In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1alpha, -6, -7, -8, -18, TNF-alpha and GM-CSF, but not TGF-beta or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen-host cell interactions.
High dietary zinc concentrations are used to prevent or treat diarrhea in piglets and humans, but long-term adaptation to high zinc supply has yet not been assessed. Intestinal zinc uptake is facilitated through members of zinc transporter families SLC30 (ZnT) and SLC39 (ZIP). Whereas in rodents, regulation of zinc homeostasis at low or adequate zinc supply has been described, such mechanisms are unclear in piglets. A total of 54 piglets were fed diets containing 57 [low dietary zinc (LZn)], 164 [normal dietary zinc (NZn)], or 2425 [high dietary zinc (HZn)] mg/kg dry matter zinc. After 4 wk, 10 piglets/group were killed and jejunal tissues taken for analysis of zinc transporters SLC30A1 (ZnT1), SLC30A2 (ZnT2), SLC30A5 (ZnT5), SLC39A4 (ZIP4), divalent metal transporter 1 (DMT1), and metallothionein-1 (MT). Weight gain was higher (P < 0.05) in pigs fed HZn than in the LZn and NZn groups during the first 2 wk. Food intake did not differ between groups. The digesta and jejunal tissue zinc concentrations were higher (P < 0.05) in the HZn pigs than in NZn and LZn pigs. Expression of ZnT1 was higher (P < 0.05) and ZIP4 lower (P < 0.05) in HZn pigs than in the 2 other groups, whereas expression of ZnT5 and DMT1 did not differ between treatments. Expression of ZnT2 was lower (P < 0.05) in the LZn group than in the HZn and NZn groups. The mRNA expression and protein abundance of MT was higher (P < 0.05) in the HZn group than in the NZn and LZn groups. Studies with intestinal porcine cell line intestinal epithelial cell-J2 confirmed the dose-dependent downregulation of ZIP4 and upregulation of ZnT1 and MT (P < 0.05) with increasing zinc concentration within 24 h. In conclusion, high dietary zinc concentrations increase intracellular zinc, promote increased zinc export from intestinal tissues into extracellular compartments, and decrease zinc uptake from the gut lumen. The adaptive process appears to be established within 24 h; however, it does not prevent tissue zinc accumulation.
The effects of luminal hyperosmolarity on Na and Cl transport were studied in rumen epithelium of sheep. An increase of luminal osmotic pressure with mannitol (350 and 450 mosm/l) caused a significant increase of tissue conductance, G (T), which is linearly correlated with flux rates of (51)Cr-EDTA and indicates an increase of passive permeability. Studies with microelectrodes revealed, that an increase of the osmotic pressure caused a significant increase of the conductance of the shunt pathway from 1.23 +/- 0.10 (control) to 1.92 +/- 0.14 mS cm(-2) (450 mosm/l) without a change of fractional resistance. Hyperosmolarity significantly increased J (sm) and reduced J (net) Na. The effect of hyperosmolarity on J (ms) Na is explained by two independent and opposed effects: increase of passive permeability and inhibition of the Na(+)/H(+) exchanger. Hypertonic buffer solution induced a decrease of the intracellular pH (pH(i)) of isolated ruminal cells, which is consistent with an inhibition of Na(+)/H(+) exchange, probably isoform NHE-3, because NHE-3-mRNA was detectable in rumen epithelium. These data are in contrast to previous reports and reveal a disturbed Na transport and an impaired barrier function of the rumen epithelium, which predisposes translocation of rumen endotoxins and penetration of bacteria.
The intention of this study was to determine the effects of mucosal osmotic pressure on transport and barrier functions of the rumen epithelium of sheep, which were fed various diets: hay ad libitum, or 600, 1200 or 1800 g day -1 of a supplemented diet plus hay ad libitum. The experiments were conducted by using the conventional Ussing chamber technique. Mucosal osmolarity was adjusted to 300 (control), 375 or 450 mosmol l -1 . Feeding of a supplemented diet led to a significant increase of mucosal to serosal Na + transport and net Na + transport, probably because of an increase of apical Na + -H + exchange activity. An increase in mucosal osmotic pressure: (a) reduced net Na + transport in all feeding groups, the remaining net Na + transport being higher in tissues of sheep fed a supplemented diet; (b) increased transepithelial tissue conductance, this rise being smallest with a high intake of the supplemented diet; and (c) enhanced the serosal to mucosal Na + transport in tissues of hay-fed sheep and sheep fed with 600 g day -1 of the supplemented diet, while higher intakes of the supplemented diet (1200 and 1800 g) did not produce any effect. All these changes indicate a diet-dependent adaptation to luminal hypertonicity.
Probiotics have shown positive effects on gastrointestinal diseases; they have barrier-modulating effects and change the inflammatory response towards pathogens in studies in vitro. The aim of this investigation has been to examine the response of intestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E. faecium), a probiotic positively affecting diarrhea incidence in piglets, and two pathogenic Escherichia coli (E. coli) strains, with specific focus on the probiotic modulation of the response to the pathogenic challenge. Porcine (IPEC-J2) and human (Caco-2) intestinal cells were incubated without bacteria (control), with E. faecium, with enteropathogenic (EPEC) or enterotoxigenic E. coli (ETEC) each alone or in combination with E. faecium. The ETEC strain decreased transepithelial resistance (TER) and increased IL-8 mRNA and protein expression in both cell lines compared with control cells, an effect that could be prevented by pre- and coincubation with E. faecium. Similar effects were observed for the increased expression of heat shock protein 70 in Caco-2 cells. When the cells were challenged by the EPEC strain, no such pattern of changes could be observed. The reduced decrease in TER and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic.
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