Impact Of Pregabalin Treatment On Synaptic Plasticity And Glial Reactivity During The Course Of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease that affects young adults. It is characterized by generating a chronic demyelinating autoimmune inflammation in the central nervous system. An experimental model for studying MS is the experimental autoimmune encephalomyelitis (EAE), induced by immunization with antigenic proteins from myelin. The present study investigated the evolution of EAE in pregabalin treated animals up to the remission phase. The results demonstrated a delay in the onset of the disease with statistical differences at the 10th and the 16th day after immunization. Additionally, the walking track test (CatWalk) was used to evaluate different parameters related to motor function. Although no difference between groups was obtained for the foot print pressure, the regularity index was improved post treatment, indicating a better motor coordination. The immunohistochemical analysis of putative synapse preservation and glial reactivity revealed that pregabalin treatment improved the overall morphology of the spinal cord. A preservation of circuits was depicted and the glial reaction was downregulated during the course of the disease. qRT-PCR data did not show immunomodulatory effects of pregabalin, indicating that the positive effects were restricted to the CNS environment. Overall, the present data indicate that pregabalin is efficient for reducing the seriousness of EAE, delaying its course as well as reducing synaptic loss and astroglial reaction.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10. RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Increased apoptosis of T lymphocytes and macrophages in the central and peripheral nervous systems of Lewis rats with experimental autoimmune encephalomyelitis treated with dexamethasone

Using light and electron microscopic histological and immunocytochemical techniques, we investigated the effects of the glucocorticoid dexamethasone on T cell and macrophage apoptosis in the central nervous system (CNS) and peripheral nervous system (PNS) of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP). A single subcutaneous injection of dexamethasone markedly augmented T cell and macrophage apoptosis in the CNS and PNS and microglial apoptosis in the CNS within 6 hours (h). Pre-embedding immunolabeling revealed that dexamethasone increased the number of apoptotic CD5+ cells (T cells or activated B cells), αβ T cells, and CD11b+ cells (macrophages/microglia) in the meninges, perivascular spaces, and CNS parenchyma. The induction of increased apoptosis was dose-dependent. Daily dexamethasone treatment suppressed the neurological signs of EAE. However, the daily injection of a dose of dexamethasone (0.25 mg/kg). which, after a single dose, did not induce increased apoptosis in the CNS or PNS, was as effective in inhibiting the neurological signs of EAE as the high dose (4 mg/kg), which induced a marked increase in apoptosis. This indicates that the beneficial clinical effect of glucocorticoid therapy in EAE does not depend on the induction of increased apoptosis. The daily administration of dexamethasone for 5 days induced a relapse that commenced 5 days after cessation of treatment, with the severity of the relapse tending to increase with dexamethasone dosage.

Immunopathology and Pathophysiology of Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS), and is widely studied as an animal model of the human CNS demyelinating diseases, including multiple sclerosis (Raine, 1984). EAE can be induced by inoculation with whole CNS tissue, purified myelin basic protein (MBP) or myelin proteolipid protein (PLP), together with adjuvants. It may also be induced by the passive transfer of T cells specifically reactive to these myelin antigens. EAE may have either an acute or a chronic relapsing course. Acute EAE closely resembles the human disease acute disseminated encephalomyelitis, while chronic relapsing EAE resembles multiple sclerosis. EAE is also the prototype for T-cell-mediated autoimmune disease in general. This chapter will focus on the immunopathology and pathophysiology of EAE, which are the subjects of investigation in my laboratory.

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis

P>According to the hygiene hypothesis, the increased incidence of allergic and autoimmune diseases in developed countries is mainly explained by the decreased contact between the human population and certain environmental agents as lactobacillus, mycobacteria and helminths. In this study, we evaluated the effect of multiple infections with Strongyloides venezuelensis on the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Multiple infections before EAE induction were not able to change the evolution of the disease. No alterations were observed in weight loss, clinical score and inflammation intensity at the central nervous system. The presence of significant levels of parasite-specific IgG1 but not IgG2b suggested a Th2 polarization. However, the percentage and absolute number of CD4+CD25+Foxp3+ T cells were not changed, being their levels in the spleen and lymph nodes of infected rats comparable to the ones found in normal animals. These results suggest that a Th2-polarized response without concomitant expansion of Foxp3+ regulatory T cells was not able to modify EAE progression. Even though these results do not threaten the hygiene hypothesis, they suggest that this paradigm might be an oversimplification. They also emphasize the need of a study to compare the immunoregulatory ability associated with different helminth spp.

The effects of pregnancy on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats: Suppression of clinical disease, modulation of cytokine expression in the spinal cord inflammatory infiltrate and suppression of lymphocyte p

Problem: The present study was performed to explore the effects of pregnancy on experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by inoculation with myelin basic protein (MBP) (MBP-EAE). Method of study: MBP-EAE was induced in pregnant and non-pregnant rats and severity of disease evaluated. Serum from pregnant and non-pregnant rats was used in standard lymphocyte proliferation assays. Real-time polymerase chain reaction (PCR) was used to investigate the expression of cytokine mRNA in the inflammatory cells obtained from the spinal cord of rats on day 15 after inoculation. Results: Pregnant rats developed less severe disease than non-pregnant rats. Serum from pregnant rats suppressed the proliferation of T lymphocytes in response to MBP. There was significantly increased expression of IL-4. IL-10 and TNF-alpha mRNA in the spinal cord infiltrate of pregnant rats. Conclusion: Circulating humoral factors and alteration in cytokine production by inflammatory cells may contribute to the suppression of EAE in pregnant rats.

Early pregnancy factor treatment suppresses the inflammatory response and adhesion molecule expression in the spinal cord of SJL/J mice with experimental autoimmune encephalomyelitis and the delayed-type hypersensitivity reaction to trinitrochlorobenzene in normal BALB/c mice

Early pregnancy factor (EPF) is a secreted protein, present in serum during early pregnancy and essential for maintaining viability of the embryo. It is a homologue of chaperonin 10 (Cpn10) but, unlike Cpn10, it has an extracellular role. EPF has immunosuppressive and growth regulatory properties. Previously we have reported the preparation of recombinant EPF (rEPF) and shown that treatment with rEPF will suppress clinical signs of MBP-EAE in Lewis rats and PLP-EAE in SJL/J mice. In the present study, these findings have been extended to investigate possible mechanisms involved in the action of EPF. Following treatment of mice with rEPF from the day of inoculation, there were fewer infiltrating CD3+ and CD4+ cells in the parenchyma of the spinal cord during the onset of disease and after the initial episode, compared with mice treated with vehicle. Expression of the integrins LFA-1, VLA-4 and Mac-1 and of members of the immunoglobulin superfamily of adhesion molecules ICAM-1 and VCAM-1 was suppressed in the central nervous system (CNS) following rEPF treatment. The expression of PECAM-1 was not affected. To determine if rEPF suppressed T cell activation in the periphery, the delayed-type hypersensitivity (DTH) reaction of normal BALB/c mice to trinitrochlorobenzene (TNCB) following treatment with rEPF was studied. The results showed that treatment with rEPF suppressed the DTH reaction, demonstrating the ability of EPF to downregulate the cell-mediated immune response. These results indicate that suppression of immunological mechanisms by rEPF plays a major role in the reduction of clinical signs of disease in experimental autoimmune encephalomyelitis (EAE). (C) 2003 Elsevier Science B.V. All rights reserved.

Early pregnancy factor suppresses the infiltration of lymphocytes and macrophages in the spinal cord of rats during experimental autoimmune encephalomyelitis but has no effect on apoptosis

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties that has been shown to suppress acute experimental autoimmune encephalomyelitis (EAE) induced with myelin basic protein (MBP) in Lewis rats. EAE is associated with infiltration of the central nervous system (CNS) with inflammatory cells. Spontaneous recovery involves the loss of T lymphocytes from the CNS and the selective apoptosis of Vbeta8.2(+) cells. In the present study, T cell, macrophage (CD11b/c(+)) and B cell (CD45RA(+)) populations in spinal cord and popliteal lymph nodes (LN) of Lewis rats with EAE were quantitated and apoptosis was studied. Rats were treated with EPF or vehicle. Following treatment on day 14 after inoculation with MBP, neither 1 x 100 mug nor 2 x 100 mug doses of EPF affected the total number of cells infiltrating the spinal cord on day 15, although the higher dose caused a decrease in the number of CD5(+) and CD11b/c(+) cells. Treatment with 2 x 100 mug/day from days 10 to 14 decreased the total number of infiltrating cells, and the numbers of CD5(+), CD11b/c(+) and CD45RA(+) cells. Apoptosis was unaffected. No alteration on the number or type of inflammatory cells in the popliteal LN was observed after treatment on days 10-14. However, treatment with EPF from days 0 to 11 increased the total number of T and B cells and CD5(+) T cells found on day 12 in the LN. Similarly, there was an increase in the frequency of MBP-reactive cells in the LN as determined by limiting dilution analysis. These results suggest that EPF treatment reduces the numbers of lymphocytes and macrophages in the CNS, possibly through an effect on cell trafficking. (C) 2003 Elsevier B.V. All rights reserved.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.

Supresión oral de la Encefalomielitis autoinmune experimental por antígenos sinaptosomales. Oral suppression of experimental autoimmune encephalomyelitis by sinaptosomal antigens.

La inducción de las manifestaciones clínicas de la encefalomielitis autoinmune experimental (EAE) involucra una reacción inmune celular contra determinantes antigénicos del sistema nervioso central (SNC), principalmente contra la proteína básica de mielina (PBM). Atento a la reactividad inmunológica cruzada previamente descrita entre la PBM y la proteína neuronal Sinapsina I, estudiaremos el efecto de la administración oral de moléculas híbridas (LTBSC, LTBSABC) entre la subunidad B de la toxina lábil al calor de Escherichia coli (LTB) con péptidos de Sinapsina (dominios C y ABC de la molécula) sobre el desarrollo de la EAE en ratas Wistar. Se administrarán oralmente los antígenos híbridos de LTB, LTB y péptidos de sinapsina no acoplados previa o posteriormente a la inducción activa de la EAE. Se estudiará la aparición de las manifestaciones clínicas de la enfermedad y se caracterizará la respuesta histopatológica e inmunológica (reacción de DTH, activación de linfocitos T, respuesta inmune humoral, vías de activación de macrófagos, patrón de citocinas) y los eventos celulares e inmunes desencadenados a nivel local luego de administrar los antígenos recombinantes en sistemas in vivo e in vitro. Estos estudios acerca de la respuesta autoinmune contra componentes de mielina y sinaptosomales en EAE tienen como objetivo poder comprender los diferentes mecanismos subyacentes involucrados en el desarrollo y regulación de esta enfermedad experimental. Específicamente este proyecto relacionado a la supresión de los síntomas clínicos como así también las alteraciones neuropatológicas del SNC de la EAE a través de un proceso de supresión oral de la enfermedad no invasivo utilizando tanto antígenos mielínicos como sinaptosomales fusionados a subunidades B (atóxicas) de la enterotoxina lábil al calor de E. coli (LTB), es de suma importancia para un estudio posterior en las patologías humanas relacionadas y su uso en el diagnóstico, pronóstico y/o terapia de las mismas.

Astrocyte-specific function of A20 and FasL in experimental autoimmune encephalomyelitis

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2015

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis.

AIMS: Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. METHODS AND RESULTS: EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. CONCLUSION: Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Innate signaling promotes formation of regulatory nitric oxide-producing dendritic cells limiting T-cell expansion in experimental autoimmune myocarditis.

BACKGROUND: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo. CONCLUSIONS: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.