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PubMed2001

Increased enterocyte apoptosis and Fas-Fas ligand system in celiac disease.

Our aim was to evaluate whether increased enterocyte apoptosis was responsible for mucosal flattening in celiac disease (CD), and, since the mechanisms responsible for tissue injury in this condition are unknown, we studied the possibility that the Fas-Fas ligand (FasL) system may be involved. Endoscopic duodenal biopsy specimens from 12 patients with untreated and 12 with treated CD and 12 control subjects were evaluated for enterocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end labeling assay and for Fas and FasL expression by immunohistochemistry. A coculture of isolated enterocytes (targets) and purified lamina propria mononuclear cells (LPMCs) (effectors) was performed in the absence or presence of an antagonistic ZB4 anti-Fas antibody. We found a significant correlation between the degree of villous atrophy, morphometrically evaluated, and the level of enterocyte apoptosis, suggesting that mucosal flattening is a consequence of exaggerated epithelial cell death. Most celiac enterocytes express Fas, and LPMCs express FasL. The abolishment of enterocyte apoptosis observed in the presence of ZB4 antibody suggests that enterocytes are potential targets of lymphocyte infiltrate. These results directly demonstrate that FasL-mediated apoptosis is a major mechanism responsible for enterocyte death in CD.

Adult↗
PubMedDate not supplied

Lectin histochemistry of enterocytes sugar residues in the gut of the chick embryo and of the newborn.

The glycoconjugates sugar residues content, distribution and changes in the enterocytes of different tracts of the developing intestine of the chick embryo and of 1-day-old chick were investigated, using a battery of seven HRP-conjugated lectins (DBA, SBA, PNA, WGA, ConA, LTA and UEAI). The results of the present research have shown the presence of a large amount of glycoconjugates sugar residues in the enterocytes of duodenal, ileal and colonic anlage, starting from the beginning of the second week of incubation. Differences were detected among the three investigated intestinal segments, as to the time of appearance of the glycoconjugates sugar residues in the enterocytes. The duodenal enterocytes showed the most precocious appearance of lectin-reactive material, followed by the ileal enterocytes and afterwards by colonic enterocytes. The duodenal enterocytes were characterised by the presence of SBA binding sites, which were not detectable in the duodenal enterocytes of the adult animal.

Aging↗
PubMed1998

Resistance to high concentrations of methotrexate and 5-fluorouracil of differentiated HT-29 colon-cancer cells is restricted to cells of enterocytic phenotype.

Adaptation of HT-29 cells to increasing concentrations of methotrexate (MTX) results in the selection of differentiated populations which show sequential dose-dependent changes of their differentiated phenotype with, at the highest concentrations (0.1 and 1 mM), a shift of differentiation from a mucus-secreting to an enterocytic phenotype coinciding with an amplification of the DHFR gene. We show here that DHFR gene amplification itself does not play a role in the shift of differentiation. An alternative explanation is the presence, within the mucus-secreting population, of an undetectable minor population of cells committed to enterocytic differentiation and able to develop resistance to higher concentrations of MTX. This was confirmed by cloning the population of cells resistant to 10 microM MTX. Out of 19 isolated clones, 17 were found to be mucus-secreting and 2 enterocytic. We tested 9 of these clones for their ability to develop resistance to 0.1 mM MTX: only 1 of enterocytic phenotype, was found to develop resistance to this higher concentration and to amplify the DHFR gene. The ability of enterocytic cells to develop resistance to elevated MTX concentration through amplification of the DHFR gene was demonstrated in another enterocytic HT-29 population selected by glucose deprivation. Enterocytic cells resistant to 10 microM MTX were also found, unlike mucus-secreting cells, to be readily adaptable to 5-fluorouracil, this occurring without amplification of the thymidylate synthase gene. Together these results highlight a previously uncharacterized relationship between commitment to enterocytic differentiation of colon-cancer cells and their ability to develop resistance to MTX and 5-fluorouracil.

Antimetabolites, Antineoplastic↗
PubMed1991

Cryofixation, ultracryomicrotomy, and X-ray microanalysis of enterocytes from chick duodenum: vitamin-D-induced formation of an apical tubulovesicular system.

New methods of tissue preparation were developed to study the morphology and distribution of calcium ions in duodenal enterocytes from normal, rachitic, and vitamin D-replete (either cholecalciferol [CC] or 1,25-dihydroxycholecalciferol [1,25-DHCC] treated) chicks. Frozen hydrated sections were prepared from cryofixed tissues by ultracryomicrotomy at -125 degrees C. Sections were subsequently freeze-dried by increasing the temperature to -100 degrees C. The latter temperature was maintained throughout both the structural and elemental analyses. In cells from normal, rachitic, and vitamin D-treated [CC] animals the brush border from lanthanum-infused tissues was electron dense and calcium-lanthanum positive by x-ray analysis. In the absence of lanthanum, i.e., sucrose-infused duodena, the microvilli were still calcium positive. In the terminal web region of normal and CC-treated enterocytes, numerous, apparently interconnected, tubules and vesicles were seen. Vacuole-like structures were also seen. Such structures were especially prominent in the enterocytes from the vitamin-treated [CC] animals. Except for the vacuoles, the tubules and vesicles were electron dense in the lanthanum-infused duodena, and clear in sucrose-infused tissues. In both instances, the structures were calcium positive. Similar, but even larger structures were seen below the terminal web. Here however, the tubules and vesicles seemed to be organized into multiple complex interconnecting networks, i.e., tubulo-vesicular complexes. Both the tubules and the vesicles seemed to be interconnected via smaller channel-like entities. The extensiveness of this structure was better appreciated in the enterocytes from lanthanum-infused tissues, where it appeared similar in structure and complexity to an en face view of the sarcoplasmic reticulum of skeletal muscle. These intestinal complexes were less well developed, decreased in number, and quite often absent, in the apical cytoplasm of absorptive cells from rachitic chicks. In the enterocytes from animals treated for 24 hours with 1,25-DHCC, the same highly developed tubulo-vesicular networks were again seen in the enterocyte apical cytoplasm. They were even more developed in the 1,25-DHCC-treated animals. All structures were intensely calcium positive in enterocytes from both the lanthanum- and the sucrose-infused preparations. Numerous endocytotic (pinocytotic) vesicles were seen at the lumenal plasmalemma. Similar structures were also apparent in the terminal web region of the 1,25-DHCC-treated enterocytes. Exocytotic vesicles were seen at the apical aspect of the lateral cell membrane, below the level of the junctional complex. All components of this unique system contained high concentrations of calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Anti-gliadin antibodies in patients with celiac disease cross-react with enterocytes and human calreticulin.

One of the characteristic features of celiac disease is an increase in anti-gliadin antibodies (Abs). Recently we found that some of the monoclonal Abs to gliadin cross-react with molecules on rat enterocytes. One of these cross-reacting molecules was identified as rat calreticulin. This study shows that the levels of serum IgA Abs to gliadin, rat, and human enterocytes; purified enterocyte antigens; and calreticulin in sera from patients with active disease were significantly higher than in patients on a gluten-free diet and healthy controls (P < 0.001). Anti-gliadin Abs were isolated by affinity chromatography from the sera of six active celiac patients. The reactivity of these anti-gliadin Abs was demonstrated to be significantly higher (P < 0.05) with human enterocytes and human calreticulin than with other antigens tested. Furthermore, using isolated patients' anti-gliadin Abs bound to Sepharose 2B, two main proteins of molecular mass 62 and 66 kDa were purified from a lysate of human enterocytes. The 62-kDa enterocyte antigen was identified as human calreticulin. These findings suggest that anti-gliadin Abs may play a pathogenic role in celiac disease by cross-reacting with enterocytes. Calreticulin in enterocytes may be one of the putative targets for autoimmune reactions.

Adolescent↗
PubMed1995

Urea synthesis in enterocytes of developing pigs.

Urea synthesis from ammonia, glutamine and arginine was determined in enterocytes from newborn (0-day-old), 2-21-day-old suckling, and 29-58-day-old post-weaning pigs. Pigs were weaned at 21 days of age. Cells were incubated for 30 min at 37 degrees C in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing (i) 0.5-2 mM NH4Cl plus 0.05-2 mM ornithine and 2 mM aspartate, (ii) 1-5 mM glutamine, or (iii) 0.5-2 mM arginine. In enterocytes from newborn and suckling pigs, there was no measurable synthesis of urea from ammonia, glutamine or arginine, and analysis of amino acids by a sensitive fluorimetric HPLC method revealed the formation of negligible amounts of ornithine from arginine. In contrast, in cells from post-weaning pigs, relatively large amounts of urea and ornithine were produced from ammonia, glutamine and arginine in a dose-dependent manner. To elucidate the mechanism of the developmental change of urea synthesis in pig enterocytes, the activities of urea-cycle enzymes were determined. The activities of enterocyte carbamoyl phosphate synthase I and ornithine carbamoyltransferase were lower in post-weaning pigs than in suckling ones, whereas there was no difference in arginino-succinate lyase. The activities of argininosuccinate synthase and arginase were increased by 4-fold and 50-100-fold, respectively, in enterocytes from post-weaning pigs compared with suckling pigs. The induction of arginase appears to be sufficient to account for the formation of urea from ammonia, glutamine and arginine in post-weaning pig enterocytes. These results demonstrate for the first time the presence of synthesis of urea from extracellular or intramitochondrially generated ammonia in enterocytes of post-weaning pigs. This hitherto unrecognized urea synthesis in these cells may be a first line of defence against the potential toxicity of ammonia produced by the extensive intestinal degradation of glutamine (a major fuel for enterocytes) and derived from diet and luminal micro-organisms.

Ammonia↗
PubMed1988

Folate transport in enterocytes and brush-border-membrane vesicles isolated from the small intestine of the neonatal goat.

1. The uptake of 5-methyltetrahydrofolate (MTHF) and folic acid (pteroylmonoglutamate) by enterocytes and brush-border-membrane vesicles prepared from the small intestine of the 6-d-old male goat was determined using a rapid-filtration assay. 2. Both MTHF and folic acid were taken up by membrane vesicles at 25 degrees and enterocytes at 37 degrees by a pH-dependent mechanism with maximum uptake when the pH of the incubation medium was 5.0. 3. Experiments in which the osmotic pressure of the medium was raised in successive increments with the non-absorbable sugar cellobiose indicated that transport rather than membrane binding was the main component of uptake. 4. Experiments at pH 5.0 showed that uptake of MTHF and folic acid was saturable and that the characteristics of folate transport were similar in both tissue preparations: (a) transport rates of both MTHF and folic acid were constant during the first 1-2 min for a given folate concentration, then declined to reach a steady-state in 10-30 min; (b) initial velocities of transport of MTHF and folic acid increased in proportion to their concentrations up to 7-10 microM, but the rate of increase slowed thereafter until saturation was reached at 20-25 microM (Km for brush-border-membrane vesicles 40.8 and 62.9 microM, Km for enterocytes 50.9 and 55.2 microM for MTHF and folic acid respectively). Values of Vmax for membrane vesicles (pmol/mg protein per min) were 46.5 MTHF or 40.3 folic acid; enterocytes Vmax (pmol/10(7) cells per min) 15.9 MTHF or 30.6 folic acid; (c) uptake of MTHF and folic acid by brush-border-membrane vesicles and enterocytes measured under steady-state conditions approached saturation at 50 microM for each analogue (Km for membrane vesicles 58.1 and 55.2 microM, Km for enterocytes 43.1 and 49.4 microM for MTHF and folic acid respectively; Vmax for membrane vesicles 49.7 and 77.8 pmol/mg protein, for enterocytes 52.8 and 54.7 pmol/10(7) cells for MTHF and folic acid respectively). 5. It was concluded that transport of MTHF and folic acid was by a similar pH-dependent mechanism both in brush-border-membrane vesicles and in intact enterocytes. At pH 5.0, transport involved a saturable carrier-mediated process located in the brush-border membrane.

Animals↗
PubMed1998

Targeting of gliadin peptides, CD8, alpha/beta-TCR, and gamma/delta-TCR to Golgi complexes and vacuoles within celiac disease enterocytes.

Celiac disease (CD) is characterized by autodestruction of enterocytes after exposure of genetically susceptible individuals to dietary gluten. To define the transport pathways of proteins involved in the celiac immune response, we wished to determine the subcellular compartments of the intestinal mucosa where wheat gliadin peptides colocalize with receptors of T lymphocytes, including alpha/beta-TCR, gamma/delta-TCR, and CD8. Semithin and ultrathin frozen section of jejunal biopsies from CD patients and controls were used to perform immunofluorescence and immunogold labeling as well as in situ hybridization experiments. In patients with active CD, we detected gliadin peptides in vacuoles and Golgi complexes of enterocytes. CD8, alpha/beta-TCR, and gamma/delta-TCR were found in vacuoles and Golgi complexes within these gliadin-containing enterocytes in addition to the surface of intraepithelial and mucosal T lymphocytes. In contrast, we observed that the localization of CD4, CD3, T cell-restricted intracellular antigen (TIA), and leukocyte common antigen (LCA) was restricted to lymphocytes in CD patients. We further detected labeling signals for gliadin peptides, CD8, alpha/beta-TCR, and gamma/delta-TCR at the basal membrane of enterocytes that were interdigitated by extensions of lymphocytes. In situ hybridization experiments revealed that CD8 and gamma/delta-TCR were not expressed by CD enterocytes. We conclude that CD8, alpha/beta-TCR, and gamma/delta-TCR are targeted to Golgi complexes and vacuoles of small intestinal enterocytes in active CD. The observed process may be involved in the pathogenesis of CD enterocytes. We propose a mechanism for the uptake of CD8, alpha/beta-TCR, and gamma/delta-TCR by the basolateral membrane of small intestinal enterocytes.

Antibodies, Monoclonal↗
PubMed1992

Time dependency of 1,25(OH)2D3 induction of calbindin mRNA and calbindin expression in chick enterocytes during their differentiation along the crypt-villus axis.

Quantitative methods of in situ hybridization and immunocytochemistry have been used to measure 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induction of calbindin mRNA and calbindin protein expressed in jejunal enterocytes at all points along the crypt-villus axis over a 24 h period. Small amounts of calbindin mRNA detected in vitamin D3 deficient (D-deficient) chick intestine increased rapidly to maximal values 8 h after hormone injection. The magnitude of this response was inversely related to age of enterocyte measured separately by injecting tritiated thymidine into D-deficient and 1,25(OH)2D3-injected birds. Enterocytes of all ages expressed small amounts of calbindin 3 h after hormone injection. This amount of calbindin then increased up to 24 h after hormone injection. Maximal calbindin expression took place in basal villus enterocytes. Later decrease in the ability of upper villus enterocytes to express calbindin was associated with a similar fall in calbindin mRNA expression. Previously it was suggested that inefficient translation to calbindin mRNA might take place in basal villus enterocytes 48 h after vitamin D injection. Present work using 1,25 (OH)2D3 shows that calbindin expression takes place at a constant rate during this early stage of enterocyte development. Secondary events limiting higher rates of calbindin synthesis in upper crypt and basal villus enterocytes remain to be identified.

Animals↗
PubMed1984

Developmental changes in intra-enterocyte cation activities in hamster terminal ileum.

Intracellular Na+ and K+ activities have been determined under visual control in enterocytes located at known positions along hamster ileal villi in vitro. The intracellular K+ activity found in enterocytes migrating over the basal third of villi was significantly less than that found in older enterocytes (83 and 113 mM respectively). The intracellular Na+ activity remained unaffected by the state of enterocyte development (32 mM throughout). Extracellular K+ and Na+ activities measured close to the luminal surface of the intestine were always similar to those measured in the bulk of the bathing medium. There was, however, a small difference in K+ activity detected between the bulk solution and solution near the luminal surface of enterocytes (3 . 4 and 3 . 6-3 . 7 mM respectively). Na+ activity in the villus core was also similar to that of the bathing medium. The brush border membrane content of sucrase-isomaltase, determined cytochemically, was found to increase rapidly as enterocytes migrated over the basal third of the villus and to plateau at a point roughly equivalent to that where intracellular K+ activities became maximal. The numerous ways by which enterocytes might control their intracellular K+ concentrations during development are emphasized. Attention is nevertheless drawn to the close correspondence seen between different aspects of brush border membrane development and the ability of enterocytes to maintain high internal concentrations of K+.

Animals↗
PubMed2000

Inducible expression of Enterococcus faecalis aggregation substance surface protein facilitates bacterial internalization by cultured enterocytes.

Aggregation substance (AS) is an Enterococcus faecalis surface protein that may contribute to virulence. Using a recently described system for controlled expression of AS in E. faecalis and the heterologous host Lactococcus lactis, experiments were designed to assess the effect of AS on bacterial internalization by HT-29 and Caco-2 enterocytes. AS expression was associated with increased internalization of E. faecalis by HT-29 enterocytes and of L. lactis by HT-29 and Caco-2 enterocytes. Compared to enterocytes cultivated under standard conditions, either cultivation in hypoxia or 1-h pretreatment of enterocytes with calcium-free medium resulted in increased internalization of both E. faecalis and L. lactis (with and without AS expression). Also, AS expression augmented these increases when E. faecalis was incubated with pretreated HT-29 enterocytes and when L. lactis was incubated with pretreated Caco-2 and HT-29 enterocytes. These data indicated that AS might facilitate E. faecalis internalization by cultured enterocytes.

Bacterial Proteins↗
PubMed1993

Effect of LPS on epithelial integrity and bacterial uptake in the polarized human enterocyte-like cell line Caco-2.

Bacterial lipopolysaccharide (LPS) has been speculated to facilitate bacterial translocation by a mechanism involving physical disruption of the gut mucosal barrier. Polarized, cultured intestinal epithelial cells (Caco-2 cells) were used to study the effect of LPS on enterocyte structure, viability, and susceptibility to bacterial invasion. Varying concentrations of biologically active LPS were incubated with enterocytes for 1 and 16 hr. LPS had no noticeable effect on enterocyte viability or morphology, as measured by uptake of vital dyes, by distribution of cytoskeletal filamentous actin, and by visualization of subcellular ultrastructure. Transepithelial electrical resistance was similar in enterocyte cultures incubated with LPS for 1 hr, but there was a noticeable decrease after 16 hr, indicating a loss of epithelial integrity after prolonged exposure to LPS. The effect of LPS on bacterial uptake was studied using six strains of enteric bacteria with varying abilities to invade Caco-2 cells: Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, Escherichia coli (2 strains), and Enterococcus faecalis. Electron microscopy showed enteric bacteria in intimate association with enterocyte apical microvilli, and internalized bacteria were consistently observed within cytoplasmic, membrane-bound vacuoles. Following a 1-hr incubation of individual strains of enteric bacteria with Caco-2 cells, numbers of viable intracellular bacteria varied significantly between individual bacterial strains, but numbers of intracellular bacteria were similar for each strain incubated with enterocytes exposed to 0, 10, and 100 micrograms LPS for 1 and 16 hr. Thus, although prolonged exposure to LPS might have some effect on enterocyte culture integrity (as measured by decreased electrical resistance), LPS had no discernible effect on enterocyte structure, viability, and susceptibility to bacterial invasion. These results suggested that LPS-induced bacterial translocation might not involve loss of epithelial viability, or facilitated entry of bacteria into intestinal epithelial cells.

Bacterial Physiological Phenomena↗
PubMed2003

Increased enterocyte apoptosis in inflamed areas of Crohn's disease.

PURPOSE: Because increased enterocyte apoptosis has been associated with the pathogenesis of several chronic inflammatory diseases, the aim of our study was to investigate epithelial cell death in Crohn's disease and the possible role of the Fas-Fas ligand system, E-cadherin, and matrix metalloproteinase-1 in modulating enterocyte apoptosis in this condition. METHODS: Endoscopic ileal and colonic biopsy specimens were collected from macroscopically involved and uninvolved areas of 20 patients with Crohn's disease and 20 subjects who proved to have functional diarrhea. Diagnosis was established by clinical and pathologic criteria. Biopsy specimens were processed for traditional histology and for the immunohistochemical evaluation of Fas, Fas ligand, E-cadherin, Ki67 antigen, and matrix metalloproteinase-1 expression. For the in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end labeling was used. RESULTS: The percentages of apoptotic enterocytes were higher in involved than in uninvolved areas of Crohn's disease patients and normal intestine. No significant difference was found between Crohn's disease uninvolved areas and normal intestine. In Crohn's disease, both enterocyte Fas and lamina propria mononuclear cell Fas ligand expression did not differ from controls. E-cadherin was strongly expressed by epithelium in both normal and inflamed intestine, except for the regenerative epithelium over the base of the ulcers, where a reduced E-cadherin expression was observed. The number of Ki67-positive proliferating epithelial cells did not differ either in involved or uninvolved areas of Crohn's disease patients compared with controls. A lamina propria overexpression of matrix metalloproteinase-1 was found in involved compared with uninvolved Crohn's disease areas and normal tissue, and a significant positive correlation between matrix metalloproteinase-1 expression and enterocyte apoptosis was found in Crohn's disease inflamed areas. CONCLUSIONS: Enterocyte apoptosis is increased in involved areas of Crohn's disease. This increase is not mediated by a Fas-Fas ligand mechanism or by an abnormal E-cadherin distribution. Increased matrix metalloproteinase-1 release from lamina propria mononuclear cells might be one of the possible mechanisms responsible for the increased enterocyte apoptosis in Crohn's disease.

Adolescent↗
PubMed2004

Preventing enterocyte apoptosis after massive small bowel resection does not enhance adaptation of the intestinal mucosa.

BACKGROUND: After massive small bowel resection (SBR), increased rates of enterocyte apoptosis are observed in the remnant bowel via a mechanism requiring bax gene expression. This study tested the hypothesis that adaptive mucosal growth could be enhanced by the novel strategy of preventing postresection enterocyte apoptosis. METHODS: Male bax-null and corresponding wild-type (WT) mice underwent a 50% proximal SBR or sham operation (bowel transaction with reanastomosis alone). Mice were killed after a full adaptation interval of 1 month. Adaptation was measured in the remnant ileum as alterations in villus height, crypt depth, and wet weight. Rates of enterocyte proliferation were derived by immunostaining of crypt enterocytes for Ki-67 and apoptosis by the presence of apoptosis bodies. RESULTS: The expected increase in enterocyte apoptosis after SBR occurred in the WT mice but was unchanged in the bax-null mice. Despite the prevention of postresection apoptosis in the bax-null mice, all parameters of adaptation and proliferation increased equally after SBR in both groups of mice. CONCLUSIONS: Bax deficiency prevents the increase in enterocyte apoptosis that occurs after massive SBR throughout the entire adaptation period. Attenuation of postresection enterocyte apoptosis does not augment mucosal adaptation to massive intestinal loss.

Adaptation, Physiological↗
PubMed2004

Antigen transport and cytoskeletal characteristics of a distinct enterocyte population in inflammatory bowel diseases.

Intestinal antigen uptake is enhanced in inflammatory bowel disease. We analyzed transcellular transport routes of antigens in different compartments of normal enterocytes and atypical intestinal epithelial cells called "rapid antigen uptake into the cytosol enterocytes" (RACE cells). These cells constitute a recently described population of enterocyte-derived cells, which are increased in inflammatory bowel disease. Mucosa of freshly resected specimens were incubated with the antigens ovalbumin or horseradish peroxidase. Ultrastructural labeling patterns of differentiation-dependent proteins, the brush-border enzyme sucrase-isomaltase and the cytoskeleton proteins villin and actin, were determined in enterocytes. Apoptosis was investigated biochemically and ultrastructurally by cleavage of caspase-3. Both antigens were transported to late endosomes and to trans-Golgi vesicles of enterocytes in inflammatory bowel disease and control specimens. Quantitative evaluation revealed a significantly increased transepithelial antigen transport in both compartments of RACE relative to normal enterocytes. Labeling densities for sucrase-isomaltase, villin, and actin were decreased in RACE relative to normal enterocytes. Caspase-3 was not increased in RACE cells relative to controls. RACE cells are characterized by increased antigen transport to late endosomes and the trans-Golgi network, a disassembled cytoskeleton and lower concentrations of proteins that are markers of cell differentiation.

Actins↗
PubMed2000

Cytochrome P450 enzyme activity and protein expression in primary porcine enterocyte and hepatocyte cultures.

1. A method for the isolation and cultivation of porcine hepatocytes and porcine duodenal enterocytes for the investigation of drug oxidation reactions has been established. 2. Hepatocytes as well as enterocytes metabolized ethoxyresorufin (EROD) and ethoxycoumarin (ECOD) effectively, the rate being 31+/-17 pmol/h x dish (EROD) and 9530+/-4062 pmol/h x dish (ECOD) in the case of hepatocytes, and 9+/-4 pmol/h x dish (EROD) and 510+/-467 pmol/h x dish (ECOD) in the case of enterocytes. Diazepam, another CYP monooxygenase substrate, was also metabolized by porcine hepatocytes but not with porcine enterocytes, thus indicating differences in the metabolic competence of the liver and the gut. 3. The ability to induce enzymes responsible for the metabolism of ethoxyresorufin and ethoxycoumarin was investigated in vitro on treatment of the cell cultures with either 50 microM 3-methylcholanthrene (3-MC) or 50 microM beta-naphthoflavone (beta-NF). With enterocyte cultures, ECOD activity was inducible up to 20-fold, whereas EROD remained unchanged following treatment with either 3-MC or beta-NF. 4. Western blotting provided additional evidence for the expression of CYP1A1 and CYP3A4 at the protein level and treatment of cultured enterocytes with 30 microM Aroclor 1254 or 50 microM beta-NF resulted in enhanced expression of the CYP1A protein, and CYP3A4 protein expression was induced following treatment with 50 microM DEX, 2 mM PB, 30 microM Aroclor 1254 or 50 microM beta-NF. 5. The metabolism of diazepam was also investigated with baculovirus-expressed human CYP enzymes (2C8, 2C9-ARG, 2C9-CYS, 2C19, 3A4, 3A4+cytochrome b5 and 3A5) and evidence was obtained to suggest the formation of temazepam and oxazepam by enzymes of the CYP3A subfamily. Small amounts (32+/-12 ng/ml) of desmethyldiazepam were additionally recovered in microsomal preparations of all CYP-transfected cell lines. 6. In conclusion, porcine duodenal enterocytes can successfully be cultured for a short period and may be used as a tool for studying intestinal metabolism, whereas porcine hepatocytes can be cultured for prolonged periods (>10 days) reliably to investigate hepatic drug oxidation reactions.

Albumins↗
PubMed2006

Molecular characterisation of non-absorptive and absorptive enterocytes in human small intestine.

BACKGROUND AND AIMS: Perturbation of differentiation of the crypt-villus axis of the human small intestine is associated with several intestinal disorders of clinical importance. At present, differentiation of small intestinal enterocytes in the crypt-villus axis is not well characterised. SUBJECTS AND METHODS: Expression profiling of microdissected enterocytes lining the upper part of crypts or the middle of villi was performed using the Affymetrix X3P arrays and several methods for confirmation. RESULTS: A total of 978 differentially expressed sequences representing 778 unique UniGene IDs were found and categorised into four functional groups. In enterocytes lining the upper part of crypts, cell cycle promoting genes and transcription/translation related genes were predominantly expressed, whereas in enterocytes lining the middle of villi, high expression of cell cycle inhibiting genes, metabolism related genes, and vesicle/transport related genes was found. CONCLUSION: Two types of enterocytes were dissected at the molecular level, the non-absorptive enterocyte located in the upper part of crypts and the absorptive enterocyte found in the middle of villi. These data improve our knowledge about the physiology of the crypt-villus architecture in human small intestine and provide new insights into pathophysiological phenomena, such as villus atrophy, which is clinically important.

Adult↗
PubMed2006

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier.

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.

Animals↗