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Biomedical subjects

I R Poxton

Publications and source records attributed to I R Poxton.

At least 37 records · Page 2Linked to original sources
PubMed1998

Burkholderia cepacia produces a hemolysin that is capable of inducing apoptosis and degranulation of mammalian phagocytes.

Burkholderia cepacia is an opportunistic pathogen that has become a major threat to individuals with cystic fibrosis (CF). In approximately 20% of patients, pulmonary colonization with B. cepacia leads to cepacia syndrome, a fatal fulminating pneumonia sometimes associated with septicemia. It has been reported that culture filtrates of clinically derived strains of B. cepacia are hemolytic. In this study, we have characterized a factor which contributes to this hemolytic activity and is secreted from B. cepacia J2315, a representative of the virulent and highly transmissible strain belonging to the recently described genomovar III grouping. Biochemical data from the described purification method for this hemolysin allows us to hypothesize that the toxin is a lipopeptide. As demonstrated for other lipopeptide toxins, the hemolysin from B. cepacia was surface active and lowered the surface tension of high-pressure liquid chromatography-grade water from 72.96 to 29.8 mN m(-1). Similar to reports for other pore-forming cytotoxins, low concentrations of the hemolysin were able to induce nucleosomal degradation consistent with apoptosis in human neutrophils and the mouse-derived macrophage-type cell line J774.2. Exposure of human neutrophils to higher concentrations of toxin resulted in increased activities of the neutrophil degranulation markers cathepsin G and elastase. Based on the results obtained in this study, we suggest a role that allows B. cepacia to thwart the immune response and a model of the events that may contribute to the severe inflammatory response in the lungs of CF patients.

Animals↗
PubMed1997

Divergent activity and function of superoxide dismutases in Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10.

Representative strains of Pasteurella haemolytica serotypes A1 and A2 and Pasteurella trehalosi serotype T10 were examined for the presence of superoxide dismutase. Visualisation of superoxide dismutase enzyme activity on polyacrylamide gels, and specific inhibition with potassium cyanide verified a copper/zinc (Cu/Zn) superoxide dismutase only in serotype A2 whereas serotypes A1 and T10 showed other superoxide dismutase activity. Using a simple freeze-thaw method the cellular location of superoxide dismutase enzyme activity was determined in all three serotypes. In serotypes A1 and A2 but not T10 superoxide dismutases were located in the periplasm. The viability of serotypes A2 and T10 cells in the presence of exogenous superoxide was unchanged over a 30 min period, whereas serotype A1 cells declined in viability between 15 and 30 min. Purified immunoglobulin from sheep convalescent serum did not reduce superoxide dismutase activity in the serotypes in an in vitro assay. The presence of this enzyme within the pasteurellae suggests a supportive role in the virulence of this major pathogen of ruminants.

Animals↗
PubMed1997

Isolation of a cell surface component of Helicobacter pylori that binds H type 2, Lewis(a), and Lewis(b) antigens.

BACKGROUND & AIMS: Individuals of blood group O and nonsecretors of ABO blood group antigens are more susceptible to peptic ulcers. The aim of this study was to determine if blood group antigens associated with group O or secretor status are epithelial cell receptors for Helicobacter pylori. METHODS: Bacterial binding and binding of monoclonal antibodies to H type 2, Lewis(a), and Lewis(b) to Kato III, buccal epithelial, and gastric mucosal cells were shown by flow cytometry. Bacterial outer membrane proteins eluted from H type 2, Lewis(a), or Lewis(b) were shown by polyacrylamide gel electrophoresis. RESULTS: Kato III and human epithelial cells bound each monoclonal antibody; O cells bound more anti-H type 2 (P < 0.05). Binding indices for H. pylori correlated with those for anti-H type 2 (P < 0.005) and anti-Lewis(b) (P < 0.001) but not anti-Lewis(a). A 61-kilodalton protein was eluted from H type 2, Lewis(a), or Lewis(b). CONCLUSIONS: Our results indicate that H type 2 is an important receptor for the 61-kilodalton bacterial adhesin, partly explaining increased susceptibility of individuals of blood group O to ulcers. Lewis(b) binds H. pylori more efficiently than Lewis(a). If these interactions occur in vivo, lack of Lewis(b) in mucosal fluids of nonsecretors may contribute to colonization by H. pylori.

Antibodies, Monoclonal↗
PubMed1997

Mucosa-associated bacterial flora of the human colon.

Biopsy samples of mucosa were taken during colonoscopy from the proximal colon and rectum of 12 patients, six with ulcerative colitis (UC) and six with non-inflammatory conditions. After anaerobic transport to the laboratory, biopsy specimens were examined by quantitative bacteriological culture on selective and non-selective media for total aerobic count, total anaerobic count, Bacteroides spp., lactobacilli, bifidobacteria and asaccharolytic, lactic acid producers. Isolates of the genus Bacteroides were identified to species level. Counts from proximal colonic and rectal biopsy samples in the same patient were not significantly different. Viable aerobic counts (aerobes and facultative organisms) ranged from 2.4 x 10(3) to 1.3 x 10(6) cfu/sample biopsy (5.6 mg) and total anaerobic counts were 10-10(2) times higher at (1.4 x 10(5))-(3 x 10(7)) cfu/sample. Bacteroides spp. predominated at both sites (range 8.6 X 10(4) to 1.4 x 10(7) cfu/sample), comprising 66% of total counts from proximal colon (range in individual patients 31-80%) and 68.5% from rectum (range 38-91%). Lactobacilli were isolated from eight biopsy samples from five patients, counts ranging from 3.6 X 10(2) to 1 X 10(5) cfu/sample; bifidobacteria were isolated from both sites from 10 of the 12 patients, counts ranging from 50 to 1.8 x 10(6) cfu/sample. From the 24 biopsy samples, 235 isolates representing 11 species of Bacteroides were identified. For any individual patient, only a few species (2-7; mean 4.4) of Bacteroides were found, with just one or two species predominating. B. vulgatus was cultured from both samples of seven patients (where it was the major isolate in four) and from single samples of two others; B. fragilis was cultured from both sites in six patients, being the major isolate in one patient and second commonest in three, but was not detected in the other six; the majority of other isolates were B. merdae/distasonis, B. ovatus, B. thetaiotaomicron and B. uniformis. B. thetaiotaomicron was isolated from both biopsy samples in all three UC patients with active inflammation (16 of the 60 isolates from these patients) but from only four of the other 18 samples from non-inflamed colonic mucosa (nine of 175 isolates).

Adult↗
PubMed1997

Phenotypic characteristics and lipopolysaccharides of human and animal isolates of Fusobacterium necrophorum.

As part of a collaborative study, six culture collection isolates and 50 coded isolates of Fusobacterium necrophorum were examined for the types of lipopolysaccharides (LPS) they contained, and to see if this related to their reactions in a range of phenotypic tests and their susceptibility to a panel of six antimicrobial compounds. The biotype B type strain, putative biotype B isolates and human isolates were predominantly coccobacillary, had rough type LPS and some of these isolates (8 of 26) required incubation for > 2 days to demonstrate lipase activity. The biotype A type strain, putative biotype AB isolates and most putative biotype A isolates (16 of 18) were predominantly rod-shaped, had either smooth LPS or low M(r) rough type LPS and all demonstrated lipase activity within 2 days. The other two putative biotype A isolates were coccobacillary and had rough type LPS, and one of these required incubation for > 2 days to demonstrate lipase activity. The results of these latter two isolates more closely resembled biotype B. A few isolates were asaccharolytic, but most fermented one or more of glucose, fructose, maltose and galactose. There was no correlation between fermentation pattern and LPS type, biotype or source of isolate (animal or human) but, with the exception of two abberant isolates, there was good correlation between cellular morphology, type of growth in liquid media and LPS type.

Animals↗
PubMed1995

Antibodies to lipopolysaccharide.

Lipopolysaccharides (LPS) are indispensable structural components of the Gram-negative bacterial outer membrane and are major determinants of virulence in pathogenic species. In the infected host LPS is better known as endotoxin where it acts as a potent stimulator of the inflammatory response. This article reviews the methods for the production and measurement of anti-LPS antibodies, and then describes the uses to which these methods have been employed. Antibodies to LPS (either monoclonal or polyclonal) may be used directly as immunotherapeutic agents for the treatment of Gram-negative sepsis or endotoxaemia, or as probes for the diagnosis and epidemiological investigation of Gram-negative bacterial infections. Antibodies are useful tools for investigation of the chemical structure of LPS, its expression on bacteria and to study the role of LPS in pathogenic mechanisms. The detection and quantitation of anti-LPS antibodies has formed the basis of classical and more recent serological studies of major bacterial infections.

Antibodies, Bacterial↗
PubMed1995

Biological activity of Bacteroides lipopolysaccharide--reappraisal.

Lipopolysaccharide (LPS) was extracted from six species of Bacteroides by the phenol/water, petroleum/chloroform/ phenol, and Triton/magnesium methods. Yields and chemical analysis demonstrated that the products were different. Biological activity (endotoxicity) was assessed by the limulus amebocyte lysate (LAL) assay, induction of tumor necrosis factor (TNF) from human mononuclear leukocytes, and a lethality model with galactosamine-sensitized mice. Results showed that endotoxicity varied greatly depending on the species and the extraction method. LPS prepared by the phenol/water method was most endotoxic and that from Bacteroides fragilis had the greatest activity. Compared with Escherichia coli LPS, the phenol/water extract of B. fragilis was sevenfold more active in the LAL assay and marginally less active (five-to seven-fold) in the bioassay for TNF induction. However, when B. fragilis LPS was added to E. coli LPS, the induction of TNF was inhibited. In the mouse model, B. fragilis LPS was 5,000-fold less toxic. If the gastrointestinal tract is the source of the endotoxin in patients with systemic inflammatory response syndrome, then the obligately anaerobic Bacteroides species, which outnumber the facultative species such as E. coli by 1,000-fold, should not be overlooked.

Animals↗
PubMed1995

A re-appraisal of the biological activity of bacteroides LPS.

Lipopolysaccharides (LPS) were extracted from seven Bacteroides strains by three different techniques: the phenol-water (PW), phenol-chloroform-petroleum (PCP) and Triton-Mg2+ methods. The strains selected included two different B. fragilis strains, one of which was grown in two different media. Yields varied between the strains, growth media and extraction technique, but generally the highest yield by weight was from the PCP method and the lowest from the PW method. The PW method was selected for the greatest amounts of carbohydrate and KDO, and the PCP method for the least. Phosphorus levels were more uniform among all extraction methods. Protein contamination was found in all Bacteroides LPS extracts, with extremely low levels in PW-LPS and the highest levels in material extracted by the PCP and Triton-Mg2+ techniques. No protein contamination could be detected after proteinase K treatment. After silver staining LPS PAGE profiles showed ladder patterns characteristics of smooth LPS for B. vulgatus, B. thetaiotaomicron and the control Escherichia coli O18:K- strains, whereas the other Bacteroides strains showed mainly rough and low M(r) material only. The PCP method did not select for high M(r) material in the B. fragilis strains; otherwise the LPS profiles for all extraction methods were identical. The biological activities of native and sodium salt form LPS were investigated on a weight for weight basis and compared to that of E. coli O18:K- PW-LPS. Amongst the LPS from Bacteroides strains, those prepared by the PW method were found to have a significantly higher activity in a galactosamine mouse lethality model, in induction of TNF and the Limulus amoebocyte lysate (LAL) assay, than LPS extracted by the PCP or Triton-Mg2+ methods. LPS from Bacteroides strains extracted by the PCP method had consistently low activity in all assays. Comparing PW-LPS from Bacteroides strains with that from E. coli O18:K- in the galactosamine mouse model, the E. coli O18:K- LPS was c. 5000-fold more active than the most active bacteroides LPS. However, in the LAL assay native PW-LPS from both the B. fragilis strains, and B. caccae had higher activities (up to 30-fold) than E. coli O18:K- LPS, with the PW-LPS from the other Bacteroides spp. being up to 15-fold less active than the E. coli O18:K- PW-LPS. In the TNF induction assay, E. coli O18:K- PW-LPS was 4-50-fold more active than bacteroides PW-LPS.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Anti-bacteroides lipopolysaccharide IgG levels in healthy adults and sepsis patients.

Members of the genus Bacteroides greatly outnumber enterobacteria in the human colon and therefore represent a vast potential pool of biologically active LPS. An enzyme-linked immunosorbent assay was developed to estimate the distribution of IgG levels to LPS from B. fragilis, B. vulgatus, B. thetaiotaomicron and to a mixture of rough LPS from three enterobacteria and Pseudomonas aeruginosa in sera from 641 adult blood donors. By inhibition ELISA some cross-reactivity was demonstrated between the different anti-bacteroides LPS IgG, but with very little between the anti-bacteroides LPS IgG and the anti-enterobacterial/Pseudomonas LPS IgG. Serum IgG was measured daily over 5-9 day periods in 12 sepsis patients (6 survivors, 6 non-survivors) and in a healthy individual. In all patients IgG levels fluctuated to a greater extent than levels in a healthy subject. Variations all followed similar overall trends and indicated that exposure to bacteroides LPS had occurred. In 5 out of 6 survivors, IgG levels were rising at the end of the period, while 4 of the 6 non-survivors showed falls, with an exception showing increasing levels to B. fragilis LPS. In 5 out of 6 non-survivors, IgG levels against B. fragilis LPS were substantially higher than those against the other LPSs. In this small sample some trends in antibody kinetics have been recognised which suggest bacteroides LPS may be significant in sepsis, and indicate that this study should be extended.

Antibodies, Bacterial↗
PubMed1995

Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide.

Burkholderia cepacia has emerged as an important multiresistant pathogen in cystic fibrosis (CF), associated in 20% of colonised patients with a rapid and fatal decline in lung function. Although knowledge of B. cepacia epidemiology has improved, the mechanisms involved in pathogenesis remain obscure. In this study, B. cepacia lipopolysaccharide (LPS) was assessed for endotoxic potential and the capacity to induce tumour necrosis factor (TNF). LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species Burkholderia gladioli exhibited a higher endotoxic activity and more pronounced cytokine response in vitro compared to preparations from the major CF pathogen Pseudomonas aeruginosa. This study help to explain the vicious host immune response observed during pulmonary exacerbations in CF patients colonised by B. cepacia and lead to therapeutic advances in clinical management.

Animals↗
PubMed1995

Prophylactic use of human endotoxin-core hyperimmune gammaglobulin to prevent endotoxaemia in colostrum-deprived, gnotobiotic lambs challenged orally with Escherichia coli.

The efficacy of human IgG polyclonal antibody to endotoxin-core in preventing endotoxaemia and subsequent disease was studied in colostrum-deprived gnotobiotic lambs challenged orally at about 5 h old with 10(9) cfu Escherichia coli. Human endotoxin-core hyperimmune gammaglobulin was given intravenously to 5 lambs at 1.9 g IgG/kg bodyweight prior to challenge. Human albumin was given intravenously to 3 control lambs. Bacteraemia was observed in all lambs, but the incidence was lower (P < 0.01) and the onset later (P < 0.05) in gammaglobulin pre-treated lambs. These lambs showed no signs of disease, whereas clinical endotoxaemia, manifesting as watery mouth disease, was diagnosed in 2 of the 3 control lambs which were killed between 18 and 22 h after challenge. Thus, prophylactic treatment of colostrum-deprived lambs with human IgG enriched in endotoxin-core antibodies was effective in reducing the degree of bacteraemia and preventing endotoxaemia, leukopenia and clinical disease following oral challenge with E. coli.

Administration, Oral↗
PubMed1995

Tumor necrosis factor induction by an aqueous phenol-extracted lipopolysaccharide complex from Bacteroides species.

The stimulation of macrophages and monocytes by lipopolysaccharide (LPS) results in the secretion of tumor necrosis factor (TNF), a cytokine which is thought to play a pivotal role in subsequent host responses. Its induction is thought to be facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. The LPS of Bacteroides species was considered a weak endotoxin; however, in a recent study we have shown that the biological activity and chemical composition of the LPS from Bacteroides species are dependent on the extraction method. The present study determines the capacity of LPS extracted by aqueous phenol (the method for producing an LPS of high endotoxic activity) from four species of Bacteroides to induce TNF. Induction was investigated from human mononuclear leukocytes (MNL), THP-1 cells (with and without enhancement by vitamin D2 for CD14), and peritoneal macrophages from C3H/HeJ (LPS nonresponder) and C3H/HeN (LPS responder) mice. Escherichia coli O18K- LPS, a typical smooth LPS of heterogeneous molecular mass, was used as a control throughout. The stimulation of TNF production by E. coli LPS was between two- and fourfold more than that by Bacteroides LPS in MNL, in THP-1 cells (with enhancement for CD14), and in peritoneal macrophages from C3H/HeN mice. In THP-1 cells (without enhancement for CD14), there was no significant difference in TNF production between E. coli and Bacteroides LPSs. In peritoneal macrophages from C3H/HeJ mice, E. coli LPS stimulated no TNF production, but there was no significant difference in TNF production from peritoneal macrophages from C3H/HeJ and C3H/HeN mice by Bacteroides LPS. In all cell populations, there was a peak of TNF production after approximately 4 h of stimulation with all LPSs tested. However, other peaks of TNF production were seen in MNL and THP-1 cells (with enhancement for CD14) after stimulation with E. coli LPS only. In stimulation assays in which Bacteroides LPS was together with but in excess of E. coli LPS, it was found that TNF production from MNL and THP-1 cells (with and without enhancement for CD14) was comparable to that of Bacteroides LPS alone and not E. coli LPS alone. An anti-CD14 monoclonal antibody did not inhibit Bacteroides LPS-stimulated TNF production. However, E. coli LPS-stimulated TNF release was inhibited by an anti-CD14 monoclonal antibody, most noticeably in MNL and THP-1 cells (with enhancement for CD14).(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1994

The influence of growth medium on serum sensitivity of Bacteroides species.

The susceptibility of 12 different Bacteroides strains (representing nine species) to the bactericidal effect of human serum complement was investigated. When grown in nutrient-rich proteose peptone-yeast extract medium, all 12 strains were, to varying degrees, sensitive to serum. However, when grown in Van Tassell and Wilkins's minimal medium, six of the 12 strains became markedly more serum resistant. Five of these six strains became totally resistant to serum when grown in heat-inactivated (56 degrees C, 30 min) sheep serum. By Percoll discontinuous density centrifugation and light microscopy, the ratio of bacteria with large and small capsules was found to vary with the growth medium used. Lipopolysaccharide (LPS) was extracted with aqueous phenol after growth in the three media. Polyacrylamide gel electrophoresis (PAGE) and silver staining of the LPS showed some differences in LPS profiles in all strains tested. Therefore, variation of growth conditions results in alterations of both the expression of surface structures and, in some cases, sensitivity to serum. The biochemical basis for these changes requires further investigation.

Bacterial Capsules↗
PubMed1994

Serum sensitivity of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis.

Bacterial strains which are sensitive to the bactericidal activity of serum are generally considered to be less virulent than serum-resistant strains and are seldom associated with bacteraemia. Burkholderia (Pseudomonas) cepacia is an important pathogen in cystic fibrosis and is associated with rapid fatal pulmonary decline and bacteraemia in 20% of colonised patients. In this study 19 isolates of B. cepacia expressing either rough or smooth LPS were investigated to determine the degree of serum sensitivity. Strains expressing rough-LPS were serum-sensitive: these included a highly transmissible strain of B. cepacia isolated from approximately 50 cystic fibrosis patients attending various U.K. regional centres and associated with cases of bacteraemia.

Antibodies, Bacterial↗
PubMed1994

Isolation of an adhesin from Staphylococcus aureus that binds Lewis a blood group antigen and its relevance to sudden infant death syndrome.

A 67 kDa protein was isolated from cell membrane preparations of Staphylococcus aureus (NCTC 10655) by affinity adsorption with synthetic Lewis a antigen conjugated to Synsorb beads. Pre-treatment of buccal epithelial cells expressing Lewis a with the purified protein reduced binding of the staphylococcal strain to a greater extent than the material not bound to the Synsorb beads. The significance of this work is discussed with reference to expression of Lewis a antigen in infants and the proposed role of toxigenic strains of staphylococci in some cases of sudden infant death syndrome.

Adhesins, Bacterial↗
PubMed1993

The accessibility of cross-reactive anti-lipopolysaccharide-core monoclonal antibodies to Escherichia coli grown in sub-MICs of temocillin and other antibiotics.

Two broadly cross-reactive anti-lipopolysaccharide core monoclonal antibodies WN1 222.5 and SZ27/150.3 were used in an ELISA system to detect the accessibility of core epitopes in Escherichia coli (four clinical isolates and NCTC 10418) grown to early stationary phase in the absence and presence of a half, a quarter and an eighth the MIC of temocillin, ampicillin, chloramphenicol, gentamicin and ciprofloxacin. The bacteria were coated on to ELISA microtitre plates. By comparing ELISA-titre ratios, temocillin induced a significantly large increase in binding of WN1 222.5 to all strains except NCTC 10418. Ampicillin and chloramphenicol induced a small increase in the binding of WN1 222.5 in some instances. Ciprofloxacin and gentamicin caused no increase in binding. Lipopolysaccharide SZ27/150.3 was only tested against temocillin-grown bacteria, but binding was not increased significantly compared with WN1 222.5. The results obtained demonstrate the potential use of temocillin to improve the clinical efficacy of immunotherapeutic monoclonal antibodies in Gram-negative sepsis.

Anti-Bacterial Agents↗