Biomedical subjects

Gerhard Rogler

Publications and source records attributed to Gerhard Rogler.

At least 19 recordsLinked to original sources

PubMed2006

Evidence for a differential expression of fibronectin splice forms ED-A and ED-B in Crohn's disease (CD) mucosa.

BACKGROUND AND AIMS: Fibronectin (FN) is an essential factor for the induction of migration of primary colonic lamina propria fibroblasts (CLPF). The FN isoform ED-A is an important inducer of migration. Recently, we have shown that CLPF isolated from inflamed Crohn's disease (CD) mucosa migrated significantly less than control CLPF. We, therefore, investigated changes in FN or integrin expression that could be relevant for CLPF migration. MATERIALS AND METHODS: mRNA of control-CLPF and CLPF isolated from fibrotic mucosa of CD patients was subtractively hybridized. Expression of FN, ED-A, and ED-B in frozen sections from intestinal mucosa was determined by immunohistochemistry. The mRNA expression of the FN isoforms in control, CD, and fibrosis biopsies was quantified by real-time polymerase chain reaction (PCR). Integrin alpha5beta1 protein and mRNA expression was analyzed by fluorescence activated cell sorting (FACS) and PCR, respectively. RESULTS: Subtractive hybridization indicated differential regulation of FN isoform expression in CD. The immunohistochemical analysis of FN protein revealed a reduction of FN isoforms in inflamed CD mucosa compared to control mucosa. In CD fistulae, the ED-A and ED-B isoforms were virtually absent. In fibrotic mucosa, both proteins were increased. Real-time PCR showed a decrease of FN and ED-A expression during mucosal inflammation in CD in contrast to UC and a significant increase of FN and isoforms in CD fibrosis. No difference was found for protein and mRNA of integrin alpha5beta1 in control, CD, and fibrosis CLPF by FACS and PCR. CONCLUSION: Downregulated expression of migration-inducing FN-isoforms in contrast to unchanged FN receptor expression may contribute to the observed alterations of CD CLPF migration.

Adult↗

PubMed2006

(E)-metanicotine hemigalactarate (TC-2403-12) inhibits IL-8 production in cells of the inflamed mucosa.

BACKGROUND: Nicotine is of therapeutic value in ulcerative colitis, but its administration is connected with adverse events. Nicotine derivatives are currently being tested to maintain the therapeutic effects and minimize adverse events. TC-2403-12 is a (E)-metanicotine hemigalactarate. The aim of this study was to determine the effectiveness of TC-2403-12 in the inhibition of TNF- and lipopolysaccharide (LPS)-induced cell activation. METHODS: Colonic epithelial cells (CEC), monocytes (MM6), granulocytes, and the intestinal epithelial cell line HT-29 were stimulated with TNF and LPS and treated with TC-2403-12. IL-8 secretion in the cell supernatants and NF-kappaB activation were determined by ELISA. Apoptosis was quantified by flow cytometry. RESULTS: In MM6 cells, IL-8 secretion was significantly decreased to 30% of control after TC-2403-12 treatment, with best results after pretreatment for 24 h. This decrease in cell activation was not due to apoptosis and was not mediated by inhibition of NF-kappaB activation. IL-8 production in neutrophils and primary CEC also tended to be decreased after TC-2403-12 treatment. TC-2403-12 had no influence on IL-8 secretion of HT-29 cells. CONCLUSION: TC-2403-12 effectively inhibited TNF- and LPS-induced IL-8 production in different cell types. No toxic effects occurred at the concentrations used. Preincubation of cells with TC-2403-12 showed the best effects.

Adjuvants, Immunologic↗

PubMed2006

[The German competence network inflammatory bowel disease (KNCED) -- network research leads to the identification of the cause of disease and to the improvement in patient care].

The competence network chronic inflammatory bowel disease (KN-CED) is one of 17 networks of competence initiated by the German Federal Ministry of Education and Research (BMBF). These networks are concerned with disease patterns which are characterized by their high frequency, high mortality rate or which present a large expense factor. The project-executing organization is the German Center for Air and Space Travel (DLR e. V.). The central structure of organization is the Telematic Platform for medical Networks (TMF e. V.). Aim of the KN-CED is to investigate, in their complexity, the incurable chronic diseases ulcerative colitis and Crohn's disease, particularly with regard to the causes of disease, the establishment of new therapy standards as well as patient care. To achieve this goal, the competence network is integrated into both national and international research associations and is also backed by the national self-help group DCCV and the pharmaceutical industry. Principal items of the competence network are the core facilities and their main focus on molecular genetics, animal and cell models and serum markers. Having stored the data of more than 4,000 patients so far, the central database of the competence network is one of the largest databases worldwide with regard to inflammatory bowel disease (IBD). The successful cooperation within the network is reflected in numerous publications. Thus, two of the three known genes of Crohn's disease were identified. Also with the participation of the competence network national guidelines for the diagnosis and therapy of IBD were generated.Furthermore, the competence network operates study centers where significant therapeutic developments in the field of biotechnological drugs are taking place. The analysis of existing structures of care as well as the development of standards of organization for patients with IBD top the research within the competence network and emphasize the claim to find comprehensive answers to the questions connected with IBD.

Clinical Trials as Topic↗

PubMed2006

Prognostic significance of NOD2/CARD15 variants in HLA-identical sibling hematopoietic stem cell transplantation: effect on long-term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination.

To assess the role of NOD2/CARD15 variants on the long-term outcome of allogeneic stem cell transplantation in a genetically homogeneous group, we extended our previous study (cohort I, n = 78) and typed DNA for NOD2/CARD15 single nucleotide polymorphisms (SNPs) from an additional 225 recipients and their HLA-identical sibling donors (cohort II) treated at four other European centers. Results of genotyping were compared with clinical outcome. The strong association of NOD2/CARD15 variants with transplantation-related mortality (TRM) was confirmed in univariate and multivariate analysis; TRM increased from 20% in cohort I/22% in cohort II in recipient/donor pairs without any NOD2/CARD15 variants to 47% in cohort I/32% in cohort II in the presence of one variant in either donor or recipient and further to 57% in cohort I/74% in cohort II in the presence of 2 or more variants (P < .002 in both cohorts). NOD2/CARD15 SNPs were not associated with relapse rate but had a strong impact on overall survival. In an analysis of center effects, the type of gastrointestinal decontamination was the only factor interfering with the prognostic significance of NOD2/CARD15 SNPs. Our data further support an interaction between gastrointestinal defense mechanisms, activation of the innate immune system, and specific transplant-related complications.

Adolescent↗

PubMed2006

IL-15 protects intestinal epithelial cells.

IL-15, a T-cell growth factor, has been shown to be increased in inflammatory bowel disease (IBD). It has been suggested that neutralization of IL-15 could protect from T cell-dependent autoimmune inflammation. On the other hand, an anti-apoptotic effect of IL-15 has been demonstrated in kidney epithelial cells during nephritis. We therefore tested the role of IL-15 in two different experimental models of colitis in vivo, and in models of intestinal epithelial cell (IEC) apoptosis in vitro. IL-15 blockade in chronic dextran sulphate sodium-induced colitis resulted in aggravation of the disease with a significantly 2.1-fold increased epithelial damage score compared to controls. TUNEL staining clearly revealed increased apoptosis. IL-6, TNF and IFN-gamma secretion by mesenteric lymph node cells were increased. In the T cell-dependent SCID transfer model of colitis IL-15 neutralization reduced the inflammatory infiltration and proinflammatory cytokine production. Despite that, the intestinal epithelial damage was not reduced. In vitro, IL-15 pre-incubation prevented up to 75% of CH11 antibody-induced apoptosis in SW-480 cells and reduced caspase-3 activity. According to this, endogenously produced IL-15 in chronic colitis does not only act as a proinflammatory cytokine but has at the same time the potential to reduce mucosal damage by preventing IEC apoptosis.

Animals↗

PubMed2006

A new organotypic model to study cell interactions in the intestinal mucosa.

BACKGROUND: Recently, we demonstrated that freshly elutriated monocytes differentiate into macrophages with a phenotype similar to that of intestinal macrophages in a three-dimensional model of intestinal epithelial cells. Here we describe a more organotypic model to study cell interactions in the intestinal mucosa. METHODS: Primary intestinal fibroblasts and freshly elutriated blood monocytes (ratio 1:1) were embedded in collagen type I gels and cultured for 5 days. At day 5, intestinal epithelial cells (HT-29) were seeded on top of the collagen gels. After another 7 days collagen gels were harvested and fixed for immunohistochemical analysis. Cryosections of the aggregates were prepared and staining for monocyte/macrophage markers and basement membrane compounds was performed. Cell interactions inside the aggregates were examined by electron microscopy. RESULTS: Intestinal fibroblasts contracted the collagen gels which formed stable three-dimensional aggregates within the first 5 days of culture. Intestinal epithelial cells formed a monolayer on top of the gels about 3 days after seeding. Intestinal fibroblasts were distributed randomly over the aggregate. Monocytes inside aggregates were localized in the vicinity to epithelial cells by positive staining for CD68. Typical monocyte/macrophage specific markers such as CD14, CD16, CD11b, CD11c and the co-stimulatory molecules CD80 and CD86 were down-regulated or not detectable on these cells after co-culture in three-dimensional aggregates. Omission of epithelial cells from the model was followed by impaired differentiation of intestinal macrophages. CONCLUSION: In the three-dimensional organotypic cell culture model monocytes differentiate into intestinal-like macrophages when co-cultured with control intestinal fibroblasts and intestinal epithelial cells. Intestinal epithelial cells may be necessary for differentiation of intestinal macrophages.

Cell Communication↗

PubMed2006

Profiling adipocytokine secretion from creeping fat in Crohn's disease.

BACKGROUND: Adipose tissue is recognized as a compartment secreting highly active molecules. Creeping fat represents a characteristic feature of Crohn's disease (CD). Proinflammatory or anti-inflammatory adipose-derived secretory products, now generally called adipocytokines, may play a role in the pathogenesis of CD. MATERIALS AND METHODS: Adipose tissue specimens were obtained from creeping fat contiguous to the involved intestine of 10 patients with CD. Mesenteric adipose tissue specimens resected from 13 patients with colon cancer (CC) and from 7 patients with diverticulitis served as controls. Three fat tissue specimen per well and 6 to 8 wells per patient were incubated ex vivo for 24 h. The release of adiponectin, resistin, leptin, interleukin-6, macrophage colony-stimulating factor, monocyte chemotactic protein-1, and migration inhibitory factor was measured by ELISA. The expression of AdipoR1 and AdipoR2 receptors was investigated by real-time quantitative polymerase chain reaction in a subset of adipose tissues. RESULTS: The secretion of adiponectin and macrophage colony-stimulating factor, as well as leptin and migration inhibitory factor, was significantly upregulated in CD compared with CC and diverticulitis or CC only, respectively. Resistin, interleukin-6, and monocyte chemotactic protein-1 were not specifically induced in CD but were associated with unspecific inflammation. Adiponectin receptor expression was not different in CC, CD, or diverticulitis. Steroid treatment in CD patients had differential effects on the ex vivo secretion of adipocytokines. CONCLUSIONS: A specific secretion pattern of proinflammatory and anti-inflammatory adipocytokines indicates the significance of adipose tissue in intestinal inflammation in CD. Future investigations of this intestinal compartment may provide novel insights into the pathophysiological role of creeping fat and CD.

Adipose Tissue↗

PubMed2006

Multiparameter immunofluorescence on paraffin-embedded tissue sections.

Immunohistochemical techniques have gained increasing importance in diagnostics and research. While formalin-fixed, paraffin-embedded human tissue retains excellent morphology, the detection of antigens by immunofluorescence in its sections and especially the demonstration of multiple simultaneous antibodies have limitations. Double immunofluorescence labeling of routinely processed paraffin sections has been described previously. The signal intensity observed after triple labeling has been reported to be significantly inferior to that obtained by application of double fluorochromes. The authors show multicolor labeling of three and four primary antibodies in routinely processed paraffin-embedded tissue sections using a standardized immunofluorescence technique. In addition, procedures to reduce background staining and to avoid nonspecific double staining are described.

Antibodies↗

PubMed2006

Secretion of RANTES (CCL5) and interleukin-10 from mesenteric adipose tissue and from creeping fat in Crohn's disease: regulation by steroid treatment.

BACKGROUND: Creeping fat represents a characteristic feature of Crohn's disease (CD) and adipose tissue is currently being recognized as a complex compartment secreting highly active molecules. Pro- or anti-inflammatory adipose tissue-derived secretory products (adipocytokines) might play a role in the pathogenesis of CD. METHODS: Adipose tissue specimens were obtained from creeping fat contiguous to the involved intestine of 10 patients with CD. Mesenteric adipose tissue specimens resected in 13 patients with colon cancer (CC) and in seven patients with diverticulitis (DIV) served as controls. Three fat tissue specimens per well and n = 6-8 wells per patient were incubated ex vivo for 24 h. The release of regulated on activation, T-cell expressed and secreted (RANTES) and interleukin (IL)-10 into the supernatant was measured by ELISA. RESULTS: Both RANTES and IL-10 secretion could be demonstrated from total adipose tissue explants. The RANTES secretion is increased from creeping fat in CD (3691 +/- 597 pg/g fat per 24 h) when compared to mesenteric adipose tissue from patients with CC (1690 +/- 191 pg/g fat per 24 h; P < 0.0001) or DIV (1672 +/- 336 pg/g fat per 24 h; P < 0.0001). In contrast, IL-10 secretion is downregulated significantly only in patients with DIV (1418 +/- 180 pg/g fat per 24 h; P = 0.016) when compared to CC patients (2368 +/- 259 pg/g fat per 24 h). Crohn's disease patients receiving steroids had a higher secretion rate of RANTES and IL-10. CONCLUSIONS: Both RANTES and IL-10 secretion can be detected from mesenteric adipose tissue and from creeping fat. The elevated RANTES and IL-10 secretion from creeping fat in CD is not due to a CD-specific effect but caused by steroid treatment.

Adipose Tissue↗

PubMed2006

Vascular endothelial growth factor secretion from mesenteric adipose tissue and from creeping fat in Crohn's disease.

BACKGROUND: Creeping fat represents a characteristic feature of Crohn's disease (CD), and adipose tissue secretes adipocytokines and chemokines/growth factors such as vascular endothelial growth factor (VEGF). Because VEGF serum levels and mucosal VEGF expression is elevated in CD patients, the aim of the present paper was to investigate creeping fat-derived VEGF secretion in CD. MATERIAL AND METHODS: Adipose tissue was obtained from creeping fat of 10 patients with CD. Mesenteric adipose tissue was resected from 13 patients with colon cancer (CC) and from seven patients with diverticulitis (DIV). Three fat tissue specimens per well, and several wells (6-8) per patient were incubated ex vivo for 24 h. The release of VEGF into the supernatant was measured by ELISA. RESULTS: There was stable VEGF secretion from mesenteric adipose tissue of patients with CC or DIV and from creeping fat of patients with CD. Whereas the VEGF secretion rate was not different between patients with CD (465 +/- 98 pg/g fat per 24 h) and CC (399 +/- 48 pg/g fat per 24 h), VEGF secretion was significantly reduced in patients suffering from DIV (115 +/- 41 pg/g fat per 24 h; P < 0.0001 and P = 0.001, respectively). The CD patients treated with steroids had significantly lower VEGF secretion rates (294 +/- 42 pg/g fat per 24 h) than CD patients not receiving steroids (607 +/- 105 pg/g fat per 24 h; P = 0.001). CONCLUSIONS: Creeping fat is an important source of VEGF secretion. The characteristics of the inflammatory changes in CD might be due to the lack of VEGF downregulation that is seen in DIV.

Adipose Tissue↗

PubMed2005

Functional expression of the interleukin-11 receptor alpha-chain in normal colonic epithelium and colon cancer.

BACKGROUND: Interleukin-11 (IL-11) has been evaluated as an anti-inflammatory and mucosa-protective therapeutic agent in inflammatory bowel diseases (IBDs). Activity of IL-11 requires binding to the alpha receptor subunit (IL-11Ralpha) that provides ligand specificity. Recently, we showed that in the intestinal mucosa, IL-11Ralpha is mainly present on epithelial cells mediating antiapoptotic effects. The aim of this study was to investigate the expression profiling of IL-11Ralpha and its downstream signaling cascade in colonic adenoma and carcinoma. MATERIALS AND METHODS: The expression of IL-11Ralpha in normal colonic mucosa, 11 colonic adenomas, and 10 carcinomas was analyzed by immunohistochemistry. In addition, IL-11Ralpha-expression and IL-11Ralpha-induced phosphorylation of signal transducer and activator of transcription (STAT)3 were investigated by Western blot analysis. RESULTS: Immunohistochemistry revealed significant IL-11-Ralpha expression in epithelial cells of normal colonic mucosa. In contrast, the expression of IL-11-Ralpha in colon adenomas and carcinomas was either absent or only detectable in very few scattered epithelial cells. Densitometric analysis of Western blots confirmed these results, showing a decrease of IL-11Ralpha-protein in cells isolated from adenomas or carcinomas. Reduced STAT3-phosphorylation in carcinoma cells indicated functional consequences of decreased IL-11Ralpha-protein expression on signal transduction. CONCLUSION: This study demonstrates a decrease of IL-11-Ralpha-protein expression in epithelial cells isolated from colon carcinomas and adenomas compared to normal colonic mucosa and a reduced STAT3 signaling. Because of reduced binding and signal transduction, it is unlikely that therapeutically administered IL-11 would contribute to colorectal carcinoma induction and growth.

Adult↗

PubMed2005

Physiological role of macrophage inflammatory protein-3 alpha induction during maturation of intestinal macrophages.

Intestinal macrophages (IMAC) are a central component in the defense of the intestinal mucosa against luminal microbes. In normal mucosa, monocytes differentiate to immunologically tolerant IMAC with a typical phenotype lacking activation markers such as CD14 and TLRs 2 and 4. CD33+ IMAC were isolated from normal intestinal mucosa by immunomagnetic beads. A subtractive hybridization subtracting mRNA from normal IMAC from those of in vitro differentiated macrophages was performed. IMAC differentiation was studied in multicellular spheroids (MCS). Functional assays on migration of CD45R0+ T cells were performed in MCS coculture models. Of 76 clones, 3 obtained by subtractive mRNA hybridization showed >99% homology to mRNA of MIP-3alpha, indicating that this chemokine is induced in IMAC compared with in vitro differentiated macrophages. MIP-3alpha protein expression was confirmed in cryostat sections of normal intestinal mucosa by immunohistochemistry. IMAC in the lamina propria stained positive for MIP-3alpha. FACS of purified IMAC clearly indicated expression of MIP-3alpha in these cells. In the MCS-in vitro differentiation model for IMAC, MIP-3alpha protein expression was absent on day 1 but detectable on day 7 of coculture, demonstrating the induction of MIP-3alpha during differentiation of IMAC. IMAC attracted CD45R0+ T cells to migrate into an MCS coculture model. In human mucosa, a close contact between IMAC and CD45R0+ T cells could be demonstrated. MIP-3alpha is induced during the differentiation of monocytes into IMAC. Our data suggest that MIP-3alpha expression could be involved in the recruitment of CD45R0+ cells into the lamina propria.

Adult↗

PubMed2005

Serum antibodies to EpCAM in healthy donors but not ulcerative colitis patients.

PURPOSE: The gastrointestinal carcinoma-associated antigen epithelial cell adhesion molecule (EpCAM) has been a target for passive and active immunotherapy of gastrointestinal carcinoma patients. The antigen is expressed by both tumor and normal tissues. The immunogenicity of EpCAM in colorectal cancer patients has been described previously. The purpose of this study was to evaluate humoral and cellular immune responses of healthy individuals and ulcerative colitis patients to EpCAM and to relate immune responses to colonic tissue expression of EpCAM. METHODS: An inhibition radioimmunoassay was used to detect anti-EpCAM serum antibodies. Anti-EpCAM antibodies of a healthy donor were expressed by phages and sequenced. (3)H-thymidine incorporation assay was used for detection of lymphoproliferative responses to stimulation with EpCAM. EpCAM tissue expression was determined by immunohistochemistry. RESULTS: We detected anti-EpCAM serum antibodies in 4 of 10, and EpCAM-specific lymphoproliferation responses in 1 of 10 healthy volunteers. The majority of anti-EpCAM antibodies derived from a healthy donor were germline-encoded. In contrast, none of the 23 patients with ulcerative colitis showed serum antibodies to EpCAM (P=0.005). Antigen expression was greatly reduced and altered in ulcerative colitis patients, whereas colon from healthy individuals and uninvolved colon of colorectal cancer patients expressed high levels of EpCAM. CONCLUSION: The results of these studies suggest an association between EpCAM antibody production and colonic EpCAM expression in healthy individuals and patients with ulcerative colitis. Decreased and altered colonic EpCAM expression in ulcerative colitis patients may be related to the disease induction, based on the previously demonstrated adhesion function of this molecule. Healthy individuals with anti-EpCAM immune responses and high risk for developing colorectal carcinoma are prime candidates for prophylactic immunization against EpCAM.

Adult↗

PubMed2005

Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro.

AIM: Eph receptor tyrosine kinases and their membrane bound receptor-like ligands, the ephrins, represent a bi-directional cell-cell contact signaling system that directs epithelial movements in development. The meaning of this system in the adult human gut is unknown. We investigated the Eph/ephrin mRNA expression in the intestinal epithelium of healthy controls and patients with inflammatory bowel disease (IBD). METHODS: mRNA expression profiles of all Eph/ephrin family members in normal small intestine and colon were established by real-time RT-PCR. In addition, differential expression in IBD was investigated by cDNA array technology, and validated by both real-time RT-PCR and immunohistochemistry. Potential effects of enhanced EphB/ephrin-B signaling were analyzed in an in vitro IEC-6 cell scratch wound model. RESULTS: Human adult intestinal mucosa exhibits a complex pattern of Eph receptors and ephrins. Beside the known prominent co-expression of EphA2 and ephrinA1, we found abundantly co-expressed EphB2 and ephrin-B1/2. Interestingly, cDNA array data, validated by real-time PCR and immunohistochemistry, showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis. Stimulation of ephrin-B signaling in ephrin-B1/2 expressing rat IEC-6-cells with recombinant EphB1-Fc resulted in a significant dose-dependent acceleration of wound closure. Furthermore, fluorescence microscopy showed that EphB1-Fc induced coordinated migration of wound edge cells is associated with enhanced formation of lamellipodial protrusions into the wound, increased actin stress fiber assembly and production of laminin at the wound edge. CONCLUSION: EphB/ephrin-B signaling might represent a novel protective mechanism that promotes intestinal epithelial wound healing, with potential impact on epithelial restitution in IBD.

Crohn Disease↗

PubMed2005

Comparison of capsule endoscopy and magnetic resonance (MR) enteroclysis in suspected small bowel disease.

BACKGROUND AND AIMS: Small bowel MR enteroclysis and wireless capsule endoscopy (WCE) are new diagnostic tools for the investigation of the small bowel. The aim of this study was to compare the diagnostic yield of WCE with MR enteroclysis in the detection of small bowel pathologies. METHODS: A total of 36 patients were included in the study. Indications for imaging of the small bowel were proven or suspected small bowel Crohn's disease (CD; n=18), obscure gastrointestinal (GI) bleeding (n=14) and tumour surveillance (n=4). RESULTS: In patients with Crohn's disease WCE detected significantly more inflammatory lesions in the first two segments of the small bowel compared with MR enteroclysis (12 patients vs. 1 patient, p=0.016). In 5 out of 14 (36%) patients with GI bleeding, angiodysplasia was detected as a possible bleeding source. Three of these patients had active bleeding sites detected by WCE. One patient had scattered inflammation of the mucosa. MR enteroclysis did not reveal any intestinal abnormalities in this patient group. MR enteroclysis provided extraintestinal pathologies in 10 out of 36 (28%) patients. CONCLUSION: In patients with Crohn's disease WCE revealed significantly more inflammatory lesions in the proximal and middle part of the small bowel in comparison to MR enteroclysis, whereas in patients with obscure GI bleeding WCE was superior to MR enteroclysis.

Adolescent↗

PubMed2005

Epidermal growth factor partially restores colonic ion transport responses in mouse models of chronic colitis.

BACKGROUND & AIMS: Epidermal growth factor receptor (EGFR) activation, which plays an important role in regulating intestinal ion transport, can alleviate clinical symptoms such as diarrhea in patients with ulcerative colitis and promote mucosal restitution in animal models of colitis. Here, we investigate whether EGFR can regulate colonic ion transport in the setting of colitis. METHODS: Distal colon from control mice and mice with colitis was retained for immunohistochemistry or mounted in Ussing chambers. Ion transport responses across the tissues to the calcium agonist carbachol and the adenosine 3',5'-cyclic monophosphate agonist forskolin were measured with or without epidermal growth factor (EGF) pretreatment. RESULTS: EGF pretreatment of normal colonic mucosa inhibited ion transport responses to carbachol and forskolin but potentiated the reduced ion transport responses seen in dextran sulfate sodium (DSS)-treated and mdr1a knockout mouse colon. Ion substitution studies and the sodium transport inhibitor amiloride showed that sodium movement primarily accounted for the potentiating effect of EGF in DSS-treated tissues, despite decreased sodium channel expression. EGF potentiation of transport responses in DSS-treated colon was completely blocked by the cytoskeletal disruptor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas the novel and conventional protein kinase C isoform inhibitor Gö6850 and the extracellular signal-regulated kinase inhibitor PD98059 partially reduced EGF effects. EGFR epithelial distribution and transforming growth factor alpha expression were also altered in DSS-treated tissues. CONCLUSIONS: Chronic inflammation uncovers a potentiating effect of EGFR activation on epithelial electrogenic sodium absorption that would be expected to ameliorate diarrheal symptoms associated with colitis.

Animals↗

PubMed2005

Strong expression of methylthioadenosine phosphorylase (MTAP) in human colon carcinoma cells is regulated by TCF1/[beta]-catenin.

Methylthioadenosine phosphorylase (MTAP) is known as a ubiquitously expressed house keeping gene important in biochemical salvage processes. The MTAP gene is localized on the human chromosomal region 9p21, a region often deleted in cancer. Recently, several groups including our own have shown that MTAP serves as a tumour suppressor gene. The aim of this study was to analyse the role of MTAP in colon carcinoma and normal colon epithelium and the regulation of gene expression. To examine MTAP RNA and protein expression, we screened six colon carcinoma cell lines and human primary colon epithelial cells by RT-PCR and immunoblotting. MTAP expression was confirmed in vivo by immunohistochemical staining of normal colon tissue compared to adenoma and colon carcinoma. Interestingly, we found strong MTAP mRNA and protein expression by colon carcinoma cell lines but no expression by colonic epithelial cells. To analyse the regulation of MTAP expression, promoter studies were performed and revealed control of MTAP expression by LEF/TCF/beta-catenin. Furthermore, we demonstrated a significant correlation between MTAP protein expression and tumour progression as the intensity of MTAP protein staining increased from normal tissue to carcinoma. In addition, the recently postulated association between MTAP activity and interferon (IFN) sensitivity was confirmed in colon epithelial cells showing only little response to IFN-gamma, in contrast to the carcinoma cell lines. In summary, these data indicate for the first time that MTAP is not expressed in normal human colonic epithelium but is strongly upregulated in colon carcinoma. This finding may be of clinical significance concerning the homeostasis of normal colon epithelium and potential treatment of colon carcinoma.

Adenocarcinoma↗

PubMed2005

CpG motifs of bacterial DNA essentially contribute to the perpetuation of chronic intestinal inflammation.

BACKGROUND & AIMS: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-induced colitis. Here, we investigated whether DNA derived from luminal bacteria plays a role in the perpetuation of chronic intestinal inflammation. METHODS: Toll-like receptor (TLR9)-deficient and wild-type (wt) control mice were used for the induction of chronic DSS colitis. Moreover, mice with established chronic colitis using different experimental models were treated with adenoviral ODN (AV-ODN) known to block CpG effects. Colonic inflammation was scored and cytokine production was quantified both in colonic tissue and draining mesenteral lymph node cells (MLC). RESULTS: Eight weeks after induction of chronic DSS colitis in TLR9-deficient mice, intestinal inflammation was significantly lower (-68%), and proinflammatory cytokine production was drastically reduced. Treatment of wt mice with chronic DSS-induced colitis with AV-ODN resulted in a significant amelioration of disease with a reduced histologic score (-43%) and reduced cytokine production of MLC (interleukin [IL]-6: -68%; interferon [IFN]-gamma: -48%) and RNA expression of the T helper (Th)1-specific transcription factor T-bet (-62%) in colonic tissue. Qualitatively, the same results were obtained in the severe combined immunodeficiency disease (SCID) transfer model of colitis and in spontaneous colitis in IL-10-deficient mice. CONCLUSIONS: Bacterial DNA derived from luminal bacteria contributes significantly to the perpetuation of chronic intestinal inflammation. Inhibition of the immune-stimulating properties of bacterial DNA using AV-ODN may offer a novel and specific tool for the treatment of inflammatory bowel disease.

Animals↗