Role of Stromal Cell-Derived Factor-1 in Neoangiogenesis in Endometriosis Lesions

Endometriosis affects 5-10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Treatment for endometriosis primarily focuses on symptom relief, is short term with severe side effects and often leads to recurrence of the condition. Establishing new blood supply is a fundamental requirement for endometriosis lesions growth. This has led to the idea that antiangiogenic therapy may be a successful approach for inhibiting endometriosis. Recent evidence indicates that endothelial progenitor cells (EPCs) contribute to neoangiogenesis of endometriotic lesions. These EPCs are recruited to the lesion site by stromal cell-derived factor-1 (SDF-1). We hypothesize that SDF-1 is central to the neoangiogenesis and survival of endometriotic lesions and that administration of SDF-1 blocking antibody will inhibit lesion growth by inhibiting angiogenesis in a murine model of endometriosis. Immunohistochemistry for SDF-1 and CD34 was performed on human endometriosis and normal endometrial samples. Quantification of SDF-1 and EPCs was performed in the blood of endometriosis patients and controls using ELISA and flow cytometry, respectively. A new mouse model of endometriosis was developed using BALB/c-Rag2-/-/IL2rg-/- mice to investigate role of SDF-1 in neoangiogenesis. Either SDF-1 blocking antibody or an isotype control was administered on a weekly basis for four weeks. Weekly samples of peripheral blood from mice were analyzed for SDF-1, other cytokines of interest and EPCs. Mice were euthanized at seven weeks to observe lesion growth and blood vessel development. Our results indicate overabundance of SDF-1 and CD34+ progenitor cells in human endometriotic lesions compared to eutopic endometrium. In the mouse model, SDF-1 and circulating EPC levels decreased from pre-treatment levels after one week, and remained constant over the course of the treatment in both SDF-1 blocking antibody and isotype control groups. In the SDF-1 blocking group, reduced vascularity of lesions, identified by immunofluorescence staining for CD31, was revealed compared to isotype controls. These findings suggest that SDF-1 may be responsible for CD34+ progenitor cell recruitment to the neoangiogenic sites in endometriosis. Blocking of SDF-1 reduces neovascularization of human endometriotic lesions in a mouse model. Further studies on blocking SDF-1 in combination with other antiangiogenic agents are needed.

Conjugação e validação de controle isotípico IgG1-FITC para uso em citometria de fluxo

Em meados da década de 50 iniciou-se o desenvolvimento da citometria de fluxo, tecnologia que permite verificar características físico-químicas de células ou partículas suspensas em meio fluido. Esta tecnologia utiliza anticorpos monoclonais marcados com fluorocromos como ferramenta de investigação em diversas análises e necessita de controles isotípicos para definição da região negativa (background). Estes controles são constituídos por imunoglobulinas de mesmo isotipo e fluorocromo dos anticorpos testes, sendo o isotiocianato de fluoresceína (FITC) o marcador fluorescente mais utilizado na conjugação de anticorpos. Os controles isotípicos têm como função definir a fluorescência inespecífica (células negativas) e as regiões fluorescentes (células positivas). No presente estudo foi selecionado anticorpo monoclonal murino (AcMm) dirigido contra antígeno eritrocitário canino, produzido no Laboratório de Anticorpos Monoclonais do Hemocentro de Botucatu, o qual reage positivamente com hemácias de cães, mas nunca com leucócitos humanos, tendo, portanto, potencial utilidade como controle negativo em citometria de fluxo. A purificação do AcMm da subclasse IgG1 foi feita por cromatografia de afinidade em Proteína-A Sepharose, e o controle da purificação realizado por eletroforese em géis de ágarose e poliacrilamida (SDS-PAGE). A imunoglobulina purificada foi conjugada ao FITC e filtrado em coluna de Sephadex G-25 para separação das proteínas marcadas e não-marcadas. O AcMm conjugado foi testado contra hemácias de cães, e o êxito da conjugação comprovado por testes de fluorescência, sendo a mediana de positividade de 94,70. Frente a leucócitos humanos a mediana de positividade foi 0,03 contra 0,50 dos reagentes comerciais. Os testes estatísticos não-paramétricos de Wilcoxon e correlação de Spearman comprovaram a eficiência e validam o controle isotípico produzido em comparação aos reagentes comerciais testados.

Estudo da resposta imune humoral em mulheres com neoplasia ovariana. -

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

The Role of IL-25 in Colitis-Associated Colon Cancer and Pulmonary Inflammation

Thesis (Ph.D.)--University of Washington, 2016-06

Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Blockade of interleukin-6 effects on cytokine profiles and macrophage activation after spinal cord injury in mice

The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. MR16-1 antibodies versus isotype control antibodies or saline alone was administered immediately after thoracic SCI in mice. MR16-1-treated group samples showed increased neuronal regeneration and locomotor recovery compared with controls. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. MR16-1 treatment promoted arginase-1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site and enhanced positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

A second order control-volume finite-element least-squares strategy for simulating diffusion in strongly anisotropic media

An unstructured mesh �nite volume discretisation method for simulating di�usion in anisotropic media in two-dimensional space is discussed. This technique is considered as an extension of the fully implicit hybrid control-volume �nite-element method and it retains the local continuity of the ux at the control volume faces. A least squares function recon- struction technique together with a new ux decomposition strategy is used to obtain an accurate ux approximation at the control volume face, ensuring that the overall accuracy of the spatial discretisation maintains second order. This paper highlights that the new technique coincides with the traditional shape function technique when the correction term is neglected and that it signi�cantly increases the accuracy of the previous linear scheme on coarse meshes when applied to media that exhibit very strong to extreme anisotropy ratios. It is concluded that the method can be used on both regular and irregular meshes, and appears independent of the mesh quality.