Small bowel adenocarcinoma copy number profiles are more closely related to colorectal than to gastric cancers.

BACKGROUND: Small bowel adenocarcinoma (SBA) is a rare cancer and consequently, the options for clinical trials are limited. As they are treated according to either a colorectal or a gastric cancer regimen and the molecular biology of a tumor is a pivotal determinant for therapy response, chromosomal copy number aberrations were compared with the colorectal and gastric adenocarcinomas. MATERIALS AND METHODS: A total of 85 microsatellite stable (MSS) adenocarcinomas from the stomach, colorectum and small bowel were selected from existing array comparative genomic hybridization (aCGH) datasets. We compared the aCGH profiles of the three tumor sites by supervised analysis and hierarchical clustering. RESULTS: Hierarchical clustering revealed substantial overlap of 27 SBA copy number profiles with matched colorectal adenocarcinomas but less overlap with profiles of gastric adenocarcinomas. DNA copy number aberrations located at chromosomes 1p36.3-p34.3, 4p15.3-q35.2, 9p24.3-p11.1, 13q13.2-q31.3 and 17p13.3-p13.2 were the strongest features discriminating SBAs and colorectal adenocarcinomas from gastric adenocarcinomas. CONCLUSIONS: We show that MSS SBAs are more similar to colorectal than to gastric cancer, based on the 27 genome-wide DNA copy number profiles that are currently available. These molecular similarities provide added support for treatment of MSS small bowel cancers according to colorectal cancer regimens.

Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-kappaB signalling.

OBJECTIVE: Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts. DESIGN: 458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1 x 10(-04) and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls). RESULTS: We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3 x 10(-08), and rs842647 p = 5.2 x 10(-07)). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression. CONCLUSIONS: Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-kappaB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain approximately 40% of the heritability of coeliac disease.

Regulation of nitric oxide synthase activity in cultured human endothelial cells: effect of antioxidants.

Nitric oxide release is induced in many cells, including vascular endothelium, as part of the host response to inflammation. Nitric oxide synthase activity is increased in patients with sepsis, associated with increased oxidant demands and decreased antioxidant protection. We used a human vascular endothelial cell line to investigate the influence of antioxidants on nitric oxide synthase activity. Cells were cultured to confluence and incubated with interferon gamma, tumor necrosis factor, and lipopolysaccharide in the combined presence of the antioxidants ascorbic acid, Trolox, catalase, or superoxide dismutase, singly and in combination, for 48 h. Additionally, some cells were incubated with hypoxanthine-xanthine oxidase or a nitric oxide donor. Nitric oxide synthase activity was upregulated by cytokine exposure (p < .0005). Ascorbic acid and superoxide dismutase/ catalase resulted in decreased enzyme activity (p < .05). Superoxide anion release from xanthine oxidase caused increased activity (p < .05) and exogenous nitric oxide tended to suppress synthase activity. We suggest that antioxidants scavenge superoxide anion, enabling feedback inhibition of nitric oxide synthase activity by nitric oxide, and thus reducing enzyme activity. Exogenous nitric oxide also has a similar effect. Superoxide generation suppresses this feedback inhibition. This study has important implications in patients with sepsis in whom nitric oxide synthase inhibitor therapy is currently under investigation.

The effects of intravenous antioxidants in patients with septic shock.

Oxidative stress is implicated in septic shock. We investigated the effect of intravenous antioxidant therapy on antioxidant status, lipid peroxidation, hemodynamics and nitrite in patients with septic shock. Thirty patients randomly received either antioxidants (n-acetylcysteine 150 mg/kg for 30 min then 20 mg/kg/h plus bolus doses of 1 g ascorbic acid and 400 mg alpha-tocopherol) or 5% dextrose. Basal vitamin C was low and redox-reactive iron was elevated in all patients. In the 16 patients receiving antioxidants, vitamin C increased (p = .0002) but total antioxidant capacity was unaffected. Lipid peroxides were elevated in all patients but did not increase further in the patients receiving antioxidants. Plasma total nitrite also increased (p = .007) in the antioxidant group. Heart rate increased in patients receiving antioxidants at 60 min (p = .018) and 120 min (p = .004). Cardiac index also increased at 60 min (p = .007) and 120 min (p = .05). Systemic vascular resistance index decreased at 120 min in the antioxidant treated patients (p = .003). The effect of antioxidants on hemodynamic variables has not previously been reported. Antioxidant administration may be a useful adjunct to conventional approaches in the management of septic shock.

Review article: management of patients with non-responsive coeliac disease.

A substantial minority of patients with coeliac disease (estimated at anything between 7 and 30%) fail to respond to treatment with a gluten-free diet. Non-responsiveness may be primary, that is when the patient fails to respond to treatment following initial diagnosis, or secondary, when a patient who has previously had a documented response to gluten exclusion becomes non-responsive to therapy. The commonest cause of non-responsiveness is continued gluten ingestion, either voluntary or inadvertent. Other causes to be considered include intolerances to dietary constituents other than gluten (e.g. milk, soya), pancreatic insufficiency, enteropathy-associated T-cell lymphoma and ulcerative jejunitis. There is some evidence that ulcerative jejunitis is, in fact, a manifestation of lymphoma. The most important steps in the management of the non-responsive coeliac patient are (a) to determine whether the patient is indeed coeliac, (b) to exclude lymphoma and (c) to establish the cause of the non-responsiveness. In those coeliac patients with no demonstrable cause for non-responsiveness, a variety of therapeutic stratagems (mostly based on small, uncontrolled studies) have been described; these include elimination diets, dietary supplementation with zinc and copper, and pharmacological therapy in the form of steroids, azathioprine and cyclosporin. In a minority of non-responsive patients, the clinical course is characterized by a rapid decline, and total parenteral nutrition is required. None of the therapies described above has been subjected to rigorous controlled studies. The precise mechanisms of non-responsiveness in coeliac patients need to be unravelled before rational therapeutic approaches can be established.

Is a raised intraepithelial lymphocyte count with normal duodenal villous architecture clinically relevant?

BACKGROUND: A raised intraepithelial lymphocyte (IEL) count with normal villous architecture is a recognised finding in latent coeliac disease. Little information is available in cases without gluten sensitive enteropathy in adults. AIMS: To assess the frequency of such a finding in routine practice and to determine whether it is clinically relevant. METHODS: Patients with subjectively increased IELs as the only abnormality were identified prospectively from a routine duodenal biopsy series over a 12 month period. The biopsy specimens in these index cases were re-examined together with two controls with normal histology for each case, and three counts of IEL/100 epithelial cells were made in all samples. The index cases were then contacted and interviewed to obtain clinical information, approximately 12 months from the initial biopsy. Further data were obtained from their clinical records. RESULTS: Fourteen of 626 (2.2%) patients who had duodenal biopsies over the 12 month period had a subjective increase in IELs with normal villous architecture. Fifteen patients with newly diagnosed gluten sensitive enteropathy were also identified during the study period. Formal counting of the index cases and controls revealed a significant difference in IELs/100 epithelial cell counts between the two (mean, 38 (SD, 6.2) v 12.4 (4.6); p < 0.0001). Three of the 14 index cases tested had a positive coeliac antibody test compared with 12 of 15 newly diagnosed patients with coeliac disease and 10 of 93 patients with normal histology. The major clinical diagnostic categories in raised IEL cases were those with positive coeliac serology (n = 3), unexplained anaemia (n = 3), and chronic liver disease (n = 3). Six of 10 patients who were interviewed had ongoing gastrointestinal symptoms one year later. Three patients had had follow up duodenal biopsies, at the discretion of their responsible clinicians, with no change in IEL counts despite the commencement of a gluten free diet in two patients. CONCLUSION: A raised IEL count with normal villous architecture is not uncommon. Six of the 14 patients may have had latent coeliac disease. The cause in at least half of cases is not obvious at present. The finding of a raised IEL count with normal villous architecture is of sufficient clinical importance to be highlighted in routine duodenal biopsy reports.

Analysis of T cell receptor beta chain CDR3 size using RNA extracted from formalin fixed paraffin wax embedded tissue.

AIMS: To isolate RNA and DNA simultaneously from formalin fixed paraffin wax embedded tissue to assess the clonality of enteropathy associated T cell lymphomas and to analyse it in detail by a non-radioactive method of T cell receptor complementarity determining region 3 (CDR3) spectratyping. METHODS: DNA and RNA were isolated simultaneously from formalin fixed paraffin wax embedded tissue blocks and subjected to the polymerase chain reaction (PCR) and semi-nested reverse transcription PCR (RT-PCR), respectively. The RT-PCR T cell receptor V beta products were analysed by CDR3 spectratyping using a denaturing polyacrylamide gel and silver staining. RESULTS: Usable DNA and RNA were isolated simultaneously from formalin fixed paraffin wax embedded tissue. The specific clonality of the tissue was successfully analysed by a non-radioactive method of T cell receptor CDR3 spectratyping of the RT-PCR products. CDR3 spectratying of the RT-PCR products demonstrated the precise clonal nature of the tumour and non-tumour tissue showing that the non-tumour tissue comprised an oligoclonal population of a number of different T cell receptor V beta families. The tumour tissue comprised two T cell subtypes of the one family, T cell receptor V beta 9. CONCLUSIONS: RNA and DNA were isolated from formalin fixed paraffin wax embedded enteropathy associated T cell lymphoma tissue. Detailed analysis of clonality can be carried out by a non-radioactive method of CDR3 spectratyping.

Expression of interleukin-6 by intestinal enterocytes.

AIMS: To investigate the cellular source of the cytokine interleukin-6 (IL-6) in the small and large intestines of patients with inflammatory bowel disease, coeliac disease, and in controls. METHODS: IL-6 was detected in frozen sections of bowel by single and double label indirect immunofluorescence using rabbit polyclonal and murine monoclonal anti-IL-6 antibodies. The murine monoclonal antibodies RFDR1 (anti-MHC class II) and UCHT1 (anti-CD3) were used to localise macrophages and T lymphocytes, respectively. Lipopolysaccharide stimulated peripheral blood monocytes were used as positive control cells for IL-6 protein. RESULTS: IL-6 was demonstrated in the small and large intestine of patients with inflammatory bowel disease, coeliac disease, and in controls. The protein was present predominantly in enterocytes and colocytes in normal and inflamed mucosa, but not in the infiltrating inflammatory cells of the lamina propria. There were no discernable differences between patients with inflammatory bowel disease or coeliac disease and controls, nor between small and large bowel mucosa. Incubation of antibody with recombinant human IL-6 protein abolished the labelling. IL-6 protein was also present in lipopolysaccharide stimulated peripheral blood monocytes. CONCLUSIONS: The data suggest that enterocytes and colocytes may play an active part in the immune response of the gut. The presence of IL-6 in both inflamed and non-inflamed small and large intestine requires further investigation into the function of this cytokine in the gut.

Helicobacter pylori serology in patients with coeliac disease and dermatitis herpetiformis.

AIMS: To investigate whether Helicobacter pylori infection or autoimmune gastritis is responsible for the reported increase in gastric pathology and abnormalities of gastric function in patients with coeliac disease and dermatitis herpetiformis (DH). METHODS: Serum H pylori IgG antibodies were assayed by enzyme linked immunosorbent assay and intrinsic factor antibodies by radioimmunoassay in 99 patients with coeliac disease and 58 patients with dermatitis herpetiformis from two geographic areas. RESULTS: H pylori positivity in patients with coeliac disease and dermatitis herpetiformis increased with age, reaching 50% and 70%, respectively, in patients over 50 years. The percentage H pylori seropositivity in coeliac disease did not differ from the percentage positivity observed in 250 similarly aged blood donors from the same geographic area (Leeds). Seropositivity in patients with dermatitis herpetiformis was not significantly different from the level of positivity observed in 98 age matched patients without dermatitis herpetiformis attending the same Edinburgh dermatology clinic. Only one patient with coeliac disease had positive intrinsic factor antibodies. H pylori seropositivity in Edinburgh control subjects under 30 years of age (41.9%) was significantly higher (p less than 0.03) than in Leeds controls (18%) of corresponding age. An increasing prevalence of H pylori seropositivity with age in coeliac disease and dermatitis herpetiformis paralleled that of the control groups. CONCLUSIONS: Gastritis in coeliac disease and dermatitis herpetiformis is largely caused by H pylori infection at a level that is no different from that of the general population. Any increase in the prevalence of gastritis in these two diseases might be caused by lymphocytic gastritis rather than pernicious anaemia.

Primary small-bowel malignancy in the UK and its association with coeliac disease.

BACKGROUND: Malignancies of the small intestine are rare, accounting for <2% of all cancers of the gastrointestinal tract. There is little information about the presentation and prognosis of these tumours, and the frequency of established risk factors. AIM: To estimate the frequency of small-bowel malignancy in the UK, and its relationship to the presence of coeliac disease. DESIGN: Survey of clinicians registered with the British Society of Gastroenterology. METHODS: Data were collected monthly from June 1998 to May 2000. Clinicians (n=1327) were asked by post to report newly diagnosed cases of primary small-bowel malignancy. A form was sent to reporting clinicians, requesting an anonymous identifier, type of malignancy, and whether coeliac disease was present. A detailed questionnaire followed, requesting further clinical and pathological details. RESULTS: Clinico-pathological data were ascertained for 395 cases, including 175 adenocarcinomas, 107 lymphomas and 79 carcinoid tumours. In 13% of adenocarcinoma cases and in 39% of lymphomas, there was a diagnosis of coeliac disease. Survival rates at 30 months for adenocarcinomas, lymphomas and carcinoid tumours were 58%, 45% and 78%, respectively. Prognosis of all tumours was inversely related to stage at presentation, and lymphomas associated with coeliac disease were associated with a poorer prognosis. DISCUSSION: This study provides additional evidence that coeliac disease confers susceptibility to adenocarcinoma of the small bowel, as well as lymphoma. The long time from the onset of symptoms to diagnosis of small bowel tumours is of concern, as this delay is reflected in the high proportion that presented with metastatic disease. Although the absolute risk of malignancy is small, coeliac disease complicated by malignancy appears to be poorly controlled.

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 detection of enteric adenoviruses by means of the polymerase chain reaction.

A simple polymerase chain reaction (PCR) assay for detecting enteric adenoviruses (Ads 40 and 41) in faecal extracts is described. A pair of PCR primers designed to hybridise to the EIB genes of Ad40 and 41 was found to amplify only Ad40 and 41 DNA but not EIB genes or viral DNA from representative numbers of the other human adenovirus subgenera. The PCR assay was tested on a panel of 10 faecal extracts, all of which contained adenovirus particles (as judged by electron microscopy) but only four of which proved amenable to serotyping. Extracts in which enteric adenoviruses had been detected serologically yielded positive results in the PCR assay. These results suggest that this PCR assay may be useful for detecting enteric adenoviruses in clinical samples.

The effect of anticoagulant choice on apparent total antioxidant capacity using three different methods.

We assessed total antioxidant capacity using three different methods, in plasma samples treated with either EDTA or heparin as anticoagulant, from 26 healthy subjects. Total antioxidant capacity was determined using an oxygen electrode (as the total peroxyl radical-trapping antioxidant parameter), by enhanced chemiluminescence, and by measurement of the antioxidant-mediated quenching of the absorbance of a radical cation. The choice of anticoagulant had a profound effect on antioxidant capacity with heparinized plasma giving consistently higher values than plasma anticoagulated with EDTA. Using the oxygen electrode the mean value was 786.5 +/- 171.5 mumol/L (heparin) compared to 681.4 +/- 160.4 mumol/L (EDTA, P < 0.01). The chemiluminescence technique gave a mean antioxidant capacity of 915.6 +/- 214.1 mumol/L in heparin samples and 714.4 +/- 195.4 mumol/L in EDTA samples (P < 0.0001). The absorbance quenching technique gave a mean value of 867.0 +/- 199.2 mumol/L (heparin) and 675.5 +/- 245.4 mumol/L (EDTA, P < 0.001). All methods tested showed comparable results for EDTA plasma, but the chemiluminescence technique gave higher apparent antioxidant capacity than either of the two techniques when heparin plasma was used. We suggest that either heparin is interacting to enhance antioxidant protection perhaps through release of superoxide dismutase, or the chelation of metal ions by EDTA is limiting the activity of antioxidant metalloenzymes. Consistency in the choice of anticoagulant is clearly extremely important.

Evidence of high sugar intake, and low fibre and mineral intake, in the gluten-free diet.

BACKGROUND: The only therapy for coeliac disease (CD) is a long-term gluten-free diet (GFD). Little is known about the detailed composition of such a diet. AIM: To clarify the nutritional composition of a GFD and to compare it with a non-GFD diet in representative non-CD populations. METHODS: A total of 139 consecutive patients with CD were invited to fill in a prospective validated 5-day food diary, of whom data from 93 were analysed. Results were compared with data from the National Diet and Nutrition Survey of Adults and the UK Women's Cohort Study (UKWCS). RESULTS: Individuals consuming a strict GFD generally had similar intakes of energy and nutrients to those of comparison populations, but a higher proportion of carbohydrate intake was obtained from nonmilk extrinsic sugars and intakes of nonstarch polysaccharides were low. Compared with the UKWCS sample, female patients adhering to a GFD had lower intakes of magnesium, iron, zinc, manganese, selenium and folate. In male patients, intakes of magnesium and selenium were particularly low. CONCLUSIONS: This study reinforces the need for clinicians to recognize that avoidance of gluten cannot be the sole focus of a gluten-free diet. Maintenance of adequate intakes of essential nutrients and in particular complex carbohydrates must also be the goal for patients.

Long-term histological follow-up of people with coeliac disease in a UK teaching hospital.

BACKGROUND: Coeliac disease is a relatively common condition which is usually managed by placing patients on a gluten free diet. Follow up biopsies to confirm histological recovery are controversial with a considerable variation in practice observed. AIM: To determine the length of time to histopathological recovery in a group of coeliac disease patients and its associations with clinicopathological data. DESIGN AND METHODS: All patients attending a specialist coeliac disease clinic prior to March 2009 were entered onto a database which recorded various clinicopathological data. The histopathology reports for all duodenal biopsies were reviewed and each biopsy was given a histopathological disease score based on a modified Marsh grade. RESULTS: Two hundred and eighty-four patients underwent index and at least one subsequent biopsy. Two-hundred and twenty-seven (80%) showed histopathological improvement and 100 (35%) returned to normal (median recovery time 1.9 years, inter-quartile range 1.0-4.8 years). Patients with less severe disease at diagnosis were more likely to show a better response (r = 0.281, P < 0.0001). Older patients demonstrated a shorter time to histopathological recovery (r = -0.200, P = 0.001). Compliance with a gluten free diet was correlated with the best biopsy score (r = -0.134, P = 0.040) and degree of histological recovery (r = 0.161, P = 0.014). CONCLUSION: Current guidelines for the timing of repeat biopsy after commencing a gluten free diet are unclear, although 4-6 months has been recommended. This study shows that time to histological recovery is longer than traditionally thought and may need to take into account the patient's age at diagnosis, the initial disease score and the level of compliance with a gluten free diet.