Biomedical subjects

E H Beachey

Publications and source records attributed to E H Beachey.

At least 73 records · Page 4Linked to original sources

PubMed1986

Opsonic antibodies evoked by hybrid peptide copies of types 5 and 24 streptococcal M proteins synthesized in tandem.

The protective immunogenicity of a hybrid peptide containing tandem copies of types 5 and 24 epitopes was investigated. Carboxy-terminal peptides of the cyanogen bromide-derived fragment 7 (CB7) of type 24 M protein were chemically synthesized, and then extended to include the first 20 residues of the amino-terminus of type 5 M protein. When emulsified in CFA and injected into rabbits without conjugation to a carrier, each of the synthetic hybrid peptides, designated S-M5(1-20)-S-CB7(23-35)C and S-M5(1-20)-S-CB(19-34), evoked opsonic antibodies against both types 5 and 24 streptococci without raising heart tissue-crossreactive immunity. These results suggest that tandem hybrid peptides may provide a new approach to the development of multivalent vaccines, not only to different serotypes of group A streptococci but perhaps also to a variety of other infectious agents.

Animals↗

PubMed1986

Localization of protective epitopes of the amino terminus of type 5 streptococcal M protein.

We have used a set of overlapping chemically synthesized peptides representing the amino terminus of type 5 streptococcal M protein to localize protective, as opposed to nonprotective and tissue-crossreactive epitopes that might be appropriate for vaccine formulations. Rabbit antisera raised against SM5(1-35) reacted in high titer with pep M5 by ELISA and opsonized type 5 streptococci. None of the antisera crossreacted with human heart tissue or myosin. Antisera against SM5(26-35) reacted with SM5(1-35) and pep M5 but failed to opsonize type 5 streptococci. Particle-phase ELISA indicated that SM5(26-35) antibodies were directed against nonprotective determinants of pep M5 that were not exposed on the surface of viable organisms. Opsonization and ELISA inhibition assays showed that, of the SM5(1-35) antibodies that reacted with M5, all were inhibited by SM5(14-35), whereas none was inhibited by SM5(26-35), suggesting that the protective epitopes of SM5(1-35) resided between residues 14 and 26. This was confirmed by subsequent chemical synthesis of this region; SM5(14-26) totally inhibited SM5(1-35) antibodies that reacted with pep M5 in ELISA, and completely inhibited opsonization of type 5 streptococci by SM5(1-35) antibodies. SM5(14-26) evoked high titers of type-specific, opsonic antibodies against type 5 streptococci, confirming the protective immunogenicity of this 13-residue peptide of type 5 M protein.

Amino Acid Sequence↗

PubMed1986

Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein.

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.

Adjuvants, Immunologic↗

PubMed1986

Influence of CONH2 or COOH as C-terminus groups on the antigenic characters of immunogenic peptides.

The influence of the presence of a terminal COOH or CONH2 on the antigenic characters of synthetic immunogenic peptides has been studied on a streptococcal synthetic vaccine model. The obtained results show that when a peptide amide is used, the antibodies raised specifically against the amide group recognize neither free COOH nor the parent protein. The carboxamide group is thus unsuitable as was postulated for raising antibodies which recognize the peptide bond.

Amides↗

PubMed1986

Localization and characterization of Sendai virus nonstructural C and C' proteins by antibodies against synthetic peptides.

Antibodies were raised in rabbits against two synthetic peptides, each 30 residues in length, one corresponding to the predicted common carboxyl termini of the nonstructural C and C' proteins of Sendai virus and the other to the unique amino terminus of the larger C protein. Each peptide was inoculated as a covalent complex with tetanus toxoid or in uncomplexed form. Only antibodies to the free carboxyl-terminal peptide precipitated both C and C' proteins made by in vitro translation of viral mRNA and reacted with the C protein from infected cells. These results confirm that the C and C' proteins are carboxyl-coterminal. Contrasting with the reported colocalization of intracellular measles virus C proteins with nucleocapsid inclusions, immunofluorescence studies revealed that Sendai virus C proteins were uniformly distributed in the cytoplasm whereas the viral P protein was present in inclusions that were mainly perinuclear. Since almost all P protein molecules are associated with viral nucleocapsids, these observations suggested that Sendai virus C protein molecules may be both nucleocapsid-associated and free in the cytoplasm. This interpretation was supported when the C and C' proteins were found in both nucleocapsid and free protein fractions of cell lysates. Anti-C antibodies did not inhibit viral RNA synthesis when added to an extract of infected cells. This result was consistent with the conclusion that the C proteins have no direct role in viral transcription, since virions lack C proteins but are transcriptionally active. Therefore, the functions of the C proteins remain undefined.

Animals↗

PubMed1986

Influence of trimethoprim and sulfamethoxazole on the synthesis, expression, and function of type 1 fimbriae of Escherichia coli.

We investigated the effects of low levels of sulfamethoxazole and trimethoprim on biosynthesis, expression at the cell surface, and hemagglutinating activity of type 1 fimbriae from a urinary tract isolate of Escherichia coli. The mannose-sensitive hemagglutination of E. coli was reduced after growth in either of the antimicrobial agents. Moreover, trimethoprim affected antigenic fimbrial expression at the bacterial cell surface. Fimbrial subunit synthesis, as detected in lysates of solubilized whole bacteria, was inhibited in bacteria grown with one-half the minimum inhibitory concentration (MIC) or trimethoprim. Organisms grown in low doses of trimethoprim and sulfamethoxazole (one-thirty-second the MIC each) together exhibited marked reduction of fimbrial synthesis, expression, and hemagglutinating activity. Neither agent induced any detectable effect when used alone at the same concentrations. These results demonstrate that the synergistic activity of these drugs at concentrations below the MIC influence the synthesis, expression, and adhesive properties of type 1 fimbriae.

Dose-Response Relationship, Drug↗

PubMed1986

Type-specific opsonic antibodies evoked with a synthetic peptide of streptococcal M protein conjugated to polylysine without adjuvant.

A chemically synthesized copy (S-CB7) of a fragment (35 amino acid residues) of type 24 streptococcal M protein was covalently linked to polylysine with carbodiimide and injected subcutaneously into rabbits without adjuvant. Although the primary immune responses as measured by enzyme-linked immunosorbent assays at biweekly intervals were weak, the secondary responses as measured by both enzyme-linked immunosorbent assays and opsonophagocytic assays were as high as those obtained previously in rabbits immunized with the peptide conjugate emulsified in complete Freund adjuvant. Injection of murabutide, a synthetic muramyl dipeptide derivative of bacterial peptidoglycan, with the initial immunizing dose of peptide conjugate had no apparent effect on the secondary immune responses. These results indicate that protective immune responses may be raised against polylysine conjugates of chemically synthesized peptide copies of streptococcal M protein without adjuvant.

Adjuvants, Immunologic↗

PubMed1986

Isolation and characterization of a 60-kilodalton salivary glycoprotein with agglutinating activity against strains of Streptococcus mutans.

A bacterial agglutinin specific for strains of Streptococcus mutans was isolated from human saliva. Physiochemical analyses showed the agglutinin to be a glycoprotein with a molecular weight of 60,000. The agglutinin aggregated four of the eight strains of Streptococcus mutans tested but did not aggregate the strains of Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis tested. Chemical modification of carbohydrate moieties of the agglutinin with sodium metaperiodate had no effect on aggregation, whereas modification of the polypeptide portion with trypsin abolished aggregating activity. A set of five murine hybridoma antibodies was employed to further analyze the agglutinin. Two carbohydrate-specific antibodies, directed against D-mannose and N-acetylgalactosamine moieties, respectively, failed to block agglutinin- or whole saliva-mediated aggregation of S. mutans cells. In contrast, two antibodies directed against pronase-sensitive antigenic sites blocked both agglutinin- and saliva-mediated aggregation of S. mutans cells. Western blot analysis with the agglutinin-specific hybridoma antibodies demonstrated the agglutinin in whole saliva and in artificial tooth pellicles formed on hydroxyapatite beads incubated with saliva. These results suggest that a 60-kilodalton glycoprotein of human saliva is a bacterial agglutinin with specificity for certain strains of S. mutans. They further suggest that aggregation is mediated by polypeptide rather than carbohydrate determinants of the glycoprotein.

Agglutination↗

PubMed1986

Relationship of critical micelle concentrations of bacterial lipoteichoic acids to biological activities.

The critical micelle concentration (CMC) of lipoteichoic acid (LTA) was investigated with two dyes, rhodamine 6G and Coomassie brilliant blue R-250. Both dyes gave similar values for the CMC of LTA. The CMC of LTA from several species of bacteria ranged from 28 to 60 micrograms/ml in phosphate-buffered saline. The CMC values for the LTAs are in the range expected for an amphiphile containing a single, 16-carbon fatty acid residue. Formation of micelles was not detected with deacylated LTA. Salt decreased the CMC of LTA from 380 micrograms/ml in distilled water to 37 micrograms/ml in 0.5 M NaCl. At concentrations greater than the CMC, LTA induced the lysis of sheep erythrocytes and was cytotoxic for Girardi heart cells. The data suggest that LTA in the micellar state may cause disruption of the erythrocyte membrane and may be cytotoxic for cells in culture.

Animals↗

PubMed1986

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.

Adhesiveness↗

PubMed1986

Inhibition of the interaction of Streptococcus sanguis with hexadecane droplets by 55- and 60-kilodalton hydrophobic proteins of human saliva.

The effect of salivary secretions on the hydrophobicity of Streptococcus sanguis was investigated. Pretreatment of the bacteria with paraffin-stimulated whole saliva resulted in a 79% inhibition of adhesion to hexadecane droplets. Column chromatography on Sepharose 4B and sodium dodecyl sulfate gel electrophoretic analysis indicated that the inhibitory activity of saliva resided in a fraction containing material of approximately 60,000 molecular weight. The active components, which we have termed the hydrophobic components (HC), bind to octyl-Sepharose beads. Pretreatment of S. sanguis with HC resulted in a dose-dependent inhibition of the streptococcus-hexadecane interaction that reached a maximum of 85%. Furthermore, HC effectively blocked the ability of S. sanguis to adhere to hydroxyapatite beads coated with either whole saliva or HC. Sodium dodecyl sulfate-polyacrylamide gel analysis indicated that HC eluted from octyl-Sepharose consisted primarily of two proteins (60 kDa and 55 kilodaltons) which could be resolved by high-pressure liquid chromatography. Both of these proteins were able to inhibit the binding of S. sanguis to hexadecane in a dose-dependent manner; however, the 60-kilodalton molecule was slightly more effective in this assay. Amino acid analysis of these proteins showed that both proteins contained a high percentage of nonpolar amino acids. These findings suggest that certain components of saliva influence the interaction of S. sanguis with hydrophobic surfaces.

Adhesiveness↗

PubMed1986

Binding of Streptococcus pyogenes to soluble and insoluble fibronectin.

The interaction of soluble and insoluble fibronectin with Streptococcus pyogenes was investigated. Soluble fibronectin bound to S. pyogenes in a dose-dependent and irreversible manner. Lipoteichoic acid competitively inhibited the binding of fibronectin to S. pyogenes but had little effect on the binding of fibronectin to staphylococci or pneumococci. The phase of growth of the streptococci had a slight effect on binding of fibronectin, with optimal binding occurring in the late log phase. S. pyogenes cells bound to fibronectin immobilized on microtiter plates in a dose-dependent and saturable manner. Both soluble fibronectin and lipoteichoic acid inhibited the binding of streptococci to immobilized fibronectin, suggesting that streptococci interact with soluble and insoluble fibronectin in a similar manner. Antibodies to fibronectin blocked the attachment of streptococci to immobilized fibronectin, whereas normal serum had no effect. Adherence of streptococci to buccal epithelial cells was inhibited by antibodies to fibronectin, but not by normal sera or by antibodies to buccal epithelial cells. The data suggest that lipoteichoic acid on the surface of S. pyogenes binds to fibronectin exposed on the host cell and that such binding mediates the attachment of streptococci to host cells.

Adhesiveness↗

PubMed1986

Interaction of a 60-kilodalton D-mannose-containing salivary glycoprotein with type 1 fimbriae of Escherichia coli.

A 60-kilodalton glycoprotein previously isolated and purified from human saliva (J. B. Babu, E. H. Beachey, D. L. Hasty, and W. A. Simpson, Infect. Immun. 51: 405-413, 1986) was found to interact with type 1 fimbriae and prevent adhesion of type 1 fimbriated Escherichia coli to animal cells in a D-mannose-sensitive manner. Purified salivary glycoprotein agglutinated type 1 fimbriated E. coli and, at subagglutinating concentrations, blocked the ability of type 1 fimbriated E. coli to attach to human buccal epithelial cells or agglutinate guinea pig erythrocytes. Both interactions were inhibited by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Complexing of the glycoprotein to type 1 fimbriae was demonstrated by molecular sieve chromatography and modified Western blots. When mixed with type 1 fimbriae, the radiolabeled salivary glycoprotein coeluted with type 1 fimbriae from a column of Sepharose 4B. When blotted from a sodium dodecyl sulfate gel to nitrocellulose sheets, the glycoprotein interacted directly with type 1 fimbriae applied to the blots. Both of the latter interactions also were blocked by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Chemical modification of the glycoprotein with sodium metaperiodate abolished its ability to interact with isolated type 1 fimbriae or type 1 fimbriated E. coli. These results suggest that the carbohydrate moiety of the 60-kilodalton glycoprotein serves as a receptor for type 1 fimbriae in the oral cavity, and we postulate that the interaction may cause agglutination and early removal of E. coli, thereby preventing colonization by these organisms of oropharyngeal mucosae and dental tissues.

Antibodies, Monoclonal↗

PubMed1986

The ability to sensitize host cells for destruction by autologous complement is a general property of lipoteichoic acid.

Previous studies have demonstrated that lipoteichoic acid (LTA) from Streptococcus pneumoniae binds to erythrocytes and renders them susceptible to lysis by autologous complement. The present study was performed to determine whether LTA from two other gram-positive bacterial species had the ability to render mammalian cells susceptible to lysis by autologous complement. Human erythrocytes were sensitized with LTA from S. pneumoniae, Streptococcus pyogenes, or Lactobacillus fermentum. Under incubation in normal autologous serum, lysis was observed with each of the LTA-sensitized erythrocyte preparations. When erythrocytes from a C2-deficient patient were sensitized with the LTA preparations and then incubated in autologous, C2-deficient serum, the erythrocytes sensitized with S. pyogenes or L. fermentum LTA demonstrated relatively little lysis, whereas the erythrocytes sensitized with S. pneumoniae LTA yielded near-total lysis. After reconstitution of the C2-deficient serum with purified human C2, lysis was observed with all three LTA preparations. When erythrocytes from an agammaglobulinemic patient were sensitized with either the S. pyogenes or the L. fermentum LTA, they were not lysed in the presence of autologous agammaglobulinemic serum, whereas the erythrocytes sensitized with S. pneumoniae LTA were completely lysed. Serum obtained from the agammaglobulinemic patient after reconstitution with intravenous pooled gamma globulin was able to lyse autologous erythrocytes sensitized with each of the three LTA preparations. These results demonstrate that the ability to render host cells susceptible to lysis by autologous complement is a general property of LTA. Whether activation of the autologous complement occurs by the classical or alternative pathways and whether it is antibody dependent depends on the nature of the bacterial LTA.

Agammaglobulinemia↗

PubMed1986

The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit.

The role of type 1 fimbriae in the mannose-sensitive attachment of Escherichia coli to eucaryotic cells was investigated by deletion mutation analysis of a recombinant plasmid, pSH2, carrying the genetic information for the synthesis and expression of functional type 1 fimbriae. A mutant, pUT2002, containing a deletion remote from the structural gene encoding the 17-kilodalton subunit protein of type 1 fimbriae failed to agglutinate guinea pig erythrocytes even though the bacteria expressed fimbriae morphologically and antigenically indistinguishable from those produced by the intact recombinant plasmid. Fimbriae isolated from pUT2002 failed to agglutinate guinea pig erythrocytes, but reacted with a monoclonal antibody specific for quaternary structural determinants of type 1 fimbriae. Moreover, the dissociated fimbrial subunits from this mutant were indistinguishable from normal fimbriae by their migration during electrophoresis in sodium dodecyl sulfate-polyacrylamide gels, by their reactivity with a monoclonal antibody directed against a subunit-specific epitope, and in enzyme-linked immunosorbent assays with monospecific antisera. These results indicate that the adhesive functions in type 1 fimbriae are dependent on a factor(s) encoded by a gene other than those required for synthesis, assembly, and expression of the structural 17-kilodalton subunit.

Adhesiveness↗

PubMed1986

Enzyme-linked immunosorbent assay for adherence of bacteria to animal cells.

Epithelial cells scraped from human oral mucosa and from pig intestines were immobilized onto the flat bottom surfaces of microtiter plates to study the adherence of various bacterial species to host cells. Bacterial adherence was quantitated either by an enzyme-linked immunosorbent assay technique with specific antibacterial serum as the first antibody followed by peroxidase-conjugated second antibody or by using biotinylated bacteria and avidin-peroxidase as the detecting agent. Unlabeled Escherichia coli and purified E. coli 987P fimbriae inhibited the adherence of biotinylated E. coli to immobilized enterocytes. The adherence of a mannose-sensitive strain of E. coli to immobilized oral epithelial cells was inhibited by mannose derivatives. The adherence of fimbriated E. coli 987P to immobilized enterocytes was approximately four times higher than the adherence of a nonfimbriated variant of the same strain. The adherence of Streptococcus pyogenes to oral cells was detected in the range of 10 to 150 bacteria per cell and was inhibited by lipoteichoic acid and albumin. The data suggest that the putative receptors which bind bacteria on the immobilized cells retain a functional form similar to that of native cells in suspension. The proposed adherence assay is easy to perform, allows the detection of specific adherence of test bacteria, and provides objective quantitation of adherence with a sensitivity of 10 bacteria per cell. Most importantly, the assay allows the testing of many variables in the same day.

Animals↗

PubMed1986

Immunochemical and ultrastructural mapping of the gelatin-binding and cell-attachment regions of human plasma fibronectin with monoclonal antibodies.

Monoclonal antibodies against fibronectin were used to locate the gelatin-binding and cell-attachment regions of plasma fibronectin at an ultrastructural level. A total of 23 hybridomas were generated using mice immunized with either intact fibronectin or a 40 000 Mr gelatin-binding fibronectin fragment. One of these antibodies (D9b) strongly inhibited the interaction of radio-labelled fibronectin with gelatin. Another antibody (IB10) inhibited the attachment of Chinese hamster ovary (CHO) cells to a fibronectin substratum by 99%. Both of these antibodies were purified by affinity chromatography on columns of fibronectin-Sepharose and were then incubated with soluble fibronectin to form antigen-antibody complexes. The complexes were separated from free antibody on a column of Sephadex G-200 and were prepared for electron-microscopic examination by spraying on mica discs and rotary shadowing with platinum. As determined by this method, the fibronectin molecules measured 124 +/- 1.7 nm in length. Monoclonal antibody IB10 was visualized as a globular projection 40 +/- 1.4 nm from one end of the fibronectin filament. Monoclonal antibody D9b, on the other hand, was visualized as a globular projection at or near one or both ends of the molecule. These data provide the first morphological localization of the gelatin-binding and cell-attachment regions of fibronectin and indicate that further studies using monoclonal antibodies directed toward other epitopes should shed light not only on function but also on the tertiary and quaternary structure of the fibronectin molecule.

Animals↗

PubMed1986

Synthetic peptide fragments of streptococcal M proteins.

The surface M proteins of group A streptococci prevent phagocytosis by the non-immune host but antibodies subsequently developed against these M proteins opsonize the organism to allow phagocytosis and killing. In some cases, antibodies developed against M proteins are cross-reactive with host tissue and have been implicated in rheumatic fever. Peptide fragments of several serotypes, namely type 24, 5 and 6 M proteins were chemically synthesized and tested for their ability to induce protective and tissue cross-reactive antibodies in rabbits. Two synthetic 35 residue peptides of type 24 M protein, S-CB3 and S-CB7 had previously been shown to evoke high ELISA titers as well as opsonic antibody titers in each of three rabbits. Neither contained host tissue cross-reactive antibodies when examined with human heart tissue. Subpeptides of CB7 were synthesized to identify the smallest protective epitope. Three synthetic subpeptides (S-CB7-(13-35), - (18-35) and - (23-35) C were covalently linked to tetanus toxoid and evoked opsonic antibodies in rabbits and thus protective immunity with no tissue cross-reactive epitopes. Synthetic peptides of the NH2-terminal region of peptide M 5, which is known to contain cardiac tissue cross-reactive epitopes, were also tested. When covalently linked to tetanus toxoid, the synthetic peptide S-M 5 (1-20), but not S-M5 (20-40), evoked antibodies which were protective against type 5 streptococci; no heart cross-reactive antibodies were evoked even when large excesses of the synthetic peptides were injected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗