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Biomedical subjects

T G Cleary

Publications and source records attributed to T G Cleary.

At least 55 records · Page 3Linked to original sources
PubMed1990

A method for detecting Shiga toxin and Shiga-like toxin-I in pure and mixed culture.

Shiga toxin and Shiga-like toxins (SLTs, syn. Verotoxins) are currently detected by tissue culture assays that are expensive, time-consuming and require specialised facilities and experienced personnel. We have developed a rapid method to detect Shiga toxin and SLT-I (Verotoxin 1) based on their binding to globotriosyl ceramide (Gb3). Bound toxin was then detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The direct detection of cytotoxins from pure culture plates and from a mixed bacterial culture was studied. Using polymyxin extraction (0.1 g/L, 30 min, 37 degrees C) and Gb3-based ELISA we detected toxin from reference strains Shigella dysenteriae 1 strain 60R (Shiga toxin) and Escherichia coli O26:H11 strain H30 (SLT-I), and from clinical isolates of E. coli O157:H7 and O26:H11 (both SLT-I) from 11 patients with diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. Toxin production by these strains was confirmed by a radiolabelled HeLa cell assay and the structural genes were detected by DNA hybridisation. The Gb3-based ELISA could detect SLT-I in extracts of a mixed culture even when the toxin-positive strains represented only 1% of the mixture. No cross-reactivity was found with bacteria that produce other cytotoxins, such as other E. coli and Shigella, Salmonella, Aeromonas and Campylobacter spp.

Bacterial Toxins↗
PubMed1989

Escherichia coli O114:nonmotile as a pathogen in an outbreak of severe diarrhea associated with a day care center.

Five infants from a day care center developed severe diarrhea associated with enteropathogenic Escherichia coli O114:nonmotile (EPEC O114:NM) and required hospitalization. Five additional cases of diarrhea associated with EPEC O114:NM subsequently occurred, four in hospital contacts of the patients and one in a household contact. Biochemically, all EPEC O114:NM isolates were sorbitol nonfermenters. All isolates produced low concentrations of cytotoxin with a mean of 10(1.23) CD50/mg of protein. Cytotoxin was not neutralized with antibody to Shiga-like toxin I or II. Heat-labile and heat-stable enterotoxins were not present by gene probe analysis. Stool isolates from 9 of 10 hospitalized infants were positive for EPEC adherence factor by colony blot DNA probe analysis. The severity of the disease, sorbitol nonfermentation, and presence of enteroadherence are unusual features of this organism.

Child Day Care Centers↗
PubMed1989

Comparative in vitro activity of ceftibuten (Sch 39720) against bacterial enteropathogens.

Ceftibuten is a new orally active cephalosporin with significant bioavailability. Its in vitro activity was compared with those of other agents against 383 strains of enteric pathogens derived from clinical specimens. Ceftibuten was very active against the strains of the family Enterobacteriaceae tested (overall MIC for 90% of strains tested, 0.25 microgram/ml) but was less active against Campylobacter jejuni (MIC for 90% of strains, 16 micrograms/ml). The MBC was one to two dilutions higher than the corresponding MICs for most pathogens tested.

Anti-Bacterial Agents↗
PubMed1989

Human milk immunoglobulin A antibodies to Shigella virulence determinants.

Because human milk is thought to protect infants from shigellosis, we evaluated milk for immunoglobulin A to Shigella virulence determinants. Milk was preincubated to remove antibodies unrelated to each locus of interest, using defined Shigella and E. coli hybrids containing known Shigella genetic segments prior to immunoblotting. The milk could not be shown to contain antibodies to chromosomally encoded virulence loci except for the expected antibodies to the products of the histidine locus. However, all the milk samples contained antibodies to antigens encoded by the large virulence plasmid. The finding of these antibodies suggests a possible mechanism by which human milk might protect infants.

Adsorption↗
PubMed1989

Rapid method to detect shiga toxin and shiga-like toxin I based on binding to globotriosyl ceramide (Gb3), their natural receptor.

Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli represent a group of very similar cytotoxins that may play an important role in diarrheal disease and hemolytic uremic syndrome. These toxins have the same biologic activities and according to recent studies also share the same binding receptor, globotriosyl ceramide (Gb3). They are currently detected, on the basis of their ability to damage several cell lines, by using expensive and tedious assays that require facilities for and experience with tissue cultures and are therefore most suitable for research laboratories. We have developed a rapid method to detect Shiga toxin and Shiga-like toxin I based on specific binding to their Gb3 natural receptor, which was coated onto microdilution plates. Bound toxin was then detected by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The sensitivity of the Gb3 ELISA was 0.2 ng (2 ng/ml) of purified toxin. The assay was positive with sonic extracts of Shigella dysenteriae serotype 1 strain 6OR (a Shiga toxin producer), E. coli serotype O26:H11 strain H30, and E. coli serotype O157:H7 (both Shiga-like toxin I producers). The assay was very specific in that no cross-reactivity was noted with purified cholera toxin, E. coli heat-labile and heat-stable enterotoxins, and Clostridium difficile cytotoxin, or sonic extracts of other cytotoxin-producing organisms, such as other shigellae, pathogenic and nonpathogenic E. coli, Salmonella spp., Campylobacter spp., and Aeromonas spp. These results were in complete agreement with a [3H]thymidine-labeled HeLa cell cytotoxicity assay and with detection of the structural genes by DNA hybridization studies with a Shiga-like toxin I probe. Quantitative analysis showed a high correlation between Gb3 ELISA and HeLa cell assay when fractions obtained at various stages of toxin purification were examined by both methods (r = 0.99, P < 0.01). This rapid Gb3 ELISA is sensitive and specific and may be diagnostically useful in cytotoxin-related infections.

Bacterial Toxins↗
PubMed1988

Anticytotoxin-neutralizing antibodies in immune globulin preparations: potential use in hemolytic-uremic syndrome.

The pathogenesis of primary (classic) hemolytic-uremic syndrome (HUS) is thought to be related to cytotoxin-producing enteric pathogens such as Shigella dysenteriae serotype 1 and Escherichia coli serotypes O157:H7 and 026:H11. The relevant cytotoxins include Shiga toxin and the closely related Shiga-like toxins (SLTs) produced by some E. coli strains. Intravenously administered immune globulin (IVIG) therapy has been reported to be beneficial in a few children with HUS. We therefore examined commercially available immune globulin preparations for the presence of anticytotoxin-neutralizing antibodies. Cytotoxicity and neutralization of the HUS-associated cytotoxins were quantitatively determined by means of a (3H)thymidine-labeled HeLa cell assay. The immune globulin preparations tested almost completely neutralized Shiga toxin (produced by S. dysenteriae 1) and SLT-I (produced by E. coli serotype 026:H11). Twofold dilutions of the preparations showed significant (p less than 0.01) neutralizing titers of 1:64 to 1:128. No significant neutralization (greater than 20%) of SLT-II (produced by E. coli strain C600 (933W] was noted. The IVIG preparation lost its inhibitory activity when passed through a protein A-Sepharose column, which bound immune globulin, indicating that its neutralizing effect is related to the antibody content. We also examined sera from 30 children without diarrhea or HUS; only one child had neutralizing titers against Shiga toxin (1:64) and SLT-I (1:128). Immune globulin preparations contain anticytotoxin-neutralizing antibodies, a finding that warrants further investigation of the therapeutic role of these preparations in early treatment of children with HUS related to Shiga toxin and SLT-I.

Adolescent↗
PubMed1988

Cytotoxin-producing Escherichia coli and the hemolytic uremic syndrome.

Recently, a new class of diarrhea-associated Escherichia coli has been linked to the hemolytic uremic syndrome. The organisms included in this group produce cell-damaging toxins (cytotoxins) related to Shigatoxin made by S. dysenteriae 1. The most common pathogen in this group is E. coli O157:H7.

Child↗
PubMed1988

Quantitative analysis and partial characterization of cytotoxin production by Salmonella strains.

The pathogenesis of the wide-spectrum human disease caused by Salmonella species is poorly understood. Cytotoxin production by other enteric pathogens has been increasingly investigated recently, and data are accumulating regarding the role of cytotoxins in enteric infections and hemolytic uremic syndrome. We studied the cytotoxic activity of 131 Salmonella strains of the major serotypes, including 94 strains of Salmonella enteritidis, 12 strains of Salmonella typhi, and 25 strains of Salmonella choleraesuis. Cytotoxicity was quantitatively determined in sonic extracts by a [3H]thymidine-labeled HeLa cell assay. All Salmonella strains examined showed some degree of cytotoxic activity. The geometric means +/- standard deviations of the amounts of cytotoxin produced (50% cytotoxic dose per milligram of bacterial protein) were 27 +/- 2 for S. typhi, 65 +/- 2 for S. enteritidis, and 117 +/- 2 for S. choleraesuis. Analysis of variance showed that the differences in cytotoxin production by the three species were significant (P less than 0.001). No significant differences were found between stool isolates and invasive strains of the same species. Neutralization studies showed that the cytotoxins produced by all Salmonella strains were immunologically distinct from Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli. DNA hybridization studies with DNA probes for Shiga-like toxins of types I and II showed no hybridization. In each species the cytotoxin was heat labile and sensitive to trypsin treatment, which indicated that its active component was probably protein in nature. Upon ultrafiltration with Amicon membranes and gel filtration chromatography, cytotoxic activity was found in the molecular weight range of 56,000 to 78,000. Our findings indicate that salmonellae produce cytotoxin(s) that may play a role in the manifestations of the various species.

Antibodies, Bacterial↗
PubMed1988

Lack of Shiga-like cytotoxin production by enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli has not been extensively studied for cytotoxin production. We evaluated 30 well-characterized enteroinvasive E. coli strains of all the known invasive serogroups from several geographic regions for their ability to produce Shiga-like cytotoxic activity assayed in a HeLa cell system. None of these strains produced cytotoxic activity that was neutralizable with antibody to Shiga-like toxin I or II.

Bacterial Toxins↗
PubMed1987

Clinical and biochemical significance of toxin production by Aeromonas hydrophila.

Production of cytotoxin and enterotoxin by Aeromonas strains obtained from stools of 50 children in Mexico and Texas and from blood of 9 children with sepsis was determined. Results were correlated with clinical features of infected children as well as with biochemical traits of Aeromonas strains. Cytotoxin was produced by 40 of 42 Aeromonas strains (95%) isolated from stools of children with diarrhea, by all 8 isolates from stools of well children, and by all 9 isolates from children with sepsis. There was no difference in the quantities (amount of cytotoxin per milligram of protein required to kill 50% of the cells) of cytotoxin produced and in clinical manifestations among the groups. None of the isolates produced a toxin that could be neutralized by antiserum raised against Shiga toxin produced by Shigella dysenteriae 1 60R. Heat-labile-like enterotoxin (LT) was produced by 26 of 42 stool isolates (62%), while only 1 of the 42 isolates (2%) produced enterotoxinlike activity in suckling mice; 65% of the cytotoxin-producing strains also produced an LT-like material. All strains from blood produced LT-like material, and 2 of 6 (33%) produced activity in suckling mice. All strains produced hemolysin; 37 of 57 (65%) were Voges-Proskauer positive; 27 of 57 (47%) were lysine decarboxylase positive by API 20E strips, none were positive for lysine decarboxylose production by lysin-iron agar slants at 24 h, but 17 of 54 (31%) were positive at 48 h. There was no correlation between biochemical reactions and enterotoxin or cytotoxin production. There appears to be no correlation between toxin production by Aeromonas spp. and gastroenteritis.

Acute Disease↗
PubMed1986

The relation between production of cytotoxin and clinical features in shigellosis.

The relation between in vitro production of HeLa cell cytotoxin by strains of Shigella and clinical symptomatology was determined for 35 travelers from the United States who developed shigellosis in Guadalajara, Mexico. There were 25 patients with Shigella sonnei, eight with Shigella flexneri, one with Shigella boydii, and one with Shigella dysenteriae. These strains were evaluated for in vitro production of cytotoxin. The amount of cytotoxin did not correlate with the number of stools passed, the severity of abdominal pain, or the presence of nausea or vomiting. However, patients with strains of Shigella that produced more cytotoxic activity were more likely to have fever (P less than .02) and occult blood in their stools (P less than .004). The cytotoxicity produced by 30 (86%) strains could not be neutralized with rabbit antiserum to purified, formaldehyde-treated Shiga toxin from S. dysenteriae type 1 strain 60 R; the cytotoxicity of five (14%) of the strains was partially neutralized. When only nonneutralizable cytotoxin was considered, the presence of fecal leukocytes (P less than .04), as well as of occult blood (P less than .002) and fever (P less than .02), correlated with the amount of cytotoxin. The amount of nonneutralizable cytotoxin produced by shigella strains was related to the clinical findings. This cytotoxic activity was infrequently attributable to "Shiga toxin".

Animals↗
PubMed1986

Production of Shiga toxin and other cytotoxins by serogroups of Shigella.

We measured the cytotoxic activity of 119 strains of Shigella by using a quantitative [3H]thymidine-labeled HeLa cell assay. We assayed 13 strains of Shigella dysenteriae 1; 18 strains of S. dysenteriae types 2 and 3; and 88 strains of Shigella sonnei, Shigella flexneri, and Shigella boydii. Strains of S. dysenteriae 1 demonstrated high levels of cytotoxicity (geometric mean, 10(5.04) CD50/mg of protein; range, 10(3.95)-10(6.10). Cytotoxic activities of the non-type 1 strains of S. dysenteriae and of the other Shigella serogroups were approximately 1/1,000 that of the S. dysenteriae 1 strains (range, 10(1.09)-10(3.11) CD50/mg of protein). Neutralization of cytotoxicity by using rabbit antiserum to purified Shiga toxin revealed that in all strains of S. dysenteriae 1, greater than or equal to 99.5% of cytotoxic activity was attributable to Shiga toxin. In contrast, 88 of the other Shigella strains produced only nonneutralizable cytotoxic activity. Six of 18 strains of non-type 1 S. dysenteriae and 12 of 88 strains from other Shigella serogroups produced both Shiga toxin and nonneutralizable toxin.

Bacterial Toxins↗
PubMed1985

Pharmacokinetics of cefotaxime in the postpartum patient.

A scarcity of pharmacokinetic data are available on cefotaxime in the obstetric patient. Fifteen patients received either 1 or 2 gm of cefotaxime after cesarean section. After intravenous administration of the designated dose, serial blood samples were analyzed for cefotaxime and its active metabolite, desacetyl cefotaxime (DCTX), by high-pressure liquid chromatography. The mean (+/- SD) peak concentrations of cefotaxime were 14.1 +/- 7.9 micrograms/ml and 40.0 +/- 32.5 micrograms/ml for the 1- and 2-gm dosage regimens, respectively. Detectable trough concentrations were 0.87 +/- 0.24 and 1.0 +/- 0.26, respectively, with many values falling below the sensitivity limits of the assay (0.5 micrograms/ml). The mean peak concentrations of DCTX for the two doses were 5.5 +/- 1.9 micrograms/ml and 10.9 +/- 6.2 micrograms/ml, respectively. Measurable trough levels for DCTX were more frequently identified, than for cefotaxime, at the end of the dosing interval due to an extended half-life as compared to its parent compound. Pharmacokinetic parameters of cefotaxime demonstrated large volumes of distribution and high clearance rates. Our data suggest altered pharmacokinetics of this agent in the obstetric patient when compared to previous studies in nonpregnant patients. This may be important in the seriously ill gravida where dosage adjustments in cefotaxime administration may be needed.

Adult↗