A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom / Nueva actividad de los timocitos estimulada por el veneno de la serpiente de cascabel (Crotalus durissus cumanensis)

Abstract. Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods. Objective: To test naïve thymocytes proliferation and monocytes stimulation. Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 105 cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages. Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.

Resumen. Introducción. El timo es activo principalmente durante los períodos neonatal y preadolescente. Objetivo. Probar la proliferación de los timocitos tempranos y la estimulación de monocitos que producen. Materiales y métodos. Se recogió tejido de timo fresco después de la cirugía de ratones recién nacidos. La suspensión de células se colocó sobre un soporte de Ficoll-Hypaque. Las células obtenidas (timocitos) se cultivaron y se midió la proliferación de células T vírgenes estimuladas por el veneno de Crotalus durissus cumanensis (Cdc) en dosis subletales (20 ng). A continuación, se agregó AlamarBlue™ a los pocillos y se incubaron durante 5 horas para evaluar la proliferación. Se recogieron células mononucleares de sangre periférica de ratones y se colocaron sobre un soporte de solución de Ficoll-Hypaque. Los timocitos que se dividieron activamente (25 x 105 células) a partir de los cultivos estimulados con veneno de Cdc (20 ng/pocillo) y se agregaron a los cultivos de monocitos recién obtenidos de la separación en la solución de Ficoll-Hypaque. Ambas poblaciones celulares se incubaron durante 36 horas hasta que los monocitos maduraron a macrófagos. Resultados Los timocitos tempranos experimentaron una rápida proliferación estimulada por el veneno de Cdc (NTCdc) y, posteriormente, indujeron la maduración y la función de las células progenitoras de monocitos, los cuales maduraron a macrófagos, que se tiñeron con tinta china. Conclusiones. Los timocitos tempranos proliferaron con la estimulación del veneno de Cdc y, posteriormente, el NT/Cdc indujo la maduración rápida y la función de las células progenitoras de monocitos, transformándose en macrófagos con sus características fenotípicas.

Polyclonal anti T-lymphocyte antibody therapy monitoring in kidney transplant recipients: comparison of CD3+ T cell and total lymphocyte counts / Monitoramento da terapia com anticorpo policlonal antilinfócitos T em transplantados renais: comparação da contagem de células T CD3+ e de linfócitos totais

ABSTRACT Objective: To investigate the correlation between total lymphocyte and CD3+ T cell counts in peripheral blood in renal transplant patients treated with anti-thymocyte globulin, and discuss related outcomes. Methods: A single-center, retrospective study involving 226 patients submitted to kidney transplant between 2008 and 2013, and treated with anti-thymocyte globulin for induction or treatment of cellular rejection. Doses were adjusted according to CD3+ T cell or total lymphocyte counts in peripheral blood. Results: A total of 664 paired samples were analyzed. The Spearman's correlation coefficient was 0.416 (p<0.001) for all samples combined; the overall Kappa coefficient was 0.267 (p<0.001). Diagnostic parameters estimated based on total lymphocyte counts were also calculated using the number of CD3+ T cells (gold standard), with a cut off of >20 cells/mm3. Conclusion: Total lymphocyte and CD3+ T cell counts in peripheral blood are not equivalent monitoring strategies in anti-thymocyte globulin therapy.

RESUMO Objetivo: Investigar a correlação entre a contagem de linfócitos totais e células T CD3+ no sangue periférico em receptores de transplante renal submetidos a tratamento com globulina antitimocitária, e discutir resultados relacionados. Métodos: Estudo retrospectivo de centro único envolvendo 226 pacientes submetidos a transplante renal entre 2008 e 2013 e tratados com globulina antitimocitária, para fins de indução ou tratamento de rejeição celular. As doses foram ajustadas de acordo com a contagem de células T CD3+ ou linfócitos totais no sangue periférico. Resultados: No total, 664 amostras pareadas foram analisadas. O coeficiente de correlação de Spearman para as amostras em geral foi de 0,416 (p<0,001) e o coeficiente Kappa, de 0,267 (p<0,001). Os parâmetros diagnósticos estimados com base na contagem de linfócitos totais foram recalculados, empregando-se o número de células T CD3+ (padrão-ouro) e adotando-se o ponto de corte >20 células/mm3. Conclusão: A contagem de linfócitos totais no sangue periférico não substitui a contagem de células T CD3+ enquanto estratégia de monitorização da terapia à base de globulina antitimocitária.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro

BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4-CD8-) and single-positive CD4+ and CD8+ thymocytes. A decrease in cell number was noted in double-positive (CD4+CD8+) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.

Modulação da expressão gênica no timo: uma perspectiva integrativa da regulação gênica no desenvolvimento de timócitos e involução tímica decorrente de infecção / Gene expression modulation in thymus: an integrative perspective of the gene regulation during thymocyte development and thymic involution due to infection

O timo é um órgão linfoide primário no qual ocorre o desenvolvimento de células T. Distúrbios nesse processo podem levar a doenças diversas inclusive à autoimunidade. [...] Nesse sentido, essa tese aborda os aspectos moleculares envolvidos na linfopoiese T sob a perspectiva das células progenitoras e das TEC. Nosso estudod e meta-análise utilizando microarranjos de timócitos em estágios de desenvolvimento definidos pela expressão de moléculas CD4 e CD8 permitiu a identificação de 14 agrupamentos gênicos,os quais se relacionam intimamente às etapas do desenvolvimento dessas células. A modulaçãodesses genes está vinculada à sinalização via receptor de células T (TCR) e pode sercorrelacionada com a afinidade pelo complexo formado entre peptídeo e o complexo principal dehistocompatibilidade (MHC). Destacam-se ainda, vias de sinalização relacionadas a outrosreceptores de superfície, evidenciando a importância dos estímulos externos no desenvolvimentode células T. Através da integração de dados, nós identificamos as vias e moléculas com maiorimpacto para a diferenciação de timócitos, bem como complementamos a compreensão de comoos eventos envolvidos nesse processo se organizam temporalmente, apresentando umaperspectiva global das ondas de modulação gênica. Já em outra perspectiva, estudamos aregulação gênica em TEC comparando células corticais e medulares, mas também focamos nainvolução tímica, uma resposta natural a diversas fontes de estresse e ao envelhecimento.Nessa tese, esse fenômeno foi estudado em modelos experimentais de fase aguda da doençade Chagas, pois existem evidências de que as alterações no timo durante a fase aguda podemser responsáveis pelo desfecho da doença anos mais tarde...

The thymus is a primary lymphoid organ, where the T cell development takes place. Disturbanceson this process can lead to a diversity of diseases, including autoimmunity. [...] In this sense, this thesis addresses the molecular aspects involved on Tlymphopoiesis under the progenitor cells and TEC perspective. Our meta-analysis study usingmicroarrays of thymocytes sorted based on the developmental stages defined by the CD4 andCD8 expression allowed to identify 14 gene clusters, which are closely related to thedevelopmental stages of these cells. Those genes modulation is linked to the T cell receptor(TCR) signaling pathway and it can be related to the TCR affinity for the complex formed betweenthe peptide and the major histocompatibility complex (MHC). Moreover, other signaling pathwaysrelated to surface receptors stand out, highlighting the importance of external stimuli on the T celldevelopment. Through data integration, we identify pathways and molecules that have morerelevant impact on the thymocyte differentiation, as well as, we improved the comprehension onhow the events are organized temporally, providing a global perspective on the gene modulationwaves. In another perspective, we have studied the gene regulation on TEC by comparingcortical and medullary cells, but also focused on thymic involution, a natural response to varioussources of stress and aging. In this thesis, this phenomenon was studied using Chagas' diseaseacute phase experimental model because there are evidences showing that the thymic alterationsduring the acute phase can be responsible for the disease outcome years later...

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.