Kouhei Fukushima

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model.

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.

Non-Pathogenic Bacteria Modulate Colonic Epithelial Gene Expression in Germ-Free Mice

Background: We established a bacterial reconstitution model to investigate epithelial cell-luminal bacteria interaction. The aim of the study was to identify the known genes directly or indirectly modulated by non-pathologic bacterial flora in the colonic epithelia of germ-free mice. Methods: Germ-free mice were orally given a bacterial suspension prepared from specific pathogen-free counterparts (bacterial reconstitution). Colonic epithelial cells were isolated, then total and poly (A) RNA were extracted. We investigated differential gene expression in colonic epithelial cells among germ-free, bacteria-reconstituted, and specific pathogen-free mice by DNA microarray. Finally, differential expression was confirmed by Northern blot or quantitative RT-PCR. Results: Thirty genes were initially selected as differentially expressed genes in DNA microarray analysis. We confirmed that genes associated with growth (Reg III #, Reg III % , guanylate nucleotide binding protein 2), apoptosis (Bcl-associated death promotor), cytoskeleton (tubulin ! 4, erythrocyte protein band 7.2), and immune response (lymphocyte antigen complex 6) were induced by bacterial reconstitution. In contrast, genes possibly participating in extracellular oxidant defence (selenoprotein P, metallothionein 1) and cellular metabolism (cytochrome P450, HMGCoA synthase 2, alcohol dehydrogenase 1 complex, aldehyde dehydrogenase family 1, carbonic anhydrase 1, glycoprotein galactosyltransferase ! 1,3) were down-regulated by bacterial challenge. Conclusion: Non-pathogenic bacteria modulated colonic gene expression in germ-free mice, suggesting that non-pathogenic bacteria possibly initiate epithelial change in genetically normal and/or abnormal hosts. The present study provides a basis for the functional study of each molecule in symbiosis with luminal bacteria in healthy and diseased colon.

Inflammatory bowel disease-associated gene expression in intestinal epithelial cells by differential cDNA screening and mRNA display.

Intestinal epithelial cells are actively involved in the pathogenesis of inflammatory bowel disease resulting in an altered functional phenotype. The modulation of epithelial gene expression may occur as a consequence of proliferative, metabolic, immune, inflammatory, or genetic abnormalities.Differential screening of epithelial-cell-derived cDNA libraries (from control, ulcerative colitis, and Crohns disease epithelial cells) and differential display of mRNA were used for investigation of disease-associated gene expression and modulation.Intestinal epithelial gene expression was successfully analyzed by both approaches. Using differential screening with clones encoding mitochondrial genes, quantitative overexpression was observed in both ulcerative colitis and Crohns disease, while a unique expression of small RNA was noticed in Crohns disease cells using Alu-homologous clones. Differential display demonstrated that several genes were differentially displayed among control, ulcerative colitis, and Crohns disease epithelial cells. This was confirmed by immunohistochemical staining of pleckstrin, desmoglein 2 and voltage-dependent anion channel in control and inflammatory bowel disease mucosal samples.In summary, several inflammatory bowel disease-related associations were found. Since both differential screening and display have advantages and limitations, the combination of both techniques can generate complementary information, facilitate search for novel genes, and potentially identify genes uniquely associated with inflammatory bowel disease.

Pouchitis atlas for objective endoscopic diagnosis.

Abstract“Pouchitis” is a term for nonspecific mucosal inflammation of the pouch after total proctocolectomy and ileal pouch–anal anastomosis for ulcerative colitis. Pouchitis is the most frequent complication of the pelvic pouch at the late stage. To improve the accuracy of the pouchitis diagnosis, sets of clinical symptoms and endoscopic findings (with or without histology of biopsy samples) have previously been evaluated. Endoscopic findings are central to the diagnosis, and a universal consensus of various endoscopic findings must be the initial step toward an objective diagnosis of pouchitis. Since a proper signpost for the endoscopic evaluation of pouchitis has been absent, we developed this pouchitis atlas to minimize the diagnostic variation inherent among individual endoscopists. We also propose new criteria for the diagnosis of pouchitis: the Japanese criteria for diagnosis of pouchitis. These criteria are based on clinical symptoms and endoscopic findings that are clearly categorized in the atlas, and exclude infectious enteritis, anastomotic insufficiency, pelvic infection, anal dysfunction, and Crohns disease. Advantages of the new criteria include ease of bedside diagnosis, without the calculation of points required by the other criteria for pouchitis. This pouchitis atlas, together with our new criteria, should contribute to the establishment of a clear-cut diagnosis for pouchitis and promote better evaluation and treatment of this novel intestinal inflammation.

Renal expression of the essential genes associated with sodium transport following a total proctocolectomy in rats.

The water and electrolyte balance is regulated by the renin-angiotensin-aldosterone system in the kidney. We previously reported that the levels of circulating aldosterone dramatically increased following a total proctocolectomy in rats. However, there is no direct evidence regarding whether renal adaptation is accelerated by the induction of aldosterone-associated molecules. To explore this question, Sprague-Dawley rats underwent total proctocolectomies and then were killed 8 weeks later. We investigated the renal expression of 11β-hydroxysteroid dehydrogenase type 2, the α-, β-, and γ-subunits of the epithelial sodium channel, and the α1- and β1-subunits of Na+,K+-ATPase mRNAs because those molecules are responsible for the aldosterone specificity for mineralocorticoid receptor, amiloride-sensitive sodium absorption, and sodium extrusion from distal tubules, respectively. A Northern blot analysis demonstrated the kidney to exhibit mRNA induction for all of these molecules, thus supporting the idea that renal adaptation following a total proctocolectomy depends, at least in part, upon the molecular induction which is principally regulated by circulating aldosterone.

An approach to analyze mechanisms of intestinal adaptation following total proctocolectomy

We hypothesized that epithelial cells of the remnant small intestine display “colonic” phenotype after total proctocolectomy. The aims of the present study were to identify preferentially expressed molecules in the colon or in the small intestine and to evaluate mRNA levels of those in the ileal pouch. Differential gene expression was investigated between the small intestine and the colon by using cDNA microarray and was confirmed by Northern blotting. Expression of three colonic mRNAs (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, deleted malignant brain tumors 1, carcinoembryonic antigen-related cell adhesion molecule 1) and one “small intestinal” (microsomal triglyceride transfer protein) mRNA were compared between the control and the ileal pouch mucosae by quantitative reverse transcriptase-polymerase chain reaction. Seventy-four clones were differentially expressed with more than a threefold difference. Differential expression was confirmed in all mRNAs examined, including 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and microsomal triglyceride transfer protein. The mucosal expression of carcinoembryonic antigen-related cell adhesion molecule 1 mRNA in the ileal pouch was enhanced in humans. The remnant ileum develops some, but not all, colonic phenotype after total proctocolectomy. Comparative study of epithelial gene expression between the small intestine and the colon enables us to analyze mechanisms of intestinal adaptation after total proctocolectomy.

A New Drug Delivery System Targeting Ileal Epithelial Cells Induced Electrogenic Sodium Absorption: Possible Promotion of Intestinal Adaptation

We previously demonstrated the induction of the epithelial sodium channel, prostasin, and 11β-hydroxysteroid dehydrogenase type 2 and activation of sodium transport mediated by those molecules in the remnant ileum after total proctocolectomy. The aims of the present study were to develop a new drug delivery system that targets ileal epithelial cells and to enhance local mineralocorticoid action without systemic effects. Orally administered d-aldosterone-containing d,l-lactide/glycolide acid copolymer microspheres are absorbed in the rat terminal ileum and released aldosterone. Blood and terminal ileal tissues were collected 2xa0weeks after the administration of the microspheres, and the aldosterone concentrations, mRNA, and protein expressions of the above molecules and sodium transport were evaluated. Significantly high levels of tissue aldosterone in the absence of elevated plasma levels were detected in the microspheres-treated rats. Epithelial mRNA and protein expression of the above molecules increased significantly in the microspheres-treated animals. Electrogenic sodium transport in the ileum was enhanced in the microspheres-treated rats. Aldosterone-containing microspheres successfully induced the expression of the above molecules and activated sodium transport in the ileal mucosa, both of which are essential for intestinal adaptation. Pre- and/or postoperative treatment with this drug may compensate for the excessive loss of sodium and water following proctocolectomy.

Fecal stream is essential for adaptive induction of glucose-coupled sodium transport in the remnant ileum after total proctocolectomy

Our previous studies demonstrated that sodium glucose cotransporter 1 (SGLT-1) was induced in the remnant ileum of total colectomized rats via the action of factors other than hyperaldosteronism. The aim of the present study was to clarify whether fecal stream is required for the enhancement of SGLT-1-mediated sodium transport. Twenty-seven pairs of ileal tissues were obtained from the proximal and distal side, respectively, of loop ileostomy after total proctocolectomy. Mucosae were mounted in an Ussing chamber to evaluate glucose-coupled sodium transport. Levels of SGLT-1 mRNA in proximal and distal mucosae were compared by Northern blotting. Villous height and crypt depth were measured to test for correlations between mucosal structure and SGLT-1-mediated sodium transport or mRNA expression levels. Both glucose-coupled sodium transport and expression of SGLT-1 mRNA were significantly lower in distal mucosae relative to proximal mucosae. In distal mucosae, villous height, but not crypt depth, was significantly lower than in proximal mucosae, demonstrating a positive correlation between villous height and SGLT-1 function and expression. Comparative studies of proximal and distal mucosae demonstrated that in addition to hormonal changes, fecal stream is required for full induction of the sodium transport system (which includes SGLT-1-mediated transport) in the remnant ileum following total proctocolectomy.

Genetic disorders are complex in inflammatory bowel disease.

Reprint requests to: K. Fukushima Familial aggregations of inflammatory bowel disease (IBD) suggest that IBD may comprise several genetic diseases. However, these genetic disorders appear to be too complex to determine the causes of IBD. It is apparent that IBD does not follow Mendelian inheritance patterns or a simple segregation of the disease. It is widely accepted that a complex interplay among genetic, environmental, and unknown factors leads to onset, progression, and/or recurrence of IBD. Several years ago three groups discovered that homozygous or compound heterozygous mutations of CARD15, a gene encoding NOD2, are a major susceptibility factor underlying a subgroup of patients with Crohn’s disease in Western countries.1–3 Functional roles of NOD2 mutations in dysregulation of mucosal immune responses have not yet been fully elucidated. However, accumulated knowledge of normal NOD2 functions promotes understanding of the consequence of NOD2 mutations. NOD2 is a member of a large protein family known as the NOD-LRR (leucine-rich repeat) protein family sharing the same structural regions: an LRR and a NOD (nucleotide oligomerization domain) and a CARD (caspase recruitment domain) or PYRIN domain.4 CARD15 suggests a proinflammatory pathway stimulated by a bacterial component–the muramyl dipeptide (MDP)–that activates NF-kB.5–7 In general, the identification of susceptibility genes for complex genetic diseases is not frequent. The difficulties may be related to the limited effects of the predisposing gene on the phenotype of the disease.8 An additional difficulty is genetic heterogeneity, which can be supported by ethnic differences. In this issue of Journal of Gastroenterology, Ozen et al.9 have attempted to establish an association between IBD (Crohn’s disease and ulcerative colitis) and polymorphisms of three genes in the Turkish population. They studied three variants in the coding region of the NOD2/CARD15 gene, two polymorphisms in the nucleotide-binding domain of the NOD1/CARD4 gene, and two polymorphisms in ICAM-1. However, they did not find any association between IBD and the gene polymorphisms examined. The discovery of NOD2/CARD15 gene mutations in Western countries was striking because this was the first susceptibility gene known for Crohn’s disease. However, patient–control studies of Japanese, Chinese, Korean, and also Turkish populations have not encountered NOD2/CARD15 polymorphisms in either patient or control groups.10–13 Those data clearly demonstrate that ethnically divergent populations do not necessarily share the same set of predisposing genes, although they may present identical phenotypes. This implies that other gene polymorphisms may also be involved in a genetic predisposition for IBD. Information regarding the frequency of genetic polymorphisms in different populations may have less impact than the first information on significant disease susceptibility. Nonetheless, evaluation of gene polymorphisms in different populations is essential, because this type of study will promote understanding of the “real” significance of each genetic variation in the pathogenesis of IBD and its contribution to disease phenotypes.