Streptococcus pyogenes T1 was previously found to produce an acidic mitogenic exotoxin, designated A, antigenically distinct from erythrogenic toxin type A (ETA) of strains T1 and NY5. Following chemical analysis and biological characterization, we have renamed this toxin streptococcal mitogenic exotoxin Z (SMEZ). Physicochemical separation of SMEZ from ETA was successfully performed on a hydrophobic chromatograph. The isoelectric point was pH 5.3, and the molecular size was estimated to be 28 kDa. These values were similar to those of ETA, but the amino acid composition and the NH 2-terminal sequence of SMEZ were distinct from those of any mitogenic exotoxins hitherto described. Its mitogenic activity was found to be more potent than that of ETA in rabbit lymphocyte cultures. A specific antiserum raised against SMEZ did not cross-react with ETA, ETB, or ETC in the neutralization tests of mitogenic and erythrogenic activities. Its superantigenic nature was evident from the reverse transcriptase PCR findings of the T-cell receptor V profiles of rabbit lymphocytes stimulated in vitro. The V 8 subfamily was unique to SMEZ, while the V 2 and 6 subfamilies were found to be common among lymphocytes stimulated with ETA, ETB, ETC, or SMEZ. The results from this study provide an additional example of the diversity that exists among mitogenic or superantigenic exotoxins of streptococcal origin.
SUMMARYThe aim of this study was to further assess the role of pooled human immunoglobulin (PHIG) on cytokine production from PBMC stimulated with a bacterial superantigen. Human PBMC were cultured with Streptococcus pyrogenic exotoxin A (SPE-A) with or without PHIG and several proinflammatory cytokine levels of culture supernatants were measured. Serum cytokine levels of KD patients before and after PHIG therapy were also examined. PHIG greatly reduced the production of IL-12, interferongamma (IFN-g) and other cytokines from SPE-A-stimulated PBMC, while exogenous IL-12, but neither IL-1 nor tumour necrosis factor-alpha (TNF-a), restored IFN-g production inhibited by PHIG. Although PHIG partially adsorbed SPE-A, its inhibitory effect on cytokine production was not played by anti-SPE-A antibody. Although purified CD4þ T cells cultured with human HLA-DR-transfected mouse L cells and SPE-A could not effectively produce IFN-g, they produced large amounts of IFN-g if exogenous IL-12 was introduced. KD patients in the acute phase had higher levels of serum IFN-g than did controls and patients with bacterial infection. Although IL-12 levels of children with or without KD were not significantly different, IL-12 levels of children were much higher than those of adults. However, serum levels of IL-12 of KD patients were transiently but significantly decreased by PHIG therapy and IFN-g amounts subsequently reverted to basal levels thereafter. These findings indicate that PHIG inhibits IL-12 production of SPE-A-activated monocytes and thereby decreases IFN-g synthesis by T cells and suggest that inhibition of IL-12 and IFN-g production is an important part of the mechanisms underlying PHIG therapy on KD.
Streptococcal erythrogenic toxin type A (ET-A) was purified from culture filtrate of Streptococcus pyogenes strain NY-5 grown in a chemically defined synthetic medium NCTC-135. We succeeded in simplifying the purification procedure, and obtained a highly purified preparation of ET-A. The purification procedure was the combination of ultrafiltration with Amicon PM-10 and YM-10 membranes, chromatofocusing with PBE-94 exchanger (pH 4.0-6.0), and gel filtration through Sephacryl S-200. The purified toxin protein showed a single band with Mr 28,000 on SDS-PAGE and had pi 5.2 on agarose IEF. HPLC chromatography pattern of the toxin revealed one symmetric peak. The result of amino acid analysis of the toxin was in accordance with that of Gerlach et al and with Weeks and Ferretti who reported the nucleotide sequence of the spe A gene. Biological activities of the purified toxin were remarkably potent. The mitogenic activity for rabbit lymphocytes and one skin test dose in rabbit were found at the lower dose of 10 pg and 1 ng of the toxin, respectively.
ABSTRACT. W e describe a new method to measure human serum antibody against streptococcal erythrogenic toxins that uses inhibition of lymphocyte mitogenicity of the toxins a s the indicator. Sera from 53% of 53 Kawasaki disease patients contained specific inhibitory activity against A toxin, whereas only 15% had serum inhibitory activity against B toxin. The specific anti-A toxin serum inhibitor was found in 10% of 118 age-matched control patients suffering from various infections and allergic diseases (p = 0.001, compared to Kawasaki disease patients).
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