Search PubMed⌕ Search

Biomedical subjects

R A Finkelstein

Publications and source records attributed to R A Finkelstein.

At least 55 records · Page 3Linked to original sources
PubMed1986

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.

Animals↗
PubMed1986

Immunity to heat-labile enterotoxins of porcine and human Escherichia coli strains achieved with synthetic cholera toxin peptides.

Antiserum to a synthetic peptide, CTP3, consisting of residues 50 to 64 of the B subunit of cholera enterotoxin (CT), a sequence which is conserved in heat-labile enterotoxins from Escherichia coli strains of human (H-LT) or porcine (P-LT) origin, cross-reacted with and neutralized all three enterotoxins. It also cross-reacted with genetically engineered chimeric B subunit proteins in which H-LT amino acids had been substituted for the corresponding residues in P-LT. Antisera against another CT peptide, CTP1, consisting of residues 8 to 20, were weakly cross-reactive with H-LT but not with P-LT, whereas antisera against four other CT peptides were not cross-reactive with the heterologous enterotoxins. A single dose of CTP3-linked tetanus toxoid, by itself nonimmunogenic, primed rabbits to a vigorous immune response to subsequent administration of single, small, subimmunizing doses of any of the three enterotoxins.

Animals↗
PubMed1986

Competitive enzyme-linked immunosorbent assay for cholera-related enterotoxins in Salmonella typhimurium.

We report a rapid competitive enzyme-linked immunosorbent assay to screen Salmonella typhimurium strains for cholera-related enterotoxin antigens. Polymyxin B extracts of bacterial cells from syncase-glucose broth cultures of 7 of 15 strains gave positive results. The specificity of the test was confirmed with known heat-labile-enterotoxin-positive and -negative Escherichia coli strains which gave significantly different values.

Antigens, Bacterial↗
PubMed1985

Monoclonal antibodies to outer membrane antigens of Vibrio cholerae.

Hybridoma-derived monoclonal antibodies were prepared against outer membrane antigens of four strains of Vibrio cholerae that were cultivated under iron-limited conditions, and these antibodies were partially characterized. We established a library of 66 hybridomas which produced monoclonal antibodies defining 16 different V. cholerae antigens. Two antigens (molecular weights, 18,000 and 112,000) were heat modifiable, whereas the reacting epitope of a third antigen (40,000-dalton-18,000-dalton doublet) was completely destroyed when it was heated at 100 degrees C. The 112,000-dalton heat-modifiable protein was an iron-regulated outer membrane protein. This protein bound 59Fe in vitro when it was combined with the V. cholerae siderophore-iron complex 59Fe-vibriobactin; it was also found in in vivo grown V. cholerae, as were three other antigens. A total of 26 hybridomas produced antibody to V. cholerae lipopolysaccharide. Of these, 12 were cross-reactive with lipopolysaccharides of other gram-negative bacteria, including 2 which recognized lipid A. Several of these anti-lipopolysaccharide monoclonal antibodies appeared to be lipopolysaccharide region specific. Some membrane antigens were strain specific, whereas others were common to both O group 1 and non-O group 1 vibrios.

Antibodies, Monoclonal↗
PubMed1985

Enzyme-linked immunosorbent assay to measure antibodies to purified heat-labile enterotoxins from human and porcine strains of Escherichia coli and to cholera toxin: application in serodiagnosis and seroepidemiology.

Serum immunoglobulin G antibodies to purified heat-labile enterotoxin (LT) from human (LTh) and porcine (LTp) Escherichia coli strains and cholera enterotoxin (CT) were measured by an enzyme-linked immunosorbent assay. Sera from patients with LTh E. coli infection showed a prominent response with LTh, an intermediate response with LTp, and a meager response with CT. Of 47 persons with clinical LTh-producing E. coli (herein shortened to LTh E. coli) infections, significant rises in antitoxin were detected against LTh in 36 (77%), against LTp in 30 (64%), and against CT in only 13 (28%) patients; seroconversions also occurred in 11 of 14 (79%) patients with subclinical LTh E. coli infections. In North Americans with experimental LTh E. coli infection, anti-Lth did not remain at high levels for more than 3 months. Persons with cholera manifested antitoxin responses that were similarly potent against all three toxin antigens; in fact, net optical density values were often slightly higher against LTh than against CT. The ratio of CT/LTh ELISA net optical density in convalescent sera proved to be a sensitive means to differentiate LT E. coli from cholera infection. All 11 cholera patients tested had CT/LTh ratios of greater than 0.70, whereas in only 1 of 47 LTh E. coli infections did the ratio exceed that value (it was 0.71) (P less than 0.0000000001). In single serum specimens, a net optical density of greater than or equal to 0.30 against LTh was shown to be a useful cutoff in screening sera for recent LTh E. coli or past cholera infection. The CT/LTh ratio was then used to differentiate definitively. Sera from healthy 3- to 5-year -olds and 15- to 19-year-olds in Maryland, Chile, and Bangladesh were tested against LTh and CT. The serological results fit known epidemiological observations. (i) LTh infections are rare in the United States (only 2 of 60 sera had LTh net optical density values of >/= 0.30. (ii) In contrast, evidence of recent LTh E. coli infections was very common in Chilean (69%) and Bangladeshi (57%) 3- to 5-year-olds and not uncommon in 15- to 19-year-olds (38 and 31%, respectively) in those countries. (iii) Only Bangladeshi sera showed serological evidence of cholera infections (CT/LTh ratios of > 0.70). The immunoglobulin G enzyme-linked immunosorbent assay measuring antibodies to purified LTh and CT represents a practical and effective tool for the serological study of LTh E. coli and cholera diarrheal infections.

Adolescent↗
PubMed1985

Application of monoclonal antibodies and genetically-engineered hybrid B-subunit proteins to the analysis of the cholera/coli enterotoxin family.

Single amino acid substitutions, introduced by genetic engineering, significantly modify the behavior of the B-subunits of the cholera/coli enterotoxin family in SDS-PAGE and also markedly affect the reactivity of the proteins with mouse hybridoma-derived monoclonal antibodies raised against H-LT. The results indicate that single amino acids play an important role in defining epitopes in these proteins.

Amino Acid Sequence↗
PubMed1984

Evaluation in humans of attenuated Vibrio cholerae El Tor Ogawa strain Texas Star-SR as a live oral vaccine.

Texas Star-SR, an A- B+ mutant derived by nitrosoguanidine treatment from Vibrio cholerae El Tor Ogawa strain 3083, was fed to 68 volunteers as an oral vaccine in doses of 10(5) to 5 X 10(10) organisms with NaHCO3. Sixteen (24%) vaccinees experienced some loose stools (unrelated to vaccine dose), but in only one did the total stool volume exceed 1.0 liter. The vaccine strain was cultured from duodenal fluid of 35 of 46 (76%) persons who ingested doses of 10(8) organisms or greater. No A+ B+ toxinogenic revertants were found among 456 clinical isolates tested. Sixty-three vaccinees (93%) manifested seroconversions of vibriocidal antibody, whereas only 20 (29%) had significant rises in serum antitoxin titers. Paired intestinal fluids from 41 volunteers showed significant rises of secretory immunoglobulin A against lipopolysaccharide (29%), Ogawa outer membrane preparation (29%), and toxin (12%) antigens. In challenge studies with pathogenic V. cholerae El Tor Ogawa and El Tor Inaba, the attack rate in vaccinees (7 of 25) was significantly lower than in controls (18 of 25) (vaccine efficacy, 61%); furthermore, the diarrheal stool volume in vaccinees was significantly less than that in controls (P less than 0.01). Texas Star-SR served as a prototype to investigate the concept of immunoprophylaxis by means of attenuated strains as oral vaccines. These observations provide an invaluable background for planning future studies with newly developed attenuated strains prepared by recombinant DNA techniques.

Administration, Oral↗
PubMed1984

Preparation of procoligenoids from Escherichia coli heat-labile enterotoxins.

Heat-labile enterotoxins from Escherichia coli strains of porcine and human origin polymerize on heating to form high-molecular-weight aggregates, "procoligenoids," analogous to procholeragenoid derived from the cholera enterotoxin. This aggregation is accompanied by loss of biological activity (toxicity). Further heating results in the release of B-subunit oligomers, coligenoids, analogous to choleragenoid. Further studies are needed to determine whether, like procholeragenoid, the procoligenoids are superior antigens in stimulating gut immunity after parenteral administration.

Animals↗
PubMed1984

Vibrio cholerae hemagglutinin/protease nicks cholera enterotoxin.

Unnicked cholera enterotoxin was isolated from culture supernatants of Vibrio cholerae 569B by either rapid processing of flask-grown cultures or by growing and processing fermentor cultures in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid, an inhibitor of the previously described V. cholerae hemagglutinin/protease. When unnicked cholera enterotoxin was incubated with purified hemagglutinin/protease, the unnicked A subunit was converted to a molecular weight consistent with that of the A1 subunit as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity for Y1 adrenal cells increased.

Cholera Toxin↗
PubMed1983

Immunological differences among the cholera/coli family of enterotoxins.

Pure enterotoxins from two strains of Vibrio cholerae and choleragen-related heat-labile enterotoxins (LTs) from strains of Escherichia coli of human and porcine origin were examined by Ouchterlony-type immunodiffusion assays and by neutralization tests in Y-1 adrenal cells using specific and immunopurified antisera. In accordance with previous findings, the results indicated that each of the toxins shared antigens with each of the others and that each, in addition, possessed unique antigenic determinants. The present study, however, indicates further that enterotoxin-type specific antibodies, in some instances, account for a major portion of the neutralizing activity. The full extent and importance of the antigenic drift in the cholera/coli family of ADP-ribosylating, adenylate cyclase-activating, heat-labile enterotoxins remains to be determined. The results will be pertinent to efforts to develop broad-spectrum antitoxic immunity as well as in the development of methods for the rapid detection and identification of enterotoxigenic strains.

Bacterial Toxins↗
PubMed1983

Vibrio cholerae hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F.M. Burnet revisited.

Cholera vibrios produce a single polymeric protein that (i) causes hemagglutination; (ii) appears to participate in their attachment to gut epithelium; (iii) may mediate their detachment from gut epithelium; and (iv) is a protease that hydrolyzes fibronectin and mucin, cleaves lactoferrin, and nicks the A subunit of the choleragen-related heat-labile enterotoxin of Escherichia coli.

Animals↗
PubMed1983

Vibrio cholerae soluble hemagglutinin/protease is a metalloenzyme.

A soluble hemagglutinin/protease produced by Vibrio cholerae, which has previously been shown to hydrolyze fibronectin and ovomucin and to cleave lactoferrin and the A subunit of the heat-labile enterotoxin of Escherichia coli, appears to be a zinc metalloendopeptidase. Both its hemagglutinative and protease functions are inhibited by chelating agents, including Zincov, a hydroxamic acid derivative specifically designed to inhibit zinc metalloproteases. Thermolysin, a known zinc-containing protease, also causes hemagglutination of responder chicken erythrocytes. This activity is inhibited by Zincov, which does not affect the hemagglutination activity of trypsin and pronase. The hemagglutinin/protease is active on furylacryloyl-Gly-Leu-NH2, a synthetic substrate for thermolysin and other similar proteases. The hemagglutination activity of V. cholerae-infected or cholera toxin-treated infant rabbit intestinal fluid is not inhibited by Zincov, which suggests that this activity is not due to the hemagglutinin/protease, as formerly proposed.

Calcium Chloride↗
PubMed1983

Vibrio cholerae expresses iron-regulated outer membrane proteins in vivo.

A comparison was made, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of the outer membrane proteins of four strains of Vibrio cholerae grown in vivo in infant rabbits and in vitro in low-iron and iron-supplemented defined media. In vivo-grown V. cholerae expressed novel outer membrane-associated proteins which, in part, were similar to those observed on V. cholerae grown in vitro under conditions of iron deprivation.

Animals↗