Jaap E. van Dijk

The heat shock response and cytoprotection of the intestinal epithelium

Abstract Following heat stress, the mammalian intestinal epithelial cells respond by producing heat shock proteins that confer protection under stressful conditions, which would otherwise lead to cell damage or death. Some of the noxious processes against which the heat shock response protects cells include heat stress, infection, and inflammation. The mechanisms of heat shock response–induced cytoprotection involve inhibition of proinflammatory cytokine production and induction of cellular proliferation for restitution of the damaged epithelium. This can mean selective interference of pathways, such as nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), that mediate cytokine production and growth responses. Insight into elucidating the exact protective mechanisms could have therapeutic significance in treating intestinal inflammations and in aiding maintenance of intestinal integrity. Herein we review findings on heat shock response–induced intestinal epithelial protection involving regulation of NF-κB and MAPK cytokine production.

Obstructive Jaundice, Bacterial Translocation and Interdigestive Small-Bowel Motility in Rats

Background/Aims: Translocation of gut bacteria occurs in obstructive jaundice, the underlying mechanisms are unclear. We designed this experimental study to investigate the association between interdigestive motility and the pathogenesis of bacterial translocation during biliary obstruction. Methods: Rats were fitted with jejunal myoelectrodes for the measurement of the interdigestive migrating motor complex (MMC) and with two cannulas in the proximal common bile duct (CBD) for exteriorization of biliary flow. This allowed measurement of MMCs under control conditions with an intact enterohepatic circulation and during 3 days of CBD obstruction without surgical intervention. Mesenteric lymph nodes, liver, spleen and segments of the duodenum, the jejunum and the caecum were removed for microbial culturing. Results: The MMC cycle length increased from 17.3 min before CBD obstruction to 31.9, 34.1, and 25.3 min on days 1, 2 and 3, respectively, after CBD obstruction (p < 0.05 for all days). Bacterial levels in the jejunum were significantly higher in CBD-obstructed rats than in control rats. The translocation incidence was significantly higher in rats with CBD obstruction (6/8) than in control rats (1/8). The bacterial levels in the jejunum correlated significantly with the MMC cycle length (r = 0.60, p <0.05). Conclusion: Experimental biliary obstruction is associated with disturbance of MMCs, small-bowel bacterial overgrowth and increased bacterial translocation.

Reduced life expectancy in rats after neonatal dexamethasone treatment.

The glucocorticoid dexamethasone (Dex) is widely used in preterm infants for the prevention of chronic lung disease. However, major concern has arisen about the long-term sequelae of this therapy. Here we report that neonatal treatment with dexamethasone significantly shortens the lifespan by 25% of male rats (28.6 ± 1.1 to 21.3 ± 0.8 mo) and by 18% of female rats (26.9 ± 1.8 to 22.0 ± 0.7 mo). Histopathological examination indicated end-stage cardiac and renal failure as the cause of premature death. Furthermore, Dex- treated rats showed symptoms of hypertension at young adult age, which worsened with increasing age. Thus, a brief period of glucocorticoid treatment during early life results in untimely death presumably due to cardiovascular and renal disease later in life. These serious, adverse long-term consequences call for prudence with glucocorticoid treatment of human preterm infants and careful follow-up of young adults with a history of neonatal glucocorticoid treatment.

Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

Abstract The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40–43°C) or to graded numbers (101–108) of bacteria and in villus-like cells exposed to S enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.

Quantitative determination of the lectin binding capacity of small intestinal brush-border membrane. An enzyme linked lectin sorbent assay (ELLSA)

A test to determine quantitatively the lectin binding sites in brush-border membranes has been developed. Highly purified bovine small intestinal brush-border membranes were prepared, and subsequently coated directly to the bottom of a microtiter plate. Soybean agglutinin conjugated with peroxidase was coupled to its binding sites in the brush-border membranes and the peroxidase activity was determined in a spectrophotometer. The number of soybean agglutinin binding sites in the brush-border membranes has been established by means of iterized computer fit analysis of the data, indicating values for maximal binding of 7.10(-7) M soybean agglutinin per mg of brush-border membrane protein and a dissociation constant of 1.5.10(-5) M.

Actin cytoskeletal lesions in differentiated human colon carcinoma Caco-2 cells after exposure to soybean agglutinin

We have investigated the effects of soybean agglutinin on the cytoskeletal element actin in differentiated Caco‐2 cells. The actin cytoskeleton of the cells was visualized by fluorescence microscopy using 7‐nitrobenz‐2‐oxa‐1, 3‐diazole phallacidin as a specific marker for F‐actin. Compared with control Caco‐2 cells no changes in the fluorescence pattern were observed after incubation with soybean agglutinin. However, using the deoxyribonuclease‐I inhibition assay a dose‐related response was noted in the increase of intracellular G‐actin after a 2‐hour incubation period with soybean agglutinin. Already after exposure for 15 min to soybean agglutinin a decrease in intracellular F‐actin was demonstrable. This apparent depolymerization could be prevented by incubating the Caco‐2 cells with soybean agglutinin and the appropriate monosaccharide simultaneously. The increase in the amount of G‐actin appeared to be correlated with a shortening of microvilli on the Caco‐2 cells.