Smad7 in intestinal CD4+ T cells determines autoimmunity in a spontaneous model of multiple sclerosis.

Environmental triggers acting at the intestinal barrier are thought to contribute to the initiation of autoimmune disorders. The transforming growth factor beta inhibitor Smad7 determines the phenotype of CD4+ T cells. We hypothesized that Smad7 in intestinal CD4+ T cells controls initiation of opticospinal encephalomyelitis (OSE), a murine model of multiple sclerosis (MS), depending on the presence of gut microbiota. Smad7 was overexpressed or deleted in OSE CD4+ T cells to determine the effect on clinical progression, T cell differentiation, and T cell migration from the intestine to the central nervous system (CNS). Smad7 overexpression worsened the clinical course of OSE and increased CNS inflammation and demyelination. It favored expansion of intestinal CD4+ T cells toward an inflammatory phenotype and migration of intestinal CD4+ T cells to the CNS. Intestinal biopsies from MS patients revealed decreased transforming growth factor beta signaling with a shift toward inflammatory T cell subtypes. Smad7 in intestinal T cells might represent a valuable therapeutic target for MS to achieve immunologic tolerance in the intestine and suppress CNS inflammation.

[Fecal microbiota transplantation in relapsing clostridium difficile colitis]. / Stuhltransplantation beim Rezidiv der Clostridium-difficile-Kolitis.

BACKGROUND: Since the turn of the millennium there has been an alarming increase in the incidence and severity of clostridium difficile infections. Stopping medication with the triggering antibiotic and switching to a recommended antibiotic leads to healing up in 80%. However, patients who relapse have a 40% risk of an additional relapse and those with 2 or more episodes face a 60% risk. Fecal microbiota transplantation (FMT) is a new therapeutic option. Up to now there only exist two randomized studies (University of Amsterdam and the Massachusetts General Hospital in Boston). METHOD: Data from 16 patients with recurrent clostridium difficile infection who had undergone FMT at a local hospital in the city of Bremen, Germany, were reviewed and compared to the results of the 2 randomized studies. RESULTS: 11 out of 16 patients got cured after the first FMT (68.75%). The remaining 5 patients received a second FMT, with cure in 3 patients. The overall response rate was 14 from 16 patients (87.5%). CONCLUSIONS: In comparison to the response rates of the University of Amsterdam (81.3% after the first and 93.8% after the second FMT) and of the Massachusetts General Hospital in Boston (70% after the first and 90% after the second FMT) we received slightly worse results. But, treatment of notably older patients and intensive care patients in our group explain these findings well. Therefore, we advocate a wide use of FMT for the treatment of recurrent clostridium difficile colitis in non-university hospitals.

Upper gastrointestinal motility and symptoms in individuals with diabetes, prediabetes and normal glucose tolerance.

AIMS/HYPOTHESIS: Type 2 diabetes has been associated with upper gastrointestinal motility dysfunction, but the relationship with diabetes duration and glucose control is less well understood. Gastric emptying, oesophageal motility and gastrointestinal symptoms were examined in volunteers with diabetes, prediabetes (impaired fasting glucose [IFG] or impaired glucose tolerance [IGT]) and normal glucose tolerance (NGT). METHODS: The study included 41 patients with type 2 diabetes, 17 individuals with IFG/IGT and 31 individuals with NGT. A gastric emptying breath test and high-resolution oesophageal manometry were performed. Gastrointestinal symptoms were assessed using questionnaires. RESULTS: Gastric emptying was delayed in individuals with IFG/IGT (p < 0.05) but was normal in the diabetic group. Amongst the diabetic patients, gastric emptying rate was fastest in those with longer diabetes duration and the highest HbA1c levels (p < 0.001). Oesophageal motility variables were similar between the groups. However, the lower oesophagus resting pressure was reduced in patients with longer diabetes duration (p = 0.01). Abdominal pain/discomfort was more frequent amongst patients with diabetes (p = 0.04) but was unrelated to gastric emptying. Significant associations between various oesophageal motility variables and gastrointestinal symptoms were observed. CONCLUSIONS/INTERPRETATION: Gastric emptying and oesophageal motility are not generally altered in patients with type 2 diabetes. In more advanced disease stages, however, gastric emptying and oesophageal motility may be disturbed, probably as a consequence of autonomic neuropathy. Delayed gastric emptying in IFG/IGT individuals might be secondary to acute hyperglycaemia. Determination of gastric emptying and oesophageal manometry should be considered for the diagnostic workup of patients with diabetes and gastrointestinal symptoms.

Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery.

BACKGROUND: Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. METHODS: In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. RESULTS: The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. CONCLUSION: The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

Defensin-mRNA expression in the upper gastrointestinal tract is modulated in children with celiac disease and Helicobacter pylori-positive gastritis.

OBJECTIVES: Defensins are expressed in epithelial cells as cationic peptides with antimicrobial properties. Because of their role as immunologically important effector molecules, their contribution in maintaining a stable microenvironment in the gastrointestinal tract has recently received much attention. The present study was designed to further characterize expression patterns of defensins in diseases of the upper gastrointestinal tract in children, particularly in Helicobacter pylori (Hp)-associated gastritis or celiac disease (CD). PATIENTS AND METHODS: Semiquantitative real-time reverse transcriptase-polymerase chain reaction (PCR) was carried out with gene-specific primers for human beta-defensin 1 to 6 (hBD1 to 6) and human alpha-defensin 5 and 6 (hD5 and 6) in mucosal biopsies of children diagnosed as having CD (n = 11; 4.2-16.2 years) or Hp gastritis (n = 18; 3.2-16.7 years). Levels of expression were compared with those of healthy individuals (n = 21; 2.8-14.6 years). Expression levels in Hp-infected specimens were furthermore compared with those with histologic inflammation not associated with Hp infection (n = 30; 3.6-15.7 years). RESULTS: Expression of hBD2 was upregulated in the antrum and corpus of patients with Hp gastritis, whereas inflammation without detection of Hp was not associated with any change in defensin gene expression. In patients with CD, expression of hBD2 was upregulated in the antrum, whereas hBD1 and 4 were downregulated in duodenal biopsies. CONCLUSIONS: Different pathological conditions of the upper gastrointestinal tract lead to specific modulations of defensin gene expression in children. Especially the pathophysiological role of hBD2 in Hp infection and hBD1 and 4 in CD warrant further attention.

Probiotics regulate the expression of COX-2 in intestinal epithelial cells.

Cyclooxygenase-2 (COX) 2 promotes intestinal wound healing but elicits also proinflammatory effects and has been implicated in colorectal carcinogenesis. Thus, a balanced expression of COX-2 is essential for intestinal homeostasis. This study was designed to evaluate the regulation of COX-2 by probiotic organisms and to characterize ligands and receptors involved. Colo320 and SW480 intestinal epithelial cells (IEC) were stimulated with gastrin or TNF-alpha and pre- or coincubated with commensales, bacterial supernatants, or distinct toll-like receptor (TLR) ligands. COX-2 promoter activity was determined by luciferase assays, protein expression by Western blotting, and secretion of prostaglandin E(2) (PGE(2)) by ELISA. Commensales differentially regulated COX-2 expression in IEC. E. coli Nissle 1917, the probiotic mixture VSL#3, and media conditioned by these organisms ameliorated induced COX-2 expression and PGE(2) secretion. Heat inactivation and DNase treatment significantly decreased these regulatory capacities. Lactobacillus acidophilus, however, significantly increased COX-2 expression and PGE(2) secretion. TLR agonists differentially ameliorated basal or induced COX-2 expression. Distinct probiotics specifically and significantly decrease induced COX-2 expression in IEC, most likely mediated by released factors and in part by bacterial DNA. A significant involvement of TLRs in these regulatory processes remains to be established.

Defensins are differentially expressed with respect to the anatomic region in the upper gastrointestinal tract of children.

Defensins are antimicrobial peptides expressed on various epithelial surfaces. Although they are believed to contribute to intestinal homeostasis, their expression pattern in children is not well characterized. As determined by real-time polymerase chain reaction, amount of human alpha-defensins (hD)-5 and -6 mRNA in duodenal biopsies were significantly higher than in biopsies taken from the gastric mucosa. On the contrary, expression of human beta-defensins (hBD)-1 and -2 mRNA showed a significantly higher expression in the stomach. Expression of hBD3 to 6 was inconsistently detected. These results suggest a distinct role for various defensins in host defense in the upper gastrointestinal tract of children.

Human beta defensin 2 promotes intestinal wound healing in vitro.

Limiting microbial threats, maintenance and re-establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human beta-defensin 2 (hBD-2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco-2 and HT-29 cells were stimulated with 0.1-10 microg/ml hBD-2 for 6-72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo-deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot-blot analysis. Furthermore, anti-apoptotic capacities of hBD-2 were studied. Over a broad range of concentrations and stimulation periods, hBD-2 was well tolerated by IECs and did not induce apoptosis. hBD-2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD-2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD-2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD-2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease.

Macrophage migration inhibitory factor (MIF) -173G/C promoter polymorphism influences upper gastrointestinal tract involvement and disease activity in patients with Crohn's disease.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with increased expression in inflammatory bowel disease. The aim of the study was to analyze the role of the MIF -173G/C single nucleotide polymorphism in Crohn's disease (CD). METHODS: Using restriction fragment length polymorphism analysis, genomic DNA of 198 patients with CD and 159 unrelated controls was analyzed for the -173G/C SNP in the MIF promoter region. Colonic MIF mRNA expression was measured by quantitative polymerase chain reaction (PCR), serum MIF levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-six of the 146 G/G wildtype carriers (24.7%) but only 3 of the 45 G/C heterozygotes (6.7%) and only 1 of the C/C homozygotes (14.3%) were diagnosed with upper gastrointestinal tract involvement (P = 0.009, odds ratio [OR] = 0.22, 95% confidence interval [CI], 0.06-0.75 for wildtype versus hetero- and homozygous carriers). This result was confirmed in a second prospective study, in which all patients diagnosed with upper gastrointestinal involvement (n = 13) were G/G wildtype carriers (P = 0.01 versus controls). All patients (n = 12; 100%) with a Crohn's disease activity index (CDAI) > 300 were G/G wildtype carriers compared to only 65.6% wildtype carriers in the group with a CDAI < 150 (P = 0.016). MIF is expressed in the colonic mucosa of CD patients and intestinal epithelial cells but its mRNA expression does not correlate with the degree of inflammation and is not upregulated by proinflammatory cytokines. In CD, MIF serum levels are not influenced by the MIF -173G/C polymorphism. CONCLUSIONS: The MIF -173G/C polymorphism appears to be a factor contributing to a particular CD phenotype characterized by protection against upper gastrointestinal tract involvement and severe disease activity.

Orlistat reduces gallbladder emptying by inhibition of CCK release in response to a test meal.

BACKGROUND AND AIMS: Orlistat is a covalent inhibitor of digestive lipase derived from lipstatin, the natural product of Streptomyces toxytricini. By blocking the active site of intestinal lipase, orlistat inhibits hydrolysis of dietary triglycerides and thus reduces the intestinal lipid absorption. It is uncertain whether intestinal inhibition of lipase by orlistat also interferes with nutrient-induced CCK release from intestinal I-cells. The aim of the present study was therefore to assess whether oral administration of orlistat inhibits CCK release in response to a test meal and thus causes impaired gallbladder emptying. METHODS: 22 healthy volunteers were given a test meal consisting of 200 ml dairy cream and two teaspoons of chocolate powder (552 kcal=2328 kJ; 56.0 g fat; 5.2 g proteins, 6.6 g carbohydrates), with and without oral application of 120 mg orlistat. Gallbladder volume was determined by ultrasound before and 5, 10, 20, 30 and 40 min after meal ingestion. In parallel, a venous blood sample was collected for the measurement of bioactive CCK. CCK activity was assessed using a bioassay with isolated rat pancreatic acini cells. RESULTS: Oral administration of orlistat significantly impairs gallbladder emptying. After ingestion of the test meal the gallbladder contracted by 78.5% in the control group, whereas the test group with orlistat only showed a contraction of 45.7% (p<0.01). Maximal contraction was reached after 35 to 40 min, the maximal gallbladder emptying was delayed up to 10 min by orlistat. Orlistat induced a significant reduction of bioactive CCK levels in response to a test meal (CCK(max) with orlistat=4.1 pmol/l; CCK(max) without orlistat=7.8 pmol/l). CCK levels were reduced by 47% and the onset of maximal CCK secretion was delayed up to 10 min. CONCLUSION: The inhibition of intestinal lipolytic activity by orlistat results in reduced gallbladder emptying through inhibition of meal-mediated CCK release. We therefore hypothesize that impaired gallbladder motility may represent a risk factor in chronic treatment of severe obesity using orlistat.

Differential regulated expression of keratinocyte growth factor and its receptor in experimental and human liver fibrosis.

BACKGROUND AND AIM: Immunomodulatory and protective properties have been identified for the keratinocyte growth factor (KGF). For hepatocytes, pro-proliferative and anti-apoptotic effects of this growth factor have been reported in vitro. This study was designed to characterize a putative role of KGF in observed histomorphological changes in both, human and experimental liver fibrosis. METHODS: Liver fibrosis and cirrhosis was induced in rats by repetitive exposure to phenobarbitone and increasing doses of carbon tetrachloride. Human samples were obtained from patients undergoing surgery for partial hepatectomy or transplantation. Organ samples were scored for inflammation and morphological changes. Expression of KGF and its receptor (KGFR) mRNA was quantified by real-time RT-PCR. Protein expression and receptor phosphorylation was determined by Western blot analysis. In-situ hybridization and immunohistochemistry were utilized to determine distribution of KGF and KGFR in the liver. RESULTS: Expression of KGF was significantly increased in damaged liver tissue in correlation to the degree of fibrosis, whereas expression of the receptor was up-regulated in early stages of liver fibrosis and down-regulated in cirrhotic organs. Protein expression of this growth factor and its receptor correlated with the alterations in mRNA. KGF expression was restricted to mesenchymal cells, whereas expression of KGFR was detected on hepatocytes only. CONCLUSION: The expression of KGF and KGFR is differentially and significantly regulated in damaged liver tissue. This growth factor might therefore not only contribute to morphological alterations but also regeneration of liver parenchyma most likely mediated by indirect mechanisms of action.

Oral administration of taurolidine ameliorates chronic DSS colitis in mice.

Taurolidine (TRD) has antimicrobial and anti-inflammatory properties. However, the anti-inflammatory effects of TRD in inflammatory bowel diseases (IBD) have not been investigated. Here, we have analyzed the toxicity of TRD after oral long-term application in mice and examined the impact of oral TRD in a dextran sulfate sodium (DSS) model of experimental colitis. Female C57/BL6 mice received TRD in various concentrations (0.1% to 0.4%) for 60 days. Toxicity was evaluated by use of a disease activity index (DAI) and histological examination of major metabolic organs. Furthermore, the impact of 0.2% TRD on a chronic DSS colitis was examined by daily DAI, histological crypt damage score (CDS), bacterial translocation into mesenteric lymph nodes (MLN), and colonic expression of tumor necrosis factor (TNF) alpha, transforming growth factor (TGF) beta, interleukin (IL)-1beta, IL-6, cytochrome oxidase (COX)-2, and monocyte chemotactic protein (MCP)-1 by real-time polymerase chain reaction (PCR). Oral TRD administration for 60 days was well tolerated by the animals and did not show any toxic effects in terms of DAI and histological changes. TRD treatment of DSS colitis led to increased survival of 100%, compared to 33% in the untreated colitis group (p < or = .005). Clinical amelioration was mirrored by significantly reduced DAI and CDS in the TRD treated colitis. Colonic cytokine expression and bacterial translocation into MLN showed no differences between both groups. We thus report for the first time that oral application of TRD results in amelioration of an experimental IBD model. We hypothesize direct intraluminal antimicrobial effects of TRD as well as anti-inflammatory effects during the acute phase of DSS colitis.

Rapid tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130Cas by gastrin in human colon cancer cells.

Although the expression of CCK(2) receptors is widely reported in human colorectal cancers, little is known on its role in mediating the proliferative effects of mature amidated gastrin (G17 amide) on colorectal cancers. The purpose of the present study was to determine the effects of G17 amide on tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and p130 Crk-associated substrate (p130(Cas)) in Colo 320 cells, a human colorectal cancer cell line which expresses CCK(2) receptors. By immunoprecipitation and immunoblotting, an increase in tyrosine phosphorylation of FAK (tyrosine-397), paxillin (tyrosine-31), and p130(Cas) was detected in a time- and dose-dependent manner. Overexpression of CCK(2) receptors in Colo 320 cells (Colo 320 WT) by stable transfection with the human CCK(2) receptor cDNA resulted in an increased tyrosine phosphorylation of FAK, paxillin, and p130(Cas). After incubation with 1 microM L-365,260, a specific CCK(2) receptor antagonist, this increase was completely inhibited. Our results demonstrate that in human colon cancer cells, gastrin caused a rapid tyrosine phosphorylation of FAK, paxillin, and p130(Cas) by activation of CCK(2) receptor. The phosphorylation of these proteins might be important in mediating gastrin effects on proliferation, apoptosis, and metastasis.

Antimicrobial peptides in innate immunity of the human intestine.

The intestinal mucosa has to withstand exposure to a variety of substances, challenges in pH, temperature, and osmolarity; and, finally, bacterial products which might induce local and systemic inflammatory responses. The mucosal integrity is conserved by a defense system which consisting of constitutive and inducible mechanisms. These include the physical barrier function; the secretion of factors into the lumen, such as mucins and antibacterial substances; the mucosal immune system; and, finally, the ability of the mucosa to reconstitute once damage has occurred. The homeostasis and integrity of the gastrointestinal mucosa ultimately depends upon the balance between defensive and aggressive factors. While the physical barrier function was formerly believed to play the major role in mucosal protection against luminal bacteria, the recent discovery of Toll-like receptors and antimicrobial peptides in the intestinal epithelium has modified the concept of intestinal defense towards a more active character, which will be discussed in this review.

Effects of the cathelicidin LL-37 on intestinal epithelial barrier integrity.

The human cathelicidin LL-37 is involved in innate immune responses, angiogenesis and wound healing. Functions in maintenance and re-establishment of intestinal barrier integrity have not been characterized yet. Following direct and indirect stimulation of human colonic HT-29 and Caco-2 cells with LL-37 the cellular viability, rate of apoptosis, proliferation and wound healing were determined. Expression of mucins and growth factors was quantified by real-time PCR and Western blotting. Direct application of LL-37 stimulated migration in Caco-2 cells expressing the proposed LL-37 receptor P2X7. Intestinal epithelial cell (IEC) proliferation was not altered. Indirectly, LL-37 significantly enhanced IEC migration via release of growth factors from subepithelial fibroblasts and IEC. Furthermore, LL-37 induced the expression of protective mucins in IEC and abated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis in IEC. LL-37 induced signaling is mediated in part by the P2X7 receptor, the epidermal growth factor receptor and the p38 mitogen-activated protein kinase (MAPK). LL-37 contributes to maintenance and re-establishment of the intestinal barrier integrity via direct and indirect pathways. These features, in addition to its known antimicrobial properties, suggest an important role for this peptide in intestinal homeostasis.

Cell differentiation dependent expressed CCR6 mediates ERK-1/2, SAPK/JNK, and Akt signaling resulting in proliferation and migration of colorectal cancer cells.

The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.

IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.

IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-alpha, IL-1beta, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation (P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration (P < 0.00001) as well as mRNA expression of TNF-alpha, IL-8, and human beta-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn's disease and correlated highly with the IL-8 expression in these lesions (r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and IEC migration.

CXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP-9 activation.

Colorectal cancer (CRC) is characterized by a distinct metastatic pattern resembling chemokine-induced leukocyte trafficking. This prompted us to investigate expression, signal transduction and specific functions of the chemokine receptor CXCR4 in CRC cells and metastases. Using RT-PCR analysis and Western blotting, we demonstrated CXCR4 and CXCL12 expression in CRC and CRC metastases. Cell differentiation increases CXCL12 mRNA levels. Moreover, CXCR4 and its ligand are inversely expressed in CRC cell lines with high CXCR4 and low or not detectable CXCL12 expression. CXCL12 activates ERK-1/2, SAPK/JNK kinases, Akt and matrix metalloproteinase-9. These CXCL12-induced signals mediate reorganization of the actin cytoskeleton resulting in increased cancer cell migration and invasion. Moreover, CXCL12 increases vascular endothelial growth factor (VEGF) expression and cell proliferation but has no effect on CRC apoptosis. Therefore, the CXCL12/CXCR4 system is an important mediator of invasion and metastasis of CXCR4 expressing CRC cells.

Expression of keratinocyte growth factor and its receptor in adaptive changes of ileorectal pouch mucosa.

OBJECTIVE: Total proctocolectomy with formation of an ileo-anal pouch is a well-established surgical procedure for patients with ulcerative colitis (UC) or familiar adenomatous polyposis (FAP). The pouch mucosa undergoes adaptive changes, with inflammation of the ileal reservoir being the most common complication. The aetiology is unknown. The keratinocyte growth factor (KGF) has not only been shown to promote intestinal wound healing and re-epithelialization but also to have some immunomodulatory properties. This study was designed to investigate a putative involvement of KGF in observed histomorphological changes in the pouch mucosa. MATERIALS AND METHODS: Multiple biopsies were obtained from age-matched and sex-matched patients. Biopsies were stained with H&E and scored for inflammation and morphological changes. mRNA expression levels of KGF and KGF-receptor (KGFR) were determined using competitive RT-PCR, protein expression and phosphorylation was analyzed by Western blotting. KGF and KGFR were localized in tissue samples by immunohistochemistry. RESULTS: Expression of KGF and KGFR was significantly increased in inflamed and adapting mucosa. Patterns of expression did not significantly differ in pouch mucosa from UC or FAP patients. Protein expression correlated with the mRNA results and KGFR was shown to be activated in adapting pouch mucosa. KGF was detected on subepithelial cells, mainly on fibroblasts, whereas expression of KGFR was restricted to epithelial cells. CONCLUSION: Expression of KGF and KGFR is significantly increased in the pouch mucosa, suggesting an involvement of this growth factor in tissue repair and adaptive changes. Topical application of KGF might alleviate the inflammatory and promote the adaptive process in the ileo-anal pouch mucosa.

IL-28A and IL-29 mediate antiproliferative and antiviral signals in intestinal epithelial cells and murine CMV infection increases colonic IL-28A expression.

Human cytomegalovirus virus (CMV) is a major cause of morbidity and mortality in immunocompromised individuals. Recently, a novel group of cytokines [interleukin (IL)-28A/B and IL-29, also termed interferon (IFN)-lambdas] has been described. Here, we demonstrate that intestinal epithelial cell (IEC) lines as well as murine and human colonic tissue express the IFN-lambda receptor subunits IL-28R and IL-10R2. IL-28A and IL-29 binding to their receptor complex activates ERK-1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase MAPKs and Akt, resulting in increased IL-8 protein expression. IFN-lambdas also induce phosphorylation of signal transducer and activator of transcription 1 and significantly increase mRNA expression of suppressor of cytokine signaling 3 and the antiviral proteins myxovirus resistance A and 2',5'-oligoadenylate synthetase. These signals result in an up to 83% reduction of cells positive for human CMV immediate-early protein after human CMV infection. In mice, IL-28A mRNA expression is upregulated after infection with murine CMV in vivo. Both IL-28A and IL-29 significantly decrease cell proliferation but have no effect on Fas-induced apoptosis. In conclusion, IECs express functional receptors for IFN-lambdas, which mediate antiviral and antiproliferative signals in IECs, suggesting a potential for therapeutic use in certain viral infections and as (antiproliferative) anticancer therapy.