K. Geboes

Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors

Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of μ-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut—similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treament of abdominal pain and irritable bowel syndrome.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis

Background and aims: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor α (anti-TNFα) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. Methods: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. Results: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. Conclusion: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Infliximab induces potent anti‐inflammatory and local immunomodulatory activity but no systemic immune suppression in patients with Crohn’s disease

Anti‐TNFα therapy with infliximab is effective for Crohn’s disease. Infliximab neutralizes the biological activities of TNFα, a cytokine involved in host‐defence against certain infections.

A new approach to the validation of tissue microarrays

Although tissue microarrays (TMA) have been widely used for a number of years, it is still not clear how many core biopsies should be taken to determine a reliable value for percentage positivity or how much heterogeneity in marker expression influences this number. The first aim of this study was to validate the human visual semi‐quantitative scoring system for positive staining of tumour tissue with the exact values determined from computer‐generated images. The second aim was to determine the minimum number of core biopsies needed to estimate percentage positivity reliably when the immunohistochemical staining pattern is heterogeneous and scored in a non‐binary way. Tissue sections from ten colorectal cancer specimens were stained for carbonic anhydrase IX (CA IX). The staining patterns were digitized and 400 artificial computer‐generated images were generated to test the accuracy of the human scoring system. To determine the minimal number of core biopsies needed to account for tumour heterogeneity, 50 (artificial) core biopsies per section were taken from the tumoural region of the ten digitally recorded full tissue sections. Based on the semi‐quantitative scores from the 50 core biopsies per section, 2500 × n (n = 1–10 core biopsies) experimental core biopsies were then generated and scores recorded. After comparison with field‐by‐field analysis from the tumoural region of the whole tissue section, the number of core biopsies that need to be taken to minimize the influence of heterogeneity could be determined. In conclusion, visual scoring accurately estimated the percentage positivity and the percentage tumour present in a section, as judged by comparison with the artificial images. The exact number of core biopsies that has to be examined to determine tumour marker positivity using TMAs is affected by the degree of heterogeneity in the expression pattern of the protein, but for most purposes at least four is recommended. Copyright

Contractile effects and intracellular Ca2+ signalling induced by motilin and erythromycin in the circular smooth muscle of human colon

Motilin has excitatory effects on the colon of the rabbit and the dog, but little is known of its effect on the human colon. The aim of this study was to investigate the effects induced by motilin and erythromycin A (EMA) on muscle strips and on single cells from primary cultures from human colon. Isotonic contraction was recorded in circular muscle strips from macroscopically normal resection specimens of patients operated on for colonic neoplasm. Agonist‐induced intracellular Ca2+ ([Ca2+]i) signalling was studied in primary cultures of colonic smooth‐muscle cells using the ratiometric Ca2+ indicator Indo 1, on a laser‐scanning confocal epifluorescence microscope. In circular muscle strips, norleucine13‐porcine motilin ([Nle13]‐pm)and EMA induced tonic contractions with an EC50 of 92u2003±u200321u2003nmolu2003L−1 and 31u2003±u200316u2003μmolu2003L−1, respectively. The maximal contraction was 21u2003±u20034% (motilin) and 33u2003±u200312% (EMA) of the response to 10−4u2003molu2003L−1 acetylcholine (ACh). The motilin antagonist OHM‐11526 (10−5.5u2003molu2003L−1) abolished the effects of both [Nle13]‐pm and EMA. Neither tetrodotoxin (10−5.5u2003molu2003L−1), L‐nitro‐D‐arginine methyl ester (L‐NAME) (10−3.5 molu2003L−1) nor guanethidine (10−5u2003molu2003L−1) interfered with the effects of [Nle13]‐pm or EMA. [Nle13]‐pm (10−11−10−6 molu2003L−1) induced rises of [Ca2+]i in cultured colonic myocytes. At 10−6u2003molu2003L−1, 94% of the cells responded, and half of the cells responded at 1.4u2003nmolu2003L−1 [Nle13]‐pm. 81% (35/43) and 95% (75/79) responded to EMA (10−6u2003molu2003L−1) and acetylcholine (ACh, 10−4u2003molu2003L−1), respectively. The motilin antagonist GM‐109 inhibited motilin‐ and EMA‐induced [Ca2+]i rises. In the absence of extracellular Ca2+, only 13% (7/52) of the cells responded to [Nle13]‐pm (10−6u2003molu2003L−1) vs. 90% (47/52) to ACh (10−4u2003molu2003L−1). Motilin and EMA have direct excitatory effects on circular smooth muscle from the human colon and these effects are mediated via a smooth‐muscle motilin receptor. These findings suggest that motilin may regulate colonic motility and that motilides may have therapeutic potential for the treatment of colonic hypomotility.

Validation of 16S rDNA sequencing in microdissected bowel biopsies from Crohn's disease patients to assess bacterial flora diversity

The bowel flora is implicated in Crohns disease (CD) pathogenesis but its precise role is still unclear. Several non‐mutually exclusive hypotheses have been proposed: an unidentified persistent pathogen; excessive bacterial translocation; an immune system abnormality in response to normal bacteria; or a breakdown in the balance between protective and harmful bacteria. These hypotheses can be tested by identifying bacteria in specific microscopic bowel structures or lesions. The present paper describes a novel technique to assess bacterial flora diversity in bowel biopsies, by combining laser capture microdissection with broad‐range 16S rDNA sequencing. Fifty‐four samples comprising histologically normal and pathological mucosa, MALT, ulcers, submucosal lymphangiectasias, epithelioid granulomas, and lymph nodes were microdissected out of 30 bowel biopsies from five CD patients. Bacterial 16S rDNA was successfully amplified by PCR in all samples, and PCR products from 15 samples were selected for cloning and sequence analysis. A total of 729 bacterial DNA sequences were analysed, which could be attributed to six different phyla (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria, and Planctomycetes). DNA from typical bowel bacteria (Enterobacteriaceae, Clostridiales, Bacteroidetes, Fusobacteria) was detected in all microdissected areas. It was thus convincingly demonstrated that 16S rDNA sequencing can be combined with microdissection to study the bowel flora. However, no specific persistent pathogen causal for CD was identified. The results suggest that Enterobacteriaceae may initiate or colonize ulcers in CD. Translocation of bacteria through established mucosal lesions or as a result of increased permeability may be involved in the evolution towards chronic inflammation and in the establishment of persistent lesions. Further study is needed to confirm these preliminary findings. Copyright

Pouchitis, similar to active ulcerative colitis, is associated with impaired butyrate oxidation by intestinal mucosa

Background: Healthy colonic mucosa uses butyrate as the major energy source. In ulcerative colitis (UC) butyrate oxidation has been shown to be disturbed, but it remains unclear whether this is a primary defect. The aim of this study was to measure mucosal butyrate oxidation in UC (involved and noninvolved colon) and in pouchitis and to study the relationship with endoscopic as well as histological disease activity. Methods: Butyrate oxidation was measured in 73 UC patients, 22 pouchitis patients, and 112 controls (95 colon, 17 ileum) by incubating biopsies with 1 mM 14C‐labeled Na‐butyrate and measuring the released 14CO2. Results: Compared with that in normal colon, butyrate oxidation was significantly impaired in endoscopically active but not in quiescent disease or uninvolved colon segments. The severity of the metabolic defect was related to histological disease activity and decreased epithelial cell height. In active pouchitis, butyrate oxidation was significantly decreased compared with that in normal ileum and excluded pouches without inflammation. The histological pouchitis score correlated significantly with butyrate oxidation. Conclusions: Active UC and pouchitis show the same inflammation‐related metabolic defect. Our data suggest that the defect is a consequence of inflammation and that pouchitis is metabolically similar to active UC.

Topical cidofovir (HPMPC) is an effective adjuvant to surgical treatment of anogenital condylomata acuminata.

AbstractPURPOSE: Human papilloma virus infections of the anogenitalnregion are very common and cause condylomata acuminata;ncervical, penile, vulvar, or perianal intraepithelial neoplasia;nand more rarely, invasive cancer. The currentlynavailable therapies often result in painful, extensive, slowhealingnulcerations and frequent early relapses. This studynwas aimed at determining the efficacy of topical applicationnof the antiviral agent cidofovir at 1 percent. METHODS:nTwenty patients treated with coagulations were comparednwith 27 patients treated with cidofovir. Lesions refractorynto cidofovir were cleared up with additional coagulations.nThe number of patients previously treated for condylomatandid not differ between the two groups. Significantly morenpatients treated with cidofovir, however, had an impairednimmune status (37 percent) compared with the patientsntreated with coagulations (5 percent). RESULTS: Cidofovirnalone cured the lesions in 32 percent of the patients andninduced partial regression in 60 percent. However, in smokers,ncomplete resolution of the condylomata occurred onlynin 16.6 percent compared with 66 percent of nonsmokersn(P = 0.03). The number of coagulation sessions was muchnlower (P < 0.0005) in the cidofovir treated group (1 ± 0.8nvs. 2.9 ± 2). Furthermore, the relapse rate was significantlynlower in the cidofovir group (3.7 vs. 55). All recurrences innthe electrocoagulation group occurred within four monthsnof confirmed lesion clearance. Topical applications of cidofovirn1 percent were well tolerated. Thirty-three percent ofnthe patients reported only mild pain caused by erosivendermatitis. In contrast, coagulations caused painful ulcerationsnthat necessitated the use of analgesics in all patientsntreated this way. CONCLUSIONS: Topical applications ofncidofovir, an antiviral compound with activity against humannpapilloma virus, is effective in the majority of patientsnwith perianal condylomata and is a valuable adjuvant tonsurgical treatment of these lesions.

Germline Mutations of the hMLH1 and hMSH2 Mismatch Repair Genes in Belgian Hereditary Nonpolyposis Colon Cancer (HNPCC) Patients

Background: Hereditary nonpolyposis colon cancer (HNPCC-Lynch syndrome) is caused by mutations in genes involved in DNA mismatch repair (MMR), mostly in the hMLH1 and hMSH2 genes. The mutation spectrum in the Belgian population is still poorly documented. Aim: To report our experience on the mutation screening in Belgian familial colorectal cancer (CRC) patients, including the investigation of the pathogenicity of the missense and splice mutations. To increase the mutation detection rate by selecting the target population. Methods: Two hundred and twenty five Belgian patients with familial clustering of CRC were genetically tested. Point mutations in the hMLH1 and hMSH2 genes were screened by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing. Genomic deletions and duplications were assessed by multiplex ligase dependent probe amplification (MLPA) and multiplex PCR. Missense mutations were examined for pathogenicity by means of cosegregation of the mutation with the disease, microsatellite instability (MSI) in tumors, immunohistochemical staining of tumors and determination of the population frequency of the particular mutation. Results: Twenty five pathogenic mutations were identified from which 16 were novel: 7 frameshifts, one in frame deletion, 5 genomic deletions, 5 splice defects, 4 nonsense (stop) mutations and 3 missense mutations which were classified as pathogenic (out of 10 missense mutations). In retrospect, a mutation detection rate of 71% was obtained if MSI was used as a supplementary selection criterion in addition to familial clustering. Conclusion: Different types of pathogenic mutations in the hMLH1 and hMSH2 genes were identified in a Belgian CRC group with familial clustering. The mutation detection yield drastically increased by preliminar selection of those familial CRC patients with a microsatellite instable tumor. Considerable attention went to the assessment of the pathogenicity of the missense mutations. In practice, the cosegregation with the disease was the most relevant criterion.