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Abstract:  The estrogenic action on pituitary cell growth is widely known. We have previously demonstrated the activation of the antioxidant pathway of phosphorylated erythroid 2-derived nuclear factor 2 (p-Nrf2) in response to DNA damage by 17β-estradiol (E2). In this study we analyzed the impact of E2 on the tumoral suppressor p53 and cell cycle regulator p21 activation, as well as the damage response in pituitary cells in vitro. Pituitary tumour development was induced in adult male Wistar rats by subcutaneous implantation of silastic capsules containing estradiol benzoate (30mg) for 10 days (E10; n = 5). The control group was implanted with empty capsules (n=5). Subsequently, pituitary glands were collected, with cells being cultured and exposed to E2 (1-10-100nM) for 15, 30 and 60 min. The p53 and p21 protein levels were determined by western blot. By immunofluorescence, the co-expression of prolactin (PRL)/p-Nrf2 and growth hormone (GH)/p-Nrf2 was evaluated to determinate the cell type involved in the activation of the oxidative damage response. Statistical analysis: ANOVA-Fischer (p <0.05). Under tumoral contexts, a significant increase in p53 protein at the cytoplasmic level was detected after 15 and 30 min of treatment with E2 (1nM). After supraphysiological concentrations (10-100nM), this response was observed after 30 and 60 min. At nuclear level, we only detected an increase in p53 at 15 min, regardless of the dose. The p2 expression showed a similar profile in both subcellular compartments, with significant increases after 15 and 30 min of exposure with 1nM E2 and 30 and 60 min with supraphysiological doses. In normal cells cultures we did not observe significant changes in the expression of both markers. The number of lactotroph tumoral cells co-expressing PRL/p-Nrf2 increased significantly at 30 min compared to supraphysiological doses of E2. No changes were detected in the expression of GH/p-Nrf2 in GH cells. In tumoral pituitary cells, the pro-oxidant action induced by E2 triggers the activation of p53 and p21 in order to repair DNA damage, through the stabilization of Nrf2. This response would mainly have an impact on PRL tumoral cells. These mechanisms could guarantee the cell viability, thus regulating pituitary tumor development.Resumen:  Es ampliamente conocida la acción estrogénica sobre el crecimiento celular hipofisario. Previamente demostramos la activación de la vía antioxidante del factor nuclear 2 derivado del eritroide 2 fosforilado (Nrf2-p) en respuesta al daño del ADN por 17β-estradiol (E2). Analizamos entonces, el impacto del E2 sobre la activación del supresor tumoral p53 y el regulador del ciclo celular p21, y la respuesta al daño en células hipofisarias in vitro. Indujimos el desarrollo tumoral hipofisario en ratas Wistar macho adultas mediante la implantación subcutánea de cápsulas de silástico conteniendo benzoato de estradiol (30 mg) durante 10 días (E10; n=5). El grupo control fue implantado con cápsulas vacía (n=5). Posteriormente, extrajimos las adenohipófisis, cultivamos sus células y fueron expuestas a E2 (1-10-100nM) por 15, 30 y 60min. Determinamos los niveles proteicos de p53 y p21 por western blot. Por inmunofluorescencia, evaluamos la co-expresión de Prolactina (PRL)/Nrf2-p y hormona de crecimiento (GH)/Nrf2-p para determinar el tipo celular involucrado con activación de respuesta al daño oxidativo. Análisis estadístico: ANOVA-Fischer (p <0,05). En contextos tumorales, detectamos un aumento significativo de la proteína p53 a nivel citoplasmático luego de 15 y 30 min de tratamiento con E2 (1nM); frente a concentraciones suprafisiológicas (10-100nM) esta respuesta se observó luego de 30 y 60 min. A nivel nuclear, detectamos un aumento de p53 solo a los 15 min, de manera independiente de la dosis. La expresión de p21 mostró un perfil similar en ambos compartimientos subcelulares con incrementos significativos luego de 15 y 30 min de exposición con E2 1nM y 30 y 60 min con dosis suprafisiológicas. En cultivos normales no observamos cambios significativos en la expresión de ambos marcadores. El número de células lactotropas tumorales que co-expresaron PRL/Nrf2-p aumentó significativamente a los 30 min frente a dosis suprafisiológicas de E2; sin detectarse modificaciones en la expresión de GH/Nrf2-p en células somatotropas. En células hipofisarias tumorales, la acción pro-oxidante inducida por E2 desencadena la activación de p53 y de p21 a fin de reparar el daño del ADN, vía estabilización de Nrf2. Esta respuesta impactaría principalmente sobre células lactotropas tumorales. Mediante estos mecanismos se garantizaría la viabilidad celular, regulando el desarrollo tumoral hipofisario.

Klebsiella pneumoniae is able to trigger epithelial-mesenchymal transition process in cultured airway epithelial cells

The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells

Therapy for metastatic melanoma: the past, present, and future

Metastatic melanoma is the most aggressive form of skin cancer with a median overall survival of less than one year. Advancements in our understanding of how melanoma evades the immune system as well as the recognition that melanoma is a molecularly heterogeneous disease have led to major improvements in the treatment of patients with metastatic melanoma. In 2011, the US Food and Drug Administration (FDA) approved two novel therapies for advanced melanoma: a BRAF inhibitor, vemurafenib, and an immune stimulatory agent, ipilimumab. The success of these agents has injected excitement and hope into patients and clinicians and, while these therapies have their limitations, they will likely provide excellent building blocks for the next generation of therapies. In this review we will discuss the advantages and limitations of the two new approved agents, current clinical trials designed to overcome these limitations, and future clinical trials that we feel hold the most promise

Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68

<p>Abstract</p> <p>Background</p> <p>Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo.</p> <p>Methods</p> <p>In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection.</p> <p>Results</p> <p>We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection.</p> <p>Conclusions</p> <p>Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection.</p

Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond.

International audienceABSTRACT: The immunological roles of B-cells are being revealed as increasingly complex by functions that are largely beyond their commitment to differentiate into plasma cells and produce antibodies, the key molecular protagonists of innate immunity, and also by their compartmentalisation, a more recently acknowledged property of this immune cell category. For decades, B-cells have been recognised by their expression of an immunoglobulin that serves the function of an antigen receptor, which mediates intracellular signalling assisted by companion molecules. As such, B-cells were considered simple in their functioning compared to the other major type of immune cell, the T-lymphocytes, which comprise conventional T-lymphocyte subsets with seminal roles in homeostasis and pathology, and non-conventional T-lymphocyte subsets for which increasing knowledge is accumulating. Since the discovery that the B-cell family included two distinct categories - the non-conventional, or extrafollicular, B1 cells, that have mainly been characterised in the mouse; and the conventional, or lymph node type, B2 cells - plus the detailed description of the main B-cell regulator, FcγRIIb, and the function of CD40+ antigen presenting cells as committed/memory B-cells, progress in B-cell physiology has been slower than in other areas of immunology. Cellular and molecular tools have enabled the revival of innate immunity by allowing almost all aspects of cellular immunology to be re-visited. As such, B-cells were found to express "Pathogen Recognition Receptors" such as TLRs, and use them in concert with B-cell signalling during innate and adaptive immunity. An era of B-cell phenotypic and functional analysis thus began that encompassed the study of B-cell microanatomy principally in the lymph nodes, spleen and mucosae. The novel discovery of the differential localisation of B-cells with distinct phenotypes and functions revealed the compartmentalisation of B-cells. This review thus aims to describe novel findings regarding the B-cell compartments found in the mouse as a model organism, and in human physiology and pathology. It must be emphasised that some differences are noticeable between the mouse and human systems, thus increasing the complexity of B-cell compartmentalisation. Special attention will be given to the (lymph node and spleen) marginal zones, which represent major crossroads for B-cell types and functions and a challenge for understanding better the role of B-cell specificities in innate and adaptive immunology

Intraperitoneal but Not Intravenous Cryopreserved Mesenchymal Stromal Cells Home to the Inflamed Colon and Ameliorate Experimental Colitis

BACKGROUND AND AIMS: Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. METHODS: After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. RESULTS: Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. CONCLUSIONS: Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis

The Role of Intestinal Microbiota in the Development and Severity of Chemotherapy-Induced Mucositis

Mucositis, also referred to as mucosal barrier injury, is one of the most debilitating side effects of radiotherapy and chemotherapy treatment. Clinically, mucositis is associated with pain, bacteremia, and malnutrition. Furthermore, mucositis is a frequent reason to postpone chemotherapy treatment, ultimately leading towards a higher mortality in cancer patients. According to the model introduced by Sonis, both inflammation and apoptosis of the mucosal barrier result in its discontinuity, thereby promoting bacterial translocation. According to this five-phase model, the intestinal microbiota plays no role in the pathophysiology of mucositis. However, research has implicated a prominent role for the commensal intestinal microbiota in the development of several inflammatory diseases like inflammatory bowel disease, pouchitis, and radiotherapy-induced diarrhea. Furthermore, chemotherapeutics have a detrimental effect on the intestinal microbial composition (strongly decreasing the numbers of anaerobic bacteria), coinciding in time with the development of chemotherapy-induced mucositis. We hypothesize that the commensal intestinal microbiota might play a pivotal role in chemotherapy-induced mucositis. In this review, we propose and discuss five pathways in the development of mucositis that are potentially influenced by the commensal intestinal microbiota: 1) the inflammatory process and oxidative stress, 2) intestinal permeability, 3) the composition of the mucus layer, 4) the resistance to harmful stimuli and epithelial repair mechanisms, and 5) the activation and release of immune effector molecules. Via these pathways, the commensal intestinal microbiota might influence all phases in the Sonis model of the pathogenesis of mucositis. Further research is needed to show the clinical relevance of restoring dysbiosis, thereby possibly decreasing the degree of intestinal mucositis

SAT0631 Inter-observer and intra-observer reliability of the omeract ultrasonographic (US) criteria for the diagnosis of calcium pyrophosphate deposition disease (CPPD) at the metacarpal-phalangeal (MCP), wrist, acromion-clavicular (AC) and hip joints

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