Cloning, expression, and physical mapping of the 3beta-hydroxysteroid dehydrogenase gene cluster (HSD3BP1-HSD3BP5) in human.

Seven members of the human 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene family (HGMW-approved symbols HSD3BP1-HSD3BP5) have been cloned and physically mapped. HSD3B1 and 2 express 3beta-HSD enzymes; HSD3Bpsi1-5 are unprocessed pseudogenes that are closely related to HSD3B1 and 2 but contain no corresponding open reading frames. mRNA is expressed from psi4 and psi5 in several tissues, but with altered splice sites that disrupt reading frames. A 0.5-Mb contig of 3 yeast artificial chromosome and 32 bacterial artificial chromosome genomic clones contained no additional members of the gene family. The seven genes and pseudogenes mapped within 230 kb in the order HSD3Bpsi5-psi4-psi3-HSD3B1-psi1-psi2 -HSD3B2. HSD3B1 and 2 are in direct repeat, 100 kb apart. Six HSD3B2 mutations involve substitutions that are present in several of the pseudogenes. In four cases, mutations arose in CpG sites that are conserved within the gene cluster. The tendency for CpG sites to mutate by transition provides an adequate explanation for these HSD3B2 mutations, which are unlikely to be due to recombination or conversion within the gene family.

Genetics of vitamin D 1alpha-hydroxylase deficiency in 17 families.

Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."

New members of the 3 beta-hydroxysteroid dehydrogenase gene family.

Several bands of hydridization are detected when southern blots of human genomic DNA are proved with cDNA of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type I. Two experimental approaches were adopted to estimate the size of the 3 beta-HSD gene family. Firstly, primer designed to amplify 3 beta-HSD type I and II genes were found on occasion to amplify DNA products of appropriate length but which were resolved as distinct sequences by denaturing gradient gel electrophoresis (DGGE). Five of these novel bands were cloned and their sequences were found to be closely related to 3 beta-HSD types I and II. Secondly, 57 genomic clones were selected from two lambda genomic libraries by hybridization with exonic probes of 3 beta -HSD type I. These were screened for novel members of the gene family by pcr amplification using various combinations of PCR primers to the type I and II genes, particularly those primers that previously amplified novel PCR products from genomic DNA. Amplification products from (lambda) clones were screened for novel sequences by DGGE. As a result of these approaches, at least five new members of the 3 beta-HSD gene family were found, one of which locates to the 3 beta -HSD type I and II gene cluster on 1p13. The existence of additional closely related but distinct members of the gene family should be recognized as a potential complication when screening PCR fragments for mutations in the type I and II genes. DGGE was found to be an exceedingly rapid means of screening amplification products from (lambda) clones to search for novel members of the gene family.

Rapid eradication of Helicobacter pylori infection.

BACKGROUND/AIMS: Current Helicobacter pylori eradication therapy for peptic ulcer disease usually involves a 2-week course of either a bismuth preparation or omeprazole in combination with antibiotics. We have studied a shorter, 7-day course of treatment to assess efficacy and tolerability. METHODS: Four hundred and thirty-six patients, in three non-randomized groups, received omeprazole (40 mg mane), amoxycillin (500 mg t.d.s.) and metronidazole (400 mg t.d.s.): 308 patients received the triple combination for 14 days; 80 patients were treated for 7 days; and 48 patients received omeprazole and amoxycillin for 7 days but metronidazole for only 5 days. RESULTS: Helicobacter pylori was eradicated in 89.5%, 91.1% and 87.5%, respectively (98.3%, 92.9% and 100% of metronidazole-sensitive isolates and 75.6% and 88.2% of metronidazole-resistant isolates in the first two groups). Side effects were significantly more frequent in patients who received 14 days (49%) compared with 7 days of treatment (33%); only 8/308 and 1/128 patients, respectively, failed to complete the course. CONCLUSIONS: On the basis of efficacy, tolerability and cost, we conclude that a 7-day course of the omeprazole (40 mg mane), amoxycillin (500 mg t.d.s.) plus metronidazole (400 mg t.d.s.) combination is effective therapy for the eradication of H. pylori.

Variation in the expression of human 3 beta-hydroxysteroid dehydrogenase.

Polymorphic genetic variation shows that the genes for human 3 beta-hydroxysteroid dehydrogenases (3 beta-HSD) types I and II are closely linked. The type II mutations A82T, S100N and L173R are associated with male pseudohermaphroditism and A82T is associated, with variable penetrance, with female premature puberty. When expressed in vitro A82T showed less than 5% of normal activity and L173R showed a 30-50% reduction in activity. PCR experiments and direct genomic cloning show that there is a larger family of 3 beta-HSD sequences which require to be tested for expression. The phenomenon of epitopic heterogeneity of 3 beta-HSD is discussed and is now shown to apply to testicular Leydig cells as well as extrauterine trophoblast. RT-PCR analyses indicate that the phenomenon is most likely to be due to post-translational modification affecting the carboxytermini 3 beta-HSD types I and II. This phenomenon may reflect a further level at which enzyme activity is regulated.

Helicobacter pylori eradication in patients with peptic ulcer disease: clinical consequences and financial implications.

We assessed clinical consequences and financial implications of Helicobacter pylori eradication in 175 patients with peptic ulceration, of whom 106 had been free from H. pylori infection for a mean of 3.2 years, while 69 remained infected. We used quarterly questionnaires to examine consumption of ulcer-healing medication and antacids. In the 106 successfully treated patients, gastrointestinal haemorrhage as a complication of peptic ulcer complications during the 344 patient years after eradication (0.003 per patient year) was 18-fold lower than during the 912 patient years before eradication (0.056 per patient year). Of the H. pylori-negative patients, 12-18% used ulcer-healing medication during any one of the three-month periods of the survey, compared with 34-51% of the patients with residual H. pylori infection. The average cost of the ulcer-healing drugs consumed by the H. pylori-negative patients was 30.59 pounds during the 12 months of the survey, compared with 99.05 pounds for H. pylori-positive patients. Consumption of antacids was also lower in the H. pylori-negative group. Successful eradication of H. pylori significantly reduced the annual cost of ulcer-healing drugs consumed by the patients with ulcer disease. Maintenance of ulcer remission following successful eradication of H. pylori also significantly reduced ulcer complications.

Helicobacter pylori eradication: efficacy and side effect profile of a combination of omeprazole, amoxycillin and metronidazole compared with four alternative regimens.

We evaluated eradication of Helicobacter pylori infection in 263 patients by a new 14-day regimen of omeprazole 40 mg mane (a gastric secretory inhibitor) plus two antibiotics: amoxycillin 500 mg three-times daily (tds) plus metronidazole 400 mg tds. The comparative groups included updated results of our previous work with a 14-day course of either standard triple therapy (STT, colloidal bismuth subcitrate 120 mg four times daily (qds) plus tetracycline 500 mg qds and metronidazole 400 mg tds), omeprazole 40 mg once daily plus amoxycillin 500 mg tds (OA), or two modified triple therapy: either Borody's (BTT) of all three components (colloidal bismuth subcitrate 120 mg, tetracycline 500 mg, metronidazole 200 mg) qds instead of tds, or Logan's (LTT) seven-day therapeutic regimen of colloidal bismuth subcitrate 120 mg qds, amoxycillin 500 mg qds and, for the last three days, metronidazole 400 mg five times daily. Omeprazole/amoxycillin/metronidazole (OAM) therapy was better tolerated than STT (course completion 98.1% vs. 81.4%, p < 0.001). H. pylori was eradicated by OAM therapy in 53/55 (96.4%) patients with metronidazole-sensitive organisms and in 54/72 (75.0%) with metronidazole-resistant isolates (p < 0.01). The respective corresponding rates for STT and OA therapy were 20/22 (90.9%) and 14/29 (48.3%), (metronidazole-sensitive organisms) and 7/21 (33.3%) and 15/31 (48.4%) (infections resistant to metronidazole). BTT and LTT were also better tolerated than STT. The eradication rate for BTT was 23/26 (88.5%) but that for LTT, the best tolerated of the five treatment regimens, was only 19/28 (67.9%) when pretreatment isolates were metronidazole-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Audit of patients' experiences after endoscopy of the upper alimentary tract.

This study comprises an audit of the experiences of upper alimentary endoscopy by 400 patients examined in 15 hospitals. The patients were asked to complete a 19-section questionnaire regarding their experience of the endoscopy before, during and after the procedure. The audit was carried out in order to evaluate the patients' understanding of the investigation and to determine the impact of the findings on the patients' level of anxiety. Of the 400 patients, 304 (76.0%) returned the questionnaire; overall satisfaction was revealed with the decision to proceed with endoscopy and with the procedure itself, as well as the reassurance provided by the investigation. The study thus supports the role of open-access endoscopy.

Reinfection or recrudescence after apparently successful eradication of Helicobacter pylori infection: implications for treatment of patients with duodenal ulcer disease.

Helicobacter pyloris is considered to be aetiologically implicated in gastritis and peptic ulceration, since if H. pyloris infection can be eradicated the risk of subsequent ulcer relapse is markedly reduced. The rate of 'reinfection' following treatment ranges from 0% to 45%, but its origin remains controversial (reappearance of uneradicated original infection or a fresh infection). To distinguish temporary suppression of H. pylori from fresh infection we conducted a retrospective analysis of the criteria used to establish eradication of the original infection in 304 patients. We used the [14C]urea breath test, in which an integrated area under the curve (AUC) value of < 40 in 2 h is considered to indicate eradication of H. pylori in patients tested 1 month after treatment. The results suggest that relapsed infection with H. pylori usually represents recrudescence of the original infection rather than a fresh infection; there was a higher relapse rate in patients with a breath test AUC > 20 < 40, compared with those with an AUC < 20. All 'reinfections' occurred within 24 months of the original treatment. 'Reinfection' was uncommon in patients receiving powerful therapeutic regimens (e.g. triple therapy) compared with those receiving monotherapy or relatively ineffective dual therapy combinations. In patients whose urea breath test remains negative 12 months after treatment the subsequent reinfection rate is only 0.44%/year. This supports the strategy of eradicating H. pylori infection from suitable peptic ulcer patients.

Short report: omeprazole plus antibiotic combinations for the eradication of metronidazole-resistant Helicobacter pylori.

Twenty-eight Helicobacter pylori-positive patients with metronidazole-resistant isolates and 25 with metronidazole-sensitive isolates were treated for 14 days with 40 mg omeprazole nocte plus 500 mg amoxycillin t.d.s. Eradication of H. pylori, defined as absence of the organism one month after cessation of treatment, was assessed using the [14C]urea breath test. The eradication rate in patients with metronidazole-resistant isolates was 14/28 (50%) while that in patients was metronidazole-sensitive isolates was 12/25 (48%). In contrast to these encouraging eradication rates, very poor results were obtained with a 7-day course of omeprazole (40 mg nocte) in combination with erythromycin ethylsuccinate (500 mg q.d.s.) and tripotassium dicitrato bismuthate tablets (120 mg q.d.s.). The latter eradication rates were 3/20 (15%) in patients taking erythromycin tablets and 3/19 (16%) in those taking a liquid formulation of erythromycin. All treatment regimens were well tolerated and all patients completed the prescribed course of therapy.

Experience with 'triple' anti-Helicobacter pylori eradication therapy: side effects and the importance of testing the pre-treatment bacterial isolate for metronidazole resistance.

At the 1990 World Congresses of Gastroenterology, the Working Party on Helicobacter pylori (H. pylori) recommended that, in suitable patients, the bacterium should be eradicated using a therapeutic regimen comprising a bismuth salt, tetracycline and metronidazole for two weeks. We have treated 40 patients infected with H. pylori with 'triple' therapy consisting of 120 mg tripotassium dicitrato bismuthate q.d.s., 500 mg tetracycline q.d.s. and 400 mg metronidazole t.d.s. for two weeks. The success rate, in terms of bacterial eradication, was 19/21 (90.5%) in patients with metronidazole-sensitive organisms, compared with only 6/19 (31.6%) in patients whose H. pylori were resistant to metronidazole (P less than 0.01). Side effects, particularly diarrhoea and vomiting/nausea, were common: 23/40 patients reported such symptoms during the 14-day course of therapy. Fifteen of these 23 patients completed the entire 14-day course, although suffering from significant side effects, while the remaining eight patients had to discontinue the treatment because side effects became intolerable. If a form of triple therapy is going to be widely used to eradicate H. pylori infection, the regimen will have to be simpler, shorter, produce fewer side effects and be more effective in patients with metronidazole-resistant bacteria.

Does a previous course of tripotassium dicitrato bismuthate affect the subsequent chances of successful Helicobacter pylori eradication?

We have performed a retrospective study of 103 patients with either peptic ulcer or non-ulcer dyspepsia, infected with metronidazole-sensitive strains of Helicobacter pylori (H. pylori), who were treated with a combination of tripotassium dicitrato bismuthate and metronidazole for a period of at least two weeks. Dual therapy with tripotassium dicitrato bismuthate plus metronidazole showed similarly high eradication rates (greater than or equal to 80%) of H. pylori from patients irrespective of age, gender or clinical diagnosis. Most importantly, dual therapy achieved a similar eradication rate of H. pylori infection in 41 patients who had previously been treated with tripotassium dicitrato bismuthate alone or in combination with an antibiotic other than metronidazole. It therefore appears that H. pylori does not become resistant to treatment with tripotassium dicitrato bismuthate.

Upper gastrointestinal endoscopy: a prospective randomized study comparing continuous supplemental oxygen via the nasal or oral route.

We have examined the efficacy of supplemental oxygen in preventing episodes of significant arterial desaturation (SpO2 less than 90%) during upper gastrointestinal endoscopy. We have compared the effects of 2 liters.min-1 of oxygen given orally via the bite-guard with the same flow rate via nasal cannulas and have also examined the effects of pre-oxygenation. Results of this study at a flow rate of 2 liters.min-1 have been compared with previously published results at a flow rate of 3 liters.min-1. Although in this study fewer episodes of desaturation were seen in the orally supplemented group compared with the nasal group, the difference observed was not statistically significant. Pre-oxygenation significantly reduced episodes of desaturation (SpO2 less than 90%, p less than 0.01) and prevented SpO2 falls below 85% in all patients studied. Supplemental oxygen given at a rate of 2 liters.min-1 was as effective as that given at a rate of 3 liters.min-1 in preventing significant desaturation, as previously defined, during the procedure. We therefore recommend the use of supplemental oxygen at a flow rate of 2 liters.min-1 in all high risk patients and conclude that the oral route has practical advantages and is at least as effective as nasal cannulas.

Ammonia production by intestinal bacteria: the effects of lactose, lactulose and glucose.

Ammonia production by eight groups of intestinal bacteria was measured, and the effect on ammonia production of lowered pH and ambient ammonia concentration was determined. Endogenous ammonia production from bacterial protoplasm was also examined. To examine the mechanisms by which fermentable substrates reduce ammonia formation in a faecal incubation system, the effect of lactose, lactulose or glucose on ammonia release by pure cultures of intestinal bacteria was studied. The largest amounts of ammonia were generated by gram-negative anaerobes, clostridia, enterobacteria, and Bacillus spp. Gram-positive non-sporing anaerobes, streptococci and micrococci formed modest amounts, and lactobacilli and yeasts formed very little ammonia. All groups of bacteria formed less ammonia at pH 5.0 than at pH 7.0 and production of ammonia was not inhibited when 30 mmol ammonia/litre was included in the medium. Small amounts of ammonia were formed due to endogenous metabolism of bacterial cells. Washed cell suspensions of four isolates of Bacteroides, one clostridial isolate and two streptococcal isolates formed less ammonia from alanine, methionine or histidine after growth in the presence of either lactose or lactulose. In contrast, the Bacteroides isolates formed more ammonia from aspartate than from either lactose or lactulose. Also, cultures of gram-negative anaerobes and enterobacteria, and to a lesser extent clostridia and streptococci, formed significantly less ammonia in nutrient broth when lactose, lactulose or glucose was included in the medium. This decrease in ammonia formation was not due to a fall in pH of the medium. Ammonia production by gram-positive non-sporing anaerobes was not affected by carbohydrate fermentation. These results suggest that gram-negative anaerobic bacteria make a major contribution to ammonia generated from peptides and amino acids in vivo, and that ammonia may be formed from bacterial cells in the colon. Fermentation of lactose and lactulose may repress the formation and inhibit the activity of enzymes responsible for ammonia release. In the human colon these substrate effects may decrease the amount of ammonia available to exert a toxic effect on the host, and thus contribute to the beneficial effects of lactulose when it is used in the treatment of portosystemic encephalopathy.