Intestinal Cell Culture Research Articles

Aberrant expression of the polarity complex atypical PKC and non-muscle myosin IIA in active and inactive inflammatory bowel disease

Epithelial barrier function is contingent on appropriate polarization of key protein components. Work in intestinal epithelial cell cultures and animal models of bowel inflammation suggested that atypical PKC (aPKC), the kinase component of the Par3–Par6 polarity complex, is downregulated by pro-inflammatory signaling. Data from other laboratories showed the participation of myosin light chain kinase in intestinal inflammation, but there is paucity of evidence for assembly of its major target, non-muscle myosin II, in inflammatory bowel disease (IBD). In addition, we showed before that non-muscle myosin IIA (nmMyoIIA) is upregulated in intestinal inflammation in mice and TNFα-treated Caco-2 cells. Thus far, it is unknown if a similar phenomena occur in patients with IBD. Moreover, it is unclear whether aPKC downregulation is directly correlated with local mucosal inflammation or occurs in uninvolved areas. Frozen sections from colonoscopy material were stained for immunofluorescence with extensively validated specific antibodies against phosphorylated aPKC turn motif (active form) and nmMyoIIA. Inflammation was scored for the local area from where the material was obtained. We found a significant negative correlation between the expression of active aPKC and local inflammation, and a significant increase in the apical expression of nmMyoIIA in surface colon epithelia in inflamed areas, but not in non-inflamed mucosa even in the same patients. Changes in aPKC and nmMyoIIA expression are likely to participate in the pathogenesis of epithelial barrier function in response to local pro-inflammatory signals. These results provide a rationale for pursuing mechanistic studies on the regulation of these proteins.

Anti-inflammatory and anti-angiogenic effect of long chain n-3 polyunsaturated fatty acids in intestinal microvascular endothelium

The role of endothelial cells in inflammatory bowel disease has been recently emphasized. Endothelial activation and expression of adhesion molecules are critical for leukocytes recruitment into the inflammatory wall. Compelling evidence demonstrated anti-inflammatory effects of long chain n-3 PUFA in inflammatory models. We previously showed that long chain n-3 PUFA (EPA and DHA) inhibited inflammatory response in epithelial and dendritic cells. As long chain n-3 PUFA treatment led to a decreased expression of adhesion molecules in endothelial cells from other organs, we have now investigated their effect on intestinal endothelial cells invitro and in colitic rats. In vitro study: Primary culture of human intestinal microvascular endothelial cells (HIMEC) were pre-treated with DHA and then incubated with IL-1β. In vivo study: Colitis was induced in 2 groups at day0 by intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS). Rats received by gavage either fish oil, rich in EPA and DHA (TNBS+n-3) or an isocaloric isolipidic oil formula for 14 days. DHA led to a decreased VCAM-1, TLR4, cyclooxygenase-2 and VEGFR2 expression and a decreased production of IL-6, IL-8 and GM-CSF and a reduced production of PGE(2) and LTB(4) (p<0.001) in IL-1β-induced HIMEC. Similarly, dietary intervention with fish oil rich in EPA and DHA significantly decreased colon production of PGE(2) and LTB(4,) endothelial VCAM-1 and VEGFR2 in rats with colitis. Data obtained from invitro and invivo studies reveal a potential anti-angiogenic role of long chain n-3 PUFA in intestinal endothelial cells. This protective effect of long chain n-3 PUFA may partly explain the observed benefit of dietary intake of long chain n-3 PUFA in IBD development.

Fibroblast Growth Factor Receptor-3 (FGFR-3) Regulates Expression of Paneth Cell Lineage-specific Genes in Intestinal Epithelial Cells through both TCF4/β-Catenin-dependent and -independent Signaling Pathways

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

Magnetic Nanoparticles: A Versatile Method for the Reductive, One‐Pot Synthesis of Bare, Hydrophilic and Hydrophobic Magnetite Nanoparticles (Adv. Funct. Mater. 8/2011)

AbstractA biocompatible, reliable, and particularly versatile synthesis of magnetic iron oxide nanoparticles (IONPs) is presented that uses iron(III) acetylacetonate Fe(acac)3 as an iron precursor and sodium borohydride as a reducing agent. Both the reaction temperature and the concentration of the reducing agent have considerable effects on the IONP size. These dependencies can be used to prepare IONPs ranging in size from 5 to 8 nm, as determined by transmission electron microscopy (TEM). Synthesis at room temperature or with higher sodium borohydride concentrations always resulted in smaller particle sizes. Powder X‐ray diffraction patterns show the presence of an iron oxide phase with a cubic unit cell and allow for the determination of the lattice parameters and average crystallite sizes for all synthesized IONPs. Transmission Mössbauer spectroscopy shows that the as‐synthesized IONPs are pure magnetite (Fe3O4) and is further used to elucidate the reaction pathway by analyzing iron intermediates formed prior to nanoparticle formation and precipitation. TEM and high‐resolution TEM reveal quasi‐spherical shapes and lattice fringes for most IONPs. With only minor modifications of the synthesis procedure, this versatile, one‐pot synthesis is proven to be suitable for the production of bare (uncoated) IONPs, IONPs with hydrophilic poly(ethylene glycol), L‐arginine, and L‐glutamic acid coatings, as well as IONPs with hydrophobic coatings such as oleic acid. All coated IONPs were characterized by FT‐IR spectroscopy. In addition, the bare IONPs could easily be modified post‐synthesis with a suitable capping agent using ultrasonication. To verify the biocompatibility of the IONPs, in vitro cytotoxicity studies were carried out on bare IONPs with intestinal (Caco2) and liver epithelial (HepG2) cell cultures using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Phase contrast microscopy after hematoxylin‐eosin staining showed the intact morphology of the Caco2 and HepG2 cells treated with IONPs.

A Versatile Method for the Reductive, One‐Pot Synthesis of Bare, Hydrophilic and Hydrophobic Magnetite Nanoparticles

AbstractA biocompatible, reliable, and particularly versatile synthesis of magnetic iron oxide nanoparticles (IONPs) is presented that uses iron(III) acetylacetonate Fe(acac)3 as an iron precursor and sodium borohydride as a reducing agent. Both the reaction temperature and the concentration of the reducing agent have considerable effects on the IONP size. These dependencies can be used to prepare IONPs ranging in size from 5 to 8 nm, as determined by transmission electron microscopy (TEM). Synthesis at room temperature or with higher sodium borohydride concentrations always resulted in smaller particle sizes. Powder X‐ray diffraction patterns show the presence of an iron oxide phase with a cubic unit cell and allow for the determination of the lattice parameters and average crystallite sizes for all synthesized IONPs. Transmission Mössbauer spectroscopy shows that the as‐synthesized IONPs are pure magnetite (Fe3O4) and is further used to elucidate the reaction pathway by analyzing iron intermediates formed prior to nanoparticle formation and precipitation. TEM and high‐resolution TEM reveal quasi‐spherical shapes and lattice fringes for most IONPs. With only minor modifications of the synthesis procedure, this versatile, one‐pot synthesis is proven to be suitable for the production of bare (uncoated) IONPs, IONPs with hydrophilic poly(ethylene glycol), L‐arginine, and L‐glutamic acid coatings, as well as IONPs with hydrophobic coatings such as oleic acid. All coated IONPs were characterized by FT‐IR spectroscopy. In addition, the bare IONPs could easily be modified post‐synthesis with a suitable capping agent using ultrasonication. To verify the biocompatibility of the IONPs, in vitro cytotoxicity studies were carried out on bare IONPs with intestinal (Caco2) and liver epithelial (HepG2) cell cultures using an 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Phase contrast microscopy after hematoxylin‐eosin staining showed the intact morphology of the Caco2 and HepG2 cells treated with IONPs.

Role of PKCb in Signal Transduction for the 1,25D3-MARRS Receptor (ERp57/PDIA3) in Steroid Hormone-Stimulated Calcium Extrusion

Protein kinase C (PKC) is involved in the rapid 1,25(OH) 2 D 3 -mediated extrusion of calcium in chick intestinal epithelial cells. A previous report demonstrated that PKC a and PKC b redistribution corresponded to the dose- response curve for calcium extrusion. We investigated the role of both PKC a and PKC b in hormone-stimulated calcium extrusion by using siRNA in primary cultures of intestinal cells. The results indicated that there was no change in calcium extrusion in cells transfected with siRNA to PKC a , whereas the siRNA to PKC b significantly decreased calcium extrusion in 1,25(OH) 2 D 3 -treated cells when compared with the corresponding hormone-treated cells in transfected and nontransfected cells ( P < 0.01). Similar results were also found using chemical inhibitors of PKC a (safingol) and PKC b ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione]) in intestinal cell suspension. The results demonstrated that PKC b inhibited the extrusion of calcium (resulting in increased calcium uptake) from intestinal cells treated with 1,25(OH) 2 D 3 when compared with vehicle controls ( P < 0.001), the PKC a blocker did not show any difference in calcium extrusion among the treatment groups. Using confocal microscopy, we reduced the hormone exposure to 30 sec in order to view redistribution of PKC a and PKC b . Rapid redistribution of PKC a was found to significantly increase in fluorescence in the apical membrane region after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH) 2 D 3 ( P < 0.01 and 0.001, respectively). By comparison, PKC b immunofluorescence was found to increase significantly in the basal lateral region of the cells, relative to controls after the exposure of cells to 300- ( P < 0.01) or 650 pM ( P < 0.05) seco-steroid. The results suggest that PKC a is the PKC isotype involved in 1,25(OH) 2 D 3 -mediated calcium efflux in intestinal epithelial cells.

Effects of cadmium pollution in soil on cadmium accumulation of cabbage and its biological effects on human intestinal cells line

The pot experiment grown with cabbage was conducted. The initial soil and six incubated soils with different total Cd (0.25, 0.78, 1.3, 3.18, 5.34 and 7.11mg Cd/kg soil) were used in the pot experiment. The present study was to examine cadmium (Cd) accumulation in edible parts of cabbage and its correlation with Cd concentrations in cabbage garden soil, and evaluate the bioavailability and toxicity of Cd accumulating in cabbage by a human intestinal cell culture model (Caco-2). The results showed that Cd accumulation of cabbage was linearly correlated with Cd concentration in grown soils. Cd uptake of Caco-2 cells increased gradually with increasing level of Cd accumulation in cabbage. MTT assay investigation suggested that viability of the Caco-2 cells reduced with Cd accumulation in cabbage. In comparison with the control group, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the experiment groups decreased as treated with Cd contaminated cabbages, and there was significant difference in partial groups (p < 0.05). At levels of 47.69mg Cd kg-1 accumulated in cabbage, the value of T-AOC of cells decreased significantly (p < 0.05), while the content of MDA in cells was significantly higher than that in other groups. The results suggested that Cd concentrations in the edible parts of test cabbage crops were linearly responded to the Cd levels in the growth soil, and toxicological effect of Cd to Caco-2 cells aggravated gradually with Cd accumulation increased in cabbage.

Endoplasmic Reticulum Stress-activated C/EBP Homologous Protein Enhances Nuclear Factor-κB Signals via Repression of Peroxisome Proliferator-activated Receptor γ

Endoplasmic reticulum (ER) stress is a causative factor of inflammatory bowel diseases. ER stress mediators, including CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP), are elevated in intestinal epithelia from patients with inflammatory bowel diseases. The present study arose from the question of how chemical ER stress and CHOP protein were associated with nuclear factor-κB (NF-κB)-mediated epithelial inflammatory response. In a human intestinal epithelial cell culture model, chemical ER stresses induced proinflammatory cytokine interleukin-8 (IL-8) expression and the nuclear translocation of CHOP protein. CHOP was positively involved in ER-activated IL-8 production and was negatively associated with expression of peroxisome proliferator-activated receptor γ (PPARγ). ER stress-induced IL-8 production was enhanced by NF-κB activation that was negatively regulated by PPARγ. Mechanistically, ER stress-induced CHOP suppressed PPARγ transcription by sequestering C/EBPβ and limiting availability of C/EBPβ binding to the PPARγ promoter. Due to the CHOP-mediated regulation of PPARγ action, ER stress can enhance proinflammatory NF-κB activation and maintain an increased level of IL-8 production in human intestinal epithelial cells. In contrast, PPARγ was a counteracting regulator of gut inflammatory response through attenuation of NF-κB activation. The collective results support the view that balances between CHOP and PPARγ are crucial for epithelial homeostasis, and disruption of these balances in mucosal ER stress can etiologically affect the progress of human inflammatory bowel diseases.

EGF signaling activates proliferation and blocks apoptosis of mouse and human intestinal stem/progenitor cells in long-term monolayer cell culture

The homeostatic renewal of the intestinal epithelium depends on regulation of proliferation and differentiation of stem/progenitor cells residing in a specific site, called the ‘stem cell niche.' Thus, the reconstitution of the microenvironment of the stem cell niche may allow us to maintain intestinal stem/progenitor cells in culture for a longer period. Although epidermal growth factor (EGF) is conventionally used as a supplement of intestinal epithelial cell culture, little has been known regarding a role of EGF signaling in a stem/progenitor cell population. In this study, we attempted to clarify the role of EGF signaling in intestinal stem/progenitor cells, and to establish a culture system in which these cells could be maintained with normal differentiation potential. We first examined the expression pattern of EGF and its receptor, EGFR, and inhibited EGF signaling in mouse intestines. Next, we cultured intestinal cells isolated from mouse and human intestines in the presence of EGF and analyzed the function of EGF signaling in cultured cells. In both embryonic and adult mouse intestines, EGFR and EGF were expressed in immature epithelial cells and adjacent fibroblasts, respectively, and EGF signaling was essential to activate proliferation and inhibit apoptosis of intestinal stem/progenitor cells. Activation of EGF signaling also stimulated proliferation and suppressed apoptosis, both of which are necessary to maintain mouse and human intestinal epithelial cells in culture. Moreover, in these cultured epithelial cells, putative intestinal stem/progenitor cells persisted longer, and gave rise to different types of differentiated intestinal epithelial cells. We conclude that EGF signaling is indispensable for activation of proliferation and inhibition of unexpected cell death, not only in the intestinal stem cell niche, but also in culture of primitive intestinal epithelial cells.

Potato protease inhibitors inhibit food intake and increase circulating cholecystokinin levels by a trypsin-dependent mechanism

ObjectiveTo investigate the mechanisms underlying the satiety-promoting effects of a novel protease inhibitors concentrate derived from potato (PPIC).MethodsAcute and prolonged effects of oral PPIC administration (100 mg/kg per day) on food intake, body weight, and gastric emptying were evaluated in healthy rats. Parameters of body weight, food intake, plasma glucose, insulin, and cholecystokinin (CCK) were measured. Duodenal proteolytic activity and CCK expression were determined in tissue extracts. Intestinal STC-1 cell culture model was used to investigate the direct effect of PPIC on CCK transcript level and secretion.ResultsAcute oral administration of PPIC reduced immediate food intake during the first two hours following the treatment, delayed gastric emptying, and decreased proteolytic activity in the duodenum. Repeated oral ingestion of PPIC reduced weight gain in male rats and significantly elevated the plasma CCK levels. Although duodenal mucosal CCK mRNA levels increased in response to PPIC administration, the concentrate failed to elevate CCK expression or release in STC-1 cells. The 14-day ascending dose range study (33 to 266 mg/kg PPIC per day) showed no adverse side effects associated with PPIC administration.ConclusionThese findings provided evidence that PPIC is effective in reducing food intake and body weight gain in healthy rats when administered orally by increasing circulating CCK levels through a trypsin-dependent mechanism.

A New Intestinal Cell Culture Model To Discriminate the Relative Contribution of P-gp and BCRP on Transport of Substrates Such as Imatinib

P-glycoprotein (P-gp/MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) play an important role in transport of a wide variety of endogenous compounds, drugs and toxins. Transport of some drugs, for example the tyrosine kinase inhibitor imatinib, is influenced by both P-gp and BCRP. Establishing an intestinal Caco-2 cell culture model with specific knock-downs of P-gp and BCRP and double knock-down of both proteins, we aimed to elucidate the impact of each transporter on transport of imatinib. Stable single and double knock-downs of P-gp and BCRP were obtained by RNA interference (RNAi). Transporter expression was measured on RNA and protein level using real-time RT-PCR and Western blot, respectively. Functional activity was quantified by transport of specific substrates across Caco-2 cells. MDR1 and BCRP mRNA expression was reduced to 75% and 90% compared to wild-type control in single MDR1- and BCRP-knock-down clones, respectively. In double knock-down clones, MDR1 expression decreased to 95% and BCRP expression to 80%. Functional activity of P-gp and BCRP was diminished as transport of the P-gp-specific substrate (3)H-digoxin and the BCRP-specific substrate (14)C-PhIP was augmented in the opposite direction, when the respective transporter was knocked down. Similar effects were observed by chemical inhibition of the respective transporter. Bidirectional transport studies with (14)C-imatinib revealed an abrogation of asymmetric transport when P-gp was knocked down, either in single or double knock-down clones compared to wild-type cells. This was not observed in single BCRP-knock-down clones. In conclusion, this newly established cell system with single and concomitant knock-down of P-gp and BCRP can be used to quantify the specific partial impact of the transporters on transport of substrates that are transported by both proteins. For imatinib transport, the contribution of P-gp seems to be more important compared to BCRP in this Caco-2 cell system.

Synergic effects of crypt-like topography and ECM proteins on intestinal cell behavior in collagen based membranes

The basement membrane of small intestinal epithelium possesses complex topography at multiple scales ranging from the mesoscale to nanoscale. Specifically, intestinal crypt-villus units are comprised of hundred-micron-scale well-like invaginations and finger-like projections; intestinal cell phenotype is related to location on this crypt-villus unit. A biomimetic intestinal cell culture system composed of type I collagen based permeable cell culture membranes incorporating both micron-scale intestinal crypt-like topography and nanometer scale topography was fabricated. Membranes were pre-incubated with either laminin (Ln) or fibronectin (Fn), inoculated with intestinal epithelial Caco-2 cells and cultured for 1–21 days to study the relative significance of influence of crypt-like topography and biomimetic substrate chemistry on cell phenotype. Crypt-like topography inhibited Caco-2 differentiation during early culture, as evidenced by slower cell spreading and lower brush border enzyme activity. For example, alanine aminopeptidase activity was lower on Ln-coated patterned collagen (∼3.4 ± 0.24 mU/mg) compared to flat collagen (10.84 ± 0.55 mU/mg) at day 7. Caco-2 cultured on Fn-coated collagen started to spread earlier (1 day vs 3 days) and formed longer protrusions than on Ln-coated collagen. Pre-coating of Ln enhanced cell differentiation, as the maximum activity of a cell differentiation marker (alkaline phosphatase) was 2–3 times higher than on Fn-coated collagen, and maintained differentiated phenotype in long term (up to 21 days) culture. In general, compared to substrate topography, coating with ECM protein had more prominent and longer effect on cell behavior. Crypt-like topography affected Caco-2 spreading and differentiation during early culture, however the effect diminished as culture progressed. This information will benefit intestinal tissue engineering scaffold design, and modification of in vitro intestinal cell models.

Metabolites of amygdalin under simulated human digestive fluids

In the present study, degradation of amygdalin in the human digestive fluids and absorption of its metabolites by the human small intestine were evaluated by simulating a gastrointestinal digestion model combined with a human intestinal cell culture. Orally administered amygdalin was degraded into prunasin by digestive enzymes after passing through the salivary and gastrointestinal phases. Prunasin, the major metabolite of amygdalin in the digestive fluids, was incubated in a caco-2 cell culture system. Prunasin was degraded into the mandelonitrile by β-glucosidase and then hydroxylated across the small intestinal wall, producing hydroxymandelonitrile (149 Da). Results from this study suggest that risk assessment of amygdalin from food consumption can be done in a more accurate way by determining a pathway of amygdalin metabolism in the simulating human upper gastrointestinal tract.

Drug Transport Across Pulmonary Epithelial Cell Monolayers: Effects of Particle Size, Apical Liquid Volume, and Deposition Technique

Pulmonary cell cultures are increasingly used to predict in vivo drug absorption after inhalation, similar to intestinal cell culture models that have already been well established to predict oral drug absorption. In contrast to the intestinal barrier, however, the so-called air-blood barrier of the lung is covered only with a thin film of liquid, on which the aerosol particles are deposited. The aim of this study was to investigate the relevance of this apical liquid film on the drug absorption rate when deposited as a dry powder formulation on pulmonary epithelial cells in vitro. Budesonide and salbutamol sulfate were chosen as model drugs, and for each drug three generic aerosol powder formulations were used. Filter-grown monolayers of the human bronchial epithelial cell line Calu-3 were used as a model, using various volumes of apical liquid. Although proven to be bioequivalent in vivo for each of the two drugs, the generic dry powder fomulations showed strikingly different epithelial transport rates in vitro, depending on the amount of apical liquid and the deposition technique, and suggesting that the dissolution of the aerosol particles in the apical liquid volume was rate limiting for the overall absorption rate. However, we found that the absorption rates of the formulations were similar after aerosolization and deposition in a multistage liquid impinger, which simulates more realistically the detachment of the drug crystals from the carrier lactose and their aerodynamic particle size-dependent deposition in the respiratory tract following inhalation from a dry powder inhaler. These data demonstrate the need for improved in vitro test systems to allow deposition of aerosol particles on the air-liquid interface cultivated cell monolayers by simultaneously taking into account aerodynamic properties.

Effects ofCurcumaExtracts and Curcuminoids on Expression of P-glycoprotein and Cytochrome P450 3A4 in the Intestinal Cell Culture Model LS180

Curcuma longa L. is a widely used spice. Its main ingredients, the curcuminoids, are used in the treatment of inflammatory diseases and cancer. Bioavailability of curcuminoids is low, and huge amounts remain in the intestine. We therefore aimed to investigate their interaction potential with the ABC-transporter P-glycoprotein (P-gp, product of the MDR1/ABCB1 gene) and cytochrome P450 3A4 (CYP3A4) in an intestinal cell line (LS180). Intestinal P-gp and CYP3A4 play a major role in drug absorption, and consequently changes in their expression level could lead to interactions. The intestinal LS180 cell line was incubated with different Curcuma extracts, the single curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin), as well as a curcuminoid mixture. Changes in mRNA expression of MDR1 and the cytochrome CYP3A4 were measured by real-time RT-PCR. MDR1 mRNA expression was significantly but not relevantly downregulated by the curcuminoids, whereas the extracts had no significant effect on it. CYP3A4 mRNA expression did not alter significantly after treatment. Curcuma extracts, the single curcuminoids, and a curcuminoid mixture had no relevant effect on MDR1 and CYP3A4 mRNA expression in our intestinal cell system. Further studies are required to evaluate their effects in vivo.

Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4(+) T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8(+) T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8(+) T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8(+) T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite.

Experimental impact of aspirin exposure on rat intestinal bacteria, epithelial cells and cell line

Aspirin, a commonly used therapeutic non-steroidal anti-inflammatory drug (NSAID) is known to cause gastric mucosal damage. Intestinal bacteria having a regulatory effect on intestinal homeostasis play significant role in NSAID-induced intestinal injury. Bacteria and specific cell lines are considered to be suitable for toxicity screening and testing of chemicals. Therefore, to evaluate and compare in vitro toxicity, cultures of rat intestinal epithelial cells (IEC), isolated bacteria and IEC-6 cell line were assessed for viability, morphometric analysis, membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producing processes and esterase activity test for intra- and extra-cellular degradation, following the post exposure to aspirin (0-50 µg mL(- 1)). Similar pattern of dose-dependent changes in these parameters were observed in three types of cells. Similar in situ effects on IEC validated the in vitro findings. These findings indicate that higher aspirin concentrations may alter cellular functions of IEC and gut bacteria. Furthermore, results suggest that gut bacteria and IEC-6 cell line can be used for the initial screening of gastrointestinal cellular toxicity caused by NSAIDs.

Thalidomide inhibits inflammatory and angiogenic activation of human intestinal microvascular endothelial cells (HIMEC)

The glutamic acid derivative thalidomide is a transcriptional inhibitor of TNF-alpha but is also known to affect human blood vessels, which may underlie its teratogenicity. Thalidomide has been used in the treatment of refractory Crohn's disease (CD), but the therapeutic mechanism is not defined. We examined the effect of thalidomide on primary cultures of human intestinal microvascular endothelial cells (HIMEC), the relevant endothelial cell population in inflammatory bowel disease (IBD), to determine its effect on endothelial activation, leukocyte interaction, and VEGF-induced angiogenesis. HIMEC cultures were pretreated with thalidomide before activation with either TNF-alpha/LPS or VEGF. A low-shear-stress flow adhesion assay with either U-937 or whole blood was used to assess HIMEC activation following TNF-alpha/LPS, and a Wright's stain identified adherent leukocytes. Expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1) was assessed using radioimmunoassay. Effects of thalidomide on NF-kappaB activation, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) expression in TNF-alpha/LPS-activated HIMEC were determined by RT-PCR and Western blotting. Thalidomide blocked adhesion of both U-937 and whole blood leukocytes by 50% in HIMEC, inhibiting binding of all classes of leukocytes. Thalidomide also blocked NF-kappaB and cell adhesion molecule expression in HIMEC. In marked contrast, thalidomide did not affect either iNOS or COX-2 expression, two key molecules that play a role in the downregulation of HIMEC activation. VEGF-induced HIMEC transmigration, growth, proliferation, tube formation, and Akt phosphorylation were significantly inhibited by thalidomide. In summary, thalidomide exerted a potent effect on HIMEC growth and activation, suggesting that it may also function via an endothelial mechanism in the treatment of CD.

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

Vascular cell adhesion molecule-1 expression in human intestinal microvascular endothelial cells is regulated by PI 3-kinase/Akt/MAPK/NF-κB: inhibitory role of curcumin

Endothelial activation and surface expression of cell adhesion molecules (CAMs) is critical for binding and recruitment of circulating leukocytes in tissues during the inflammatory response. Endothelial CAM expression plays a critical role in the intestinal microvasculature in inflammatory bowel disease (IBD), as blockade of leukocyte alpha4-integrin binding by gut endothelial CAM ligands has therapeutic benefit in IBD. Mechanisms underlying expression of vascular cell adhesion molecule (VCAM)-1, a ligand for alpha4-integrin in primary cultures of human intestinal microvascular endothelial cells (HIMEC) has not been defined. We investigated the effect of curcumin, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and mitogen-activated protein kinase (MAPK) inhibitors on VCAM-1 expression and function in HIMEC. CAM expression was assessed and HIMEC-leukocyte adhesion was visualized under static and flow conditions. Western blotting and in vitro kinase assays were used to assess Akt and MAPK activation. Nuclear factor-kappaB (NF-kappaB) activation and nuclear translocation of its p65 subunit were determined. Tumor necrosis factor (TNF)-alpha/lipopolysaccharide (LPS)-induced VCAM-1 expression in HIMEC was suppressed by Akt small-interfering RNA, curcumin, and inhibitors of NF-kappaB (SN-50), p38 MAPK (SB-203580) and PI 3-kinase/Akt (LY-294002). VCAM-1 induction was partially suppressed by p44/42 MAPK (PD-098059) but unaffected by c-Jun NH2-terminal kinase (SP-600125) inhibition. Curcumin inhibited Akt/MAPK/NF-kappaB activity and prevented nuclear translocation of the p65 NF-kappaB subunit following TNF-alpha/LPS. At physiological shear stress, curcumin attenuated leukocyte adhesion to TNF-alpha/LPS-activated HIMEC monolayers. In conclusion, curcumin inhibited the expression of VCAM-1 in HIMECs through blockade of Akt, p38 MAPK, and NF-kappaB. Curcumin may represent a novel therapeutic agent targeting endothelial activation in IBD.