Release of fibronectin-lipoteichoic acid complexes from group A streptococci with penicillin.

Fibronectin binds to Streptococcus pyogenes, and this binding is inhibited by lipoteichoic acid (LTA). Previous studies have shown that LTA can be released from S. pyogenes by treatment with penicillin. Penicillin released LTA from both S. pyogenes and Staphylococcus aureus; however, the binding of fibronectin correlated with the amount of LTA released only in the case of S. pyogenes. Contrarily, clindamycin decreased the ability of S. aureus to bind fibronectin without affecting the binding of fibronectin to S. pyogenes. Further studies indicated that LTA does not inhibit the binding of fibronectin to S. aureus. Fibronectin bound to S. pyogenes could be released from the cell surface by penicillin. Immunological analysis of the released fibronectin indicated that LTA was the only surface component which could be detected as a soluble complex with the released fibronectin. These studies suggest that LTA plays a central role in the binding of fibronectin to S. pyogenes and is not involved in the binding of fibronectin to S. aureus.

Cellular immune responses in mice challenged with an amyocarditic variant of Coxsackievirus B3.

An amyocarditic variant of a temperature-sensitive (ts) mutant derived from the parent myocarditic variant Coxsackievirus B3 (CVB3m) was studied in a murine model of CVB3m-induced myocarditis to assess virus-induced antigens and their possible role in the disease process. Amyocarditic variant ts5R induced a heart tissue antigen(s), extractable by hypertonic KC1, which inhibited migration of peritoneal exudate cells from CVB3-inoculated myocarditic mice in an agarose droplet cell-migration-inhibition assay. The ts5R variant was amyocarditic at inoculum doses of 10(3) to 10(8) plaque-forming units per mouse, but in cyclophosphamide-immunosuppressed mice, ts5R induced myocarditis. Viable ts5R served as a vaccine and protected mice against CVB3m-induced myocarditis. Murine neonatal skin fibroblasts (MNSF) infected with either virus served as in vitro targets and were lysed by splenic cytotoxic T lymphocytes from mice inoculated with either virus variant. ts5R and CVB3m replicated to similar titers in murine neonatal skin fibroblasts (MNSF) at 24 hr postinoculation (pi), but differences in titers were found by 72 hr pi. Levels of natural killer cell activities in spleens of ts5R-inoculated mice were slightly lower than in spleens of CVB3m-inoculated mice at 7 days pi. The data suggest that viral induction of new antigens on target cells and viral induction of specific cytotoxic T lymphocytes that recognize these antigenic changes do not always result in induction of myocarditis.

Kinetic and chemical analyses of the biologic significance of lipoteichoic acids in mediating adherence of serotype III group B streptococci.

The mechanism(s) involved in the binding of lipoteichoic acid (LTA), isolated from virulent, asymptomatic, or avirulent serotype III strains of group B streptococci, to human embryonic epithelial cells (HEC), human fetal epithelial cells (HFC), and human adult buccal epithelial cells was investigated. It was determined that the binding of purified [3H]LTA to human adult buccal epithelial cells differed from the binding to HEC and HFC. LTA from all group B streptococcus strains bound to human adult buccal epithelial cells in a similar manner and was enhanced by the lipid portion of the polymer; in contrast, [3H]LTA binding to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate backbone of LTA. Binding avidity of the LTAs to HEC and HFC varied depending on the bacterial strain. Polymers from asymptomatic and avirulent strains were easily dissociated from cell surfaces with unlabeled virulent LTA through competitive interactions; however, 10-fold greater levels of the same material were required to displace virulent [3H]LTA from HEC and HFC surfaces. These observed differences in binding avidity were shown to be due to longer LTA chains (30 to 35 glycerolphosphate units) in virulent strains when compared with LTA chains (10 to 12 glycerolphosphate units) of asymptomatic and avirulent strains. Thus, LTA appears to enhance the ability of virulent group B streptococci to bind to HEC and HFC with stronger avidity by virtue of the increased length of the cell-associated polymers synthesized by these strains. Mild enzymatic treatment of HEC and HFC with trypsin or periodate abolished LTA binding, which suggests the presence of a certain glycoprotein receptor(s) for LTA which does not appear to be present on human adult buccal epithelial cells. These data may therefore partially explain the increased susceptibility of newborn infants to group B streptococcal infections.

Role of cellular lipoteichoic acids in mediating adherence of serotype III strains of group B streptococci to human embryonic, fetal, and adult epithelial cells.

Lipoteichoic acids (LTA) of serotype III strains of group B streptococci (GBS) were shown to mediate adherence of these organisms to human embryonic (HEC), fetal (HFC), and adult buccal (HBEC) epithelial cells. The binding of GBS was temperature dependent, and maximum attachment occurred at 37 degrees C. HEC, HFC, and HBEC preincubated with purified LTA significantly inhibited attachment of GBS, whereas the group B and type III antigens had no effect. Under phosphate-limiting conditions in which cell-associated LTA could not be detected in these organisms, bacterial adherence did not take place. GBS (virulent) that were isolated from infected infants and previously shown to have significantly higher quantities of cell-associated LTA in comparison to GBS strains from asymptomatically colonized infants adhered with greater binding avidity to HEC and HFC and in greater numbers than to HBEC. It was determined that the mechanism of LTA-mediated adherence of GBS to HBEC differed from adherence to embryonic and fetal cells for both virulent and asymptomatic GBS strains bound to HBEC in a similar manner, enhanced by the lipid portion of the LTA. In contrast, the binding of GBS to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate polymer of LTA. These results indicate that possible receptor sites for LTA present on cells in prenatal stages of development may differ from those of adult cells, which may result in increased susceptibility of newborn infants to group B streptococcal disease. The implications of LTA-mediated adherence of GBS and their possible role as virulence factors are discussed.

Association of elevated levels of cellular lipoteichoic acids of group B streptococci with human neonatal disease.

Cell-associated lipoteichoic acids (LTAs) from late-exponential-phase cultures (serotypes Ia, Ib, Ic, II, and III) of group B streptococci isolated from infected and asymptomatically colonized infants were quantitated and characterized by growing the organisms in a chemically defined medium containing [3H]glycerol and [14C]acetate. Cell pellets were extracted with 45% aqueous phenol and chloroform-methanol and subjected to DEAE-Sephacel anion-exchange chromatography. Elution profiles resolved three major peaks, I, II, and III, with glycerol and phosphate present in a 1:1 molar ratio in each peak, and results obtained by Ouchterlony immunodiffusion analysis confirmed the presence of poly(glycerol phosphate). Saponification indicated that [14C]acetate was incorporated into fatty acids of peaks I and II only, suggesting that these were cell-associated LTAs. Peak II was of small molecular weight (less than 10,000) and probably represented another species of LTA. Peaks I and II were further demonstrated to be LTA by their ability to sensitize human type O erythrocytes. Peak III lacked fatty acids and was shown to probably be deacylated LTA. Quantitation of cell-associated teichoic acid material produced by the group B streptococcal strains indicated that the clinical isolates from infants with early- or late-onset disease possessed significantly higher levels than did the asymptomatic (clinical isolates from infants without symptoms of disease) group B streptococcal strains.

Temperature-sensitive mutant of coxsackievirus B3 establishes resistance in neonatal mice that protects them during adolescence against coxsackievirus B3-induced myocarditis.

Inoculation of neonatal CD-1 mice by multiple routes with an amyocarditic temperature-sensitive (ts) mutant (ts 1) derived from a myocarditic parent variant of coxsackievirus B3 (CVB3(m)) resulted in approximately half of the neonates surviving to adolescence. Challenge of the ts 1 survivors with CVB3(m) did not induce myocarditis, as assessed by histological examination of heart tissues. Virus was not detected in heart tissues of adolescent ts 1 survivors, but inoculation of these mice with CVB3(m) resulted in virus concentrations similar in titers to those found in CVB3(m)-inoculated normal adolescent mice. The ts 1 survivors did not contain detectable levels of anti-CVB3(m) neutralizing antibody, but upon challenge with CVB3(m) they produced antibody more rapidly and to higher titers than did normal CD-1 adolescents after primary inoculation with CVB3(m). Cell-mediated immunity in ts 1 survivors was compared with that of normal mice after challenge with CVB3(m). The capacity for production of migration inhibitory factor was assessed by the agarose droplet cell migration inhibition assay, using peritoneal exudate cells and a CVB3(m) cell lysate or KCl-extracted antigens from heart tissues of CVB3(m)-inoculated mice. Migration inhibitory factor activity was not detected in cultures of splenic leukocytes from ts 1 survivors of CVB3(m)-inoculated ts 1 survivors, but it was readily detected in cultures of splenic leukocytes from CVB3(m)-inoculated normal adolescent mice. The [(3)H]thymidine stimulation assay, performed with splenic lymphoid cells and purified CVB3(m) particles, revealed that lymphocytes from normal mice, whether inoculated with CVB3(m) or not, were not stimulated by CVB3(m) particle antigens, whereas lymphoid cells from a significantly higher proportion of ts 1 survivors, whether inoculated with CVB3(m) or not, responded with a stimulation index >/=2.0. The cells responding with positive stimulation were T lymphocytes. A higher proportion of normal mice and ts 1 survivors, both inoculated with CVB3(m), contained splenic cytotoxic T lymphocytes with higher reactivity against CVB3(m)-infected neonatal skin fibroblasts than against normal skin fibroblasts, as assessed by a (51)Cr release assay. The group of uninoculated ts 1 survivors present as a high proportion of individuals with cytotoxic T-lymphocyte reactivity against both uninoculated and CVB3(m)-inoculated skin fibroblasts. However, ts 1 survivors and normal mice possessed the same proportions of splenic lymphocytes carrying either allele for Lyt 1 and Lyt 2 surface markers. The results suggest two mechanisms by which ts 1 survivors exhibit resistance to CVB3(m) induction of myocarditis, namely, the rapid production of high-titered anti-CVB3(m) neutralizing antibody in response to CVB3(m) inoculation and altered cell-mediated immune responses against CVB3(m)-induced viral or novel cellular antigens. The data are compatible with the notion that an immune deviation mechanism, thought to be controlled through a mechanism requiring suppressor cell activity which inhibits macrophage activation in ts 1 survivors, protects these mice from induction of myocarditis.

Immunogenic fractions of Cryptococcus neoformans.

Cryptococcus neoformans cell and culture supernatant extracts were fractionated by ion exchange and gel filtration column chromatography. Various fractions were used to immunize mice, and to assess the release of migration inhibition factor, delayed type hypersensitivity, and protective immunity after challenge with C. neoformans. Results suggest that the C. neoformans fractions, which protect mice, contain a high molecular weight, predominantly carbohydrate antigen that can be distinguished from the capsular polysaccharide.

Assessment of cell-mediated immunity against coxsackievirus B3-induced myocarditis in a primate model (Papio papio).

Delayed hypersensitivity and myocarditis were induced in five baboons (Papio papio) after inoculation with myocarditic murine coxsackievirus B3 (CVB3m). By means of the [3H]thymidine incorporation assay and the agarose droplet cell migration inhibition assay, specific cell-mediated immunity was detected in all five animals against viral antigens and/or KCl-extractable soluble antigens extracted from the heart of a CVB3m-inoculated baboon. KCl-extracted antigens from normal baboon heart tissues, as well as KCl-extracted antigens from spleen or liver tissues from a CVB3m-inoculated baboon, failed to stimulate baboon peripheral blood lymphocytes or inhibit the migration of baboon peritoneal exudate cells. The appearance of dissimilar antigen(s) in extracts of heart tissues from CVB3m-inoculated baboons which did not contain infectious CVB3m parallels the appearance of novel KCl-extractable antigen(s) induced by CVB3m in murine heart tissues (Paque et al., J. Immunol. 120:1672-1678). The animal model described represents the first systematic evaluation of cell-mediated immunity in CVB3m-induced myocarditis in primates and suggests that a similar phenomenon may occur in humans.

Protease production by clinical isolates of type III group B streptococci.

Six strains of serotype III group B streptococci isolated from confirmed cases of neonatal disease were examined for their ability to produce proteolytic enzymes. Three neuraminidase-producing strains and three non-neuraminidase-producing strains were employed in this study. Protease production was examined in 1,000-fold concentrated filtrates of stationary-phase cells with an insoluble substrate derived from horse hide powder labeled covalently with Remazol brilliant blue. Protease activity was not detected in any cultural supernatant fluids until they were fractionated on Sephadex G-100. After fractionation, the neuraminidase-producing strains were shown to elaborate approximately sixfold more protease than the non-neuraminidase-producing strains. The finding that clinical isolates of group B streptococci that elaborated high levels of neuraminidase also produced elevated levels of extracellular protease may indicate that the production of several different factors may determine the virulence of these organisms.

Fractionation and immunologic assessment of KCl-extracted cardiac antigens in coxsackievirus B3 virus-induced myocarditis.

Hypertonic salt extracts prepared from the heart tissues of adolescent CD-1 mice were fractionated on Sephadex G-100 columns. Two separate fractions were obtained. Fraction I, containing the antigenic immunoreactive activity, was able to inhibit the migration of CVB3-PPD immune mouse peritoneal exudate cells (IMPEC) as well as PEC from mice infected with CVB3 virus alone. Fraction II did not have antigenic activity as assessed by the agarose droplet cell migration inhibition assay. As controls, Fraction I prepared from the livers of spleens of CVB3-infected CD-1 mice was unable to inhibit the migration of CVB3 IMPEC. Unimmunized or "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited by Fraction I. Antibodies prepared against Fractions I and II were unable to neutralize CVB3m virus in the plaque reduction test, and polyacrylamide gel analysis revealed multiple bands in 10% SDS gels.

Assessment of cell-mediated hypersensitivity against coxsackievirus B3 viral-induced myocarditis utilizing hypertonic salt extracts of cardiac tissue.

Hypertonic KCl extracts prepared from heart tissues of adolescent CD-1 mice inoculated with coxsackievirus B3 (CVB3) were tested for antigenicity in evaluating cell-mediated sensitivity to CVB3 virus utilizing the agarose droplet cell-migration-inhibition assay. Immune mouse peritoneal exudate cells (IMPEC) from mice immunized against CVB3 virus and Freund's complete adjuvant were specifically inhibited in the cell-migration-inhibition assay with graded doses of KCl-extracted antigen and purified protein derivative (PPD). Unimmunized for "normal" mouse peritoneal exudate cells (NMPEC) were not inhibited in the presence of the CVB3 KCl extracts. KCl heart extracts from mice inoculated with a cardiotropic strain of antigenically distinct mengovirus failed to inhibit CVB3 IMPEC, and noncardiac KCl extracts of liver and spleen from CVB3-inoculated mice also failed to inhibit cellular migration of CVB3 IMPEC. Reciprocal specificity experiments utilizing KCl-extracted antigens from mice infected with antigenically distinct cardiotropic mengovirus failed to inhibit cellular migration of IMPEC from mice immunized against the mengovirus. Serum-blocking power experiments indicate the antigenic KCl extracts failed to bind virus-neutralizing antibodies, indicating absence of detectable quantities of virion antigens. The results indicate that inoculation of mice with CVB3 virus results in the appearance of a new antigen(s) in cardiac tissue reacting with CVB3-IMPEC, but not with mengovirus IMPEC.