Suite à leur activation par des cellules présentatrices d’antigène, les lymphocytes T expriment le récepteur de chimiokines CXCR3 et peuvent alors infiltrer les tissus enflammés. Pour ce faire, CXCR3 permet aux cellules de détecter et de chimiotaxer vers des gradients de concentration croissants des chimiokines CXCL11 et CXCL10 retrouvées dans le milieu extracellulaire. Les gradients de chimiokines doivent être régulés afin d’assurer une réponse immunitaire adéquate. Toutefois, comment ces gradients sont formés, maintenus et éliminés n’est pas très bien compris. Il a été montré que certaines cellules participent à la régulation des gradients de chimiokines dans d’autres contextes. Pour ce faire, elles expriment des récepteurs de chimiokines spécialisés qui séquestrent les chimiokines, réduisant leur niveau dans le milieu extracellulaire. Ces récepteurs sont classés comme étant atypiques et n’induisent pas la chimiotaxie. Dans ce travail, nous proposons un mécanisme de régulation pour les chimiokines CXCL11 et CXCL10 qui est en ligne avec un mécanisme d’auto-génération de gradients de chimiokines par les lymphocytes T activés. / During the immune response, T lymphocytes are activated in lymph nodes by antigen-presenting cells. Following activation, T cells up-regulate the chemokine receptor CXCR3, which allows them to infiltrate inflamed tissues by a migratory process named chemotaxis. To do so, CXCR3 allows the cells to detect concentration gradients of the chemokines CXCL11 and CXCL10 which are secreted locally, at the inflammation site. The chemokine gradient represents a directional cue, indicating to cells in which direction to migrate. Chemokine gradients must be regulated to assure an appropriate immune response. However, how these gradients are formed, maintained and eliminated is not well understood. It has been shown in other contexts that cells participate in gradient shaping. To do so, they express specialised chemokine receptors who scavenge chemokines, reducing chemokine levels in the environment. These receptors are considered atypical because they do not induce chemotaxis. In this work, we provide evidence for the regulation of the chemokines CXCL11 and CXCL10 that is in line with self-generation of chemokine gradients by chemotaxing activated T cells.

Nef is an accessory protein encoded by human and simian immunodeficiency viruses (HIV and SIV), and is critical for viral pathogenicity in vivo.The structure of Nef has been resolved and the major cellular activities of Nef are generally described as down-regulation of cell surface molecules, enhancement of virus infectivity and regulation of cell signaling and activation. Macrophages represent a key target of HIV-1 infection and may contribute significantly to viral pathogenesis by facilitating viral propagation, maintaining a viral reservoir and regulating viral replication. During HIV-1 infection, various cytokines and chemokines are induced for viral advantages more than for host defense. We have previously demonstrated that HIV-1 Nef regulates the release of chemokines, MIP-1α and MIP-1ß, from infected macrophages and have proposed that this may enhance conditions for viral replication by promoting recruitment of substrate lymphocytes to sites of infection (1). However, the molecular basis for this Nef activity remains to be defined. The main goals of this thesis are to identify the functional motif in Nef that is responsible for chemokine induction in macrophages and to elucidate the relevance of this motif to other Nef functions. Using a mutagenesis approach, we have eventually identified a novel motif (KEK) that regulates chemokine production in infected macrophages after we excluded several previously described Nef motifs. This motif is conserved in both HIV-1 and SIV Nef proteins. Mutations in this domain abrogated MIP-1ß induction as well as the Nef-dependent release of other secretory factors by macrophages. However, disruption of this motif did not affect other Nef-ascribed activities such as CD4 and MHC-I down-regulation. In addition, we have determined the involvement of viral Env proteins in Nef-induced chemokine production. Distinct signaling pathways that regulate chemokine release in macrophage will also be described. Finally, several possible roles of the KEK motif are proposed and some preliminary results of co-immunoprecipitation experiments will be presented which aim to characterize cellular proteins involved in chemokine regulation by Nef. Collectively, our studies reveal a specific determinant within Nef that is critical for chemokine release by Nef. Identification of this motif paves the way for future studies to explore the molecular machanisms of Nef-regulated cell signaling pathways. Such knowledge may point to new therapeutic strategies that interrupt Nef function and limit the course of HIV-1 infection.

The chemokine receptor CCR5 has been implicated in the pathogenesis of prostate cancer (PCa). A novel series of piperazine derivatives have been designed and synthesized as CCR5 antagonists and their activity as inhibitors of PCa cell lines proliferation was explored. A lead compound has been identified which induced 100% inhibition of PCa cell proliferation at micromolar concentrations. Anibamine, the only natural product CCR5 antagonist, was also examined for its anti-proliferative activity and was found to inhibit proliferation of PCa cells at micromolar concentrations as well. The expression of RANTES mRNA was observed in DU-145, M12 and P69 cells via RT-PCR, while the expression of CCR5 mRNA was observed only in M12 cells. A CHO-CCR5 stable cell line was prepared for the CCR5 ligand competition binding assays. Both anibamine and the newly identified lead compound will serve as leads in the development of novel CCR5 antagonists as anti-prostate cancer agents.

Previously, the CCR5 receptor was found to be a good target for treating prostate cancer (PCa). Dr. Yan Zhang’s laboratory designed several CCR5 antagonists, which were screened for their inhibitory effect on the growth and invasion of the M12, DU145 and PC-3 PCa cell lines. Primary in vitro screening showed one compound (Drug 17) significantly inhibited the proliferation of PCa cells at 1μM concentration, with a half-maximal inhibitory concentration of 237.68 nM. Further in vitro assays including a proliferation, cytotoxicity and invasion assay confirmed the inhibitory effect of drug 17. The physiological effect of drug 17 was tested by the Ware laboratory in vivo by subcutaneous injection of M12 cells into male, athymic nude mice. Tumor growth was slowed in mice receiving injections of drug 17 compared to sham injected controls. Thus, in vitro and in vivo assays suggest drug 17 might be an effective therapy to block PCa progression.

A doença enxerto contra hospedeiro (GVHD) é uma complicação comum nos pacientes submetidos ao transplante de células-tronco hematopoiéticas (TCTH), sendo considerada a maior causa de morbidade e mortalidade nesses pacientes. O principal objetivo do presente estudo foi relacionar a concentração de células de Langerhans em mucosa bucal de pacientes com GVHDc bucal com a expressão da quimiocina CCL20 e de seu receptor CCR6 no epitélio bucal, a fim de elucidar os mecanismos biológicos envolvidos no recrutamento das células de Langerhans na GVHDc. Foram selecionados fragmentos obtidos por biópsia de mucosa bucal de 60 pacientes onco-hematológicos e hematológicos submetidos previamente ao transplante de células tronco hematopoiéticas no Hospital Amaral Carvalho, Jaú SP, onde 30 pacientes desenvolveram GVHDc em mucosa bucal (Grupo 1) e 30 não desenvolveram GVHDc (Grupo 2). Amostras obtidas a partir de 30 biópsias de lesões não inflamatórias em mucosa bucal constituíram o Grupo Controle (Grupo 3). Cortes microscópicos foram avaliados em coloração de rotina Hematoxilina e Eosina, e submetidos à técnica imuno-histoquímica, utilizando-se anticorpos monoclonais anti-CD1a e anti-CCR6, e anticorpos policlonais anti-CCL20. As células de Langerhans CD1a+ foram quantificadas no epitélio da mucosa bucal, e os resultados demonstraram um maior número destas células nos pacientes com GVHDc quando comparados àqueles sem GVHDc e ao Grupo Controle (p<0,001). A análise da imunomarcação das moléculas CCR6 e CCL20 foi subjetiva com aplicação de escores. Quanto à molécula CCR6, houve maior expressão no Grupo 1 (p<0,001) em comparação aos outros Grupos; porém, quanto à expressão de CCL20, não houve diferença estatística entre os três Grupos (p=0,108). Estes resultados sugerem que o aumento das células de Langerhans, na doença enxerto contra hospedeiro crônica, em mucosa bucal, pode estar associado a maior expressão do receptor CCR6. Possivelmente, o maior recrutamento de células de Langerhans até a mucosa bucal, em pacientes transplantados de medula óssea, colabora para o desenvolvimento da GVHDc bucal. / The graft versus host disease (GVHD) is a common complication in patients undergoing hematopoietic stem cell transplantation (HSCT), and considered a major cause of morbidity and mortality in these patients. The main objective of this study was to compare the concentration of Langerhans cells in oral mucosa of patients with oral chronic GVHD (GVHDc) with the expression of the chemokine CCL20 and its receptor CCR6 in oral epithelium, in order to clarify the biological mechanisms involved in the recruitment of Langerhans cells in GVHDc. We selected 60 biopsies of oral mucosa from onco-hematological and hematological patients submitted to prior hematopoietic stem cell transplantation at Hospital Amaral Carvalho, Jaú - SP from which 30 patients developed GVHDc in the oral mucosa (Group 1) and 30 did not develop GVHDc (Group 2). The Control Group (Group 3) was obtained from 30 biopsies of non-inflammatory lesions of oral mucosa. Microscopic sections were evaluated in routine Hematoxylin and Eosin staining, and submitted to immunohistochemistry using anti-CD1a and anti-CCR6 monoclonal antibodies, and anti-CCL20 polyclonal antibody. The Langerhans cells (CD1a+) were quantified in the epithelium of the oral mucosa, and the results showed a greater number of these cells in patients with GVHDc compared to those without GVHDc and the Control Group (p<0.001). Analysis of immunostaining of molecules CCL20 and CCR6 were subjective with application of scores. The expression of CCR6 molecule was more significant in Group 1 (p<0.001) compared to other groups, but in relation to CCL20 expression, there was no statistical difference between the three groups (p=0.108). These results suggest that the increase of Langerhans cells in GVHDc affecting oral mucosa may be associated with increased expression of the receptor CCR6. We suggest that the increased recruitment of Langerhans cells to the oral mucosa in patients with transplanted bone marrow contributes to the development of oral GVHDc.

Toxoplasma gondii é um protozoário intracelular obrigatório que causa a toxoplasmose. Em modelo experimental, camundongos C57BL/6 infectados por via oral com 100 cistos de T. gondii, cepa ME-49, desenvolvem sérias lesões intestinais similares as observadas em doenças inflamatórias intestinais. Ao invadir as células epiteliais intestinais, o parasito induz uma resposta inflamatória de padrão T helper (Th) 1 elevada, ativada pela produção de quimiocinas e citocinas envolvidas na migração e ativação celular. Para que ocorra essa migração celular para o sítio de infecção é necessário a presença de receptores de quimiocinas. O receptor de quimiocinas CCR5 é muito importante para o recrutamento celular em algumas infecções e está envolvido com a migração de vários subtipos celulares como células dendríticas, células T e, em particular, células T reguladoras. CCR5 pode estar relacionado também a mecanismos independentes da migração celular, no qual a sinalização intracelular e ativação de NF-B podem levar a intensificação da resposta imunológica. Ainda não está claro o papel do receptor CCR5 no modelo de infecção oral por T. gondii. Dessa forma, animais C57BL/6 e deficientes em CCR5 foram infectados por via oral com 5 cistos de T. gondii, cepa ME-49, e alguns parâmetros imunológicos e bioquímicos foram avaliados no 8º dia de infecção. Os resultados mostraram que animais CCR5-/- apresentaram alta suscetibilidade à infecção oral por T. gondii, exibindo um intenso infiltrado inflamatório no íleo e regiões de ulceração epitelial, quando comparados com animais C57BL/6. Independentemente de serem deficientes ou não de CCR5, os camundongos apresentaram focos inflamatórios dispersos pelo parênquima do fígado, entretanto camundongos CCR5-/- apresentaram uma extensiva vacuolização dos hepatócitos, com excessivo acúmulo de lipídeos no órgão e elevada concentração sérica de triglicérides e de transaminases. A carga parasitária foi significativamente mais elevada no intestino delgado e no fígado dos animais CCR5-/- em comparação com animais C57BL/6. Foi observada também uma menor migração de células NK no intestino delgado, bem como um aumento na frequência de células T CD4+ neste órgão e uma menor concentração de IFN- e IL-12p40 no macerado do fígado dos animais CCR5-/- em comparação com C57BL/6. Análise de expressão gênica no fígado revelou redução na formação de transcritos para PPAR nos animais deficientes em CCR5, e quando os camundongos foram tratados com Gemfibrozil, um agonista de PPAR, houve reversão na vacuolização hepática e na concentração de triglicérides no soro dos animais CCR5-/-. Estes dados sugerem que a migração celular dependente de CCR5 é essencial para a modulação da resposta inflamatória induzida por T. gondii no intestino delgado. Além do mais, a ausência de CCR5 compromete a integridade hepática durante a infecção oral por T. gondii e os mecanismos moleculares envolvidos podem estar relacionados à expressão de PPAR. / T. gondii is an obligate intracellular protozoan parasite which is the causative agent of toxoplasmosis. In experimental model, C57BL/6 mice orally infected with a high parasitic load develop serious intestinal lesions, whose injuries are similar to those observed in Inflammatory Bowel Disease. This inflammation is caused due to parasite invasion of intestinal epithelial cells that elicit a robust Th1 type immune response. Moreover, chemokines produced by intestinal epithelial cells are involved in the migration and activation of inflammatory cells. In particular, the chemokine receptor CCR5 is important for cell recruitment in some infections and is involved with the migration of various cells subsets such as dendritic cells, T cells and, in particular regulatory T cells. CCR5 may also be related to mechanisms independent of cell migration, in which the intracellular signaling and activation of NF-B may lead to intensification of the immune response. The role of CCR5 has not been clear in the experimental oral T. gondii infection. Thus, wild type C57BL/6 mice and CCR5-/- littermates were infected with T. gondii by gavage and immune and biochemical parameters, were analyzed at day 8 after infection. The CCR5-/- mice showed to be highly susceptible to the parasite, with intense inflammatory infiltration in the ilea and regions of epithelial ulcerations in comparison with WT mice. Both strain of mice presented inflammatory foci scattered by parenchyma of the liver, however the CCR5-/- mice presented an extensive hepatocyte vacuolization with an excessive accumulation of lipids in the organ and elevated serum triglycerides and transaminases concentration. The parasite load was significantly higher on small intestine and liver samples of CCR5-/- in comparison with WT mice. There was also a minor migration of NK cells in the small intestine, as well as greater frequency of CD4+ T cells in this organ and a lower IFN- and IL-12p40 levels in liver homogenate samples in the CCR5-/- mice compared with WT mice. Gene expression analysis revealed a reduction in the formation of transcripts for PPAR in mice deficient in CCR5, and when the animals were treated with Gemfibrozil, a PPAR agonist, there was an improvement in the level of vacuolization and reduced triglycerides. These data suggest that a CCR5-dependent cell migration is essential for the modulation of T. gondii-induced inflammatory response in the small intestine. In addition, hepatic integrity during T. gondii oral infection is compromised in the absence of CCR5, and the molecular mechanisms involved can be related to PPAR expression.

No description available.

A doença enxerto contra hospedeiro (GVHD) é uma complicação comum nos pacientes submetidos ao transplante de células-tronco hematopoiéticas (TCTH), sendo considerada a maior causa de morbidade e mortalidade nesses pacientes. O principal objetivo do presente estudo foi relacionar a concentração de células de Langerhans em mucosa bucal de pacientes com GVHDc bucal com a expressão da quimiocina CCL20 e de seu receptor CCR6 no epitélio bucal, a fim de elucidar os mecanismos biológicos envolvidos no recrutamento das células de Langerhans na GVHDc. Foram selecionados fragmentos obtidos por biópsia de mucosa bucal de 60 pacientes onco-hematológicos e hematológicos submetidos previamente ao transplante de células tronco hematopoiéticas no Hospital Amaral Carvalho, Jaú SP, onde 30 pacientes desenvolveram GVHDc em mucosa bucal (Grupo 1) e 30 não desenvolveram GVHDc (Grupo 2). Amostras obtidas a partir de 30 biópsias de lesões não inflamatórias em mucosa bucal constituíram o Grupo Controle (Grupo 3). Cortes microscópicos foram avaliados em coloração de rotina Hematoxilina e Eosina, e submetidos à técnica imuno-histoquímica, utilizando-se anticorpos monoclonais anti-CD1a e anti-CCR6, e anticorpos policlonais anti-CCL20. As células de Langerhans CD1a+ foram quantificadas no epitélio da mucosa bucal, e os resultados demonstraram um maior número destas células nos pacientes com GVHDc quando comparados àqueles sem GVHDc e ao Grupo Controle (p<0,001). A análise da imunomarcação das moléculas CCR6 e CCL20 foi subjetiva com aplicação de escores. Quanto à molécula CCR6, houve maior expressão no Grupo 1 (p<0,001) em comparação aos outros Grupos; porém, quanto à expressão de CCL20, não houve diferença estatística entre os três Grupos (p=0,108). Estes resultados sugerem que o aumento das células de Langerhans, na doença enxerto contra hospedeiro crônica, em mucosa bucal, pode estar associado a maior expressão do receptor CCR6. Possivelmente, o maior recrutamento de células de Langerhans até a mucosa bucal, em pacientes transplantados de medula óssea, colabora para o desenvolvimento da GVHDc bucal. / The graft versus host disease (GVHD) is a common complication in patients undergoing hematopoietic stem cell transplantation (HSCT), and considered a major cause of morbidity and mortality in these patients. The main objective of this study was to compare the concentration of Langerhans cells in oral mucosa of patients with oral chronic GVHD (GVHDc) with the expression of the chemokine CCL20 and its receptor CCR6 in oral epithelium, in order to clarify the biological mechanisms involved in the recruitment of Langerhans cells in GVHDc. We selected 60 biopsies of oral mucosa from onco-hematological and hematological patients submitted to prior hematopoietic stem cell transplantation at Hospital Amaral Carvalho, Jaú - SP from which 30 patients developed GVHDc in the oral mucosa (Group 1) and 30 did not develop GVHDc (Group 2). The Control Group (Group 3) was obtained from 30 biopsies of non-inflammatory lesions of oral mucosa. Microscopic sections were evaluated in routine Hematoxylin and Eosin staining, and submitted to immunohistochemistry using anti-CD1a and anti-CCR6 monoclonal antibodies, and anti-CCL20 polyclonal antibody. The Langerhans cells (CD1a+) were quantified in the epithelium of the oral mucosa, and the results showed a greater number of these cells in patients with GVHDc compared to those without GVHDc and the Control Group (p<0.001). Analysis of immunostaining of molecules CCL20 and CCR6 were subjective with application of scores. The expression of CCR6 molecule was more significant in Group 1 (p<0.001) compared to other groups, but in relation to CCL20 expression, there was no statistical difference between the three groups (p=0.108). These results suggest that the increase of Langerhans cells in GVHDc affecting oral mucosa may be associated with increased expression of the receptor CCR6. We suggest that the increased recruitment of Langerhans cells to the oral mucosa in patients with transplanted bone marrow contributes to the development of oral GVHDc.

This dissertation represents efforts to determine the functional consequences acquired by vascular smooth muscle cells (SMC) in response to CD40 ligation by activated CD154+ T cells, and to elucidate components of the signaling pathway(s) activated in response to CD40 signaling in both monocytes and SMC. To study the consequences of CD40 stimulation, primary human monocytes and aortic SMC were treated with plasma membranes purified from CD154 + , CD4+ T cells. The results presented in this dissertation demonstrate that SMC, like monocytes/macrophages, are capable of interacting with T cells in a manner that results in reciprocal activation events. SMC were shown to present antigen to, and activate T cells. In turn T cell stimulus resulted in the activation of proinflammatory function in SMC initiated through the CD154:CD40 interaction. CD40 stimulation of SMC resulted in the production of the chemokines interleukin 8 (IL-8) and macrophage chemotactic protein-1 (MCP-1), and the upregulation of intercellular adhesion molecule (ICAM). Examination of the intracellular signaling pathways activated through CD40 signaling revealed the involvement of MAPKs in the pathway leading to induction of proinflammatory activity. Evaluation of CD40 signaling in monocytes demonstrated the activation of the MAPK family members ERK1/2, but not the MAPK family members p38 or c-jun-N-terminal kinase (JNK). In contrast, CD40 signaling in SMC was shown to result in ERK1/2 and p38 activation, and both of these kinases were shown to play a critical role in the induction of chemokine synthesis. An examination of the ability of anti-inflammatory cytokines to modulate CD40 signaling in monocytes and SMC demonstrated that the anti-inflammatory cytokines IL-4 and IL-10 abrogate CD40-mediated induction of inflammatory cytokine production by monocytes. This inhibition was shown to be a result of a negative influence of IL-4 and IL-10 on CD40 mediated ERK1/2, activation in monocytes. However, IL-4 and IL-10 did not inhibit SMC proinflammatory responses indicating a difference in the intracellular responses to these cytokines by the two cell types. (Abstract shortened by UMI.)

Mice infected with neurotropic variants of the murine coronavirus, mouse hepatitis virus, (strains JHMV or J2.2–V–1) develop acute and chronic CNS infections, and provide a model system to study the pathogenesis of virus–induced neuroinflammation, mechanisms of virus persistence, and anti–viral immune responses in the CNS. Using the J2.2–V–1 model of CNS infection, we addressed the role of sustained CCL2 production during viral infection using mice in which CCL2 was expressed transgenically in oligodendrocytes. Tonic CCL2 expression in the CNS resulted in delayed kinetics of virus clearance, and converted what is typically a mild, nonlethal disease to acutely lethal encephalitis, with the majority of mice succumbing to the infection. CCL2 induced a rapid and dysregulated inflammatory response that was no longer protective and was unable to efficiently clear virus from the CNS. Infected CCL2 Tg mice had increased numbers of Foxp3–expressing CD4 T cells (Tregs) and of macrophages and microglia expressing elevated levels of YM–1, a marker for alternatively activated macrophages, and nitric oxide. Our results showed that CCL2 has effects beyond serving as a chemoattractant for leukocytes, and has effects on the composition and function of inflammatory cells at sites of infection. In a separate set of experiments, I identified and characterized two additional heteroclitic variants of the JHMV epitope S598 that induced CD8 T cells with greater antigen sensitivity to the native S598 determinant relative to the cells primed by the native epitope. One of these heteroclitic epitopes elicited a T cell response with nearly complete cross–reactivity towards the native peptide. The structural data show that these heteroclitic epitopes induced modest conformational changes in the local environment of the peptide–MHCI complex. I also provide data to support the notion that heteroclitic determinants augment functional avidity by increasing surface epitope density. Collectively, these data will help guide the design of heteroclitic epitopes in the setting of vaccine development. Lastly, I examined the consequences of oxidative stress induced by viral infection on antigen presentation. The brains of JHMV–infected mice were found to have signs of oxidative stress, with significantly decreased ratios of reduced (GSH) to oxidized (GSSG) glutathione, suggesting that there is an environment that is conducive for cysteine modification with oxidized glutathione. We found that virus–induced oxidative stress resulted in the presentation of both native and S–glutathionylated forms of the JHMV epitope S510 by infected cells. A subset of the S510–specific CD8 T cells failed to recognize the modified form of the epitope, suggesting that GSH–modification of a cysteine–containing viral epitope might interfere with T cell recognition. Further, GSH-modified peptides were identified in stressed human cells, including herpes virus–transformed B cells, suggesting that the modification is not limited to mouse cells. Collectively these findings have implications for both anti–viral immunity and anti–tumor immunity, where oxidative stress has been shown to play a role during infection and tumorgenesis.