O lúpus eritematoso discoide (LED) é a variante mais comum do Complexo Lúpus Eritematoso, sendo considerado uma das dermatopatias imunemediadas mais evidenciadas em cães, ficando muito próximo à casuística do pênfigo foliáceo. É afecção, relativamente, benigna sem envolvimento sistêmico e, clinicamente, caracterizada por leucodermia e/ou eritema associado à perda do aspecto em \"calçamento de pedras\" do plano nasal e, imunologicamente, pela deposição de autoanticorpos (IgA, C3, IgM e IgG) na zona da membrana basal (ZMB). O presente estudo visa correlacionar os achados clínicos e histopatológicos com aqueles da imunofluorescência direta (IFD), objetivando aumentar a acurácia do diagnóstico do LED. Foram incluídos 11 cães (Grupo I) com o quadro lesional tegumentar característico do LED e, cinco caninos (Grupo II), dermatologicamente hígidos, para o controle negativo da IFD. Fragmentos cutâneos oriundos do plano nasal foram submetidos ao exame histopatológico e à reação da IFD e, os resultados obtidos em ambos os Grupos, I e II, foram analisados estatisticamente pelo Índice kappa (k) visando a determinação do grau de concordância ou confiabilidade entre os dois métodos diagnósticos. No Grupo I, o diagnóstico de LED foi firmado em 100% (11/11) dos animais, pela reação de IFD, enquanto que o exame histopatológico estabeleceu o diagnóstico em 81,8% (9/11) dos casos. A positividade da IFD para o LED canino foi estabelecida, em sua totalidade, a partir da fluorescência esverdeada evidenciada na ZMB e/ou em seus anexos cutâneos e, os imunoreagentes mais frequentemente encontrados foram a IgM e C3. Já, em relação ao Grupo II, na totalidade dos animais não se evidenciou histopatologicamente qualquer alteração tegumentar.Também, pela IFD os resultados mostraram-se negativos. O substancial grau de concordância entre a IFD e a histopatologia (k= 0,738 e p= 0,002) comprova que a reação de imunofluorescência direta é exequível e útil no estabelecimento do diagnóstico do LED canino. / The discoid lupus erythematosus (DLE) is the most common variant of the Complex Lupus Erythematosus, considered one of auto-immune skin diseases more evident in dogs, getting very close to the casuistry of pemphigus foliaceus. The disease is relatively benign and without systemic involvement, clinically characterized by leukoderma and / or erythema associated with loss of the normal planum nasale \"cobblestone \" architecture and immunologically by deposition of autoantibodies (IgA, C3, IgM and IgG) at the basement membrane zone (BMZ). This study aims to correlate the clinical and histopathological findings with those of direct immunofluorescence (DIF), aiming to increase the accuracy of diagnosis DLE. We included 11 dogs (Group I) with cutaneous lesions characteristic of DLE and five dogs (Group II), with no cutaneous alterations, for the negative DIF. Cutaneous fragments derived from the planum nasale were submitted to histopathological examination and DIF, and the results obtained in both Groups I and II were analyzed by kappa index (k) in order to determine the degree of agreement or reliability among two methods. In Group I, the diagnostic DLE was established in 100 % (11/11) of animals, by the reaction of DIF, while the histopathological examination confirmed the diagnosis in 81.8 % (9/11) of cases. The positive DIF for DLE canine was established, in its totality, from the greenish fluorescence evident in the BMZ and / or on their skin annexes and the immunoreagents most frequently found were IgM and C3. Now, in relation to the Group II, all animals did not reveal any histopathological tegumentar changes. Also, by the DIF results were negative. The substantial degree of agreement between the DIF and histopathology (k = 0.738 and p = 0.002) shows that the direct immunofluorescence assay is feasible and useful in establishing the diagnosis of canine DLE.

Herpetic stromal keratitis (HSK) is an immunopathological and tissue destructive corneal lesion caused by herpes simplex virus (HSV) infection, which induces an intense inflammatory response and finally leads to blindness. Accumulating evidence using the murine model has shown that Th-1 phenotype CD4+ T cells orchestrating the inflammation mainly contribute to the immunopathological reaction in HSV-1 infected cornea. Initially various innate immune cells recruit and produce numerous inflammatory and angiogenic molecules into the corneal stroma those in turn drive the corneal immunopathology. While the basic principles of immunity to the influenza A viruses (IAV) are probably similar for all vertebrates, detailed understanding is based largely on experiments in laboratory mice. Virus clearance is normally mediated via CD8+ effector T cells but, in their absence, the class-switched antibody response can ultimately achieve the same goal. Influenza virus-specific plasma cells and CD8+ T cells persist in the long term and the recall of the CD8+ T cell response can lead to earlier virus clearance. The first part (Part I) of this dissertation focuses on the understanding of HSV-1 induced immunoinflammatory processes in the cornea and the secondary lymphoid tissues and the involvement of immuno-modulatory mechanisms following acute viral infections such as HSV and IAV. The next three parts (Part II-IV) focus on different inflammatory and counter-inflammatory mechanisms that are activated following acute viral infections. Results in Part II evaluate the role of small molecule inhibitors of VEGFR2/src kinase inhibitors in controlling the progression of the inflammatory lesions after ocular HSV infection. Results of the third section show that the host counter inflammatory mechanisms inhibit tissue damage but these may also act to constrain the effectiveness of immunity to acute infections. The fourth section describes the functional significance of HVEM expression on regulatory T cell in their expansion following HSV-1 infection. In this study, experiments were designed to understand the mechanisms involved in the regulation of immunity and resultant immunopathology using HSV-1 and IAV as the model systems and that modulation of these processes can enhance immune response and diminish immunopathology following acute infections.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children in sub-Saharan Africa. Patients with CM are comatose and often convulse, develop retinal haemorrhages and motor abnormalities. Recent histological studies on brain tissue obtained from patients who have died from CM have identified heterogeneity in brain pathology. As a result, CM is considered to be a complex disease that may be comprised of a number of syndromes. Patients admitted to hospital with CM are treated with anti-malaria drugs; however, even in the best equipped hospitals, a large number of CM patients die within the first 24-hours following hospital admission before the anti-malarial treatment can have an effect. For this reason, it is critical that the mechanisms leading to CM are elucidated in order to develop effective adjunct therapies. Experimental cerebral malaria (ECM) caused by P. berghei ANKA (PbA) infection of susceptible mice displays many features of human CM. A key feature of this model is the pivotal role of the host immune response in pathogenesis, particularly the involvement of T cells. Evidence, predominantly from ECM studies, suggests that tumour necrosis factor (TNF) superfamily (TNFSF) members play critical roles in the immunopathology associated with CM. The first hypothesis investigated in this thesis was that key immune response pathways contribute to the development of CM and, despite the heterogeneity observed between CM patients, common pathways exist that may be targeted to prevent CM. The second hypothesis tested was that members of the TNF superfamily modulate the immune response to infection and are involved in the development of pathology observed in severe malaria (SM). In order to investigate the above hypotheses, three projects were carried out. First, we examined the great heterogeneity in brain expression profiles between ECM-susceptible CBA/CaH (CBA) and C57BL/6 (B6) mice at the peak of disease, as well as the significant differences in circulating cytokine expression and expansion of microglia in brain tissue. We found that, despite these differences, common therapeutic and preventative strategies existed to disrupt the development of ECM in the two ECM-susceptible mouse strains. Second, studies in ECM mice have identified T cells and TNFSF members, TNF and lymphotoxin (LT)-a, as critical mediators of ECM pathology. We extend these studies to examine the role of the TNFSF member LIGHT in ECM. Specific blockade of LIGHT signalling through its receptor, LTβR, in PbA-infected B6 mice abrogated the hallmark features of ECM brain pathology and improved the control of parasite growth. Importantly, specific blockade of LIGHT-LTβR signalling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process antigen during infection. Together, this study discovered a novel pathogenic role for LIGHT and LTβR in ECM and identified this TNF family receptor-ligand interaction as a potential target for therapeutic intervention in SM. Finally, we investigated the role of LTa in human SM and, more specifically, CM. We tested whether the polymorphisms within the gene encoding LTa (LTA) were associated with susceptibility to SM in Papuan Highland children and adults who had migrated from an area without malaria pressure to a region where malaria is endemic. Despite a lack of association between single nucleotide polymorphisms (SNPs) in the LTA/TNF locus and susceptibility to SM in Papuan Highland children and adults, we found a significant association between a SNP in the LTa-related gene encoding galactin-2 (LGALS2) and susceptibility to CM in children, but not adults in this study population. Interestingly, no association was found between this SNP and susceptibility to CM in Tanzanian children originating from and living in a malaria endemic region. These results suggest that there may be differences in the mechanisms leading to CM in adults and children, as well as between individuals from malaria endemic and non-endemic areas. Together, the findings outlined in this thesis are important to both the understanding of the underlying mechanisms leading to CM and to the development of improved interventions and adjunct therapies.

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of schistosome-induced pathology, including granuloma formation, fibrosis and splenomegaly, is essential for understanding how schistosomes influence the immune system of the mammalian host. I report on the first whole genome microarray analysis of the murine liver and spleen during the progression of Schistosoma japonicum infection and of S. japonicum-Soluble Egg Antigen (SEA)-stimulated macrophages. My analyses of the infected liver revealed a distinct temporal relationship between the expression of chemokines and the recruitment of cells to the liver. T-cell and B-cell chemoattractants were up-regulated earlier reflecting the recruitment of these cells to the liver as illustrated by flow cytometry. The later phases of the response corresponded with peak accumulation of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11, members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the hepatic stellate cell/myofibroblast chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively-activated macrophages (e.g. Retnla) during this later phase provided further evidence for a role for these cells in schistosome-induced pathology. Additionally, I demonstrated that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggested the involvement of neutrophils in S. japonicum¬-induced hepatic fibrosis. The transcriptional profile of the spleen was closely related to changes in cellular composition illustrated by flow cytometry and immunohistochemistry. Significant up-regulation of genes associated with progression through the cell cycle, proliferation makers and genes involved in lymphocyte proliferation, paralleled the initial expansion of T-cells and B-cells and the increased cellularity of the spleen overtime. Accumulation of eosinophils, neutrophils and macrophages was paralleled by enhanced expression of markers for these cells and the declining proportion of B- and T-cells in the spleen over time was reflected in the decreased expression of B- and T-cell markers. Significant up-regulation of Chi3l3 and F4/80+ macrophages suggested the presence of alternatively activated macrophages in the spleen, where these cells could play an immunoregulatory role. Comparison of the liver and spleen profiles revealed divergent expression of chemokines and cell adhesion molecules. Expression of lymphocyte chemokines including the homeostatic chemokines, CXCL13, CCL19 and CCL21, were significantly up-regulated in the liver while down regulated in the spleen. Expression of chemokines with activity for eosinophils (CCL11, CCL24), neutrophils (CXCL1) and monocytes (CXCL14, CCL12) and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver while unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Divergent expression of chemokines and cell adhesion molecules likely contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver. The results of liver and spleen microarrays suggested an important role for alternatively activated macrophages in the development of schistosome-induced pathology. This led me to investigate the in vivo transcriptional profile of S. japonicum SEA-stimulated peritoneal macrophages. The transcriptional profile of these cells was characterised by up-regulation of alternatively activated macrophage makers (Chi3l3, Chi3l4, Arg1). Retnla was not significantly induced in these macrophages suggesting that the specific function of these cells may differ to those induced by S. mansoni and other parasites. Other features of the transcriptional profile of these cells included modulated expression of T-cell co-stimulatory molecules and chemokines which may confer immunomodulatory activity. S. japonicum-stimulated alternative activation of macrophages was additionally associated with deactivation of classical activation pathways and altered expression of cell surface receptors and complement components that may alter phagocytic activity. Together these data significantly enhance our understanding of the mechanisms associated with alternative activation of macrophages and provide significant insight into the role of these cells in schistosomiasis japonica. The findings presented in this thesis represent the most comprehensive description to date of the molecular mechanisms, and especially chemotactic signalling pathways, regulating the development of schistosome-induced granulomas, fibrosis, splenomegaly and alternative macrophage activation in the murine host. In summary, my data have revealed that co-ordinated gene expression of chemokines in the liver and spleen regulates the recruitment of cells to the liver during schistosome infection. My results provide additional evidence for a role for neutrophils and alternatively activated macrophages in the development of schistosome-induced pathology and provide further insight to the molecular basis of alternative macrophage activation during infection. Furthermore, my data serve to highlight clear differences in the pathogenesis of schistosomiasis mansoni and schistosomiasis japonica. Together these findings further our understanding of the systemic, local, cellular, and especially, chemokine signalling pathways that regulate the development of S. japonicum-induced pathology and offer correlative insight into the pathogenesis of other chronic inflammatory diseases where fibrosis, splenomegaly and alternative activation of macrophages are common features.

Dengue virus (DENV) is an arthropod-borne virus and has become a worldwide problem with steadily rising annual infection rates. Patients present with a range of symptoms from mild fever to, in some cases, life-threatening hemorrhagic fever and shock syndrome. The most severe cases require emergency hospital care and currently, there is no effective drug treatment or vaccine for dengue. As severe symptoms appear post-peak viremia, immuno-pathology is thought to be the cause and a potential trigger of this is differential activation of the immune response upon recognition of DENV. This could be due to a combination of factors including varying receptors, signalling pathways and immune regulation mechanisms. In order to understand DENV infection better, it is imperative to study the mechanisms of activation and control of immune responses triggered by the virus. Very early events in viral infection (after 10 min stimulation) were studied aiming to identify proteins involved in differential activation of immune responses. Phosphorylated proteins were isolated from cells post-stimulation and analysed by mass spectrometry. More than 200 proteins were differentially regulated by phosphorylation in response to DENV stimulation as compared to Mock, Influenza A virus and LPS stimulation. The effect of two specific proteins, namely Calpain-2 and Importin-5, identified to be differentially phosphorylated was investigated further. Calpain-2 was seen to be vital in the efficient production of progeny virions and the transcription of Mx1, an anti-viral interferon stimulated gene. Importin-5 is known to transport DENV NS5 into the nucleus during infection and was seen to co-precipitate with many host proteins. In summary, it is imperative that novel treatments and vaccines are developed for dengue as it is one of the world’s most prevalent arthropod-borne viruses. It was discovered here that many proteins undergo phosphorylation/de-phosphorylation in response to DENV stimulation to a differing degree than other stimuli. Calpain-2 plays a vital role DENV infection, potentially influencing the potency of immune response. Importin-5 associates with various host proteins during DENV infection, potentially altering their function or the function of Importin-5 itself. Research into targeted inhibition of Calpain-2 function or Importin-5 interaction with DENV NS5 could lead to a successful anti-viral treatment for DENV infection.

abstract: Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

O lúpus eritematoso discoide (LED) é a variante mais comum do Complexo Lúpus Eritematoso, sendo considerado uma das dermatopatias imunemediadas mais evidenciadas em cães, ficando muito próximo à casuística do pênfigo foliáceo. É afecção, relativamente, benigna sem envolvimento sistêmico e, clinicamente, caracterizada por leucodermia e/ou eritema associado à perda do aspecto em \"calçamento de pedras\" do plano nasal e, imunologicamente, pela deposição de autoanticorpos (IgA, C3, IgM e IgG) na zona da membrana basal (ZMB). O presente estudo visa correlacionar os achados clínicos e histopatológicos com aqueles da imunofluorescência direta (IFD), objetivando aumentar a acurácia do diagnóstico do LED. Foram incluídos 11 cães (Grupo I) com o quadro lesional tegumentar característico do LED e, cinco caninos (Grupo II), dermatologicamente hígidos, para o controle negativo da IFD. Fragmentos cutâneos oriundos do plano nasal foram submetidos ao exame histopatológico e à reação da IFD e, os resultados obtidos em ambos os Grupos, I e II, foram analisados estatisticamente pelo Índice kappa (k) visando a determinação do grau de concordância ou confiabilidade entre os dois métodos diagnósticos. No Grupo I, o diagnóstico de LED foi firmado em 100% (11/11) dos animais, pela reação de IFD, enquanto que o exame histopatológico estabeleceu o diagnóstico em 81,8% (9/11) dos casos. A positividade da IFD para o LED canino foi estabelecida, em sua totalidade, a partir da fluorescência esverdeada evidenciada na ZMB e/ou em seus anexos cutâneos e, os imunoreagentes mais frequentemente encontrados foram a IgM e C3. Já, em relação ao Grupo II, na totalidade dos animais não se evidenciou histopatologicamente qualquer alteração tegumentar.Também, pela IFD os resultados mostraram-se negativos. O substancial grau de concordância entre a IFD e a histopatologia (k= 0,738 e p= 0,002) comprova que a reação de imunofluorescência direta é exequível e útil no estabelecimento do diagnóstico do LED canino. / The discoid lupus erythematosus (DLE) is the most common variant of the Complex Lupus Erythematosus, considered one of auto-immune skin diseases more evident in dogs, getting very close to the casuistry of pemphigus foliaceus. The disease is relatively benign and without systemic involvement, clinically characterized by leukoderma and / or erythema associated with loss of the normal planum nasale \"cobblestone \" architecture and immunologically by deposition of autoantibodies (IgA, C3, IgM and IgG) at the basement membrane zone (BMZ). This study aims to correlate the clinical and histopathological findings with those of direct immunofluorescence (DIF), aiming to increase the accuracy of diagnosis DLE. We included 11 dogs (Group I) with cutaneous lesions characteristic of DLE and five dogs (Group II), with no cutaneous alterations, for the negative DIF. Cutaneous fragments derived from the planum nasale were submitted to histopathological examination and DIF, and the results obtained in both Groups I and II were analyzed by kappa index (k) in order to determine the degree of agreement or reliability among two methods. In Group I, the diagnostic DLE was established in 100 % (11/11) of animals, by the reaction of DIF, while the histopathological examination confirmed the diagnosis in 81.8 % (9/11) of cases. The positive DIF for DLE canine was established, in its totality, from the greenish fluorescence evident in the BMZ and / or on their skin annexes and the immunoreagents most frequently found were IgM and C3. Now, in relation to the Group II, all animals did not reveal any histopathological tegumentar changes. Also, by the DIF results were negative. The substantial degree of agreement between the DIF and histopathology (k = 0.738 and p = 0.002) shows that the direct immunofluorescence assay is feasible and useful in establishing the diagnosis of canine DLE.

Les espèces de Plasmodium induisent une réponse immunitaire spécifique, qui stimulent la libération de cytokines et entraînent des réponses protectrices ou pathologique. Dans les modèles murins, ses réponses dépendent des combinaisons de la souche de souris et de l’espèce Plasmodium utilisés. Les souris C57BL/6 infectées, avec un nombre relativement faible de Plasmodium chabaudi AS, expérimentent une infection qui n’est pas fatale et une résistance à l'infection associée avec une réponse inflammatoire forte qui implique l'interleukine-12 et l'activation précoce des cellules T CD4+ avec une production élevée de IFN-γ et TNF-α. Cette réponse pro inflammatoire est par la suite contrôlée par l'IL-10 qui mène au contrôle et à la résolution de l'infection. Cependant, les souris qui succombent à l’infection développent la parasitémie fulminante et une infection mortelle. La cause des différences de résolution n'est pas bien comprise, bien que les principaux composants de protection et les réponses immunitaires pathologiques sont bien connus. Cette étude utilise le modèle de Plasmodium chabaudi dans le souris C57BL/6, pour: 1) comparer la résolution de l'infection et de la pathologie entre le P. chabaudi AS qui est relativement non virulentes et la plus virulentes de P. chabaudi CB; 2) déterminer si les différences dans la réplication des parasites, charge parasitaire ou les réponses immunitaire de l'hôte contribuent a des différences pathologiques; 3) ) chercher à déterminer ce qui mène les différences dans la réponse immunitaire de l'hôte. P. chabaudi CB provoque une infection plus sévère chez les souris C57BL/6 par rapport à P. chabaudi AS, avec une mortalité de ±40% endéans 12 jours et une moyenne des pics de 50% par rapport à 30% observé dans des souris infectées avec P. chabaudi AS. Aucune différence n’a été observé entre la charge corporelle des parasites durant les infections de P. chabaudi AS comparée a celle de P. chabaudi CB. Un taux élevé de cellules NK a été constaté dans les rate des souris infectées par P. chabaudi AS au niveau maximum de parasitémie, tandis que le taux des cellules NK dans les rates des souris infectées par P. chabaudi CB est resté constant tout au long de l'infection. Par conséquent, la poussée du taux des cellules NK contribue a l’infection non virulente des clones P. chabaudi AS. Ceci est du à la capacité cytolytique des cellules NK ainsi qu’à la production des IFN- γ. Le nombre de lymphocytes T CD4+ présents dans les rates des souris C57BL/6 infectées par P. chabaudi CB était inférieur a celui des lymphocytes T CD4+ présent dans les rates des souris infectées par P. chabaudi AS ; 1 fois moins de cellules T CD4+l'IFN-γ+ et 2 fois moins de cellules l'IL-10 +T CD4+ ont été observés. Une analyse plus approfondie a illustré que les cellules l'IL-10+ IFN-γ+ T CD4+ sont plus fréquentes chez les souris infectées par P. chabaudi AS. L'absence des cellules d'IL-10+ IFN-γ+ T CD4+ chez les souris infectées par P. chabaudi CB contribue à la pathologie, parce que ces cellules limitent les réactions inflammatoires mortelles et améliorent la perte de poids, l'hypothermie et l'anémie, en régulant l'immunopathologie. Les différences observées dans les réponses immunitaires ne sont pas contrôlées par l'interaction des parasites spécifiques tel que glycophosphatidylinositol avec TLR2, mais plutôt par l'IFN-α/β. Bien qu’ IFN-α/β n’ont pas été détecté dans le plasma, des niveaux plus élevés d'IFN-α/β d'ARNm transcrits ont été trouvés dans la rate de C57BL/6 infectées par P. chabaudi CB au cinquième jour d’infection. Le manque d’IFN-α/βR limite la pathologie et l’infection P. chabaudi CB virulentes sans affecter les infections P. chabaudi AS non virulentes. / Plasmodium species induces a specific immune response, stimulating the release of cytokines, resulting in either protective or pathological responses. In mouse models, this is depended on mouse strain or parasite combination used. C57BL/6 infected with relatively low numbers of Plasmodium chabaudi AS pRBC experience a non-lethal infection and resistance is associated with robust inflammatory responses that involves IL-12, early activation of CD4+Th1 cells with production of high levels of IFN-γ and TNF-α, this pro-inflammatory response is subsequently controlled by IL-10 leading to control and resolution of infection. However, mice that succumb to infection develop fulminant parasitaemia and increase mortality. The cause of the differences in infection outcome is not well understood, although the principal components of protective and pathological immune responses are well known. Using the model of Plasmodium chabaudi in C57BL/6, this study addresses the following; 1) compares the course of infection and pathology between the relatively avirulent P. chabaudi AS and the more virulent P. chabaudi CB; 2) investigates whether differences in parasite replication, parasite load or host immune responses contribute to differences in pathology; 3) what mediates the differences in host immune response? P. chabaudi CB causes a more severe infection in C57BL/6 compared to P. chabaudi AS, with ±40% mortality within 12 days and mean peak parasitaemia of 50% compared to 30% in P. chabaudi AS infected mice. There was no difference in total body parasite load between the P. chabaudi AS and CB infections. A High number of NK cells was found in the spleen of P. chabaudi AS infected mice at peak parasitaemia, whereas NK cells numbers in spleen of P. chabaudi CB infected mice remained constant throughout infection. Hence, the elevated NK cells contribute to parasite clearance in the avirulent P. chabaudi AS clone, because of its IFN-γ production and cytolytic activity. P. chabaudi CB infected C57BL/6 were found to have reduced number of CD4+T cells in the spleen, with 1-fold decrease in IFN-γ+CD4+T cells and 2-fold decrease in IL-10+ CD4+T cells when compared to CD4+T cells from P. chabaudi AS infected mice. Further analysis showed that IL-10+IFN-γ+CD4+T cells were more prevalent in P. chabaudi AS infected mice. The absence of IL-10+IFN-γ+CD4+T cells in P. chabaudi CB infected C57BL/6 contributes to pathology, because these cells limit lethal inflammatory responses and ameliorate weight loss, hypothermia and anemia, by regulating immunopathology. The differences observed in immune responses are not mediated by interaction of parasite specific GPIs with TLR2, but rather by IFN-α/β. Although IFN-α/β were not detected in plasma, higher levels of IFN-α/β mRNA transcripts were found in the spleen of P. chabaudi CB infected C57BL/6 on day 5. IFN-α/βR deficiency limits virulent P. chabaudi CB infections with no effect on avirulent P. chabaudi AS infections.

A paracoccidioidomicose é uma micose sistêmica caracterizada por ser uma doença granulomatosa. As formas benignas da doença são caracterizadas por uma infecção localizada, contendo granulomas compactos com poucos fungos; já nas formas mais graves, ocorre um processo granulomatoso frouxo com focos de necrose e intensa disseminação fúngica. Objetivou-se avaliar o desenvolvimento de lesões granulomatosas em baço, fígado, pulmão e epiplon de camundongos, após infecção pela via intraperitoneal com o isolado de alta virulência, Pb18, em diferentes períodos de infecção após tratamento com fármacos, os quais possuem mecanismo de ação relacionado com a alteração no balanço entre a síntese e degradação dos produtos do colágeno, interferindo diretamente na formação do granuloma A citocina IFN-<font face=\"Symbol\">g, o antibiótico Tetraciclina e as drogas antiinflamatórias Lumiracoxib e Celecoxib. Avaliamos a presença de alguns componentes do granuloma (colágeno, células do infiltrado inflamatório, de citocinas primordiais para sintese/degradação da MEC do granuloma, presença de P. brasiliensis). / Paracoccidioidomycosis (PCM) is a systemic mycosis that is endemic in Latin America, whose causative agent is the thermal dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is a granulomatous disease, and the formation of granulomas can be understood as a mechanism of the body to block and limit the invasiveness of the fungus or its antigenic components, once unable to lyse them. Bening forms of the disease are characterized by a localized infection, where granulomasa are compact and contain few fungi. More severe forms present loose granulomatous processes with foci of necrosis and severe fungal. Studies in which granulomatous response was developed in resistant (A/J) and susceptible (B10.A) mice to the high virulence isolate Pb18 showed the presence of different patterns of injuries related to the type of extracellular matrix (ECM) components and the different cells types in the area, suggesting a important role of these elements in the formation and constitution of the granuloma and thus the outcome of infection. In our project, we aimed to evaluate the development of granulomatous lesions in the spleen, liver, lung and omentum of mice susceptible to PCM after intraperitoneal infection with Pb18, at different periods of infection (acute and chronic) with or without treatment with drugs. These drugs have mechanisms of action closely related to the change in the balance between synthesis and degradation of collagen Thus, they interfere directly in the granuloma formation and in maintaining the viability of fungi and also with the development of fibrosis. Which is a common and devastating sequelae of numerous infections including the PCM, with the characteristic proliferation of fibroblasts and deposition of ECM. The treatments were chosen based on prior knowledge on their effects on the course of experimental murine PCM. IFN-<font face=\"Symbol\">g was chosen due to its antifibrotic effect, being an activator of macrophages in infection by P. brasiliensis and increasing the fungicidal effect of neutrophils. The antibiotic tetracycline was used because of its inhibitory effect on the synthesis of extracellular matrix, limiting antimicrobial activity and the ability of collagenase to degrade ECM. Finally, the antiinflammatory drugs Celecoxib and Lumiracoxib (inhibitors of the COX-2 enzyme) 11 were used because they cause an increase in the expression of collagen type III and type IV. We analyzed the components of the granuloma (collagen, inflammatory cells, cytokines essential for synthesis / degradation of the ECM of the granuloma, the presence of P. brasiliensis). Among the cytokines analyzed, we studied the importance of TNF-<font face=\"Symbol\">&#945 in the formation of granulomas and regulation of matrix metalloproteinases (MMP) synthesis and function. We analyzed TGF-<font face=\"Symbol\">b because it negatively modulate the secretion of nitric oxide by macrophages and promote the accumulation of ECM and is believed to be the central mediator of the process of fibrosis in several pathologies. IFN-<font face=\"Symbol\">g was studied because of its correlation to the preferential Th1 immune response in diseases and infectious processes of fungal and bacterial infections, and also because it modulates fibroblast function.

Chronic Lymphocytic Leukemia (CLL) is defined by the accumulation of clonally expanded CD5+ and CD19+ B lymphocytes in blood and secondary lymphoid organs with impaired apoptotic mechanisms. CLL represents one third of all leukemia cases with an average age of 72 years at diagnosis making it the most common adult leukemia. The Eµ-Tcl1 mouse serves as an excellent model to study the development of CLL as they progress to a CLL like disease by 9-14 months of age, due to overexpression of an oncogene, T cell Leukemia 1(Tcl1), specifically in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to the development of a CLL like disease within 3-8 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, spleen ultrasonography and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 protein (Par-4), a known pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers and has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. We found that CLL cells have constitutively active B-cell receptor signaling (BCR) and that inhibition of BCR signaling with FDA approved drugs causes a decrease in Par-4 protein, mRNA levels, and an increase in apoptosis. In particular, activities of Src family kinases, spleen tyrosine kinase and Bruton’s tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling in both Eµ-Tcl1 CLL cells and primary human CLL samples. Consistent with this, lenti-viral shRNA mediated knockdown of Lyn kinase leads to a decrease in Par-4 expression in MEC-1 cells, a human CLL derived cell line. Igα (CD79a) silencing in primary human CLL cells also results in down regulation of Par-4 expression. Additionally, we knocked down expression of Par-4 in MEC-1 cells which resulted in a decrease in cell growth that could be attributed to an increase in p21 expression and a reduction in the G1/S cell cycle transition. We have also observed this phenomenon by crossing mice deficient in Par-4 with the Eµ-Tcl1 mouse where lack of Par-4 delays CLL growth in the mouse significantly (time to euthanization due to poor body condition - Eµ-Tcl1: 8.9mo vs Par4-/-EµTcl1: 11.97 mo, p = 0.0472) and splenic B-CLL cells from these mice also have increased expression of p21. Since mice in this cohort are whole body knockout for Par-4, the difference in survival times between the Par-4 +ve and Par-4 –ve EµTcl1 mice could be due to the influence of Par-4 on CLL cells as well as the effect of Par-4 secreted by the CLL cells on the microenvironment. There could be other potential roles for Par-4 in the context of CLL which are under further investigation. We have also investigated the site of CLL growth in mouse models to determine that the spleen is the primary organ to accumulate the CLL tumor burden. We have found that splenectomy significantly delays the development of CLL in the primary Eμ-Tcl1 mouse model and prevents growth and development in the adoptive transfer model. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for Par-4 compared to C57BL/6 wild type mice. Par-4 appears to regulate a specific microenvironment required for CLL growth. Current studies are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.