Characterizing osmolyte chemical class hierarchies and functional group requirements for thermal stabilization of proteins.

Osmolytes are naturally occurring organic compounds that protect cellular proteins and other macromolecules against various forms of stress including temperature extremes. While biological studies have correlated the accumulation of certain classes of osmolytes with specific forms of stress, including thermal stress, it remains unclear whether or not these observations reflect an intrinsic chemical class hierarchy amongst the osmolytes with respect to effects on protein stability. In addition, very little is known in regards to the molecular elements of the osmolytes themselves that are essential for their functions. In this study, we use differential scanning fluorimetry to quantify the thermal stabilizing effects of members from each of the three main classes of protecting osmolytes on two model protein systems, C-reactive protein and tumor necrosis factor alpha. Our data reveals the absence of a strict chemical class hierarchy amongst the osmolytes with respect to protein thermal stabilization, and indicates differential responses of these proteins to certain osmolytes. In the second part of this investigation we dissected the molecular elements of amino acid osmolytes required for thermal stabilization of myoglobin and C-reactive protein. We show that the complete amino acid zwitterion is required for thermal stabilization of myoglobin, whereas removal of the osmolyte amino group does not diminish stabilizing effects on C-reactive protein. These disparate responses of proteins to osmolytes and other small molecules are consistent with previous observations that osmolyte effects on protein stability are protein-specific. Moreover, the data reported in this study support the view that osmolyte effects cannot be fully explained by considering only the solvent accessibility of the polypeptide backbone in the native and denatured states, and corroborate the need for more complex models that take into account the entire protein fabric.

Clinical trial: oral prednisolone metasulfobenzoate (Predocol) vs. oral prednisolone for active ulcerative colitis.

BACKGROUND: Systemic corticosteroids are effective in ulcerative colitis but commonly cause side effects. AIM: To compare the safety and efficacy of a sparingly absorbed formulation of prednisolone metasulfobenzoate (Predocol) with a conventional tapering course of oral prednisolone. METHOD: In a double-blind randomized study, 59 active ulcerative colitis patients received Predocol 40 mg/day for 6 months, 61 received Predocol 60 mg/day for 6 months and 61 received prednisolone 40 mg/day for 2 weeks, tapered to week 8, followed by placebo until 6 months. RESULTS: Steroid-related side effects assessed using a 10-cm visual analogue scale were fewer at 2 months with Predocol 40 mg [VAS 8.1 cm (2.6), mean (s.d.)], or 60 mg [8.1 (2.1)] compared with prednisolone [6.7 (2.7); P = 0.01]. Mood changes affected 43% receiving prednisolone at 4 weeks vs. 8% for Predocol 40 mg (P = 0.001). Remission rates (Powell-Tuck < or =2) at 2 months were Predocol 40 mg 46%, Predocol 60 mg 28% and tapering prednisolone 41% (P = 0.13). Visual analogue scale for efficacy also showed non-inferiority for Predocol 40 mg/day. Remission rates at 6 months were Predocol 40 mg 51%, Predocol 60 mg 38% and tapering prednisolone 32% (P = 0.08). CONCLUSION: Predocol 40 mg/day has similar efficacy but markedly fewer side effects than a conventional tapering prednisolone regimen (ISRCTN14133410).

Regression of columnar-lined (Barrett's) oesophagus with omeprazole 40 mg daily: results of 5 years of continuous therapy.

BACKGROUND: We have previously reported the effect of 2 years of omeprazole 40 mg daily on columnar-lined (Barrett's) oesophagus (CLO). AIMS: In the present study, follow-up has been extended to 5 years to assess the macroscopic and microscopic effects of continuing therapy. PATIENTS AND METHODS: The 23 patients have been followed for up to a further 3 years. Endoscopy with multiple biopsies was performed at the end of years 3, 4 and 5. RESULTS: Although there had been a statistically significant regression in the length of CLO after 2 years, there was no overall further measurable change after 5 years. However, one patient showed complete macroscopic and microscopic regression. The number and size of macroscopic squamous islands within the CLO continued to increase, and there was a further increase in microscopic squamous re-epithelialization of surface mucosa, gland ducts and Barrett's gland tissue. Low-grade dysplasia was found consistently in one patient in biopsies taken up to the end of year 3 but it could not be detected thereafter. CONCLUSIONS: Omeprazole 40 mg daily appears to have beneficial effects on CLO, although it rarely induces a complete regression. Whether the benefits will reduce the risk of malignant transformation is unknown.

Treatment options for cirrhotic ascites.

Ascites due to cirrhosis can usually be managed successfully by dietary sodium restriction and carefully monitored diuretic therapy. However, paracentesis with an albumin infusion is a relatively safe alternative that has gained widespread acceptance. Other plasma expanders offer a cheaper alternative to albumin. Other recently developed techniques include peritoneovenous shunt and transjugular intrahepatic portosystemic stent shunt. Each of these is associated with an operative mortality and substantial complications, but for diuretic-resistant ascites a peritoneovenous shunt has comparable results to paracentesis/albumin. For patients with spontaneously occurring renal failure the prognosis is poor. None of the above treatments improves renal function. Management should therefore be symptomatic with paracentesis as necessary.

The histopathology of treated Barrett's esophagus: squamous reepithelialization after acid suppression and laser and photodynamic therapy.

Columnar metaplasia of the lower esophageal epithelium (Barrett's esophagus) occurs in response to acid reflux, and its most important long-term complication is malignancy. In view of this, techniques are being explored for the eradication of Barrett's esophagus, and histopathologists will increasingly be required to assess response to these therapies in esophageal biopsy samples. The histopathologic features before and after treatment were studied in biopsy samples from 16 patients receiving omeprazole only, 10 treated by KTP laser photoablation, and five who underwent photodynamic therapy. All the treatment modalities resulted in histologic changes with at least partial squamous reepithelialization of the metaplastic columnar epithelium. The histologic findings suggest three main mechanisms for this: encroachment of adjacent squamous epithelium at the squamocolumnar junction, extension of epithelium from the submucosal gland duct to form squamous islands, and squamous metaplasia within the Barrett's columnar mucosa itself. The latter mechanism implies the existence of pluripotential stem cells within Barrett's mucosa. A relatively common finding was residual glandular mucosa, nonneoplastic and dysplastic, beneath squamous epithelium indicating the requirement for histologic confirmation of endoscopically suspected complete squamous reepithelialization with sufficiently deep biopsies.

Histological assessment of the Sydney classification of endoscopic gastritis.

To determine the significance of the endoscopic classification of gastritis proposed by a working party at the World Congress of Gastroenterology in Sydney 1990, 167 patients undergoing upper alimentary endoscopy were prospectively assessed by comprehensive endoscopic and histological methods. Ninety eight patients had endoscopic mucosal changes of gastritis according to the Sydney classification. Twenty six (27%) of these had histologically normal biopsy specimens. This was not statistically significantly different to the 26 (38%) of 69 with normal endoscopies whose biopsy specimens were histologically normal (chi 2 = 1.857, p > 0.1). Forty three (62.5%) patients with normal endoscopies had histological gastritis. No histological counterpart was found for the macroscopic appearances of the gastric mucosa said to show inflammation proposed by the Sydney classification of gastritis. These findings confirm the inappropriateness of an endoscopic diagnosis of gastritis and it is suggested such a term should be reserved for the histological findings.

Omeprazole in the treatment of asthmatics with nocturnal symptoms and gastro-oesophageal reflux: a placebo-controlled cross-over study.

Eleven adult patients with nocturnal asthma, and gastro-oesophageal reflux documented by endoscopy or ambulatory oesophageal pH monitoring completed a double-blind cross-over study (4 week treatment, one week run-in and cross-over periods) comparing the effects of omeprazole 20 mg daily and placebo on asthma control assessed by symptoms, peak expiratory flow rate and bronchodilator usage. Omeprazole treatment did not improve asthma symptoms during the day or night, or peak expiratory flow rate readings. There was no difference in bronchodilator inhaler usage during omeprazole therapy. Treatment of gastro-oesophageal reflux with omeprazole in patients with nocturnal asthma and gastro-oesophageal reflux does not improve asthma symptoms or peak expiratory flow rate. This suggests that gastro-oesophageal reflux does not exacerbate bronchoconstriction in nocturnal asthma.

Preservation of gastric antral mucus is associated with failure of eradication of Helicobacter pylori by bismuth, metronidazole and tetracycline.

METHODS: Forty-three patients positive for Helicobacter pylori by histology and culture of antral biopsies (n = 40) or histology alone (n = 3) were investigated. They received either regimen 1--tripotassium dicitrato bismuthate 120 mg q.d.s. and tetracycline 250 mg q.d.s. for 4 weeks, with metronidazole 200 mg q.d.s. for the first 2 weeks, or regimen 2--omeprazole 20 mg b.d., amoxycillin 500 mg t.d.s., tetracycline 500 mg q.d.s. each for 3 weeks. Gastric antral biopsies were scored (0-3) histologically for mucus depletion, polymorphonuclear and mononuclear cell infiltrate. H. pylori eradication was assessed by biopsy and culture 1 month after the cessation of treatment. RESULTS: With regimen 1, pre-treatment mucus depletion was significantly higher where eradication was successful (median score 2) compared to where it was not (median score 1, P < 0.01); there were no differences in the scores for polymorphonuclear or mononuclear cell infiltrates. In patients receiving regimen 2, there were no differences in either mucus depletion or polymorphonuclear or mononuclear cell infiltrate, between those where eradication was successful and those where it was not. Metronidazole minimum inhibitory concentrations rose when eradication with regimen 1 was unsuccessful (median before 0.19 mg/L, median after treatment 16 mg/L; P = 0.04). CONCLUSION: Pre-treatment mucus depletion is identified as a factor affecting H. pylori eradication. Preservation of mucus may facilitate acquisition of metronidazole resistance.

Regression of columnar lined (Barrett's) oesophagus with continuous omeprazole therapy.

Twenty-three adult patients with a columnar lined (Barrett's) oesophagus are being treated with long-term omeprazole, 40 mg daily. Twelve had never undergone anti-reflux surgery (Group 1), the other eleven having previously had insertion of an Angelchik anti-reflux prosthesis (Group 2). Endoscopy was carried out six months before, immediately before and six months, one year and two years into treatment. Multiple and standardized biopsies were taken at each endoscopy. Results from the two groups were similar. During the 6-month run-in period there was a statistically non-significant increase in the linear extent of the columnar mucosa, but this showed a progressive, statistically significant decrease during the two years of treatment. Other evidence for regression of the Barrett's mucosa includes the emergence of large numbers of macroscopic squamous islands within the abnormal mucosa, an increase in the number of microscopic squamous islands, and microscopic squamous encroachment of the abnormal mucosa at the squamo-columnar junction. Histological assessment showed a reduction in the proportion of sulphomucin-rich intestinal metaplasia, but this only achieved statistical significance in Group 1. The results substantiate the importance of acid in the pathogenesis of Barrett's oesophagus. Omeprazole may have a therapeutic role in bringing about regression of the metaplastic epithelium.

Distribution of mucosal pathology and an assessment of colonic phenotypic change in the pelvic ileal reservoir.

The mucosa of the pelvic ileal reservoir undergoes adaptive changes--inflammatory, architectural, and metaplastic--on exposure to the faecal stream. Twenty three quadruple loop ileal pouches constructed for ulcerative colitis (20 patients) and familial adenomatous polyposis (FAP) (three patients) were studied. No patient fulfilled clinical, endoscopic, or histopathological criteria for pouchitis. Standard duplicate biopsy specimens were taken from the proximal limb, the anterior wall, the posterior wall, and the body of the reservoir. An established scoring system was used and showed a highly significant increase in inflammatory scores in posterior wall biopsy specimens compared with those from the anterior wall. These results suggest that the adaptive changes are the direct result of contact with static faecal contents. One patient only showed significant inflammation in the proximal limb. There was no evidence of mucosal prolapse in any anterior wall biopsy specimen. Patients with colitis showed substantially more inflammatory and architectural changes than those with FAP. Ninety six per cent of pouches showed some colonic phenotypic expression as defined by mucin histochemical and PR 3A5 immunohistochemical studies. Our results suggest, however, that there may not be complete colonic metaplasia and that the mucin changes and other phenotypic alterations may represent a non-specific response to pouch inflammation and not a prerequisite for the development of pouchitis. The focal nature of the inflammatory and architectural changes, which may be the result of direct contact with static faecal residue, are clearly shown. A single random biopsy specimen of pouch mucosa is of limited value in assessing pathological changes and screening for potential neoplastic change within the reservoir.

Endoscopic crescentic fold disease of the sigmoid colon: the clinical and histopathological spectrum of a distinctive endoscopic appearance.

In this paper we describe an endoscopic appearance of the sigmoid colon characterised by mucosal swelling, erythema and haemorrhage strictly localised to the crescentic mucosal folds. In a 5-year period these changes were seen in 34 (1.42%) of 2380 colonoscopies and fibreoptic sigmoidoscopies. The majority of patients were middle-aged or elderly. Diverticular disease was present in most (82%) but the abnormalities were confined to the crescentic mucosal folds with sparing of the diverticular orifices. The majority of patients presented with a history of bleeding per anum. Histologically there was a spectrum of changes varying from minor vascular congestion to florid active inflammatory disease with crypt architectural abnormalities mimicking ulcerative colitis, but rectal biopsies were invariably normal. Three patients later progressed to typical distal ulcerative colitis and two other patients presenting to us with endoscopic crescentic fold disease had a previous histologically documented history of distal ulcerative colitis. In three patients the histological features were of mucosal prolapse. About half the patients required some form of therapy to control their symptoms. Steroids and/or sulphasalazine were of value although two patients subsequently underwent sigmoid resection, one to control bleeding and the second for a diverticulosis-associated stricture. Whilst endoscopic crescentic fold disease represents a specific endoscopic appearance the clinical and histological features indicate a wide spectrum of disease.

Use of the SeHCAT test in the investigation of diarrhoea.

The SeHCAT test was used to investigate possible bile acid malabsorption in 166 patients presenting to a district general hospital with chronic diarrhoea of uncertain cause. Eighty-four (51%) patients had impaired SeHCAT retention. These included 23 of 28 patients with a possible type I abnormality (terminal ileal resection or disease, previous pelvic radiotherapy), 20 of 74 with a possible type II abnormality (idiopathic diarrhoea), 32 of 45 with a possible type III abnormality (post-cholecystectomy, post-vagotomy), and 9 of 19 with diarrhoea associated with diabetes. Patients with severe bile acid malabsorption demonstrated a good response to cholestyramine whereas the response in patients with a mildly abnormal SeHCAT retention was variable. Bile acid malabsorption is an important cause of diarrhoea in patients presenting with unexplained chronic diarrhoea.

An Eudragit-coated prednisolone preparation for ulcerative colitis: pharmacokinetics and preliminary therapeutic use.

Prednisolone metasulphabenzoate, a steroid with poor colonic absorption, was coated with the pH-dependent acrylic resin Eudragit S, as a means of delivering an orally administered preparation to the proximal colon. The therapeutic potential of delivering this steroid with potentially less systemic side-effects to the proximal colon was assessed in extensive ulcerative colitis. Plasma and urine prednisolone profiles in 6 healthy volunteers confirmed minimal absorption from Eudragit S-coated prednisolone metasulphabenzoate compared to prednisolone acetate: peak plasma prednisolone concentrations 29 +/- 21 ng/ml vs. 570 +/- 185 ng/ml (P less than 0.01), area under curve measurements 204 +/- 214 vs. 2724 +/- 1236 ng.h/ml (P less than 0.01). Prednisolone metasulphabenzoate coated with Eudragit S (30-60 mg daily) was then administered for 12 weeks to 12 patients with colonoscopically proven extensive ulcerative colitis in relapse. Symptoms, sigmoidoscopic appearances and rectal histological abnormalities all improved during therapy. Complete clinical remission occurred in 7 patients, a partial response in 2 patients and no response in 3 patients. Cortisol responses to tetracosactrin demonstrated no significant adrenal suppression following treatment. Eudragit S-coated prednisolone metasulphabenzoate may be a useful treatment for extensive ulcerative colitis, without risk of systemic steroid side-effects.

Long-term follow up of patients with gastritis associated with Helicobacter pylori infection.

The aim of this study was to evaluate the long-term prognosis for patients suffering from gastritis associated with Helicobacter pylori infection, and in particular the proportion of cases progressing to peptic ulcer. The study was carried out in one urban general practice. One hundred and three patients who had presented with dyspepsia over the 1973-80 period and who were found to have a macroscopically normal endoscopy were reassessed between seven and 14 years later. Gastric antral biopsies had been taken routinely at endoscopy and were subsequently re-examined for the presence of H pylori. The patients' medical records were examined to establish their consulting rates over the follow-up period and whether they suffered from any other medical conditions. Patients were interviewed to assess the course of their dyspeptic symptoms. Comparison of patients who were unequivocally H pylori positive with those who were negative revealed no significant differences in the consultation rate for gastroenterological symptoms, in the proportion of patients referred to a hospital consultant or for further gastroenterological investigations or in the proportion reporting that their symptoms had improved. However, a statistically highly significant relationship was found between H pylori infection and hypertension. The results of this study have shown that there is a good prognosis for non-ulcer dyspepsia whether or not H pylori infection is present. The association between H pylori gastritis and hypertension clearly merits further investigation.

Urinary steroid profiles and alcohol-related blood pressure elevation.

1. From an earlier cross-sectional survey of 343 public servants, 15 pairs of non-smoking teetotallers and heavy drinkers (alcohol intake more than 350 mL/week) were matched for age and adiposity and utilized for a case-control study of the effects of alcohol on 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) activity and blood pressure. 2. Two successive 24 h urine collections were analysed by radio-immunoassay (RIA) for cortisol excretion, and for the cortisol and cortisone metabolites, tetrahydrocortisol (THC), allo-tetrahydrocortisol (allo-THC) and tetrahydrocortisone (THE), by capillary column gas chromatography. 3. Heavy drinkers had higher systolic and diastolic blood pressure (BP) than teetotallers (132.6 +/- 2.5 vs 123.2 +/- 1.3 and 78.7 +/- 1.6 vs 71.7 +/- 1.4, respectively; unpaired t-test, P less than 0.01). Twenty-four-hour urinary sodium and cortisol excretion were similar in the two groups. 4. The THC plus allo-THC:THE ratio was similar in drinkers and teetotallers (1.81 +/- 0.20 vs 2.03 +/- 0.20), consistent with no effect of alcohol on 11 beta-OHSD activity. The ratio of THC to allo-THC was increased in drinkers compared with teetotallers (1.49 +/- 0.18 vs 1.05 +/- 0.13; unpaired t-test, P less than 0.05), consistent with either a decrease in 5 alpha-reductase activity or an increase in 5 beta-reductase activity. 5. This study provides no evidence for alcohol-related inhibition of 11 beta-OHSD, despite substantially higher blood pressures in heavy drinkers compared to teetotallers. Such an effect is, therefore, unlikely to contribute significantly to the mechanism of alcohol-related hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of omeprazole and cimetidine in reflux oesophagitis: symptomatic, endoscopic, and histological evaluations.

Symptomatic patients with endoscopically verified reflux oesophagitis were randomised to a double blind trial in which they received either omeprazole (20 mg once daily) or cimetidine (400 mg four times daily) for four, and if necessary, eight weeks. In an 'intention to treat' analysis, oesophagitis was found to have healed after four weeks in 77 of 137 (56%) in the omeprazole group and in 34 of 133 (26%) in the cimetidine group (p less than 0.001). By eight weeks these values were 71% and 35% respectively; p less than 0.001. Histological assessments were available for 73% of the patients. At entry, 63% (66 of 104) in the omeprazole group and 60% (56 of 94) in the cimetidine group (ns) had abnormal histology. After the study, the proportions of patients who initially had had abnormal histology but who then progressed to normal were 67% (44 of 66: omeprazole) and 48% (27 of 56: cimetidine) respectively (p less than 0.001). All patients had reflux symptoms at entry. After four weeks, 46% in the omeprazole group and 22% (p less than 0.001) in the cimetidine group were asymptomatic. Diary cards completed for the first two weeks showed that patients treated with omeprazole experienced fewer reflux symptoms by day and night and used fewer antacids. Omeprazole, 20 mg once a day for four to eight weeks, healed a greater proportion of patients with reflux oesophagitis than cimetidine, 1.6 g per day, assessed endoscopically and histologically, and relieved more patients' symptoms.