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D L Kasper

Publications and source records attributed to D L Kasper.

At least 109 records · Page 6Linked to original sources
PubMed1989

Intraperitoneal host cellular responses and in vivo killing of Bacteroides fragilis in a bacterial containment chamber.

A bacterial containment chamber was used to evaluate the peritoneal cellular response to Bacteroides fragilis during intraperitoneal challenge. This containment system was also used to determine the fate of bacteria within the peritoneal cavities of animals immunized, either actively or through adoptive transfer of cells or cell lysates, with the capsular polysaccharide of B. fragilis. This system demonstrated that the dominant cell types in the peritoneal cavities within 48 h of implantation of the containment chambers containing B. fragilis were neutrophils and macrophages. However, the early cellular response in immunized animals included an increase in the lymphocyte population within 4 h of challenge which was not detected in naive animals. In immunized animals, a later dramatic increase in the lymphocyte population at approximately 4 to 6 days following implantation of the containment chambers occurred. This increase in the lymphocyte population in immunized animals coincided with a decline in the viable bacterial counts within the chambers from 10(8) to 10(9) CFU/ml to less than 10(2) CFU/ml. A similar decline was not seen in naive animals challenged in the same manner. Killing of B. fragilis within containment chambers occurred when spleen cells, T cells, or lysates of T cells from actively immunized animals were passively transferred to naive recipient animals. It was shown that the factor responsible for bacterial killing was not antibody mediated, since bacteria contained within dialysis sacs with an exclusion of 50 kilodaltons were still killed in this model. Moreover, removal of T cells from adoptively transferred cell populations before transfer abrogated the decline in viable bacterial populations. The postulated mechanisms by which this bacterial killing occurred are discussed.

Animals↗
PubMed1989

Isolation and characterization of type IV group B Streptococcus capsular polysaccharide.

An antigenically distinct serotype, type IV, has recently been added to the recognized serotypes of group B streptococci (GBS). We isolated and purified the capsular polysaccharide antigen from a prototype type IV GBS strain. The type IV capsular polysaccharide formed a precipitin line with rabbit antiserum to type IV GBS organisms but not with antiserum to organisms of GBS serotype Ia, Ib, II, or III. Enzyme-linked immunosorbent assay inhibition experiments showed no cross-reaction between type IV antiserum and other GBS serotypes. Capsular polysaccharide released from the bacterial cells with mutanolysin and that isolated from the culture supernatant had similar elution profiles on Sepharose CL-6B, with a Kav of 0.30 and an estimated Mr of 200,000. The purified type IV polysaccharide was found to contain galactose, glucose, N-acetylglucosamine, and N-acetylneuraminic acid (sialic acid) as exclusive sugars. The polysaccharide contained 23% (by weight) sialic acid and galactose, glucose, and N-acetylglucosamine in a relative ratio of (1):1.10:0.55. These results are compatible with a repeating structure of six monosaccharide residues containing galactose, glucose, N-acetylglucosamine, and sialic acid in a molar ratio of 2:2:1:1. Unlike type Ia, II, and III GBS polysaccharides, desialylation of the type IV polysaccharide produced an antigen which formed a line of identity with the native type IV antigen in double diffusion in agar against homologous antiserum. This result suggests that sialic acid is not as critical to the immunodeterminant structure of the type IV antigen as it is for other GBS capsular types.

Animals↗
PubMed1989

Immune mechanisms in the prevention of intra-abdominal abscess formation.

Bacteroides fragilis is the most commonly isolated anaerobe from intraabdominal infections. In experimental models of intraabdominal sepsis, B. fragilis has been shown to be uniquely virulent. Some of these virulence traits are due to the capsular polysaccharide of this organism. Immunity to infection secondary to B. fragilis seems to involve both arms of the immune system. Humoral immunity (complement, antibody and PMNs) is critical to clearance of these bacteria from the blood stream. Cellular immune mechanisms predominate against intraabdominal abscess formation. Adoptive transfer experiments have shown that a CD8+, IJ+, non H2 restricted immune T cell or lysate from this T cell confers protection to immunocompetent, naive mice. An in vivo system has been developed to begin defining the mechanism of protection. B. fragilis placed inside a filter containment chamber within the peritoneum of immune mice are specifically killed over an 8-day period in the absence of white blood cells. This killing phenomenon was also observed inside filter chambers within mice receiving adoptively transferred immune T cells or lysates of these T cells. Furthermore, killing is specific to B. fragilis. These results support a T cell dependent mechanism for killing this bacteria and provide an interesting model for further exploration.

Abscess↗
PubMed1988

Immunization of pregnant women with a polysaccharide vaccine of group B streptococcus.

Immunization of pregnant women with a polysaccharide vaccine of group B streptococcus is a promising strategy for the prevention of perinatal infections caused by group B streptococci. To explore the feasibility of this strategy, we vaccinated 40 pregnant women at a mean gestation of 31 weeks with a single 50-microgram dose of the Type III capsular polysaccharide of group B streptococcus. The only adverse effect detected was a mild local reaction in nine women (22 percent). Of the 35 women with low or unprotective antibody levels before immunization (less than 2 micrograms per milliliter), 20 (57 percent) responded to the vaccine. The geometric mean antibody level rose from 1.3 to 7.1 micrograms per milliliter four weeks after vaccination (P less than 0.02), and these levels persisted at delivery and three months post partum. Sixty-two percent of the vaccine-induced immunoglobulin in the mothers was IgG, which readily crosses the placenta. Infant antibody levels in cord serum correlated directly with maternal antibody levels at delivery (r = 0.913, P less than 0.001). Of the 25 infants born to women who responded to the vaccine, 80 percent continued to have protective levels of antibody at one month of age and 64 percent had protective levels at three months. Serum samples from infants with greater than or equal to 2 micrograms of antibody to Type III group B streptococcus per milliliter uniformly promoted efficient opsonization, phagocytosis, and bacterial killing in vitro of Type III strains. This effect could be mediated exclusively by the alternative complement pathway. Although this vaccine with an overall response rate of 63 percent is not optimally immunogenic, we conclude that maternal immunization is feasible and can provide passive immunity against systemic infection with Type III group B streptococcus in the majority of newborns. Larger trials with better vaccines will be required to evaluate the safety and clinical effectiveness of this strategy.

Adult↗
PubMed1988

Multiantennary group-specific polysaccharide of group B Streptococcus.

The group-specific antigen of group B Streptococcus is composed of four different oligosaccharide units of Mw 766 (III), 1277 (II), 1462 (IV), and 1788 (I). The major constituent sugars of the oligosaccharides are alpha-L-rhamnopyranose, alpha-D-galactopyranose, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, and D-glucitol except that III does not contain alpha-D-galactopyranosyl or 2-acetamido-2-deoxy-beta-D-glucopyranosyl residues and IV contains no D-glucitol but has one additional beta-L-rhamnopyranosyl residue. The structures of II and III have been previously elucidated [Michon, F., Katzenellenbogen, E., Kasper, D. L., & Jennings, H. J. (1987) Biochemistry 26, 476-486]. In the group B antigen all the oligosaccharides are linked by one type of phosphodiester bond from O6 of the D-glucitol residue of one oligosaccharide to O6 of the alpha-D-galactopyranosyl residue of the next to form a complex and highly branched multiantennary structure. However, despite the heterogeneous nature of its component oligosaccharides, some order has been identified in the biosynthesis of the group B antigen from chemical and enzymatic sequence studies. Because III lacks an alpha-D-galactopyranosyl residue but has a D-glucitol residue, it is situated at the reducing terminus of all the branches of the group B antigen where it is always adjacent to a II moiety. Conversely, IV has an alpha-D-galactopyranosyl residue but has no D-glucitol and is therefore located at the reducing terminus of the group B antigen where it probably functions as a linker molecule between the group B polysaccharide and the cell wall peptidoglycan of the group B streptococcal organisms. Oligosaccharide I contains two alpha-D-galactopyranosyl residues and one D-glucitol residue and thus constitutes the branch point in the group B antigen, whereas II contains one of each of the above residues and therefore is situated in linear interchain positions. The group B antigen is highly branched and probably has a unique multiantennary structure.

Carbohydrate Conformation↗
PubMed1988

Bacteroides vulgatus outer membrane antigens associated with carrageenan-induced colitis in guinea pigs.

Previous experiments with the carrageenan model for ulcerative colitis demonstrated that the inflammatory response in guinea pigs can be enhanced by immunization with Bacteroides vulgatus and subsequent feeding of this organism to experimental animals. The studies reported here show that antigens extractable from the bacterial outer membrane by EDTA are responsible for this effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the outer membrane proteins from various strains as well as the lipopolysaccharides (LPS) extractable by the phenol-water method. Although the observed pattern of outer membrane proteins was complex, the strains could be divided into two electrophoretic types (phenons) on the basis of immunoblotting against a panel of antisera. Cross-absorbed antisera used in immunoblotting experiments identified four outer membrane proteins uniquely associated with the phenon capable of enhancing the colitis inflammatory response. These proteins had molecular weights of 100,000, 57,000, 34,000, and 21,000 when measured in 8% to 12% acrylamide gradient sodium dodecyl sulfate gels. Other antigens identified included at least one type of smooth LPS, three types of rough LPS, and a common antigen of 30,000 molecular weight among the strains of B. vulgatus tested. The outer membrane preparations were used in animal immunization and challenge experiments, and the severity of colitis was correlated with one electrophoresis type. The potential role of membrane proteins in the enhancement of colitis is discussed.

Animals↗
PubMed1987

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen.

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.

Carbohydrate Conformation↗
PubMed1987

Structure of the complex group-specific polysaccharide of group B Streptococcus.

The group-specific antigen was isolated from a type Ia group B streptococcal strain and is a complex polysaccharide composed of alpha-L-rhamnopyranosyl, alpha-D-galactopyranosyl, 2-acetamido-2-deoxy-beta-D-glucopyranosyl, D-glucitol, and phosphate residues. The complexity of the group B polysaccharide antigen is evident from the fact that when depolymerized by basic hydrolysis it yielded three structurally related, but nevertheless significantly different, oligosaccharides. These oligosaccharides were obtained in different molar quantities as their monophosphate esters. This evidence strongly suggests that they are linked by phosphodiester bonds in the original group B antigen. If these oligosaccharides are in fact randomly situated throughout the linear polysaccharide, then this type of heterogeneous repeating unit is unusual for a polysaccharide of bacterial origin. However, this structural arrangement of the oligosaccharides has yet to be unambiguously established because the alternate explanation of there being three different polysaccharides in the group B antigen cannot be discounted in the evidence presented here. The oligosaccharides were enzymatically dephosphorylated, and the structures of two of the three oligosaccharides are (formula: see text) Despite their structural differences, the two oligosaccharides are related by the smaller being an integral part of the larger. In the structural analysis of the group B antigen, methylation analysis, periodate oxidation, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, fast atom bombardment mass spectrometry, and various specific chemical and enzymatic degradations were the principal methods used. Of particular interest was the use of an alpha-rhamnosidase to selectively degrade the larger oligosaccharide. This facilitated the assignment of signals in its 1H and 13C NMR spectra.

Carbohydrate Conformation↗
PubMed1987

4,8-anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination.

A new, sialic-acid-derived compound was isolated from the acid hydrolysate of edible bird's nest by ion-exchange chromatography. Combined use of mass spectroscopy and 1H- and 13C-NMR spectroscopy established that it is the 4,8-anhydro derivative of N-acetylneuraminic acid and that in solutions it exists in two tautomeric forms. The formation of the new compound supports and earlier findings that in the glycoprotein of edible bird's nest at least a portion of N-acetylneuraminic acid is acetylated at HO-4.

Animals↗
PubMed1987

Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence.

The capsular polysaccharide of type III group B Streptococcus (GBS) is thought to be a major factor in the virulence of this organism. Transposon mutagenesis was used to obtain isogenic strains of a GBS serotype III clinical isolate (COH 31r/s) with site-specific mutations in the gene(s) responsible for capsule production. The self-conjugative transposon Tn916 was transferred to strain COH 31r/s during incubation with Streptococcus faecalis strain CG110 on membrane filters. Eleven transconjugant clones did not bind type III GBS antiserum by immunoblot. Immunofluorescence, competitive ELISA, and electron microscopy confirmed the absence of detectable GBS type III capsular polysaccharide in one of the transconjugants, COH 31-15. Southern hybridization analysis with a Tn916 probe confirmed the presence of the transposon sequence within each mutant. A 3.0-kilobase EcoRI fragment that flanked the Tn916 sequence was subcloned from mutant COH 31-15. This fragment shared homology with DNA from the other GBS serotypes, suggesting a common sequence for capsulation shared by organisms of different capsular types. Loss of capsule expression resulted in loss of virulence in a neonatal rat model. We conclude that a gene common to all capsular types of GBS is required for surface expression of the type III capsule and that inactivation of this gene by Tn916 results in the loss of virulence.

DNA Transposable Elements↗
PubMed1987

A model of high-affinity antibody binding to type III group B Streptococcus capsular polysaccharide.

We recently reported that the single repeating-unit pentasaccharide of type III group B Streptococcus (GBS) capsular polysaccharide is only weakly reactive with type III GBS antiserum. To further elucidate the relationship between antigen-chain length and antigenicity, tritiated oligosaccharides derived from type III capsular polysaccharide were used to generate detailed saturation binding curves with a fixed concentration of rabbit antiserum in a radioactive antigen-binding assay. A graded increase in affinity of antigen-antibody binding was seen as oligosaccharide size increased from 2.6 repeating units to 92 repeating units. These differences in affinity of antibody binding to oligosaccharides of different molecular size were confirmed by immunoprecipitation and competitive ELISA, two independent assays of antigen-antibody binding. Analysis of the saturation binding experiment indicated a difference of 300-fold in antibody-binding affinity for the largest versus the smallest tested oligosaccharides. Unexpectedly, the saturation binding values approached by the individual curves were inversely related to oligosaccharide chain length on a molar basis but equivalent on a weight basis. This observation is compatible with a model in which binding of an immunoglobulin molecule to an antigenic site on the polysaccharide facilitates subsequent binding of antibody to that antigen.

Antibodies, Bacterial↗
PubMed1986

Cellular control of abscess formation: role of T cells in the regulation of abscesses formed in response to Bacteroides fragilis.

Although abscesses are a major sequela of infection, little is known about which cellular events initiate and which prevent this pathologic response. These studies are the first to indicate a role for T cells in the important pathogenic process of abscess development and also in immunity to abscesses induced by Bacteroides fragilis. We have shown that T cells initiate the formation of abscesses in mice after i.p. challenge with B. fragilis. These T cells bear both Ly-1 and Ly-2 surface markers. Nude mice (which have been shown by others to have T cell or T cell precursors) are also able to form abscesses. Cyclophosphamide-treated mice (with depressed T cell function) were not capable of developing abscesses. Reconstitution with normal or nude mouse spleen cells restored this ability. However, reconstitution with anti-Thy-1.2-treated, anti-Ly-1, or anti-Ly-2-treated spleen cells (or a mixture of the two cell populations) failed to allow abscess formation after bacterial challenge. Immunity to abscesses caused by B. fragilis requires two T cells. The first Ly-1-2+ T cell has an IJ surface marker and has been shown to release a small m.w. soluble factor (ITF) that is antigen specific. Immunity to abscesses, however, depends on the interaction of ITF with a second Ly-1-2+ T cell, demonstrated in reconstitution experiments with nude mice. The data presented document a critical role for T cells in abscess induction and suggest the existence of a suppressor-like T cell circuit in immunity to abscesses.

Abscess↗
PubMed1986

Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway.

The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in antibody-independent binding of C1 and activation of the classic complement pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of 3H-type Ia GBS with purified F(ab')2 to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab')2 blocking was shown after adsorption of F(ab')2 with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab')2 had a 70% decrease in ability to block the association of bacteria with PMN. Evidence for the requirement of the capsular polysaccharide in classic complement pathway activation came from a C1 transfer assay with the use of neuraminidase-digested type Ia GBS. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysaccharide of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH.

Adsorption↗
PubMed1986

Isolation of a C (Ibc) protein from group B Streptococcus which elicits mouse protective antibody.

The C (Ibc) proteins of group B Streptococcus (GBS) have been shown to induce mouse protective antibodies when present as immunogens on whole organisms. However, characterization of specific proteins responsible for inducing protection has not been reported. We have grown type Ic GBS in a dialysate of Todd Hewitt broth and analyzed the proteins extruded into the broth. Multiple proteins of varying size were visualized by SDS-PAGE. Ultrafiltration was used to separate the GBS components by molecular weight (MW) into 2 pools, those below 30,000 MW but above 10,000 MW (P10) and those above 30,000 MW (P30). The P10 contained 4 major proteins, including a 14,000 MW protein. Balb-c mice were immunized with the P10 fraction and the antisera used in mouse protection studies. This immune sera protected 100% of mice against challenge with type Ib GBS and protection was not altered by prior absorption of the sera with type Ia or Ib capsular polysaccharide. The P10 was fractionated by column chromatography and eluted proteins examined by SDS-PAGE and Western blot with the mouse protective antisera elicited to the P10. There was one major immunologically reactive protein at 14,000 MW which eluted in a partially purified form from the column. The 14,000 MW protein was reisolated from preparative SDS-PAGE gels and used to elicit antiserum in a rabbit. In mouse protection studies this rabbit antiserum protected mice against subsequent challenge with type Ib GBS (89% protection). Surface antigens were extracted from 125I-labelled type Ic GBS and immunoprecipitated with antiserum to the 14,000 MW protein. The 14,000 MW protein and multiple higher molecular weight proteins were immunologically cross-reactive suggesting the presence of shared epitopes. Thus the 14,000 MW protein from type Ic GBS that is antigenic and elicits mouse protective antibodies against the heterologous type Ib GBS fulfills the criteria for a C protein of GBS.

Animals↗
PubMed1986

Effect of subinhibitory doses of clindamycin on the virulence of Bacteroides fragilis: role of lipopolysaccharide.

The capsular polysaccharide (CP) of Bacteroides fragilis is an important virulence factor in the formation of experimental intraabdominal abscesses. Incubation of this organism with subinhibitory doses of clindamycin induced morphological changes in the bacteria, including elongation and loss of CP, detected by ferritin-labeled antibody to capsule. Pretreatment of bacteria with subinhibitory doses of clindamycin, however, did not affect the ability of live or heat-killed organisms to produce intraabdominal abscesses in a mouse model of intraabdominal sepsis. Dose-response experiments with purified CP as well as lipopolysaccharide (LPS) from B. fragilis ATCC strain 23745 mixed with sterile cecal contents as adjuvant revealed that both surface components of the organism were capable of causing abscesses in the mouse model. The dose of LPS required to induce abscesses was five times higher than the required dose of CP. Nevertheless, these studies suggested that B. fragilis LPS is another virulence factor in the formation of intraabdominal abscesses.

Bacteroides fragilis↗