Previous studies suggested that circulating tumor necrosis factor alpha (TNF-a) may have a pathophysiologic role in experimental neonatal sepsis induced by group B streptococci (GBS). This study was undertaken to investigate the ability of the type III and group-specific polysaccharides of GBS to induce TNF-a production and TNF-a-dependent lethality in neonatal rats. The cytokine was detected in plasma samples by the L929 cytotoxicity assay. Intracardiac injections of either polysaccharide induced dose-dependent, transient elevations in plasma TNF-a levels that returned to baseline values after 5 h. The group-specific antigen induced significantly higher mean peak TNF-a levels than the type III antigen (125 ± 47 versus 44 ± 15 U/ml with 70 mg/kg of body weight). Glycogen (70 mg/kg), used as a negative control, did not induce TNF-a. The lipopolysaccharide-neutralizing agent polymyxin B did not decrease TNF-a levels induced by either polysaccharide, ruling out contamination with endotoxin as a possible cause of TNF-ot induction. Fifty percent lethal doses of the type III and group-specific antigens given as intracardiac injections were 105 and 16 mg/kg, respectively. Salmonella endotoxin, used as a positive control, had a 50%o lethal dose of 0.1 mg/kg. The lethal activities of GBS polysaccharides, as well as endotoxin, were completely prevented by pretreatment of neonatal rats with the respective specific antibodies or anti-murine TNF-a serum. To assess the relative importance of the type-specific substance in TNF-aL induction by whole bacteria, two unrelated GBS transposon mutants devoid of only the type-specific capsular polysaccharide (COH1-13 and COH31-15) were employed. Each of the heat-killed unencapsulated mutants was able to produce plasma TNF-a level elevations or TNF-ea-dependent lethality but was significantly less efficient in these activities than the corresponding encapsulated wild-type strain. These data suggest that the presence of type-specific material on GBS is not necessary for the stimulation of TNF-a production. Type III capsular polysaccharide, however, can significantly increase the ability of GBS to induce TNF-a. Further studies will be needed to assess the importance of TNF-a induction by the group-and type-specific antigens in the pathophysiology of GBS disease.

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-␣), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration-and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 g/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-␣ was released before the other cytokines. TNF-␣, however, did not appear to directly induce IL-1␤, as shown by blockade experiments with anti-TNF-␣ antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-␣. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-␣, IL-1␤, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.

We describe the use of lectins as specific stabilizing agents for the polysaccharide capsular components of two Gram-positive bacteria, Streptococcus agalactiae and Streptococcus bovis. Treatment of bacterial suspensions with wheatgerm agglutinin and concanavalin A allowed better morphological preservation as well as immunoelectron microscopic localization of a capsular component (lipoteichoic acid) by employing specific antibodies and the protein A-gold technique. Data obtained indicate that lectins are useful agents in preserving highly water-soluble capsular components during the electron microscopy procedures for both unembedded and embedded samples.

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Infection by group A streptococci is often associated with highly invasive diseases resulting in sepsis and shock. Recently, this species has renewed interest in the mechanism by which some strains are better able to invade mucosal epithelia and to penetrate into the bloodstream than are others. In this work we have evaluated the invasive ability of eight group A Streptococcus pyogenes strains isolated either from patients with severe invasive diseases, or with pharyngitis, or from healthy carrier. Five out of the eight strains studied were efficiently internalized within HeLa cells and, of these, four produced the M6 protein. A recombinant S.gordonii strain constitutively expressing the M6 protein failed to invade HeLa cell monolayers, suggesting that the expression of the M6 protein is not sufficient to allow the non-invasive S.gordonii to be internalized within Hela cells. As fibronectin-binding proteins have been implicated as primary adhesins in host-parasite interactions, we assayed the adhesiveness and the invasiveness of five invasive GAS strains in competition experiments where HeLa cells were infected with bacteria in the presence of purified fibronectin. The results obtained indicated that fibronectin moderately inhibits bacterial adhesion, while it does not affect internalization. These results indicate that other factors, together with fibronectin-binding proteins, participate in the adhesion of streptococci, and that fibronectin-mediated adhesion does not seem to be important in the internalization process of streptococci within HeLa cells.