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Abstract: The aim of this work was to develop mAbs against porcine CD205 and to conduct a comparative analysis of the CD205 protein expression on lymphoid tissues, monocyte-derived dendritic cells (DCs) and DCs isolated from the porcine skin. To conduct this study, we generated a monoclonal antibody, designated 1.F6F6, against the C-type lectin-like domain-5 of the porcine CD205 and showed that it recognizes a protein band of ∼200kDa by Western Blot analysis in mesenteric lymph nodes cells. Flow cytometric analysis showed that the mAb 1.F6F6 recognized 28.5%, 28.1% and 34.1% of cells from tonsil, inguinal and mesenteric lymph nodes, respectively, and 6% of cells from thymus. Analysis of monocyte-derived DCs showed that approximately 20% were positive and activation of the cells with LPS increased the positive population to 36%. Analysis of DCs isolated from the porcine skin showed that approximately 70% of the cell population expressed the CD205 receptor. The development of a monoclonal antibody capable of recognizing the CD205 receptor in swine opens up possibilities of applying new strategies for enhancing vaccine efficacy by using the anti-CD205 antibody for DC antigen-targeting to enhance priming of immune responses. [Copyright &y& Elsevier]

Abstract: Dendritic cells express a type cell-surface receptor, CD205 that plays a role in antigen capture and delivery to the endocytic pathway. Besides DCs, high CD205 expression is also detected on thymic epithelial cells but B cells, macrophages, and T cells have limited or no expression. CD205 has been characterized in several animal species except swine. The aim of this work was to characterize porcine CD205 and mRNA expression on different cells and tissues involved in immune responses. A complete porcine CD205 sequence of 5175bp was obtained from porcine thymus cDNA by PCR gene-walking strategy and this gene encoded a protein of 1723 amino acids. The multi-domain structure reported for murine, human, and bovine CD205 was also conserved in porcine with an overall amino acid identity of 74, 81, and 85%, respectively. Quantitative RT-PCR analysis of the CD205 mRNA profiles in normal porcine tissues and cells showed that thymus and Langerhans cells expressed the highest levels. Further characterization of porcine CD205 will lead to better understanding of the role of this receptor and development of contemporary strategies for antigen targeting to DCs in swine. [Copyright &y& Elsevier]

Abstract: Delayed development of virus-specific immune response has been observed in pigs infected with the porcine reproductive and respiratory syndrome virus (PRRSV). Several studies support the hypothesis that the PRRSV is capable of modulating porcine immune system, but the mechanisms involved are yet to be defined. In this study, we evaluated the induction of T regulatory cells by PRRSV-infected dendritic cells (DCs). Our results showed that PRRSV-infected DCs significantly increased Foxp3+CD25+ T cells, an effect that was reversible by IFN-α treatment, and this outcome was reproducible using two distinct PRRSV strains. Analysis of the expressed cytokines suggested that the induction of Foxp3+CD25+ T cells is dependent on TGF-β but not IL-10. In addition, a significant up-regulation of Foxp3 mRNA, but not TBX21 or GATA3, was detected. Importantly, our results showed that the induced Foxp3+CD25+ T cells were able to suppress the proliferation of PHA-stimulated PBMCs. The T cells induced by the PRRSV-infected DCs fit the Foxp3+CD25+ T helper 3 (Th3) regulatory cell phenotype described in the literature. The induction of this cell phenotype depended, at least in part, on PRRSV viability because IFN-α treatment or virus inactivation reversed these effects. In conclusion, this data supports the hypothesis that the PRRSV succeeds to establish and replicate in porcine cells early post-infection, in part, by inducing Th3 regulatory cells as a mechanism of modulating the porcine immune system. [Copyright &y& Elsevier]

Dendritic cells (DC) are considered the most important antigen presenting cells of the immune system. Its anatomical location (skin, mucosa and peripheral blood), the expression of receptors to recognize pathogens, the expression of co-stimulatory molecules (CD80/86), the major histocompatibility complex (MHC) class I and II, and the production of cytokines (such as IFN-α, IL-10, IL-12) confers to these cells the characteristic to regulate innate and adaptive immune responses. The objective of this work was to evaluate the effects of the porcine reproductive and respiratory virus (PRRS) in mature DC. DC were generated from blood monocytes using IL-4 and GM-CSF and were stimulated with lipopolysaccharide (LPS) to induce their maturation. The results show that the expression of CD14 and CD172a molecules in infected DC was not affected, while MHC II and CD80/86 expression was diminished. This decrease seems to affect the allogenic proliferation of lymphocytes stimulated with infected DC. On the other hand, the virus increases mRNA expression of IL-10 and TNF-α, and diminishes that for IL-1β and IL-6. The results obtained could explain, in part, the immunophatology of the disease. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to characterize the immune responses of DCs after infection with four different EU strains of PRRSV and whether they show any ability to immunomodulate T cells activation. Our results show that all EU strains can efficiently infect and replicate in DCs. Nevertheless, SLA-II levels remained unaltered in DC infected by all EU PRRSV strains, whereas SLA-I expression was only reduced when strain 2992 was used. IL-10 production was induced by three EU PRRSV strains, being strain 2992 the highest inducer. However, no induction of Treg cells, measured by CD25 and Foxp3 expression on lymphocytes co-cultured with infected DCs, was found. TGF-β induction was not detected in DC infected with any EU strain tested. In conclusion, DCs infected with EU PRRSV strains exhibited an unbalanced ability to stimulate T cell response and was strain dependent. However, Treg cells were not induced, at least in vitro. [Copyright &y& Elsevier]