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J B Dale

Publications and source records attributed to J B Dale.

At least 55 records · Page 3Linked to original sources
PubMed1988

Protective and heart-crossreactive epitopes located within the NH2 terminus of type 19 streptococcal M protein.

M protein was purified to homogeneity from limited pepsin digests of intact type 19 streptococci (pep M19). The purified pep M19 when emulsified in CFA and injected into rabbits evoked type-specific and crossreactive opsonic antibodies, as well as heart-crossreactive antibodies. The NH2-terminal primary structure of pep M19 was determined and a peptide copying the first 24 amino acids [SM19(1-24)C] was chemically synthesized. Rabbits that were immunized with the unconjugated peptide developed antibodies that recognized the native pep M19, as determined by ELISA, and opsonic antibodies against type 19 streptococci, as determined by in vitro opsonophagocytosis tests. The synthetic peptide also evoked antibodies that crossreacted with a 60-kD sarcolemmal membrane protein of human myocardium. By using overlapping synthetic subpeptides as immunoinhibitors, the opsonic and heart-crossreactive epitopes of SM19(1-24)C were localized to SM19(11-24)C. Our data confirm the presence of heart-crossreactive epitopes within the primary structure of pep M19 and show that these potentially harmful autoimmune epitopes may be located in the NH2-terminal regions of certain M proteins. We conclude that continued efforts to identify the primary structures of protective and heart-crossreactive epitopes will be necessary to elucidate the pathogenesis of acute rheumatic heart disease and to develop safe and effective streptococcal vaccines.

Amino Acid Sequence↗
PubMed1988

Comparison of the leader sequences of four group A streptococcal M protein genes.

The 5' portions and flanking sequences of genes encoding types 1, 12, 24, and 6 M proteins were compared. Although the DNA sequences encoding the amino-termini of the mature M proteins had no obvious similarity, upstream sequences, and those encoding the signal peptides (leader sequences) of the four M protein genes had considerable similarity. In general, the 5' ends of all the leader sequences were more conserved than the 3' ends, although the M6 and M24 leader sequences had identical 3' ends. Sequence similarity among the deduced amino acid sequences of the four signal peptides was more extensive than the corresponding DNA sequences. We found that strict DNA similarity among all four sequences extended only to the ends of the hydrophilic amino-terminal regions of the signal peptides, but that amino acid sequence conservation continued to the ends of the respective hydrophobic cores. With the exception of the M6 and M24 sequences, the regions adjacent to the signal peptidase cleavage sites were highly variable.

Amino Acid Sequence↗
PubMed1988

Protective and autoimmune epitopes of streptococcal M proteins.

Several rheumatogenic serotypes of streptococcal M protein have been shown to contain both protective and cardiac tissue crossreactive epitopes. By synthesizing peptides copying different regions of M protein polypeptides, we were able to localize the protective and heart crossreactive epitopes. Some epitopes are only opsonic, some are only crossreactive, whereas others are both opsonic and tissue crossreactive. Multivalency of vaccines can be obtained by synthesizing protective peptides of one M serotype in tandem with protective peptides of other M serotypes. Such hybrid peptides evoke protective immune responses against the related streptococci without evoking tissue crossreactive immunity.

Animals↗
PubMed1987

Human cytotoxic T lymphocytes evoked by group A streptococcal M proteins.

Purified group A streptococcal M proteins were used to stimulate peripheral blood lymphocytes from normal adult volunteers. The activated lymphocytes were cytotoxic against cultured human heart cells, as well as liver cells, fibroblasts, and K562 cells, but showed only minimal cytotoxicity against several animal cell types. The cytotoxic activity evoked by type 5 M protein was dose and time dependent. Rabbit antisera against pep M5 that contained heart-crossreactive antibodies partially inhibited cytotoxicity against heart cells, but had no effect on other target cells, suggesting that a fraction of the effector lymphocytes may be recognizing M protein-crossreactive cell surface antigens. All of the cytotoxic activity was recovered from a CD3+ population of lymphocytes obtained from a fluorescence-activated cell sorter, and CD4+ and CD8+ cells were also cytotoxic. M protein-responsive T cell clones were generated that showed specificity for heart and K562 cells, in addition to clones that were cytotoxic against both cell lines. Our data show that streptococcal M protein evokes cytotoxic T lymphocytes against multiple human but not animal target cells. Some of the effector cells may be specific for cultured myocardial cells, but their role in the pathogenesis of rheumatic carditis will require further studies of lymphocytes from patients with acute rheumatic fever and carditis.

Antigens, Bacterial↗
PubMed1987

Sequence and type-specific immunogenicity of the amino-terminal region of type 1 streptococcal M protein.

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.

Amino Acid Sequence↗
PubMed1987

Protective immunogenicity and T lymphocyte specificity of a trivalent hybrid peptide containing NH2-terminal sequences of types 5, 6, and 24 M proteins synthesized in tandem.

The protective immunogenicity of a hybrid peptide containing tandem copies of types 5, 6, and 24 M protein epitopes was investigated. An NH2-terminal peptide of type 24 M protein was chemically synthesized and then extended to include NH2-terminal peptides of types 6 and 5 M proteins yielding a 34-residue hybrid peptide containing a cysteine residue at its COOH-terminus. When conjugated via the cysteine residue to keyhole limpet hemocyanin (KLH), emulsified in CFA, and injected into rabbits, the synthetic hybrid evoked opsonic antibodies against types 5, 6, and 24 streptococci without stimulating tissue crossreactive immunity. The trivalent hybrid also was capable of priming T lymphocytes in vivo that responded to each of the native serotypes of M protein as well as to the synthetic hybrid peptide in vitro. The primed T cells failed to respond to the individual component peptides contained in the hybrid peptide, suggesting that the hybrid peptide confers conformations resembling the presentations of each of the subpeptides in the respective serotypes of M protein. The brisk immune responses to the type 6 peptide contained in the middle of the tandem hybrid indicates that with judicious placement between proline residues, potentially hidden peptides are readily accessible to the immune system. These results suggest that synthetic tandem peptides can be tailored in a fashion in which each of the component sets of protective epitopes can be made optimally immunoaccessible and immunogenic.

Amino Acid Sequence↗
PubMed1987

Sequence of protective epitopes of streptococcal M proteins shared with cardiac sarcolemmal membranes.

Synthetic peptide fragments spanning the entire amino acid sequence of pep M5 were used to detect epitopes cross-reactive with heart tissue components other than myosin. Heart-cross-reactive pep M5 antibodies were affinity purified by absorption to and elution from purified sarcolemmal membranes. Only one of the synthetic peptides, SM5(164-197)C, inhibited reactivity of the affinity-purified antibodies with pep M5 by ELISA. SM5(164-197)C linked to KLH evoked both opsonic and heart-cross-reactive antibodies in rabbits. In addition to type 5, the immune sera opsonized M types 6, 18, 19, and 49 streptococci. The antisera reacted strongly with isolated cardiac sarcolemmal membranes by immunofluorescence. In Western blots of cardiac tissue, the anti-SM5(164-197)C reacted with a 40 kDa protein but not with myosin. The reaction was inhibited by pep M5 and SM5(164-197)C but not by any of the other peptides spanning pep M5. The cross-reactive anti-SM5(164-197)C affinity purified on sarcolemmal membranes opsonized types 5, 6, and 19 but not type 24 streptococci. These results indicate that SM5(164-197)C contains heart-cross-reactive, opsonic epitopes that are shared among heterologous serotypes of group A streptococci.

Amino Acid Sequence↗
PubMed1987

Stimulation of S-adenosylmethionine synthetase in human lymphocytes by streptococcal M protein.

The effects of the specific antigen M5 protein of group A streptococci on AdoMet synthetase activity and AdoMet levels in peripheral blood (PB) lymphocytes were studied and were compared with the effects of the nonspecific polyclonal T cell mitogen PHA. M5 protein stimulated AdoMet synthetase activity, whereas PHA had a biphasic effect with an early inhibitory effect and a later stimulatory effect on AdoMet synthetase activity. S-Carbamyl-L-cysteine (SCC), an inhibitor of human lymphocyte AdoMet synthetase, reduced AdoMet levels and inhibited the blastogenic response of PB lymphocytes to both M5 protein and PHA. Inhibition of the response to M5 protein was stronger than that to PHA. However, the inhibitory effects of SCC were totally reversible by washing the cells. It is our hypothesis that such differences in the biochemical events triggered by specific antigen as opposed to a polyclonal mitogen may determine the direction of the functional differentiation of T lymphocytes.

Antigens, Bacterial↗
PubMed1987

Monoclonal antibody to human renal glomeruli cross-reacts with streptococcal M protein.

Two murine monoclonal antibodies (immunoglobulin M) evoked against human kidney tissue were examined for cross-reactivity with group A streptococci. A glomerulus-specific antibody, PMII.40.H.2, cross-reacted in an enzyme-linked immunosorbent assay with purified pepsin-extracted M proteins from type 6 and 12 streptococci, but not type 1, 3, 5, 19, and 24 M proteins. The cross-reactive monoclonal antibody also opsonized type 6 and 12 streptococci, indicating that it was directed against protective M protein epitopes that were exposed on the surfaces of these organisms. A control antibody, which was tubule specific, did not cross-react with any of the purified M proteins, nor did it opsonize any of the serotypes of streptococci tested. Western immunoblot experiments identified the glomerular cross-reactive antigen as a 43-kilodalton protein. The results demonstrate that M protein of group A streptococci and glomerular antigens of the human kidney possess cross-reactive determinants.

Animals↗
PubMed1986

Sequence of myosin-crossreactive epitopes of streptococcal M protein.

Group A streptococcal M proteins contain epitopes that crossreact with sarcolemmal membrane proteins of human myocardium and myosin. In the present study, synthetic peptide copies spanning the entire 197-residue pepsin extracted fragment of type 5 M protein were used to localize the myosin-crossreactive epitopes of the molecule. Peptide 84-116 inhibited by 75% the binding of myosin-crossreactive antibodies evoked by pep M5, as determined by ELISA. Immunoblot inhibition studies confirmed that peptide 84-116 almost totally inhibited the binding of pep M5 antibodies to the heavy chain of human cardiac myosin. None of the remaining synthetic peptides, including peptide 1-35, which contains protective epitopes, inhibited antibodies binding to myosin. Two of three rabbits immunized with peptide 84-116 developed low but significant levels of antibodies crossreactive with myosin. Identification of the primary structures containing tissue-crossreactive as opposed to protective epitopes should not only allow the development of safe and effective M protein vaccines, but may also provide insights into the pathogenesis of rheumatic heart disease.

Amino Acid Sequence↗
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

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↗
PubMed1985

Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid.

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Epitopes of streptococcal M proteins shared with cardiac myosin.

We present evidence that M proteins from three different serotypes of group A streptococci share epitopes with cardiac myosin. Rabbit antisera evoked by a purified fragment of type 5 M protein crossreacted with myosin, but not alpha-tropomyosin, actin, or myosin light chains. In enzyme-linked immunosorbent assays, the myosin-crossreactive antibodies were totally inhibited by type 5 M protein and partially inhibited by types 6 and 19 M proteins. The affinity-purified myosin antibodies opsonized type 5 streptococci, indicating that they were directed against protective M protein epitopes on the surface of the organisms. Immunoblot analyses demonstrated the binding of the crossreactive antibodies to myosin heavy chains. Sera from patients with acute rheumatic fever showed significantly stronger reactions with myosin than did sera from their siblings, hospitalized controls, or patients with poststreptococcal glomerulonephritis.

Antibodies, Bacterial↗
PubMed1985

Multiple, heart-cross-reactive epitopes of streptococcal M proteins.

We present evidence that a highly purified pepsin extract of type 5 streptococcal M protein (pep M5) contains at least three epitopes that are cross-reactive with sarcolemmal membrane proteins of human myocardium. The tissue-cross-reactive determinants of pep M5 are also partially shared with pep M6 and pep M19. Three rabbits immunized with a single 300 micrograms dose of pep M5 developed significant levels of heart-cross-reactive antibodies, as determined by indirect immunofluorescence tests. All three sera also contained antibodies that cross-reacted with pep M6 and pep M19. The heart tissue--specific antibodies that were eluted from sarcolemmal membranes opsonized types 5, 6, and 19 streptococci, indicating that they were directed against protective M protein epitopes on the surface of virulent organisms. Immunofluorescence inhibition tests, using purified M proteins as soluble inhibitors of heart-cross-reactive antibodies, revealed the number and M protein serotype distribution of the tissue-cross-reactive epitopes. Immunoblot analyses demonstrated the sarcolemmal membrane proteins containing the various cross-reactive antigenic determinants.

Animals↗
PubMed1985

Expression of protective and cardiac tissue cross-reactive epitopes of type 5 streptococcal M protein in Escherichia coli.

The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.

Antigens, Bacterial↗