Azathioprine and 6-mercaptopurine were considered effective in approximately 40% of IBD patients after 5 years of treatment. A quarter of the patients discontinued thiopurines within 3 months, mostly due to adverse events. A high 6-MMP concentration or 6-MMP/6-TGN ratio was associated with therapeutic failure. If thiopurine use was successfully initiated in the first months, its use was usually extended over many years, as long-term use was associated with continuation of therapy.

These results suggest that coeliac disease and ulcerative colitis may share a common aetiology through tight junction-mediated barrier defects, although the observations need further replication.

The use of real-time quantitative PCR (5 nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of Escherichia coli and Bacteroides vulgatus. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5 nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of E. coli and 9 CFU of B. vulgatus could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to E. coli and B. vulgatus and with eight other gut microflora species. Mucosal samples spiked with known amounts of E. coli or B. vulgatus DNA showed no PCR inhibition. We conclude that the 5 nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.The human intestinal tract harbors a large and complex community of microbes which is involved in maintaining human health by preventing colonization by pathogens and by producing nutrients. At least 400 bacterial species are thought to be present in the human gastrointestinal tract (4, 9, 14, 19). Microorganisms are not randomly distributed throughout the intestine. The flora of the stomach and proximal small intestine differs significantly from that of the terminal ileum and colon. The numerically predominant species of bacteria are obligate anaerobes and are represented by both gram-positive and gram-negative genera. The most frequently identified anaerobic microorganisms are Bacteroides spp., Bifidobacterium spp., Eubacterium spp., Peptostreptococcus spp., and Fusobacterium spp. (17). The total number of bacterial cells, as determined by fecal culture methods, increases gradually from the small intestine to the large bowel and reaches 10 11 to 10 12 cells/ml of contents. Conventional bacteriological methods like microscopy, culture, and identification are used for the analysis and/or quantification of the intestinal microflora (7,16,20). Limitations of conventional methods are their low sensitivities (5), their inability to detect noncultivatable bacteria and unknown species, their time-consuming aspects, and their low levels of reproducibility due to the multitude of species to be identified and quantified. In addition, the large differences in growth rates and growth requirements of the different species present in the human gut indicate that quantification by culture is bound to be inaccurate. To overcome the problems of culture, techniques based on 16S ribosomal DNA (rDNA) genes were developed (2, 22). These include fluorescent in situ hybridization (8,11,12,13,21), denaturing gradient gel electrophoresis (15, 18), and temperature gradient gel electrophoresis (6, 23). These techniques have low sensitivities and are laborious and technically demanding. We evaluated the suitability of a rea...

ABSTRACT

Linskens RK, Huijsdens XW, Savelkoul PHM, Vandenbroucke-Grauls CMJE, Meuwissen SGM. The bacterial flora in inflammatory bowel disease: current insights in pathogenesis and the influence of antibiotics and probiotics. Scand J Gastroenterol 2001;36 Suppl 234:29-40.The pathogenesis of in ammatory bowel disease (IBD) remains unknown, although in recent years more data have become available. The contribution of genetic and environmental factors is evident, and the luminal bacterial ora plays a major role in the initiation and perpetuation of chronic IBD. Animal models of IBD have shown that colitis does not occur in a germ-free environment. In human IBD, in ammation is present in parts of the gut containing the highest bacterial concentrations. Moreover, the terminal ileum, caecum and rectum are areas of relative stasis, providing prolonged mucosal contact with luminal contents.Enhanced mucosal permeability may play a pivotal role in maintaining a chronic in ammatory state, due to a genetic predisposition or as a result of direct contact with bacteria or their products. A defective epithelial barrier may cause a loss of tolerance to the normal enteric ora. Furthermore, an increased mucosal absorption of viable bacteria and bacterial products is found in IBD. Serum and secreted antibodies are increased and mucosal T-lymphocytes that recognize luminal bacteria are present. However, there is evidence that the immune system reacts over aggressively towards the normal luminal ora rather than the ora being altered in IBD. Several approaches have been used in attempts to discover a speci c microbial agent in the cause of IBD. These include demonstration of the presence of organisms or speci c antigens in affected tissues, culture of microbes from the affected tissues, demonstration of serological responses to several agents, and localization and detection of individual pathogen-speci c nucleic acid sequences in affected tissue by in situ hybridization and polymerase chain reaction. So far, no speci c micro-organism has been directly associated with the pathogenesis of IBD. Analysis of the luminal enteric ora, however, has revealed differences in the composition of this ora compared to healthy controls. In Crohn disease, concentrations of Bacteroides, Eubacteria and Peptostreptococcus are increased, whereas Bi dobacteria numbers are signi cantly reduced. Furthermore, in ulcerative colitis, concentrations of facultative anaerobic bacteria are increased. The arrival of new molecular techniques qualifying and quantifying the complex intestinal ora has induced a revival of interest in this micro ora.Therapeutic approaches geared towards changing the environment at the mucosal border have been attempted by the use of elemental diets, total parenteral nutrition, surgical diversion of the faecal stream and antibiotics. Over the past few years, the use of probiotics in IBD and other intestinal disorders has gained attention. Strengthened by promising experimental data and commercial interests, research in this eld is rapidly expandin...