Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that causes motor, visual, and sensory symptoms. Patients also experience constipation, which is not yet understood, but could involve dysfunction of the enteric nervous system (ENS). Autoimmune targeting of the ENS occurs in other autoimmune diseases that exhibit gastrointestinal (GI) symptoms, and similar mechanisms could lead to GI dysfunction in MS. Here, we characterize GI dysmotility in the experimental autoimmune encephalomyelitis (EAE) model of MS and test whether autoantibodies targeting the ENS are present in the serum of MS patients. Male SJL or B6 mice were induced with EAE by immunization against PLP139-151, MOG35-55, or mouse spinal cord homogenate, and monitored daily for somatic motor symptoms. EAE mice developed GI symptoms consistent with those observed in MS. In vivo motility analysis demonstrated slower whole GI transit, and decreased colonic propulsive motility. EAE mice had faster rates of gastric emptying, with no changes in small intestinal motility. Consistent with these results, ex vivo evaluation of isolated colons demonstrated that EAE mice have slower colonic migrating myoelectric complexes and slow wave contractions. Immunohistochemistry of EAE colons exhibited a significant reduction in GFAP area of ENS ganglia, with no changes in HuD, S100, or neuron numbers. To test whether antibodies in MS bind to ENS structures, we collected serum samples from MS patients with constipation and without constipation, and healthy control patients without constipation. Immunoreactivity was tested using indirect immunofluorescence by applying serum samples to guinea pig ENS tissue. MS serum exhibited significantly higher immunoreactivity against guinea pig ENS than control patients, which was particularly evident in MS patients who did not experience constipation. There was no significant difference in immunoreactivity between MS patients with and without constipation. Targets of human MS and mouse EAE serum include enteric glia and neurons. Taken together, these data validate EAE as a model for constipation in MS, and support the concept that this symptom involves changes within the neuromuscular system of the colon. EAE mice develop symptoms consistent with constipation that affects functional ENS networks and may result in structural or phenotypic changes at the cellular level. Serum immunoreactivity suggests that autoantibodies could play a role in the development of constipation in MS by targeting the ENS itself.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) with characteristic multifocal lesions or ‘ plaques’ of demyelination mainly in the white matter of the brain ( involving cerebral cortex, cerebellar, brain stem and spinal cord). These MS plaques vary in size and shape, and are composed of infiltrates of lymphocytes and macrophages - which contain myelin debris. CD8 T cells are more prevalent in CNS lesions and display oligoclonal expansion. However, their role in disease remains unclear with studies showing both protective and pathogenic roles for myelin-specific CD8 T cells in the experimental autoimmune encephalomyelitis (EAE) model. Our studies have demonstrated a disease-suppressive function for CNS-specific CD8 T cells in a model where the antigen is exogenously administered in vivo and used for in vitro CD8 activation. My studies focus on probing the nature of the CD8 response elicited by endogenously presented myelin antigens in vivo utilizing a novel approach, infection with Listeria monocytogenes (LM) encoding for myelin proteolipid protein peptide PLP178-191 (LM-PLP). I show that LM-PLP infection preferentially induces PLP-specific CD8, but not CD4, T cell responses. Despite this, infection does not result in autoimmunity. In fact, routinely induced EAE is significantly ameliorated in LM-PLP-infected mice, compared to controls. Disease suppression is dependent on the presence of CD8 T cells, and the effector molecules IFN-g and perforin. CNS T cell infiltration and inflammatory responses are reduced in LM-PLP-protected mice, and CD4 T cells from LM-PLP-protected mice are less inflammatory than those from controls. Importantly, infection with LM-PLP ameliorates already established disease. My studies indicate that myelin-specific CD8 T cells induced by endogenous presentation of antigen attenuate CNS autoimmunity in multiple mouse models of EAE, implicating the potential of this approach as a novel immunotherapeutic strategy.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Orientador: Alexandrina Sartori / Banca: Ângela Maria Victoriano de Campos Soares / Banca: Virmondes Rodrigues Júnior / Resumo: A única vacina disponível contra a tuberculose (TB) é o bacilo de Calmette- Guérin (BCG), que é constituída por uma cepa de Mycobacterium bovis vivo atenuado, mas que possui variabilidade de proteção de 0 a 80%. Portanto é necessário o desenvolvimento de novas vacinas para o controle dessa infecção. Dentre as novas formulações profiláticas em teste para TB destacam-se as vacinas gênicas, sendo a DNAhsp65 a vacina investigada por nosso grupo. A DNAhsp65 é uma construção que contém o gene que codifica a proteína de choque térmico de 65KDa do M. leprae. Em virtude da elevada homologia entre a hsp65 micobacteriana e a hsp60 dos mamíferos, pode ocorrer mimetismo antigênico, ou seja, reatividade cruzada entre anticorpos e células T específicas para hsp65 micobacteriana e hsp60 humana. Nesse contexto, nosso grupo tem estudado o possível efeito colateral da DNAhsp65 no desenvolvimento de doenças autoimunes. Neste projeto foi investigado o efeito (proteção, exacerbação ou inocuidade) de diferentes formulações vacinais para TB na encefalite autoimune experimental (EAE). Os objetivos foram caracterizar a resposta imune específica induzida pela DNAhsp65 em ratos Lewis e avaliar o efeito imunomodulador tanto da estratégia clássica de imunização (3 doses de DNAhsp65 por via im) quanto da estratégia prime-boost BCG / DNAhsp65, nas características clínicas, imunológicas e histológicas da EAE. Quanto à imunogenicidade da DNAhsp65, foram testadas duas doses da vacina administradas via intramuscular (100 e 300 μg), sendo que somente a maior dose induziu IgG2b específica para hsp65 e produção de IFN- em cultura de células esplênicas estimuladas in vitro com rhsp65. Para avaliar o efeito da imunização com DNAhsp65 ou do prime-boost BCG /DNAhsp65 no desenvolvimento da EAE, os animais foram previamente imunizados com 3 doses de 300 μg de DNAhsp65 ou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: BCG is the only accepted vaccine against tuberculosis (TB) but its protective ability is very limited. Many new vaccines are therefore being evaluated. Our group has been working with DNAhsp65 that is a genetic construction containing the gene of hsp65 from Mycobacterium leprae. In previous experimental work we demonstrated that both, DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with 3 doses of DNAhsp65 or primed with BCG followed by 2 DNAhsp65 boosters. The animals were then immunized with myelin associated with Complete Freund Adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, inflammation at the central nervous system (CNS) and immune response against myelin. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and inflammation at the CNS did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN- and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate MS, being therefore... (Complete abstract click electronic access below) / Mestre

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Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-26T14:08:12Z No. of bitstreams: 1 marcioluizdepaula.pdf: 356960 bytes, checksum: 5867e0339760e8cd97d12ed8a70ed57e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-12-15T12:53:21Z (GMT) No. of bitstreams: 1 marcioluizdepaula.pdf: 356960 bytes, checksum: 5867e0339760e8cd97d12ed8a70ed57e (MD5) / Made available in DSpace on 2016-12-15T12:53:21Z (GMT). No. of bitstreams: 1 marcioluizdepaula.pdf: 356960 bytes, checksum: 5867e0339760e8cd97d12ed8a70ed57e (MD5) Previous issue date: 2007-11-13 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A Esclerose Múltipla é uma doença autoimune que acomete o Sistema Nervoso Central. O modelo animal mais amplamente utilizado para o estudo desta patologia é o da encefalomielite autoimune experimental (EAE), comumente induzida em camundongos. Em estudos anteriores, observou-se que o estrógeno representa um papel central na resposta imune e nas doenças imunomediadas. Os fitoestrógenos representam uma família de compostos vegetais com propriedades estrogênicas, pois apresentam muitas similaridades estruturais com os estrógenos endógenos, inclusive ligando-se aos seus receptores. O presente trabalho teve como objetivo testar o fitoestrógeno genisteína em camundongos da linhagem C57Bl/6 com EAE. Para isto, foi inicialmente padronizado o modelo de EAE e, posteriormente, o efeito imunomodulador da isoflavona genisteína foi analisado não somente através da avaliação clínica dos camundongos como também pela produção de citocinas Th1 e Th2, além da interação dos leucócitos com o endotélio cerebral. / Multiple sclerosis (MS) is the most common non-traumatic, disabling neurological human inflammatory demyelinating disease of the CNS. Experimental autoimmune encephalomyelitis (EAE) models MS and is characterized as a CD4+ Th1 cell-mediated autoimmune disease. Recent data show that estrogen plays a pivotal role in the immune response as well as in immune-mediated diseases. Isoflavones are found in a variety of plants, especially in soy, and make up the most common form of phytoestrogens which demonstrate estrogenic properties due to their structural similarities with estrogens. Genistein is the major bioactive isoflavone. In the present report, we investigated the use of genistein for the treatment of the murine model of MS in C57Bl/6 mice. The genistein’s modulatory effects were evaluated after induction of EAE with MOG35-55, assessing not only clinical symptoms but also pro- and anti-inflammatory cytokines, and the leukocyte rolling and adherence in the CNS by performing intravital microscopy.

Resultados obtidos pelo nosso grupo demonstraram, in vitro, que a PGE2 é capaz de modular a sobrevivência de linfócitos TCD4+ protegendo essas células da morte. Dentro do modelo de EAE, nossa hipótese é que a PGE2 liberada pelas APCs, durante a fase de indução, module a sobrevivência de linfócitos autorreativos específicos induzindo a doença. Realizamos o tratamento de camundongos submetidos à EAE com indometacina durante 5 dias e notamos que houve redução da EAE associada à redução de linfócitos produtores de IFN-γ, IL-17 e GM-CSF, e macrófagos infiltrantes e microglias ativadas, no SNC. O tratamento alterou a freqüência de células em proliferação e a frequência de células produtoras de IFN-γ e IL-17 na periferia e a concentração dessas citocinas. Esses resultados sugerem que a indometacina reduz o desenvolvimento da EAE e sua resposta antígeno-específica demonstrando a sua importância na modulação das respostas de linfócitos T na autoimunidade. / Results obtained by our group demonstrated in vitro that PGE2 is able to modulate CD4+ T cells survival protecting these cells from death. Within the EAE model, we hypothesized that PGE2 released by APCs during the induction phase, modulate survival of autoreactive specific lymphocytes by induction the disease. We carried out the treatment of EAE in mice subjected to indomethacin for 5 days and noticed that there is reduction of EAE associated with decreased IFN-γ, IL-17 and GM-CSF producing T cells, and infiltrating macrophages and activated microglia in the CNS. The results suggest that indomethacin reduces EAE and its antigen-specif response demonstrating their importance in the modulation of T lymphocyte responses in autoimmunity.

As células T exercem papel crucial nas respostas imunes adaptativas e em doenças autoimunes, como a esclerose múltipla. O glutamato, neurotransmissor mais abundante no SNC, age por meio de duas famílias de receptores: metabotrópico e ionotrópico. As células T são alvo do glutamato durante a ativação e apresentação de antígenos, pois está presente nas sinapses imunológicas, porém, pouco se sabe a respeito de seu papel na função das células T. Nós estudamos o papel do NMDAR nas respostas mediadas por células T. In vitro, o uso do antagonista MK801 reduziu a linfoproliferação e a síntese de IFN-γ e IL-17A, bem com o NMDA reduziu a proliferação e produção de IFN-γ e IL-17A. In vivo, o MK801 reduziu a gravidade da EAE, resultado da menor infiltração de linfócitos Th1 e Th17 no SNC. Além disso, o MK801 reduziu a expressão de Rorc, Il17a, Stat4, Ccr4, Ccr6 e Ifna2 no SNC. Em suma, esses dados confirmam que o NMDAR exerce papel nas funções mediadas por células T, indicando que as células T são alvos do excesso do glutamato via NMDAR em doenças neuroinflamatórias. / T cells play a crucial role in adaptive immune responses and autoimmune diseases, such as multiple sclerosis. Glutamate is the most abundant neurotransmitter in the CNS, and it acts through two receptor families: metabotropic and ionotropic. T cells are target of glutamate during activation and antigen presentation, because glutamate is also present in the immunological synapses, however, little is known about its role on T cell functions. We investigated the role of NMDAR in immune-mediated T cell responses. In vitro, the use of the antagonist MK801 reduced T cell proliferation and cytokine production (IFN-γ e IL-17A), as well as NMDA reduced lymphocyte proliferation and IFN-γ e IL-17A production, in a dose dependent manner. In vivo, MK801 diminished severity of EAE, result of the minor Th1 and Th17 infiltration in the CNS. In addition, MK801 reduced Rorc, Il17a, Stat4, Ccr4, Ccr6 and Ifna2 expression in the CNS. In short, our data confirm that the NMDAR play a role in T cell-mediated functions, indicating that T cells are target of glutamate excess via NMDAR in neuroinflammatory diseases.