Minimal Gluten Exposure Alters Urinary Volatile Organic Compounds in Stable Coeliac Disease.

Coeliac disease (CD) patients are distinguishable from healthy individuals via urinary volatile organic compounds (VOCs) analysis. We exposed 20 stable CD patients on gluten-free diet (GFDs) to a 14-day, 3 g/day gluten challenge (GCh), and assessed urinary VOC changes. A control cohort of 20 patients continued on GFD. Urine samples from Days 0, 7, 14, 28 and 56 were analysed using Lonestar FAIMS and Markes Gas Chromatography-Time of Flight-Mass Spectrometer (GC-TOF-MS). VOC signatures on D (day) 7-56 were compared with D0. Statistical analysis was performed using R. In GCh patients, FAIMS revealed significant VOC differences for all time points compared to D0. GC-TOF-MS revealed significant changes at D7 and D14 only. In control samples, FAIMS revealed significant differences at D7 only. GC-TOF-MS detected no significant differences. Chemical analysis via GC-MS-TOF revealed 12 chemicals with significantly altered intensities at D7 vs. D0 for GCh patients. The alterations persisted for six chemicals at D14 and one (N-methyltaurine) remained altered after D14. This low-dose, short-duration challenge was well tolerated. FAIMS and GC-TOF-MS detected VOC signature changes in CD patients when undergoing a minimal GCh. These findings suggest urinary VOCs could have a role in monitoring dietary compliance in CD patients.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Selective measurement of anti-tTG antibodies in coeliac disease and IgA deficiency: an alternative pathway.

OBJECTIVE: To determine the ability of selective antibody testing to screen for coeliac disease in the presence of IgA deficiency and to define the sensitivity of a pathway using this method. METHOD: All IgA and IgG anti-tTG tests performed at our centre between January 2008 and December 2009, using the Immunocap 250 analyser, were retrospectively reviewed. Positive results were correlated with histology. Results were used to validate our diagnostic pathway. RESULTS: 12 289 consecutive serological tests were reviewed. IgA deficient patients gave either an 'error' reading or very low response on the Immunocap 250 analyser. Subsequent testing of this sub-group demonstrated raised IgG anti-tTG antibodies in those with histologically proven coeliac disease. CONCLUSIONS: Using our antibody screening pathway, which involves the selective use of IgG anti-tTG, sensitivity increased from 87% to 92% in those with IgA deficiency. Adoption of this pathway for coeliac screening would negate the routine screening of immunoglobulin levels, with resultant cost saving.

Application of a novel tool for diagnosing bile acid diarrhoea.

Bile acid diarrhoea (BAD) is a common disease that requires expensive imaging to diagnose. We have tested the efficacy of a new method to identify BAD, based on the detection of differences in volatile organic compounds (VOC) in urine headspace of BAD vs. ulcerative colitis and healthy controls. A total of 110 patients were recruited; 23 with BAD, 42 with ulcerative colitis (UC) and 45 controls. Patients with BAD also received standard imaging (Se75HCAT) for confirmation. Urine samples were collected and the headspace analysed using an AlphaMOS Fox 4000 electronic nose in combination with an Owlstone Lonestar Field Asymmetric Ion Mobility Spectrometer (FAIMS). A subset was also tested by gas chromatography, mass spectrometry (GCMS). Linear Discriminant Analysis (LDA) was used to explore both the electronic nose and FAIMS data. LDA showed statistical differences between the groups, with reclassification success rates (using an n-1 approach) at typically 83%. GCMS experiments confirmed these results and showed that patients with BAD had two chemical compounds, 2-propanol and acetamide, that were either not present or were in much reduced quantities in the ulcerative colitis and control samples. We believe that this work may lead to a new tool to diagnose BAD, which is cheaper, quicker and easier that current methods.

Why patients take part in the Royal College of Physicians Practical Assessment of Clinical Examination Skills (MRCP PACES).

The aim of this study was to examine patients' motivation to participate in the Royal College of Physicians Practical Assessment of Clinical Examination Skills (PACES). An exploratory cross-sectional study was performed with data collected via telephone interviews. All patients aged 18+ who participated in PACES at University Hospitals Coventry and Warwickshire in the last two years were invited to take part; 28 patients were interviewed. Data were analysed using thematic content analysis. Motivational factors identified included the opportunity to give something back for the care received, contributing to doctors' learning processes, altruism and being able to learn more about one's own condition. Patients believed that they offered real-life experiences that cannot be provided by actors. The social environment during PACES played a large part in volunteer retention. Recruitment of patient volunteers needs to emphasise the altruistic nature of the role in assisting the learning process for doctors and the subsequent benefit for future patients.

The detection of patients at risk of gastrointestinal toxicity during pelvic radiotherapy by electronic nose and FAIMS: a pilot study.

It is well known that the electronic nose can be used to identify differences between human health and disease for a range of disorders. We present a pilot study to investigate if the electronic nose and a newer technology, FAIMS (Field Asymmetric Ion Mobility Spectrometry), can be used to identify and help inform the treatment pathway for patients receiving pelvic radiotherapy, which frequently causes gastrointestinal side-effects, severe in some. From a larger group, 23 radiotherapy patients were selected where half had the highest levels of toxicity and the others the lowest. Stool samples were obtained before and four weeks after radiotherapy and the volatiles and gases emitted analysed by both methods; these chemicals are products of fermentation caused by gut microflora. Principal component analysis of the electronic nose data and wavelet transform followed by Fisher discriminant analysis of FAIMS data indicated that it was possible to separate patients after treatment by their toxicity levels. More interestingly, differences were also identified in their pre-treatment samples. We believe these patterns arise from differences in gut microflora where some combinations of bacteria result to give this olfactory signature. In the future our approach may result in a technique that will help identify patients at "high risk" even before radiation treatment is started.

Dysregulation of plasma ghrelin in alcoholic cirrhosis.

OBJECTIVE: Abnormalities in circulating ghrelin have been reported in chronic liver disease. This study assessed the response of anabolic peptides ghrelin, growth hormone (GH) and insulin-like growth factor 1 (IGF-1) in patients with alcoholic cirrhosis and healthy subjects to oral glucose. In a previous study, using oral glucose we identified loss of ghrelin regulation in nonalcoholic steato-hepatitis. PATIENTS/DESIGN/MEASUREMENTS: Fourteen patients with alcoholic cirrhosis were compared with 11 healthy subjects. Patients with cirrhosis were studied when adjudged clinically stable in hospital. After an overnight fast, they ingested 100-g glucose dissolved in 250 ml of water. Blood was sampled before and every 20 minutes after ingestion for 120 minutes. Plasma acylated and des-acyl ghrelin, GH, IGF-1 and insulin were assayed by ELISA. RESULTS: Expressed as median (95% CI): 120-minutes integrated acylated ghrelin was 26 (19-66) in controls compared to 170 (129-252) pg/ml per hour in patients with cirrhosis; P < 0.001. Both groups exhibited a normal postglucose plasma total ghrelin profile. Among patients with cirrhosis (compared to controls), growth hormone was increased 15-fold and IGF-1 decreased 4-fold. Acylated ghrelin correlated with GH (Spearman r = 0.69, P = 0.0015) in control subjects but not in patients with cirrhosis. CONCLUSIONS: Acylated ghrelin is markedly increased in alcoholic cirrhosis, with apparent preservation of normal postprandial mechanisms of gastric ghrelin secretion. GH is also increased; however, its correlation with acylated ghrelin (confirmed in healthy subjects) is absent in patients with cirrhosis. Despite increased ghrelin and GH, patients with alcoholic cirrhosis remain anorexic and catabolic suggesting potential tissue resistance to the actions of these anabolic peptides.

Pre-analytical and analytical variables that influence urinary volatile organic compound measurements.

There has been rapidly accelerating interest in the utilization of volatile organic compounds (VOCs) as non-invasive methods for rapid point-of-care medical diagnostics. There is widespread variation in analytical methods and protocols, with little understanding of the effects of sample storage on VOC profiles. This study aimed to determine the effects on VOC profiles of different storage times, at room temperature, prior to freezing, of sealed urine samples from healthy individuals. Analysis using Field Asymmetric Ion Motility Spectrometry (FAIMS) determined the alterations in VOC and total ion count profiles as a result of increasing room temperature storage times. Results indicated that increasing exposure time to room temperature prior to freezing had a threefold effect. Firstly, increased urinary VOC profile variability, with a plateau phase between 12 and 48 hours, before further degradation. Secondly, an increase in total ion count with time exposed to room temperature. Finally, a deterioration in VOCs with each sample run during the analysis process. This provides new insight into the effect of storage of urine samples for VOC analysis using FAIMS technology. Results of this study provide a recommendation for a 12-hour maximum duration at room temperature prior to storage.

A Randomized, Double-blind, Placebo-controlled, Parallel-group, Pilot Study of Cannabidiol-rich Botanical Extract in the Symptomatic Treatment of Ulcerative Colitis.

Background: Cannabidiol (CBD) exhibits anti-inflammatory properties that could improve disease activity in inflammatory bowel disease. This proof-of-concept study assessed efficacy, safety and tolerability of CBD-rich botanical extract in ulcerative colitis (UC) patients. Methods: Patients aged 18 years or older, with left-sided or extensive UC, Mayo scores of 4-10 (endoscopy scores ≥1), and on stable 5-aminosalicylic acid dosing, were randomized to 10-weeks' CBD-rich botanical extract or placebo capsules. The primary endpoint was the percentage of patients in remission after treatment. Statistical testing was 2-sided, using a 10% significance level. Results: Patients were less tolerant of CBD-rich botanical extract compared with placebo, taking on average one-third fewer capsules, and having more compliance-related protocol deviations (principally insufficient exposure), prompting identification of a per protocol (PP) analysis set. The primary endpoint was negative; end of treatment remission rates were similar for CBD-rich botanical extract (28%) and placebo (26%). However, PP analysis of total and partial Mayo scores favoured CBD-rich botanical extract (P = 0.068 and P = 0.038, respectively). Additionally, PP analyses of the more subjective physician's global assessment of illness severity, subject global impression of change, and patient-reported quality-of-life outcomes were improved for patients taking CBD-rich botanical extract (P = 0.069, P = 0.003, and P = 0.065, respectively). Adverse events (AEs) were predominantly mild/moderate with many in the CBD-rich botanical extract group potentially attributable to the ∆9-tetrahydrocannabinol content. A greater proportion of gastrointestinal-related AEs, indicative of UC worsening, was seen on placebo. Conclusion: Although the primary endpoint was not reached, several signals suggest CBD-rich botanical extract may be beneficial for symptomatic treatment of UC.

An assessment of candidate genes to assist prognosis in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the fourth commonest cancer worldwide, with the second highest mortality rate. Its poor mortality is linked to delayed presentation. There is a drive towards non-invasive biomarker screening and monitoring of many different types of cancer, although with limited success so far. We aimed to determine if any genes from a 32-gene panel could be used to determine GC prognosis. METHODS: We carried out a retrospective study on the expression of 32 genes, selected for their proven or potential links to GC, on historic formalin fixed paraffin-embedded (FFPE) GC specimens from our unit. Gene expression was measured using quantitative nuclease protection assays (qNPA) technology. Following statistical analysis of the results, immunohistochemical staining for eight genes, both discriminating and non-discriminating, was conducted in seven age and sex matched non-metastatic: metastatic GC pairings. The stained samples were reviewed by two blinded consultant histopathologists. RESULTS: Multivariate Cox analysis of the gene expression data revealed metastatic status, age, sex and five genes appeared to influence GC survival. Genes negatively influencing survival included BCAS1, P53 and HSP90AA1 (relative risks 2.20, 3.73 and 7.53 respectively). Genes conveying survival benefit included CASP3 and TERT (relative risks 0.10 and 0.24 respectively). Immunohistochemical staining of seven age and sex matched non-metastatic: metastatic pairs revealed no association between gene expression and protein expression. CONCLUSIONS: Our study found several genes whose expression may affect GC prognosis. However, immunohistochemical analysis revealed no association between gene expression and protein expression. It remains to be determined whether gene expression or protein expression are reliable means of assessing GC prognosis.

Immunohistochemical and quantitative mRNA assessment of ghrelin expression in gastric and oesophageal adenocarcinoma.

BACKGROUND: Ghrelin is an orexigenic gut peptide produced predominantly by the stomach. Gastric mucosal ghrelin production could be compromised by an infiltrating adenocarcinoma. AIMS: To assess the expression of ghrelin mRNA and peptide in oesophagogastric adenocarcinomas and adjacent non-neoplastic mucosa. METHODS: 10 gastric and 22 oesophageal adenocarcinoma archival samples were randomly selected from a database. The presence of ghrelin-positive cells was assessed in cancer and corresponding non-neoplastic mucosa by immunohistochemistry. Quantitative reverse transcriptase polymerase chain reaction (PCR) for ghrelin mRNA was also performed on 24 gastric and 8 oesophageal adenocarcinoma specimens and adjacent non-neoplastic mucosa. RESULTS: Immunohistochemistry and reverse transcriptase PCR confirm a negligible expression of ghrelin in adenocarcinoma specimens. By contrast, non-neoplastic gastric mucosa was rich in ghrelin-positive cells and ghrelin mRNA. The number (median and range) of ghrelin-positive cells per 2 mm section of non-neoplastic mucosa was 73 (45-215) in the corpus; this was significantly higher than in cardia mucosa (9 (0-64), p<0.001) and antral mucosa (5 (0-14), p<0.001). CONCLUSIONS: Gastric and oesophageal adenocarcinomas have no ghrelin-producing cells. The highest level of ghrelin expression was noted in the non-neoplastic mucosa of the gastric corpus. Disruption of the gastric ghrelin-producing mechanism may occur during oesophagogastric malignancy.

Extended lifespan and long telomeres in rectal fibroblasts from late-onset ulcerative colitis patients.

OBJECTIVES: Ulcerative colitis (UC) is characterized by damage to the intestinal epithelium and connective tissue. The causes of this damage could include changes in the ability of colonic fibroblasts to heal wounds and maintain epithelial cell proliferation. Telomeres shorten with each cell division and eventually signal senescence. The aim of this study is to investigate whether the impaired function of rectal fibroblasts in UC is due to accelerated telomere shortening, oxidative stress and premature senescence. METHODS: We isolated rectal fibroblasts from eight UC patients and nine non-colitis controls, and recorded their in-vitro lifespans. Telomere lengths and superoxide dismutase mRNA expression were also measured by real-time polymerase chain reaction and peroxide levels were measured by flow cytometry. RESULTS: The fibroblast lifespan decreased as patient age increased (R2=0.68, P=0.003) in control patients, but this relationship was absent in UC fibroblasts. We identified a group of patients who were diagnosed later in life than a second group (59 versus 35 years, P=0.002). Fibroblasts from these late-onset UC patients underwent significantly more population doublings before senescence than age-matched controls (25 versus 15, P=0.02). Slower in-vitro telomere shortening rates (32 versus 344, P=0.006) and trends towards longer telomeres at explant were also observed in late-onset UC fibroblasts. Peroxide levels correlated positively with telomere shortening rate (r=0.581, P=0.078). CONCLUSIONS: Some UC-predisposed individuals may have more efficient antioxidant systems that protect the telomeres from oxidative damage. This may allow their rectal fibroblasts to live longer, function better and thus delay the onset of the disease until later life.

Perioperative peripheral parenteral nutrition for patients undergoing esophagectomy for cancer: a pilot study of safety, surgical, and nutritional outcomes.

At esophagectomy, cancer patients may be malnourished. Nutrition administered central venously is associated with complications, potentially negating nutritional benefits. We aimed to determine the safety of nutrition administered by the peripheral parenteral route (PPN) and record changes in nutritional and surgical outcome. Ivor-Lewis esophagectomy was performed by a single experienced surgeon. Consecutive patients received either 7 days of PPN perioperatively (n = 16) or oral diet reintroduction on the fourth postoperative day (n = 11). Mortality, complications, measures of body composition, protein metabolism, and biochemistry were assessed. Thirty-day mortality was 0% and 18% in the PPN and standard group, respectively. By the 90th day, mortality had increased to 36% in the standard group (P < 0.05). Perioperative PPN can be administered safely in cancer patients undergoing esophagectomy. This form of nutritional support merits further examination by larger, multicenter studies to confirm or refute the observations made in this pilot study.

A systematic review of the role of DNA methylation on inflammatory genes in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is an idiopathic disease of the large intestine with evidence pointing to the role of epigenetic changes. METHODS: Searches were performed in three databases (EMBASE, MEDLINE and Web of Science), following PRISMA protocol. DNA methylation was the only epigenetic mechanism affecting genes linked to inflammatory response in UC. RESULTS: A total of 25 differentially methylated inflammatory genes were identified. Hypermethylation of miR-1247 significantly correlates (p = 0.0006) with refractory UC while PAR2 hypermethylation correlates (p = 0.007) with corticosteroid dependence. CONCLUSION: Evidence points to a step-wise increase in methylation status of the genome between a healthy colon, quiescent UC and when inflamed. Inflammatory genes (which are aberrantly methylated), have also been implicated in cancer development in UC.

Synchronous Upper and Lower Gastrointestinal Mucosa-Associated Lymphoid Tissue Lymphomas.

Mucosa-associated lymphoid tissue lymphoma (MALToma) is a subtype of B-cell non-Hodgkin's lymphoma, comprising ∼17% of all gastrointestinal (GI) tract lymphomas. It is associated with chronic inflammation and autoimmunity, for example Helicobacter pylori gastritis and Sjogren's syndrome, respectively. Approximately 50% of GI MALTomas occur in the stomach, with small bowel and colonic lesions being less frequent. Synchronous upper and lower GI MALTomas occur rarely, with few cases reported. We present the case of a 73-year-old patient who presented with change in bowel habit and was found to have synchronous multifocal upper and lower GI MALTomas, which did not respond to H. pylori cure or to rituximab therapy, but did respond to a combination of surgery and chemotherapy with rituximab and bendamustine.

Clinical outcomes at 12†months and risk of inflammatory bowel disease in patients with an intermediate raised fecal calprotectin: a 'real-world' view.

OBJECTIVES: A recent systematic review confirmed the usefulness of fecal calprotectin (FC) in distinguishing organic (inflammatory bowel disease (IBD)) from non-organic gastrointestinal disease (irritable bowel syndrome (IBS)). FC levels <50 µg/g have a negative predictive value >92% to exclude organic gastrointestinal (GI) disease. Levels >250 µg/g correlate with endoscopic IBD disease activity; sensitivity 90%. We aimed to determine clinical outcomes in intermediate raised FC results (50-250 µg/g). SETTING: Primary care general practices in Coventry and Warwickshire, and 3 secondary care hospitals. PARTICIPANTS: 443 FC results in adults (>16 years old) were reviewed from July 2012 to October 2013. Clinical data was collected from hospital databases and general practitioners. Long-term clinical data was available in 41 patients (out of 48). PRIMARY AND SECONDARY OUTCOME MEASURES: The number of new diagnoses of IBD, IBS and other diagnoses for the intermediate group. The number referred and discharged from secondary care. RESULTS: A new IBD diagnosis was made in 19% (n=8) of intermediate results (1% of normal and 38% of raised results). 5% (n=2) of intermediate results had known IBD in remission. A new IBS diagnosis was made in 27% (n=11) of intermediate results, while 34% (n=14) remained undiagnosed, although 8 of these were not referred to secondary care. CONCLUSIONS: FC testing remains useful in aiding diagnosis of organic GI conditions. However, unlike negative and strongly positive FC results, intermediate FC results lead to a mixture of diagnoses. The OR of a new diagnosis of IBD for an intermediate result compared to normal FC result was 26.6, while an intermediate FC result gave an OR of 0.54 for a new IBS diagnosis compared to normal FC. For intermediate FC results, 1 in 3 patients remained in secondary care after 12 months with an OR of 3.6 compared to a normal FC result.