Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.

Modulation of fibronectin expression in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis

Fibronectin (FN), a large family of plasma and extracellular matrix (ECM) glycoproteins, plays an important role in leukocyte migration. In normal central nervous system (CNS), a fine and delicate mesh of FN is virtually restricted to the basal membrane of cerebral blood vessels and to the glial limitans externa. Experimental autoimmune encephalomyelitis (EAE), an inflammatory CNS demyelinating disease, was induced in Lewis rats with a spinal cord homogenate. During the preclinical phase and the onset of the disease, marked immunolabelling was observed on the endothelial luminal surface and basal lamina of spinal cord and brainstem microvasculature. In the paralytic phase, a discrete labelling was evident in blood vessels of spinal cord and brainstem associated or not with an inflammatory infiltrate. Conversely, intense immunolabelling was present in cerebral and cerebellar blood vessels, which were still free from inflammatory cuffs. Shortly after clinical recovery minimal labelling was observed in a few blood vessels. Brainstem and spinal cord returned to normal, but numerous inflammatory foci and demyelination were still evident near the ventricle walls, in the cerebral cortex and in the cerebellum. Intense expression of FN in brain vessels ascending from the spinal cord towards the encephalon preceded the appearance of inflammatory cells but faded away after the establishment of the inflammatory cuff. These results indicate an important role for FN in the pathogenesis of CNS inflammatory demyelinating events occurring during EAE.

Baicalin reduces the severity of experimental autoimmune encephalomyelitis

Scutellaria baicalensis Georgi is one of the important medicinal herbs widely used for the treatment of various inflammatory diseases in Asia. Baicalin (BA) is a bioactive anti-inflammatory flavone found abundantly in Scutellaria baicalensis Georgi. To explore the therapeutic potential of BA, we examined the effects of systemic administration of the flavone (5 and 10 mg/kg, ip) on relapsing/remitting experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein 139-151 in SJL/J mice, an experimental model of multiple sclerosis. The mice treated with PBS or BA at day -1 and for 3 consecutive days were observed daily for clinical signs of disease up to 60 days after immunization. In the PBS-EAE group, neurological scores were: incidence (100%), mean day of onset (8.0 ± 0.73), peak clinical score (3.0 ± 0.4), and cumulative disease index (141.8 ± 19.4). In the BA-EAE group (5 or 10 mg kg-1 day-1, respectively), incidence (95 or 90%), mean day of onset (9.0 ± 0.80 or 9.2 ± 0.75; P = 0.000), peak clinical score (2.2 ± 0.3 or 2.0 ± 0.3; P = 0.000), and cumulative disease index (75.9 ± 10.1 or 62.9 ± 8.4; P = 0.000) decreased, accompanied by the histopathological findings (decrease of dense mononuclear infiltration surrounding vascellum) for the spinal cord. Additionally, the in vitro effects of BA (5, 10, and 25 µM) on mononuclear cells collected from popliteal and inguinal lymph nodes of day-10 EAE mice were evaluated using an MTT reduction assay for cell proliferation, and ELISA to measure IFN-g and IL-4 cytokines. Compared with the control group, BA caused an increase in IL-4 (EAE-DMSO: 3.56 ± 0.42 pg/mL vs EAE-BA (5, 10, and 25 µM): 6.03 ± 1.1, 7.83 ± 0.65, 10.54 ± 1.13 pg/mL, respectively; P < 0.001); but inhibited IFN-g (EAE-DMSO: 485.76 ± 25.13 pg/mL vs EAE-BA (5, 10, and 25 µM): 87.08 ± 9.24, 36.27 ± 5.44, 19.18 ± 2.93 pg/mL, respectively; P < 0.001) and the proliferation of mononuclear cells (EAE-DMSO: 0.73 ± 0.021 vs EAE-BA (5, 10, and 25 µM): 0.41 ± 0.015, 0.31 ± 0.018, 0.21 ± 0.11, respectively; P < 0.001) in a concentration-dependent manner. The results suggest that BA might be effective in the treatment of multiple sclerosis.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40% of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

Schistosomiasis protects against multiple sclerosis

The incidences of schistosomiasis and multiple sclerosis (MS) are mutually exclusive worldwide suggesting that schistosomiasis may offer protection against the induction of the immune-mediated disease, MS. Recent studies using the mouse model of MS, experimental autoimmune encephalomyelitis, support a direct suppression of the onset of MS by chronic Schistosoma mansoni infection. Self-reactive Th1 but not Th2 responses develop in infected mice immunized with myelin oligodendrocyte glycoprotein albeit at reduced levels indicating that the induction of auto-reactive T cells is not abolished nor phenotypically altered. CNS infiltration by inflammatory cells, particularly macrophages, is significantly reduced in S. mansoni-infected, immunized mice compared to uninfected, immunized mice. Because activated macrophages are crucial to the induction of clinical disease, these findings support the hypothesis that differences in macrophage activation may contribute to the reduced incidence and delayed progression of experimental autoimmune encephalomyelitis during schistosomiasis.

The immunomodulator glatiramer acetate influences spinal motoneuron plasticity during the course of multiple sclerosis in an animal model

The immunomodulador glatiramer acetate (GA) has been shown to significantly reduce the severity of symptoms during the course of multiple sclerosis and in its animal model - experimental autoimmune encephalomyelitis (EAE). Since GA may influence the response of non-neuronal cells in the spinal cord, it is possible that, to some extent, this drug affects the synaptic changes induced during the exacerbation of EAE. In the present study, we investigated whether GA has a positive influence on the loss of inputs to the motoneurons during the course of EAE in rats. Lewis rats were subjected to EAE associated with GA or placebo treatment. The animals were sacrificed after 15 days of treatment and the spinal cords processed for immunohistochemical analysis and transmission electron microscopy. A correlation between the synaptic changes and glial activation was obtained by performing labeling of synaptophysin and glial fibrillary acidic protein using immunohistochemical analysis. Ultrastructural analysis of the terminals apposed to alpha motoneurons was also performed by electron transmission microscopy. Interestingly, although the GA treatment preserved synaptophysin labeling, it did not significantly reduce the glial reaction, indicating that inflammatory activity was still present. Also, ultrastructural analysis showed that GA treatment significantly prevented retraction of both F and S type terminals compared to placebo. The present results indicate that the immunomodulator GA has an influence on the stability of nerve terminals in the spinal cord, which in turn may contribute to its neuroprotective effects during the course of multiple sclerosis.

Role of Th1 and Th2 cells in autoimmune demyelinating disease

Evidence is accumulating that Th1 cells play an important role in the development of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), whereas Th2 cells contribute to recovery from disease. A major determinant in the development of Th1 and Th2 cells is the type of antigen-presenting cell (APC) involved and its functional characteristics, e.g., the production of interleukin-12. Therefore, modulation of APC might interfere with the development of Th1 type responses and as such be beneficial for MS and EAE. The potential of cytokines, in particular interleukin-10, and glucocorticoids to exert a selective effect on APC, and as a consequence to affect the Th1-Th2 balance in EAE, is discussed

Nitric oxide synthase activity and endogenous inhibitors in rats recovered from allergic encephalomyelitis

We have previously reported that in comparison with normal rats, the presence of experimental allergic encephalomyelitis (EAE) leads to decreased endogenous inhibitory activity (EIA) of Ca2+-dependent nitric oxide synthase (NOS) in both brain and serum, and increased expression of protein 3-nitrotyrosine (NT) in brain. In this work we show that animals recovered from the clinical signs of EAE are not different from controls in terms of either brain NOS activity, EIA of NOS, or NT expression. These results suggest that parallel to the reversal of the disease symptoms, a normalization of the production of nitric oxide and related species occurs.

Acquired and Congenital Ocular Toxoplasmosis Experimentally Induced in Calomys callosus (Rodentia, Cricetidae)

An experimental model for acquired and congenital ocular toxoplasmosis as well as a model to induce experimental autoimmune uveitis (EAU) was investigated in Calomys callosus. Toxoplasma gondii, ME-49 strain, was used to infect males and pregnant- and not pregnant-females while S-antigen, a major glycoprotein of the retinal photoreceptor cell, was used to induce EAU. The ocular lesions elicited by T. gondii were characterized by the presence of cysts, free tachyzoites and inflammatory cells in the retina or related tissues. In the congenital form, 40% of the fetus presented ocular lesions, i.e., presence of cysts in the retina, vitreous, and extra-retinal tissues. In the acquired form, 75% of the females and 50% of the males presented unilateral ocular cysts both at 21 and 47 days post-infection. It was also demonstrated that S-antigen was not uveitogenic in the C. callosus model. No lesion was observed in the animals exclusively immunized with this retinal component, even when jacalin was used as additional adjuvant for polyclonal response to the retinal antigen. It can be concluded that C. callosus may constitute in a promising model for study both acquired and congenital ocular toxoplasmosis, particularly when it is important to make sure that a non autoimmune process is involved in the genesis of the ocular infection.

Immunopathology in ocular toxoplasmosis: facts and clues

Although parasite-mediated host cell lysis is deemed to be an important cause of tissue destruction in ocular toxoplasmosis (OT), the severity of the disease is probably correlated with hypersensitivity and inflammation. Notwithstanding, the mechanisms that regulate the inflammatory process in recurrent OT are poorly understood. Recent evidence has identified interleukin (IL) 17 as a marker for disease severity. The ocular and cerebral presence of this cytokine is generally associated with the induction of autoimmune responses in the brain and the eye. Indeed, there are indications that autoimmunity may contribute to clinical variability in the activity of OT. IL-23, which induces the proliferation of IL-17-producing cells and IL-27, which is a counterplayer to IL-17, may regulate T(H)-1-cell-mediated responses in OT. The importance of these cytokines in experimental models of uveitis and encephalitis has been recently reported. CD25(+) regulatory T-cells may control the local inflammatory response and protect the host against collateral inflammatory tissue damage. The responses of these cells to OT may be suitably tailored to cope with either an acquired or a congenital aetiology. Knowledge relating to immunoreactivity in OT has grown impressively during the past few years. Its characteristic and variable features have been identified and the potential relevance of autoimmunity has been assessed. In light of this knowledge, potential future treatment options have been considered.

Differential expression of adhesion moleculesshaping the T-cell subset prevalence during the early phase of autoimmune and Trypanosoma cruzi-elicited myocarditis

The participation of cell adhesion molecules (CAMs) in the establishment of autoimmune and infectious myocarditis is an important matter of investigation and may have therapeutic implication. Trypanosoma cruzi infection induces a CD8-mediated myocarditis in patients with severe cardiomyopathy and experimental animals. Previously, we have proposed that this predominance of CD8+ T-cells is, at least in part, consequence of the differential expression of CAMs on circulating CD8+ lymphocytes. In the present study we investigated the participation of CAMs in shaping the phenotypic nature of the autoimmune CD4-mediated myosin-induced and the CD8-mediated T. cruzi-elicited myocarditis. We provide evidence that the prevalence of a certain T-cell subset inside the inflamed heart reflects the differential profile of the adhesion molecules VLA-4, LFA-1, and ICAM-1 displayed on a large proportion of this particular T-cell population in peripheral blood during the early phase of inflammation. Further, the expression of VCAM-1, ligand for VLA-4, and ICAM-1, counter-receptor for LFA-1, was up-regulated on vascular endothelium and paralleled the entrance of inflammatory cells into the cardiac tissue. Thus, this up-regulated expression of receptors-counter-receptors that regulate T-cell transmigration through the vascular endothelium may have an important role in the pathogenesis of the early phase of both autoimmune and infectious myocarditis.

Cytokine profiles during experimental Chagas' disease

People infected with Trypanosoma cruzi remain so for life, yet only 30-40% of these individuals develop characteristic chagasic cardiomyopathies. Similarly, when infected with the Brazilian strain of T. cruzi, DBA/2 mice develop severe cardiac damage while B10.D2 mice do not. To better understand the immunological parameters that may be involved in the disease process, we have used this murine model (DBA/2 vs B10.D2) and compared the changes in cytokine production during the course of infection with T. cruzi. Concanavalin A (Con A) stimulation of spleen cells harvested during the acute phase (day 30) resulted in similarly high levels of IFN-g in both mouse strains. However, the amount of IFN-g in supernatants from cultures of B10.D2 spleen cells initiated during the chronic phase (day 72) was at subacute levels, whereas secretion by chronic DBA/2 spleen cells remained high. In addition, Con A-stimulated spleen cells from acute DBA/2 mice produced approximately twice as much IL-10 and significantly more IL-4 than cells from B10.D2 mice. IL-4 secretion remained low by cells from chronic B10.D2 mice, but when using cells from chronic DBA/2 mice, levels continued to increase beyond the already high levels secreted by cells harvested during the acute phase. Proliferative responses to Con A stimulation by spleen cells from DBA/2 mice were significantly higher than those from B10.D2 mice in both the acute and chronic phases. These data suggest that enhanced responses in DBA/2 mice, which may be related to a higher parasite burden, a lack of down-regulation, and/or the onset of autoimmune phenomena, correlate with the more severe cardiomyopathy seen in pathopermissive mice.

Histologia da camada superficial da lâmina própria da prega vocal ao se aplicar retalho pediculado de mucosa: estudo experimental em cães

Os resultados conseguidos até hoje para a correção de sulcos vocais e lesões cicatriciais não são universalmente aceitos. A Técnica do Retalho Pediculado de Mucosa de Prega Vocal consiste na colocação de um retalho de mucosa de prega vocal com pedículo anterior na camada superficial da lâmina própria, abaixo da borda livre. OBJETIVO: Descrever os achados histológicos pós-operatórios ocorridos na camada superficial da lâmina própria de cães ao se aplicar a técnica em questão, tomando-se como parâmetro a variação dos colágenos total, tipo I, tipo III e número de núcleos celulares. FORMA DE ESTUDO: experimental. MATERIAL E MÉTODO: Foram utilizados 15 cães. Numa das pregas foi realizada a intervenção e a contralateral foi deixada como controle. Cada grupo de três cães foi sacrificado em 10, 30, 90, 180 e 360 dias após a cirurgia. As colorações utilizadas foram: H.E. e Syrius Red. RESULTADOS: Os níveis de colágeno total e tipo I apresentaram uma tendência a aumento nos grupos de intervenção nos 90º e 180º dias de pós-operatório, contudo só houve significância estatística no 180º dia (p<0,05). A área do colágeno tipo III alcançou níveis inferiores ao do grupo controle no 180º dia (p<0,05). O número de núcleos atingiu maiores níveis no grupo teste no 10º dia de pós-operatório, seguido de decréscimo após o 30º dia. DISCUSSÃO: Os resultados encontrados quanto ao colágeno total, tipo I e tipo III e número de núcleos sugerem semelhanças ao processo cicatricial pós-operatório em laringe encontrado em outros estudos experimentais. Os presentes achados deverão ser complementados por experimentos.

Achados otomicroscópicos e histológicos da miringoesclerose induzida em ratos: estudo crítico de um modelo experimental

A miringoesclerose é caracterizada por hialinização da lâmina própria da membrana timpânica. Trabalhos experimentais têm utilizado a otomicroscopia ou a histologia para estudar a miringoesclerose em animais, porém sem correlacionar precisamente estes dois métodos de avaliação. OBJETIVO: Este estudo procura avaliar a acurácia da otomicroscopia no diagnóstico de miringoesclerose em modelo experimental com ratos. FORMA DE ESTUDO: experimental. MATERIAL E MÉTODO: Foi induzida miringoesclerose por inoculação transtimpânica de Streptococcus pneumoniae em 25 ratos Wistar, que foram examinados quinzenalmente por otomicroscopia e sacrificados após oito semanas, tendo suas membranas timpânicas examinadas por histologia. RESULTADOS: Da confrontação das alterações otomicroscópicas com os achados histológicos, houve uma sensibilidade de 80% e especificidade de 75%. CONCLUSÃO: Pelos resultados obtidos neste estudo, a otomicroscopia não representou um bom método para avaliar a miringoesclerose neste modelo experimental.