BackgroundDemyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage.Methods/Principal FindingsTo monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage.ConclusionThe model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.

T regulatory cells type 1 (Tr1 cells) are excellent candidates for cell therapy in multiple sclerosis (MS). The aim of our study was to assess the functional state of Tr1 cells and IL-10R signaling in patients with MS. Tr1 cells were induced in vitro by activation with anti-CD46 antibodies in controls and patients with MS. Cells were phenotyped by cytometry and suppression assays, and the expression of cytokines and transcription factors was evaluated by real-time PCR, ELISA, cytometry and Western blotting. We found that the activity of Tr1 cells and IL-10R signaling is impaired in MS patients since Tr1 cells isolated from MS patients produced less IL-10 than those obtained from controls. Indeed, the supernatants from Tr1 cells from controls did not suppress the proliferation of stimulated CD4 + cells from patients with MS. Furthermore, the IL-10R signaling pathway was not fully active in CD4 + cells from MS patients and these cells had higher baseline levels of SOCS3 transcripts than controls. Indeed, after in vitro IL-10 stimulation, the expression levels of the STAT1, STAT3 and IL-10RA genes were higher in MS patients than in controls. Moreover, Stat-3 phosphorylation was lower in controls than in patients after IL-10 stimulation. These results indicate that IL-10 regulatory function is impaired in patients with MS.

Background: Methylthioadenosine (MTA) is a natural metabolite with immunomodulatory properties. MTA improves the clinical course and pathology of the animal model of multiple sclerosis, even when therapy is started after disease onset. Objective: Our aim was to compare the efficacy of MTA in ameliorating experimental autoimmune encephalomyelitis (EAE) compared with first line approved therapies, to develop an oral formulation of MTA and to assess its pharmacokinetic profile. Methods: EAE was induced in C57BL/6 mice by immunization with MOG35-55 peptide in Freund’s Adjuvant. Animals were treated with MTA, interferon-beta or Glatiramer acetate starting the day of immunization and the clinical score was collected blind. Pharmacokinetic studies were performed in Sprague Dawley rats by administering MTA by intraperitoneal injection and orally, and collecting blood at different intervals. MTA levels were measured by high-performance liquid chromatography. Results: We found that MTA ameliorated EAE in a dose—response manner. Moreover, the highest dose of MTA (60 mg/kg) was more efficacious than mouse interferon-beta or Glatiramer acetate. We developed a salt of MTA for oral administration, with similar dose—response effect in the EAE model. Combination therapy assays between MTA and interferon-beta or Glatiramer acetate were more effective than the individual therapies. Finally, oral MTA half-life was 20 min, with a Cmax of 80 mg/L and without signs of obvious toxicity (animal death, behavioural changes, liver enzymes). Conclusions: In the EAE model MTA is more efficacious than first line therapies for multiple sclerosis, with a dose— response effect and higher efficacy when combined with interferon-beta or Glatiramer acetate. Oral MTA was also effective in the animal model of multiple sclerosis.

Background5′-deoxy-5′-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo.MethodsNeuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy.ResultsMTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region.ConclusionMTA may potentially offer therapeutic neuroprotection.

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