thymus epithelium and skin epidermis. The presence of antibodies to the cells of the epithelial tissue of the thymus in the sera of patients suffering from myasthenia gravis permits to suppose the existence of an immunopathological process against the thymus tissue antigens (including the heteroorganic structures of its epithelium) in this disease.     

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.     

Structure of the group G streptococcal polysaccharide

The structure of the group-specific polysaccharide of group G Streptococcus was determined by means of methylation analysis and selective chemical degradations. The anomeric configurations and conformations of the sugar residues were studied by 1H- and 13C-n.m.r. spectroscopy. The tetrasaccharide repeating unit, ----3)-alpha-D-Galp-(1----2)-[alpha-L-Rhap-(1----3)-beta-D-GalpNAc - (1----4)]-alpha-L-Rhap-(1----, was determined.     

Production of hybridomas synthesizing monoclonal antibodies to streptococcal group A polysaccharide

Nine stable hybridomas synthetizing monoclonal antibodies to the antigenic determinants of polysaccharide A of group A streptococcus were obtained. Three monoclonal antibodies possessed precipitating properties. The formation of hybridomas was found to be influenced by the presence of immune splenocytes and the standard conditions of cell fusion. The highest yield of hybridomas was observed under the conditions ensuring the growth of cell in 80-100% of the wells. Rapid and specific screening was found to be an important stage in obtaining hybridomas.     

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.     

Suppression of cytotoxic cellular reactions by the production of antibodies to rhamnose determinants of streptococcal group A polysaccharide cross-reacting with antigens of the skin epithelium]

By the BALB/c mice after different periods of immunization with the streptococci group A, treated with pepsin, antibodies belonging to autoantibodies to the determinants (DT) of polysaccharide (A-PS), cross-reactive (CR) with the epithelial skin cells, were investigated. In one of the mice groups, in the autologous system, on the target cells--macrophages of lymph nodes, the suppression of cytotoxic (CT) reactions was obtained. The CR are bound with the delayed type hypersensitivity appearing after the sensibilization with BCG. The suppression effect correlate (z-0.95) with the presence in the sera antibodies to the rhamnose DT'S of A-PS, which cross-react with the cells of basal and superbasal layers of skin epithelium. Antibodies to the group specific of the A-PS, cross-react only with the basal skin layer and not produce the suppression of CT reactions. It is possible that they also prevent the suppression of CT reactions, bound with the CR antibodies to the rhamnose DT-S of A-PS. The obtained data corroborate the earlier supposition that the autoantibodies to the CR DT'S of A-PS reacting with the skin epithelial cells as a rule common the thymus epithelial cells. It is possible that different IRD'S can prevent or stimulate the development of autoimmune processes by the infections with the streptococci group A.     

A peptide, containing the universal antigenic determinant of tryptophanyl-tRNA-synthetase]

Among clostripain hydrolysate peptides of beef pancreas tryptophanyl-tRNA synthetase the peptide Ile-Ser-Phe-Pro-Ala-Ile-Asn-Gln-Phe-Ala-Ala-Pro-Ser-Gln-Phe-Ser-Ile-Arg was revealed which contains the continuous antigenic determinant for monoclonal antibody Am1. This antibody specifically cross-reacts with tryptophanyl-tRNA synthetases of procaryotes, eucaryotes and archebacteriae. The synthetic peptide with identical amino acid sequence plus N-terminal Arg residue (S-peptide), being immobilized on enzyme immunoassay (EIA) microtitration plate, also binds with Am1. Am1 affinity constant (M-1) measured by non-competitive EIA was (3.0 +/- 0.3).10(7) for S peptide and (1.4 +/- 0.3).10(9) for the native enzyme. The sequence of immunoreactive peptide adopts with high probability the secondary structure including beta-turn(s) and antiparallel beta-sheet composed of inverted repeats. At the same time, the analysis of circular dichroism spectrum (in the far UV) of the peptide dissolved in water comes closest to 16% beta-turn and only 8% beta-sheet. The binding of Am1 with peptide was not observed in aqueous solution.     

Streptolysin S contributes to group A streptococcal translocation across an epithelial barrier

Group A Streptococcus pyogenes (GAS) is a human pathogen that causes local suppurative infections and severe invasive diseases. Systemic dissemination of GAS is initiated by bacterial penetration of the epithelial barrier of the pharynx or damaged skin. To gain insight into the mechanism by which GAS penetrates the epithelial barrier, we sought to identify both bacterial and host factors involved in the process. Screening of a transposon mutant library of a clinical GAS isolate recovered from an invasive episode allowed identification of streptolysin S (SLS) as a novel factor that facilitates the translocation of GAS. Of note, the wild type strain efficiently translocated across the epithelial monolayer, accompanied by a decrease in transepithelial electrical resistance and cleavage of transmembrane junctional proteins, including occludin and E-cadherin. Loss of integrity of intercellular junctions was inhibited after infection with a deletion mutant of the sagA gene encoding SLS, as compared with those infected with the wild type strain. Interestingly, following GAS infection, calpain was recruited to the plasma membrane along with E-cadherin. Moreover, bacterial translocation and destabilization of the junctions were partially inhibited by a pharmacological calpain inhibitor or genetic interference with calpain. Our data indicate a potential function of SLS that facilitates GAS invasion into deeper tissues via degradation of epithelial intercellular junctions in concert with the host cysteine protease calpain.     

Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide

Immunization of rabbits with group B type III streptococcus organisms induces two distinct populations of antibodies with a specificity for determinants on the native capsular polysaccharide antigen of these organisms. Some of the structural and conformational features of the two determinants responsible for the formation of these antibodies were elucidated by (13)C NMR and serological studies on the native type III polysaccharide and some of its structurally modified analogues. The specificity of the determinant corresponding to the major population of antibodies is dependent of the presence of sialic acid residues on the native type III antigen, and although these residues are not an integral part of the determinant, they exert conformational control over it. The carboxylate groups of the sialic acid residues are an important factor in this control mechanism which could possibly involve intramolecular hydrogen bonding. The terminal sialic acid residues control the orientation of the penultimate beta-d-galactopyranose residues with respect to the backbone of the native antigen. The orientation of these residues is critical to the determinant because the determinant is probably small and is located precisely at the junction of the same beta-d-galactopyranose residues with the backbone of the native type III antigen. The determinant corresponding to the other population of antibodies is not sialic acid dependent. This determinant is located on the backbone of the native antigen in the vicinity of the other determinant but on the opposite side to the oligosaccharide branches. In this position, its conformation is unaffected even by the removal of the oligosaccharide branches from the native antigen.     

Evidence suggesting the presence of common antigenic determinant between dynein and tubulin.

The present experiments showed that the guinea pig antiserum prepared against the main polypeptides of 14 S dynein from Tetrahymena cilia reacted with sea urchin sperm flagellar dynein and with bovine brain high molecular weight protein to give rise to a precipitin line confluent with that formed between the antiserum and Tetrahymena dynein. Furthermore, it was found that this antiserum also reacted with tubulins from Tetrahymena cilia, sea urchin sperm flagella and bovine brain to give rise to the confluent precipitin line. Among muscle proteins, only actin preparation from rabbit skeletal muscle reacted with the anti-Tetrahymena dynein serum, whereas neither rabbit skeletal muscle myosin, chicken skeletal muscle tropomyosin nor chicken skeletal muscle troponin reacted with the antiserum. These results suggest that dynein and tubulin and probably actin share an antigenic determinant regardless of different protein species and of different animal species. The common antigenic determinant was detected only when the proteins denatured with urea/sodium dodecyl sulfate/beta-mercaptoethanol/N-ethylmaleimide were used, but it was not detected at all when the native proteins were used. This implies that a certain common antigenic determinant which is involved in the precipitin line formation exists in the primary structures of dyneins and tubulins and probably actin, and is hidden inside the tertiary structures of the native protein molecules.     

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.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties

The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.