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Biomedical subjects

V S Chadwick

Publications and source records attributed to V S Chadwick.

At least 19 recordsLinked to original sources
PubMed2006

Carriage, quantification, and predominance of methanogens and sulfate-reducing bacteria in faecal samples.

AIMS: To determine carriage rates and densities of methanogens and sulfate-reducing bacteria in adults and children using molecular methods, and to also determine if a reciprocal relationship exists between these organisms. METHODS AND RESULTS: Real-time PCR was used to detect and quantify methanogens and sulfate-reducing bacteria. Real-time PCR was more sensitive than breath methane measurements. Real-time PCR assays were applied to faecal DNA samples collected from 40 children and 12 adults. Methanogens were present in 25% of the children and 42% of the adults studied, and sulfate-reducing bacteria were detected in 15% of the children and 58% of the adults. High levels of sulfate-reducing bacteria were found in two methanogenic adults. CONCLUSIONS: Carriage rates and densities of methanogens and sulfate-reducing bacteria are greater in adults than in children. Competition does not necessarily lead to the predominance of one group in the faecal microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes sensitive, molecular assays that could be used to monitor these organisms in gastrointestinal disease. Therapeutic exclusion of one group from the bowel would not necessarily lead to the expansion of the other, as there does not appear to be a reciprocal relationship between these groups.

Adult↗
PubMed2001

High prevalence of Mycoplasma pneumoniae in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls.

Intestinal microflora are believed to play an important role in the pathogenesis of inflammatory bowel disease (IBD). Mycoplasma have been suggested previously as organisms of ubiquitous distribution with the potential to cause inflammatory diseases, including IBD in susceptible individuals. The aim of this study was to determine the frequency of the presence of M. pneumoniae DNA in intestinal biopsies from patients with IBD and non-IBD controls using a microplate polymerase chain reaction-hybridization assay (PCR-ELISA). A total of 260 endoscopic biopsies (49 from 19 patients with Crohn's disease, 76 from 27 patients with ulcerative colitis, and 135 from 43 non-IBD controls) were used in this study. Overall, M. pneumoniae-specific DNA was detected in 100 endoscopic biopsy samples (38.5%). Among them, the detection rate of M. pneumoniae DNA was significantly higher in biopsies from patients with CD (59.2%) than in those from patients with UC (26.3%) or non-IBD controls (37.7%) (chi2 = 13.65, P < or = 0.001). The high prevalence of M. pneumoniae in both IBD patients and controls suggest this organism is ubiquitous and may persist in the intestinal mucosa. Epidemiological studies in IBD suggest acquisition of some agents early in life probably during epidemics in temperate latitudes. M. pneumoniae could be one of the ubiquitous agents implicated in the pathogenesis of IBD.

Adult↗
PubMed2001

Helicobacter pylori infection: mechanism of colonization and functional dyspepsia Reduced colonization of gastric mucosa by Helicobacter pylori in mice deficient in interleukin-10.

BACKGROUND AND AIMS: Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine. Mice deficient in IL-10 production (IL-10-/-mice) develop a spontaneous chronic enterocolitis, suggesting that IL-10 is an important regulator of the mucosal immune response in vivo. The objective of this study was to determine the role of endogenous IL-10 in the host defense against gastric colonization by Helicobacter pylori by using IL-10-deficient mice. METHODS: The IL-10-/-mice were inoculated intragastrically with a mouse-adapted H. pylori isolate (Sydney Strain 1). Gastric colonization by H. pylori (biopsy urease test and bacterial colony counts), serum levels of H. pylori-specific immunoglobulin (Ig) M, A, G, isotypes of IgG, and the gastric mucosal inflammatory scores were determined 6 weeks after inoculation. Results were compared with those obtained from H. pylori-infected control mice (IL-10+/-mice). RESULTS: The colonization of gastric mucosa by H. pylori was reduced approximately 100-fold (P < 0.0001) in IL-10-/-mice (log10 4.87 +/- 0.26CFU/g tissue) as compared to IL-10+/-mice (log10 6.64 +/- 0.22 CFU/g tissue). Furthermore, IL-10-/-mice infected with H. pylori had significantly higher H. pylori-specific IgA and IgG antibodies in serum (P < or = 0.01), and developed much more severe chronic active gastritis than infected IL-10+/-mice. The median scores of the infiltration of gastric mucosa by mononuclear cells and neutrophils were up to threefold higher in IL-10-/-mice than they were in IL-10+/-mice. CONCLUSION: Our studies suggest that endogenous IL-10 is an inhibitor of the protective immune response to H. pylori infection. Interleukin-10 participates in the downregulation of H. pylori-induced gastric inflammatory responses, which apparently confers a survival advantage to the organism promoting more effective colonization of gastric mucosa.

Animals↗
PubMed2000

Inhibition of mitogen-induced murine lymphocyte proliferation by Helicobacter pylori cell-free extract.

BACKGROUND AND AIMS: Previous studies have shown that lysates of Helicobacter pylori inhibit mitogen-induced proliferation of human peripheral blood mononuclear cells. The objective of the present study was to determine whether H. pylori cell-free extract (HPCE) has similar effects on murine lymphoid cells and could, therefore, be used to further delineate the mechanisms of alteration of lymphocyte function by H. pylori. METHODS: The HPCE was prepared from a H. pylori reference strain and from five clinical strains with varying status of cagA and vacA. Mouse splenic and mesenteric lymph node cells were cultured in microwell plates in the presence or absence of varying concentrations of HPCE (0.625-12.5 microg/mL). T cell mitogens were added into the culture 2 h later and the cells were cultured at 37 degrees C in 5% CO2 for a further 72 h. Cell proliferation was determined by a non-radioactive rapid dye assay and the percentage inhibition caused by HPCE was calculated. RESULTS: Pre-exposure to HPCE significantly inhibited concanavalin A-induced proliferation of murine spleen and mesenteric lymph node cells (up to 100% inhibition; P < or = 0.01). The HPCE also inhibited lymphocyte proliferation stimulated by mitogens phorbol-myristate-acetate and ionomycin and by the anti-CD3epsilon monoclonal antibody (P < or = 0.05). The inhibition was dose-dependent, but independent of the presence of virulence genes cagA or vacA. Treatment of HPCE at 80 degrees C for 30 min, but not at 55 degrees C for 60 min, completely abolished its inhibitory action. The HPCE, pretreated with pronase E, proteinase K, trypsin, acid or alkali also completely lost its inhibitory effect (P < or = 0.01), while in contrast, treatment with carboxypeptidase and leucine aminopeptidase had no effect. CONCLUSION: Helicobacter pylori produces heat-labile proteins or peptides that suppress T cell mitogen-induced proliferation of murine lymphoid cells in a similar manner to that observed with human peripheral blood mononuclear cells. The mouse cell culture system can, therefore, be used as a model to further study the mechanisms of action and antigen specificity of these immunomodulatory factors.

Animals↗
PubMed2000

Detection of Listeria monocytogenes by polymerase chain reaction in intestinal mucosal biopsies from patients with inflammatory bowel disease and controls.

BACKGROUND AND AIMS: Components of the intestinal microflora are believed to play an important role in the pathogenesis of inflammatory bowel disease (IBD) in genetically susceptible hosts acting either as a non-specific antigenic stimulus or as a specific pathogen. Listeria monocytogenes has been suggested as an organism with the potential to cause IBD. The objective of the present study was to investigate the prevalence of L. monocytogenes DNA in intestinal biopsies from patients with IBD and from non-IBD controls by using nested polymerase chain reaction (PCR). METHODS: The DNA was extracted from 274 colonoscopic biopsies, which were obtained from 23 patients with Crohn's disease (CD), 28 with ulcerative colitis (UC) and 39 non-IBD control patients. Nested PCR amplification was used to detect the presence of the L. monocytogenes listeriolysin O (hly) gene. The sequences of positive PCR products were determined and compared with databases. RESULTS: The sensitivity of our nested PCR was 10 fg L. monocytogenes DNA. Overall, L. monocytogenes DNA was detected in 13.0% patients with CD, 17.9% patients with UC and 25.6% non-IBD control patients or in 29 of 274 (10.6%) endoscopic biopsies. Among them, L. monocytogenes DNA was detected in four of 67 (6%) biopsies from patients with CD, five of 94 (5.3%) biopsies from patients with UC and 20 of 113 biopsies (17.7%) from non-IBD control patients. Sequence analysis of positive PCR products demonstrated more than 95% similarity to the hly gene sequence of L. monocytogenes, confirming the authenticity of our PCR products. CONCLUSION: Listeria monocytogenes DNA was detected in the intestine of both patients with IBD and in non-IBD control patients, probably reflecting the widespread presence of this organism in the environment. The low yield of positive biopsies in our IBD patients (5-6%) and the fact that the detection rate of L. monocytogenes DNA was similar in endoscopic biopsies from IBD patients and non-IBD controls does not support a direct role for L. monocytogenes in the pathogenesis of IBD, at least in New Zealand patients.

Adolescent↗
PubMed1999

Rapid elimination of Helicobacter pylori and reduction of histocompatibility leucocyte antigen-DR expression 12 h after a single dose of omeprazole, amoxycillin and metronidazole triple therapy.

BACKGROUND: Studies of eradication of Helicobacter pylori and subsequent resolution of H. pylori-related gastritis, have focused mainly on medium and long-term change following eradication therapies. Results from those studies have shown that both acute and chronic inflammatory changes found in gastric mucosa eventually return to normal. However, the early events in the stomach, particularly the effects on bacterial density and acute inflammatory markers of anti-H. pylori treatment, are largely unknown. The objective of this study was therefore to examine changes in the number of H. pylori, and the severity of gastric mucosal inflammation in the gastric biopsy specimens of patients before (0 h group, n = 14) and 12 h (12 h group, n = 14) after initiating anti-H. pylori treatment. METHODS: Biopsies were assessed, either quantitatively or semi-quantitatively, for the presence of H. pylori, neutrophils, mast cells, intraepithelial lymphocytes and the expression of histocompatibility leucocyte antigen (HLA)-DR by gastric epithelium and the results were compared between groups. RESULTS: Median H. pylori scores were 5 (range 2-5) and 0 (range 0-2) in biopsies from untreated and 12 h post-treatment groups, respectively (P < 0.001). In most 12 h post-treatment biopsies, H. pylori organisms could not be identified. There was a significant reduction in HLA-DR expression by gastric epithelium (median 3.5 with range 2-4 at 0 h group vs median 2 with range 0-4, P < 0.05), but there was no significant difference in the number of intraepithelial lymphocytes, CD3+ cells, mast cells or the distribution and density of neutrophils (all P > 0.05). Furthermore, the severity of gastritis as scored with the Sydney system was similar in both untreated and treated groups. CONCLUSIONS: The results of this study indicate that elimination of H. pylori organisms and resolution of some inflammatory markers occurs as early as 12 h following a single dose of omeprazole 40 mg, amoxycillin 1.0 g and metronidazole 400 mg, which suggests that rational therapeutic strategies with shorter duration using the currently available drugs may be possible.

Amoxicillin↗
PubMed1999

Helicobacter pylori infection in interleukin-4-deficient and transgenic mice.

BACKGROUND: Interleukin (IL)-4 is a potent anti-inflammatory and Th2-type immunoregulatory cytokine. Helicobacter pylori infection in humans induces a polarized Th1 immune response characterized by increased production of interferon-gamma and absence of IL-4. This study was designed to determine the role of endogenous IL-4 in the host defence against gastric colonization by H. pylori using IL-4-deficient (IL-4-/-) and transgenic (IL-4 Tg) mice. METHODS: IL-4-/- mice and IL-4 Tg mice were inoculated intragastrically with H. pylori Sydney Strain 1. Gastric colonization by H. pylori (biopsy urease test and bacterial colony counts), serum levels of H. pylori-specific immunoglobulin M, A, G, isotypes of IgG, and the gastric mucosal inflammatory scores were determined 6 weeks after inoculation. Results were compared with those obtained from H. pylori-infected IL-4+/+ (controls for IL-4-/- mice) and IL-4 WT (controls for IL-4 Tg) mice. RESULTS: Colonization of the gastric mucosa by H. pylori in IL-4-/- mice was similar to that of control IL-4+/+ mice. There was no significant difference in titres of H. pylori-specific antibodies or gastric inflammatory scores between the two groups of mice. Colonization of gastric mucosa by H. pylori was consistently lower in IL-4 Tg mice (log10 6.40+/-1.09 CFU/g tissue) compared with IL-4WT mice (log10 7.20+/-0.34 CFU/g tissue), although the difference was not significant. Nevertheless, IL-4 Tg mice did have significantly higher titres of H. pylori-specific IgA and IgG (P< or =0.01). CONCLUSION: These results show that endogenous IL-4 is not a major contributor to host resistance to H. pylori, and enhanced IL-4 production has little if any effect on gastric colonization by this organism, despite increased specific antibody production.

Animals↗
PubMed1995

Flexible sigmoidoscopy or colonoscopy for colorectal screening: a randomized trial of performance and acceptability.

In a randomized clinical trail to assess acceptability, yields, costs, and unwanted effects of screening procedures, 232 subjects (137 with family history of colorectal carcinoma or adenoma, 95 without) were offered either flexible sigmoidoscopy or colonoscopy. Subjects with polyps found on sigmoidoscopy were followed up by colonoscopy. The two procedures were similar in compliance (65%) and yield (19% adenoma, 15% hyperplastic polyps). Polyps of either type were more common in those with a family history (prevalence: 41% compared with 24% without family history, p = 0.04). Costs per procedure were 60% lower for sigmoidoscopy, but follow-up colonoscopy reduced this cost advantage to 20% per subject. The subjects found the preparation for sigmoidoscopy easier, but the procedure more uncomfortable and embarrassing, as colonoscopy was performed under sedation. In this hospital-based study, colonoscopy was as acceptable to subjects, and only slightly more costly than sigmoidoscopy. Advantages of sigmoidoscopy would be greater for use outside hospitals and with less intensive follow up.

Aged↗
PubMed1995

Mycobacterium paratuberculosis and inflammatory bowel disease: frequency distribution in serial colonoscopic biopsies using the polymerase chain reaction.

An association between Mycobacterium paratuberculosis and Crohn's disease is suspected but the evidence remains controversial. Using a one-step DNA extraction procedure with the thermophilic protease PRETAQ and amplification by the polymerase chain reaction, M. paratuberculosis DNA was detected in 22% of patients with Crohn's disease, and in 13% of patients with ulcerative colitis. M. paratuberculosis DNA was not found in any biopsy tissue from control non-inflammatory bowel disease patients. The biopsy tissues in which M. paratuberculosis was detected all came from regions which were inflamed when viewed microscopically. Overall, 7.7% of biopsies from such inflamed areas were positive. This low frequency of detection could be explained on the basis of extremely low abundance of the organism in relation to the area of mucosa sampled, or be consistent with a non-aetiological role for M. paratuberculosis in inflammatory bowel disease.

Animals↗
PubMed1994

Anti-inflammatory effects of LPS, MDP and FMLP on carrageenan pleurisy in the rat.

Bacterial products fmet-leu-phe (FMLP), muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were assayed for their ability to alter the inflammatory response to lambda carrageenan-induced pleurisy in Hooded Surgery rats. Continuously infused FMLP, or one initial i.v. dose of FMLP, MDP or LPS either ablated or partially suppressed the pleurisy. Total circulating leucocytes and neutrophils were suppressed by 55-65% when compared to the normal circulating leucocyte response to carrageenan pleurisy, excepting the protocol incorporating a single i.v. dose of FMLP where suppression was intermediate at 30%. There were also significant changes in the expression of FMLP receptors on circulating neutrophils. MDP and LPS induced a receptor number increase of 2 and 1.7 times initial value respectively, whilst a continuous FMLP infusion caused a receptor decrease to 0.3 times the initial value. The introduction of bacterial products at an alternative site to that of the pleurisy had an anti-inflammatory effect and the pleurisy was reduced.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1993

Formylmethionyl-leucylphenylalanine and the SOS operon in Escherichia coli: a model of host-bacterial interactions.

To determine the biological significance of the existence of highly specific receptors for the bacterial chemotactic peptide formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) on neutrophil leucocytes, we investigated the role of this peptide in bacterial metabolism. The UmuD protein of the Escherichia coli SOS operon was identified as having an N-terminal fMet-Leu-Phe sequence and a recombinant E. coli with the umuD gene on plasmid pSB13 was shown to be an over-producer of both UmuD and fMet-Leu-Phe. Activation of SOS genes in conventional wild-type E. coli (K12) by u.v. light or hydrogen peroxide increased fMet-Leu-Phe production up to 4-fold. A RecA- strain, incapable of SOS activation, was a low basal producer of fMet-Leu-Phe and showed no increased production with u.v. light or oxidant stress. We propose that host phagocytes respond to fMet-Leu-Phe and closely related peptides because they are generated by bacteria under oxidant stress. Increased fMet-Leu-Phe production may signal to the host a change in the organism's biological status from commensal to pathogen because of the invasion into tissues exposing bacteria to high pO2 levels and oxidant stress.

Amino Acid Sequence↗
PubMed1992

Flow cytometry analysis of the expression of neutrophil FMLP receptors.

The number of cell surface FMLP receptors expressed by peripheral blood neutrophils taken from healthy individuals was quantitated using a direct fluorescence label analysed on the FACScan. Receptor numbers, assayed on cells from heparinised blood prepared at 4 degrees C or 37 degrees C or in the presence of LPS (30 ng/ml) were 12,540 +/- SD 2413, 18,588 +/- 4938 and 45,041 +/- 10,744 respectively. Anticoagulation with EGTA resulted in 25-30% greater basal receptor numbers but had no effect on receptor level after maximal stimulation with LPS. Double labelling with a neutrophil-specific mAb showed that the entire neutrophil population expressed FMLP receptors.

Antibodies, Monoclonal↗
PubMed1992

Isolation and purification of N-formylmethionine aminopeptidase from rat intestine.

The intestinal mucosal epithelium is exposed to products of intestinal bacteria including potent inflammatory N-formylmethionyl oligopeptides. An N-formylmethionine aminopeptidase has been purified 2300-fold from rat intestine and was shown to degrade natural fMet oligopeptides from Escherichia coli culture supernatants with loss of bioactivity (release of specific granule constituents from human polymorphonuclear leucocytes) and immuno-reactivity (assessed using a polyclonal anti-fMet-Leu-Phe antiserum). The enzyme which was specific for N-terminal acyl-methionine residues had a native Mr of 340,000 and comprised four sub-units of Mr 82,000. The presence of this enzyme in intestinal mucosa could prevent absorption of intact bioactive fMet peptides produced by commensal bacteria in the gut lumen.

Amino Acid Sequence↗
PubMed1992

Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 +/- SEM 2.2 vs. 27.4 +/- 2.5, P less than .005) a left shift in dose-response curves after feeding compared to fasting (P less than .005), spontaneous up-regulation of responses in blood incubated at 37 degrees C for 1 h, and marked upregulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.

Cytochalasin B↗
PubMed1992

Hepatobiliary excretion of bacterial formyl-methionyl peptides in rat. Structure activity studies.

The bacterial chemotactic peptide formyl-met-leu-phe and its radioiodinated analog formyl-met-leu-[125I]tyr are rapidly excreted by the liver into bile following portal or systemic venous infusions in rats or after absorption from the gut lumen. To determine the molecular structural requirements for hepatobiliary excretion of formyl-methionyl peptides, structure-activity studies using portal venous infusions of 24 structural analogs of formyl-met-leu-tyr were performed in rats with biliary cannulae. Hepatic extraction of peptides was studied in vivo using external gamma counting after portal infusion. Efficient hepatobiliary excretion was not restricted to bioactive formyl peptides, but showed a broad specificity for different amino-acylated (formyl, acetyl, propionyl, carbobenzoxy) di- and tripeptides and no requirement for methionine in position one or for a free carboxy terminus. However, nonacylated peptides and an acyl-amino acid showed little excretion. Hepatic extraction of peptide was also related to N-acylation. Hepatic extraction and excretion of N-acyl peptides were also related to hydrophobicity. Thus, the presence of an N-acyl group is the key determinant of biliary excretion of inflammatory bacterial f-met peptides in the rat.

Animals↗
PubMed1992

Identification of formyl Met-Leu-Phe in culture filtrates of Helicobacter pylori.

Helicobacter pylori synthesizes and secretes a substance which co-chromatographs and is antigenically cross-reactive with the bacterial chemotactic peptide fMet-Leu-Phe. Using reverse phase and affinity chromatography this substance has now been purified. Carboxypeptidase Y microsequencing has verified that this material is fMet-Leu-Phe. The infiltration of polymorphonuclear leucocytes to sites of H. pylori infection may be a response to mucosal permeation of soluble, diffusable bioreactive substances such as fMet-Leu-Phe.

Amino Acid Sequence↗
PubMed1990

Fourier analysis of biliary and pancreatic excretion in man based on data obtained by a duodenal perfusion/aspiration technique.

A standard duodenal perfusion/aspiration technique was used to continuously monitor biliary and pancreatic excretion in young healthy human subjects, and the excretory patterns were examined by Fourier power spectral analysis. Experiments were carried out in the fasting state, either without or during a continuous parenteral (i.v.) stimulation by secretin and the cholecystokinin analogue ceruletide. The duodenal content aspirated was either discarded after sampling or reinfused into the jejunum. In the fasting state, significant biliary and pancreatic excretion was detected, fluctuating with a periodicity of about 60 min. During parenteral infusion with ceruletide/secretin, to simulate a postprandial state, the rate of biliary and pancreatic excretion increased as compared with fasting levels alone (basal levels). A dominant period of about 60 min was still detected but second periods of approximately 45 min and approximately 95 min, respectively, were also observed. The peak power and the total power of the biliary excretion signals were reduced. Reinfusion of aspirated duodenal fluid into the intestine (jejunum) led to a further decrease in peak power and total power of the known biliary signals. Trypsin excretion into the duodenum revealed mainly insignificant changes in peak and total power upon hormone stimulation despite a definite increase in total amount of trypsin excreted. The results indicate that parenteral ceruletide/secretin stimulation has a stabilizing effect on biliary excretion in man, and that reinfusion of aspirated duodenal content into the intestine further stabilizes the excretion.

Adult↗