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Biomedical subjects

D W Acheson

Publications and source records attributed to D W Acheson.

16 recordsLinked to original sources
PubMed1992

Detection by ELISA of low numbers of Shiga-like toxin-producing Escherichia coli in mixed cultures after growth in the presence of mitomycin C.

Techniques currently available to detect Shiga-like toxin (SLT)-producing Escherichia coli lack sensitivity or require specialised equipment and facilities, and in some cases detect only strains belonging to serotype O157. We have used an ELISA technique, capable of detecting both SLTI and SLTII with crude P1 glycoprotein from hydatid cysts, in combination with enhancement of toxin production by culture with mitomycin C. Supernates of Tryptone Soya Broth cultures containing mitomycin C 200 ng/ml were tested for SLTII. For SLTI, cell lysates pre-treated with polymyxin B were tested. In tests with E. coli O157:H7 in mixed culture with E. coli strain C600 alone, or with E. coli C600, Proteus mirabilis and Enterococcus faecalis, SLTI could be detected when the proportion of toxigenic organisms represented 1% of the mixture, and SLTII when the proportion was 0.025%. When faecal samples with added E. coli O157:H7 were examined in this system, SLTII-producing strains were detected when they comprised less than 0.1% of the coliform population. This technique is a sensitive and specific assay for detecting low numbers of SLT-producing organisms in mixed culture such as occurs in cases of haemolytic uraemic syndrome and haemorrhagic colitis.

Bacterial Toxins↗
PubMed1990

Enzyme-linked immunosorbent assay for Shiga toxin and Shiga-like toxin II using P1 glycoprotein from hydatid cysts.

Shiga toxin from Shigella dysenteriae type 1 strains and Shiga-like toxins (SLT) I and II from Escherichia coli bind to terminal alpha-D-Galp-(1----4)-D-Galp containing glycolipids. Hydatid cyst fluid isolated from sheep infected with Echinococcus granulosus contains a glycoprotein (P1gp) with a terminal alpha-D-Galp-(1----4)-D-Galp disaccharide. Preparations of P1gp were shown to interact directly with Shiga toxin and to inhibit the binding and cytotoxicity of Shiga toxin to HeLa cells. A sandwich ELISA was developed using preparations of P1gp as the toxin capture molecule, which, with an appropriate polyclonal antibody, was capable of detecting as little as 80 pg/well Shiga toxin and 132 pg/well SLT-II. Thus, the P1gp-toxin interaction forms the basis for a simple antigen-capture ELISA that may be useful clinically for the rapid detection and quantitation of Shiga and Shiga-like toxins.

Animals↗
PubMed1990

A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit.

We have constructed a plasmid expression vector (pSBC32) that encodes the B subunit of Shiga toxin/Shiga-like toxin I under control of the inducible trc promoter. The encoded B subunit is transported to the periplasmic space, allowing single-step purification of milligram amounts of this protein from periplasmic extracts by using receptor analog affinity chromatography. The purified B subunit interacts normally with both polyclonal antiserum to Shiga toxin and a monoclonal antibody specific for B subunit. B subunit purified in this system is pentameric (as in native holotoxin) and biologically active in blocking binding of Shiga holotoxin to HeLa cells. This expression system may allow rapid purification of sufficient amounts of Shiga toxin B subunit to attempt crystallization or to study its efficacy as a vaccine, either by itself or coupled to an appropriate polysaccharide antigen.

Animals↗
PubMed1989

Factors contributing to the accelerated clearance of theophylline and antipyrine in adults with exocrine pancreatic disease.

1. Oxidative drug metabolizing capacity has been assessed by oral antipyrine and/or theophylline tests in consecutive patients with chronic pancreatitis (CP; alcoholic 24, idiopathic 47), acute pancreatitis (AP; 28) and pancreatic cancer (CA; 11). Most of the patients had drastically reduced their alcohol consumption and dietary fat intake for variable periods before the tests. Excellent bioavailability of theophylline was confirmed from paired oral and intravenous tests in seven subjects, including two with exocrine pancreatic failure. 2. The clearance of theophylline in the patients was faster than in 15 controls with a 'healthy lifestyle' [median 104 (range 18-320) ml h-1 kg-1 vs median 68 (range 50-97) ml h-1 kg-1, P less than 0.01]. The difference was especially apparent in the groups with alcoholic CP (P less than 0.001 and idiopathic CP (P less than 0.01), but not in the AP and CA group as a whole, although drug clearance in some 50% of those cases exceeded the reference range. 3. There was good correlation between theophylline and antipyrine clearance in a subset of 91 subjects who had both tests (15 controls, 76 patients), but antipyrine was much less sensitive as a marker of enzyme induction. This suggests that enzyme induction in pancreatic disease preferentially involves the polycyclic aromatic hydrocarbon-inducible subfamily of cytochrome P-450. 4. The lack of correlation between pancreatic secretory capacity in 56 cases, judged by a secretin-pancreozymin test, and theophylline clearance suggests that enzyme induction is not secondary to pancreatic dysfunction. 5. Multivariate regression analysis identified approximately 50% of variability in clearance of each probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.

Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.

Animals↗
PubMed1988

Antioxidants, enzyme induction, and chronic pancreatitis: a reappraisal following studies in patients on anticonvulsants.

Our published dietary and pharmakokinetic studies in 15 patients with idiopathic chronic pancreatitis and 15 age- and sex-matched controls suggested that a combination of subnormal antioxidant intakes and chronic induction of the cytochromes P450 facilitates the pancreatic problem. We have now attempted to determine the relative importance of these two factors by studying a group of 15 institutionalized patients with epilepsy (EP), but without abdominal pain, who were on long-term treatment with anticonvulsant inducers of cytochromes P450 so that their clearance of theophylline (which reflects cytochromes P450 activities, and thereby provides an index of antioxidant demand) was as high as in the patients with chronic pancreatitis (CP) (mean +/- s.d., 123 +/- 59 ml/kg/h versus 120 +/- 62 respectively), and significantly higher than in controls (74 +/- 16 ml/kg/h, P less than 0.02). Canonical variate analysis of the drug kinetic and dietary data provided two functions with which to separate the three groups. The first function, heavily weighted on selenium, separated the controls from the other two groups whose values were lower; the second function, equally weighted on methionine and vitamin C, separated the EP group from the CP group whose values were generally lower. The results suggest that enzyme induction per se is not the critical factor in the development of CP. Instead, suboptimal availability of antioxidants in the face of increased demand--in particular of those substances that protect cells against non-biological free radicals--may be the key consideration, a deduction reinforced by observations in patients with epilepsy who went on to develop chronic pancreatitis.

Adult↗
PubMed1987

Theophylline and antipyrine disposition in smoking and non-smoking epileptic subjects.

Theophylline and antipyrine disposition has been compared in smoking epileptic patients, non-smoking epileptic patients and non-smoking healthy volunteers. Although clear differences in drug clearance and half-life were evident as a result of anticonvulsant drug therapy, no effect of smoking was discernible. Thus, additive effects from induction of the hepatic microsomal monooxygenase system in man by anticonvulsant drugs and polycyclic aromatic hydrocarbons (in cigarette smoke) were not evident.

Adult↗
PubMed1986

Dietary antioxidants and chronic pancreatitis.

Fifteen patients with idiopathic chronic pancreatitis (aged 17-78 years), who had not altered their diet since their first symptoms, completed 7-d weighed dietary records at home. The computed information was compared with that from 15 age- and sex-matched volunteers. Attention was focussed on the intakes of antioxidants and unsaturated fatty acids. The patients ingested less selenium, vitamin E, vitamin C and riboflavin than did controls (P less than 0.001, P less than 0.02, P less than 0.001 and P less than 0.05 respectively, using paired t-tests): selenium was by far the best discriminator on step-wise analysis. When the selenium intakes were examined alongside the results of theophylline tests--which reflect cytochromes P450 activities and, thereby, provide an index of antioxidant demand--a line of discrimination separated the majority of patients (with faster drug clearances and lower selenium intakes) and controls. There were no differences in the intakes of individual unsaturated fatty acids, C14:1 through to C24:6, between the two groups. However, amongst six subjects in the overlap zone, three with chronic pancreatitis habitually ate greater amounts of highly unsaturated fatty acids C20:4 to C24:6 inclusive (1970, 1049, 750 mg/d) than did three controls (329, 320, 82 mg/d). Animal experiments show that suboptimal intakes of dietary antioxidants and/or excessive intakes of highly unsaturated fatty acids and/or induction of cytochromes P450 facilitate peroxidation of cellular lipid membranes by free radicals. Our dietary data, taken in conjunction with pharmacokinetic data, thus suggest that a similar situation--favouring lipid peroxidation--may underlie human chronic pancreatitis.

Adolescent↗
PubMed1985

Induction of cytochromes P-450 in pancreatic disease: consequence, coincidence or cause?

We have examined the pharmacokinetics of antipyrine and of theophylline--validated probes for cytochromes P-450 activities--in a series of patients with pancreatic disease. The half-life of each drug was significantly lower, and its clearance faster, in patients than in controls and this pattern was detected in the subgroups with acute pancreatitis (6), chronic pancreatitis (22), or pancreatic cancer (4). These data suggest induction of cytochromes P-450 in all forms of exocrine pancreatic disease. Enzyme induction is unlikely to be secondary to pancreatic malfunction since there was no correlation between prevailing exocrine status, as assessed by secretin-pancreozymin tests, and the half-life or clearance of either drug. The corollary is that induction of the mono-oxygenases by environmental agents, both recognised and unidentified, is a primary event in pancreatic disease. The possible relevance of this finding is discussed.

Adolescent↗
PubMedDate not supplied

Shiga toxin: purification, structure, and function.

Shiga toxin is a potent toxin produced by Shigella dysenteriae type 1 strains. The toxin has three biologic activities--cytotoxicity, enterotoxicity, and neurotoxicity--and one known biochemical effect: inhibition of protein synthesis. It consists of two polypeptide chains, an A chain (molecular weight, 32,225) and a B chain (molecular weight, 7,691). These two peptides associate with a stoichiometry of one A and five B subunits to form the holotoxin. The A chain is responsible for the biochemical effect of the holotoxin: cleavage of the N-glycosidic bond of adenine at nucleotide position 4324 in the 28S rRNA of the 60S ribosomal subunit. The B chain mediates binding of toxin to cell surface receptors. Shiga toxin is the prototype of a family of toxin molecules that have been termed Shiga-like in terms of both structural and functional analysis.

Bacterial Toxins↗