No polymorphism in the tissue transglutaminase gene detected in coeliac disease patients.

BACKGROUND: The autoantigen for the anti-endomysial antibody (AEA) found in coeliac disease has recently been reported to be the enzyme tissue transglutaminase (tTG). Polymorphisms in the gene for tTG would result in different enzymic isoforms being expressed. Certain isoforms may interact with gliadin to create antigenic neoepitopes, which could then generate an immune response in genetically predisposed individuals possessing major histocompatibility complex (MHC) class II DQ2. METHODS: We have sequenced the coding region of tTG in coeliac patients and normal controls. Total RNA was extracted from mucosal biopsies from eight AEA-positive histologically proven coeliac disease patients and four control patients with a histologically normal duodenum and a negative AEA. The 2-kb coding region of tTG was amplified in overlapping fragments by reverse transcription polymerase chain reaction (PCR), using five sets of PCR primers. Each overlapping PCR fragment was sequenced using the fmol DNA sequencing system. RESULTS: tTG transcripts were detected in all samples. There was no difference in the coding sequence of tTG between the four control and eight coeliac patients, even though we observed differences in sequence between our study and the original published sequence. These differences have also been reported in sequences published subsequent to the original description. CONCLUSIONS: Polymorphisms in the tTG gene have not been observed in coeliac disease patients and therefore cannot explain the creation of neoepitopes.

Intestinal permeability and liver disease.

Increased intestinal permeability has for many years been implicated as a possible contributory factor in the development of encephalopathy and spontaneous bacterial peritonitis (SBP) seen in patients with cirrhosis. The majority of studies indicate that there is an increase in small intestinal permeability in cirrhotic patients and there is also evidence of an increase in patients with acute liver failure. The cause of these changes is unknown and whether they are related to the development of SBP and encephalopathy is unclear.

Rapid determination of the complexity of cDNA bands extracted from DDRT-PCR polyacrylamide gels.

A band extracted from a differential display polyacrylamide gel often represents a composite of heterogeneous products. We have developed a non- radioactive method to simply and rapidly analyse its complexity. A fluorescent restriction enzyme fingerprint of the composite mixture is generated. The number of individual bands observed in this fingerprint indicates the complexity of the re-amplified cDNA mixture. Restriction fingerprints of the inserts of cDNA subclones derived from the re-amplified cDNA mixture are compared to the composite fingerprint to select those representing the most intense bands in the composite. This dramatically reduces the number of clones required for further characterisation.

Automated differential display using a fluorescently labeled universal primer.

We have modified the automated differential display reverse transcription polymerase chain reaction technique (DDRT-PCR) such that a single fluorescently labeled universal primer (d(F)CTCACG-GATCCGTCGATTTT) is used in all PCRs together with a selection of arbitrary primers. We term this fluorescent detection procedure FDDRT-PCR. Anchoring primers of general structure dTGGTCTCACGGATCCTCGA-(T)12 VN (where N can be any deoxynucleoside and V can be any deoxynucleoside other than thymidine) are used for the RT step, and the universal primer together with selected arbitrary primers are then used for the PCR amplification. Advantages of this approach are: (i) the fluorescently labeled universal primer is a constant feature in every PCR, so that changes in banding profile are highly likely to reflect the incorporation of different arbitrary 10-mer primers; (ii) artifacts that result from arbitrary 10-mer to arbitrary 10-mer primer amplifications are not observed by fluoresence detection on an automated gene scanner because such products are not fluorescently labeled; (iii) sample throughput and ease of data handling are increased when compared with the conventional radioactive/manual approach and (iv) using a single fluorescently labeled primer in all PCRs is highly cost-effective.

Hepatic disorders. Features and appropriate management.

The spectrum of liver disease is extremely wide, with many of the underlying disorders having acute and chronic presentations. Most of the underlying pathogenetic mechanisms are accounted for by autoimmune disease, viral infection and toxic insult. The management strategy of any liver disease is a combination of treating the symptoms and complications that arise, as well as drug therapies relevant to the specific underlying diagnosis. Encephalopathy, ascites, spontaneous bacterial peritonitis, variceal bleeding and pruritus are the main complications at which drug therapy is directed, although in some cases it represents only 1 aspect of the overall management. Drug therapy per se is largely ineffective in acute liver failure with the possible exception of acetylcysteine, but many drugs are used in the management of the constituent components of this complex medical emergency. Treatments for specific liver conditions are expanding, especially in the areas of autoimmune and viral disease. The increasing availability and success of liver transplantation has tended to change the emphasis of management, and it is often not appropriate to exhaust the treatment options before referring the patient for transplantation. A comprehensive review of all liver disease is beyond the scope of this article, but hopefully the important principles of management and commonly occurring clinical decisions are discussed.

Patterns of acid reflux in complicated oesophagitis.

Oesophageal manometry and 24 hour ambulatory pH recordings from the distal oesophagus were carried out in 25 patients with complications of oesophagitis (stricture, Barrett's oesophagus or oesophageal ulcer) and compared with 25 patients with uncomplicated oesophagitis. Acid reflux was more severe in the complicated group with 26.2% of time below pH 4 compared with 11.3% in uncomplicated patients (p less than 0.01). This difference was most marked at night, when complicated patients had long periods of acid reflux with 35.6% time less than pH 4 compared with 5.2% uncomplicated (p less than 0.001). The mean duration of nocturnal acid reflux was 15.4 minutes (2.1 minutes uncomplicated, p less than 0.001). Oesophageal motility was markedly abnormal in all groups, but with no demonstrable differences in lower oesophageal sphincter pressure or peristalsis between the groups. Patients with complications of oesophagitis have different patterns of acid reflux from uncomplicated patients, with prolonged nocturnal bathing of the oesophageal mucosa, which may be the cause of stricture formation, metaplasia, or ulceration.

H2 antagonists in the treatment of reflux oesophagitis: can physiological studies predict the response?

Ambulatory oesophageal pH, oesophageal manometry and fasting serum gastrin concentrations were carried out on 28 patients with reflux oesophagitis, before and during treatment with ranitidine 300 mg bd. Fourteen patients healed endoscopically at six weeks (group A) and 14 had residual oesophagitis (group B). Group A were characterised by a lower serum gastrin concentration before treatment (4.52 pmol/l; 2.4-10: mean and range) than group B (11.1 pmol/l; 3.5-21: p less than 0.05) and showed a marked reduction in acid reflux on treatment to near normal values. Mean per cent time below pH4 fell from 14.9 to 4.2 in group A (p less than 0.05) but was not affected in group B (14.2-15.6, not significant). Abnormal oesophageal motility was found in 13 patients from each group. This did not inhibit the response to ranitidine, and was not improved by healing of oesophagitis.