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Biomedical subjects

F A Klipstein

Publications and source records attributed to F A Klipstein.

At least 19 recordsLinked to original sources
PubMed1986

Immunisation of volunteers with a synthetic peptide vaccine for enterotoxigenic Escherichia coli.

A completely synthetically produced peptide vaccine, consisting of the 18-aminoacid Escherichia coli heat-stable toxin and the 26-aminoacid epitope of the heat-labile toxin B subunit, was given orally to thirteen volunteers. It raised antitoxin titres to both toxin components four-fold in serum samples and seven-fold in jejunal aspirates over preimmunisation control titres. Jejunal aspirates taken after immunisation from vaccinees, but not controls, neutralised the secretory activity of both toxins in appropriate biological assays. These findings show that synthetically produced vaccines are immunogenic in human beings. The peptide used is a promising vaccine for diarrhoeal disease caused by enterotoxigenic strains of E coli.

Adult↗
PubMed1986

Enzyme-linked immunosorbent assays for virulence properties of Campylobacter jejuni clinical isolates.

To evaluate the capacity of enzyme-linked immunosorbent assays (ELISAs) to identify pathogenic strains among clinical fecal isolates of Campylobacter jejuni, 40 consecutively obtained strains from 39 sick patients and 1 asymptomatic person were tested by respective ELISAs for enterotoxin production in culture filtrates and for the invasive virulence antigen of bacterial cells. Of the 40 strains, 14 produced the enterotoxin; 15 strains, two of which were also enterotoxigenic, were invasive; and 11 strains had no detectable virulence property. The presence or absence of these virulence properties was confirmed by the demonstration that viable cells of all 12 randomly selected enterotoxigenic or invasive strains tested, but none of 9 nonpathogenic strains tested, caused fluid secretion in rat ligated ileal loops. All 12 patients examined who were infected with an invasive strain had grossly or microscopically evident blood cells in their stools or both, whereas none of those infected with an enterotoxigenic strain had overtly bloody diarrhea, and only 1 of 8 patients examined had microscopically evident blood cells in the stool. Twelve of the invasive, five of the enterotoxigenic, and three of the nonpathogenic strains also produced small amounts of cytotoxin, but there was no correlation between cytotoxin production and an abnormal response in rat ligated ileal loops. These observations show that enterotoxin production or invasiveness or both can be detected by ELISAs in three-fourths of C. jejuni fecal isolates and that there is usually a relationship between the specific pathogenic property of the infecting strain and the clinical mainfestations.

Adolescent↗
PubMed1985

Immunological relationship of the B subunits of Campylobacter jejuni and Escherichia coli heat-labile enterotoxins.

The application of dissociation techniques, involving gel filtration in the presence of guanidine, to a semipurified preparation of Campylobacter jejuni heat-labile enterotoxin yielded a material whose functional and immunological properties resemble those of the B subunits of cholera toxin and Escherichia coli heat-labile toxin (LT). The C. jejuni toxin B subunit reacted with GM1 ganglioside in an enzyme-linked immunosorbent assay, but lacked the holotoxin's cytotonic activity in the Chinese hamster ovary tissue culture assay and its ability to cause fluid secretion in rat ileal ligated loops. The C. jejuni toxin B subunit showed lines of partial identity with the B subunits of both cholera toxin and LT in gel immunodiffusion; it appeared to be more closely related immunologically to the LT B subunit than to the cholera toxin B subunit in enzyme-linked immunosorbent assays that used antisera either to LT or to its B subunit. Rats immunized with LT B subunit were significantly protected against challenge with either the semipurified C. jejuni toxin or a viable enterotoxigenic strain of C. jejuni, although twice the immunization dosage was required to achieve protection comparable to that against the homologous toxin or viable bacteria. These observations indicate that the C. jejuni enterotoxin contains a B subunit that bears an immunological relationship with the B subunits of cholera toxin and LT.

Animals↗
PubMed1985

A completely synthetic toxoid vaccine containing Escherichia coli heat-stable toxin and antigenic determinants of the heat-labile toxin B subunit.

The immunodeterminant regions of the Escherichia coli heat-labile toxin B subunit were identified by determining the antigenicity, by using enzyme-linked immunosorbent assays, of synthetically produced peptides corresponding to various segments of its 124-amino-acid sequence. The addition of the 18-amino-acid sequence of heat-stable toxin (ST) to some of these peptides enhanced their B subunit antigenicity. Peptide residues containing the 26 amino acids of B subunit sequence 58 to 83 joined to the 18-amino-acid sequence of ST yielded a 44-amino-acid peptide whose antigenicity was 50% that of both native B subunit and ST. This peptide was completely nontoxic when tested in Chinese hamster ovary tissue culture, suckling mouse, and rat ligated ileal loop assays. Peroral immunization of rats with the polymeric form of this peptide yielded a dose-dependent response of intestinal immunoglobulin A antitoxin titers to both the ST and B subunit components and provided strong protection against challenge with viable ST- and heat-labile toxin-producing E. coli strains. The immunogenicity of the synthetic peptide in rats was the same as that of ST and about 50% that of native B subunit. The completely synthetic peptide vaccine has the following advantages over previously described toxoid vaccines that consist of synthetic ST chemically cross-linked to native B subunit derived from bacterial cultures: it is produced by a single synthetic process, it is completely nontoxic, and it is immunogenic for both ST and B subunit.

Amino Acid Sequence↗
PubMed1985

Mucosal antitoxin response in volunteers to immunization with a synthetic peptide of Escherichia coli heat-stable enterotoxin.

Peroral immunization of volunteers on four weekly occasions with 750 micrograms of a conjugate containing 3,000 antigen units of a synthetically produced peptide of hyperantigenic Escherichia coli heat-stable (ST) toxin, conjugated with the heat-labile toxin B subunit as a carrier, raised serum immunoglobulin G antitoxin titers to ST by fourfold and intestinal immunoglobulin A antitoxin titers to ST by sevenfold over control values at five weeks postimmunization. The ability of jejunal aspirates from the immunized volunteers to neutralize ST in the suckling mouse assay correlated with the intestinal immunoglobulin A ST antitoxin response determined by enzyme-linked immunosorbent assay.

Adult↗
PubMed1985

Pathogenic properties of Campylobacter jejuni: assay and correlation with clinical manifestations.

The pathogenic properties of 20 strains of Campylobacter jejuni isolated from persons with clearly defined clinical manifestations were determined. Cell-free broth filtrates were examined for (i) enterotoxin production by Chinese hamster tissue culture assay and an enzyme-linked immunosorbent assay (ELISA) employing GM1 ganglioside and affinity-purified antiserum to Escherichia coli heat-labile toxin, (ii) cytotoxin production by Vero and HeLa cell tissue culture lines, and (iii) their ability to cause fluid secretion in rat ligated ileal loops. Viable bacteria were examined for invasive properties by an ELISA with the immunoglobulin fraction of antiserum to Formalin-killed bacteria of an invasive strain, and by their effect on fluid secretion and morphology in rat ligated ileal loops. None of the eight isolates obtained from asymptomatic carriers had any detectable pathogenic properties. All six strains isolated from persons with bloody invasive-type diarrhea elaborated a cytotoxin; their viable bacteria had high titers in the ELISA for invasive properties and caused fluid secretion in ligated ileal loops, although consistent morphologic abnormalities and evidence of mucosal invasion, examined by immunofluorescence techniques, were not detected. All six strains isolated from persons with watery secretory-type diarrhea produced an enterotoxin, one elaborated a cytotoxin, and broth filtrates of all strains caused fluid secretion in ligated ileal loops; viable bacteria had low titers in the ELISA for invasive properties and evoked fluid secretion in ligated loops by means of enterotoxin production. These observations show (i) that a correlation exists between the pathogenic properties of the infective C. jejuni strain and gastrointestinal manifestations in the infected host, and (ii) that these pathogenic properties can be identified by in vitro assays, including ELISAs.

Animals↗
PubMed1984

Chemical synthesis of an octadecapeptide with the biological and immunological properties of human heat-stable Escherichia coli enterotoxin.

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid phase peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography. This material was pure and identical to native ST by analytical high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the suckling mouse assay respectively.

Amino Acid Sequence↗
PubMed1984

Differences in cross-protection in rats immunized with the B subunits of cholera toxin and Escherichia coli heat-labile toxin.

Although cholera toxin (CT), Escherichia coli heat-labile toxin (LT), and their B subunits are known to be immunologically related, the ability of each to raise an antitoxin response that provides equally strong cross-protection against active challenge with pure heterologous toxin has not been examined previously. We immunized rats with pure preparations of the B subunits of human LT, porcine LT, and CT. Immunization with either of the LT B subunits raised greater than or equal to fourfold increases in specific mucosal immunoglobulin A antitoxin titers to homologous and heterologous LT and CT B subunits, thereby providing strong protection against active challenge in ligated ileal loops with all three respective holotoxins and with a viable LT-producing E. coli strain. In contrast, immunization with the CT B subunit raised a greater than or equal to fourfold increase in antitoxin titers only to itself and provided strong protection only against challenge with the CT holotoxin. Conjugation of the CT B subunit with the E. coli heat-stable toxin by the carbodiimide reaction yielded a cross-linked immunogen with equal antigenicity for both components; immunization with this conjugate raised greater than or equal to fourfold increases in antitoxin titers to both components, but it provided significant protection only against challenge with a viable heat-stable toxin-producing E. coli strain and not to an LT-producing E. coli strain. These observations indicate that immunization with the LT B subunits raises a heterologous antitoxin response that extends to the CT B subunit, thereby providing equally strong protection against LT and CT; however, immunization with the CT B subunit raises principally a homologous antitoxin response, so that this immunogen provides strong protection only against CT.

Animals↗
PubMed1984

Properties of cross-linked toxoid vaccines made with hyperantigenic forms of synthetic Escherichia coli heat-stable toxin.

The ability of hyperantigenic preparations of synthetically produced Escherichia coli heat-stable toxin (ST) to provide an immunogenically more potent vaccine when cross-linked by the glutaraldehyde reaction to the heat-labile toxin B subunit was assessed. Three synthetic ST preparations were evaluated: ST(S) had the same antigenicity and toxicity (secretory potency in the suckling mouse assay) as native ST, ST 1056 had 3.5-fold more antigenicity and 1% toxicity, and ST(C) had 15-fold greater antigenicity and 31% toxicity. Vaccines that contained equal antigenic proportions of ST and B subunit, as determined by enzyme-linked immunosorbent assays, consisted by weight of 52% ST(S), 25% ST 1056, and 9% ST(C). The initially lower toxicity and smaller proportions by weight of hyperantigenic ST preparations yielded vaccines that had nearly 10-fold less residual ST toxicity than the ST(S) vaccine. Immunization of rats with graded dosages of vaccines containing 9% ST(C) and 51% ST(S) by weight, but equal amounts of ST(S) antigenicity, raised to the same degree dose-dependent increases in mucosal immunoglobulin A antitoxin titers to ST(S) which correlated with the amount of protection against challenge with a viable LT-/ST+ strain. These observations indicate that hyperantigenic synthetic ST preparations provide immunologically more potent vaccines than those obtained with the previously used synthetic ST(S) preparation, which has the same biological properties as native ST.

Animals↗
PubMed1984

Properties of crude Campylobacter jejuni heat-labile enterotoxin.

The amount of crude Campylobacter jejuni enterotoxin present in culture products was quantitated by comparing the response of these preparations with that of pure Escherichia coli heat-labile toxin (LT) in the Chinese hamster ovary assay and in enzyme-linked immunosorbent assays that used GM ganglioside or antisera to LT or both. Maximum C. jejuni enterotoxin production was achieved by growth at 42 degrees C for 24 h under agitation in supplemented GC medium. Adding polymyxin separately to either the broth supernatant or the cells enhanced the recovery of toxin; the yield from cell lysates was much lower. The quantity of C. jejuni enterotoxin produced by clinical isolates obtained locally or provided from Mexico varied widely, over a spectrum from none to large amounts; quantitative values for the amount of C. jejuni enterotoxin determined by the Chinese hamster ovary and enzyme-linked immunosorbent assays correlated with the degree of secretory potency of this material in ligated rat ileal loops. The cytotonic activity of C. jejuni enterotoxin in Chinese hamster ovary cells was abolished by heating at 96 degrees C for 10 min and by preincubation either with GM ganglioside or with LT or cholera toxin antisera. The secretory activity of C. jejuni enterotoxin in ligated rat ileal loops was passively neutralized by antiserum to LT, and immunizing rats with either LT or its B subunit significantly (P less than 0.001) reduced fluid response to active challenge with C. jejuni enterotoxin in ligated ileal loops. These observations indicate that strains of C. jejuni vary in their capacity to elaborate a heat-labile enterotoxin that has close immunological homology with LT and cholera toxin.

Animals↗
PubMed1984

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect pure native Escherichia coli heat-stable toxin (ST) and to identify ST-producing strains among clinical isolates was determined. Two synthetically produced ST preparations were used to raise hyperimmune antisera in rabbits and goats: ST(S), which has the same antigenicity as native ST; and ST(C), which is 15-fold more immunogenic. These antisera were used in the double-sandwich technique as either crude double-species antisera or pure single-species antibody. The sensitivity of the assay was increased by using either a purer antibody preparation or the antiserum to the more potent immunogen; the assay in which pure antibody to ST(C) was used was 2,857-fold more sensitive in detecting ST than the assay in which crude antiserum to ST(S) was used. The minimum amount of ST detectable by the ST(C) ELISA was 140 pg/ml, which was an amount 285-fold smaller than that detectable by the suckling mouse assay. Among 50 human E. coli isolates examined by both the ST(C) ELISA and an ELISA for heat-labile toxin (LT), which had a sensitivity of 290 pg/ml for LT, the respective toxins were consistently identified in broth cultures of 10 LT+ and ST-, 15 LT+ and ST+, and 10 LT- and ST+ strains, and there were no false-positive responses. The ST(C) ELISA also detected ST in all of seven ST - producing E. coli strains tested of human origin, which had been shown elsewhere by DNA hybridization probes to have ST-coding genes of either human or porcine origin, and in all of three ST-producing E. coi strains tested of porcine origin. These results indicate that the sensitivity of the ST(C) ELISA is the same as that of previously described LT ELISAs. The concomitant use of both ST and LT ELISAs provides a rapid, simple, and sensitive method for identifying among clinical isolates enterotoxigenic strains of E. coli which produce either toxin.

Animals↗
PubMed1983

Vaccine for enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-linked to the B subunit of heat-labile toxin.

Synthetically produced Escherichia coli heat-stable toxin (ST) was conjugated to the nontoxic B subunit of the heat-labile toxin (LT) by the carbodiimide reaction. Modifying the molar ratio of toxins mixed and the ratio of carbodiimide added to the toxins permitted synthesis of conjugates with any desired degree of proportional antigenicity for each toxin component. Immunization of rats by the parenteral/peroral routes with cross-linked vaccine containing 39% ST and 61% B subunit antigenicity, with 0.06% residual ST toxicity, evoked fourfold to sevenfold increases over control values of serum IgG and mucosal secretory IgA antitoxin titers to each of the component toxins, thus providing significant (P less than 0.001) protection against challenge with either LT or ST or with viable heterologous strains which produce these toxins. These observations show that cross-linking synthetic ST to the B subunit results in a nontoxic vaccine that provides protection against all types of enterotoxigenic E. coli.

Animals↗
PubMed1983

Properties of synthetically produced Escherichia coli heat-stable enterotoxin.

The properties of a synthetically produced peptide composed of the same primary structure of 18 amino acids described for human Escherichia coli heat-stable enterotoxin were compared with those of purified heat-stable toxin obtained by bacterial growth. The dosage required to evoke fluid secretion in the suckling mouse and rat ligated ileal loop assays was the same for both toxins. The antigenicity of the two toxins was similar when assayed by enzyme-linked immunosorbent assay with hyperimmune antiserum to either toxin. The secretory effect of the two toxins in the suckling mouse assay was seroneutralized by the same dilutions of hyperimmune antiserum to either toxin. Immunization of rats with the synthetic toxin coupled to a large-molecular-weight carrier raised serum and mucosal antitoxin responses which provided protection against challenge with either the synthetic or biological toxin as well as with viable heat-stable enterotoxin-in-producing organisms. These observations indicate that synthetically produced heat-stable toxin has the same properties as the toxin derived by bacterial culture. The availability of the more readily made synthetic form of heat-stable toxin should facilitate the production of a vaccine based on cross-linking this toxin with either the heat-labile toxin or its nontoxic B subunit.

Animals↗
PubMed1983

Peroral immunization of rats with Escherichia coli heat-labile enterotoxin delivered by microspheres.

The antigenicity of the Escherichia coli heat-labile enterotoxin was not protected against the adverse effect of gastric acidity when the toxin was given together with bicarbonate for peroral immunization to rats, but immunization with the heat-labile enterotoxin encapsulated in pH-dependent microspheres aroused the same strong degree of serum and mucosal antitoxin responses and of protection against challenge as was achieved by peroral immunization after ablation of gastric secretions by pretreatment with cimetidine.

Administration, Oral↗
PubMed1983

Cloning and molecular characterization of the B subunit of Escherichia coli heat-labile enterotoxin.

We have constructed a plasmid containing the gene for production of the B subunit of the heat-labile enterotoxin (LT-B) from a human isolate of Escherichia coli, strain H10407. The 0.8-kilobase gene fragment encoding synthesis of LT-B was cloned onto plasmid pBR322 after sequential digestion of the enterotoxin plasmid of strain H10407 with restriction endonucleases PstI and HindIII. LT-B was isolated by agarose affinity chromatography from cell lysates of recombinant clones expressing the B subunit. The B subunit was isolated in its oligomeric form, was structurally identical to native B subunit when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dissociated to monomeric B in the presence of 5 M guanidine, was immunologically identical to native B subunit in an enzyme-linked immunosorbent assay, and contained no demonstrable A subunit in any of the assays.

Antibodies, Bacterial↗