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Biomedical subjects

F A Klipstein

Publications and source records attributed to F A Klipstein.

At least 37 records · Page 2Linked to original sources
PubMed1983

Protection in rabbits immunized with a vaccine of Escherichia coli heat-stable toxin cross-linked to the heat-labile toxin B subunit.

Rabbits and rats were immunized with a vaccine consisting of synthetically produced Escherichia coli heat-stable toxin cross-linked by the carbodiimide reaction to the B subunit of biologically produced porcine heat-labile toxin. The vaccine contained 50% of each toxin component by weight and antigenicity; the toxicity of the heat-stable enterotoxin component was reduced by greater than 600-fold. Two or three peroral immunizations with vaccine containing 1,000 antigen units of each component raised greater-than-threefold increases in specific mucosal immunoglobulin A antitoxin titers to each component in all animal groups. Protection index values for challenge with either heat-labile or heat-stable toxins in ligated ileal loops were 3.4 to 4.0 in rats immunized by a parenteral primary immunization followed by two peroral booster immunizations, greater than 9 in rabbits immunized by these routes, and greater than 8 in rabbits given just three peroral immunizations. The antigenicity of the B-subunit component of the peroral vaccine was protected equally well against gastric acidity either by pretreatment with cimetidine or by delivery of the vaccine encapsulated in pH-dependent microspheres. The vaccine did not cause diarrhea when given perorally to any of the experimental animals or evoke fluid secretion when instilled into rabbit ligated ileal loops. These observations (i) confirm the effectiveness of this vaccine as an immunogen in a second animal model, (ii) establish that it is effective when given exclusively by the peroral route, and (iii) provide further evidence regarding its lack of toxicity.

Administration, Oral↗
PubMed1983

Protection against human and porcine enterotoxigenic strains of Escherichia coli in rats immunized with a cross-linked toxoid vaccine.

To compare their relative immunogenicities, we used synthetically produced Escherichia coli heat-stable toxin coupled to a protein carrier and the B subunit of porcine heat-labile toxin separately in graded dosages to immunize rats. Equivalent antigen unit dosages of each toxin raised approximately the same level of mucosal immunoglobulin A (IgA) antitoxin response and degree of protection against a challenge with respective heat-stable- or heat-labile-toxin-producing viable bacteria. Conjugation conditions were identified, therefore, which yielded a vaccine of these toxins, cross-linked by the carbodiimide reaction, that consisted of equal antigenic proportions of each toxin component as determined by enzyme-linked immunosorbent assay and expressed in antigen units. The dose-related response to immunization with this vaccine was the same as the response to its components given separately. The toxicity of the heat-stable toxin component was reduced greater than 600-fold. Immunization with optimal antigen unit dosages of the vaccine gave greater than or equal to sixfold increases in mucosal IgA antitoxin titers and provided significant (P less than 0.001) protection against challenge with heterologous serotypes of viable strains, of either human or porcine origin, that produce heat-stable or heat-labile toxin or both.

Adjuvants, Immunologic↗
PubMed1983

Immunological properties of purified Klebsiella pneumoniae heat-stable enterotoxin.

Klebsiella pneumoniae heat-stable enterotoxin was purified to apparent homogenicity by the same techniques used to purify Escherichia coli heat-stable enterotoxin. The two toxins had the same potency in the suckling mouse assay and showed immunological cross-reactivity in enzyme-linked immunosorbent assay, neutralization of secretory activity by specific hyperimmune antisera, and protection against active challenge in rats immunized with a vaccine containing synthetically produced E. coli heat-stable enterotoxin.

Animals↗
PubMed1982

Development of a vaccine of cross-linked heat-stable and heat-labile enterotoxins that protects against Escherichia coli producing either enterotoxin.

A vaccine of cross-linked heat-stable (ST) and heat-labile (LT) toxins that protects against heterologous serotypes of strains of Escherichia coli which produce either the LT or ST enterotoxin was developed by conjugating ST to LT by the carbodiimide reaction. Three interrelated factors were found to affect the composition and properties of the final conjugate: (i) the amount of carbodiimide added to the toxins, (ii) the initial ratio of ST to LT, and (iii) the duration of the conjugation reaction. Optimal conjugation conditions were identified as a carbodiimide-to-toxin ratio of 10:1 by weight, an initial molar ratio of ST to LT of 100:1, and a conjugation reaction time of 96 h. This approach yielded a conjugate that contained 96% by moles and 36% by weight pure ST, determined with radioiodinated pure ST, and 34% by weight semi-pure ST, determined by the Lowry protein method. The retained antigenicities of the conjugated toxins, as determined by enzyme-linked immunosorbent assays, was greater than or equal to 82%, and their toxicities, as determined by the Y1 adrenal cell assay for LT and by the suckling mouse assay for ST, were reduced to less than or equal to 0.15%. Immunization of rats with this cross-linked ST-LT vaccine provided strong protection against challenge with either the LT or the ST toxin or with viable heterologous strains which produce these toxins, either singly or together. These observations indicate that conjugation of ST to LT results in a unique new immunogen in that ST acquires immunogenicity as a function of the reaction, LT retains most of its antigenicity, and the toxic properties of each individual toxin are greatly reduced.

Animals↗
PubMed1982

Arousal of mucosal secretory immunoglobulin A antitoxin in rats immunized with Escherichia coli heat-labile enterotoxin.

Specific serum and mucosal antitoxin levels were determined by enzyme-linked immunosorbent assays in rats immunized with Escherichia coli heat-labile enterotoxin (LT). Immunization by means of a parenteral prime followed by peroral boosts was the only approach that aroused titers of both serum immunoglobulin G (IgG) antitoxin and mucosal secretory IgA antitoxin that were increased fourfold or more over control values. Primary parenteral immunization was effective when given either intraperitoneally or subcutaneously with either Freund complete adjuvant or alum as the adjuvant. The magnitude of the nucosal secretory IgA antitoxin response and the degree of protection against challenge with either LT or viable LT-producing organisms were related to the number and dosage of peroral boosts. LT antigenicity, as determined by enzyme-linked immunosorbent assay, was progressively reduced by toxoiding it with increasing amounts of glutaraldehyde or a carbodiimide; when LT antigenicity was reduced by greater than 50%, the effectiveness of the toxoid in stimulating mucosal antitoxin and providing protection was compromised. Strong protection extended for more than 6 weeks only in rats immunized with a sufficient peroral dosage of LT to arouse mucosal secretory IgA antitoxin titers at least fourfold greater than those of controls. These observations indicate that the ability of LT to stimulate a mucosal secretory IgA antitoxin response is dependent on the antigenicity, route, and dosage of this immunogen; they suggest that the duration of protection in animals immunized by the peroral route is related to the extent of arousal of mucosal secretory IgA antitoxin.

Adjuvants, Immunologic↗
PubMed1981

Protective effect of immunization of rats with holotoxin or B subunit of Escherichia coli heat-labile enterotoxin.

The relative immunogenicities of three forms of the Escherichia coli heatlabile enterotoxin (LT), the holotoxin, its B subunit, and the polymyxin-release form (PM LT) were compared by immunizing rats with various dosages of each given exclusively by the parenteral (IP/IP) or peroral (PO/PO) routes or by a combination of the two (IP/PO). The degree of protection was evaluated by challenge in ligated ileal loops, and the serum antitoxin response was determined by an enzyme-linked immunosorbent assay with homologous antigens. When given by the PO/PO route, each LT antigen provided only weak protection against the toxin and virtually none against viable LT-producing strains; serum antitoxin titers were not significantly increased. When the toxins were given after a parental primary immunization by either the IP/IP or the IP/PO routes, each LT antigen provided a dose-related increase in serum antitoxin titers and in the degree of protection against the toxin as well as against viable strains which produce LT alone (LT(+)/ST(-)) or in combination with the heat-stable toxin (LT(+)/ST(+)). The degree of protection against viable bacteria, particularly the LT(+)/ST(+) strain, was stronger in animals which received booster immunizations by the PO route. When expressed on the basis of molar equivalents, holotoxin provided significant protection (a protection index of >5 against toxin challenge and >50% reduced secretion with bacterial challenge) with 4 to 15 times fewer moles than PM LT and up to 50 times fewer moles than the B subunit. These observations indicate that, on the basis of molar equivalents, the holotoxin (which contains one A plus five or six B subunits) is a more potent immunogen than either PM LT (which contains one A and probably one B subunit) or the B subunit.

Animals↗
PubMed1981

Respective contributions to protection of primary and booster immunization with Escherichia coli heat-labile enterotoxin in rats.

The respective contributions to protection of the route and dosage of primary and booster immunizations with Escherichia coli heat-labile enterotoxin were evaluated in rats. The degree of protection was determined by challenge with toxin and viable bacteria in ligated ileal loops, and the serum antitoxin response was assayed by enzyme-linked immunosorbent assay. Primary immunization was effective only when given by the parenteral route. The degree of protection was enhanced a fivefold dosage increase in the primary parenteral immunization in rats given constant dosages of booster immunizations either parenterally or perorally, but not by further dosage increases. In contrast, the degree of protection rose when dosages of the booster immunizations were increased over a 25-fold range. Four weekly peroral, but only two biweekly parenteral, booster immunizations were necessary to achieve strong protection; biweekly combined parenteral and peroral booster immunizations yielded both strong, immediate and extended protection. The degree of protection against the toxin correlated with that against viable bacteria and with elevated serum antitoxin titers: all seven groups with a protection index of greater than 5 against the toxin had strong protection against heat-labile toxin-producing strains and fourfold or greater increases in the antitoxin titers, whereas none of the nine groups with a protection index of less than 3 was protected against bacteria or had an equivalent antitoxin response. These observations show that once an adequate parenteral primary immunization is given, the degree of protection is influenced primarily by the dosage of the booster immunizations, the necessary number of which is dependent on their route of administration.

Administration, Oral↗
PubMed1981

Immunization of rats with heat-labile enterotoxin provides uniform protection against heterologous serotypes of enterotoxigenic Escherichia coli.

Rats immunized with Escherichia coli heat-labile (LT) enterotoxin, either in the form of the holotoxin derived from a transformed K-12 strain or the polymyxin-release form obtained from human strains which produce LT toxin alone (LT+/ST- [ST is heat-stable toxin)] or together with ST toxin (LT+/ST+), were challenged with viable organisms of 10 different serotypes, 5 LT+/ST- and 5 LT+/ST+. The serum antitoxin response was monitored by enzyme-linked immunosorbent assay, and the degree of protection was determined by challenge in ligated ileal loops. Immunization with the holotoxin provided a strong antitoxin response and protection against all 10 challenge strains. Immunization with toxin from the LT+/ST+ strain provided equally strong protection against all strains, but immunization with toxin from the LT+/ST- strain yielded only a weak antitoxin response, moderate protection against challenge with LT+/ST- strains, and no protection against LT+/ST- strains, increasing by fivefold the immunization dosage of the LT+/ST- toxin failed to enhance protection. These observations (i) establish the fact that immunization with the LT holotoxin provides uniformly strong protection against heterologous serotypes and (ii) indicate that, for reasons which remain to be determined, the immunogenicity of the polymyxin-release LT from an LT+/ST+ strain differs from that of an LT+/ST- strain.

Animals↗
PubMed1981

Protection in rats immunized with Escherichia coli heat-stable enterotoxin.

Rats immunized with a semipurified preparation of the Escherichia coli heat-stable (ST) enterotoxin conjugated with a protein carrier were protected against challenge with semipurified or purified ST and viable organisms of multiple heterologous serotypes that produce only ST (LT-/ST+), but they were not protected against heal-labile (LT) toxin or viable strains which produce LT either alone (LT+/ST-) or together with ST (LT+/ST+).

Animals↗
PubMed1981

Tropical sprue in travelers and expatriates living abroad.

Changes in the world political situation, the rapidity of transportation, and the availability of effective therapy have altered the pattern of sprue in persons going to the tropics. Gone, for the most part, are the days when expatriates liver for years in tropical areas, progressed on the full-blown pattern of debilitating disease when they acquired sprue, and then were never totally cured either by return home or by the then-available forms of therapy. Today, visitors to the tropics usually return home by jet aircraft within weeks or months after acquiring the disease, and thus they present just with manifestations of small bowel disease in the absence of nutritional deficiencies. In this circumstance, the differential diagnosis usually lies between sprue and giardiasis. Both of these disorders are caused by chronic contamination of the small bowel by enteric pathogens, and both can be cured by specific therapy directed at eradicating these organisms. In contrast to the situation in travelers, sprue among the indigenous population of the tropics remains largely unchanged: a chronic debilitating disorder that represents a significant contributory factor to the pathogenesis of morbidity and malnutrition in some areas.

Adult↗
PubMed1980

Influence of route of administration on immediate and extended protection in rats immunized with Escherichia coli heart-labile enterotoxin.

The effect of route of administration, dosage, and number of boosts employed during immunization with the polymyxin-release form of Escherichia coli heat-labile (LT) enterotoxin on the degree and duration of protection afforded was evaluated in rats which were challenged by the ligated loop technique. Increasing the boosting dosage by fivefold from 50 to 250 mug resulted in a marked increase in protection against challenge with toxin in rats immunized either just by the parenteral route (i.p./i.p.) or by a parenteral prime, followed by peroral boosts (i.p./p.o.) in rats pretreated with cimetidine to ablate gastric secretions; such was not the case, however, even with a 50-fold increase in dosage in rats immunized just by the peroral route (p.o./p.o.). Four weekly peroral boosts were required to achieve the strongest degree of protection. Increasing the boosting dosage also increased the degree of protection against challenge with viable LT(+)/ST(-) and LT(+)/ST(+) strains (ST indicates heat-stable enterotoxin) in rats immunized by the i.p./p.o., but not by the i.p./i.p., route; no protection was evident against an LT(-)/ST(+) strain. Protection was lost within 3 weeks after immunization in rats immunized by the i.p./i.p. route. In contrast, protection was extended over the 3-month observation period in those immunized by the i.p./p.o. route; the degree of protection was enhanced in rats which received an additional boost at 2 months. These observations establish the fact that immunization with LT is similar to that with cholera toxin in that arousal of the local immune intestinal response by means of peroral immunization provides maximal extended protection.

Animals↗
PubMed1980

Protective effect of immunization with heat-labile enterotoxin in gnotobiotic rats monocontaminated with enterotoxigenic Escherichia coli.

The protective effect of active immunization with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile enterotoxin (LT), administered using a parenteral prime and peroral boosts given after ablation of gastric secretion by means of cimetidine, was assessed in gnotobiotic rats which were challenged by monocontamination with enterotoxigenic strains of E. coli. Water transport was evaluated by the in vivo marker perfusion technique at weekly intervals over a 3-week period after contamination. Water transport in unimmunized control rats was consistently in absorption in those contaminated by a nontoxigenic strain, in secretion during only week 2 in those contaminated by an LT(+)/- strain, in secretion during weeks 2 and 3 in those contaminated by an LT(+)/ST(+) (heat-stable enterotoxin) strain, and consistently in absorption in those contaminated by an -/ST(+) strain. Rats immunized with a booster dosage of 250 mug had a significant increase (P < 0.001) in net water absorption as compared to unimmunized rats, with values in the borderline range of absorption, when challenged with either the LT(+)/- or LT(+)/ST(+) strains. Rats immunized with a 10-fold-higher boosting dosage had a significant increase (P < 0.001) in net water absorption as compared to those boosted at the lower dosage; water absorption was within the normal range. There was no difference between the ileal bacterial counts of unimmunized and immunized rats challenged by the various strains. These observations indicate that this immunization program provides complete protection in an animal model against challenge by intestinal contamination with enterotoxigenic strains of E. coli which produce LT, either alone or in combination with ST.

Animals↗
PubMed1980

Immunological cross-reactivity of heat-labile enterotoxins produced by enterotoxigenic and enteropathogenic strains of Escherichia coli.

Weanling rats were immunized with a heat-labile enterotoxin contained in whole cell lysate (WCL) ultrafiltrate preparations of enteropathogenic (EPEC) and enterotoxigenic (ETEC) strains or with a purified preparation of heat-labile toxin (LT) from the ETEC strain and then challenged either with viable bacteria of each strain or the purified ETEC LT by means of the ileal ligated loop technique. Immunization with the WCL toxin preparations of either the EPEC or ETEC strain conferred protection against challenge with viable organisms of both strains; immunization with a similar preparation from a nontoxigenic strain did not yield protection. Immunization with either the WCL or purified LT toxin from ETEC strain afforded protection against challenge with the ETEC LT toxin, but immunization with the EPEC WCL preparation did not. The antigenicity of all of the toxin preparations was destroyed by heat-treatment. Possible contributory protective effects of somatic or colonization factor (CFA) antigens present in the WCL were excluded by the findings that protection was afforded against a heterologous somatic serotype, ileal bacterial counts were not reduced in protected animals, and WCL preparations of strains containing or lacking CFA yielded equal protection. These observations indicate that the heat-labile enterotoxin of EPEC strains is antigenic and is immunologically related to a heat-labile toxin present in similarly prepared material from an ETEC strain but not to the conventional LT toxin of ETEC strains. They suggest that the WCL preparation of the ETEC strain contains two heat-labile enterotoxins, one of which is conventional LT and the other of which resembles the EPEC toxin.

Animals↗
PubMed1979

Protective effect of active immunization with purified Escherichia coli heat-labile enterotoxin in rats.

The protective effect of active immunization by different routes with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile toxin was evaluated in rats. Immunized animals were challenged by placing toxin into ligated ileal loops at dosages which produced either 50% or the maximum secretory response in unimmunized rats. Immunization exclusively by the parenteral route yielded significant protection. Rats were also protected when parenteral priming was followed by boosting given either directly into the duodenum or perorally 2 h after intragastric cimetidine, but not when the peroral boosts were given with bicarbonate. Immunization administered entirely by the peroral route with cimetidine yielded protection but only when the immunizing dosage was fivefold greater than that found effective in the parenteral-peroral approach. Rats immunized exclusively by the parenteral route and those boosted perorally with cimetidine were also tested, and found to be protected, against challenge with viable organisms of strains that produce either heat-labile toxin alone or both heat-labile and heat-stable toxin, but they were not protected against a strain which produces just heat-stable toxin. Geometric mean serum antibody titers were increased by 16-fold or more over control values in those groups of rats in which protection was achieved, with the exception of those immunized exclusively by the peroral route. These observations demonstrate that (i) active immunization with purified E. coli heat-labile toxin results in significant protection against both this toxin as well as viable organisms which produce it, but not against viable strains which produce heat-stable toxin only, and (ii) concomitant ablation of gastric secretion by the use of cimetidine renders the peroral route of immunization effective. They suggest that prophylactic immunization against diarrheal disease caused by heat-labile toxin-producing strains of E. coli may be feasible in humans.

Animals↗
PubMed1979

Comparison of assay of coliform enterotoxins by conventional techniques versus in vivo intestinal perfusion.

Thirty-six strains of coliform bacteria were tested for enterotoxigenicity both by conventional assays, including the Y-1 adrenal and Chinese hamster ovary cell assays for heat-labile toxin and the suckling mouse assay for heat-stable toxin, and by determining the ability of graded concentrations of ultrafiltrate high- or low-molecular-weight toxin preparations to induce water secretion during in vivo perfusion in the rat jejunum. The ultrafiltrates of all 18 strains isolated from persons with infectious diarrheal disease, including seven of Escherichia coli, seven of Klebsiella pneumoniae, and four of Enterobacter cloacae, contained one (nine strains) or two (nine strains) potent toxin fractions (resembling either heat-labile or heat-stable toxin in terms of apparent molecular weight and heat lability characteristics) that induced water secretion at perfusion concentrations of 10 ng/ml or less. Unconcentrated broth filtrates of five of the E. coli strains and two of Klebsiella reacted positively in one or more of the conventional assay systems. Concentrated ultrafiltrates from two strains that were negative in the in vitro assays for heat-labile toxin were tested and also proved to be inactive in these test systems. None of 18 strains isolated from control sources produced, in the ultrafiltrates, enterotoxins capable of inducing water secretion at low concentrations, and none reacted positively in the conventional assays. These results indicate that some strains of coliform bacteria elaborate potent toxin materials that are capable of inducing water secretion and can be detected by perfusion of concentrated ultrafiltrates but not by conventional assay systems for enterotoxigenicity. Whether this represents quantitative or qualitative differences between the toxin materials that stimulate these different test systems remains to be established.

Animals↗