http://repub.eur.nl/res/aut/1943/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/32030/ 2002-11-20T00:00:00Z In the first part of this thesis the role of the colonic epithelium and in particular its associated mucus-layer during IBD and in several experimental colitis models is discussed (Chapter 2). In Chapter 3-5 our investigations regarding the colonic epithelium in rat during the different phases ofDSS-induced acute colitis are described. Studied are: A. clinical symptoms, morphology, proliferation and apoptosis (Chapter 3). B. enterocyte-specific gene expression, goblet cell-specific gene expression, and the Muc2 - and TFF3 secretion capacity of goblet cells, as a measure of enterocyte and goblet cell functioning (Chapters 4 and 5). C. goblet cell-specific Muc2 biosynthesis, Muc2 sulfation, and Muc2 secretion (Chapter 5). These parameters were studied in conjunction during the onset of disease, active disease and the regenerative phase of DSS-induced colitis, in the proximal and distal colon to obtain insight in the specific colonic functions during damage and regeneration In the second part of this thesis the small intestinal epithelium after treatment with the cytostatic drug MTX is investigated. Described are: A. clinical symptoms, morphology, proliferation, apoptosis, and enterocyte-, goblet cell-, and Paneth cell specific gene expression in the 'normal' small intestinal epithelium, i.e. the epithelium distant from the Peyer's Patches (Chapter 6). B. the above described parameters in the PP-associated epithelium, and compared these with alterations seen in the small intestinal located more distantly from PP (Chapter 7). These parameters were studied on various days after MTX-treatment in the duodenum, jejunum, ileum, and colon to obtain a complete picture of the epithelial functions during MTX-induced damage and regeneration. Finally, by comparing the DSS-induced colonic damage with the MTX-induced small intestinal damage, the obtained insight in the types of damage and damage control in the colon and small intestine are discussed (Chapter 8). http://repub.eur.nl/res/pub/10009/ 2002-01-01T00:00:00Z The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase (-62%) and CPS (-82%) were correlated. Correlations were also found between lactase (-76%) and SGLT1 (-77%) and between I-FABP (-52%) and L-FABP (-45%). Decreases in GLUT5 (-53%), MUC2 (-43%), and TFF3 (-54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased (+76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 (+115%) and TFF3 (+81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected. http://repub.eur.nl/res/pub/13015/ 2002-01-01T00:00:00Z To gain insight into mucin 2 (Muc2) synthesis and secretion during dextran sulfate sodium (DSS)-induced colitis, rats were treated with DSS for 7 days. Colonic segments were excised on days 0 (control), 2 (onset of disease), 7 (active disease), and 14 (regenerative phase) for histological evaluation. Explants were metabolically labeled with (35)S-labeled amino acids or [(35)S]sulfate followed by chase incubation. Homogenates were analyzed by SDS-PAGE and (35)S-labeled Muc2 was quantified. Also, total Muc2 protein and mRNA were quantified. DSS-induced crypt loss, ulcerations, and concomitant goblet cell loss were most pronounced in the distal colon. Muc2 precursor synthesis increased progressively in the proximal colon but was unaltered in the distal colon during onset and active disease. During the regenerative phase, Muc2 precursor synthesis levels normalized in the proximal colon but increased in the distal colon. Total Muc2 levels paralleled the changes seen in Muc2 precursor synthesis levels. During each disease phase, total Muc2 secretion was unaltered in the proximal and distal colon. [(35)S]sulfate incorporation into Muc2 only decreased in the proximal colon during active disease and the regenerative phase, whereas secretion of [(35)S]sulfate-labeled Muc2 increased. During the regenerative phase, Muc2 mRNA levels were downregulated in both colonic segments. In conclusion, DSS-induced loss of goblet cells was accompanied by an increase or maintenance of Muc2 precursor synthesis, total Muc2 levels, and Muc2 secretion. In the proximal colon, Muc2 became undersulfated, whereas sulfated Muc2 was preferentially secreted. Collectively, these data suggest specific adaptations of the mucus layer to maintain the protective capacities during DSS-induced colitis. http://repub.eur.nl/res/pub/13063/ 2002-01-01T00:00:00Z In the present study, we aimed to investigate enterocyte- and goblet cell-specific functions during the different phases of acute colitis induced with dextran sulfate sodium (DSS). Rats were treated with DSS for 7 days, followed by a 7-day recovery period. Colonic tissue was excised on days 2 (onset of disease), 7 (active disease), and 14 (regenerative phase). Enterocyte functions were studied by the expression of carbonic anhydrases (CAs), sodium/hydrogen exchangers (NHEs) and intestinal fatty acid-binding protein (iFABP) and by alkaline phosphatase (AP) activity. The expression and secretion of the mucin Muc2 and trefoil factor family peptide-3 (TFF3) were used as parameters for goblet cell function. DSS induced a downregulation of the CAs, NHEs, and iFABP in some normal-appearing surface enterocytes and in most of the flattened-surface enterocytes during disease onset and active disease. During the regenerative phase most enterocytes expressed these genes again. Quantitative analysis revealed a significant decrease in CAs, NHEs, and iFABP expression levels during onset and active disease. During the regenerative phase, the expression levels of the CAs were restored, whereas the expression levels of the NHEs and iFABP remained decreased. In contrast, enterocyte-specific AP activity was maintained in normal and flattened enterocytes during DSS-induced colitis. Goblet cells continued to express MUC2 and TFF3 during and after DSS treatment. Moreover, Muc2 and TFF3 expression and secretion levels were maintained or even increased during each of the DSS-induced disease phases. In conclusion, DSS-induced colitis was associated with decreased expression of CAs, NHEs, and iFABP. The loss of these genes possibly accounts for some of the pathology seen in colitis. The maintenance or upregulation of Muc2 and TFF3 synthesis and secretion levels implies that goblet cells at least maintain their epithelial defense and repair capacity during acute inflammation induced by DSS. http://repub.eur.nl/res/pub/9506/ 2000-01-01T00:00:00Z Proliferation, differentiation, and cell death were studied in small intestinal and colonic epithelia of rats after treatment with methotrexate. Days 1-2 after treatment were characterized by decreased proliferation, increased apoptosis, and decreased numbers and depths of small intestinal crypts in a proximal-to-distal decreasing gradient along the small intestine. The remaining crypt epithelium appeared flattened, except for Paneth cells, in which lysozyme protein and mRNA expression was increased. Regeneration through increased proliferation during days 3-4 coincided with villus atrophy, showing decreased numbers of villus enterocytes and decreased expression of the enterocyte-specific genes sucrase-isomaltase and carbamoyl phosphate synthase I. Remarkably, goblet cells were spared at villus tips and remained functional, displaying Muc2 and trefoil factor 3 expression. On days 8-10, all parameters had returned to normal in the whole small intestine. No methotrexate-induced changes were seen in epithelial morphology, proliferation, apoptosis, Muc2, and TFF3 immunostaining in the colon. The observed small intestinal sparing of Paneth cells and goblet cells following exposure to methotrexate is likely to contribute to epithelial defense during increased vulnerability of the intestinal epithelium.