Clinical Significance of Claudin Expression in Oral Squamous Cell Carcinoma

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC

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

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

Macromolecule Translocation across the Intestinal Mucosa of HIV-Infected Patients by Transcytosis and through Apoptotic Leaks

Based on indirect evidence, increased mucosal translocation of gut-derived microbial macromolecules has been proposed as an important pathomechanism in HIV infection. Here, we quantified macromolecule translocation across intestinal mucosa from treatment-naive HIV-infected patients, HIV-infected patients treated by combination antiretroviral therapy, and HIV-negative controls and analyzed the translocation pathways involved. Macromolecule permeability was quantified by FITC-Dextran 4000 (FD4) and horseradish peroxidase (HRP) flux measurements. Translocation pathways were addressed using cold inhibition experiments. Tight junction proteins were characterized by immunoblotting. Epithelial apoptosis was quantified and translocation pathways were further characterized by flux studies in T84 cell monolayers using inducers and inhibitors of apoptosis and endocytosis. In duodenal mucosa of untreated but not treated HIV-infected patients, FD4 and HRP permeabilities were more than a 4-fold increase compared to the HIV-negative controls. Duodenal macromolecule permeability was partially temperature-dependent and associated with epithelial apoptosis without altered expression of the analyzed tight junction proteins. In T84 monolayers, apoptosis induction increased, and both apoptosis and endocytosis inhibitors reduced macromolecule permeability. Using quantitative analysis, we demonstrate the increased macromolecule permeability of the intestinal mucosa in untreated HIV-infected patients. Combining structural and mechanistic studies, we identified two pathways of increased macromolecule translocation in HIV infection: transcytosis and passage through apoptotic leaks

Intestinal Barrier in Post-Campylobacter jejuni Irritable Bowel Syndrome

Background: Campylobacter jejuni (C. jejuni) is one of the most common causes of bacterial gastroenteritis worldwide. One sequela of this infection is the development of post-infectious irritable bowel syndrome (PI-IBS). It has been suggested that a dysfunctional intestinal barrier may promote IBS development. We aimed to test this hypothesis against the background of the leaky gut concept for low-grade inflammation in PI-IBS. Methods: We identified patients with persistent PI-IBS symptoms after C. jejuni infection. During sigmoidoscopy, forceps biopsies were obtained for electrophysiological measurements of epithelial transport and barrier function in miniaturized Ussing devices. C. jejuni absence was checked by PCR and cytokine production with immunohistochemistry. Results: In PI-IBS, the epithelial resistance of the colon epithelium was unaltered, reflecting an intact paracellular pathway. In contrast, temperature-dependent horseradish peroxidase (HRP, 44 kDa) permeation increased. Short-circuit current (Isc) reflecting active anion secretion and ENaC-dependent electrogenic sodium absorption was unaffected. Early endosome antigen-1 (EEA1) and IL-4 levels increased. C. jejuni is not incorporated into the resident microbiota of the colon mucosa in PI-IBS. Conclusions: In PI-IBS after C. jejuni infection, macromolecule uptake via endocytosis was enhanced, leading to low-grade inflammation with pro-inflammatory cytokine release. The findings will allow C. jejuni-induced pathomechanisms to be targeted during infection and, thereafter to reduce sequelae such as PI-IBS

Vitamin D in Acute Campylobacteriosis-Results From an Intervention Study Applying a Clinical Campylobacter jejuni Induced Enterocolitis Model

Human Campylobacter infections are progressively rising and of high socioeconomic impact. In the present preclinical intervention study we investigated anti-pathogenic, immuno-modulatory, and intestinal epithelial barrier preserving properties of vitamin D applying an acute campylobacteriosis model. Therefore, secondary abiotic IL-10-/- mice were perorally treated with synthetic 25-OH-cholecalciferol starting 4 days before peroral Campylobacter jejuni infection. Whereas, 25-OH-cholecalciferol application did not affect gastrointestinal pathogen loads, 25-OH-cholecalciferol treated mice suffered less frequently from diarrhea in the midst of infection as compared to placebo control mice. Moreover, 25-OH-cholecalciferol application dampened C. jejuni induced apoptotic cell responses in colonic epithelia and promoted cell-regenerative measures. At day 6 post-infection, 25-OH-cholecalciferol treated mice displayed lower numbers of colonic innate and adaptive immune cell populations as compared to placebo controls that were accompanied by lower intestinal concentrations of pro-inflammatory mediators including IL-6, MCP1, and IFN-γ. Remarkably, as compared to placebo application synthetic 25-OH-cholecalciferol treatment of C. jejuni infected mice resulted in lower cumulative translocation rates of viable pathogens from the inflamed intestines to extra-intestinal including systemic compartments such as the kidneys and spleen, respectively, which was accompanied by less compromised colonic epithelial barrier function in the 25-OH-cholecalciferol as compared to the placebo cohort. In conclusion, our preclinical intervention study provides evidence that peroral synthetic 25-OH-cholecalciferol application exerts inflammation-dampening effects during acute campylobacteriosis

Expression of tricellular tight junction proteins and the paracellular macromolecule barrier are recovered in remission of ulcerative colitis

Background: Ulcerative colitis (UC) has a relapsing and remitting pattern, wherein the underlying mechanisms of the relapse might involve an enhanced uptake of luminal antigens which stimulate the immune response. The tricellular tight junction protein, tricellulin, takes charge of preventing paracellular passage of macromolecules. It is characterized by downregulated expression in active UC and its correct localization is regulated by angulins. We thus analyzed the tricellulin and angulin expression as well as intestinal barrier function and aimed to determine the role of tricellulin in the mechanisms of relapse. Methods: Colon biopsies were collected from controls and UC patients who underwent colonoscopy at the central endoscopy department of Campus Benjamin Franklin, Charite - Universitatsmedizin Berlin. Remission of UC was defined basing on the clinical appearance and a normal Mayo endoscopic subscore. Intestinal barrier function was evaluated by electrophysiological and paracellular flux measurements on biopsies mounted in Ussing chambers. Results: The downregulated tricellulin expression in active UC was recovered in remission UC to control values. Likewise, angulins were in remission UC at the same levels as in controls. Also, the epithelial resistance which was decreased in active UC was restored in remission to the same range as in controls, along with the unaltered paracellular permeabilities for fluorescein and FITC-dextran 4 kDa. Conclusions: In remission of UC, tricellulin expression level as well as intestinal barrier functions were restored to normal, after they were impaired in active UC. This points toward a re-sealing of the impaired tricellular paracellular pathway and abated uptake of antigens to normal rates in remission of UC

Epithelial Barrier Dysfunction in Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D) via Downregulation of Claudin-1

Background: In patients with diarrhea-predominant irritable bowel syndrome (IBS-D), the diarrheal mechanisms are largely unknown, and they were examined in this study on colon biopsies. Methods: Electrophysiological measurements were used for monitoring functional changes in the diarrheic colon specimens. In parallel, tight junction protein expression was analyzed by Western blot and confocal laser-scanning microscopy, and signaling pathway analysis was performed using RNA sequencing and bioinformatics. Results: Epithelial resistance was decreased, indicating an epithelial leak flux diarrheal mechanism with a molecular correlate of decreased claudin-1 expression, while induction of active anion secretion and impairment of active sodium absorption via the epithelial sodium channel, ENaC, were not detected. The pathway analysis revealed activation of barrier-affecting cytokines TNF-alpha, IFN-gamma, IL-1 beta and IL-4. Conclusions: Barrier dysfunction as a result of epithelial tight junction changes plays a role in IBS-D as a pathomechanism inducing a leak flux type of diarrhea

Resveratrol Alleviates Acute Campylobacter jejuni Induced Enterocolitis in a Preclinical Murine Intervention Study

The polyphenolic compound resveratrol has been shown to exert health-beneficial properties. Given globally emerging Campylobacter infections in humans, we addressed potential anti-pathogenic, immuno-modulatory and intestinal epithelial barrier preserving properties of synthetic resveratrol in the present preclinical intervention study applying a murine acute campylobacteriosis model. Two days following peroral C. jejuni infection, secondary abiotic IL-10-/- mice were either subjected to resveratrol or placebo via the drinking water. Whereas placebo mice suffered from acute enterocolitis at day 6 post-infection, resveratrol treatment did not only lead to improved clinical conditions, but also to less pronounced colonic epithelial apoptosis as compared to placebo application. Furthermore, C. jejuni induced innate and adaptive immune cell responses were dampened in the large intestines upon resveratrol challenge and accompanied by less colonic nitric oxide secretion in the resveratrol versus the placebo cohort. Functional analyses revealed that resveratrol treatment could effectively rescue colonic epithelial barrier function in C. jejuni infected mice. Strikingly, the disease-alleviating effects of resveratrol could additionally be found in extra-intestinal and also systemic compartments at day 6 post-infection. For the first time, our current preclinical intervention study provides evidence that peroral resveratrol treatment exerts potent disease-alleviating effects during acute experimental campylobacteriosis