Mitochondrial respiration in peripheral arterial disease depends on stage severity.

Peripheral arterial disease (PAD) is an increasing cause of morbidity and its severity is graded based on clinical manifestation. To investigate the influence of the different stages on myopathy of ischemic muscle we analysed severity-dependent effects of mitochondrial respiration in PAD. Eighteen patients with severe PAD, defined as chronic limb-threatening ischemia, 47 patients with intermittent claudication (IC) and 22 non-ischemic controls were analysed. High-resolution respirometry (HRR) was performed on muscle biopsies of gastrocnemius and vastus lateralis muscle of patients in different PAD stages to investigate different respiratory states. Results from HRR are given as median and interquartile range and were normalized to citrate synthase activity (CSA), a marker for mitochondrial content. In order to account for inter-individual differences between patients and controls, we calculated the ratio of O2-flux in gastrocnemius muscle over vastus muscle ('GV ratio'). CSA of the gastrocnemius muscle as a proxy for mitochondrial content was significantly lower in critical ischemia compared to controls. Mitochondrial respiration normalized to CSA was higher in IC compared to controls. Likewise, the GV ratio was significantly higher in IC compared to control. Mitochondrial respiration and CSA of PAD patients showed stage-dependent modifications with greater changes in the mild PAD stage group (IC).

Muscarinic Acetylcholine Receptor M3 Expression and Survival in Human Colorectal Carcinoma-An Unexpected Correlation to Guide Future Treatment?

Muscarinic acetylcholine receptor M3 (M3R) has repeatedly been shown to be prominently expressed in human colorectal cancer (CRC), playing roles in proliferation and cell invasion. Its therapeutic targetability has been suggested in vitro and in animal models. We aimed to investigate the clinical role of MR3 expression in CRC for human survival. Surgical tissue samples from 754 CRC patients were analyzed for high or low immunohistochemical M3R expression on a clinically annotated tissue microarray (TMA). Immunohistochemical analysis was performed for established immune cell markers (CD8, TIA-1, FOXP3, IL 17, CD16 and OX 40). We used Kaplan-Meier curves to evaluate patients' survival and multivariate Cox regression analysis to evaluate prognostic significance. High M3R expression was associated with increased survival in multivariate (hazard ratio (HR) = 0.52; 95% CI = 0.35-0.78; p = 0.001) analysis, as was TIA-1 expression (HR = 0.99; 95% CI = 0.94-0.99; p = 0.014). Tumors with high M3R expression were significantly more likely to be grade 2 compared to tumors with low M3R expression (85.7% vs. 67.1%, p = 0.002). The 5-year survival analysis showed a trend of a higher survival rate in patients with high M3R expression (46%) than patients with low M3R expression CRC (42%) (p = 0.073). In contrast to previous in vitro and animal model findings, this study demonstrates an increased survival for CRC patients with high M3R expression. This evidence is highly relevant for translation of basic research findings into clinically efficient treatments.

Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment.

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8.

Myrrh exerts barrier-stabilising and -protective effects in HT-29/B6 and Caco-2 intestinal epithelial cells.

PURPOSE: Myrrh, the oleo-gum resin of Commiphora molmol, is well known for its anti-inflammatory properties. In different animal models, it protected against DSS-, TNBS- and oxazolone-induced colitis. To date, no information concerning the effect of myrrh on barrier properties are available. Thus, this study investigates the effect of myrrh on paracellular barrier function in the absence or presence of the pro-inflammatory cytokine TNFα. METHODS: Monolayers of human colon cell lines HT-29/B6 and Caco-2 were incubated with myrrh under control conditions or after challenge with the pro-inflammatory cytokine TNFα. Barrier function was analysed by electrophysiological and permeability measurements, Western blotting, immunostaining in combination with confocal microscopy, and freeze-fracture electron microscopy. RESULTS: In Caco-2 cells, myrrh induced an increase in transepithelial resistance (TER) which was associated with downregulation of the channel-forming tight junction (TJ) protein claudin-2 via inhibition of the PI3 kinase signalling pathway. In HT-29/B6 cells, myrrh had no effect on barrier properties under basic conditions, but protected against barrier damage induced by TNFα, as indicated by a decrease in TER and an increase in fluorescein permeability. The TNFα effect was associated with a redistribution of the sealing TJ protein claudin-1, an increase in the expression of claudin-2 and a change in TJ ultrastructure. Most importantly, all TNFα effects were inhibited by myrrh. The effect of myrrh on claudin-2 expression in this cell line was mediated via inhibition of the STAT6 pathway. CONCLUSIONS: This study shows for the first time that myrrh exerts barrier-stabilising and TNFα-antagonising effects in human intestinal epithelial cell models via inhibition of PI3K and STAT6 signalling. This suggests therapeutic application of myrrh in intestinal diseases associated with barrier defects and inflammation.

Yersinia enterocolitica Affects Intestinal Barrier Function in the Colon.

Infection with Yersinia enterocolitica causes acute diarrhea in early childhood. A mouse infection model presents new findings on pathological mechanisms in the colon. Symptoms involve diarrhea with watery feces and weight loss that have their functional correlates in decreased transepithelial electrical resistance and increased fluorescein permeability. Y. enterocolitica was present within the murine mucosa of both ileum and colon. Here, the bacterial insult was of focal nature and led to changes in tight junction protein expression and architecture. These findings are in concordance with observations from former cell culture studies and suggest a leak flux mechanism of diarrhea.

A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation.

New treatment strategies for inflammatory bowel disease are needed and parasitic nematode infections or application of helminth components improve clinical and experimental gut inflammation. We genetically modified the probiotic bacterium Escherichia coli Nissle 1917 to secrete the powerful nematode immunomodulator cystatin in the gut. This treatment was tested in a murine colitis model and on post-weaning intestinal inflammation in pigs, an outbred model with a gastrointestinal system similar to humans. Application of the transgenic probiotic significantly decreased intestinal inflammation in murine acute colitis, associated with increased frequencies of Foxp3(+) Tregs, suppressed local interleukin (IL)-6 and IL-17A production, decreased macrophage inflammatory protein-1α/ß, monocyte chemoattractant protein -1/3, and regulated upon activation, normal T-cell expressed, and secreted expression and fewer inflammatory macrophages in the colon. High dosages of the transgenic probiotic were well tolerated by post-weaning piglets. Despite being recognized by T cells, secreted cystatin did not lead to changes in cytokine expression or macrophage activation in the colon. However, colon transepithelial resistance and barrier function were significantly improved in pigs receiving the transgenic probotic and post-weaning colon inflammation was reduced. Thus, the anti-inflammatory efficiency of a probiotic can be improved by a nematode-derived immunoregulatory transgene. This treatment regimen should be further investigated as a potential therapeutic option for inflammatory bowel disease.

Myrrh protects against IL-13-induced epithelial barrier breakdown in HT-29/B6 cells.

The oleoresin myrrh has been used for centuries as an anti-inflammatory remedy for a variety of diseases and is said to have a protective effect on the intestinal epithelium. An intact epithelial barrier function is the prerequisite for a healthy gut. Inflammatory and infectious diseases of the intestine, in particular, lead to barrier impairment resulting in leak-flux diarrhea and mucosal immune responses. Therefore, the aim of the present study was to investigate the protective effect of myrrh in an experimental inflammatory situation, namely, under the influence of IL-13, one of the key cytokines in ulcerative colitis. We used human intestinal epithelial HT-29/B6 cell monolayers for functional and molecular assessment of the epithelial barrier under IL-13 and myrrh treatment. IL-13 induced a loss in barrier function that was fully restored with myrrh treatment, as shown by transepithelial electrical resistance measurements. The molecular correlate of the IL-13-mediated barrier dysfunction could be assigned to an upregulation of the channel-forming tight junction (TJ) protein claudin-2 and to a subcellular redistribution of the TJ protein tricellulin, loosening the sealing of tricellular TJs. Moreover, IL-13 exposure leads to an increase in the number of apoptotic cells, contributing to the leak pathway of barrier dysfunction. Myrrh protected against changes in TJ deregulation and decreased the elevated apoptotic ratio under IL-13. The protective effects are mediated through the inhibition of the STAT3 and STAT6 pathway. In conclusion, our results demonstrate that myrrh exhibits antagonizing effects against IL-13-induced barrier impairment in a human intestinal cell model. These data suggest the use of myrrh as a promising option in the treatment of inflammatory bowel disease.

Gp130 is expressed in pancreatic cancer and can be targeted by the small inhibitor molecule SC144.

PURPOSE: Interleukin 6 (IL-6), Oncostatin M (OSM), and downstream effector STAT3 are pro-tumorigenic agents in pancreatic ductal adenocarcinoma (PDAC). Glycoprotein 130 (gp130) is a compound of the IL-6 and OSM receptor complex that triggers STAT3 signaling. SC144 is a small molecule gp130 inhibitor with anticancer activity. This study examines the gp130 expression in human PDAC specimens and the in vitro effects of SC144 in PDAC cell lines. METHODS: Tissue micro-arrays were constructed from 175 resected human PDAC. The gp130 expression in tumor epithelium and stroma was determined by immunohistochemistry, and survival analysis was performed. Growth inhibition by SC144 was assessed in vitro using BrdU and MTT assays. Western blotting was performed to evaluate the SC144 effect on IL-6 and OSM signaling. RESULTS: Gp130 was expressed in the epithelium of 78.8% and the stroma of 9.4% of the tumor samples. The median overall survival for patients with or without epithelial gp130 expression was 16.7 months and 15.9 months, respectively (p = 0.830). Patients with no stromal gp130 expression showed poorer survival than patients with stromal gp130 expression (median 16.2 and 22.9 months, respectively), but this difference did not reach significance (p = 0.144). SC144 inhibited cell proliferation and viability and suppressed IL-6- and OSM-stimulated STAT3Y705 phosphorylation in PDAC cells. CONCLUSION: Gp130 is expressed in the epithelium of most human PDAC, but stromal expression is rare. The small molecule gp130 inhibitor SC144 potently inhibits PDAC progression in vitro and may abrogate IL-6 or OSM/gp130/STAT3 signaling. These results suggest gp130 as a novel drug target for pancreatic cancer therapy.

The Impact of TRAIL on the Immunological Milieu during the Early Stage of Abdominal Sepsis.

Despite intensive scientific efforts, the therapy of peritonitis is presently limited to symptomatic measures, including infectious source control and broad-spectrum antibiotics. Promising therapeutic approaches to reduce morbidity and mortality are still missing. Within the early phase of abdominal sepsis, apoptosis of neutrophil granulocytes is inhibited, which is linked to tissue damage and septic shock. TNF-related apoptosis-inducing ligand (TRAIL) is a promising agent to stimulate neutrophil apoptosis. However, the underlying mechanisms have not been elucidated so far. The objective of the present study was to characterize the molecular mechanisms of TRAIL-stimulated apoptosis in early abdominal sepsis. Therefore, the murine sepsis model Colon ascendens stent peritonitis (CASP) was applied in wild type (WT) and TRAIL knock-out (TRAIL-/-) C57/BL6j mice. Neutrophil granulocytes were isolated from spleen, blood, bone marrow, and peritoneal lavage using magnetic-activated cell sorting. Neutrophil maturation was analyzed by light microscopy, and apoptotic neutrophils were quantified by fluorescence-activated cell sorting (FACS). Western blot and FACS were used to investigate expression changes in apoptotic proteins and TRAIL receptors. The impact of TRAIL-induced apoptosis was studied in vitro. In septic mice (CASP 6 h), the number of neutrophils in the BM was reduced but increased in the blood and peritoneal lavage. This was paralleled by an increased maturation of neutrophils from rod-shaped to segmented neutrophils (right shift). In vitro, extrinsic TRAIL stimulation did not alter the apoptosis level of naïve neutrophils but stimulated apoptosis in neutrophils derived from septic WT and TRAIL-/- mice. Neutrophils of the bone marrow and spleen showed enhanced protein expression of anti-apoptotic Flip, c-IAP1, and McL-1 and reduced expression levels of pro-apoptotic Bax in neutrophils, which might correlate with apoptosis inhibition in these cells. CASP increased the expression of intrinsic TRAIL in neutrophils derived from the bone marrow and spleen. This might be explained by an increased expression of the TRAIL receptors DR5, DcR1, and DcR2 on neutrophils in sepsis. No differences were observed between septic or naïve WT and TRAIL-/- mice. In conclusion, the present study shows that neutrophil granulocytes are sensitive to TRAIL-stimulated apoptosis in the early stage of abdominal sepsis, emphasizing the promising role of TRAIL as a therapeutic agent.

Targeting Interleukin-6/Glycoprotein-130 Signaling by Raloxifene or SC144 Enhances Paclitaxel Efficacy in Pancreatic Cancer.

Interleukine-6 plays a key role in the progression and poor survival in pancreatic ductal adenocarcinoma (PDAC). The present study aimed to clarify if targeting the interleukin-6/glycoprotein-130 signaling cascade using the small-molecule gp130 inhibitor SC144 or raloxifene, a non-steroidal selective estrogen receptor modulator, enhances paclitaxel efficacy. MTT/BrdU assays or TUNEL staining were performed to investigate cell viability, proliferation and apoptosis induction in L3.6pl and AsPC-1 human pancreatic cell lines. In vivo, effects were studied in an orthotopic PDAC mouse model. Tumor specimens were analyzed by qPCR, immunohistochemistry and ELISA. Combination of paclitaxel/raloxifene, but not paclitaxel/SC144, enhanced proliferation and viability inhibition and increased apoptosis compared to single treatment in vitro. Synergy score calculations confirmed an additive influence of raloxifene on paclitaxel. In the PDAC mouse model, both combinations of raloxifene/paclitaxel and SC144/paclitaxel reduced tumor weight and volume compared to single-agent therapy or control. Raloxifene/paclitaxel treatment decreased survivin mRNA expression and showed tendencies of increased caspase-3 staining in primary tumors. SC144/paclitaxel reduced interleukin-6 levels in mice's tumors and plasma. In conclusion, raloxifene or SC144 can enhance the anti-tumorigenic effects of paclitaxel, suggesting that paclitaxel doses might also be reduced in combined chemotherapy to lessen paclitaxel side effects.

Immune Phenotypic Characterization of a TRAIL-Knockout Mouse.

The TNF-superfamily member TRAIL is known to mediate selective apoptosis in tumor cells suggesting this protein as a potential antitumor drug target. However, initial successful pr-clinical results could not be translated into the clinic. Reasons for the ineffectiveness of TRAIL-targeting in tumor therapies could include acquired TRAIL resistance. A tumor cell acquires TRAIL resistance, for example, by upregulation of antiapoptotic proteins. In addition, TRAIL can also influence the immune system and thus, tumor growth. We were able to show in our previous work that TRAIL-/- mice show improved survival in a mouse model of pancreatic carcinoma. Therefore, in this study we aimed to immunologically characterize the TRAIL-/- mice. We observed no significant differences in the distribution of CD3+, CD4+, CD8+ T-cells, Tregs, and central memory CD4+ and CD8+ cells. However, we provide evidence for relevant differences in the distribution of effector memory T-cells and CD8+CD122+ cells but also in dendritic cells. Our findings suggest that T-lymphocytes of TRAIL-/- mice proliferate at a lower rate, and that the administration of recombinant TRAIL significantly increases their proliferation, while regulatory T-cells (Tregs) from TRAIL-/- mice are less suppressive. Regarding the dendritic cells, we found more type-2 conventional dendritic cells (DC2s) in the TRAIL-/- mice. For the first time (to the best of our knowledge), we provide a comprehensive characterization of the immunological landscape of TRAIL-deficient mice. This will establish an experimental basis for future investigations of TRAIL-mediated immunology.

Near-infrared Fluorescence Imaging for Detecting Pancreatic Liver Metastasis in an Orthotopic Athymic Mouse Model.

BACKGROUND/AIM: Evidence of metastatic disease precludes oncological resection of pancreatic cancer. Near-infrared (NIR) fluorescent labels, such as indocyanine green (ICG), assist in the intraoperative detection of occult and micrometastatic liver disease. The present study aimed to analyse the role of NIR fluorescence imaging using ICG for pancreatic liver disease as proof of concept in an orthotopic athymic mouse model. MATERIALS AND METHODS: Pancreatic ductal adenocarcinoma was induced by injecting L3.6pl human pancreatic tumour cells into the pancreatic tail of seven athymic mice. After four weeks of tumour growth, ICG was injected into the tail vein and NIR fluorescence imaging was performed at harvest to determine tumour-to-liver ratios (TLR) using Quest Spectrum® Fluorescence Imaging Platform. RESULTS: Pancreatic tumour growth and liver metastasis could be visually confirmed for all seven animals. None of the hepatic metastases showed any detectable ICG-uptake. ICG-staining failed to visualize the liver metastases or to increase fluorescence intensity of the rim around the hepatic lesions. CONCLUSION: ICG-staining fails to visualize liver metastases induced by L3.6pl pancreatic tumour cells in athymic nude mice by NIR fluorescence imaging. Further studies are necessary to delineate the underlying mechanism for insufficient ICG uptake in these pancreatic liver metastases and for the lack of a fluorescent rim around the liver lesions.

Determinants of colonic barrier function in inflammatory bowel disease and potential therapeutics.

Intestinal barrier dysfunction is a main feature of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Leak flux diarrhoea and a facilitated uptake of noxious antigens are the two consequences resulting from an impaired epithelial barrier. Barrier perturbations in IBD comprise alterations in epithelial tight junctions (TJ), i.e. a reduced number of horizontal TJ strands and an altered TJ protein expression and subcellular distribution. Moreover, increased incidence of apoptotic events as well as erosions and ulcerations can add to that leakiness. These barrier defects are attributed to enhanced activity of pro-inflammatory cytokines like TNFα, INFγ, IL-1ß and IL-13, which are highly expressed in the chronically inflamed intestine. Although the aetiology of IBD is far from being clear, chronic inflammation is believed to result from an inadequate immune response as a consequence of genetic predisposition as well as changes in, and altered responses to, the intestinal microbiota. On the other hand, an insufficient mucosal response to bacterial stimuli results in an insufficient immune response towards intestinal pathogens. However, detailed characterization of barrier defects offers the opportunity to consider and test therapeutic interventions. Beside cytokine antagonists, different plant compounds and probiotics have been shown to stabilize the barrier function by affecting TJ protein expression and distribution.

Effect of Revascularization on Intramuscular Vascular Endothelial Growth Factor Levels in Peripheral Arterial Disease.

Vascular endothelial growth factor (VEGF) is a potent driver of angiogenesis, which may help to relieve ischemia in peripheral arterial disease (PAD). We aimed to investigate the role of intramuscular VEGF in ischemic and non-ischemic skeletal muscle in PAD patients before and after surgical or endovascular revascularization and different stages of PAD. Biopsies of the gastrocnemius and vastus muscles from twenty PAD patients with stenosis or occlusion of the superficial femoral artery were obtained both during revascularization and 8 weeks postoperatively. The gastrocnemius muscle was considered ischemic, while vastus muscle biopsies served as intraindividual controls. The levels of vascular endothelial growth factor in muscle lysates were then determined by ELISA. Preoperative VEGF levels were significantly higher in ischemic muscles compared to the controls (98.07 ± 61.96 pg/mL vs. 55.50 ± 27.33 pg/mL, p = 0.004). Postoperative values decreased significantly (p = 0.010) to 54.83 ± 49.60 pg/mL in gastrocnemius biopsies. No significant change was observed in vastus muscle biopsies, with mean postoperative VEGF values found at 54.16 ± 40.66 pg/mL. Since all patients still had indications for revascularization, impairment of angiogenesis mechanisms can be assumed. More research about angiogenesis in PAD is needed with the ultimate goal to improve conservative treatment.

Yersinia enterocolitica induces epithelial barrier dysfunction through regional tight junction changes in colonic HT-29/B6 cell monolayers.

Yersinia enterocolitica is a common cause of acute gastroenteritis. This study aimed to clarify the mechanisms leading to barrier dysfunction and diarrhea. Exposure of human colonic HT-29/B6 cells to Y. enterocolitica resulted in a decrease in transepithelial resistance from 404±23 to 163±21 Ω cm² (P<0.001) in parallel with an increase in mannitol (182 Da) and fluorescein (332 Da) permeability, whereas short circuit current did not change. This effect was time dependent, required the presence of living bacteria, could not be triggered by bacterial supernatants and was not due to Yersinia outer proteins. Concomitantly, Y. enterocolitica induced necrosis as indicated by an increase in lactate dehydrogenase-release, whereas epithelial apoptosis was not upregulated. Local changes in conductivity were detected by conductance scanning, indicating 'leaky regions' within the epithelium that were visualized by biotinylation and confocal microscopy. In these regions, claudin-3 and -4 and, especially claudin-8, were redistributed off the tight junction (TJ) into the cytoplasm. In addition, the expression of claudin-2, -3, -8, -10 and ZO-1 was diminished as quantified by immunoblotting. Moreover, we found claudin-8 to be regulated by the c-Jun N-terminal kinase, the inhibition of which attenuated the Y. enterocolitica-induced decrease in transepithelial resistance and restored claudin-8 protein level. In conclusion, barrier dysfunction in Y. enterocolitica infection is due to circumscribed epithelial TJ protein changes and necrotic cell loss, as a consequence of which leak flux diarrhea and antigen-uptake provoking extraintestinal arthritis may be triggered.

Transforming growth factor-ß, a whey protein component, strengthens the intestinal barrier by upregulating claudin-4 in HT-29/B6 cells.

TGFß (isoforms 1-3) has barrier-protective effects in the intestine. The mechanisms involved in regulating tight junction protein expression are poorly understood. The aim of this study was to elucidate TGFß-dependent protective effects with special attention to promoter regulation of tight junction proteins using the HT-29/B6 cell model. In addition, the effects of whey protein concentrate 1 (WPC1), a natural source of TGFß in human nutrition, were examined. For this purpose, the claudin-4 promoter was cloned and tested for its activity. It exhibited transactivation in response to TGFß1, which was intensified when Smad-4 was cotransfected, indicating a Smad-4-dependent regulatory component. Shortening and mutation of the promoter altered and attenuated this effect. WPC1 induced an increase in the claudin-4 protein level and resistance of HT-29/B6 cell monolayers. Anti-TGFß(1-3) antibodies blocked these whey protein effects, suggesting that a main part of this function was mediated through TGFß. This effect was observed on intact monolayers as well as when barrier function was impaired by preexposure to IFNγ. In conclusion, TGFß1 affects claudin-4 gene expression via Smad-4-dependent and -independent transcriptional regulation, resulting in barrier protection, a cytokine effect that is also found in whey protein concentrates used in enteral nutrition.

The Punicalagin Metabolites Ellagic Acid and Urolithin A Exert Different Strengthening and Anti-Inflammatory Effects on Tight Junction-Mediated Intestinal Barrier Function In Vitro.

Scope: Ellagitannins are polyphenols found in numerous fruits, nuts and seeds. The elagitannin punicalagin and its bioactive metabolites ellagic acid and urolithins are discussed to comprise a high potential for therapeutically or preventive medical application such as in intestinal diseases. The present study characterizes effects of punicalagin, ellagic acid and urolithin A on intestinal barrier function in the absence or presence of the proinflammatory cytokine tumor necrosis factor-α (TNFα). Methods and Results: Transepithelial resistance (TER), fluorescein and ion permeability, tight junction protein expression and signalling pathways were examined in Caco-2 and HT-29/B6 intestinal epithelial cell models. Punicalagin had less or no effects on barrier function in both cell models. Ellagic acid was most effective in ileum-like Caco-2 cells, where it increased TER and reduced fluorescein and sodium permeabilities. This was paralleled by myosin light chain kinase two mediated expression down-regulation of claudin-4, -7 and -15. Urolithin A impeded the TNFα-induced barrier loss by inhibition of claudin-1 and -2 protein expression upregulation and claudin-1 delocalization in HT-29/B6. Conclusion: Ellagic acid and urolithin A affect intestinal barrier function in distinct ways. Ellagic acid acts preventive by strengthening the barrier per se, while urolithin A protects against inflammation-induced barrier dysfunction.

Increased proinflammatory cytokines in mesenteric fat in major surgery and Crohn's disease.

BACKGROUND: Proinflammatory cytokines play an important role in abdominal surgery and are often associated with the development of postoperative ileus, especially in Crohn's disease. The aim of this study was to investigate proinflammatory cytokine levels in mesenteric fat in Crohn's disease and patients without Crohn's disease. METHODS: Human mesenteric tissue specimen were divided into 3 patient groups (n = 10 each): minor surgery (laparoscopic cholecystectomy), major surgery (colectomy) in patients without Crohn's disease, and major surgery (colectomy) in patients with Crohn's disease. Levels of interleukin 6, interleukin 1-ß, and tumor necrosis factor α were determined by cytometric bead array, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. The Kruskal-Wallis and the Mann-Whitney U test were used to compare continuous variables. For categorical variables, the χ2 test or Fisher exact test was used. RESULTS: In minor surgery, cytokines levels of interleukin 6, interleukin 1-ß and Tumor necrosis factor α were low (ie, interleukin 6: 1 pg/mL [0-36], interleukin 1-ß: 0 fg/mL [0-18], tumor necrosis factor α: 157 fg/mL [91-237]) compared with major surgery in patients with and without Crohn's disease. Cytokines were significantly higher in major surgery (ie, interleukin 6: 147 pg/mL [29-347], interleukin 1-ß: 660 fg/mL [0-2580], tumor necrosis factor α: 532 fg/mL [289-1647]; P = .02 and major surgery with CD (cytometric bead array: interleukin 6: 94 pg/mL [24-627], interleukin 1-ß: 708 fg/mL [0-1664], tumor necrosis factor α: 733 fg/mL [209-1,354]; P < .05). Cytokine levels in major surgery with Crohn's disease showed a further increase of interleukin 6 in polymerase chain reaction in comparison to major surgery in patients without Crohn's disease (1.2 vs 4, P = .04). CONCLUSION: Proinflammatory cytokines are increased in the mesenteric fat in major operations compared to minor operations, which indicates local mesenteric inflammation. In Crohn's disease, levels of proinflammatory cytokines are even higher, which may put the patients at risk for postoperative ileus.

Effect of bowel preparation on intestinal permeability and inflammatory response during postoperative ileus in mice.

BACKGROUND: Postoperative ileus entails pathophysiological changes in mucosal permeability and an intestinal inflammatory immune response. We hypothesized that preoperative selective decontamination of the digestive tract combined with preoperative mechanical bowel preparation might be advantageous to prevent or reduce permeability changes and immune response in postoperative ileus. METHODS: Postoperative ileus was induced in mice by standardized small bowel manipulation. Intervention groups received selective decontamination and/or intestinal lavage with normal saline simulating mechanical bowel preparation before postoperative ileus induction. At 1, 3, and 9 hours after surgery, ileum samples were harvested for measurements of fluorescein (332 Da) permeability, quantification of tumor necrosis factor α-mRNA level, and leukocyte infiltration of the intestinal wall. RESULTS: Mucosal fluorescein permeability increased at 1 hour (8.6 ± 1.1 vs 5.9 ± 0.9 10-6 cm/s; P < .01) and 3 hours (8.5 ± 0.6 vs 6.5 ± 0.2 10-6 cm/s; P < .05) after induction of postoperative ileus. This increase was prevented by mechanical bowel preparation and selective decontamination+mechanical bowel preparation interventions at both points in time. Expression of tumor necrosis factor α was more than 2-fold increased (P < .05) in the very early phase after induction of postoperative ileus but did not occur in mechanical bowel preparation-pretreated animals. Myeloperoxidase staining revealed that mechanical bowel preparation inhibited postoperative ileus-associated leukocyte infiltration of the intestinal muscularis at 3 and 9 hours after surgery, but not selective decontamination + mechanical bowel preparation treatment. The number of leukocytes after mechanical bowel preparation-only treatment remained at the level of sham-controls. CONCLUSION: Mechanical bowel preparation prevents permeability and leukocyte infiltration of the intestinal wall in the early phase of postoperative ileus in mice.

Raloxifene inhibits pancreatic adenocarcinoma growth by interfering with ERß and IL-6/gp130/STAT3 signaling.

PURPOSE: Currently, the exact role of estrogen receptor (ER) signaling in pancreatic cancer is unknown. Recently, we showed that expression of phosphorylated ERß correlates with a poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). Here, we hypothesized that raloxifene, a FDA-approved selective ER modulator (SERM), may suppress PDAC tumor growth by interfering with ERß signaling. To test this hypothesis, we studied the impact of raloxifene on interleukin-6/glycoprotein-130/signal transducer and activator of transcription-3 (IL-6/gp130/STAT3) signaling. METHODS: Human PDAC cell lines were exposed to raloxifene after which growth inhibition was assessed using a BrdU assay. ER knockdown was performed using siRNAs specific for ERα and ERß. The effects of raloxifene on IL-6 expression and STAT3 phosphorylation in PDAC cells were assessed by ELISA and Western blotting, respectively. In addition, raloxifene was administered to an orthotopic PDAC tumor xenograft mouse model, after which tumor growth was monitored and immunohistochemistry was performed. RESULTS: Raloxifene inhibited the in vitro growth of PDAC cells, and this effect was reversed by siRNA-mediated knockdown of ERß, but not of ERα, indicating ER isotype-specific signaling. We also found that treatment with raloxifene inhibited the release of IL-6 and suppressed the phosphorylation of STAT3Y705 in PDAC cells. In vivo, we found that orthotopic PDAC tumor growth, lymph node and liver metastases as well as Ki-67 expression were reduced in mice treated with raloxifene. CONCLUSIONS: Inhibition of ERß and the IL-6/gp130/STAT3 signaling pathway by raloxifene leads to potent reduction of PDAC growth in vitro and in vivo. Our results suggest that ERß signaling and IL-6/gp130 interaction may serve as promising drug targets for pancreatic cancer and that raloxifene may serve as an attractive therapeutic option for PDAC patients expressing the ERß isotype.