Sonja Dullat

Epithelial to Mesenchymal Transition: Expression of the Regulators Snail, Slug, and Twist in Pancreatic Cancer

Purpose: Epithelial to mesenchymal transitions are vital for tumor growth and metastasis. Several inducers of epithelial to mesenchymal transition are transcription factors that repress E-cadherin expression, such as Snail, Slug, and Twist. In this study, we aimed to examine the expression of these transcription factors in pancreatic cancer. Experimental Design: The expression of Snail, Slug, and Twist was detected by immunohistochemistry in tissue samples from patients with pancreatic ductal adenocarcinoma. Five human pancreatic cancer cell lines (AsPC-1, Capan-1, HPAF-2, MiaPaCa-2, and Panc-1) were analyzed by reverse transcription-PCR, real-time PCR, and Western blotting. An orthotopic nude mouse model of pancreatic cancer was applied for in vivo experiments. Results: Seventy-eight percent of human pancreatic cancer tissues showed an expression of Snail, and 50% of the patients displayed positive expression of Slug. Twist showed no or only weak expression. Snail expression was higher in undifferentiated cancer cell lines (MiaPaCa-2 and Panc-1) than in more differentiated cell lines (Capan-1, HPAF-2, AsPC-1). Expression of Slug was detected in all cell lines with different intensities. Twist was not expressed. After exposure to hypoxia, the Twist gene was activated in all five pancreatic cancer cell lines. Conclusions: The transcription factors Snail and Slug are expressed in pancreatic cancer but not in normal tissue, suggesting a role in the progression of human pancreatic tumors. Twist, activated by hypoxia, may play an important role in the invasive behavior of pancreatic tumors.

Expression of catalytic proteasome subunits in the gut of patients with Crohn's disease

Background and purposeActivation of the transcription factor NF-κB by proteasomes and subsequent nuclear translocation of cytoplasmatic complexes play a crucial role in the intestinal inflammation. Proteasomes have a pivotal function in NF-κB activation by mediating degradation of inhibitory IκB proteins and processing of NF-κB precursor proteins. This study aims to analyze the expression of the human proteasome subunits in colonic tissue of patients with Crohn’s disease.Materials and methodsThirteen patients with Crohn’s disease and 12 control patients were studied. The expression of immunoproteasomes and constitutive proteasomes was examined by Western blot analysis, immunoflourescence and quantitative real-time PCR. For real-time PCR, AK2C was used as housekeeping gene.ResultsThe results indicate the influence of the intestinal inflammation on the expression of the proteasomes in Crohn’s disease. Proteasomes from inflamed intestine of patients with Crohn’s disease showed significantly increased expression of immunosubunits on both protein and mRNA levels. Especially, the replacement of the constitutive proteasome subunit β1 by inducible immunosubunit β1i was observed in patients with active Crohn’s disease. In contrast, relatively low abundance of immunoproteasomes was found in control tissue.ConclusionsOur data demonstrate that in contrast to normal colonic tissue, the expression of immunoproteasomes was evidently increased in the inflamed colonic mucosa of patients with Crohn’s disease. Thus, the chronic intestinal inflammation process in Crohn’s disease leads to significant alterations of proteasome subsets.

Permanently increased mucosal permeability in patients with backwash ileitis after ileoanal pouch for ulcerative colitis

Objective. Backwash ileitis (BI) has not been identified as a risk factor for pouchitis. The aim of this study was to investigate the barrier function of the ileoanal pouch depending on the presence of BI. The incidence of pouchitis in a population of ulcerative colitis patients with BI is also reported. Material and methods. Biopsies were taken from 80 patients with ulcerative colitis: a) terminal ileum prior to pouch creation (pre-IAP); b) 16 months after ileostomy closure (intact pouch); and c) during pouchitis. Patients were stratified into the BI group and the non-BI (ØBI) group. Barrier function was determined in Ussing-chambers as epithelial resistance by impedance analysis and as mannitol permeability from 3H-mannitol fluxes. Na+-glucose co-transport was measured as a change in short-circuit current (ISC) after addition of glucose. Relative risk of developing pouchitis was calculated by corrected χ2 test. Results. In 13/21 (BI/ØBI) pre-IAP patients, 23/37 (BI/ØBI) with an intact pouch, and 35/7 (BI/ØBI) with pouchitis, epithelial resistance in BI/ØBI was 13.5±1.6/14.3±0.9 Ω·cm2 for pre-IAP, 12.7±1.3/16.8±1.2 Ω·cm2 (p<0.05 BI versus ØBI) for the intact pouch, and 10.1±1.1/9.9±1.8 Ω·cm2 for pouchitis (p<0.05 BI versus ØBI with an intact pouch). No differences were found for electrogenic chloride secretion and active Na+-glucose co-transport between BI/ØBI in the three groups. In patients with BI, pouchitis was more common (35 versus 7 patients, odds ratio 33.0 (95% CI 8.3–143.9; p<0.0001)). Conclusions. Ulcerative colitis patients with BI show impaired barrier function in the further course of the ileoanal pouch. Thus, BI has a long-term impact on epithelial barrier function.

Increased bacterial permeation in long‐lasting ileoanal pouches

Background and Aims: Bacterial overgrowth appears to play an important role in the pathogenesis of ileoanal pouches. Therefore, the capability of bacterial permeation and its determinants is of great interest. The aim of this study was to examine bacterial permeation in the ileoanal pouch and to correlate the results with the degree of inflammation, the epithelial resistance, the mucosal transport function, and the age of the ileoanal pouches. Materials and Methods: Biopsies were taken from 54 patients before colectomy (n = 13; preileal pouch‐anal anastomosis [IPAA]), and closure of ileostomy (n = 7; deviation), <1 year after closure of ileostomy (n = 8; intact pouch I), >1 year after closure of ileostomy (n = 16; intact pouch II), in the case of pouchitis (n = 11), and in 11 controls. Tissues were mounted in a miniaturized Ussing chamber. Escherichia coli was added to the mucosal side of the Ussing chamber, and the permeation was proven by serosal presence of E. coli. Epithelial and subepithelial resistance was determined by transmural impedance analysis. Active Na+‐glucose cotransport and active Cl− secretion were measured. Specimens were analyzed by fluorescent in situ hybridization with oligonucleotide probes targeting the bacterial 16s ribosomal RNA. The bacteria in and on the tissue were enumerated. Results: Bacterial permeation occurred in 2 of 13 pre‐IPAA, 2 of 7 deviations, 0 of 8 intact pouch I, 9 of 16 intact pouch II, 5 of 11 pouchitis specimens, and 0 of 11 ileum controls. The frequency of bacterial permeation in the intact pouch II group is higher than in the intact pouch I group (P < 0.001). Epithelial resistance, mannitol fluxes, electrogenic chloride secretion, sodium‐glucose cotransport of the bacterially permeated specimens versus nonpermeated of the intact pouch II group, and the pouchitis group and subepithelial resistance remained unchanged. Intramural bacteria could be detected by fluorescence in situ hybridization mainly in long‐lasting pouches, but there was no correlation with bacterial permeation. Conclusions: The long‐lasting ileoanal pouch is associated with increased bacterial permeability. This is not correlated with a disturbed function of the pouch mucosa but could be a precursor of pouchitis.

Association Between Activation of Atypical NF-kappa B1 p105 Signaling Pathway and Nuclear beta-Catenin Accumulation in Colorectal Carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1.

NALP expression in Paneth cells provides a novel track in IBD signaling.

PurposePaneth cells are part of the innate mucosal immunity of the gut with possible regulatory function. This study intends to identify the gene expression pattern of the orthotopic and metaplastic Paneth cells, searching for differences between metaplastic occurrence between Crohns disease and ulcerative colitis.MethodsPaneth cells were collected in RNAse-free conditions via micro dissection. RNA isolation and super amplification was followed by microarray analysis of whole genome expression activity of the orthotopic and metaplastic Paneth cells. Immunohistology of β-catenin and Frizzled-5 receptor was performed.ResultsHistological analysis showed no morphological or secretory change (Frizzled-5 receptor and β-catenin) in orthotopic and metaplastic Paneth cells. Microarray analysis indicated an increased, but not mutant activation of Wnt/β-catenin signaling and firstly showed expression of NALP 1, 7, 8 and 11 in metaplastic Paneth cells.ConclusionsPaneth cells might play a NALP-mediated role in the pathogenesis of IBD.

Metaplastische Paneth Zellen bei chronisch-entzündlichen Darmerkrankungen

The pathogenesis of the inflammatory bowel disease comes out to be complex and multifactor event. Pathological changes seem to occur already in gene pool. The existent term of IBD genetics suggest several known susceptibility genes like NOD2, IL23R, PTPN2 for Crohn’s disease (CD), or IBD5 and ARG16L1 for ibd. Recent studies suggest reduced TCF4 expression in paediatric patients with CD [1]. The mucosal wall plays a pivotal role as high immune-competent barrier. Its function depends on the integrity of the epithelia, tide junctions, submucosal macrophages and dendritic cells. Particular interest merits the Paneth cells, as they are able to produce specific constitutive α-5 and α-6 defensins with antimicrobial activity. Next to the secretor function Paneth cells might participate in the regulation mucosal immunity, these functions are poor understood. The β-catenin/TFC signalling way regulates the epithelial cell differentiation in the crypt-villus-axis. The position and orientation of the Paneth cells depends on availability of the WNT-receptor-frizzled-5. In IBD Paneth cells occur in atypical gut segments of colon and show increased count in ileum in Crohn’s disease. The multiprotein complexes NALP are involved in activating MHC and seem to be novel track in the studies of IBD immunity disorder. NALP1 is promoting through caspase-1 activation the building of proinflammatory cytokine IL-1β, well known to be increased in Crohn’s disease and is usually expressed next to NALP3 and NOD2 in antigen presenting cells. NALP 8 is believed to have antiviral activity in reproductive cells, though no further details are known about NAPL’s 7, 8 or 11 [2].

Enhanced activity of immunoproteasomes in patiens with Crohn’s disease

The eukaryotic 20S proteasome is the main protease in the cytoplasm and nucleus composed of seven different α and seven different β subunits which form four rings stacked on top of each other. It is now known that the constitutive proteasome is the predominant form in most tissue. Our current study demonstrates that the second form, inducible immunoproteasome is more abundant in inflamed ileum and colon of Crohn’s disease. Especially, immunosubunit β1 i is increasingly expressed in the inflamed intestinal tissue of patients with Crohn’s disease. We show here that this subunit exchange alters the cleavage pattern of proteasomes in the inflamed intestine of patients with Crohn’s disease contributing directly to the activation of NF-kB by increasing the processing of p105 and degradation of IkBα. Thus, the immunoproteasome is involved in the enhanced activity of NF.-kB which is one of the most important regulators of proinflammatory gene expression in intestinal inflammation.

Möglichkeit der Pouchitis-Therapie durch Interleukin-10 im ileoanalen Pouch nach Colitis ulcerosa?

Apart of the immunological reaction pouchitis shows many similarities to ulcerative colitis. Concerning the cytokine expression it is well known that during pouchitis the proinflammatory interleukines IL 1-β, IL-8, and TNF-α are upregulated whereas the counterinflammatory interleukine IL-10 is downregulated. The aim of this study was to identify in the long lasting intact ileoanal pouch cytokines which could have an protective effect or could lead to a new therapeutic option. MethodsSpecimens of each 10 were obtained endoscopically from the statuses: pre ileoanal pouch, pre closure of ileostomy, pouch 1 year in function, pouchitis, and controls. IL-6 and TNF-α as proinflammatory and IL-10 as counter inflammatory cytokines were determined. Specimens were conserved at −80 °C. After PCR amplification the DNA concentration was measured and after further dilution the amplification served as a standard under the use of hybridisation probes. After RNA isolation and transcription into cDNA further quantification was performed against GAPDH via realtime-PCR. ResultsIL-6 was reduced in the status of the intact pouch 1 year compared to intact pouch < 1 year, pre closure of ileostomy, and pouchitis (225.9 ± 15.6 vs. 61.03 ± 9.6, 61.03 ± 9.7). Conclusion1. Upregulation of IL-6 or TNF-α during pouchitis could not be observed. 2. Increased IL-10 in the mature pouch might be protective against pouchitis. 3. Topical IL-10 therapy could be a new issue for pouchitis treatment.