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

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-alpha (TNF alpha). 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 TNF alpha-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

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

Oral and Fecal Campylobacter concisus Strains Perturb Barrier Function by Apoptosis Induction in HT-29/B6 Intestinal Epithelial Cells

Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (Rt) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in Rt either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10−6 cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction

Evaluating expert-based habitat suitability information of terrestrial mammals with GPS-tracking data

Aim Macroecological studies that require habitat suitability data for many species often derive this information from expert opinion. However, expert-based information is inherently subjective and thus prone to errors. The increasing availability of GPS tracking data offers opportunities to evaluate and supplement expert-based information with detailed empirical evidence. Here, we compared expert-based habitat suitability information from the International Union for Conservation of Nature (IUCN) with habitat suitability information derived from GPS-tracking data of 1,498 individuals from 49 mammal species. Location Worldwide. Time period 1998-2021. Major taxa studied Forty-nine terrestrial mammal species. Methods Using GPS data, we estimated two measures of habitat suitability for each individual animal: proportional habitat use (proportion of GPS locations within a habitat type), and selection ratio (habitat use relative to its availability). For each individual we then evaluated whether the GPS-based habitat suitability measures were in agreement with the IUCN data. To that end, we calculated the probability that the ranking of empirical habitat suitability measures was in agreement with IUCN's classification into suitable, marginal and unsuitable habitat types. Results IUCN habitat suitability data were in accordance with the GPS data (> 95% probability of agreement) for 33 out of 49 species based on proportional habitat use estimates and for 25 out of 49 species based on selection ratios. In addition, 37 and 34 species had a > 50% probability of agreement based on proportional habitat use and selection ratios, respectively. Main conclusions We show how GPS-tracking data can be used to evaluate IUCN habitat suitability data. Our findings indicate that for the majority of species included in this study, it is appropriate to use IUCN habitat suitability data in macroecological studies. Furthermore, we show that GPS-tracking data can be used to identify and prioritize species and habitat types for re-evaluation of IUCN habitat suitability data

Mechanisms of Y. enterocolitica and E. coli Nissle

Yersinia enterocolitica ist ein häufiger Durchfallerreger. Um die der Diarrhö und Barrierestörung zugrundeliegenden Mechanismen aufzuklären, wurden die erregerspezifischen Pathomechanismen in einem humanen Kolonkarzinom- Zellkulturmodell detailliert analysiert. Die Infektion der als epithelialer Monolayer wachsenden humanen Kolonkarzinom-Zelllinie HT-29/B6 führte zu einer ausgeprägten Reduktion des transepithelialen Widerstands (Rt, TER) und einer Zunahme der Permeabilität für Mannitol (182 Da) und Fluorescein (332 Da). Der als Kurzschlussstrom gemessene aktive Ionentranport war unverändert. Die Veränderungen erfolgten zeit- und dosisabhängig und konnte nur durch den lebenden Erreger selbst, nicht aber durch bakterielle Kulturüberstände induziert werden. Typische Yersinia-Virulenzfaktoren, wie Invasin oder die Expression des Yersinia-Virulenzplasmids pYV waren nicht ursächlich. Y. enterocolitica induzierte in HT-29/B6-Zellen Nekrosen, was durch eine gesteigerte LDH-Freisetzung nachgewiesen wurde. Apoptosen traten nicht vermehrt auf. Mit Hilfe der Conductance-Scanning-Technik konnten in infizierten Monolayern lokale Leitfähigkeitsänderungen detektiert sowie durch Biotinylierung und anschließende Laserscanning-Mikroskopie visualisiert werden. Innerhalb dieser als leaky regions definierten Bereiche waren die barrierebildenden Claudine 3, 4 und 8 aus der Tight Junction in das Zytoplasma umverteilt. Zusätzlich zeigten Western Blot-Analysen eine verminderte Expression von Claudin-2, -3, -8, -10 und ZO-1. Darüber hinaus konnte eine Regulation der Claudin-8-Expression über die terminale c-Jun-Kinase nachgewiesen werden. Die Inhibition der Kinase bewirkte eine Abschwächung des durch Y. enterocolitica verursachten Rt-Abfalls sowie eine vollständige Wiederherstellung der Proteinexpression von Claudin-8. Somit wird die von Y. enterocolitica induzierte Barrierestörung durch lokale Tight Junction-Defekte und Nekroseinduktion hervorgerufen. Im Gesamtorganismus würden diese Mechanismen einerseits zu einem gesteigerten Übertritt von Antigenen aus dem Lumen in die Blutbahn führen und andererseits einen vermehrten Übertritt von Soluten und Wasser vom Blut ins Lumen verursachen, der zu einer Leckflux- Diarrhö führen kann. Der Escherichia coli-Stamm Nissle 1917 (EcN) kommt seit Jahren als Probiotikum bei der Behandlung von Diarrhöen und inflammatorischen Darmerkrankungen zum Einsatz. Um seine die Barriere stärkenden Mechanismen aufzuklären, wurde in dieser Arbeit der Einfluss des hochwirksamen EcN- Kulturüberstands auf die Tight Junction-mediierte Barrierefunktion in vitro am HT-29/B6-Zellkulturmodell untersucht. Aus EcN-Kulturen präparierte Überstände (EcNK) verursachten eine Verdopplung des Rt und eine Verminderung der Permeabilitäten für Mannitol und NaCl und somit eine Abdichtung der epithelialen Barriere. Das bekanntermaßen immunstimulatorisch wirkende EcN- Flagellin war hierfür nicht verantwortlich. Messungen mittels Zwei-Wege- Impedanzspektroskopie offenbarten insbesondere eine Zunahme des parazellulären Widerstands. Die für verschiedene Tight Junction-Proteine (Occludin, Tricellulin und Claudine sowie das TJ-assoziierte Protein ZO-1) durchgeführten Expressionsanalysen zeigten ausschließlich eine Zunahme der Protein- und mRNA- Level von Claudin-14 durch EcNK. Mittels Hitzeinaktivierung des EcNK konnten die Effekte auf Rt und Claudin-14-Expression aufgehoben werden. Die barriererelevante Funktion des bisher kaum charakterisierten Claudin-14-Proteins bestätigte sich abschließend im siRNA-Transfektionsansatz. Somit ist die im HT-29/B6-Modell durch EcNK induzierte Barriereprotektion auf eine Hochregulation von Claudin-14 zurückzuführen.Yersinia enterocolitica is a common cause of acute gastroenteritis. In order to clarify the mechanisms leading to diarrhea and barrier dysfunction the pathomechanisms of this infection were studied in detail. Exposure of human colonic HT-29/B6 cells to Y. enterocolitica resulted in a decrease in transepithelial resistance (Rt, TER) with a parallel increase in mannitol (182 Da) and fluorescein (332 Da) permeability. Active ion transport measured as short circuit current was unchanged. The Y. enterocolitica induced changes were time-dependent, required the presence of living bacteria, could not be triggered by bacterial supernatants, and were not due to Yersinia invasin or expression from the virulence plasmid pYV. Concomitantly, Y. enterocolitica induced necrosis as indicated by an increase in LDH-release, while epithelial apoptosis was not upregulated. Local changes in conductivity were detected by conductance scanning, indicating leaky regions within the epithelium which were visualized by biotinylation and confocal laser scanning microscopy. In these regions, sealing claudin-3, -4 and -8 were redistributed off the tight junction into the cytoplasm. In addition, the expression of claudin-2, -3, -8, -10 and ZO-1 was diminished as quantified by immunoblotting. Moreover, claudin-8 protein expression was found to be regulated via the c-Jun N-terminal kinase, the inhibition of which partially prevented the Y. enterocolitica induced decrease in Rt and restored claudin-8 protein level. In conclusion, barrier dysfunction in Y. enterocolitica infection is due to circumscribed epithelial tight junction protein changes and necrotic cell loss. As a consequence leak flux diarrhea and antigen-uptake may be triggered in human infection. Escherichia coli Nissle 1917 (EcN) is a probiotic bacterium used for the treatment of inflam-matory bowel disorders. To study its barrier strengthening mechanisms the in vitro effect of highly active EcN supernatant (EcNK) on tight junction mediated barrier function was assessed here using the HT-29/B6 cell culture model. EcNK dissected from bacterial culture, improved barrier function measured by a two-fold increase in Rt and a reduction of permeability for mannitol and NaCl. This was not due to flagellin. Measurements by two-path-impedance spectroscopy revealed a strong effect on paracellular resistance. When changes of tight junction proteins as claudins, occludin, tricellulin as well as the scafold protein ZO-1 were examined, claudin-14 was the only one found to be affected by EcNK. Claudin-14 was elevated on protein- and mRNA-level. Heat treatment of active EcNK abolished the increase in Rt and the upregulation of claudin-14 protein level. By silencing claudin-14 in a siRNA-transfection assay, the sealing function of this so far less well characterized tight junction protein was confirmed. In conclusion, EcNK improves barrier function in HT-29/B6 by upregulating claudin-14

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

Raloxifene inhibits pancreatic adenocarcinoma growth by interfering with ERβ 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 ERβ 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 ERβ 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 ERβ. 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 ERβ, 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 ERβ 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 ERβ 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 ERβ isotype

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