Acamprosate modulates experimental autoimmune encephalomyelitis.

OBJECTIVE: This pilot study aimed to determine the efficacy of acamprosate (N-acetyl homotaurine) in reducing the pathological features of experimental autoimmune encephalomyelitis (EAE) which is an animal model for multiple sclerosis (MS). BACKGROUND: The amino acid taurine has multiple biological activities including immunomodulation and neuromodulation. The synthetic acetylated taurine derivative, acamprosate, which crosses the blood-brain barrier more readily compared to taurine, is currently being used for the prevention of alcohol withdrawal symptoms associated with enhanced glutamatergic receptor function and GABA receptor hypofunction. METHODS: EAE was induced in C57BL/6 female mice with myelin oligodendrocyte glyocoprotein, amino acid 35-55. Mice were treated with 20, 100 and 500 mg/kg acamprosate for 21 days. RESULTS: Neurological scores at disease peak were reduced by 21, 64 and 9% in the 20, 100 and 500 mg/kg groups, respectively. Neurological improvement in the 100 mg/kg group correlated with a reduction in numbers of inflammatory lesions and the extent of CNS demyelination. Blood TNF-α levels were significantly reduced in the 500 mg/kg group. DISCUSSION: Acamprosate and other taurine analogs have a potential for future MS therapy.

Temporal expression of inflammatory mediators in brain basilar artery vasculitis and cerebrospinal fluid of rabbits with coccidioidal meningitis.

Strokes due to transmural vasculitis associated with coccidioidal meningitis result in significant morbidity and mortality. The immunological and inflammatory processes responsible are poorly understood. To determine the inflammatory mediators, i.e. cytokines, chemokines, iNOS, matrix metalloproteinase-9 (MMP-9), that possibly contribute to vasculitis, temporal mRNA expression in brain basilar artery samples and MMP-9 protein in the CSF of male NZW rabbits infected intracisternally with 6.5 x 10(4) arthroconidia of Coccidioides immitis were assessed. Five infected and 3 sham-injected rabbits at each time point were euthanized 4, 9, 14 and 20 days post infection. All infected rabbits had neurological abnormalities and severe vasculitis in the basilar arteries on days 9-20. In basilar arteries of infected animals versus controls, mRNAs encoding for IL-6, iNOS, IFN-gamma, IL-2, MCP-1, IL-1beta, IL-10, TNF-alpha, CCR-1, MMP-9, TGF-beta, as well as MMP-9 protein in CSF, were found to be significantly up-regulated. Thus, this study identified inflammatory mediators associated with CNS vasculitis and meningitis due to C. immitis infection. Assessment of the individual contribution of each mediator to vasculitis may offer novel approaches to the treatment of coccidioidal CNS infection. This study also provides unique methodology for immunology studies in a rabbit model.

Mild hypothermia increases Bcl-2 protein expression following global cerebral ischemia.

Mild hypothermia protects the brain against experimental ischemia, but the reasons are not well known. We examined whether the protective effects of mild hypothermia could be correlated with alterations in expression of Bcl-2, an anti-apoptotic protein in a rat model of transient global ischemia. Following 10 min of forebrain ischemia, hippocampal neurons were examined 72 h later for survival, expression of Bcl-2 family proteins and apoptosis. Intraischemic mild hypothermia was applied for 3 h (33 degrees C, isch-33) or normal body temperature was maintained (37 degrees C, isch-37). Survival of CA1 neurons was significantly improved in the isch-33 group compared to the isch-37 group (90 vs. 53% survival; P<0.01). The proportion of Bcl-2-positive cells among surviving CA1 neurons in the isch-33 group was increased compared to that of sham and isch-37 groups (P<0.01). Bax expression in CA1 was no different between sham and isch-33 groups, but was significantly decreased in isch-37 (P<0.05). TUNEL staining was positive in many isch-37 CA1 neurons, but absent in isch-33. Utilizing electron microscopy, more cells meeting criteria for apoptosis were observed in the isch-37 than isch-33. These data suggest that mild hypothermia attenuates apoptotic death, and that this protection may be related to increases in Bcl-2.

Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity.

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

The extracellular matrix in multiple sclerosis: an update

Extracellular matrix (ECM) molecules play important roles in the pathobiology of the major human central nervous system (CNS) inflammatory/demyelinating disease multiple sclerosis (MS). This mini-review highlights some recent work on CNS endothelial cell interactions with vascular basement membrane ECM as part of the cellular immune response, and roles for white matter ECM molecules in demyelination and remyelination in MS lesions. Recent basic and clinical investigations of MS emphasize axonal injury, not only in chronic MS plaques, but also in acute lesions; progressive axonal degeneration in normal-appearing white matter also may contribute to brain and spinal cord atrophy in MS patients. Remodeling of the interstitial white matter ECM molecules that affect axon regeneration, however, is incompletely characterized. Our ongoing immunohistochemical studies demonstrate enhanced ECM versican, a neurite and axon growth-inhibiting white matter ECM proteoglycan, and dermatan sulfate proteoglycans at the edges of inflammatory MS lesions. This suggests that enhanced proteoglycan deposition in the ECM and axonal growth inhibition may occur early and are involved in expansion of active lesions. Decreased ECM proteoglycans and their phagocytosis by macrophages along with myelin in plaque centers imply that there is "injury" to the ECM itself. These results indicate that white matter ECM proteoglycan alterations are integral to MS pathology at all disease stages and that they contribute to a CNS ECM that is inhospitable to axon regrowth/regeneration

Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the size, reactivity, and function of a primed pool of CD4+ T cells.

We examined how cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates heterogeneous CD4(+) T cell responses by using experimental autoimmune encephalomyelitis (EAE), a CD4(+) T cell-mediated disease that is subject to regulation by CTLA-4. Disease incidence and severity were used as measures of in vivo CD4(+) T cell responses. The frequency, cytokine production, and reactivity of primed T cells were determined from animals immunized with proteolipid protein (PLP)-139-151 (disease agonist), PLP-Q (disease antagonist), or both peptides, and treated with control or anti-CTLA-4 antibody to analyze the responding population. CTLA-4 blockade exacerbated disease in PLP-139-151-primed animals and overcame disease antagonism in coimmunized animals, but did not permit disease induction in PLP-Q-primed animals. Experimental autoimmune encephalomyelitis enhancement was associated with increased frequencies of cytokine-producing cells and increased ratios of IFN-gamma to IL-4 secretors responsive to PLP-139-151. Priming with PLP-Q elicited IL-4 and IL-2, but not IFN-gamma secretors cross-reactive with PLP-139-151. Strikingly, CTLA-4 blockade was found to decrease rather than increase the frequencies of cross-reactive IL-4 and IL-2 secretors. Thus, CTLA-4 engagement limits the size, but increases the breadth, of reactivity of a primed pool of CD4(+) T cells, consequently regulating its function.

Endothelial cell class II major histocompatibility complex molecule expression in stereotactic brain biopsies of patients with acute inflammatory/demyelinating conditions.

To determine if central nervous system (CNS) microvessel endothelial cells express class II major histocompatibility complex (MHC) molecules in early demyelinating lesions in humans, cerebral white matter (WM) biopsies from patients with acute inflammatory/demyelinating conditions, including 4 with multiple sclerosis (MS), were immunostained for class II MHC and other antigens. Eight of 9 biopsies showed focal MHC class II-positive endothelial cells; there were none in the CNS of 1 of the MS patients at autopsy. There were more vessels with class II-positive endothelial cells in areas with intact WM and gliosis than in areas with active demyelination or control WM; class II-positive endothelial cells in small venules and capillaries were adjacent to transmigrating and perivascular CD4-positive cells. By immunoelectron microscopy, class II molecules were localized to vesicles in endothelial cell cytoplasm, suggesting the potential for antigen processing. Perivascular cells, parenchymal microglia, mononuclear cells and the perinuclear cytoplasm but not the processes of astrocytes were also class II-positive. These data indicate that in acute CNS inflammatory/demyelinating lesions, endothelial cells focally and apparently transiently express class II MHC molecules. This expression implies potential antigen-specific interactions, immunoregulatory or signalling functions in endothelial cells, or it may render them susceptible to CD4-positive cell-mediated cytotoxicity. Thus, class II-positive endothelial cells may have pivotal immunologic roles in initial stages of T cell responses in human CNS WM, particularly in acute MS lesions.

Tobacco and alcohol use in G-rated children's animated films.

CONTEXT: Tobacco and alcohol use among youth are major public health problems, but the extent to which children are routinely exposed to tobacco and alcohol products in children's films is unknown. OBJECTIVE: To identify the prevalence and characteristics associated with tobacco and alcohol use portrayed in G-rated, animated feature films. Design All G-rated, animated feature films released between 1937 and 1997 by 5 major production companies (Walt Disney Co, MGM/United Artists, Warner Brothers Studios, Universal Studios, and 20th Century Fox) that were available on videotape were reviewed for episodes of tobacco and alcohol use. MAIN OUTCOME MEASURES: Presence of tobacco and alcohol use in each film, type of tobacco or alcohol used, duration of use, type of character using substance (bad, neutral, or good), and any associated effects. RESULTS: Of 50 films reviewed, 34 (68%) displayed at least 1 episode of tobacco or alcohol use. Twenty-eight (56%) portrayed 1 or more incidences of tobacco use, including all 7 films released in 1996 and 1997. Twenty-five films (50%) included alcohol use. Smoking was portrayed on screen by 76 characters for more than 45 minutes in duration; alcohol use was portrayed by 63 characters for 27 minutes. Good characters use tobacco and alcohol as frequently as bad characters. Cigars and wine are shown in these films more often than other tobacco or alcohol substances. CONCLUSIONS: More than two thirds of animated children's films feature tobacco or alcohol use in story plots without clear verbal messages of any negative long-term health effects associated with use of either substance.

IL-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic mice.

Experimental autoimmune encephalomyelitis (EAE) and other organ-specific autoimmune diseases are induced by autoantigen-specific Th1 cells. In contrast, transfer of autoantigen-reactive Th2 cells that produce IL-4 and IL-10 can prevent and/or reverse EAE. The relative roles of these two Th2 cytokines in the regulation of EAE has not been evaluated. Utilizing IL-4 and IL-10 knockout mice deficient for these cytokines and IL-10 and IL-4 transgenic mice overexpressing these cytokines, we demonstrate that IL-10-deficient mice (IL-10(-/-)) are more susceptible and develop a more severe EAE when compared with IL-4-deficient mice (IL-4(-/-)) or wild-type mice. T cells from IL-10(-/-) mice exhibit a stronger Ag-specific proliferation, produce more proinflammatory cytokines (IFN-gamma and TNF-alpha) when stimulated with an encephalitogenic peptide, and induce very severe EAE upon transfer into wild-type mice. In contrast, while IL-4 transgenic mice develop similar disease compared with their nontransgenic littermates, mice transgenic for IL-10 are completely resistant to the development of EAE. Taken together, our data suggest that IL-10 plays a more critical role in the regulation of EAE by regulating autopathogenic Th1 responses.

Altered peptide ligand modulation of experimental allergic encephalomyelitis: immune responses within the CNS.

An altered peptide ligand (analog) of the encephalitogenic epitope of proteolipid protein residues 139-151 (p139-151) in which residues 144 and 147 are substituted with leucine and arginine, respectively (LR), protects from clinical but not histological experimental allergic encephalomyelitis (EAE). To understand in situ events associated with this protection, T cells from brains of mice immunized with either native p139-151, the analog LR or a combination of the two were isolated and characterized. High proportions of cells from co-immunized mice (38%) and LR-immunized mice (58%) reacted to both p139-151 and LR, whereas fewer cells from p139-151 immunized mice (7%) were cross-reactive. T cell clones derived from brains of LR- and co-immunized mice were also cross-reactive in vitro. By reverse transcriptase-based polymerase chain reaction, higher levels of TGF-beta mRNA, and lower levels of TNF-alpha and IFN-gamma mRNA were found in the central nervous system (CNS) tissue of LR and co-immunized mice. Immunohistochemistry demonstrated greater TGF-beta immunoreactivity in CNS inflammatory foci in co-immunized and LR-immunized mice. There were no significant differences in CD4+ or CD8+ cell infiltrates among the groups and differences in other cytokines were not identified by immunocytochemistry. Protection from clinical EAE in LR and co-immunized mice was partially abolished by anti-TGF-beta antibody treatment. Thus, protection from clinical disease following immunization with the analog LR is associated with infiltration into the CNS of a T cell population that could potentially recognize the native PLP peptide and with enhanced TGF-beta production by cells within CNS inflammatory foci.

Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease inducible in susceptible animals by myelin Ag-specific CD4+ Th1 cells. The mechanisms by which these cells induce inflammation and demyelination in the central nervous system (CNS) are incompletely understood. To determine the roles of Fas and FasL in the involvement of CNS autoimmune injury, we determined susceptibility to EAE of Fas-or FasL-deficient mice. Compared with wild-type mice, mice expressing lpr (Fas) and gld (FasL) mutations were relatively resistant to the development of clinical EAE, and this correlated with fewer inflammatory infiltrates and cells undergoing apoptosis in the CNS of the mutant mice. The gld and lpr mice, however, developed significant T cell responses with production of Th1 cytokines in response to the encephalitogenic myelin peptide. These results suggest that the Fas/FasL pathway plays a critical role in the development of EAE probably by mediating apoptosis within the target tissue.

Specific blockade of CTLA-4/B7 interactions results in exacerbated clinical and histologic disease in an actively-induced model of experimental allergic encephalomyelitis.

In addition to an antigen-specific signal, T cell activation requires an antigen-independent costimulatory signal provided by interaction of CD28 with B7 (CD80 and CD86) on the APC. By blocking B7 interactions, previous studies demonstrated the requirement for costimulation in the induction of experimental allergic encephalomyelitis (EAE). Recent studies suggest that unlike CD28, CTLA-4 (a second B7 ligand) delivers an inhibitory signal. To address the regulatory role of CTLA-4 in EAE, we used an antibody directed against CTLA-4 administered at the time of disease induction. This resulted in a significantly more severe clinical course and more inflammatory and demyelinating lesions in the CNS of anti-CTLA-4-treated mice. These data suggest that CTLA-4-mediated inhibitory signals can regulate the clinical severity and histologic parameters of neuroautoimmune disease.

Inhibition of nitric oxide synthase for treatment of experimental autoimmune encephalomyelitis.

Aminoguanidine (AG), a selective inhibitor of inducible nitric oxide synthase, prevented the clinical development of experimental autoimmune encephalomyelitis (EAE) with a reduction in inflammation and demyelination. Administration of AG reduced the expression of nitrosotyrosine in inflammatory lesions in the central nervous system. Cytokine expression, determined by semiquantitative PCR, revealed increased expression of IFN-gamma, IL-10, and TGF-beta, which was associated with protection from EAE, and reduced TNF-alpha, associated with the development of EAE. Furthermore, AG blocked the secretion of nitric oxide, TNF-alpha, and PGE2 in astrocyte cultures. AG did not influence the proliferation response of T cells to a pathogenic epitope of myelin basic protein. Down-regulation of nitric oxide by AG has widespread consequences for cytokine production in central nervous system inflammation and prevents EAE.

Immunogenic and encephalitogenic epitope clusters of myelin proteolipid protein.

To understand and develop strategies to intervene in autoimmune responses to myelin proteolipid protein (PLP), encephalitogenic epitopes must be identified. To expedite the identification of potentially immunogenic and encephalitogenic epitopes of PLP, overlapping synthetic 20-mer PLP peptides covering the whole PLP molecule were screened for their ability to bind to purified mouse I-Ad, I-Ak, and I-As molecules. The peptides that bound to the I-A molecules were tested for their ability to induce immune responses in corresponding inbred mouse strains. Immunogenic peptides were then tested for their ability to induce experimental autoimmune encephalomyelitis. Moderate to strong I-A binding was essential for development of immune responses, but immunogenicity was not sufficient for encephalitogenicity. Rather, encephalitogenic epitopes clustered in three regions of the molecule, namely within residues 40-70, 100-119, and 178-209. These were also the regions of the PLP that showed the greatest promiscuity in binding to I-A molecules. Except for PLP 139-151, which is an encephalitogenic determinant in mice expressing I-As, all encephalitogenic epitopes of PLP previously identified, regardless of their MHC class II restriction, are located within or adjacent to these epitope clusters. None of the encephalitogenic epitopes occur in regions of the molecule that have a high degree of homology with the neuronal M6a protein, a member of the DM20/PLP superfamily. Atypical clinical and histologic patterns of experimental autoimmune encephalomyelitis were observed in some strains of mice sensitized with certain PLP peptides and may reflect induction of T cells with different disease-inducing potentials.

An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, we have previously shown that the disease is mediated by Th1 cells, which recognize tryptophan 144 as the primary TCR contact point. Here we describe an altered peptide ligand (APL), generated by a single amino acid substitution (tryptophan to glutamine) at position 144 (Q144), which inhibits the development of EAE induced with the native PLP 139-151 peptide (W144). We show that the APL induces T cells that are cross-reactive with the native peptide and that these cells produce Th2 (IL-4 and IL-10) and Th0 (IFN gamma and IL-10) cytokines. Adoptive transfer of T cell lines generated with the APL confer protection from EAE. These data show that changing a single amino acid in an antigenic peptide can significantly influence T cell differentiation and suggest that immune deviation may be one of the mechanisms by which APLs can inhibit an autoimmune disease.

B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy.

CD4 T helper precursor cells mature along two alternative pathways, Th1 and Th2. Here we show that these pathways are differentially activated by two costimulatory molecules, B7-1 and B7-2. Using anti-B7 antibodies, this developmental step was manipulated both in vitro and in vivo in experimental allergic encephalomyelitis (EAE). Anti-B7-1 reduced the incidence of disease while anti-B7-2 increased disease severity. Neither antibody affected overall T cell induction but rather altered cytokine profile. Administration of anti-B7-1 at immunization resulted in predominant generation of Th2 clones whose transfer both prevented induction of EAE and abrogated established disease. Since co-treatment with anti-IL-4 antibody prevented disease amelioration, costimulatory molecules may directly affect initial cytokine secretion. Thus, interaction of B7-1 and B7-2 with shared counterreceptors CD28 and CTLA-4 results in very different outcomes in clinical disease by influencing commitment of precursors to a Th1 or Th2 lineage.

A single TCR antagonist peptide inhibits experimental allergic encephalomyelitis mediated by a diverse T cell repertoire.

Previously, six T cell clones, which are specific for an encephalitogenic determinant of myelin proteolipid protein (PLP) peptide residues 139 to 151 (HSLGKWLGHPDKF), were derived from SJL mice and shown to use diverse TCR genes. To design TCR antagonist peptides that could interfere with the activation of these clones in vitro and inhibit experimental allergic encephalomyelitis (EAE) in vivo, we first determined the TCR and MHC contact residues of the encephalitogenic peptide. The analysis indicated that residues 144 (tryptophan) and 147 (histidine) were the TCR binding sites and that residues 145 (leucine) and 148 (proline) were important for MHC class II (IAs) binding. On the basis of this information, a peptide analogue (leucine 144/arginine 147), in which both of the major TCR contact residues were substituted, was synthesized. This analogue acts as a TCR antagonist for the panel of PLP 139-151-specific T cell clones, does not cause EAE by itself, blocks the induction of disease by the native 139-151 peptide, and prevents clinical disease progression if administered at the first signs of disease. Thus, although multiple TCR genes are used by PLP 139-151-specific clones, a single peptide analogue can interfere with the disease process. This approach should be feasible for designing peptide analogues that can be tested for therapeutic efficacy in human autoimmune diseases in which the pathogenic Ags are known and TCR use is diverse.