Biomedical subjects

D M Lyerly

Publications and source records attributed to D M Lyerly.

51 records · Page 3Linked to original sources

PubMed1990

Molecular characterization of the Clostridium difficile toxin A gene.

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Amino Acid Sequence↗

PubMed1989

Clostridium difficile toxin A. Interactions with mucus and early sequential histopathologic effects in rabbit small intestine.

Clostridium difficile produces two toxins, A (enterotoxic) and B (cytopathic), that are implicated in the pathogenesis of pseudomembranous colitis. However, the relationship of the secretory effect and the early histopathologic events is still unclear. We examined the early histopathologic effect of purified C. difficile toxin A in rabbit ileal loops and correlated the mucosal damage with the secretory response. As early as 2 hours after inoculation, toxin A at 1.0 micrograms caused cytolysis of the basal portion of the apical epithelia cells. Electron microscopy showed the basal portions of the absorptive epithelial cells necrotic. No significant fluid accumulation occurred after 2 or 4 hours in ileal loops inoculated with either 0.1 or 1.0 micrograms toxin A. In contrast, with 3-10 micrograms toxin A there was significant hemorrhagic fluid accumulation after 2 hours that correlated with the severity of the histologic lesions. An early mucoid exudate was demonstrated not to be related to a mucus secretagogue effect of toxin A. Prior exposure of toxin A to rabbit intestinal mucus preparations did not alter its subsequent enterotoxic or cytopathic (Chinese hamster ovary cells) effects. These studies demonstrate that the early intestinal tissue damage by toxin A was dose and time dependent and involved disruption of the basal portions of the absorptive epithelial cells with marked separation of the cells along the mucosal surfaces that preceded the secretory response.

Animals↗

PubMed1988

Clostridium difficile: its disease and toxins.

Clostridium difficile is the etiologic agent of pseudomembranous colitis, a severe, sometimes fatal disease that occurs in adults undergoing antimicrobial therapy. The disease, ironically, has been most effectively treated with antibiotics, although some of the newer methods of treatment such as the replacement of the bowel flora may prove more beneficial for patients who continue to relapse with pseudomembranous colitis. The organism produces two potent exotoxins designated toxin A and toxin B. Toxin A is an enterotoxin believed to be responsible for the diarrhea and mucosal tissue damage which occur during the disease. Toxin B is an extremely potent cytotoxin, but its role in the disease has not been as well studied. There appears to be a cascade of events which result in the expression of the activity of these toxins, and these events, ranging from the recognition of a trisaccharide receptor by toxin A to the synergistic action of the toxins and their possible dissemination in the body, are discussed in this review. The advantages and disadvantages of the various assays, including tissue culture assay, enzyme immunoassay, and latex agglutination, currently used in the clinical diagnosis of the disease also are discussed.

Animals↗

PubMed1988

Effects of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro.

Clostridium difficile is recognized as the major cause of antibiotic-associated colitis. C. difficile produces two toxins, A (enterotoxin) and B (cytotoxin), that are implicated in the pathogenesis of the colitis. We examined the dose responses, time course, and synergism of these two toxins in ligated rabbit intestinal loops and in tissue culture. In rabbit small intestinal loops, toxin A caused histologically demonstrable intestinal tissue damage as early as 2 h. The secretory response greater than or equal to 8 h was similar to that of a cholera toxin control. The effect of toxin A on tissue damage or secretion was seen even if toxin was removed after 5 min. Purified toxin A caused significant net accumulation of sodium, chloride, potassium, and total protein and slightly increased osmolality of the fluid content at 6 h; these effects were similar to those caused by crude C. difficile culture filtrates containing toxins A and B. Crude C. difficile toxin caused fluid accumulation with a delayed time course in the rabbit large intestine, and in contrast to its effect in small intestine, crude toxin caused net accumulation of bicarbonate and increased pH. In tissue culture, toxin A caused a rounding up of CHO and T-84 colonic carcinoma cells. A monoclonal antibody (PCG-4) that has no effect on tissue culture cytotoxicity with toxins A and B completely inhibited the secretory and tissue-damaging effects in the intestine. Toxins A and B were synergistic in the gut only at high doses of toxin B (greater than or equal to 10 micrograms/ml), and they were additive in tissue culture. The cytopathic effect in tissue culture was not consistently associated with trypan blue uptake. The cytopathic effect of toxin A in tissue culture did not appear to involve inhibitable Ca2+-dependent or prostaglandin synthesis pathways or intact microfilament or microtubule function for its activity and was not inhibited by reducing or lysosomotropic agents. Our results suggest that toxins A and B have independent and distinct effects in vivo and in vitro.

Animals↗

PubMed1988

Characterization of cross-reactive proteins detected by Culturette Brand Rapid Latex Test for Clostridium difficile.

Clostridium sporogenes, Peptostreptococcus anaerobius, and Bacteroides asaccharolyticus have been reported to react in the Culturette Brand Rapid Latex Test (Marion Scientific, Div. Marion Laboratories, Inc., Kansas City, Mo.) for Clostridium difficile. From the results of this study we showed that C. sporogenes and P. anaerobius produce a protein which is very similar biochemically and immunologically to the protein of C. difficile that is detected by the test. Thus, the positive latex reactions observed with C. sporogenes and P. anaerobius are due to a cross-reactive protein. We did not detect this cross-reactive protein in filtrates from B. asaccharolyticus, indicating that this bacterium reacts with the latex reagent by some other mechanism. We cloned the C. difficile gene that codes for the cross-reactive protein and showed that the protein produced by the recombinant organism is nontoxic and distinct from toxin A, thus confirming our earlier findings.

Antigens, Bacterial↗

PubMed1986

Stimulation of enzyme secretion from isolated pancreatic acini by Clostridium difficile toxin B.

Exposure of isolated rat pancreatic acini to increasing concentrations (10 ng - 800 ng/ml) of toxin B from Clostridium difficile produced a biphasic effect on the rate of secretion of amylase, trypsinogen, and chymotrypsinogen. Whereas doses of toxin B from 10-30 ng/ml increased enzyme secretion by 15-20%, doses between 30 ng and 60 ng/ml showed a regression of this effect, whereafter the rate of secretion of amylase, trypsinogen, and chymotrypsinogen increased with increasing concentrations of the toxin. Toxin B concentration of 800 ng/ml enhanced amylase, trypsinogen and chymotrypsinogen secretion by 119%, 185% and 195%, respectively, when compared with the basal level. Stimulation of enzyme secretion by toxin B was not affected by the presence of either actinomycin-D or cycloheximide, at a concentration which inhibited acinar RNA or protein synthesis by 80-90%. Although toxin B as well as CCK8, carbachol and secretin by themselves caused significant stimulation in amylase, trypsinogen and chymotrypsinogen secretion from isolated pancreatic acini, toxin B together with either CCK8, carbachol or secretin produced no further augmentation in enzyme secretion than what was observed with the secretagogues alone. It is concluded that toxin B of Cl. difficile exerts a direct effect on pancreatic acinar cells as evidenced by stimulation of enzyme secretion.

Amylases↗

PubMed1986

Comparison of culture, cytotoxicity assays, and enzyme-linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease.

Clostridium difficile culture, test tube, and microtiter cytotoxicity assays, and enzyme-linked immunosorbent assays (ELISAs) for toxin A and toxin B, were simultaneously performed on 113 fresh diarrheal stool specimens randomly selected from those submitted to our clinical laboratory for routine C. difficile testing. The performance of these tests in diagnosing C. difficile-related enteric disease (CDRED) was based on a clinical assessment of the likelihood of CDRED as determined by a systematic review of case histories blinded from the test results. Among 61 antibiotic recipients, both the microtiter cytotoxicity assay and the toxin A ELISA were highly specific for CDRED (95% and 100%, respectively). Specificities for the other procedures were much lower (tube cytotoxicity assay, 79%; culture, 74%; and toxin B ELISA, 56%). The high sensitivities of the culture (89%) and toxin B ELISA (83%) were somewhat negated by their low specificities. The only test that was both specific and had acceptable sensitivity (78%) was the microtiter cytotoxicity assay. This study indicates that ELISAs for detection of C. difficile toxins are not as reliable as the cytotoxicity assay in the laboratory diagnosis of CDRED, and that clinical correlation is essential in the evaluation of any new test for CDRED.

Adult↗

PubMed1986

Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies.

Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B. One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A. This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin. The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide. Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components. The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C. difficile by immunoaffinity chromatography. The second antibody, the G-2 MAb, cross-reacted with toxins A and B. The cross-reaction was confirmed by immunoblot analysis. These results show that toxins A and B share an epitope and suggest that they have a common subunit. The G-2 MAb did not neutralize or precipitate either toxin. The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.

Antibodies, Monoclonal↗

PubMed1986

Commercial latex test for Clostridium difficile toxin A does not detect toxin A.

The rapid latex test recently marketed by Marion Scientific (Div. Marion Laboratories, Inc., Kansas City, Mo.) for the detection of Clostridium difficile toxin A does not react with the toxin, based on the following findings: culture filtrates from nontoxigenic strains of C. difficile gave positive reactions in the test, culture filtrate in which toxin A had been removed gave positive reactions, purified toxin A did not react in the test, and the latex reagent bound an antigen which is distinct from toxin A and which is produced in various amounts by both toxigenic and nontoxigenic strains of C. difficile.

Antigens, Bacterial↗

PubMed1986

Immunization against experimental Pseudomonas aeruginosa and Serratia marcescens keratitis. Vaccination with lipopolysaccharide endotoxins and proteases.

Rabbits vaccinated with lipopolysaccharide endotoxins or with purified protease preparations from Pseudomonas aeruginosa and Serratia marcescens before corneal challenge with the viable bacteria exhibited significantly less corneal damage than rabbits not vaccinated with the bacterial products. However, the rabbits vaccinated with the lipopolysaccharide endotoxin preparations were significantly better protected than rabbits vaccinated with the bacterial proteases. Rabbits vaccinated with antisera raised against the proteases showed significantly less corneal damage than rabbits vaccinated with normal rabbit serum, and the passive protection was not significantly different than that elicited by active immunization against the bacterial proteases. The ability of the antiserum raised against the pseudomonas elastolytic protease to passively protect against severe corneal damage produced by experimentally induced pseudomonas keratitis was confirmed in mice. These findings support the idea that the bacterial endotoxins and proteases are virulence factors during the development of pseudomonas and serratia keratitis.

Animals↗

PubMed1985

Effects of Clostridium difficile toxins given intragastrically to animals.

We examined the activities of Clostridium difficile toxin preparations given intragastrically to hamsters, mice, and rats. The culture filtrate from a highly toxigenic strain of C. difficile caused hemorrhage and accumulation of fluid in the small intestine and cecum, diarrhea, and death in hamsters and mice. In rats, the culture filtrate caused only a small amount of fluid accumulation and slight hemorrhage along the small intestine. When toxin A was removed from the culture filtrate, the filtrate lost its activity. Preparations of homogeneous toxin A caused a response similar to that observed after the administration of culture filtrate. Hamsters were more sensitive to toxin A than mice or rats were. When hamsters were given multiple low doses of toxin A 1 week apart at a concentration which singly caused no response, they became ill and died, indicating that the toxin may have long-term effects. High amounts of toxin B did not cause any significant response when given intragastrically, unless initially mixed with low amounts of toxin A or given to hamsters with bruised ceca. These results suggest that toxins A and B act synergistically and that the action of toxin B may occur via the tissue damage caused by toxin A.

Administration, Oral↗

PubMed1985

Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A.

Monoclonal antibody, affinity-purified antibody, and monospecific antiserum against toxin A were produced. The monoclonal antibody was an immunoglobulin G2a kappa chain isotype that immunoprecipitated toxin A, as shown by crossed immunoelectrophoresis. These antibodies were compared by counterimmunoelectrophoresis, latex agglutination, and indirect enzyme-linked immunosorbent assay for their sensitivity in detecting toxin A. Our findings indicate that these antibodies may be useful as immunodiagnostic reagents for Clostridium difficile disease.

Animals↗

PubMed1983

Importance of serratia protease in the pathogenesis of experimental Serratia marcescens pneumonia.

The results of studies to evaluate the possible importance of serratia proteases in the development of experimental Serratia marcescens pneumonia revealed the following. (i) Administration of a highly purified serratia protease to the lungs of guinea pigs and mice resulted in extensive pulmonary edema and hemorrhage similar to that observed in animals having an experimentally induced, acute serratia pneumonia. (ii) Guinea pigs subcutaneously vaccinated with the protease developed low levels of antiprotease antibodies and were partially protected against serratia pneumonia, as demonstrated by a significant increase in survival time. Mice intranasally vaccinated with the protease also developed antiprotease antibodies and were protected against serratia pneumonia, as demonstrated by a significant increase in survival time and an increase in the number of survivors. (iii) Serratia protease was detected in lung tissue extracts prepared from the lungs of guinea pigs dying of serratia pneumonia. Our findings support the idea that serratia protease(s) is involved in the pathogenesis of experimental serratia pneumonia.

Animals↗

PubMed1983

Enzyme-linked immunosorbent assay for Clostridium difficile toxin A.

Antibodies against Clostridium difficile toxin A were purified by affinity chromatography from antiserum prepared against crude C. difficile toxin preparations. The affinity-purified antibody preparation was free of detectable amounts of antibodies to other C. difficile antigens, as demonstrated by crossed immunoelectrophoresis, and specifically neutralized the cytotoxicity of toxin A. An indirect enzyme-linked immunosorbent assay (ELISA) was subsequently developed using the antibody preparation for the specific detection of toxin A. The ELISA, which could detect 1 ng (5 ng/ml) of toxin A, was used to quantitate the toxin in the culture supernatant fluids of strains of C. difficile. The ELISA values for toxin A closely correlated with the toxin A and B cytotoxic titers of the supernatant fluids. In addition, toxin A was detected by ELISA in human fecal specimens from persons with antibiotic-associated colitis, demonstrating that this toxin is produced during C. difficile colitis.

Animals↗

PubMed1982

Biological activities of toxins A and B of Clostridium difficile.

Examination of the biological activities of the two known toxins of Clostridium difficile revealed that one of the toxins (toxin A) elicited a hemorrhagic fluid response in rabbit intestinal loops and a positive fluid response in infant mice. The other toxin (toxin B) did not produce a significant fluid response in either model, although the toxin was more lethal in infant mice. Both toxins elicited erythematous and hemorrhagic skin reactions and increased vascular permeability in rabbit skin.

Animals↗