Increased titers of antibodies against streptococcal M12 and M19 proteins in patients with Tourette's syndrome.

It has been suggested that a post-streptococcal autoimmune process may be involved in the pathogenesis of a subgroup of children with tics and obsessive-compulsive symptoms (PANDAS). Elevated antibody titers against streptococcal antigens have also been described in adult patients suffering from Tourette's syndrome (TS). In order to characterise further streptococcal antigens, we focussed on M proteins. M proteins are a major virulence factor of group A streptococci and known to evoke an immunologic cross-reaction with diverse epitopes of human tissue including brain tissue. Therefore, antibodies against M proteins may play a role in the pathophysiology of at least a subgroup of TS patients. Antibodies against M proteins were studied in 25 adult patients suffering from TS and 25 healthy controls after careful medical examination. The antibody titers against the peptides M1, M4, M6, M12 and M19 were estimated by ELISA. Our results show increased titers of antibodies against the streptococcal M12 and M19 proteins in TS patients as compared with controls, while antibody titers against M1, M4 and M6 did not differ between the TS and control groups. Elevated serum titers of antibodies against M12 and M19 proteins support the view that a streptococcus-induced autoimmune process may be involved in TS. The finding of a possible autoimmune origin of TS has implications for both pathophysiology and future therapeutic strategies.

Estimation of type-specific anti-streptococcal M-protein antibodies with biotinylated peptides in human sera.

The design of a novel enzyme-linked immunosorbent assay for the estimation of antibodies directed against the N-terminus of the M-protein of Streptococcus pyogenes is described. The ELISA employs biotinylated peptide antigens of the types 1, 4, 12 and 19 immobilized by (strept-)avidin on the surface of polystyrene microtiter wells. In rabbit hyperimmune sera and in human serum samples, antibodies against the corresponding serotype could be detected with high sensitivity and specificity.

Antibody responses of splenectomized patients with non-Hodgkin's lymphoma to immunization with polyvalent pneumococcal vaccines.

The serum antibody responses of splenectomized patients with non-Hodgkin's lymphoma (NHL) who had been immunized with a polyvalent pneumococcal vaccine (Pneumovax 23) were evaluated by an enzyme-linked immunosorbent assay with the 23-valent pneumococcal vaccine as the antigen. A response to immunization, defined as a twofold-or-higher rise of the prevaccination titer of antibodies against Streptococcus pneumoniae polysaccharide, was elicited in 5 of 11 patients with NHL. No significant difference in the level of antibodies against S. pneumoniae polysaccharide between lymphoma patients and patients who had undergone splenectomy for other reasons was detected (P = 0.83 and 0.87 before and after vaccination, respectively). NHL patients who did not respond to the first immunization received a booster dose of the polysaccharide vaccine. This injection did not increase the pneumococcal-antibody titer significantly (P = 0.7). We conclude that vaccination with pneumococcal polysaccharides in splenectomized patients with NHL elicits an adequate antibody response in 45.4% of the cases and should therefore be administered. Revaccination of the nonresponders does not further increase the pneumococcal-antibody levels.

Pneumococcal vaccine in children and young adults with chronic renal disease.

BACKGROUND: Pneumococcal vaccination has been recommended for immunocompromised children over 2 years including patients with chronic renal disease. However, the effect of vaccination and revaccination is variable and the indication for immunization is a subject of controversy. METHODS: Forty children and young adults with chronic renal diseases (including the idiopathic nephrotic syndrome, chronic renal failure, patients undergoing dialysis and after transplantation) were vaccinated with a 23-valent pneumococcal vaccine. The efficacy of the vaccine was evaluated by measuring antibody titres before and 4 weeks, 6 months, and 12 months after vaccination. Twenty-two patients were submitted to a revaccination 1 year after the first vaccination. RESULTS: A sufficient immune response, defined as an at least fourfold increase of postvaccinal antibody titres and an antibody titre > 200, was observed in 83% of the patients 4 weeks after vaccination, but only in 68% after 6 months, and in 48% after 1 year. Revaccination produced a significant immune response in 11/22 patients (50%) followed by a rapid decline of antibody levels within 6 months. Both vaccinations were well tolerated. CONCLUSIONS: The currently available vaccine is without major side-effects and effective in producing a significant immune response. Antibody levels should be monitored in vaccinated patients with chronic renal diseases considering the rapid decline as early as 6 months after vaccination. Evaluation of the efficacy of revaccination in these patients requires further investigations.

Serum antibody responses to vaccination with 23-valent pneumococcal vaccine in splenectomized patients.

Sixty-three patients that have been splenectomized for various disorders were vaccinated with Pneumovax 23, the currently available pneumococcal vaccine. Before and one month after splenectomy, IgG antibodies against 8 pneumococcal polysaccharide antigens (types 3, 4, 6B, 7F, 10A, 14, 19F, and 20) were determined by a highly reproducible and type-specific enzyme-linked immunosorbent assay (ELISA). In order to increase the specificity of the assay, this method involved the use of a capture antibody (F(ab')2-fragments of a type-specific rabbit antipneumococcal hyperimmune serum) to bind the pneumococcal antigens to the plastic surface of microtiter plates and the absorption of these sera with the cross-reacting C polysaccharide. Rates of patients showing a two-fold antibody increase were dependent on pneumococcal type, ranging from 12.7% to 33.3%. Antibody responses of single patients were not uniform for all pneumococcal serotypes investigated. Only one patient responded to all of the eight antigens tested. In spite of relatively low response rates, splenectomized patients should be routinely vaccinated with the pneumococcal vaccine, especially when the low rate of adverse reactions is taken into consideration. It is emphasised that the results of the present study and those reported in the literature have to be compared and interpreted with caution, because the available data on the antibody response to pneumococcal vaccination are based on assays that differ substantially in methodology.

Recurrent systemic pneumococcal infection in an immunocompromised patient.

A case is reported of a splenectomized patient who experienced four episodes of systemic pneumococcal infection after receiving appropriate therapy in every instance. Serogrouping showed at least two different strains to be responsible for these infections. Immunological investigations showed a reduction in immunoglobulins, a decrease in in vitro immunoglobulin synthesis and a disorder of immunoregulatory T-cells, but immunological changes could not be readily explained by splenectomy alone.

[Infections by Streptococcus pyogenes: new aspects of diagnosis, epidemiology, clinical practice, and therapy]. / Infektionen durch Streptococcus pyogenes: Neuere Aspekte zur Diagnostik, Epidemiologie, Klinik und Therapie.

Essential procedures for the cultural and serological laboratory diagnosis of Streptococcus pyogenes are described. A reliable and rapid species identification is achieved by serologic or biochemical tests. Direct antigen detection tests from throat swabs have broadened the diagnostic possibilities; however, they are often not satisfactory in terms of sensitivity. Typing of strains (either by classic serologic techniques or by molecular methods) is of high value for the study of clinical and therapeutic questions. In recent years, the epidemiology of the prevalent group A streptococcal strains has changed in Europe as well as in the U.S.: an increase of those M protein serotypes that are associated with higher virulence is being recognized. There is good evidence that the unexpected resurgence of rheumatic fever in the U.S. and the emerging number of life-threatening invasive and toxic manifestations of disease (including the toxic shock syndrome) in the U.S. and in Europe are related to the changing epidemiology. The existence of strains with rheumatogenic and nephritogenic potency has been established, but genetic host factors are a prerequisite for the sequelae as well. Although, based on experimental data, there are a lot of hypotheses concerning the nonsuppurative sequelae and the toxic shock syndrome, the exact pathogenesis of these diseases remains to be clarified. A thorough knowledge of the various clinical manifestations of group A streptococcal infections is necessary in order to apply the therapeutic and preventive measures in a rational and sophisticated manner. For most infections penicillins are considered the drug of choice, although no final conclusions regarding optimal dosages or the reasons for treatment failures can be made.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of group A type 1 streptococcal M protein gene by a non-radioactive oligonucleotide detection method.

An oligonucleotide probe of 30 nucleotides length has been constructed, spanning the codons of amino acids 2 to 11 of the mature M1 protein of group A streptococci (Streptococcus pyogenes). It was labeled with digoxigenin-dUTP and visualized after hybridization with an anti-digoxigenin-antibody/alkaline phosphatase conjugate. It definitely detected the emm1 gene in dot-blotted 20-micrograms amounts of total nucleoid acid extracts. When tested with 27 type M1 group A streptococci of several epidemiologically unrelated outbreaks and 24 non-M1 strains, its sensitivity and specificity of detection reached 100% even at a hybridization temperature 35 degrees C below the calculated Tm. A detailed protocol for the construction and use of this oligonucleotide probe is given.

Type 1 and 3 M-proteins of Streptococcus pyogenes: peptic extraction and fibrinogen binding properties.

The pepsin extraction of group A type 1 streptococci for the isolation of M protein fragments was studied at different pH values and at different time intervals. The extracts were compared by SDS PAGE and fused rocket immunoelectrophoresis. Type 1 M protein fragments were prepared in preparative scale by pepsin extraction of type 1 streptococci at pH 5.5 for 60 min. The fragments were separated by affinity chromatography on immobilized fibrinogen and finally purified for sequence studies by gel chromatography. Pepsin extraction of group A type 3 streptococci was also studied at different pH values. In contrast to type 1, the SDS PAGE pattern changed drastically in dependence on the pH. Affinity chromatography on immobilized fibrinogen is also effective in the separation of the pH 5.5 type 3 streptococcal pepsin extract.

A screening of streptococci freshly isolated from human and animal sources for binding of human IgG.

Human isolates of groups A, C and G streptococci as well as animal isolates of group C were investigated with respect to their binding capacity for human IgG by using the direct fluorescence technique and the Mancini test. From each serological group of human isolates, more than 100 strains were tested. The results were evaluated statistically with respect to serological group, type and source of isolates. Between human isolates of groups A, C and G, no statistical differences concerning IgG binding were found. However, group C streptococci isolated from pigs showed a significantly higher number of strains with high IgG binding than the human isolates of groups A, C and G. Group A streptococci isolated from suppurating lesions showed an increased IgG uptake when compared with isolates from scarlet fever patients or patients with throat infections. However, strong IgG binding by group A streptococci seems not to be restricted to certain types. By using selected streptococcal strains, it was found that IgG absorption from a solution of purified IgG was much higher than from IgG solutions containing albumin or from diluted serum. The results are discussed in connection with the competition of different plasma proteins for binding sites on the streptococcal cell surface and with a possible influence of IgG receptors on the virulence of streptococci.

Binding of fibrinogen fragment D to group A streptococci causes strain dependent decrease in cell surface hydrophobicity as measured by the salt aggregation test (SAT) and cell clumping in polyethylene glycol.

Group A streptococcal reference strains of various M-types as well as three clinical isolates, and clinical isolates of group B, C and G streptococci were compared with respect to their agglutination by fibrinogen, specific binding of monovalent fibrinogen fragment D, and their hydrophobicity measured by salt aggregation in ammonium sulfate dilutions (SAT). Fibrinogen fragment D binding to M positive group A streptococci and clinical isolates of group A reduced salt aggregation of these strains from 0.06 molar to 2 molar in the ammonium sulfate dilution series. Group A, M protein negative and group B, C and G control strains used aggregated only at high ammonium sulfate concentration (2 molar) or not at all. Treatment of these strains with fibrinogen fragments did not influence their weak salt aggregation. By elution of specific retained fragment D from the type 1 M positive strain 40/58II by treatment of cells with 0.1 molar citric acid, 6 molar urea, buffer, pH 3.0, surface hydrophobicity could partially be restored (SAT titer 0.5 molar). Treatment of streptococcal cells with fibronectin or albumin at much higher protein concentrations had a moderate effect on surface hydrophobicity. These results indicate that binding of fibrinogen fragment D to the receptor can block a hydrophobic surface domain of the receptor or induce a sterical hindrance affecting surface exposure of associated surface molecules. A fibrinogen receptor-also responsible for binding of fibrinogen fragment D-is in M positive group A streptococci covalently linked to M protein. Decrease of salt aggregation tendency after mild pepsin treatment to release M protein supported these findings.

Type 1 M protein of Streptococcus pyogenes. N-terminal sequence and peptic fragments.

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of Mr 20270 as the main product which covers the N-terminal part of the M protein. The amino acid sequence was determined of the N-terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N-terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N-terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.

Interaction of group A type 1 streptococcal M protein with fibrinogen.

Adsorption chromatography of streptococcal extracts on immobilized fibrinogen allows isolation of components that are linked to the corresponding receptors. In this study it is shown by an indirect bactericidal test that fibrinogen binds the M proteins of the streptococcal strains used. Phage-associated lysin extracts of group A type 1 streptococci precipitated with fibrinogen in a double-diffusion test. Fibrinogen reactive components of other streptococcal types inhibited this precipitation reaction. This suggests that the fibrinogen receptors in different types of group A streptococci have identical activity. The interaction between M protein and fibrinogen does not interfere with the interaction between M protein and the corresponding type specific antibodies. The streptococcal antigen components isolated by immobilized fibrinogen showed mitogenic activity in a lymphocyte transformation test.

Tissue cages for study of experimental streptococcal infection in rabbits. II. Humoral and cell-mediated immune response to erythrogenic toxins.

Infection of rabbits with erythrogenic toxin producing streptococcal strains caused a marked increase of humoral antibodies, which was detected by immunoprecipitation and ELISA. An antibody response directed towards the erythrogenic toxin type A was demonstrated by fused rocket immunoelectrophoresis. All toxinogenic reference strains produced ET type A under in vivo conditions despite that this toxin was not always demonstrated under in vitro conditions. The infection resulted in an increase of mitogenic response of peripheral lymphocytes to the initial nonspecific mitogenic erythrogenic toxins, whereas the Con A stimulation was depressed starting 14 days after infection and lasting during a period of 90 days. Since a normal antibody response was evoked, it seems likely that the T helper cell function was not affected.

On the interaction between beta 2-microglobulin and group A streptococci.

beta 2-microglobulin (beta 2m) was found to interact with many group A streptococcal strains. The interaction appeared to require multipoint attachment, since monomeric beta 2m in solution showed no binding, whereas both beta 2m monomers bound to liposomes, and beta 2m in aggregates showed affinity for the bacteria. Aggregated HLA antigens (-A, -B and -C) and aggregated beta 2m exhibited the same binding patterns when tested in binding experiments with various group A streptococcal strains. Furthermore, beta 2m aggregates in excess completely blocked the binding of aggregated HLA antigens, thereby demonstrating that beta 2m is able to interact with streptococcal surface structures also when it is part of the HLA antigen complex. M protein-positive group A streptococcal strains bound significantly more beta 2m than M protein-negative variants of these strains. Purified M 12 protein partly inhibited the binding of radiolabelled beta 2m aggregates to whole streptococci, and in gel filtration and affinity chromatography experiments, the M 12 protein interacted with beta 2m. These various data suggest that the interaction between beta 2m and group A streptococci could be mediated by M protein. Lipoteichoic acid (LTA) is a constituent of the streptococcal cell wall that has been reported to form complexes with M protein at the bacterial cell surface. However, LTA did not influence the interaction between beta 2m and streptococci, suggesting that the binding of beta 2m to streptococcal M protein represents a pure protein-protein interaction. In vivo such an interaction could be established between infecting streptococci and host cells. Among 45 strains of different M types large differences in beta 2m binding were recorded, whereas among 60 strains of the classical nephritogenic M types 12 and 49, all were highly beta 2m-reactive, which points towards a role for beta 2m in streptococcal pathogenicity.

Comparative studies on surface hydrophobicity of streptococcal strains of groups A, B, C, D and G.

Cell surface hydrophobicity of group A, B, C, D and G streptococcal strains has been studied and compared in a new test based on the fact that the degree of bacterial aggregation in ammonium sulphate depends on amphiphilic surface antigens. M-positive group A strains showing good growth in normal human blood aggregated in the standard salt aggregation test at very low concentrations of ammonium sulphate, while M-negative strains, which were killed in normal human blood, usually aggregated at high salt concentrations. Agents such as 2 M-KSCN, 2 M-guanidine. HC1 or 2 M-urea decreased the aggregation of the M-positive strains in the salt aggregation test while non ionic detergents such as Tween 20 (1%, w/v) and ethylene glycol (2 M) did not affect cell aggregation. Binding of fibrinogen and albumin resulted in a decrease of surface hydrophobicity of the group A M-positive strains. Group B strains possess a hydrophilic surface character and did not aggregate, while group C and G strains behaved in the salt aggregation test like M-negative strains of group A streptococci. Group D strains did not aggregate even at high ammonium salt concentrations. The results are discussed in relation to the influence of lipoteichoic acid and other surface antigens on strains of the various groups, and it is suggested that M protein and possibly also other surface proteins contribute to the high surface hydrophobicity of group A strains.

Quantitative differences in specific binding of fibrinogen fragment D by M-positive and M-negative group-A streptococci.

Selected M-positive and M-negative group-A streptococcal strains were investigated with respect to their selective absorption of plasmin fibrinogen degradation products (FDP) in a simple batch technique. After incubation of killed streptococci with the FDP mixture, the centrifuged supernatants were investigated by SDS-electrophoresis and the binding capacity of the strains was calculated by evaluation of the scanning curves of stained gels. It was found that there is a specific uptake of the C-terminal fragment D by both M-positive and M-negative strains. Although the M-positive strains bound more fragment D (30%-80%) than did the M-negative strains (10%-15%), it could be clearly shown that the loss of M-protein was not necessarily linked with a total disappearance of fibrinogen binding activity. Fragment D blocked the agglutination of streptococci by fibrinogen. Washing the streptococci preincubated with FDP with a 0.1 M citric acid, 6 M urea buffer, pH 3.0, restored agglutination. Treatment of FDP-incubated bacteria with this buffer was found to be a means of recovering pure fragment D from streptococcal cells. It is suggested that M-positive and M-negative streptococci have qualitatively similar binding sites. These receptors might be reduced in the M-negative streptococci. Human serum albumin and Tween 20 did not influence the interaction between streptococcus and fibrinogen.

[Investigations about the immunobiology of m-proteins of Streptococcus pyogenes. IV. Cell-mediated immunity of rhesus monkeys (Macaca mulatta) against streptococcal antigens (author's transl)]. / Untersuchungen zur Immunbiologie von M-Proteinen des Streptococcus pyogenes IV. Mitteilung: Zellvermittelte Immunität mit Streptokokkenangigenen ei Rhesusaffen (Macaca mulatta).

Cell-mediated immunity was tested in monkeys (Macaca mulatta) immunized with highly purified M-proteins by means of the lymphocyte transformation reaction. Lymphocytes from immunized and nonimmunized monkeys were stimulated with PAL-M-Proteins. IC-M-proteins were completely free of mitogenicity and no lymphocyte stimulation was to be found after immunization with 1.6 mg. Erythrogenic toxins of strain NY5 were able to stimulate lymphocytes of monkeys unspecifically.