Tom C. Martinsen

Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demonstrates lack of major differences between Crohn's disease and ulcerative colitis.

Background In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn’s disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns. Methods Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores. Results Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls. Conclusions There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.

Serum gastrin and chromogranin A levels in patients with fundic gland polyps caused by long-term proton-pump inhibition

Objective. Use of proton-pump inhibitors (PPIs) causes hypergastrinemia, and it is well known that gastrin has a trophic effect on the oxyntic mucosa. Some PPI users develop fundic gland polyps. The purpose of this study was to determine whether patients developing fundic gland polyps have a more pronounced gastric hypoacidity, hypergastrinemia or increased serum chromogranin A (CgA), which is an enterochromaffin-like (ECL) cell marker. Material and methods. Five PPI users who developed multiple fundic gland polyps during PPI use were included in the study. PPI users without fundic gland polyps (n=6) as well as healthy individuals (n=6) were used as controls. In PPI users, we measured 24-h gastric pH, serum gastrin and CgA during one day, with standardized meals, whereas only gastrin and CgA were measured in the healthy individuals. Helicobacter pylori status was determined. Results. Gastric pH, serum gastrin and CgA did not differ significantly between PPI users with and those without fundic gland polyps. All patients with fundic gland polyps were H. pylori negative, whereas 4 out of 6 PPI users without fundic gland polyps were H. pylori positive. Fasting CgA levels were elevated in all PPI users, and CgA more than doubled during the day in all groups. Conclusions. Fundic gland polyps induced by PPIs are not related to the level of hypergastrinemia. Serum CgA is markedly affected by meals and should be measured in samples from fasting patients.

Gastric neuroendocrine carcinoma after long-term use of proton pump inhibitor

Abstract We present a case of a gastric neuroendocrine carcinoma in a patient with a history of long-term proton pump inhibitor (PPI) use. A 49-year-old man using PPI for the last 15 years due to gastroesophageal reflux disease developed progressive dysphagia, dyspepsia and weight loss. Upper gastrointestinal endoscopy, endoscopic ultrasonography and abdominal CT diagnosed a malignant tumor localized to a hiatal hernia. Fasting serum chromogranin A and gastrin concentrations were elevated (32 nmol/l and 159 pmol/l, respectively). Helicobacter pylori PCR analysis of antral biopsies was negative. Biopsies from endoscopically normal oxyntic mucosa showed enterochromaffin-like (ECL) cell hyperplasia. Tumor biopsies revealed a poorly differentiated neuroendocrine carcinoma. Sevier-Munger staining, immunohistochemistry and electron microscopy indicated ECL cell as origin of the tumor cells. Concerns have previously been raised about the safety of long-term PPI use due to a possible increased risk of cancer. This case illustrates a patient with a poorly differentiated neuroendocrine carcinoma with ECL cell characteristics probably induced by hypergastrinemia secondary to long-term PPI use.

ECL-Cell Derived Gastric Cancer in Male Cotton Rats Dosed with the H2-Blocker Loxtidine

Spontaneously hypergastrinemic cotton rats (Sigmodon hispidus) develop tumors that have the phenotype of an adenocarcinoma but most likely originate from the enterochromaffin-like (ECL) cells. Among inbred animals ∼50% of the females, but <1% of males develop spontaneous gastric carcinomas. Gastrin is the principle carcinogen in this model, as >4 months of hypergastrinemia results in carcinoma, but a gastrin receptor antagonist prevents carcinomas. Carcinomas can also be induced by partial corpectomy. In the present study, the insurmountable H2-receptor antagonist loxtidine (200 mg/kg/day) was given to male cotton rats for 6 months. The loxtidine-dosed animals developed hypergastrinemia, whereas control animals remained normogastrinemic. At termination, 4 of 5 cotton rats had cancer located to the oxyntic mucosa, whereas 1 animal had dysplasia. The gastric mucosa of all of the control animals was normal. In the dysplastic mucosa of loxtidine-dosed animals there was a marked increase in chromogranin A-positive cells, where numerous groups of cells also stained positive with the Sevier-Munger technique. In areas of high proliferation and cancer there were also histidine decarboxylase, chromogranin A, and Sevier-Munger-positive cells, altogether indicating an ECL cell origin of the tumors. This represents an interesting animal model where ECL cell-derived gastric cancer can be induced by pharmacological acid inhibition in 6 months.

Expression of neuroendocrine markers in non-small cell lung cancer : A biochemical, immunohistochemical and ultrastructural study

Neuroendocrine (NE) differentiation is reported in some cases of non‐small cell lung cancer (NSCLC). The aim of this study was to evaluate the expression of NE markers in NSCLC using novel sensitive methods. 20 cases of NSCLC were examined using immunohistochemical (IHC) and immunoelectron microscopy (IEM) methods. In addition, circulating levels of the NE markers chromogranin A (CgA) and neuron‐specific enolase (NSE) were measured. Using conventional IHC methods, two tumours (10%) showed immunoreactivity for synaptophysin (SYN), one (5%) for Cg and four (20%) for neural cell adhesion molecule (NCAM). Adding the tyramide signal amplification (TSA) technique, the number of immunoreactive tumours for both SYN and CgA increased to five (25%). No increased immunoreactivity was achieved for NCAM. Nine tumours (45%) were immunoreactive for SYN, CgA or NCAM. Using IEM, one of five representative samples that revealed IHC reactivity for CgA showed immunogold labelling of CgA in cytoplasmic vesicles. Elevated levels of circulating CgA or NSE did not correlate with positive IHC findings. In conclusion, using sensitive IHC methods NE differentiation was seen in a greater proportion of NSCLC than previously reported. Sensitive methods may improve our understanding of the tumour biology and represent an important diagnostic tool for future therapeutic modalities.

Ultrastructure and chromogranin A immunogold labelling of ECL cell carcinoids

Poorly differentiated neuroendocrine cells can be difficult to recognise. Sensitive methods are needed to label cells that have lost their ultrastructural features and have reduced concentrations of neuroendocrine markers. In gastric neoplasms, enterochromaffin‐like cells might dedifferentiate and lose their characteristic granules and secretory vesicles, making detection of such cells increasingly difficult. However, chromogranin A (CgA) immunogold labelling could provide sensitive and specific detection of gastric neuroendocrine cells. We present ultrastructural findings, CgA immunogold labelling as well as conventional immunohistochemical findings of two human enterochromaffin‐like cell carcinoids. Electron‐dense granules of poorly differentiated cells were less intensely labelled than granules in well‐differentiated cells. Granules with atypical shape as well as punctuate granules previously found in neuroendocrine neoplasms were also CgA labelled. The CgA labelling efficacy after antigen retrieval in an alkaline solution was higher after heating in an autoclave at 135 °C compared to a microwave at 100 °C for both granules and secretory vesicles without significant deterioration of the ultrastructure. In conclusion, the use of CgA immunogold labelling could ensure a specific classification of cells with neuroendocrine granules and be a supplement to immunohistochemical examination of poorly differentiated tumours.

Clinical experience with infliximab and adalimumab in a single-center cohort of patients with Crohn's disease

Abstract Background and aims: Patients with Crohns disease (CD) may need anti-inflammatory drugs for decades. Anti-TNF-α agents have good efficacy and adverse events similar to placebo in randomized controlled trials (RCTs), but there are still questions about long-term safety and efficacy. In this respect, reports from clinical practice may be useful. We currently report on the clinical experience with infliximab and adalimumab in a single-center cohort of patients with CD. Material and Methods: Patients with CD treated with infliximab or adalimumab from 2000 to 2010 were reviewed. Patient and disease characteristics at start, reason for discontinuation, and adverse events were recorded retrospectively. Corticosteroid use, the need for hospitalization, and surgeries before and during anti-TNF-α therapy were recorded. Results: Eighty-three patients had received anti-TNF-α treatment against CD, median treatment duration was 11.4 months (0.2–99.5), and follow-up time 59 months (8–135). Eighteen of 43 patients using corticosteroids at treatment start discontinued corticosteroids during TNF-α therapy. Need for hospitalizations (6.13 vs. 3.28 days/year, p < 0.001) and surgeries (0.56 vs. 0.16 operations/year, p < 0.001) were lower during anti-TNF-α therapy than before treatment. Twenty-six percent discontinued therapy due to adverse events and 26% due to lack or loss of response. Two of four deaths observed during follow-up were believed to be related to anti-TNF-α treatment. Conclusions: Anti-TNF-α therapy was beneficial in many patients with CD, but the majority of patients discontinued treatment during follow-up. Reports from clinical experience with anti-TNF-α treatment may be valuable for clinicians treating patients with CD.

5-Aminosalicylic acid, a specific drug for ulcerative colitis

Abstract Objective. The etiology of the inflammatory bowel diseases is unknown, although genetic factors play a role, and tobacco smoking has opposite effect on the two entities. Inflammation is central in the pathogenesis, and treatment is aiming to suppress it. The active part of salazopyrin, the oldest drug in use in the treatment of ulcerative colitis, is 5-aminosalicylic acid (5-ASA). In the present paper, we wanted to discuss the etiology and pathogenesis of ulcerative colitis in relation to the beneficial effects of 5-ASA and particularly whether this compound has a specific effect on ulcerative colitis. Methods/Results. 5-ASA seems to have a selective positive effect on ulcerative colitis in inducing remission, preventing relapse and possibly reducing the risk of cancer. In contrast to other agents used in the treatment of ulcerative colitis, 5-ASA does not have any known anti-inflammatory effect on other organs or other colonic inflammatory diseases like diverticulitis. Moreover, the effect on experimental colitis in rodents is not convincing. Conclusion. 5-ASA seems to have a specific effect on the inflammation in ulcerative colitis. Research on the mechanism of its action may give information on the etiology of ulcerative colitis. 5-ASA is a first-line treatment that should be given once daily in high doses and for long term to reduce the possibility of recurrence and risk of colonic cancer. Side effects with 5-ASA are rare, and every patient with ulcerative colitis who tolerate this drug, should be treated with 5-ASA.

Inhibitors of gastric acid secretion increase the risk of prion infection in mice.

Abstract Gastric juice is a unique combination of hydrochloric acid and the proteolytic enzyme pepsin. Its main function is to inactivate ingested microorganisms. Prions cause fatal transmissible degenerative encephalopathies in animals and man. These diseases have attracted attention due to the proposed link between bovine spongiform encephalopathy in cattle and the occurrence of a new variant Creutzfeldt-Jakob disease in humans where the most probable route of transmission is via contaminated food. The role of gastric juice in the protection against these agents is not settled. Objective. The aim of this study was to examine if drug-induced gastric hypoacidity increases the susceptibility of prion infection transmitted by the oral route. Material and methods. Forty-six mice (tg338) were given brain homogenates contaminated with scrapie by gastric intubation. Twenty-two of these animals were concomitantly dosed with omeprazole increasing the median gastric pH from 1.2 to 5.3. After 381 days, the animals were sacrificed and all the brains were examined for detection of pathogenic prion proteins by enzyme-linked immunosorbent assay and western blot. Results. Drug-induced decrease in gastric acidity more than doubled the rate (59% vs. 25%, p < 0.035) of brain infection compared to controls with normal gastric acidity at the time of inoculation. Conclusions. Our results demonstrate that the normal gastric juice constitutes a significant defense against prion disease in mice. Thus, gastric hypochlorhydria would be expected to enhance the susceptibility to prion infection by the oral route. This finding may have relevance to the pathogenesis of the new variant Creutzfeldt-Jakob disease and prion diseases in general.

Neuroendocrine cells in diffuse gastric carcinomas: an ultrastructural study with immunogold labeling of chromogranin A.

Neuroendocrine differentiation is often found in gastric carcinomas, but the relevance of these cells in gastric carcinogenesis is debated. We applied immunolabeling at the electron microscopic level to study the ultrastructure of neuroendocrine cells in gastric carcinomas to ensure correct cellular classification of dedifferentiated cells. The immunogold labeling at electron microscopic level was compared with an established sensitive immunohistochemical method using light microscopy. Thirteen human gastric adenocarcinomas of the diffuse type were examined for neuroendocrine differentiation by chromogranin A (CgA) labeling at both the light and electron microscopic level. The ultrastructure of CgA-positive cells was compared with CgA-positive cells from controls. Nine of 13 tumors showed CgA-positive cells both at the light and electron microscopic level. The CgA-positive cells displayed altered ultrastructural features compared with controls. Some of the CgA-positive tumor cells had granules typical for enterochromaffin-like cells. Immunoelectron microscopy seems to provide both significant immunolabeling and sufficient ultrastructure to enhance classification of cells in neoplastic tissue.