Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.     

Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis

Chronic progression of two T cell-mediated central nervous system (CNS) demyelinating models of multiple sclerosis, relapsing EAE (R-EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is dependent on the activation of T cells to endogenous myelin epitopes (epitope spreading). Using transfer of carboxyfluorescein succinyl ester (CFSE)-labeled T-cell receptor (TCR)-transgenic T cells and mixed bone marrow chimeras, we show that activation of naive proteolipid protein (PLP)139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE or TMEV-IDD occurs directly in the CNS and not in the cervical lymph nodes or other peripheral lymphoid organs. Examination of the antigen-presentation capacity of antigen-presenting cell (APC) populations purified from the CNS of mice with PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi dendritic cells (DCs) efficiently present endogenous antigen to activate naive PLP139-151-specific T cells in vitro. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia activate a PLP139-151-specific helper T cell line. The data suggest that naive T cells enter the inflamed CNS and are activated by local APCs, possibly DCs, to initiate epitope spreading.     

Remyelination induced by a DNA aptamer in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler's encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler's virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.     

Effects of progesterone in the spinal cord of a mouse model of multiple sclerosis

The spinal cord is a target of progesterone (PROG), as demonstrated by the expression of intracellular and membrane PROG receptors and by its myelinating and neuroprotective effects in trauma and neurodegeneration. Here we studied PROG effects in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis characterized by demyelination and immune cell infiltration in the spinal cord. Female C57BL/6 mice were immunized with a myelin oligodendrocyte glycoprotein peptide (MOG_40–54). One week before EAE induction, mice received single pellets of PROG weighing either 20 or 100 mg or remained free of steroid treatment. On average, mice developed clinical signs of EAE 9–10 days following MOG administration. The spinal cord white matter of EAE mice showed inflammatory cell infiltration and circumscribed demyelinating areas, demonstrated by reductions of luxol fast blue (LFB) staining, myelin basic protein (MBP) and proteolipid protein (PLP) immunoreactivity (IR) and PLP mRNA expression. In motoneurons, EAE reduced the expression of the alpha 3 subunit of Na,K-ATPase mRNA. In contrast, EAE mice receiving PROG showed less inflammatory cell infiltration, recovery of myelin proteins and normal grain density of neuronal Na,K-ATPase mRNA. Clinically, PROG produced a moderate delay of disease onset and reduced the clinical scores. Thus, PROG attenuated disease severity, and reduced the inflammatory response and the occurrence of demyelination in the spinal cord during the acute phase of EAE.     

Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis

Immunization with myelin antigens leads to the development of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. The disease can also be induced by the transfer of encephalitogenic CD4+ T helper (T(H)) lymphocytes into naive mice. These T cells need to re-encounter their cognate antigen in the context of major histocompatibility complex (MHC) class II-bearing antigen-presenting cells (APCs) in order to recognize their target. The cell type and location of the APC mediating T-cell entry into the central nervous system (CNS) remain unknown. Here, we show that APCs of the lymphoreticular system and of the CNS parenchyma are dispensable for the immune invasion of the CNS. We also describe that a discrete population of vessel-associated dendritic cells (DCs) is present in human brain tissue. In mice, CD11c+ DCs alone are sufficient to present antigen in vivo to primed myelin-reactive T cells in order to mediate CNS inflammation and clinical disease development.     

Glutamate excitotoxicity in a model of multiple sclerosis

Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.     

Axon damage and repair in multiple sclerosis

It is well known that within long-standing multiple sclerosis (MS) lesions there is axonal loss but whether it is an early or late event has been more difficult to establish. The use of immunocytochemical methods that reveal axonal end-bulbs is a valuable approach to investigating acute axonal injury in human pathological material. The application of these techniques to multiple sclerosis tissue reveals evidence of axonal injury in acute lesions; the distribution of the end-bulbs in acute and active-chronic lesions is associated with regions of maximal density of infiltrating macrophages. Axon injury within the MS lesion will result in both Wallerian degeneration of the axon and also retrograde degeneration of the cell body. The functional consequences of the axon injury will depend upon numbers of axons injured and the topographical organization of the fibres coursing through the lesion. The molecular mechanisms by which the recruited leucocytes damage or transect the axons are not known. However, investigations in the Wld mutant mouse with very slow Wallerian degeneration demonstrate that axon degeneration is not simply a passive disintegration of the axon but has clear parallels with the active processes of programmed cell death. The presence of early axon injury and the consequences of an ever increasing load of neuronal damage has important implications not only for when therapy should be initiated in MS but also the therapeutic target.     

Co-expression of multiple transgenes in mouse CNS: a comparison of strategies

The introduction of two transgenes into one animal is increasingly common as transgenic experiments become more sophisticated. In this study we examine two strategies for creating double transgenic founders from a single microinjection. In the first approach, two constructs, each with its own promoter element, were coinjected into the pronucleus. In the second approach, both transgenes were cloned into one vector, separated by an internal ribosomal entry site (IRES), and placed under control of a single promoter. Both strategies save time and increase the percentage of double transgenic offspring over the standard method of mating single transgenic lines. However, despite high transgene copy numbers, the bicistronic lines did not show robust expression of either protein. Copy number and protein expression correlated much better in the coinjected lines, with expression levels in one line approaching that observed in some of our best single transgenic controls. Thus we recommend coinjection of individual plasmids for the generation of multiply transgenic founders.     

Microsomal prostaglandin E synthase-1 aggravates inflammation and demyelination in a mouse model of multiple sclerosis

Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme required for prostaglandin E₂ (PGE₂) biosynthesis. In this study, we examined the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We induced EAE with myelin oligodendrocyte glycoprotein_35–55 peptide in mPGES-1-deficient (mPGES-1^−/−) and wild-type (WT) mice. First, we examined the histopathology in the early and late phases of EAE progression. Next, we measured the concentration of PGE₂ in the spinal cord and investigated the expression of mPGES-1 using immunohistochemistry. In addition, we examined the progression of the severity of EAE using an EAE score to investigate a correlation between pathological features and paralysis. In this paper, we demonstrate that WT mice showed extensive inflammation and demyelination, whereas mPGES-1^−/− mice exhibited significantly smaller and more localized changes in the perivascular area. The mPGES-1 protein was induced in vascular endothelial cells and microglia around inflammatory foci, and PGE₂ production was increased in WT mice but not mPGES-1^−/− mice. Furthermore, mPGES-1^−/− mice showed a significant reduction in the maximum EAE score and improved locomotor activity. These results suggest that central PGE₂ derived from non-neuronal mPGES-1 aggravates the disruption of the vessel structure, leading to the spread of inflammation and local demyelination in the spinal cord, which corresponds to the symptoms of EAE. The inhibition of mPGES-1 may be useful for the treatment of human MS.     

Cerebrospinal fluid levels of immunoreactive substance P and somatostatin in patients with multiple sclerosis and inflammatory CNS disease.

Cerebrospinal fluid (CSF) levels of substance-P like immunoreactivity (SPLI) and somatostatin-like immunoreactivity (SLI) were measured in 43 patients with multiple sclerosis (MS), differentiated according to course and activity of the disease, in 23 patients with inflammatory disease of known bacterial or viral etiology and in 16 control patients using specific radioimmunoassay. SPLI and SLI levels were not significantly different from controls in MS patients whereas SLI was significantly increased in patients with infectious disease of central nervous system and/or subarachnoidal space. It is assumed that CSF SPLI and SLI cannot serve as a diagnostic or prognostic indicator of disease state in multiple sclerosis. Analysis of immunoreactivity by reverse phase HPLC-RIA revealed marked molecular heterogeneity of both neuropeptides.     

VCAM-1-targeted magnetic resonance imaging reveals subclinical disease in a mouse model of multiple sclerosis

Diagnosis of multiple sclerosis (MS) currently requires lesion identification by gadolinium (Gd)-enhanced or T(2)-weighted magnetic resonance imaging (MRI). However, these methods only identify late-stage pathology associated with blood-brain barrier breakdown. There is a growing belief that more widespread, but currently undetectable, pathology is present in the MS brain. We have previously demonstrated that an anti-VCAM-1 antibody conjugated to microparticles of iron oxide (VCAM-MPIO) enables in vivo detection of VCAM-1 by MRI. Here, in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, we have shown that presymptomatic lesions can be quantified using VCAM-MPIO when they are undetectable by Gd-enhancing MRI. Moreover, in symptomatic animals VCAM-MPIO binding was present in all regions showing Gd-DTPA enhancement and also in areas of no Gd-DTPA enhancement, which were confirmed histologically to be regions of leukocyte infiltration. VCAM-MPIO binding correlated significantly with increasing disability. Negligible MPIO-induced contrast was found in either EAE animals injected with an equivalent nontargeted contrast agent (IgG-MPIO) or in control animals injected with the VCAM-MPIO. These findings describe a highly sensitive molecular imaging tool that may enable detection of currently invisible pathology in MS, thus accelerating diagnosis, guiding treatment, and enabling quantitative disease assessment.     

In Vivo imaging reveals distinct inflammatory activity of CNS microglia versus PNS macrophages in a mouse model for ALS

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1(G93A) mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ～180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.     

Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis

Human amniotic epithelial cells (hAEC) have stem cell-like features and immunomodulatory properties. Here we show that hAEC significantly suppressed splenocyte proliferation in vitro and potently attenuated a mouse model of multiple sclerosis (MS). Central nervous system (CNS) CD3(+) T cell and F4/80(+) monocyte/macrophage infiltration and demyelination were significantly reduced with hAEC treatment. Besides the known secretion of prostaglandin E2 (PGE2), we report the novel finding that hAEC utilize transforming growth factor-β (TGF-β) for immunosuppression. Neutralization of TGF-β or PGE2 in splenocyte proliferation assays significantly reduced hAEC-induced suppression. Splenocytes from hAEC-treated mice showed a Th2 cytokine shift with significantly elevated IL-5 production. While transferred CFSE-labeled hAEC could be detected in the lung, none were identified in the CNS or in lymphoid organs. This is the first report documenting the therapeutic effect of hAEC in a MS-like model and suggest that hAEC may have potential for use as therapy for MS.     

Endocannabinoids control spasticity in a multiple sclerosis model.

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.     

The role of deimination in ATP5b mRNA transport in a transgenic mouse model of multiple sclerosis

Deimination refers to conversion of protein-bound arginine into citrulline. An mRNA carrier, RNA binding export factor (REF), present on mitochondria undergoes loss of deimination with impaired ATP5b mRNA transport in ND4 mice (model of multiple sclerosis) compared with the controls. We present evidence of (1) reduced ATP5b mRNA binding strength of non-deiminated REF compared with deiminated REF, (2) impaired ATP5b mRNA transport in ND4 mice and (3) reduced mitochondrial ATP synthase activity on inhibition of deimination in PC12 cells. Impaired deimination of REF and defect in mitochondrial mRNA transport are critical factors in mitochondrial dysfunction in ND4 mice.     

Nature of monocyclic beta-lactam antibiotic Nocardicin A to beta-lactamases

Nocardicin A is the antibiotic which was first found to possess a monocyclic beta-lactam ring. This antibiotic was inactivated by the cleavage of its beta-lactam ring. The direct spectrophotometric assay was applied to measure the rate of enzymatic hydrolysis of Nocardicin A. Nocardicin A was highly stable to both chromosomal and plasmid-mediated beta-lactamases. Of the nine beta-lactam antibiotics including cefoxitin and cefuroxime, Nocardicin A was the most stable to the beta-lactamases tested excluding those from Klebsiella oxytoca and Proteus vulgaris. The latter broad-spectrum beta-lactamases hydrolyzed Nocardicin A rather intensively. Extreme stability of Nocardicin A to beta-lactamases was suggested to be due to the combination of its low affinity to the enzymes and stabilization of its monocyclic beta-lactam ring. Nocardicin A was shown to have inducing ability toward beta-lactamases.     

ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia

Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.     

Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis

Background

Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ).

Methodology/Principal Findings

The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS.

Conclusion/Significance

Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.     

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.