A myelin basic protein fragment induces sexually dimorphic transcriptome signatures of neuropathic pain in mice.

In the peripheral nerve, mechanosensitive axons are insulated by myelin, a multilamellar membrane formed by Schwann cells. Here, we offer first evidence that a myelin degradation product induces mechanical hypersensitivity and global transcriptomics changes in a sex-specific manner. Focusing on downstream signaling events of the functionally active 84-104 myelin basic protein (MBP(84-104)) fragment released after nerve injury, we demonstrate that exposing the sciatic nerve to MBP(84-104) via endoneurial injection produces robust mechanical hypersensitivity in female, but not in male, mice. RNA-seq and systems biology analysis revealed a striking sexual dimorphism in molecular signatures of the dorsal root ganglia (DRG) and spinal cord response, not observed at the nerve injection site. Mechanistically, intra-sciatic MBP(84-104) induced phospholipase C (PLC)-driven (females) and phosphoinositide 3-kinase-driven (males) phospholipid metabolism (tier 1). PLC/inositol trisphosphate receptor (IP3R) and estrogen receptor co-regulation in spinal cord yielded Ca2+-dependent nociceptive signaling induction in females that was suppressed in males (tier 2). IP3R inactivation by intrathecal xestospongin C attenuated the female-specific hypersensitivity induced by MBP(84-104). According to sustained sensitization in tiers 1 and 2, T cell-related signaling spreads to the DRG and spinal cord in females, but remains localized to the sciatic nerve in males (tier 3). These results are consistent with our previous finding that MBP(84-104)-induced pain is T cell-dependent. In summary, an autoantigenic peptide endogenously released in nerve injury triggers multisite, sex-specific transcriptome changes, leading to neuropathic pain only in female mice. MBP(84-104) acts through sustained co-activation of metabolic, estrogen receptor-mediated nociceptive, and autoimmune signaling programs.

Anti-CD52 antibody treatment depletes B cell aggregates in the central nervous system in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) for which several new treatment options were recently introduced. Among them is the monoclonal anti-CD52 antibody alemtuzumab that depletes mainly B cells and T cells in the immune periphery. Considering the ongoing controversy about the involvement of B cells and in particular the formation of B cell aggregates in the brains of progressive MS patients, an in-depth understanding of the effects of anti-CD52 antibody treatment on the B cell compartment in the CNS itself is desirable. METHODS: We used myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 (B6) mice as B cell-dependent model of MS. Mice were treated intraperitoneally either at the peak of EAE or at 60 days after onset with 200 µg murine anti-CD52 vs. IgG2a isotype control antibody for five consecutive days. Disease was subsequently monitored for 10 days. The antigen-specific B cell/antibody response was measured by ELISPOT and ELISA. Effects on CNS infiltration and B cell aggregation were determined by immunohistochemistry. Neurodegeneration was evaluated by Luxol Fast Blue, SMI-32, and Olig2/APC staining as well as by electron microscopy and phosphorylated heavy neurofilament serum ELISA. RESULTS: Treatment with anti-CD52 antibody attenuated EAE only when administered at the peak of disease. While there was no effect on the production of MP4-specific IgG, the treatment almost completely depleted CNS infiltrates and B cell aggregates even when given as late as 60 days after onset. On the ultrastructural level, we observed significantly less axonal damage in the spinal cord and cerebellum in chronic EAE after anti-CD52 treatment. CONCLUSION: Anti-CD52 treatment abrogated B cell infiltration and disrupted existing B cell aggregates in the CNS.

Role of perforin secretion from CD8+ T-cells in neuronal cytotoxicity in multiple sclerosis.

OBJECTIVES: Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system, and is characterized by inflammation and myelin damage. The immune system initiates the autoimmune response, although the mechanisms of neuronal damage have not been elucidated. The purpose of the present study was to investigate autoreactive CD4+ and CD8+ T lymphocytes, in conjunction with other inflammatory cells and cytokines in active MS lesions. METHODS: EAE animal models was established by plantar injections of MBP (200 µg per rat). Purified CD4+ or CD8+ T-cells were isolated from heparinized peripheral blood (EAE animals and control animals) via negative selection. To examine effects of presence of autoreactive CD4+ and CD8+ T lymphocytes, we carried out ELISA, Western blot analysis and TUNEL. In addition, we examined the direct effects of various factors on neuronal cell death using MTT assay. RESULTS: The data revealed that CD8+ T-cells were more toxic to neurons compared to CD4+ T-cells, in both the MBP and EAE conditions. Bax was greater increased when neurons were co-cultured with CD8+ T-cells in the MBP group. There is a significant increase in IL-17 secretion by CD4+ T-cells in both the MBP group and EAE group. Neuronal viability were affected by Perforin (1.5 µg/mL). CONCLUSION: The present study extends previous research by demonstrating the role of CD8+ T-cells in MS and supports perforin secretion by CD8+ T-cells as a potential therapeutic factor. Furthermore, we determined that CD4+ T-cells can enhance CD8+ T-cell neuronal cytotoxicity via induction of intense inflammation.

Lewis Rat Model of Experimental Autoimmune Encephalomyelitis.

In this unit, we describe in detail the most common methods used to break immunological tolerance for central myelin antigens and induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats as an animal model of multiple sclerosis. The resulting disease course ranges from an acute monophasic disease to a chronic relapsing or chronic progressive course, which strongly resembles the human disease. These models enable the study of cellular and humoral autoimmunity against major antigenic epitopes of the myelin basic protein, myelin oligodendrocyte glycoprotein, or proteolipid protein. We provide an overview of common immunization protocols for induction of active and passive EAE, assessment and analysis of clinical score, preparation and purification of myelin basic protein, and derivation of neuroantigen-specific rat T cell lines. Finally, we describe the major clinical characteristics of these models. © 2017 by John Wiley & Sons, Inc.

Differential effects of FTY720 on the B cell compartment in a mouse model of multiple sclerosis.

BACKGROUND: MP4-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), which enables targeted research on B cells, currently much discussed protagonists in MS pathogenesis. Here, we used this model to study the impact of the S1P1 receptor modulator FTY720 (fingolimod) on the autoreactive B cell and antibody response both in the periphery and the central nervous system (CNS). METHODS: MP4-immunized mice were treated orally with FTY720 for 30 days at the peak of disease or 50 days after EAE onset. The subsequent disease course was monitored and the MP4-specific B cell/antibody response was measured by ELISPOT and ELISA. RNA sequencing was performed to determine any effects on B cell-relevant gene expression. S1P1 receptor expression by peripheral T and B cells, B cell subset distribution in the spleen and B cell infiltration into the CNS were studied by flow cytometry. The formation of B cell aggregates and of tertiary lymphoid organs (TLOs) was evaluated by histology and immunohistochemistry. Potential direct effects of FTY720 on B cell aggregation were studied in vitro. RESULTS: FTY720 significantly attenuated clinical EAE when treatment was initiated at the peak of EAE. While there was a significant reduction in the number of T cells in the blood after FTY720 treatment, B cells were only slightly diminished. Yet, there was evidence for the modulation of B cell receptor-mediated signaling upon FTY720 treatment. In addition, we detected a significant increase in the percentage of B220+ B cells in the spleen both in acute and chronic EAE. Whereas acute treatment completely abrogated B cell aggregate formation in the CNS, the numbers of infiltrating B cells and plasma cells were comparable between vehicle- and FTY720-treated mice. In addition, there was no effect on already developed aggregates in chronic EAE. In vitro B cell aggregation assays suggested the absence of a direct effect of FTY720 on B cell aggregation. However, FTY720 impacted the evolution of B cell aggregates into TLOs. CONCLUSIONS: The data suggest differential effects of FTY720 on the B cell compartment in MP4-induced EAE.

The enteric nervous system is a potential autoimmune target in multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.

Effects of active immunisation with myelin basic protein and myelin-derived altered peptide ligand on pain hypersensitivity and neuroinflammation.

Neuropathic pain is a debilitating condition in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Specific myelin basic protein (MBP) peptides are encephalitogenic, and myelin-derived altered peptide ligands (APLs) are capable of preventing and ameliorating EAE. We investigated the effects of active immunisation with a weakly encephalitogenic epitope of MBP (MBP87-99) and its mutant APL (Cyclo-87-99[A(91),A(96)]MBP87-99) on pain hypersensitivity and neuroinflammation in Lewis rats. MBP-treated rats exhibited significant mechanical and thermal pain hypersensitivity associated with infiltration of T cells, MHC class II expression and microglia activation in the spinal cord, without developing clinical signs of paralysis. Co-immunisation with APL significantly decreased pain hypersensitivity and neuroinflammation emphasising the important role of neuroimmune crosstalk in neuropathic pain.

Age-related changes in spleen of Dark Agouti rats immunized for experimental autoimmune encephalomyelitis.

The study was undertaken considering age-related changes in susceptibility to experimental autoimmune encephalomyelitis (EAE) and a putative role of spleen in pathogenesis of this disease. The phenotypic and functional characteristics of T splenocytes were examined in young (3-month-old), middle-aged (8-month-old) and aged (26-month-old) Dark Agouti rats immunized for EAE with rat spinal cord in complete Freund's adjuvant. The rat susceptibility to EAE induction, as well as the number of activated CD4+CD134+ lymphocytes retrieved from their spinal cords progressively decreased with aging. To the contrary, in rats immunized for EAE the number of activated CD4+ splenocytes, i.e., CD4+CD134+, CD4+CD25+FoxP3- and CD4+CD40L+ cells, progressively increased with aging. This was associated with age-related increase in (i) CD4+ splenocyte surface expression of CD44, the molecule suggested to be involved in limiting emigration of encephalitogenic CD4+ cells from spleen into blood and (ii) frequency of regulatory T cells, including CD4+CD25+FoxP3+ cells, which are also shown to control encephalitogenic cell migration from spleen into the central nervous system. In favor of expansion of T-regulatory cell pool in aged rats was the greater concentration of IL-10 in unstimulated, Concanavalin A (ConA)- and myelin basic protein (MBP)-stimulated splenocyte cultures from aged rats compared with the corresponding cultures from young ones. Consistent with the age-related increase in the expression of CD44, which is shown to favor Th1 effector cell survival by interfering with CD95-mediated signaling, the frequency of apoptotic cells among CD4+ splenocytes, despite the greater frequency of CD95+ cells, was diminished in splenocyte cultures from aged compared with young rats. In addition, in control, as well as in ConA- and MBP-stimulated splenocyte cultures from aged rats, despite of impaired CD4+ cell proliferation, IFN-γ concentrations were greater than in corresponding cultures from young rats. This most likely reflected increased abundance of IFN-γ-producing cells in splenocyte cultures from aged compared with young rats. The diminished CD4+ cell proliferation in response to ConA and MBP in splenocyte cultures from aged compared with young rats could be, at least partly, associated with an enhanced splenic expression of iNOS mRNA in aged rats. Thus, the study suggests that age-associated changes leading to entrapping of activated CD4+ cells in the spleen could contribute to the restriction in development of EAE in aged rats.

Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane.

The central nervous system (CNS) insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP). MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage.

Acute treatment with valproic acid and l-thyroxine ameliorates clinical signs of experimental autoimmune encephalomyelitis and prevents brain pathology in DA rats.

Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the central nervous system (CNS) in young adults. Chronic treatments with histone deacetylase inhibitors (HDACis) have been reported to ameliorate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by targeting immune responses. We have recently shown that the HDAC inhibition/knockdown in the presence of thyroid hormone (T3) can also promote oligodendrocyte (OL) differentiation and expression of myelin genes in neural stem cells (NSCs) and oligodendrocyte precursors (OPCs). In this study, we found that treatment with an HDACi, valproic acid (VPA), and T3, alone or in combination, directly affects encephalitogenic CD4+ T cells. VPA, but not T3, compromised their proliferation, while both molecules reduced the frequency of IL-17-producing cells. Transfer of T3, VPA and VPA/T3 treated encephalitogenic CD4+ T cells into naïve rats induced less severe EAE, indicating that the effects of these molecules are persistent and do not require their maintenance after the initial stimuli. Thus, we investigated the effect of acute treatment with VPA and l-thyroxine (T4), a precursor of T3, on myelin oligodendrocyte glycoprotein-induced EAE in Dark Agouti rats, a close mimic of MS. We found that a brief treatment after disease onset led to sustained amelioration of EAE and prevention of inflammatory demyelination in the CNS accompanied with a higher expression of myelin-related genes in the brain. Furthermore, the treatment modulated immune responses, reduced the number of CD4+ T cells and affected the Th1 differentiation program in the brain. Our data indicate that an acute treatment with VPA and T4 after the onset of EAE can produce persistent clinically relevant therapeutic effects by limiting the pathogenic immune reactions while promoting myelin gene expression.

Upregulation of ß-1,4-galactosyltransferase I in rat spinal cord with experimental autoimmune encephalomyelitis.

Inflammatory infiltration has been recently emphasized in the demyelinating diseases of the central nervous system including multiple sclerosis. ß-1,4-Galactosyltransferase I (ß-1,4-GalT-I) is a major galactosyltransferase responsible for selectin-ligand biosynthesis, mediating rolling of the inflammatory lymphocytes. In the present study, Western blot showed that expression of ß-1,4-GalT-I was low in normal or complete Freund's adjuvant (CFA) control rats' spinal cords, and it began to increase since early stage and peaked at E4 stage of experimental autoimmune encephalomyelitis (EAE) and restored approximately at normal level in the recovery stage. Immunohistochemisty revealed that upregulation of ß-1,4-GalT-I was predominantly distributed in the white matter of spinal cord , while there was also some increased staining of ß-1,4-GalT-I in the grey matter. Meanwhile, the expression of E-selectin, the substrate of ß-1,4-GalT-I, was significantly increased, with a peak at E4 stage of EAE, and gradually decreased thereafter. Lectin blot showed that the protein bands with molecular weights of 65-25 kDa reacted a remarkable increase at the peak stage of EAE when compared with the normal and CFA control. Ricinus Communis Agglutinin-I (RCA-I) histochemistry revealed that RCA-Ι-positive signals were most intense in white matter of lumbosacral spinal cord at the peak stage of EAE (E4). Immunohistochemistry showed that ß-1,4-GalT-I and CD62E, a marker for E-selectin stainings located in a considerable number of ED1 (+) macrophages in perivascular or in the white matter in EAE lesions, and a good co-localization of ED1 (+) cells with CD62E was observed. All these results suggest that ß-1,4-GalT-I might serve as an inflammatory mediator regulating adhesion and migration of inflammatory cells in EAE, possibly through influencing the modification of galactosylated carbohydrate chains to modulate selectin-ligand biosynthesis and interaction with E-selectin.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Differential levels of resistance to disease induction and development of relapsing experimental autoimmune encephalomyelitis in two H-2b-restricted mouse strains.

Besides the major histocompatibility complex (MHC) genes, background genes are believed to influence the encephalitogenicity of SJL(H-2(s)) and B10.S (H-2(s)) mice responding to myelin basic protein (MBP). A new mouse strain was constructed to study the effects of the SJL genetic background in mice responding to H-2(b)-restricted neuroantigens. Although the SJL.B (H-2(b)) mouse remained resistant to MBP in active EAE induction, the disease severity was uniformly higher in MOG-induced active EAE and in MBP-induced adoptive EAE when compared to those of B6 (H-2(b)) mice. Treatment of mice with anti-CD25 antibodies prior to immunization caused 60% of SJL.B mice to become susceptible to MBP-induced EAE while only 14% of B6 mice were converted. In addition, MOG-induced EAE in SJL.B mice followed a remitting-relapsing disease course while B6 mice only exhibited monophasic or chronic episodes. The new SJL.B mouse strain provides a valuable tool for studying EAE resistance and remitting-relapsing disease in H-2(b) mice.

Myelin basic protein-reactive T cells persist in an inactive state in the bone marrow of Lewis rats that have recovered from autoimmune encephalomyelitis.

Lewis rats immunized with guinea pig myelin basic protein residues 68-86 develop acute experimental autoimmune encephalomyelitis and recover. The predominant T cell receptor expressed by the encephalitogenic T cells is TCRBV8S2. They persist in bone marrow many weeks after recovery. CD3 is down-regulated, but >90% express CD4. They fail to proliferate to GPMBP68-86 unless a nitric oxide synthase inhibitor is added to the cultures. Perhaps these are memory T cells that are maintained in a suppressed state in BM by a nitric oxide-dependent mechanism.

A public T cell clonotype within a heterogeneous autoreactive repertoire is dominant in driving EAE.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. Immunization of B10.PL mice with the Ac1-9 peptide, the immunodominant determinant of myelin basic protein (MBP), produced a single episode of EAE followed by recovery and resistance to reinduction of disease. Using the CDR3 length spectratyping technique, we characterized the clonal composition of the Ac1-9-specific T cell repertoire from induction through onset and resolution of disease. Two clonally restricted subsets within a heterogeneous self-reactive repertoire were found in mouse lymph nodes, spleen, and spinal cord soon after immunization, before any sign of EAE. These clonotypes, designated BV8S2/BJ2S7 and BV16/BJ2S5, were present in all mice examined and thus considered public. BV8S2/BJ2S7 was found in far greater excess; was exclusively Th1 polarized; disappeared from the spinal cord, spleen, and lymph nodes concomitantly with recovery; and transferred disease to naive recipients. In contrast, BV16/BJ2S5 and numerous private clonotypes were either Th1 or Th2 and persisted following recovery. These results are consistent with the hypothesis that the public clonotype BV8S2/BJ2S7 is a driver of disease and necessary for its propagation.

Design, synthesis, and analysis of a polyethelene glycol-modified (PEGylated) small molecule inhibitor of integrin {alpha}4{beta}1 with improved pharmaceutical properties.

Integrin alpha4beta1 plays an important role in inflammatory processes by regulating the migration of leukocytes into inflamed tissues. Previously, we identified BIO5192 [2(S)-{[1-(3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl}-amino)-pentanoylamino]-butyric acid], a highly selective and potent (K(D) of 9 pM) small molecule inhibitor of alpha4beta1. Although BIO5192 is efficacious in various animal models of inflammatory disease, high doses and daily treatment of the compound are needed to achieve a therapeutic effect because of its relatively short serum half-life. To address this issue, polyethylene glycol modification (PEGylation) was used as an approach to improve systemic exposure. BIO5192 was PEGylated by a targeted approach in which derivatizable amino groups were incorporated into the molecule. Two sites were identified that could be modified, and from these, five PEGylated compounds were synthesized and characterized. One compound, 2a-PEG (K(D) of 19 pM), was selected for in vivo studies. The pharmacokinetic and pharmacodynamic properties of 2a-PEG were dramatically improved relative to the unmodified compound. The PEGylated compound was efficacious in a rat model of experimental autoimmune encephalomyelitis at a 30-fold lower molar dose than the parent compound and required only a once-a-week dosing regimen compared with a daily treatment for BIO5192. Compound 2a-PEG was highly selective for alpha4beta1. These studies demonstrate the feasibility of PEGylation of alpha4beta1-targeted small molecules with retention of activity in vitro and in vivo. 2a-PEG, and related compounds, will be valuable reagents for assessing alpha4beta1 biology and may provide a new therapeutic approach to treatment of human inflammatory diseases.

T cell and antibody responses in remitting-relapsing experimental autoimmune encephalomyelitis in (C57BL/6 x SJL) F1 mice.

To characterize T cell and antibody responses in remitting-relapsing experimental autoimmune encephalomyelitis (RR-EAE), we compared myelin oligodendrocyte glycoprotein (MOG)-induced RR-EAE in C57BL/6 (B6) x SJL (F1) mice and chronic-progressive EAE (CP-EAE) in B6 mice at week 8 p.i. when clinical scores were comparable. Although these two strains exhibited similar inflammation/demyelination pattern and MOG-induced T cell responses, RR-EAE mice produced significantly higher levels of anti-MOG IgG1/IgG2a antibodies. Further, lymphocytes of RR-EAE mice proliferated vigorously to the secondary epitope myelin basic protein (MBP) 1-11. These results support a potential involvement of anti-MOG antibodies and epitope spreading in T cell responses in the development of MOG-induced RR-EAE model.

Protection against experimental autoimmune encephalomyelitis generated by a recombinant adenovirus vector expressing the V beta 8.2 TCR is disrupted by coadministration with vectors expressing either IL-4 or -10.

Adenovirus vectors are increasingly being used for genetic vaccination and may prove highly suitable for intervention in different pathological conditions due to their capacity to generate high level, transient gene expression. In this study, we report the use of a recombinant adenovirus vector to induce regulatory responses for the prevention of autoimmune diseases through transient expression of a TCR beta-chain. Immunization of B10.PL mice with a recombinant adenovirus expressing the TCR Vbeta8.2 chain (Ad5E1 mVbeta8.2), resulted in induction of regulatory type 1 CD4 T cells, directed against the framework region 3 determinant within the B5 peptide (aa 76-101) of the Vbeta8.2 chain. This determinant is readily processed and displayed in an I-A(u) context, on ambient APC. Transient genetic delivery of the TCR Vbeta8.2 chain protected mice from Ag-induced experimental autoimmune encephalomyelitis. However, when the Ad5E1 mVbeta8.2 vector was coadministered with either an IL-4- or IL-10-expressing vector, regulation was disrupted and disease was exacerbated. These results highlight the importance of the Th1-like cytokine requirement necessary for the generation and activity of effective regulatory T cells in this model of experimental autoimmune encephalomyelitis.

Inhibition of Rho GTPases with protein prenyltransferase inhibitors prevents leukocyte recruitment to the central nervous system and attenuates clinical signs of disease in an animal model of multiple sclerosis.

The ICAM-1-mediated brain endothelial cell (EC)-signaling pathway induced by adherent lymphocytes is a central element in facilitating lymphocyte migration through the tight endothelial barrier of the brain. Rho proteins, which must undergo posttranslational prenylation to be functionally active, have been shown to be an essential component of this signaling cascade. In this study, we have evaluated the effect of inhibiting protein prenylation in brain ECs on their ability to support T lymphocyte migration. ECs treated in vitro with protein prenylation inhibitors resulted in a significant reduction in transendothelial T lymphocyte migration. To determine the therapeutic potential of this approach, an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis, was induced in Biozzi ABH mice. Animals treated before disease onset with protein prenylation inhibitors exhibited a dramatic and significant reduction in both leukocyte infiltration into the CNS and clinical presentation of disease compared with untreated animals. These studies demonstrate, for the first time, the potential for pharmacologically targeting CNS EC signaling responses, and particularly endothelial Rho proteins, as a means of attenuating leukocyte recruitment to the CNS.

Experimental autoimmune encephalomyelitis (EAE).

The procedures described in this unit utilize murine models exclusively since murine EAE often results in a relapsing/remitting disease, similar to the early phase of most MS patients. EAE in the Lewis rat is a monophasic illness in which animals experience a single episode of paralysis from which most recover completely. This unit presents two methods for inducing EAE in mice: active induction and adoptive transfer.