Intestinal Mucosal Epithelial Cells Research Articles

Helicobacter pylori and colorectal cancer

Recent researches of the association between helicobacter pylori (Hp) and colorectal adenocarcinoma have found that Hp infection rate is higher in the patients with colorectal cancer and other related colorectal adenoma,which suggest that Hp probably influence on local and distant intestinal mucosa epithelial cells achieve carcinogenesis by its own virulence and the role of elevated serum gastrin levels.Hp infection may be a potential risk factor of colorectal cancer. Key words: Helicobacter pylori; Colorectal neoplasms

Carbamoyl Phosphate Synthetase 1 deficiency in Italy: Clinical and genetic findings in a heterogeneous cohort

Carbamoyl Phosphate Synthetase 1 deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder, potentially leading to lethal hyperammonemia. Based on the age of onset, there are two distinct phenotypes: neonatal and late form. The CPS1 enzyme, located in the mitochondrial matrix of hepatocytes and epithelial cells of intestinal mucosa, is encoded by the CPS1 gene. At present more than 220 clear-cut genetic lesions leading to CPS1D have been reported. As most of them are private mutations diagnosis is complicated. Here we report an overview of the main clinical findings and biochemical and molecular data of 13 CPS1D Italian patients. In two of them, one with the neonatal form and one with the late form, cadaveric auxiliary liver transplant was performed. Mutation analysis in these patients identified 17 genetic lesions, 9 of which were new confirming their “private” nature. Seven of the newly identified mutations were missense/nonsense changes. In order to study their protein level effects, we performed an in silico analysis whose results indicate that the amino acid substitutions occur at evolutionary conserved positions and affect residues necessary for enzyme stability or function.

Partial Restraint Stress Induces Increased Permeability and Low Grade Inflammation in the Rat Ileum, but Not in the Jejunum

Mixed-lineage kinase-3 (MLK3) activates multiple MAPK pathways and has been shown to initiate apoptosis, proliferation, migration or differentiation in several cell types. MLK3 signaling is also required for breast cancer cell invasion. However, the role of MLK3 signaling in regulation of intestinal epithelial cell sheet migration In Vivo and In Vitro is not known. Given its role in cancer cell migration, we sought to investigate whether MLK3 signaling is important in intestinal mucosal healing and epithelial cell motility. We compared the healing of jejunal mucosal ulcers induced by topical serosal acetic acid in different age groups of MLK3 null mice with that in age-matched wild type mice and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial migration across type I collagen substrates. In older mice (30-42 weeks of age), ulcer healing at 5 days after the initial insult was reduced by 47% (p<0.05) in MLK3 null and by 29% in MLK3 heterozygous mice when compared with wild-type littermate controls. In younger animals (8-12 weeks), ulcer healing in MLK3 null mice was 25% less at day 3 (n=7, p<0.05) and 23% less at day 5 (n=7, p<0.05) than in wild-type mice. In Vitro, an MLK3 inhibitor significantly decreased closure of circular wounds in Caco-2 cell monolayers after 24 hours compared with to vehicle-treated control by 16.3% and 20.1 % respectively (p<0.05). Proliferative blockade using hydroxyurea (30mM) did not abolish the effect of MLK3 inhibitor on Caco-2 wound closure, suggesting a true effect on cell motility rather than an effect on cell proliferation. The reduction in cell motility by MLK3 inhibitor reduced level of pERK by 79% (p<0.05) and pJNK by 12% (p<0.05) revealing suppression of ERK and JNK signaling. Total MLK3 was also compared in confluent Caco-2 cells to cell that have been migrating for 96 hours. MLK3 was 53% (p<0.05) higher in migrating vs confluent cells further indicating the importance of MLK3 signaling in migrating intestinal epithelial cells. These studies reveal a novel role for MLK3 signaling in the regulation of intestinal epithelial migration and suggest that MLK3 may be an important target to influence intestinal mucosal healing.

Avaliação dos níveis de peroxidação lipídica em células da mucosa cólica após aplicação de enemas com peróxido de hidrogênio: estudo experimental em ratos

A aplicação de clisteres contendo peróxido de hidrogênio (H2O2) determina o aparecimento de quadros graves de colite, algumas vezes de evolução fatal. É possível que a colite induzida por H2O2 possa ocorrer pela quebra da barreira funcional do epitélio cólico por estresse oxidativo. Objetivo: Avaliar os níveis de peroxidação lipídica em células da mucosa cólica após instilação de H2O2 no reto excluso de trânsito fecal. Método: Vinte seis ratos Wistar machos foram submetidos a colostomia proximal terminal no cólon descendente e fístula mucosa distal. Os animais foram randomizados em dois grupos segundo o sacrifício ter sido realizado duas ou quatro semanas após a derivação intestinal. Cada grupo experimental foi dividido e dois subgrupos segundo aplicação de clisteres, em dias alternados, contendo solução fisiológica a 0,9% ou H2O2 a 3%. O diagnóstico de colite foi estabelecido por estudo histopatológico e os níveis de dano oxidativo tecidual pela dosagem de malondialdeído por espectrofotometria. Os resultados foram analisados com os testes de Mann-Whitney e Kruskal-Wallis, estabelecendo-se nível de significância de 5% (p&lt;0,05). Resultados: Os níveis de malondialdeído nos irrigados com SF nos cólons com e sem trânsito fecal após duas e quatro semanas de irrigação foram de: 0,05 ± 0,006; 0,06 ± 0,006 e 0,05 ± 0,03, 0,08 ± 0,02, respectivamente. Os níveis de malondialdeído nos irrigados com H2O2, nos cólons com e sem trânsito, após duas e quatro semanas de irrigação foram de 0,070 ± 0,006; 0,077 ± 0,01 e 0,052 ± 0,01, 0,08 ± 0,04, respectivamente. Após duas semanas os níveis de malondialdeído foram maiores nos animais irrigados com H2O2 em relação ao grupo controle (p= 0,007 e p= 0,01, respectivamente). Após quatro semanas não houve diferenças significantes Não ocorreu variação nos níveis de malondialdeído com o decorrer tempo de irrigação. Conclusão: Clisteres com H2O2, podem determinar o aparecimento de colite por ocasionarem estresse oxidativo nas células epiteliais da mucosa intestinal.

Effects of QWBZP on T-cell subsets and their cytokines in intestinal mucosa of HRV infection suckling mice

Aim of this study Qiwei Baizhu Powder (QWBZP) is a traditional herbal prescription that has been used traditionally for the treatment of infantile diarrhea, including the infantile diarrhea caused by Human Rotavirus (HRV). In this study, we investigated the pharmacological activity of QWBZP extract. Materials and methods NIH suckling mice with HRV induced diarrhea were used. Density of CD3 +, CD4 + and CD8 + T cells, mRNA expression of IL-2, IFN-γ, IL-4 and IL-10 in intestinal mucosa epithelial cells were assayed. Results QWBZP extract promoted the expressions of mRNA of IL-2, IL-4, IL-10 and IFN-γ in intestinal mucosa epithelial cells. Also, we found that the density of CD8 + cells in intestinal mucosa epithelial cells was significantly lower in QWBZP group than in Model group, while the density of CD8 + cells was significantly higher in QWBZP group than in Model group. Conclusion These data suggest that QWBZP extract may exhibit antiviral effects through modulating the densities of T-cell subsets and the expressions of their cytokines in small intestinal mucosa epithelial cells.

Effect of actovegin on intestinal mucosa and expression of bcl-2/bax genes in rats with acute radiation enteritis

To evaluate the effect of actovegin (Nycomed, deproteinized hemoderivative of calf blood injection) on intestinal mucosa in rats with acute radiation enteritis, and observe the changes of expression of apoptosis-related bcl-2/bax genes. An abdominal irradiation in a dose of 9.0 Gy X-ray of linear accelerator was performed once on a group of Wistar rats to establish a model of acute intestinal radiation enteritis. The experimental rats were randomly divided into five groups. Group 1 was normal control group; group 2 was model control group; groups 3, 4 and 5 were treated with low, middle and high dose of actovegin, respectively. After the model was established, actovegin injection was given intraperitoneally for successive 4 days. Corresponding intestinal tissues were taken for morphological examination with an image analysis system. The expression of apoptosis related bax and bcl-2 protein in the intestinal mucosal epithelial cells was determined by immunohistochemistry. The groups 4 and 5 had significantly higher height of intestinal villi, the depth of crypt, the thickness of the mucosa and entire wall (254.66/261.71 microm, 166.47/165.41 microm, 510.44/511.71 microm, 610.38/608.98 microm), compared with those of the model control group (239.12 microm, 151.45 microm, 420.27 microm and 579.32 microm), respectively (P < 0.05). Treatment with middle and high doses of actovegin also significantly down-regulated the expression of activating apoptosis protein bax (24.54/23.24) compared with that of model control group (59.32) (P < 0.05) and up-regulated the expression of inhibiting apoptosis protein bcl-2 (55.54/52.21) compared with that of model control group (20.32) (P < 0.05). The ratio of bcl-2/bax was significantly higher in the groups 4 and 5 (2.2632, 2.1275) compared with that in the model control group (0.3425) (P < 0.01). Actovegin accelerates the recovery of the acute radiation-injured intestinal mucosal epithelium by decreasing apoptosis via down-regulation of the expression of activating apoptosis protein bax and up-regulation of inhibiting apoptosis protein bcl-2.

Transcellular relocation of tetrahydrobiopterin across Caco‐2 cells: A model study of tetrahydrobiopterin absorption through epithelial cells of intestinal mucosa

Oral administration of tetrahydrobiopterin (BH(4)) has been known to be effective in treating BH(4)-deficient patients. It has long been established that BH(4) is absorbed by the intestinal mucosa. However, the mechanism for translocation of BH(4) across epithelial cells has not been elucidated. In order to study BH(4) transport mechanisms, Caco-2 cells were employed in this study as an epithelial cell model. Caco-2 cells were cultured (2 x 10(4) cells/0.3 cm(2) well) for 21 days in a 24-well format using Transwell, a porous membrane-based culture dish, at which point they had established themselves as a tight sheet with a definite polarity. When BH(4) (100 micromol/L) was given to cells from the apical side, a considerable translocation toward their basolateral side was noted. The rate of BH(4) movement was around 150 pmol/h per well. This was comparable to the highest rate of BH(4) uptake or its release so far obtained using a monolayer culture of Caco-2 cells on an ordinary plastic plate. The transcellular movement of BH(4) across the polar culture on the porous membrane was effectively prevented by benzbromarone (10 micromol/L), a well known inhibitor of a group of transporters including urate transporter (URAT1), organic anion transporters (OATs), and multidrug-resistance-associated proteins (MRPs). It was thus concluded that in Caco-2 cells, BH(4) moved across the cell interior in a rapid ligand-specific manner that was driven by a transporter.

Effects of recombinant human growth hormone on enterocutaneous fistula patients

To explore the effects of recombinant human growth hormone (rhGH) on intestinal mucosal epithelial cell proliferation and nutritional status in patients with enterocutaneous fistula. Eight patients with enterocutaneous fistulas received recombinant human growth hormone (10 microg/d) for 7 d. Image analysis and immunohistochemical techniques were used to analyse the expression of proliferating cell nuclear antigen (PCNA) in intestinal mucosal epithelial cells in biopsy samples from the patients who had undergone an endoscopic biopsy through the fistula at day 0, 4 and 7. Body weights, nitrogen excretion, serum levels of total proteins, albumin, prealbumin, transferrin and fibronectin were measured at day 0, 4 and 7. Significant improvements occurred in the expression of PCNA in the intestinal mucosal epithelial cells at day 4 and 7 compared to day 0 (24.93 +/- 3.41%, 30.46 +/- 5.24% vs 12.92 +/- 4.20%, P < 0.01). These changes were accompanied by the significant improvement of villus height (500.54 +/- 53.79 microm, 459.03 +/- 88.98 microm vs 210.94 +/- 49.16 microm, P < 0.01), serum levels of total proteins (70.52 +/- 5.13 g/L, 74.89 +/- 5.16 g/L vs 63.51 +/- 2.47 g/L, P < 0.01), albumin (39.44 +/- 1.18 g/L, 42.39 +/- 1.68 g/L vs 35.74 +/- 1.75 g/L, P < 0.01) and fibronectin (236.3 +/- 16.5 mg/L, 275.8 +/- 16.9 mg/L vs 172.5 +/- 21.4 mg/L, P < 0.01) at day 4 and 7, and prealbumin (286.38 +/- 65.61 mg/L vs 180.88 +/- 48.28 mg/L, P < 0.05), transferrin (2.61 +/- 0.12 g/L vs 2.41 +/- 0.14 g/L, P < 0.05) at day 7. Nitrogen excretion was significantly decreased at day 7 (3.40 +/- 1.65 g/d vs 7.25 +/- 3.92 g/d, P < 0.05). No change was observed in the body weight. Recombinant human growth hormone could promote intestinal mucosal epithelial cell proliferation and protein synthesis in patients with enterocutaneous fistula.

Effects of insulin-like growth factor -I on bax and bcl-2 mRNA expression in intestinal mucosal epithelial cells of rats with severe acute pancreatitis

Effects of insulin-like growth factor -I on <i>bax</i> and <i>bcl-2</i> mRNA expression in intestinal mucosal epithelial cells of rats with severe acute pancreatitis

Proteinase-activated receptors in the gastrointestinal system: a functional linkage to prostanoids

Proteinase-activated receptors (PARs), G protein-coupled receptors, play critical roles in the alimentary system. Increasing evidence suggests that endogenous prostaglandins (PGs) mediate some of PARs' gastrointestinal functions. Systemic administration of the PAR1 agonist protects against gastric mucosal injury through PG formation in rats. PGs also appear to contribute, at least in part, to enhancement of gastric mucosal blood flow and suppression of gastric acid secretion by PAR1 activation. There is also evidence for involvement of PGs in modulation of gastrointestinal motility by PAR1 or PAR2. Importantly, modulation of ion transport by PAR1 or PAR2 in the intestinal mucosal epithelium is largely mediated by PGs. Studies using gastric and intestinal mucosal epithelial cell lines imply that the PAR1-triggered formation of PGs involves multiple signaling pathways including Src, EGF receptor trans-activation and activation of MAP kinases. Collectively, a functional linkage of PAR1 and/or PAR2 to PGs is considered important in the gastrointestinal system.

Nimesulide Inhibits Crypt Epithelial Cell Proliferation at 6 Hours in the Small Intestine in CD-1 Mice

To determine whether the gut-sparing selectivity of cyclooxygenase-2 inhibitors is related to early crypt kinetic mechanisms, this study compared the primary effects on small intestinal mucosal epithelial cell proliferation and morphometry of a nonselective dual cyclooxygenase inhibitor, indomethacin, with a cyclooxygenase-2 selective inhibitor, nimesulide. Indomethacin downregulated the crypt cell production rate in the proximal small intestine, and nimesulide reduced cell proliferation in the proximal and distal small intestine. Compared to controls, there were smaller proliferating compartments in the crypts in midintestinal segments in both indomethacin- and nimesulide-treated groups, but more dividing cells in the distal intestine in indomethacin-treated group. Crypt cellularity, numbers, and width were unchanged from control values in both treated groups, suggesting a reduction in crypt cell emigration. Despite its selectivity for inhibiting cyclooxygenase-2, nimesulide induces similar but widespread initial effects on intestinal cell kinetics when compared to indomethacin.

A primary dysregulation in the immunoregulatory role of the intestinal mucosal epithelial cell in inflammatory bowel disease pathogenesis?: Biology of inflammatory response as tissue pattern entities in Crohn's versus ulcerative colitis

Within a framework of dual involvement of mucosa and submucosa on the one hand, and of the muscularis propria of the bowel wall on the other, it might be valid to consider involvement of the vascular supply as the essential means in itself of not only causing the morphologic lesions in inflammatory bowel disease, but also especially in accounting for persisting patterns of inflammatory response both in ulcerative colitis and in Crohn's disease. Inflammatory bowel disease as a group constitutes a spectrum of biologic and pathobiologic manifestations in terms not only of inflammatory involvement of the bowel wall but also in terms of how the bowel in its turn deals with inflammation as a pathologic lesion in its own right. Parameters of inflammatory bowel activity transcend simple concepts of etiology and pathogenesis as applicable to category disorders such as infections or bowel ischemia. Indeed, the strictly characterized initiation of the inflammatory bowel response as a function of defective regulation of the antigenicity of the luminal contents on the one hand, and on interactions between nitric oxide and free oxygen radicals on the other, might help determine a persistence of tissue damage in inflammatory bowel disease that is either relapsing/remitting or chronic in progression. In a final analysis, perhaps, there might be involved a single central form of pathway induction of dysregulated immune reactivity arising from an early disturbance in activation patterns as induced by the onset of luminal antigenicity at an early or specific-stage, further characterized perhaps by specific forms of intestinal epithelial defects of the bowel mucosa in patients subsequently developing inflammatory bowel disease. Specific genetic markers for disease susceptibility and for therapeutic responsiveness are particularly of interest. The Nucleotide binding oligomerization Domain 2 (NOD2) would recognize microbial lipopolysaccharide or else mark systemic responses to pathogens that are pathogenic to evolving inflammatory bowel disease.

Structural organization of the human carbamyl phosphate synthetase I gene (CPS1) and identification of two novel genetic lesions.

Carbamyl Phosphate Synthetase I deficiency (CPSID) is a rare autosomal recessive urea cycle disorder usually characterized by potentially lethal neonatal hyperammonemia. The large (5215 bp) CPS1-cDNA, expressed only in liver and epithelial cells of intestinal mucosa, has been cloned. Until now the CPS1 genomic organization was unknown. Taking advantage of the phylogenetic lineage between the CPS1 gene of Homo sapiens and Rattus norvegicus, we determined the intron-exon organization of the human CPS1 gene. Starting from the ATG codon, the CPS I gene is organized in 38 exons spanning from 50bp to 200 bp. We also report the molecular studies on an Italian patient affected by neonatal CPSD. Two novel genetic lesions (c.1370T>G and c.2429A>G) that lead to the novel amino acid substitutions V457G and Q810R, and the known N1406T polymorphism, were detected in the patient's CPS1 RNA and in genomic DNA isolated from peripheral blood lymphocytes. The characterization of the CPS1 genomic organization will allow the identification of the genetic lesions of CPSD patients, the detection of carriers, better genetic counseling and a more certain, less invasive method of prenatal diagnosis.

Expression of heme oxygenase-1 in rat ontogeny.

Heme oxygenase (HO), the heme-degrading enzyme, plays an important role in heme catabolism. Among three isozymes, HO-1 is an inducible form expressed mainly in macrophages. In rat ontogeny, HO-1 immunoreactivity was detected in mononuclear cells in the yolk sac at 10 days of gestation. HO-1-expressing cells were then detected in the fetal liver and their numbers increased during the gestational period. The numbers of HO-1-positive cells and HO-1 mRNA levels in the liver peaked at 18 days of gestation. Most of the macrophages expressed both HO-1 and a macrophage scavenger receptor. Macrophages in the fetal liver showed marked hemophagocytosis. Macrophages in the lung, spleen, bone marrow, and other tissues also expressed HO-1. HO-1 immunoreactivity was also observed in syncytial cells of the chorionic villi, the endodermal layer of the yolk sac, and renal tubules of the fetus. Intestinal mucosal epithelial cells expressed HO-1 after birth. These findings imply that HO-1 is crucial for macrophages in heme catabolism from an early stage of ontogeny. HO-1 expression in non-macrophagic cells may be required for other purposes such as protection from oxidative stress and various stimuli.

The Conserved TFLK Motif of Mammary-Associated Serum Amyloid A3 Is Responsible for Up-regulation of Intestinal MUC3 Mucin Expression In Vitro

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.

The conserved TFLK motif of mammary-associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in vitro.

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.

Oligosaccharides in Human Milk and Bacterial Colonization

Oligosaccharides in Human Milk and Bacterial Colonization

Effect of alum on intestinal microecological balance in mice

To observe the degree of microecological imbalance induced by alum in normal intestine of mice, and the bacterial adherence activity in the intestine of the mice administered orally with alum. The mice were medicated orally with alum of small and large doses (0.25 g/kg and 1 g/kg) for 8 weeks, then 5 weeks after stopping administration of alum, microflora analysis and bacterial adherence to intestinal mucosal epithelial cells were carried out respectively. Eight weeks after administration with alum, the counts of Bifidobacteria and Lactobacilli decreased significantly, but the numbers of pathogenetic E. Coli increased significantly. The adherence rate of Bifidobacteria to the enterocytes of mice reduced markedly, but E. Coli was on the contrary of Bifidobacteria. Five weeks after the ceasing of medication, the intestinal flora were balanced, the adherence rate of both strains as mentioned above recovered to normal level. The imbalance of intestinal flora of mice administered with alum for a long time were only the transient change of bacterial counts. The imbalance of intestinal flora and the adherence rate of bacteria regained normal status five weeks after cancelling action factor.

A thermal injury-induced circulating factor(s) compromises intestinal cell morphology, proliferation, and migration.

The effects of a 60% body surface area thermal injury in rats on the morphology and proliferation of the epithelium of the small intestine and the in vitro effects of serum collected from scalded rats on intestinal epithelial cells were investigated. Scald injury caused significant reductions in duodenal villus width and crypt dimensions, villus enterocytes changed in shape from columnar to cuboidal, and the number of goblet cells decreased. The proportion of bromodeoxyuridine-labeled S phase cells in crypts was also diminished. In vitro, incubation of intestinal epithelial cells (IEC-6) with scalded rat serum (SRS) collected at either 12 or 24 h after injury caused a disruption in the integrity of the confluent culture and induced the appearance of large denuded areas. SRS also decreased DNA synthesis and delayed wound closure in an in vitro wound-healing model. The thermal injury-induced changes in intestinal mucosal morphology and epithelial cell growth characteristics described in this study may underlie, in part, the mechanism(s) involved in the diminished absorption of nutrients, increased intestinal permeability, and sepsis in patients with thermal injury.

Keratinocyte Growth Factor Down-regulates Expression of the Sucrase-Isomaltase Gene in Caco-2 Intestinal Epithelial Cells

The molecular mechanisms that regulate the proliferation and differentiation of intestinal mucosal epithelial cells are not well understood. Keratinocyte growth factor (KGF) is an epithelial cell-specific growth factor that may be involved in the maintenance of mucosal epithelial populations and in mediating epithelial repair after injury. The sucrase-isomaltase (SI) gene, which encodes an enterocyte brush border disaccharidase, has served as a model for study of intestinal-specific gene expression and differentiation. KGF down-regulated SI mRNA and protein expression in Caco-2 intestinal epithelial cells but not the expression of other brush border enzymes. The down-regulation was dose- and time-dependent and specifically blocked by anti-KGF antibodies. Transfection experiments using SI promoter constructs demonstrated that KGF decreased SI gene transcription. In contrast, the stability of SI mRNA was not affected by incubation of Caco-2 cells with KGF. Electrophoretic mobility shift analysis demonstrated that binding of nuclear proteins to the SI footprint (SIF) 3 and SIF4 regulatory elements within the SI promoter region was increased in Caco-2 cells that had been incubated with KGF. In transfection experiments using a construct in which tandem copies of the SIF4-binding site were inserted upstream of the SV40 promoter and luciferase gene, incubation with KGF resulted in a significant decrease in luciferase activity. However, transfection with a similar construct containing tandem copies of SIF3 had no significant effect on SV40 promoter activity following KGF treatment. SIF4 may bind E4BP4, a previously identified transcriptional repressor protein. This factor may in part mediate the decrease in SI transcription by KGF in Caco-2 cells.