Biomedical subjects

Ian R Poxton

Publications and source records attributed to Ian R Poxton.

15 recordsLinked to original sources

PubMed2006

"Dirty little secrets"--endotoxin contamination of recombinant proteins.

The identification of Toll-like receptors has revolutionised our understanding of innate immunity. TLR4 transduces the LPS signal and that of a number of structurally and functionally unrelated agonists. However, recent evidence adds to longstanding concerns that endotoxin contamination of bacterially derived recombinant TLR4 agonists is responsible for effects attributed to these molecules. We highlight key factors in differentiating specific agonist effects from those of endotoxin and emphasize why conventional methods of detecting and eliminating LPS may lead to erroneous results. We propose that considerable caution is needed in the investigation of TLR4 agonists, particularly when using proteins produced in a bacterium that also houses the most ideal TLR4 agonist, LPS.

Animals↗

PubMed2005

Molecular techniques in the diagnosis and management of infectious diseases: do they have a role in bacteriology?

The diagnosis and management of bacterial diseases has been done by traditional methods for a century or more. With the advent of molecular methods, however, these traditional approaches are being challenged. This review examines the pros and cons of traditional versus modern methods and tries to answer the question: when are molecular methods useful or essential? The following topics are addressed with appropriate examples: diagnosis; identification, typing and fingerprinting; pathogenesis; patient management; susceptibility to disease, and resistance to antimicrobial agents. It was concluded that there is still a place for both traditional and modern molecular methods, and training of staff must include both methodologies. Innovation is encouraged--but new technologies must be thoroughly tested before introduction into the routine lab. Liaison between laboratory scientist and physician is important, but above all experience is paramount.

Bacteriology↗

PubMed2004

Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via toll-like receptor 2.

Recognition of bacterial lipopolysaccharide (LPS) is critical in the host defence against Gram-negative infection. While enterobacterial LPS signals via Toll-like receptor 4 (TLR4), it has recently been reported that the LPS of Leptospira interrogans, Legionella pneumophila, Rhizobium species Sin-1 and at least one strain of Porphyromonas gingivalis are capable of signalling via TLR2. Using a TLR transfection assay and measurement of an NF-kappaB-sensitive promoter region, the results show that the LPS of Bacteroides fragilis NCTC-9343, Chlamydia trachomatis LGV-1 and Pseudomonas aeruginosa PAC-611 also signal via TLR2 and it is pointed out that all TLR2-signalling LPS discovered to date demonstrate relatively weak endotoxicity in some models and structural features distinct from those LPS shown to signal via TLR4.

Adaptation, Physiological↗

PubMed2004

The distribution of, and antibody response to, the core lipopolysaccharide region of Escherichia coli isolated from the faeces of healthy humans and cattle.

There are five different core types of Escherichia coli lipopolysaccharide (LPS), and enterohaemorrhagic E. coli tend to have the R3 core type. It has been hypothesized that increased carriage of bacteria with a specific core type will induce higher levels of antibodies and protect against disease caused by bacteria carrying that specific LPS core. Approximately 320 isolates of E. coli, half from healthy human faeces and half from healthy bovine faeces have been core typed both by core-specific monoclonal antibodies, and by PCR for genes encoding the enzymes responsible for the biosynthesis of the specific core structures. Results showed that E. coli possessing R1 core LPS were most frequently detected in both human and cattle populations (63 and 49%, respectively). Compared to the human isolates a significantly higher level of bacteria with R3 core LPS was detected among the bovine commensal E. coli (11% compared to 4%; P < 0.05). Antibody levels to each of the specific core types were measured in serum samples from healthy humans (n = 91) and healthy cattle (n = 39). In each population the highest level of antibody detected was reactive to the R4 core. In cattle the level of anti-R3 core antibody was significantly higher than the level of anti-R1, -R2 and -K12 antibodies (P < 0.01). In summary there was a greater proportion of E. coli with R3 core type in cattle, together with a corresponding higher anti-R3 antibody level. This suggests that cattle may have greater immunity to E. coli strains with an LPS of R3 core type.

Animals↗

PubMed2003

Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no deficit in lipopolysaccharide signalling.

Toll-like receptor 4 (TLR4)-mediated recognition of lipopolysaccharide (LPS) is required for efficient recognition of Gram-negative bacterial infections. Two commonly occurring mutations in the human TLR4 gene (Asp299Gly and Thr399Ile) have recently been shown to be associated with blunted physiological responses to inhaled LPS, and with increased risk of Gram-negative bacteraemia in sepsis patients and reduced risk of atherosclerosis in an Italian population. Here we show that monocytes from individuals heterozygous for both mutations in the TLR4 gene exhibit no deficit in recognition of LPS of Escherichia coli, Neisseria meningitidis, Bacteroides fragilis, Yersinia pestis, Chlamydia trachomatis, Porphyromonas gingivalis, or Pseudomonas aeruginosa. We propose that the relatively high frequency of these mutations in the Caucasian population may reflect modified responses of carriers to alternative TLR4 agonists.

Aspartic Acid↗

PubMed2003

Risk factors for Clostridium difficile colonisation and toxin production.

OBJECTIVES: to examine risk factors for patients becoming Clostridium difficile culture and toxin positive. DESIGN: prospective cohort study. SETTING: two medicine for the elderly wards. PARTICIPANTS: patients admitted to the wards over a 17-month period. MEASUREMENT: presence of Clostridium difficile on culture of stool specimens and toxins A and/or B. Patient's age, sex, source of admission, antibiotic, laxative, antacid and steroid use, presence/absence of colonic disease, neoplasia, leukaemia and nasogastric or percutaneous endoscopic gastrostomy tube feeding. RESULTS: 390 of 865 patients admitted provided a total of 1003 faecal specimens. Age (OR 1.04, 95% CI 1.001-1.07 per year), admission from another hospital (OR 2.13, 95% CI 1.29-3.50), non-cephalosporin antibiotics (OR 2.08, 95% CI 1.25-3.46) and cephalosporin use (OR 8.45, 95% CI 2.99-23.9) increased risk of becoming Clostridium difficile culture positive. Becoming toxin-positive was associated with antibiotic use only (OR 3.02, 95% CI 1.15-7.92), specifically amoxycillin (OR 8.72, 95% CI 1.66-45.9) and cephalosporins other than ceftriaxone (OR 7.28, 95% CI 1.34-39.6). CONCLUSION: different risk factors are important for the two stages leading to Clostridium difficile diarrhoea. Age, source of admission and third generation cephalosporins increase risk of becoming culture positive, whilst only antibiotic use is associated with the step of becoming toxin-positive. Understanding these differential risks may aid infection control strategies.

Aged↗

PubMed2003

Changes in sensitivity patterns to selected antibiotics in Clostridium difficile in geriatric in-patients over an 18-month period.

Clostridium difficile-associated disease continues to be a major problem in hospitals and long-term care facilities throughout the developed world. Administration of certain antibiotics such as amoxycillin, oral cephalosporins and clindamycin is associated with the greatest risk of developing C. difficile disease. The two antibiotics used for treatment of C. difficile disease are vancomycin and metronidazole, to which there is currently very little resistance. Randomly selected isolates (186) from 90 patients being investigated during an 18-month epidemiological study into the disease were tested for their susceptibility to vancomycin, metronidazole, amoxycillin, clindamycin, cefoxitin and ceftriaxone by the NCCLS agar dilution method. There was a narrow range of MIC for the two treatment agents (vancomycin and metronidazole), from 0.5 to 4 microg ml(-1), with no evidence of resistance. All strains were resistant to cefoxitin (MIC 64-256 microg ml(-1)), the antibiotic used in most selective media. All strains were of similar sensitivity to amoxycillin (MIC(90)= 4 microg ml(-1)). Most strains were resistant to ceftriaxone (MIC > or = 64 microg ml(-1)) or of intermediate resistance (MIC > or = 32 microg ml(-1)), with only two sensitive strains (MIC 16 microg ml(-1)). Clindamycin resistance was common, with 67 % of strains resistant (MIC > or = 8 microg ml(-1)), 25 % with intermediate resistance (MIC > or = 4 microg ml(-1)) and only 8 % sensitive (MIC < or = 2 microg ml(-1)). Twelve isolates from six different patients had very high resistance to clindamycin (MIC > or = 128 microg ml(-1)). Multiple isolates from the same patient, taken at different times, showed changes in susceptibility patterns over time. The only major change in susceptibility over the time-period was in clindamycin resistance; some strains appeared to become more resistant while others became less resistant. No differences were seen in the MIC(50) and MIC(90) of the different S-types of C. difficile identified, although some S-types were present in very small numbers. There was no correlation between the antibiotics prescribed and susceptibility.

Aged↗

PubMed2003

Clostridium difficile in a geriatric unit: a prospective epidemiological study employing a novel S-layer typing method.

Clostridium difficile is the major identifiable cause of antibiotic-associated diarrhoea in the UK. The aim of this study was to employ traditional culture, toxin detection and a novel typing method to determine the level of C. difficile colonization and disease in a population of elderly patients and to investigate the association between strains in the patients and their environment. Three hundred and ninety patients between 62 and 101 years of age admitted to a geriatric unit in the Royal Victoria Hospital (RVH), Edinburgh, were investigated for the presence of C. difficile. C. difficile was cultured from 100 (26 %) patients using pre-reduced cycloserine-cefoxitin egg yolk agar, and toxin(s) was detected in the faeces of 34 of these patients using the Techlab ELISA test kit for the detection of C. difficile toxins A and/or B. Toxin(s) was detected in a further 18 patients from whom no C. difficile was detected in culture. Of the patients in whom C. difficile was detected, 49 % had diarrhoea, with the highest proportion of patients with diarrhoea being both culture- and toxin-positive for C. difficile. Environmental sampling of the patient environment yielded C. difficile from 14 % of samples. The organism was most frequently isolated from floors, sluice-rooms and toilet areas. The variation in the molecular mass of the C. difficile S-layer proteins was exploited as the basis of a novel typing method for C. difficile. Isolates from patients in the RVH were given a four-digit 'S-type' number based on their S-layer protein profile. A total of seven S-types were identified, with one type, toxigenic S-type 5236, accounting for 73 % of all clinical isolates and 91 % of environmental isolates.

Aged↗

PubMed2003

Effects of sub-MIC concentrations of antibiotics on growth of and toxin production by Clostridium difficile.

Effects on growth and toxin A production of sub-MIC concentrations of six different antibiotics were investigated in three strains of Clostridium difficile: reference strain NCTC 11223, a fully sequenced strain (630) and a locally endemic isolate (strain 338a). The antibiotics chosen for investigation were the agents used to treat C. difficile-associated disease (CDAD), i.e. vancomycin and metronidazole, and four antibiotics that are commonly involved in precipitating CDAD (amoxycillin, clindamycin, cefoxitin and ceftriaxone). Strains were cultured in sublethal concentrations of antibiotics (1/2, 1/4 and 1/8 MIC) over 104 h and growth and toxin A production were measured three times a day. Effects varied between strain and antibiotic. The most common effect on growth of the strains was to increase the initial lag period by approximately 4 h, compared with antibiotic-free controls; however, strain NCTC 11223, which has high-level clindamycin resistance (> or = 512 microg ml(-1)), showed no lag whatsoever in comparison with the controls when grown in this antibiotic. The most common effect on production of toxin A was in the onset of toxin elaboration. Normally, toxins began to appear at low levels in the early stationary phase, before accumulating to high levels by the start of decline. In the presence of sub-MIC antibiotics, this onset appeared before that of the antibiotic-free controls. This effect was seen with metronidazole, amoxycillin and clindamycin, rarely with vancomycin and never with cefoxitin. These results suggest a very complex, strain-dependent relationship between the effects of growth and toxin production.

Anti-Bacterial Agents↗

PubMed2002

A comparative study of the intestinal microbiota of healthy horses and those suffering from equine grass sickness.

This study compares quantitatively the microbiota of the gastrointestinal tract of healthy horses with that of horses with equine grass sickness (EGS). Faecal and ileal samples were cultured quantitatively on selective and non-selective media. Confirmed anaerobes were identified to species level. Overall faecal counts gave a ratio of aerobes:anaerobes of approximately 1:1. However, the mean counts in healthy horses of 4.4x10(8) aerobes:3.7x10(8) anaerobes per gram wet weight were different from counts in EGS (means were 10-100-fold higher), with statistically significant differences for the anaerobes (p=0.04). There were 10-100-fold more anaerobic cocci in EGS samples compared to healthy controls. Most of the seven species of anaerobic cocci were found in both healthy horses and EGS. Differences in clostridia isolated between health and disease were notable: fourteen species were isolated from EGS cases, compared to only one (C. bifermentans) in controls. The mean faecal clostridial counts in chronic disease were higher than in controls (10-fold) and in acute EGS (100-fold). In contrast, mean counts for ileal samples from acute cases, showed a 10-fold increase for clostridia compared to 1000-fold reduction in chronic cases (compared to faecal counts). Results indicate an increase in the bacterial numbers in the GI tract of animals with EGS compared to the controls and clostridia are prominent in EGS. Whether the increase in clostridia is the cause of GI stasis or a consequence remains uncertain.

Animals↗

PubMed2002

Proinflammatory responses to lipo-oligosaccharide of Neisseria meningitidis immunotype strains in relation to virulence and disease.

Inflammatory responses to lipo-oligosaccharide (LOS) contribute to the severity of meningococcal disease. Strains that express the L(3,7,9) LOS immunotypes are isolated from the majority of patients, but other immunotypes are isolated predominantly from carriers. Inflammatory responses elicited from a human monocytic cell line (THP-1) that had been pretreated with vitamin D3 (VD3) were compared after stimulation with purified LOSs from standard immunotype strains. The neutralizing effects of normal human serum and serum from mice immunized with strain B:2a:P1.5,2:L3 were compared. LOSs of immunotypes L3, L7, L8, and L9 induced significantly higher levels of tumor necrosis factor-alpha and interleukin-6, compared with other immunotypes. Normal human serum neutralized the proinflammatory responses to LOSs of all immunotypes tested. Immune mouse serum neutralized inflammatory responses against LOSs from immunotypes with epitopes cross-reactive with L(3,7,9) moieties. Antibodies found in normal human serum and immune mouse serum to the oligosaccharide, core, and lipid A moieties of meningococcal endotoxin contribute to neutralizing activity.

Animals↗

PubMed2002

Structure and function of lipopolysaccharides.

The lipopolysaccharides of Gram-negative bacteria have a profound effect on the mammalian immune system and are of great significance in the pathophysiology of many disease processes. Consideration is given in this review to the relationship between structure and function of these lipopolysaccharides.

Carbohydrate Conformation↗

PubMed2002

The biological activity of a liposomal complete core lipopolysaccharide vaccine.

A vaccine that induces humoral immunity to lipopolysaccharide (LPS), while remaining non-pyrogenic should be beneficial, as high levels of antibodies against LPS are associated with a reduced risk of adverse outcome. However, pure LPS or bacteria expressing LPS are generally considered too toxic to be used as vaccines. Recently, a novel, immunogenic complete core lipopolysaccharide vaccine has been described, which has been designed to prevent endotoxin-related inflammatory reactions in surgical and high-risk hospitalized patients. In vivo studies have shown that while administration of the vaccine to rabbits results in no toxicity over 7 days, it does induce significantly enhanced antibody responses towards a broad range of clinically relevant Gram-negative LPSs. Here we show that encapsulation of the four complete core LPS types Escherichia coli K12, Escherichia coli R1, Bacteroides fragilis and Pseudomonas aeruginosa into liposomes greatly reduces the ability of a given amount of LPS to induce TNF-alpha production in vitro from human monocytes. In contrast to previous studies of liposomal LPS, we demonstrate a reduction in activity of approximately 100,000-fold; a reduction approximately 100-1,000-fold more than that previously described. The signalling by the liposomal LPS appears to be entirely dependent on serum factors, though this can be partially restored by soluble CD14 or, to a lesser extent, by lipopolysaccharide binding protein. Time-course experiments reveal that liposomal LPS signalling shows similar kinetics to pure LPS signalling. Therefore, as well as inducing specific antibody responses, liposomal LPS demonstrates characteristics suitable for use as a vaccine to be used in human beings.

Animals↗

PubMed2001

Mucosal and systemic antibody responses to the lipopolysaccharide of Escherichia coli O157 in health and disease.

Mucosal immunity in the gastrointestinal (GI) tract is a primary defence against GI pathogens. We hypothesise that a mucosal response to lipopolysaccharide (LPS), especially to the common (core) determinants of GI pathogenic Escherichia coli strains, is protective. The aims of this study were to investigate the specificities, levels and development of humoral responses in health and GI disease to the R3 LPS core and O-polysaccharide of E. coli O157. The purpose was to try to predict whether vaccination or passive immunisation might induce protection. Wherever possible, paired whole gut lavage fluid (WGLF) and serum samples were collected for comparison of the mucosal and systemic responses. Matched saliva samples were also collected from some study groups. The patient groups included those with acute E. coli O157 disease (serum only), patients convalescing after E. coli O157 infections, and patients undergoing routine investigation for GI conditions but subsequently shown to be immunologically normal. Some samples of WGLF from patients with Crohn's disease (CRO) and ulcerative colitis (UC) were included to allow comparisons with patients with inflammatory conditions known to alter antibody secretion in the GI tract. The healthy groups from whom serum and saliva only were taken included blood donors, healthy volunteers and a group of slaughterhouse workers. This latter group was likely to have been exposed regularly to faecal bacteria from animals and antibody specificities might have been expected to be different from other healthy individuals. Levels and classes of antibodies were determined by ELISA with microtitration plates coated with polymyxin complexes of whole LPS extracted from E. coli O157 and LPS from the E. coli R3 rough mutant. Antibodies of IgG and IgM classes were measured in serum and IgA was measured in WGLF and saliva. IgG antibodies to the O157 LPS and the R3 core oligosaccharide were detected in the serum of healthy blood donors. Patients with acute E. coli O157 disease showed elevated levels of serum IgM to O157 LPS and R3, with IgG levels raised only to R3. In serum from convalescent patients, IgG to O157 LPS was significantly above the control groups only in the period 6-16 weeks after infection. Total IgA levels were similar in WGLF specimens from all groups, except the patients with UC, whose levels were much higher. Specific IgA levels were higher in the E. coli O157 convalescent group, but there were no significant correlations overall. UC patients had significantly lower levels of IgA to O157 and CRO patients had higher O157 IgA levels than UC patients and healthy volunteers. In serum, inhibition of ELISA showed that the response to the O157 LPS was due in part to a response to the R3 oligosaccharide component. This response was much more pronounced in the healthy and non-O157 groups than in convalescent patients. There was no correlation between specific IgA antibody levels in saliva and matched specimens of WGLF, and levels in sequential saliva specimens fluctuated widely. The significant IgG and IgA responses to the R3 core suggest that there is immunological memory to this oligosaccharide LPS component which may have a role in protection against E. coli LPS both systemically and locally in the GI tract. Boosting of this mucosal response to the LPS core, either naturally through exposure or by active or passive immunisation, may confer protection. Finally, antibody responses to E. coli O157 must be interpreted with caution, as the response detected is a sum of responses to the O-specific polysaccharide and the R3 core.

Adolescent↗

PubMed1999

Lipopolysaccharide chemotypes of Burkholderia cepacia.

Burkholderia cepacia is an important pathogen in patients with cystic fibrosis (CF) and much is now known of its epidemiology. In contrast, its virulence mechanisms are poorly understood. The lipopolysaccharide (LPS) of B. cepacia, a well-recognised virulence factor of other gram-negative bacteria, is known to be strongly endotoxic in vitro. The aim of this study was to observe if there were any links between the structure of B. cepacia LPS and virulence. This has been investigated by polyacrylamide gel electrophoresis and immunoblotting to define the chemotype and antigenic cross reactivity of B. cepacia LPS. Strains (16) belonging to different genomovars of the B. cepacia complex were selected to represent epidemic and non-epidemic clinical isolates and environmental strains. All strains belonging to genomovars I and II (the latter now renamed B. multivorans) had smooth LPS. However, isolates belonging to genomovar III, the group to which most of the epidemic CF isolates belong - including the highly transmissible strain (ET 12) which has been found in both the UK and North America - were of either rough or smooth LPS chemotype. In this study, B. cepacia J2315 represents the ET 12 lineage, and has a rough chemotype. Rabbit antiserum raised to strain J2315 revealed that the LPS core of this strain was antigenically related to some but not all other genomovar III strains, but it also cross-reacted strongly with all B. multivorans (genomovar II) and most genomovar I strains. Intra-strain phenotypic variation was demonstrated between bacteria grown in broth or on solid agar with a concomitant variation in antigenic cross reactivity. There was no clear evidence to associate any particular LPS phenotype with epidemic or non-epidemic strains, but changes in phenotype in vitro may provide clues to the survival and adaptability of B. cepacia in hostile environments and possibly to its ability to produce an inflammatory response in vivo.

Animals↗