PIG TONSIL CELLS AS A MODEL TO EVALUATE ORAL, LOW-DOSE CYTOKINE TREATMENTS

Cytokines are low molecular weight mediators of the immune system. Because of their properties and biological roles, they have been used as diagnostic reagents for human and animal diseases, as well as for prognostic tests and prophylactic treatments. Concerning this kind of treatments, there is uncertainty as to how cytokines are effective after oral administration. They probably act on the oral lymphoid tissues (palatine and pharyngeal tonsils) and trigger a cascade of events leading to activation of the immune system and control of the inflammatory cascade in tissues and organs. Owing to above, the aim of our project was to develop a reliable in vitro model related to the crucial interactions between cytokines and oral lymphoid cells, in terms of homeostatic regulation of the inflammatory response and antibody production. In this project, we focused our attention on the IFN-\u3b1 system. The study was divided into 4 steps: 1) Isolation and culture of pig tonsil lymphocytes; 2) evaluation of IPEC-J2 cells as a reporter system of the anti-inflammatory control actions of interferon-alpha; 3) A pig tonsil cell culture model for evaluating oral, low-dose IFN-\u3b1 treatments; 4) Disease-dependent modulation of tonsil cell phenotypes. In the first steps we develop reliable procedures for isolation and culture of pig tonsil cells, which were validated for use in functional immunoassays. Our results indicate that pig tonsil cells can be employed within 2 months from freezing to maintain suitable conditions in terms of recovery, vitality and release of antibody in vitro. Tonsil mononuclear cells also showed the ability to secrete antimicrobial peptides and to respond in vitro to immunological stimuli. In the second part of the project, IPEC-J2 (a continuous cell line of porcine intestinal epithelial cells) was validated as reporter system of the biological properties of IFN-\u3b1. Three different experimental conditions (oxidative stress, inflammatory response, and amplification of lymphoid cell signals) were selected to evaluate the effects of porcine recombinant IFN-\u3b11 (rIFN-\u3b1) and 2 natural porcine IFN-\u3b1 preparations (nIFN-\u3b1). The IFNs under study showed significantly different control actions in IPEC-J2 cells. In particular, rIFN-\u3b1 was shown to down-regulate interleukin (IL)-8, IL-1\u3b2, tumor necrosis factor (TNF)-\u3b1, and \u3b2-defensin 1 genes either directly, or indirectly through second messengers released by IFN-\u3b1-treated lymphoid cells. With regard to IL-6, only second messengers from IFN-\u3b1-treated lymphoid cells could regulate the expression of this cytokine. Our results indicate that IPEC-J2 cells can be a useful tool for investigating the regulatory actions of type I IFNs and second messengers thereof. In the third step we developed an in vitro model of interaction between different types of human and porcine IFNs-\u3b1 at low / moderate concentrations and pig tonsil cells. The IFNs-\u3b1 under study showed different properties with respect to three fundamental control actions: 1) IgA release in culture (up and down-regulation, respectively), 2) release of natural antimicrobial compounds, and 3) homeostatic regulation of the inflammatory response. Some IFNs-\u3b1 caused a significant inhibition of IL-8 (protein release and gene expression) and \u3b2-Defensin 1 (gene expression) through second messengers released by IFN \u3b1-treated tonsil cells. Interestingly, a human lymphoblastoid IFN-\u3b1 under study caused the decrease of polyclonal IgA release by pig tonsil cells and significantly stimulated the in vitro recall antibody response of swine PBMC to Foot-and-Mouth Disease virus. The modulation of IgA and antibacterial compounds was accompanied by an anti-inflammatory control action at the same, low to moderate IFN-\u3b1 concentrations (1 to 100 U / ml). This highlights the very foundation of the homeostatic control actions performed by Type I IFNs: to promote an effective host response to infectious and non-infectious stressors and to turn off noxious inflammatory responses associated to tissue damage and waste of metabolic energy. The described tonsil cell model in vitro can be conducive to a further development of oral cytokine treatments in human and animals in the \u201cone health\u201d conceptual framework

In welfare we trust? Political trust in Portugal and Spain, 2008-2014

info:eu-repo/semantics/publishedVersio

Epithelial–mesenchymal transition in an EcPV2-positive vulvar squamous cell carcinoma of a mare

BackgroundVulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. ObjectiveTo describe a case of naturally occurring EcPV2-induced VSCC, by investigating tumour ability in undergoing the epithelial-to-mesenchymal transition (EMT). Study designCase report. MethodsA 13-year-old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was consistent with a VSCC. Real-time qPCR, real-time reverse transcriptase (RT)-qPCR and RNAscope were carried out to detect EcPV2 infection and to evaluate E6/E7 oncogenes expression. To highlight the EMT, immunohistochemistry (IHC) was performed. Expression of EMT-related and innate immunity-related genes was investigated through RT-qPCR. ResultsReal-time qPCR, RT-qPCR and RNAscope confirmed EcPV2 DNA presence and expression of EcPV2 oncoproteins (E6 and E7) within the neoplastic vulvar lesion. IHC highlighted a cadherin switch together with the expression of the EMT-related transcription factor HIF1 & alpha;. With RT-qPCR, significantly increased gene expression of EBI3 (45.0 & PLUSMN; 1.62, p < 0.01), CDH2 (2445.3 & PLUSMN; 0.39, p < 0.001), CXCL8 (288.7 & PLUSMN; 0.40, p < 0.001) and decreased gene expression of CDH1 (0.3 & PLUSMN; 0.57, p < 0.05), IL12A (0.04 & PLUSMN; 1.06, p < 0.01) and IL17 (0.2 & PLUSMN; 0.64, p < 0.05) were detected. Main limitationsLack of ability to generalise and danger of over-interpretation. ConclusionThe results obtained were suggestive of an EMT event occurring within the neoplastic lesion