Solid organ transplantation is the gold standard treatment for a variety of conditions that result in organ failure. However, despite considerable advances in clinical transplantation in recent decades, the almost ubiquitous requirement of life-long immunosuppression of transplant recipients persists and is complicated by graft loss to rejection in the long term and multiple serious adverse effects that are frequently life limiting. Helminths currently infect more than one quarter of the world’s population and it is now well established that their success as parasites is the result of active immunomodulation of the host immune response. Whilst this primarily secures ongoing survival of the parasites, in some cases helminth-induced immunomodulation can be beneficial to the infected host and is not associated with the adverse sequelae of pharmacological immunosuppression. An emerging body of evidence suggests that harmful immune responses to alloantigens can be suppressed by helminths, but little mechanistic data exists and the active immunomodulators involved have remained hitherto unidentified. The hypothesis behind this thesis is that the model intestinal nematode, Heligmosomoides polygyrus, produces immunomodulatory molecules that can suppress responses to allo- and auto-antigens in animal models of transplantation and autoimmunity, and that some of these molecules could potentially be exploited as novel therapeutic agents. Full-thickness skin grafting was performed between fully-allogeneic mouse strains (BALB/c to C57BL/6). Recipient mice infected with H. polygyrus immediately prior to transplantation showed significantly prolonged allograft survival. Likewise, protection from allograft rejection could be replicated in recipient mice in which H. polygyrus excretory-secretory products (HES) (isolated from culture of adult worms) were delivered by continuous infusion via surgically implanted osmotic minipumps. A number of potential mechanisms underlying allograft protection were identified including induction of CD4+CD25+Foxp3+ regulatory T cells (Treg) and suppression of Th1 and Th17 effector CD4+ T cell phenotypes. H. polygyrus and HES were further shown to ameliorate disease in murine (pMOG) experimental autoimmune encephalomyelitis and colitis induced by T cell transfer. In addition to expansion of Treg, H. polygyrus-mediated protection against EAE was found to be almost completely lost in IL-4 receptor deficient mice, indicating a protective role of Th2 immune responses in this context. Finally, the mechanisms of action of the newly-identified TGF-β mimic, TGM, contained within HES were investigated. Despite bearing no sequence homology or structural resemblance to TGF-β, TGM was shown to act through the TGF-β receptor complex to induce Treg in human and mouse CD4+ T cells in vitro and to suppress murine allogeneic skin graft rejection in vivo. TGM may represent the origin of a safe, effective and long-overdue novel alternative to current immunosuppression therapy.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-05-03T12:33:08Z No. of bitstreams: 1 luancristiandasilva.pdf: 1815758 bytes, checksum: 791b50741ba8fa9606a7586b32ad6f4b (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-07T15:12:24Z (GMT) No. of bitstreams: 1 luancristiandasilva.pdf: 1815758 bytes, checksum: 791b50741ba8fa9606a7586b32ad6f4b (MD5) / Made available in DSpace on 2016-06-07T15:12:24Z (GMT). No. of bitstreams: 1 luancristiandasilva.pdf: 1815758 bytes, checksum: 791b50741ba8fa9606a7586b32ad6f4b (MD5) Previous issue date: 2015-02-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Esclerose Múltipla (EM) é uma doença inflamatória autoimune desmielinizante que afeta o sistema nervoso central (SNC). A Encefalomielite Autoimune Experimental (EAE) é um modelo animal para o estudo da EM. A Mitoxantrona (MIT) é um agente usado para o tratamento da EM, entretanto, a cardiotoxicidade é um dos efeitos adversos do tratamento acumulativo. O presente estudo investigou o efeito do novo derivado antraquinônico,1-Metoxi-4-((2-hidroxitetradecil)amino)-antraceno-9,10-diona, denominado MTA, em modelo de EAE. Camundongos C57BL/6 foram imunizados com o peptídio da Glicoproteína Mielínica de Oligodendrócitos (MOG35-55) em adjuvante completo de Freund, suplementado com Mycobacterium tuberculosis H37RA e tratados com 1mg/Kg de MIT ou MTA do dia 14 ao dia 20 pós-imunização. Os efeitos tóxicos provenientes dos tratamentos com a MIT e o MTA, após 7 dias de tratamento em camundongos C57BL/6 imunizados, foram analisados a partir de curvas de sobrevivência, massa corporal e dosagem sérica da proteína CK-MB. No 21º dia após a imunização, as medulas espinhais foram removidas e submetidas a análises como dosagem de citocinas pró-inflamatórias IFN-γ, IL-17, IL-6, regulatórias, IL-10 e TGF-β e quimiocinas CCL5 e CCL20 através de ELISA. Foi feita a fenotipagem celular através da expressão dos marcadores CD4, CD8, CD19, CD11b, CD11c e F4/80 por citometria de fluxo e avaliação da presença de infiltrados inflamatórios e desmielinização em amostras de tecido. O tratamento com MTA reduziu o escore clínico da doença em camundongos C57BL/6 imunizados com o MOG35-55, o que foi associado com a redução da desmielinização e de células pró-inflamatórias, secreção das citocinas IL-17, IFN-γ, IL-6 e aumento de IL-10 e TGF-β na medula espinhal, de forma similar à MIT. Porém, o tratamento com MIT reduziu a capacidade de sobrevivência, massa corporal dos animais e ainda elevou a secreção da proteína CK-MB no soro, sugerindo estresse muscular cardíaco durante o período de tratamento, o que não foi observado no grupo MTA. Os resultados sugerem um importante papel do MTA na modulação da resposta imune envolvida na EM. A atenuação da inflamação e consequentemente a redução do escore clínico envolvendo a redução de citocinas pró- inflamatórias, indicam esse composto como um tratamento alternativo para doenças autoimunes no sistema nervoso central. / Multiple sclerosis (MS) is an autoimmune demyelinating inflammatory disease that affects the central nervous system (CNS). The experimental autoimmune encephalomyelitis (EAE) is an animal model for the study of MS. Mitoxantrone (MIT) is an agent used for treatment of MS, however, the cardiotoxicity is a cumulative adverse effects of treatment. This study investigated the effect of the new derivative anthraquinone, 1-methoxy-4 - ((2-hydroxytetradecyl) amino) anthracene-9,10-dione, called MTA in EAE model. C57BL/6 mice were immunized with the peptide of myelin oligodendrocyte glycoprotein (MOG35-55) in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37RA and treated with 1 mg/kg of MIT or MTA the day 14 to day 20 post-immunization. The toxic effects arising from treatment with MTA and MIT after 7 days of treatment in C57BL/6 mice immunized were analyzed using survival curves, body weight and serum protein CK-MB. At day 21 after immunization, the spinal cords were removed and subjected to analysis such as determination of pro-inflammatory cytokines IFN-γ, IL-17, IL-6 and regulatory cytokines IL-10 and TGF-β and CCL5, CCL20 chemokines using ELISA, cellular phenotype through expression of markers CD4, CD8, CD19, CD11b, CD11c, F4/80 by flow cytometry and the presence of inflammatory infiltrates and demyelination by histopathology. Treatment with MTA reduced the clinical score of the disease in C57BL /6 mice immunized with MOG35-55, which was associated with the reduction of demyelination, presence of pro-inflammatory cells, release of cytokines IL-17, IFN-γ, IL-6 and increased of IL-10 and TGF-β in the spinal cord, as well as the MIT. However, treatment with MIT reduced survivability, the body weight and further increased the production of CK-MB in serum protein, suggesting cardiac muscle stress during the treatment, which was not observed in the MTA group. The results suggest an important role of MTA in modulating the immune response involved in MS. The attenuation of inflammation and thereby reduce the clinical score involving the reduction of pro-inflammatory cytokines indicate this compound as an alternative treatment for autoimmune diseases in the central nervous system.

Abstract : Multiple Sclerosis (MS) is an organ-specific autoimmune disease characterized by the presence of demyelinating plaques throughout the central nervous system (CNS) as a result of an abnormal inflammatory response. During MS, activated microglia can play the role of antigen presenting cells and can, therefore, skew T cell responses towards a pro-inflammatory phenotype. Once activated, microglia upregulate the expression of pro-inflammatory molecules. In addition to microglial responses during MS, astrocytes are also implicated in the development of MS lesions. Upon injury and nearby neuronal death, astrocytes undergo astrogliosis. To date, several molecular pathways were identified as targets for therapeutic interventions for MS such as NF-kB & Nlrs. Nlrs are regulatory proteins of the immune system capable of regulating both innate and adaptive responses. Nlrp12 is a pyrin-containing intracellular protein, largely expressed in cells of myeloid origin. Nlrp12 plays an important role in immune inflammatory responses by negatively regulating the NF-κB pathway and modulatory roles, such as dendritic cell migration. The focus of this study was to evaluate the hypothesis where Nlrp12 plays an anti-inflammatory role in Experimental Autoimmune Encephalomyelitis (EAE), a well-characterized mouse model to study MS. Over a course of 9 weeks, Nlrp12 KO mice demonstrated increased severity in disease levels compared to WT mice. In both genotypes, the disease was observed to peak around the 3rd week post immunization A significant increase in Nlrp12 mRNA was observed in diseased WT compared to healthy WT mice. A significant increase in the expression of CCR5, COX-2, and IL-1 β in Nlrp12 KO mice relative to WT mice, was observed. Interestingly, no differences in the percentage of gliosis was seen at 3 weeks post injection in both genotypes, however after 9 weeks of diseases, in Nlrp12 KO mice we observed a significant increase in the percentage of reactive gliosis compared to WT mice. A significant activation of NF-kB-dependent inflammation was seen in primary microglial cell cultures from Nlrp12 KO relative to WT following LPS stimulation. Moreover, supernatants of analysis for the level of nitrates with Griess reagent and with ELISA for TNFα and IL-6, demonstrated an increase in the pro-inflammatory phenotype from microglia from Nlrp12 KO mice compared to WT mice. These results suggest a critical role of Nlrp12 in suppressing inflammation during the development of the disease given that in its absence, we observed an increase in the inflammatory response. / La sclérose en plaques est une maladie auto-immune déclenchée par une réaction inflammatoire anormale et caractérisée par la dégradation de myéline au niveau du système nerveux central. Durant la sclérose en plaques, la microglie promeut l’expression de molécules pro-inflammatoires et joue le rôle de cellules présentatrices d’antigènes pour forcer les cellules T à adopter un phénotype pro-inflammatoire. Outre les réponses associées à la microglie, les astrocytes sont aussi impliqués dans le développement des lésions. Jusqu’à présent, plusieurs voies moléculaires ont été identifiées comme cibles pour des interventions thérapeutiques tel que les voies de NF-kB et Nlrs. Les récepteurs Nlrs sont des protéines régulatrices du système immunitaire inné et adaptatif. Nlrp12 joue un rôle important dans les réponses inflammatoires immunes en régulant négativement la voie NF-kB et la migration de cellules dendritiques. L’objectif de cette étude est d’étudier l’hypothèse dans laquelle Nlrp12 joue un rôle anti-inflammatoire dans l’encéphalopathie expérimentale autoimmune (EAE), un modèle murin de la sclérose en plaques. Durant 9 semaines, des souris n’exprimant pas Nlrp12 ont démontré un état sévère de la maladie comparativement aux souris de type sauvage (WT). Dans les deux types de génotypes, la maladie était observée à son maximum autour de la 3ème semaine après immunisation. Une augmentation significative de l’expression d’ARNm de Nlrp12 était observée dans les souris contrôles malades comparativement aux souris saines. Une augmentation significative de l’expression de Ccr5, COX-2 ainsi qu’IL-1β était détectée dans les souris Nlrp12 KO par rapport aux souris WT. De plus, aucune différence dans le pourcentage de gliose était observée dans les deux génotypes à 3 semaines post-injection. Par contre, le pourcentage de gliose activée augmentait dans les souris Nlrp12 KO après 9 semaines de maladie. Nous avons remarqué une activation prononcée de l’inflammation dépendante de NF-kB dans des cultures cellulaires primaires de microglie provenant de souris Nlrp12 KO soumise à une stimulation au LPS. Finalement, la quantification des niveaux de nitrates et des cytokines TNFα et IL-6 traduisait une signature pro-inflammatoire de la microglie des souris Nlrp12 KO comparativement aux souris WT. Ces résultats suggèrent un rôle antiinflammatoire de Nlrp12 durant le développement de la sclérose en plaques considérant la réponse inflammatoire accrue en absence de Nlrp12.

No description available.

L'inhibition du protéasome a un effet anti-inflammatoire en bloquant l'activation de NF-êB. Dans ce travail, nous avons étudié cette propriété anti-inflammatoire dans deux modèles animaux de maladies neurologiques: la sclérose en plaques et l'AVC. Nous avons utilisé le ritonavir, un antiprotéase du VIH et qui a des effets de modulation sur le protéasome. Ainsi l'administration du ritonavir a permis d'inhiber l'EAE modèle animal de SEP. D'autre part, un effet anti-ischémique cérébral a été obtenu ches les animaux ayant subi une occlusion de l'artère cérébrale moyenne, modèle animal d'AVC. Ces effets neuroprotecteurs ont été corrélés à une inhibition de la réponse inflammatoire chez les animaux traités. Ces résultats ouvrent des nouvelles perspectives thérapeutiques dans les maladies inflammatoires basées sur la modulation du protéasome / The inhibition of the proteasome has an anti-inflammatory effect by blocking the activation of NF-êB. In this work, we studied this anti-inflammatory property in two animal models of neurological diseases: the multiple sclerosis and stroke. We used the ritonavir, an antiprotease of the HIV and which has effects of modulation on the proteasome. So the administration of the ritonavir allowed to inhibit the animal model EAE of MS. On the other hand, an anti-ischemic effect was obtained in animal model of stroke. These neuroprotective effects were correlated with an inhibition of the inflammatory response. These results open new therapeutic perspectives in the inflammatory diseases based on the modulation of the proteasome.

Recentemente demonstrou-se o papel de células T produtoras de IL-17 na patogênese da esclerosa múltipla e de seu modelo, a EAE. Através da produção desta e de outras citocinas, a população chamada Th17 promove o rompimento da barreira hematoencefálica e a conseqüente infiltração de células patogênicas para dentro do SNC. Nesse contexto, em nosso trabalho utilizamos o fenômeno da tolerância oral para avaliar a capacidade deste em suprimir a resposta imune durante o modelo de EAE, mais especificamente as células Th17. Nossos dados demonstram uma diminuição de IL-17 tanto na periferia como no SNC dos animais tolerados. Além disso, detectamos menos CCL2 e IL-6 em células extraídas do CNS dia 10 pós-imunização. Não observarmos diferença na produção de IL-4,5,10, 13, IL-12p70, TNF-<font face=\"symbol\">a, e IFN-<font face=\"symbol\">g entre os grupos. Em suma, nossos resultados mostram que o fenômeno da tolerância oral é capaz de suprimir parâmetros de EAE devido a uma menor capacidade linfoproliferativa associada a uma supressão de células patogênicas Th17 tanto na periferia como no SNC. / It has recently been shown the role of IL-17 secreting cells on the pathogenesis of multiple sclerosis and also in its model, EAE. Due to the secretion of this and other cytokines, the population so called Th17, promotes the disruption of the blood-brain barrier and the following infiltration of pathogenic cells into the CNS. In this context, in our work we used the oral tolerance phenomenon to evaluate its supressive capacity, more specifically over the Th17 cells. We showed that oral tolerated mice has a diminished production of IL-17 both in the periphery and in the CNS. Futhermore, we detected lower levels of CCL2 and IL-6 also from brain and spinal cord extracted mononucear cells at day 10th post-immunization. We were not able to detect differences on IL-4,5,10, 13, IL-12p70, TNF-<font face=\"symbol\">a, e IFN-<font face=\"symbol\">g between the groups. Thus, our results show that the oral tolerance phenomenon suppresses EAE findings, mainly due to a lower lymphoprolipherative response associated to a supression over the expansion of Th17 pathogenic T cells both in the periphery and inside the CNS.

Recentemente demonstrou-se o papel de células T produtoras de IL-17 na patogênese da esclerosa múltipla e de seu modelo, a EAE. Através da produção desta e de outras citocinas, a população chamada Th17 promove o rompimento da barreira hematoencefálica e a conseqüente infiltração de células patogênicas para dentro do SNC. Nesse contexto, em nosso trabalho utilizamos o fenômeno da tolerância oral para avaliar a capacidade deste em suprimir a resposta imune durante o modelo de EAE, mais especificamente as células Th17. Nossos dados demonstram uma diminuição de IL-17 tanto na periferia como no SNC dos animais tolerados. Além disso, detectamos menos CCL2 e IL-6 em células extraídas do CNS dia 10 pós-imunização. Não observarmos diferença na produção de IL-4,5,10, 13, IL-12p70, TNF-<font face=\"symbol\">a, e IFN-<font face=\"symbol\">g entre os grupos. Em suma, nossos resultados mostram que o fenômeno da tolerância oral é capaz de suprimir parâmetros de EAE devido a uma menor capacidade linfoproliferativa associada a uma supressão de células patogênicas Th17 tanto na periferia como no SNC. / It has recently been shown the role of IL-17 secreting cells on the pathogenesis of multiple sclerosis and also in its model, EAE. Due to the secretion of this and other cytokines, the population so called Th17, promotes the disruption of the blood-brain barrier and the following infiltration of pathogenic cells into the CNS. In this context, in our work we used the oral tolerance phenomenon to evaluate its supressive capacity, more specifically over the Th17 cells. We showed that oral tolerated mice has a diminished production of IL-17 both in the periphery and in the CNS. Futhermore, we detected lower levels of CCL2 and IL-6 also from brain and spinal cord extracted mononucear cells at day 10th post-immunization. We were not able to detect differences on IL-4,5,10, 13, IL-12p70, TNF-<font face=\"symbol\">a, e IFN-<font face=\"symbol\">g between the groups. Thus, our results show that the oral tolerance phenomenon suppresses EAE findings, mainly due to a lower lymphoprolipherative response associated to a supression over the expansion of Th17 pathogenic T cells both in the periphery and inside the CNS.

No description available.

Multiple sclerosis is a complex disease and one for which the aetiology remains largely unanswered. Anti-dsDNA antibodies have been found intrathecally and bordering lesions in multiple sclerosis patients and in view of their known pathogenity in lupus nephritis the aim of this project was to further investigate their role in multiple sclerosis. Using the acute experimental allergic encephalomyelitis (EAE) model in the Lewis rat, the inflammatory phase of disease was profiled using immunohistological and ELISA methods and was related to clinical sign severity. The parameters of interest were central nervous system deposits of IgM, IgG, B cells and C3 and anti-DNA antibodies in sera, cerebrospinal fluid and in situ. In situ evaluation of anti-dsDNA antibodies was also performed in tissue taken from Biozzi (AH) mice (relapsing/remitting EAE model) and from a multiple sclerosis patient. Inflammatory deposits specifically at sites of perivascular cuffing were found to increase with increasing clinical sign severity. At the time clinical signs had plateaued in the Lewis rat, intrathecal anti-dsDNA antibodies were at their highest level and anti-ssDNA antibodies at their lowest. The latter possibly due to their involvement in the 'clearing-up' process following tissue damage. Using novel DNA probes fluorescence suggestive of the presence of anti-dsDNA iii antibodies was seen in both animal and human tissue. Within human tissue the antibodies appeared to accumulate around active lesions and within vessels, raising the question of these antibodies having differing location dependent functions. EAE models have the potential to investigate these findings further and to evaluate new therapies.

Multiple sclerosis (MS) is a neurodegenerative disorder characterized by CNS inflammation and axonal demyelination. In addition, axonal pathology has also been reported in MS and may be responsible for the functional deficits associated with this disease. Based on preliminary data from our laboratory, we propose that a specific domain of the neuron, known as the axon initial segment (AIS), is targeted in MS. Consistent with our work from the human tissue, we have also observed disruption of AIS integrity in a murine CNS inflammatory model and observations strongly implicate reactive microglia as mediators of AIS disruption. In contrast, a murine model of demyelination did not exhibit AIS pathology but reactive microglia were prevalent. Since we propose that reactive microglia drive AIS disruption in our inflammatory CNS model but observe no AIS pathology following demyelination even in the presence of reactive microglia, we propose that reactive microglia in these models exhibit different interactions and molecular profiles. To test this hypothesis, we employed immunofluorescence labeling combined with confocal microscopy to quantify microglia reactivity and microglia-AIS interaction. Additionally, we conducted a microarray using RNA isolated from microglia from both the inflammatory and demyelinating models. Our findings show that microglia are reactive prior to pathology in both models and that the extent of AIS-microglial contact is similar between the models but significantly increased as compared to naïve mice. Our microarray data reveal a substantial difference in gene expression indicating functional differences between the reactive microglia in the inflammatory and demyelinating models. Finally, following functional enrichment analysis of microarray data, the complement pathway emerged as a potential contributor to the AIS pathology observed in EAE.