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Human intestinal ischemia-reperfusion (IR) is a frequent phenomenon carrying high morbidity and mortality. Although intestinal IR-induced inflammation has been studied extensively in animal models, human intestinal IR induced inflammatory responses remain to be characterized. Using a newly developed human intestinal IR model, we show that human small intestinal ischemia results in massive leakage of intracellular components from ischemically damaged cells, as indicated by increased arteriovenous concentration differences of intestinal fatty acid binding protein and soluble cytokeratin 18. IR-induced intestinal barrier integrity loss resulted in free exposure of the gut basal membrane (collagen IV staining) to intraluminal contents, which was accompanied by increased arteriovenous concentration differences of endotoxin. Western blot for complement activation product C3c and immunohistochemistry for activated C3 revealed complement activation after IR. In addition, intestinal IR resulted in enhanced tissue mRNA expression of IL-6, IL-8, and TNF-alpha, which was accompanied by IL-6 and IL-8 release into the circulation. Expression of intercellular adhesion molecule-1 was markedly increased during reperfusion, facilitating influx of neutrophils into IR-damaged villus tips. In conclusion, this study for the first time shows the sequelae of human intestinal IR-induced inflammation, which is characterized by complement activation, production and release of cytokines into the circulation, endothelial activation, and neutrophil influx into IR-damaged tissue.

BACKGROUND: In vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells, and TE 671 muscle cells were included too, the latter two as negative controls. RESULTS: Two-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed. CONCLUSIONS: Numerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of in vitro obtained findings when translating them to the in vivo situation. AU - Lenaerts K AU - Bouwman FG AU - Lamers WH AU - Renes J AU - Mariman EC LA - ENG PT - JOURNAL ARTICLE DEP - 20070403 TA - BMC Genomics JID - 100965258

Food deprivation results in metabolic, structural, and functional changes in the small intestine that influences gut mucosal integrity, epithelial cell proliferation, mucin synthesis, and other processes. The underlying mechanisms are still unclear, which lead to the study of molecular effects of short-term and long-term starvation in the intestine of mice. A comparative proteomics approach, combining two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, was used to identify intestinal proteins whose expression is changed under different starvation conditions (0, 12, 24, and 72 h). In total, the expression levels of 80 protein spots changed significantly between the different groups. The results demonstrate that after 12 h of starvation, mainly proteins involved in glycolysis and energy metabolism show decreased expression levels. Starvation for 24 h results in a down-regulation of proteins involved in protein synthesis and amino acid metabolism. Simultaneously, proteins with a protective role, e.g., reg I and II, glutathione peroxidase 3, and carbonic anhydrase 3, are clearly up-regulated. The last starvation phase (72 h) is characterized by increased ezrin expression, which may enhance villus morphogenesis critical for survival. Together, these results provide novel insights in the intestinal starvation response and may contribute to improved nutritional support during conditions characterized by malnutrition.

Introduction We hypothesised that the development of intestinal failure associated liver disease (IFALD) caused by an enterocutaneous fistula in which the enterohepatic circulation is interrupted without obstructive cholestasis leads to reduced farnosoid X receptor (FXR) signalling, ultimately leading to liver damage. Previous studies have primarily been performed in models of bile duct obstruction. Aim Therefore we aimed to develop a biliary diversion model in the rat with long-term continuous biliary drainage and determined its effect on hepatic function and lipid metabolism. Methods In rats (n=7–9), the bile duct was cannulated and externalised to accomplish biliary drainage. Sham controls underwent laparotomy without cannulation. Rats were sacrificed after 3 and 7 days, and liver samples were collected for histological assessment (necrosis, inflammation, bile duct proliferation). Sirius red staining and Oil Red O staining were used for analysis of fibrosis and steatosis, respectively. Quantitative PCR was performed for genes involved in the FXR pathway (FXR, SHP, BSEP, CYP7A1 and CYP8B1), lipid and cholesterol metabolism (FAS, SREBF, ACACA, PPARg, SCD1, LDLr and HMG-CoA reductase). AST, ALT, AP, GGT and bilirubin (total and direct) were assessed. Results Continuous biliary diversion resulted in increased hepatic inflammation and necrosis at day 3 and day 7 (p Conclusion Biliary diversion in rats induced hepatic inflammation, necrosis, fibrosis and bile duct proliferation. Biliary diversion had evident hepatic effects with respect to FXR signalling and lipid metabolism. This model is suitable to assess long-term effects of continuous bile drainage to test therapeutic interventions aimed to reduce IFALD.

Introduction Enterocutaneous fistula are often associated with development of intestinal failure associated liver disease (IFALD), ultimately leading to liver damage. We hypothesise that this is caused by reduced farnosoid X receptor (FXR) stimulation, due to interruption of the enterohepatic circulation and consequent impact on bile acid synthesis. Aim We aimed at investigating the effect of specific stimulation of the farnosoid X receptor (FXR) with INT-747, a synthetic agonist, in a rat biliary diversion model. Methods Four groups of rats (n=6–8) were studied; two groups underwent bile duct cannulation and externalisation to achieve continuous biliary drainage, daily receiving either vehicle or INT-747 (Intercept Pharm LtD); two groups underwent laparotomy without cannulation also receiving either vehicle or INT-747. Loss of bile was recorded daily. After 7 days, plasma, serum and liver and intestinal tissue were collected. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), γ glutamyl transpeptidase (GGT) and total bilirubin were assessed, together with histological scoring of necrosis, inflammation, bile duct proliferation, fibrosis and steatosis. Expression of genes involved in the FXR pathway, and lipid and cholesterol metabolism were quantified by qPCR. Results Loss of bile was significantly reduced in the INT-747 group compared to the vehicle group. Serum levels of ASAT, ALAT, AP, GGT, bilirubin (total and direct) were all significantly increased in the bile drainage group when compared to controls (p Conclusion The present data demonstrate that stimulation of FXR with INT-747 can attenuate hepatocellular damage in an experimental model of IFALD. The results suggest a role of FXR in the development of hepatocellular damage, hepatic fibrosis and necrosis during biliary diversion. FXR stimulation has the potential to be a novel therapy for patients with IFALD.

BACKGROUND & AIMS: In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. METHODS: Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-alpha and interleukin-6 levels were assessed. RESULTS: In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. CONCLUSIONS: ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.