Skeletal development is a tightly regulated homeostatic process that requires proper functioning of osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells. Improper functioning of either of these cell types results in diseases such as osteoporosis, osteogenesis imperfecta as well as Paget's disease. Crucial for the proper maintenance of the skeleton is the bone matrix, which encompasses both organic and inorganic components. Osteopontin (Opn) is an example of a major non-collagenous protein present in bone. Its expression is crucial for bone remodeling since it functions in recruiting osteoclasts for bone resorption and facilitates their adhesion to the bone matrix. Osteopontin is expressed in variety of cells and functions in facilitating signal transduction upon engagement of integrin. Osteopontin binds to the αvß3 (vitronectin receptor) the major integrin expressed on osteoclasts thereby mediating cell adhesion and migration. As a model to study osteopontin-mediated adhesion we have employed commercially available osteopontin and the HEK 293 cells that stably overexpress the vitronectin receptor (Vnr cells). We studied the ability of the Vnr cells to adhere to different extracellular matrices including osteopontin. / Bioengineering

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. Recent studies show that the complete genome sequence of Orientia tsutsugamushi have been determined. However, the early signaling events involved in the entry of O.tsutsugamushi into mammalian cells remains a challenge. In this study, we demonstrate that adherence ability and comparison of three major outer membrane protein TSA56, TSA47 and TSA22 of O.tsutsugamushi. Through expression and purification of three type-specific antigen 56-kDa (include TSA56-antigen domain I, TSA56-antigen domain III), 47-kDa and 22-kDa of O. tsutsugamushi , antiserum immunoblots from 22 clinical O. tsutsugamushi-infected patients and in vitro adhesion assay of E.coli overexpression outer membrane protein of O. tsutsugamushi , the antiserum titer and adherence ability of bacterial outer membrane proteins are determined. The data show that antiserum titer against three major outer membrane proteins of O. tsutsugamushi was markedly higher in TSA56 compared to TSA47 and TSA22. In adhesion assay, adhesion of host cells by TSA56 was readily than TSA47 and TSA22. Furthermore, adhesion experiment and antiserum titer against antigen-domain I (ADI) region (19-114 aa) in the extracellular domain of TSA56 was also significantly higher than previously reported antigen-domain III(ADIII) region (237-366 aa) which facilitates the invasion of O. tsutsugamushi through interaction with fibronectin .Taken together, these results clearly indicate that O. tsutsugamushi exploits TSA56-mediated bacterial adhesion, abundant antiserum titer and ADI region of TSA56 may draw another adhesion site (except for previously reported ADIII) to invade eukaryotic host cells.

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O crosstalk entre timócitos e células epiteliais tímicas é crucial para o desenvolvimento das células T e estabelecimento da tolerância central. Células tímicas epiteliais medulares (mTECs) contribuem para a autotolerância por meio da expressão ectópica de antígenos restritos aos tecidos (TRAs). A expressão de TRAs em mTECs é altamente dependente do gene Autoimmune Regulator (Aire). Por meio do reconhecimento de TRAs com alta afinidade, células T autoreativas são selecionadas negativamente do pool de timócitos em desenvolvimento. Apesar do papel de Aire na indução da tolerância central ser bem conhecido, os mecanismos celulares e moleculares precisos do processo permanecem obscuros. Nesse estudo, hipotetizamos que perturbações na expressão do gene Aire influenciam a adesão entre mTECs e timócitos, o que poderia resultar em um desequilíbrio na imunotolerância a antígenos próprios. Um ensaio funcional realizado com timócitos frescos, extraídos de um timo normal de camundongo e cocultivados com células epiteliais tímicas medulares da linhagem mTEC 3.10, demonstrou que a inibição do gene Aire por meio de RNA de interferência reduziu significativamente a capacidade das mTECs de promover a adesão dos timócitos. Análises por microarray revelaram que o silenciamento do gene Aire nas células mTEC 3.10 causou a modulação de mais de 1000 genes, alguns que codificam TRAs, outros que codificam proteínas envolvidas na adesão celular, como VCAM-1, e também outros que codificam moléculas coestimuladoras como CD80. Esses resultados contribuem para uma melhor compreensão do papel de Aire no controle da adesão mTEC-timócitos, a qual constitui um processo essencial para a seleção negativa de timócitos autoreativos / The crosstalk between thymocytes and thymic epithelial cells is critical for T cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through the ectopic expression of tissuerestricted antigens (TRAs) in the thymus. TRAs expression in mTECs is largely dependent on Autoimmune Regulator (Aire) gene. Through the recognition of TRAs with high affinity, developing autoreactive T cells are negatively select from the pool of developing thymocytes. Although the role of Aire in the induction of central tolerance is well known, the precise cellular and molecular mechanisms remain unclear. In this study, we hypothesize that disturbance in Aire gene expression influences adhesion between mTECs and thymocytes, which could result in an imbalance in immune-tolerance to self-antigens. A functional assay performed with fresh thymocytes dissociated from a normal mouse thymus and co-cultured with a medullary thymic epithelial cell line named mTEC 3.10, demonstrated that Aire RNAi knockdown significantly decreased the ability of mTECs to promote thymocyte adhesion. Microarray analysis revealed that Aire knockdown of the murine mTEC 3.10 cell line led to the modulation of more than 1000 genes, some of them coding for TRAs, others for proteins involved in cell adhesion like VCAM-1 and also for costimulatory molecules like CD80. These results contribute to a better understanding of the role of Aire in the control of mTEC-thymocyte adhesion, which is an essential process for negative selection of autoreactive thymocytes

O crosstalk entre timócitos e células epiteliais tímicas é crucial para o desenvolvimento das células T e estabelecimento da tolerância central. Células tímicas epiteliais medulares (mTECs) contribuem para a autotolerância por meio da expressão ectópica de antígenos restritos aos tecidos (TRAs). A expressão de TRAs em mTECs é altamente dependente do gene Autoimmune Regulator (Aire). Por meio do reconhecimento de TRAs com alta afinidade, células T autoreativas são selecionadas negativamente do pool de timócitos em desenvolvimento. Apesar do papel de Aire na indução da tolerância central ser bem conhecido, os mecanismos celulares e moleculares precisos do processo permanecem obscuros. Nesse estudo, hipotetizamos que perturbações na expressão do gene Aire influenciam a adesão entre mTECs e timócitos, o que poderia resultar em um desequilíbrio na imunotolerância a antígenos próprios. Um ensaio funcional realizado com timócitos frescos, extraídos de um timo normal de camundongo e cocultivados com células epiteliais tímicas medulares da linhagem mTEC 3.10, demonstrou que a inibição do gene Aire por meio de RNA de interferência reduziu significativamente a capacidade das mTECs de promover a adesão dos timócitos. Análises por microarray revelaram que o silenciamento do gene Aire nas células mTEC 3.10 causou a modulação de mais de 1000 genes, alguns que codificam TRAs, outros que codificam proteínas envolvidas na adesão celular, como VCAM-1, e também outros que codificam moléculas coestimuladoras como CD80. Esses resultados contribuem para uma melhor compreensão do papel de Aire no controle da adesão mTEC-timócitos, a qual constitui um processo essencial para a seleção negativa de timócitos autoreativos / The crosstalk between thymocytes and thymic epithelial cells is critical for T cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through the ectopic expression of tissuerestricted antigens (TRAs) in the thymus. TRAs expression in mTECs is largely dependent on Autoimmune Regulator (Aire) gene. Through the recognition of TRAs with high affinity, developing autoreactive T cells are negatively select from the pool of developing thymocytes. Although the role of Aire in the induction of central tolerance is well known, the precise cellular and molecular mechanisms remain unclear. In this study, we hypothesize that disturbance in Aire gene expression influences adhesion between mTECs and thymocytes, which could result in an imbalance in immune-tolerance to self-antigens. A functional assay performed with fresh thymocytes dissociated from a normal mouse thymus and co-cultured with a medullary thymic epithelial cell line named mTEC 3.10, demonstrated that Aire RNAi knockdown significantly decreased the ability of mTECs to promote thymocyte adhesion. Microarray analysis revealed that Aire knockdown of the murine mTEC 3.10 cell line led to the modulation of more than 1000 genes, some of them coding for TRAs, others for proteins involved in cell adhesion like VCAM-1 and also for costimulatory molecules like CD80. These results contribute to a better understanding of the role of Aire in the control of mTEC-thymocyte adhesion, which is an essential process for negative selection of autoreactive thymocytes