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Biomedical subjects

E D Gray

Publications and source records attributed to E D Gray.

At least 37 records · Page 2Linked to original sources
PubMed1982

Characterization of the human cellular immune response to purified group A streptococcal blastogen A1.

The human mononuclear cell response to purified extracellular streptococcal protein, blastogen A, was compared to the response of these cells to PHA and tetanus antigen. Blastogen A induced peak uptake of thymidine during day 6 of tissue culture whereas PHA induced peak uptake during day 5 and tetanus during day 8. Like PHA, blastogen A transformed human umbilical cord lymphocytes and those of nonimmune animals. Also like PHA, blastogen A transformed primarily T lymphocytes. However, unlike PHA, the ability of T lymphocytes to respond to blastogen A was almost completely dependent on the presence of viable non-T lymphocytes. Monocytes were not as effective in facilitating the response to blastogen A as they were for PHA. Thus, blastogen A behaves most like a polyclonal T lymphocyte mitogen, although the degree of dependence of the transformation response on the presence of non-T lymphocytes is much greater than that of PHA.

Animals↗
PubMed1981

Cellular immune responses to extracellular streptococcal products in rheumatic heart disease.

The lymphocyte transformation responses to purified preparations of two extracellular products of group A streptococci (blastogen A and nuclease B), to phytohemagglutinin, and to Candida albicans antigen were measured in tonsillar and peripheral blood lymphocytes from patients with rheumatic heart disease (RHD) and suitably matched nonrheumatic (control) subjects. The mean phytohemagglutinin dose responses of tonsillar and peripheral lymphocytes from RHD patients were essentially indistinguishable from those of controls. In contrast, the responses of tonsillar and peripheral blood lymphocytes to the two extracellular products of group A streptococci were significantly lower in RHD patients than in nonrheumatic control subjects. Candida antigen produced very little stimulation of lymphocytes in any of the subjects. The geometric means of antibody levels against streptolysin O, nuclease B, and nicotinamide adenine dinucleotidase showed no consistent differences between the control group and the group of RHD subjects. Group A streptococci were isolated from the tonsils of approximately 25% of both groups of subjects. The RHD patients clearly had a depressed cellular immune response to the two purified streptococcal extracellular antigens. The equal frequency in recovery of group A streptococci from tonsils and the absence of consistent difference in titers of humoral antibodies to streptococcal extracellular antigens, particularly nuclease B, suggest that this differential response is not due to a lower level of stimulation by repeated exposure to group A streptococcal products.

Antigens, Bacterial↗
PubMed1980

Biochemical and immunological characterization of the extracellular nucleases of group B streptococci.

Nearly all group B streptococcal strains representing the five major serotypes were found to produce extracellular nucleases by screening with an agar-well-diffusion technique in DNA-methyl green agar plates. Three different nucleases have been isolated and partially purified by DEAE-and carboxymethyl-cellulose chromatography. They possessed different mobilities on polyacrylamide gel electrophoresis and different molecular weights. These nucleases, designated I, II, and III, are optimally activated by cations of calcium and manganese and exhibited RNase as well as DNase activity. Despite differences in their physical and biochemical properties, nucleases II and III appear antigenically similar, but distinct from nuclease I. These group B streptococcal nucleases are immunologically different from the nucleases of group A streptococci. Neutralizing activity, probably antibody, to nucleases II and III was found in human sera, and was most prevalent in sera of pregnant women colonized with group B streptococci and in their newborn infants.

Bacterial Proteins↗
PubMed1980

Isolation and characterization of cell-associated nucleases related to streptococcal extracellular deoxyribonuclease D.

A group of at least four distinct nucleases designated DcI through DcIV were isolated from cellular extracts of group A streptococcal strain S43 and shown to be antigenically similar to streptococcal extracellular deoxyribonuclease (DNase) D. These cellular endonucleases degraded single- and double-stranded deoxyribonucleic acid (DNA) as well as ribonucleic acid (RNA) to acid-soluble oligonucleotides. The products of digestion of DNA bore 5'-terminal phosphates, and in partial digests pdX-pdG linkages were most susceptible and pdA-pdX linkages were most resistant to nuclease action. The enzymes had pH optima of 8.0 to 8.5, were inhibited by NaCl, were unaffected by sulfhydryl modifying reagents, and absolutely required a divalent cation. Nucleases DcIII and DcIV were apparently hydrophobic in nature since they required the presence of detergents for migration on nondenaturing polyacrylamide gels. All four nucleases were electrophoretically distinct on such gels, from each other, and from DNase D. Molecular weights of DcI and DcII were similar to that of DNase D, suggesting that the mobility differences of these enzymes at least are reflections of differing net charges. It is suggested that the cellular nucleases represent a group of processing intermediates in the maturation and excretion of DNase D.

Deoxyribonucleases↗
PubMed1980

Enzyme-linked immunosorbent assay of antibodies in human sera to streptococcal DNase B.

An ELISA method for determining the streptococcal anti--DNase B titers in human sera is presented. The details of this technique, a method for converting A493 readings into titers, and a standardization procedure are discussed. Anti--DNase B titers of 20 sera determined by ELISA were compared with titers determined by the microtechnique. A correlation coefficient of 0.96 between the two methods was obtained. The reproducibility of the ELISA method was established by comparing titers obtained from two separate determinations on the 20 sera. Twenty-four pairs of acute and convalescent sera were assayed for anti--DNase B titers by both ELISA and microtechnique to compare the ability of the two techniques to identify significant titer rises. The ELISA was as specific and possibly more sensitive than the microtechnique in identifying significant anti--DNase B titer rises. This ELISA method is simple and rapid and has the potential for automation.

Adolescent↗
PubMed1979

Purification and properties of an extracellular blastogen produced by group A streptococci.

An extracellular product of group A streptococci which induces lymphocyte blastogenesis has been purified to homogeneity by DEAE-cellulose and CM-cellulose chromatography. The protein, termed streptococcal blastogen A, has a mol wt of approximately or equal to 17,500 and is inactivated by protease treatment and by heating at 100 degrees C. The purified blastogen gave rise to multiple protein bands on nondenaturing polyacrylamide gel electrophoresis, only two of which possessed blastogenic activity. Treatment of the protein with dithiothreitol before electrophoresis resulted in the apparent conversion of the multiple forms to a single active species. Blastogen A differs in electrophoretic mobility from the streptococcal pyrogenic exotoxins and its lymphocyte stimulating activity is not inhibited by rabbit antisera to the exotoxins. An enzyme immunoassay has been developed to measure human antibodies against blastogen A. A selection of sera with varying levels of anti-DNase B contained antiblastogen A-IgG.

Antibodies, Bacterial↗
PubMed1979

Immunological properties of hyaluronidases associated with temperate bacteriophages of group A streptococci.

The antigenic relationships of hyaluronidases, bound and free, associated with temperate bacteriophages of group A streptococci were examined with antibody against purified whole phage and with antibody against phage-bound enzyme released by urea and purified to homogeneity. Studies performed by double diffusion in agar (ouchterlony) with antibody against the homologous purified enzyme from a temperate phage of a type 49 streptococcus indicated that the bound and free enzyme gave a single line of identity and that the free hyaluronidase activities in induced lysates of four strains of M type 49 streptococci were immunologically indistinguishable but different from the enzyme in induced lysates of a heterologous type. The four M type 49 strains were from widely different geographical or temporal sources and of different phage subtypes as determined by lyxic patterns. These findings were confirmed in studies that employed a functional assay of enzyme neutralization. An immunoglobulin preparation of antiserum against the purified enzyme as well as one against homologous purified whole phage neutralized the hyaluronidase activity produced by induction of the M type 49 strains and present either phage-bound or soluble in phage-free lysates. These immunoglobulin preparations had little effect on the hyaluronidase activities present in phage-lysates of other M types of group A streptococci. Inhibition of propagation of temperate phages by antibody against the purified phage hyaluronidase paralleled the neutralization of phage-associated enzyme activity by this antibody, indicating that antibody to the purified enzyme can inhibit phage infection. Antibody preparations against the purified phage-bound enzyme or against purified whole phage did not neutralize the extracellular hyaluronidase in the supernate of an uninduced culture of M type 4 streptococci. A human serum strongly inhibitory for the extracellular enzyme of this strain or on the purified phage enzyme from an M type 49 strain. The results support the view that the hyaluronidases associated with the temperate bacteriophages from various M types of group A streptococci do not share common antigenic determinants but that an immunological specificity exists that parallels the serologic specificity of the M protein of the host strains.

Antibody Specificity↗
PubMed1979

Bactericidal effect of oleic acid on group A streptococci: mechanism of action.

In contrast to Staphylococcus aureus and coagulase-negative staphylococci, group A streptococci are infrequently present on normal human skin, except in certain populations with endemic impetigo. This has been attributed to differences in susceptibility to the bactericidal effect of skin surface lipids, particularly unsaturated fatty acids. When an M type 6 strain group A streptococcus was exposed to 500 mug of oleic acid per ml, viable counts decreased by 4 logs in 5 min. The rank order of killing was 35 > 20 > 4 degrees C. Oleic acid did not kill a strain of S. aureus, a strain of coagulase-negative staphylococcus, or a strain of Escherichia coli, but bound rapidly to these bacteria as well as to the group A streptococcus. The loss of [(3)H]uridine from labeled oleic acid-treated group A streptococcal cells was greater than 100 times that of controls. There was no loss of [(3)H]-thymidine from group A streptococci or of [(3)H]uridine or [(3)H]thymidine from identically exposed coagulase-negative staphylococci. When [(3)H]uridine was added to group A streptococci during mid-log-phase growth, cessation of uptake occurred within 5 min of addition of 50 mug of oleic acid per ml. Electron microscopic changes seen within 5 min included condensation of the nucleoid and distortion of the streptococcal surface by numerous clumps and blebs. Coagulase-negative staphylococci, S. aureus, and E. coli similarly exposed showed no comparable electron microscopic changes. We propose that oleic acid kills group A streptococci by altering the integrity of the cell membrane with resulting loss of ribonucleic acid but not deoxyribonucleic acid.

Escherichia coli↗
PubMed1978

Serological inhibition of blast transformation to purified streptococcal antigens by planned immunization in HLA (A,B) compatible unrelated individuals.

Sera obtained from planned immunizations between unrelated donors and recipients, identical or compatible at HLA-A and B, were assessed for their capacity to alter the in vitro response of a test panel of lymphocytes to PHA and a purified streptococcal antigen (PAS). In the case of PHA, no serum effects were apparent. The response to PAS, however, significantly inhibited by two sera. When tested for their complement-dependent cytotoxicity on enriched populations of T and B lymphocytes, none of the sera manifested cytotoxicity against T cells nor did serological inhibition correlate with the capacity to lyze B cells. The data suggest that inhibition of the PSA response is mediated by blocking antibodies specific for a subset of lymphocytes, possibly T cells. While the precise mechanism governing the response to PSA is not known, the data are compatible with the idea that an HLA-linked Ir gene, expressed on a subset of T lymphocytes, controls immune responsiveness to PSA.

Antibody Specificity↗
PubMed1978

Interrelationships of isoacceptor phenylalanine tRNA species of Rhodopseudomonas sphaeroides.

The phenylalanine tRNA of Rhodopseudomonas sphaeroides was fractionated on benzoylated diethylaminoethyl-cellulose into four isoaccepting species (tRNAPheI to IV). tRNAPheIII represented 80% of the total tRNAPhe in anaerobic, photosynthetically grown organisms, whereas in cultures grown aerobically for prolonged periods, tRNAPheII represented 80% of the total. In cultures adapting to aerobic growth, the addition of rifampin resulted in a tRNAPhe profile characteristic of anaerobic-photosynthetic conditions due to the conversion of tRNAPheII to tRNAPheIII. In fully adapted aerobic cultures, this conversion was inhibited in the presence of chloramphenicol or rifampin. The conversion of tRNAPheIII to tRNAPheII was not observable in vivo. It is proposed that an enzymic activity synthesized during anaerobic-photosynthetic growth was responsible for the conversion.

Aerobiosis↗
PubMed1978

Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci.

Urea treatment of a temperate bacteriophage from a type 49 strain of group A streptococcus (Streptococcus pyogenes) followed by ammonium sulfate fractionation, ion exchange, and affinity chromatography of solubilized proteins provided for the recovery (12%) and purification (44-fold) of the phage-associated hyaluronidase. The molecular weight of the homogeneous, purified enzyme was estimated to be 71,000 by polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) and 75,000 by gel filtration with Sephacryl S-200. The enzyme has a pH optimum of 5.5, a Vmax of 0.1 absorbance unit/min per microgram of protein, and a Km of 4.8 X 10(-2) mg/ml with umbilical cord hyaluronic acid as substrate. Of the cations tested, calcium and magnesium were the only effectors of the enzyme. The enzyme is a glycoprotein (7.25% carbohydrate) containing glucose, galactose, and glucosamine. Analysis of the amino acid composition revealed a predominance of acidic amino acids and a relatively high content of cysteine. The partial specific volume, estimated from the amino acid and sugar analyses, was 0.725 cm3/g.

Amino Acids↗
PubMed1977

Hyaluronidase activity of bacteriophages of group A streptococci.

A sensitive dye-binding assay was employed to study the hyaluronidase associated with temperate and virulent phages infected group A streptococci. Some enzyme was detectable in each purified phage preparation examined, but differences of several orders of magnitude separated the lower enzyme levels in virulent phages that required the addition of hyaluronidase for plaque formation and the higher levels in temperate phages that did not. Infection by virulent phage A25 was accompanied by the production of levels of hyaluronidase proportionate to the average burst size. Hyaluronidase was produced during infection by temperate phages at a much higher level than could be accounted for by the number of phage particles formed. The major portion of this hyaluronidase was free and apparently unassociated with phage or phage fragments. The phage-associated enzyme was tightly bound but could be released and solubilized by treatment with urea.

Bacteriophages↗
PubMed1975

Association of HL-A 5 and immune responsiveness in vitro to streptococcal antigens.

Lymphocytes, from randomly selected individuals having normal immune function, when incubated in vitro with varying concentrations of streptococcal antigens, responded in three ways: (a) response over the entire antigen concentration range, i.e., responders; (b) low response to only the highest antigen concentrations; and (c) no response at any antigen concentration. Frequency distribution analysis of these groups indicated that a significant association occurred between the ability to respond and HL-A 5.

Adult↗
PubMed1975

Isolation of streptococcal nuclease B by batch adsorption.

A method has been developed for the preparation of streptococcal nuclease B by batch adsorpton to diethylaminoethyl-cellulose. The enzyme is homogeneous with respect to nuclease activity and is suitable for use as an antigen in measurement of anti-deoxyribonuclease B levels in sera.

Adsorption↗