Purified pyrogenic exotoxin from Group A streptococcal filtrates (Streptococcus pyogenes, type 10, strain NY-5) has been characterized primarily as a protein complexed with hyaluronic acid. Amino acid composition and analysis revealed a typical acidic protein with an average molecular weight of 29,000. The purified exotoxin was free of streptolysins O and S, nicotinamide adenine dinucleotidases (NADases), deoxyribonucleases (DNases), mucopeptide, and endotoxins. The biological activity was destroyed when the exotoxin was heated at 65°C for 30 min or boiled for 2 min. The biological activities investigated were pyrogenicity in rabbits (minimal pyrogenic dose-3 hr, 0.07 µg/kg), lethality in rabbits (LD50, 3500 µg/kg), skin test dose in human skin (> 109 skin test doses, per mg toxin), cytotoxicity of rabbit spleen macrophage (Cytotoxic Index 0.5–10 µg/ml), enhancement of susceptibility to endotoxin shock (in rabbits > 100,000-fold), and antigenic analysis (A-type toxin). The exotoxin was immunogenic and it was possible, therefore, to immunize animals against the various toxic activities. The immunity was specific for the A-type toxin. The clinical implications of the highly significant enhancement effect of these exotoxins are discussed. It is suggested that clinical or subclinical infection with Group A streptococci could prepare the host for fatal shock from Gram-negative infections or the inadvertent injection of small amounts of Gram-negative bacterial endotoxins.

The mechanism of tolerance to the pyrogenic activity of Gram-negative bacterial endotoxins is most often attributed to a non-specific increase in the activity of the reticuloendothelial system (RES) (1). Recently, it was shown that Group A streptococcal exotoxins produced biphasic fever responses in rabbits (2). In contrast to the non-specific tolerance induced by endotoxins, three distinct toxins were identified based on their ability to induce specific pyrogenic tolerance; in addition, the pyrogenic activity was neutralized specifically with antiserum.These observations suggested that, in addition to the non-specific RES activity, specific immune mechanisms may contribute to pyrogenic tolerance to Gram-negative bacterial endotoxins. Because endotoxins from organisms of different species and families induce non-specific pyrogenic tolerance, it is assumed that specific immunological mechanisms are not involved. If, however, there exist unsuspected common or cross-reactive antigens contributing to the pyrogenic activity, the non-specific nature of the mechanism would be more apparent than real. Our approach involved, therefore, an attempt to demonstrate specificity by the use of cross-tolerance tests as applied to the streptococcal pyrogenic toxins (2). Purified endotoxins were selected for this purpose on the basis of suspected chemical differences.In addition, there is evidence to implicate the immunological state of the host in many of the biological activities of endotoxins. From birth, animals are continually exposed to Gram-negative bacterial endotoxins derived from organisms growing in the gastrointestinal tract. Of particular interest is the enhanced susceptibility of animals to endotoxins after colonization with Gramnegative bacteria (3); here there was evidence of specificity because the induced susceptibility was greater when the endotoxin was derived from the

No abstract

Well-characterized Salmonella mutants formerly used in biosynthetic studies of lipopolysaccharides were used to study the toxic portion of the complex endotoxin. Endotoxins prepared from wild types and their mutants were tested for their biological activities, including pyrogenicity, lethality, and immunogenicity. There was little difference either in the endotoxin yields or in the toxicities between endotoxins from the wild-type and 0-antigen deficient mutants. Endotoxin containing mostly lipid A and keto-deoxyoctonate (KDO) prepared from the mutant deficient in both 0-and R-antigens and the backbone sugar, heptose, was biologically active. Possibly because of the difference in solubility in water, the yield of endotoxin from the heptoseless mutant was about 10% of the wild type. There was complete reciprocal cross-immunity between all endotoxins tested. These observations suggest that the common toxic moiety is not present in the 0-and R-polysaccharides or the backbone sugar heptose, but rather is associated with the lipid portion of the molecule which includes mostly lipid A and KDO.

Pig endothelial cells are the first cells to interact with human immune components after organ xenotransplantation, which is a procedure currently considered to be the best treatment option for end-stage organ failure. It is, therefore, essential to study the mechanisms of molecular interaction between pig endothelial cells and human immune components, in order to overcome xenograft rejection. The aim of this study was to establish immortalized pig aortic endothelial cell lines, in order to facilitate future in vitro studies of human anti-pig immune responses. Endothelial cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the Minnesota miniature pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. The immortalized cell lines showed a relatively rapid doubling time (17.6h) and the endothelial cell phenotype, as indicated by the formation of typical cobblestone monolayers and by the constitutive expression of PECAM-1 and the von Willebrand factor. Flow cytometric analysis demonstrated the constitutive expression of SLA class I and CD86, whereas the expression of E-selectin and SLA class II was only induced after stimulation with human TNF-alpha and pig IFN-gamma, respectively. On the other hand, no CD80 expression was detected in the primary cells or cell lines in the presence or absence of either human TNF-alpha or pig IFN-gamma. A vigorous human T cell proliferation against these cell lines was observed in the mixed lymphocyte-endothelial cell culture. These results suggest that pig endothelial cells, immortalized by the introduction of SV40 T, retain their original characteristics, except for the acquired property of immortalization, and that they may be useful for future in vitro studies of xenogeneic human anti-pig immune responses.