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D Y Graham

Publications and source records attributed to D Y Graham.

At least 73 records · Page 4Linked to original sources
PubMed2001

Furazolidone- and nitrofurantoin-resistant Helicobacter pylori: prevalence and role of genes involved in metronidazole resistance.

The prevalence of furazolidone, nitrofurantoin, and metronidazole resistance among Helicobacter pylori strains was assessed with 431 clinical isolates. Fifty-two percent were metronidazole resistant, compared to 2% (7 of 431) with resistance to furazolidone and nitrofurantoin. All seven furazolidone- and nitrofurantoin-resistant isolates were also metronidazole resistant. rdxA, frxA, and fdxB knockouts did not result in furazolidone or nitrofurantoin resistance. These data suggest that furazolidone and nitrofurantoin may be good alternatives to metronidazole for treating H. pylori infection.

Anti-Infective Agents, Urinary↗
PubMed2001

DNA sequence analysis of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant clinical Helicobacter pylori isolates.

We previously reported that inactivation of rdxA and/or frxA converted Helicobacter pylori from metronidazole sensitive to metronidazole resistant. To examine the individual roles of rdxA and frxA in the development of metronidazole resistance in H. pylori, we examined the status of rdxA and frxA from 12 pairs of metronidazole-sensitive and -resistant H. pylori isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazole-sensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of H. pylori J99 (MIC, 1 microg/ml). All strains with high-level metronidazole resistance (MICs, 128 microg/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 microg/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazole-resistant strains (MICs, 8 microg/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the rdxA and frxA genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-H. pylori therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.

Amino Acid Sequence↗
PubMed2001

Serum immunoglobulin G immune response to Helicobacter pylori antigens in Mongolian gerbils.

The Mongolian gerbil model for Helicobacter pylori infection is an animal model that mimics human disease. We examined the serum immune response to H. pylori infection in gerbils by enzyme-linked immunosorbent assay (ELISA) and Western blotting, both with whole-cell (H. pylori) extracts. A total of 66 7-week-old specific-pathogen-free male gerbils were inoculated orogastrically with H. pylori strain ATCC 43504. Sera were collected 1, 2, 4, 8, 12, 26, 38, and 52 weeks after H. pylori inoculation. Sixty-nine noninfected gerbils and their sera were used as controls. The specificity of the ELISA was 95.7%. The frequency of seropositivity increased over time: 2 of 10 (20%), 7 of 10 (70%), and 7 of 7 (100%) samples of sera from inoculated gerbils were positive for H. pylori at 2, 4, and 8 weeks postinoculation, respectively. Western blot assays showed that the primary immunoglobulin G (IgG) response against low-molecular-mass (25-, 30-, and 20-kDa) proteins appeared after a lag period of 2 to 8 weeks after inoculation. Antibodies against 160-, 150-, 110-, 120-, 80-, 66-, and 63-kDa proteins were observed 12 weeks after inoculation. The early reactive 30-kDa protein was identified as a urease alpha subunit by N-terminal amino acid sequencing. After 26 weeks, two groups of animals could be distinguished: one group developed ulcers (n = 5), and the other developed hyperplastic polyps without ulcers (n = 19). Gerbils in the gastric ulcer group showed significantly higher serum anti-H. pylori IgG levels than did gerbils in the hyperplastic group (P = 0.001) as measured by ELISA. Furthermore, a higher proportion of animals developed antibodies to H. pylori proteins of 26, 25, and 20 kDa in the ulcer group than those animals with hyperplastic polyps (75 to 100% versus 17 to 50%) in Western blot assays. These results highlight the importance of the immune response of the host in the development of H. pylori-related gastric lesions.

Animals↗
PubMed2001

The phenotype of gastric mucosa coexisting with Barrett's oesophagus.

BACKGROUND/AIMS: Barrett's oesophagus complicates the gastro-oesophageal acid reflux. Helicobacter pylori infection, particularly with cagA positive strains, induces inflammatory/atrophic lesions of the gastric mucosa, which may impair acid output. No systematic study has investigated the phenotype of the gastric mucosa coexisting with Barrett's oesophagus. This study was designed to identify the phenotype of gastric mucosa associated with Barrett's oesophagus. METHODS: In this retrospective case control study, the phenotype of the gastric mucosa was histologically characterised in 53 consecutive patients with Barrett's oesophagus and in 53 (sex and age matched) non-ulcer dyspeptic controls. Both patients and controls underwent extensive sampling of the gastric mucosa (two antral, one incisural, and two oxyntic biopsies). Intestinal metaplasia (IM) was categorised (type I, complete IM; types II and III, incomplete IM) by the high iron diamine stain; cagA status was ascertained by genotyping. RESULTS: Helicobacter pylori was present in 19 of the 53 patients with Barrett's oesophagus and in 30 of the 53 controls (p < 0.02); eight of the 19 patients with Barrett's oesophagus and 28 of the 35 controls harboured cagA positive H pylori (p < 0.03). The histological severity of non-atrophic gastritis detected in the controls was significantly higher than that detected in the patients with Barrett's oesophagus (p < 0.0001). Multifocal atrophic gastritis was present in 4% of the patients with Barrett's oesophagus and in 23% of controls (p < 0.01). The odds ratio for the association between multifocal atrophic gastritis and Barrett's oesophagus was 0.20 (95% confidence interval, 0.006 to 0.60). Gastric IM was detected in 13.2% of the patients with Barrett's oesophagus and in 30.1% of the controls (p < 0.03). Type III IM at the gastric mucosa was only detected among controls. CONCLUSIONS: Barrett's oesophagus is associated with a low prevalence of H pylori cagA positive infection and multifocal atrophic gastritis. This pathobiological pattern is considered to be associated with a low risk of distal gastric cancer.

Adult↗
PubMed2001

Gastric intestinal metaplasia: subtypes and natural history.

BACKGROUND: It has been suggested that the subtyping of intestinal metaplasia in the stomach is useful in stratifying patients with regard to risk of developing gastric cancer. AIM: To determine whether subtyping intestinal metaplasia provided useful information regarding the natural history of intestinal metaplasia. METHODS: The study used large cup gastric biopsy specimens from predetermined locations (gastric mapping). Follow up biopsies were obtained at one, two, and/or nine years. Biopsies with intestinal metaplasia were stained with high iron diamine/Alcian blue (HID/AB) to determine whether they expressed neutral mucins, sialomucins, or sulphomucins. RESULTS: Seventy nine patients with intestinal metaplasia were studied and characterised with regard to the most advanced subtype of intestinal metaplasia. The most severe type of intestinal metaplasia was type II in 33 patients and type III in 34 patients. Helicobacter pylori was cured in 67 patients. Follow up showed that changes in type of metaplasia (apparent regression or progression) occurred in both directions and were independent of H pylori status. For example, biopsy sites with "loss" of metaplasia at a follow up visit might have it "reappear" at a subsequent visit. During follow up, no patient developed gastric dysplasia or died from gastric cancer. CONCLUSION: HID subtyping did not provide useful information to the clinician or the pathologist. The data are consistent with the notion that the pattern, extent, and severity of atrophy with/without intestinal metaplasia is a far more important predictor of increased cancer risk than intestinal metaplasia subtype.

Adult↗
PubMed2001

Treatment of Helicobacter pylori infection.

Helicobacter pylori is a serious, chronic, progressive, and transmissible infection associated with a significant morbidity and mortality, which alone emphasizes the priority of developing adequate prophylactic or therapeutic measures. What was previously termed "asymptomatic H. pylori infection" is now recognized as a latent infection, and it is now accepted that the presence of an H. pylori infection is an indication for eradication therapy. Successful cure of H. pylori infection requires 2 or more antibiotics. Antibiotic resistance is the major impediment of cure. The ideal duration of therapy is unknown, but in general, 1 week therapy is less effective than longer durations. Compliance is important for the success of treatment; therefore, the favored regimen should have the least side effects. At present, a proton pump inhibitor (or ranitidine bismuth citrate)-clarithromycin triple therapy with either amoxicillin or metronidazole, for at least 10 days is considered first-line therapy. The alternative is quadruple therapy containing a proton pump inhibitor, bismuth, tetracycline, and a higher dose of metronidazole. Quadruple therapy is the best choice after failure of proton pump inhibitor-clarithromycin triple therapy. Confirmation of successful therapy with a urea breath test or a stool antigen test is now the standard of practice.

Anti-Bacterial Agents↗
PubMed2000

Specific seroreactivity of Crohn's disease patients against p35 and p36 antigens of M. avium subsp. paratuberculosis.

Crohn's disease (CD) is a chronic inflammatory bowel disease that is similar to Johne's disease in ruminants. Recent data have strengthened the association of M. avium subsp. paratuberculosis (M. paratuberculosis) with CD. To provide more evidence of an etiological association, antibody reactivities from CD patients were tested by immunoblotting against recombinant antigens that were identified previously from our M. paratuberculosis genomic library. Two clones (designated pMptb#40 (3.2-kb insert) and #48 (1.4-kb insert) expressing a 35K (p35)- and 36K(p36)-antigens showed specific reactivities with serum samples from CD patients. Serum samples from 75% of 53 CD patients, 14% of 35 normal individuals and 10% of 10 ulcerative colitis patients reacted to p35 antigen. Reactivities were also observed with serum samples from 89% of 89 CD patients, 14% of 50 normal controls and 15% of 29 ulcerative colitis patients reacted with p36 antigen. When the reactivity results from p35 and p36 were combined, the background from the controls was eliminated, i.e. only the CD patients reacted to both p35 and p36. The positive predictive value was 98% with specificity of 98% and the negative predictive value was 76% with sensitivity of 74% (39 positive out of 53). A statistical significance (p<0.0001) was observed when the results from CD serum samples reacting with either or both antigens were compared to the controls. The reactivity of anti-M. paratuberculosis (specifically against p35 and p36 antigens) antibodies in a significant proportion of CD patients would suggest a causal role for the organism in CD.

Adolescent↗
PubMed2000

In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples.

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria. Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.

Animals↗
PubMed2000

Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance.

Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer. It has been recently reported that metronidazole resistance from H. pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA). We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations. To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H. pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504. All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole. The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H. pylori 2600. In addition, the frxA gene cloned from the H. pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E. coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates.

Amino Acid Sequence↗
PubMed2000

A M(r) 34,000 proinflammatory outer membrane protein (oipA) of Helicobacter pylori.

The complete genome sequence revealed a family of 32 outer membrane proteins (OMPs) in Helicobacter pylori. We examined the effect of four OMPs (HP0638, HP0796, HP1501, and babA2) on the production of the proinflammatory cytokine, IL-8. Mutants of the four OMPs, as well as cagE and galE from H. pylori from the U.S. and Japan, were constructed by inserting a chloramphenicol-resistant cassette into the gene. Twenty-two pairs of parental and mutant H. pylori strains, as well as 160 clinical isolates (80 from Japanese and 80 from U.S.), were cocultured with gastric cancer cell lines. IL-8 production in the supernatant and adhesion was assayed by ELISA. HP0796, HP1501, babA2, and galE gene knockouts had no significant effect on IL-8 production. Knockout of the HP0638 gene in 81% of cag-positive strains reduced IL-8 production approximately 50%. The three cag-positive strains in which IL-8 levels were unchanged by HP0638 knockout had five or seven CT dinucleotide repeats in the 5' region, resulting in a frame shift and truncation. Strains with naturally inactive HP0638 gene were all from the U.S.; Japanese strains were always "on" and thus, on average, may be more virulent. Although cag-negative isolates produced a limited IL-8 response, cag-negative strains that contained a functional HP0638 gene produced more than 3-fold greater IL-8 than cag-negative nonfunctional HP0638 strains. We hypothesize that functional HP0638 gene may be an important virulence factor in relation to the risk of clinically significant outcomes of H. pylori infection. We denote HP0638 gene as outer inflammatory protein (oipA).

Amino Acid Sequence↗
PubMed2000

Conservation of Helicobacter pylori genotypes in different ethnic groups in Houston, Texas.

This study was concerned with whether the Helicobacter pylori strains circulating among ethnic groups living in the same region differ. The polymerase chain reactions to genotype (cagA, vacA, and iceA) H. pylori isolates from healthy volunteers from 4 ethnic groups (black, n=35; white Hispanic, n=31; whites, n=30; Vietnamese, n=29) residing in Houston were examined. The Vietnamese volunteers had the "East Asian"-type cagA 3' repeat region structure, and the others had the "non-Asian" type. The most common genotypes were delineated as follows: blacks and Hispanics, cagA+, vacA s1b-m1, and iceA2; whites, cagA+, vacA s1a-m2, and iceA2; and Vietnamese, cagA+, vacA s1c-m2, and iceA2. Two Hispanic families were also examined. H. pylori isolates from the children and their mothers had the same genotype and were different from those associated with the children's fathers or brothers-in-law. Conservation of an H. pylori genotype within ethnic groups over the course of generations will prove useful for epidemiological study of the coevolution of humans and H. pylori.

Adult↗
PubMed2000

Are genetic influences on peptic ulcer dependent or independent of genetic influences for Helicobacter pylori infection?

BACKGROUND: Genetic factors play a role or roles in the etiology of peptic ulcer disease and the acquisition of Helicobacter pylori infection. OBJECTIVE: To evaluate the relative importance of genetic and environmental influences as well as the importance of H. pylori on peptic ulcer disease. DESIGN: Cross-sectional study on monozygotic (MZ) and dizygotic (DZ) twins, reared apart or together. PARTICIPANTS: Twins of the subregistry of the Swedish Twin Registry included in the Swedish Adoption/Twin Study of Aging. MEASUREMENTS: Peptic ulcer disease and H. pylori status were assessed in MZ and DZ twin pairs reared apart or together. A total of 258 twin pairs had information regarding H. pylori status and history of peptic ulcer. Helicobacter pylori status was assessed as the presence of anti-H. pylori IgG. RESULTS: The intraclass correlations for peptic ulcer disease for MZ twins reared apart and together and DZ twins reared apart and together were 0.67, 0.65, 0.22, and 0.35, respectively, which indicates that genetic effects are important for liability to peptic ulcer. The correlation coefficient for MZ twins reared apart (0.67) provides the best single estimate of the relative importance of genetic effects (heritability) for variation in liability to peptic ulcer disease, and structural model fitting analyses confirmed this result (heritability, 62%). The cross-twin cross-trait correlations for MZ and DZ twins were examined to determine whether genetic effects for peptic ulcer were shared with or independent of genetic influences for H. pylori. The cross-correlations for MZ and DZ twins were almost identical (0.25 and 0.29, respectively), suggesting that familial environmental rather than genetic influences mediate the association between peptic ulcer disease and H. pylori infection. CONCLUSIONS: Genetic influences are of moderate importance for liability to peptic ulcer disease. Genetic influences for peptic ulcer are independent of genetic influences important for acquiring H. pylori infection.

Adult↗
PubMed2000

Microsatellite instability in gastric intestinal metaplasia in patients with and without gastric cancer.

The role and significance of microsatellite instability (MSI) in gastric carcinogenesis remain unknown. This study determined the chronology of MSI in gastric carcinogenesis by examining intestinal metaplasia (IM) from patients with and without gastric cancer. DNA was obtained from gastric specimens of 75 patients with gastric IM (30 cancer, 26 peptic ulcer, and 19 chronic gastritis patients) and was amplified with a set of eight microsatellite markers. Eight (26. 7%) tumors and seven (9.3%) IM samples (three from cancer-free patients) displayed high-level MSI (three or more loci altered). Low-level MSI (one or two loci altered) was detected in 50% of the tumors, in 40% of IM samples coexisting with cancer, and in 38% of IM tissues of cancer-free individuals. Among the 30 cancer patients, microsatellites were more frequently altered in IM coexisting with tumors that showed MSI (P = 0.003). In addition, patients with low-level MSI in the tumor tissues were more likely to have active Helicobacter pylori infection than those with stable tumors (P = 0.02). In conclusion, this study indicates that MSI occurs not only in gastric IM of patients with gastric carcinoma, but also in IM of cancer-free individuals. These data suggest that the progressive accumulation of MSI in areas of IM may contribute to gastric cancer development, representing an important molecular event in the multistep gastric carcinogenesis cascade.

Aged↗
PubMed2000

Helicobacter pylori.

H. pylori infection is now recognized as causing serious and life threatening disease in 20% to 30% of those infected. Reliable therapy is problematic. This article addresses the current approach to diagnosis and therapy and new considerations regarding whom to treat. The emphasis of the association of the gastric cancer phenotype of H. pylori infection (cagA positive H. pylori corpus gastritis) and protection against gastroesophageal reflux disease is an example of epidemiology without regard to the biology. Improvements in health and diet and an increase in body mass have accompanied the natural loss of H. pylori from the population. The search for virulence factors to help direct therapy to those who would most benefit has, to date, proved fruitless. Whenever H. pylori is diagnosed, it should be treated.

Anti-Bacterial Agents↗