New insights into the nature of the Cibacron brilliant red 3B-A – Chitosan interaction

Cibacron brilliant red 3B-A (CBR) has been introduced to determine chitosan (CS) concentrations in solution, and several studies applied it to measure chitosan content in pharmaceutical formulations. So far, studies have relied on the absorbance band shift to 570 nm to determine the extent of the CBR – CS interaction. In this study, we show that CBR forms micro- to nanometer sized aggregates with CS, depending on their charge ratio and that other photophysical changes in CBR are induced by this interaction. We found that, besides the bathochromic band shift, aggregation induces emission at 600 nm and emission quenching at 360 nm. We compared changes CS induced in absorbance and fluorescence emission of CBR with the CS monomer glucosamine and poly(allylamine) hydrochloride, which both contain amino groups, and found that similar but less intense photophysical changes also occur. Furthermore, CS-induced circular dichroism in CBR suggests a twisted, chiral structure of these aggregates that should match with the previously published in silico simulations of the structure of CS in solution. The low linear charge density of CS and its chiral conformation are considered responsible for the enhanced photophysical response of CBR interacting with the polycation

Mechanisms of action of Methylthioadenosine: pathways implicated in neuroprotection in models of Multiple Sclerosis and other neurological diseases

From 5th European Workshop on Immune-Mediated Inflammatory Diseases (Sitges-Barcelona, Spain. 1-3 December 2010)Background Methylthioadenosine (MTA) has anti-oxidant and anti-proliferative properties and was shown to induce cell protection in hepatic cells. We previously demonstrated that exert immunomodulatory and neuroprotective effects in the animal model of Multiple Sclerosis (MS) and other neurological diseases like Parkinson disease, stroke and Epilepsy. Objective To study the mechanisms of action and different pathways implicated in the neuroprotective effect of MTA in neurological diseases. Methods RN22 (Schwnoma cell line) and PC12 (Pheochromocytoma cell line) were used to test the neuroprotective activity of MTA against stress in RN22 and to differentiate neurites in PC12. BV2 cells were used to test the effect of MTA in microglia. Organotypic cerebellum cultures were used to determine MTA effect in demyelination/remyelination. Luminex technology, western blot and ELISA were used in order to study the phosphorylated state of different pathways (AkT/PKB, ERK/MAPK, P38/SAPK or STAT3) and to determine the amount of different cytokines (IL-1β and TNF-α). Ros determination was also done by fluorescence determination. Results In vitro studies revealed that MTA protection against different stresses and its capacity to differentiate neurites implies pathways like ERK/MAPK, P38/SAPK or STAT3. MTA neuroprotective capacity is also related with its ability to reduce ROS production and oxidative stress. MTA was shown to protect against demyelination in cerebellum organotypic cultures treated with LPS or Lysolecithin. Conclusions MTA is neuroprotective in models of MS, Parkinson disease, stroke or Epilepsy. This neuroprotective effect depends on its capacity to protect against demyelination, its anti-oxidant effect and the activation of pathways related with protection against stress and production of neurite differentiation

An easy synthetic way to exfoliate and stabilize MWCNTs in a thermoplastic pyrrole-containing matrix assisted by hydrogen bonds

This work focuses on the design of an engineered thermoplastic polymer containing pyrrole units in the main chain and hydroxyl pendant groups (A-PPy-OH), which help in achieving nanocomposites containing well-distributed, exfoliated and undamaged MWCNTs. The thermal annealing at 100 °C of the pristine nanocomposite promotes the redistribution of the nanotubes in terms of a percolative network, thus converting the insulating material in a conducting soft matrix (60 μΩ m). This network remains unaltered after cooling to r.t. and successive heating cycles up to 100 °C thanks to the effective stabilization of MWCNTs provided by the functional polymer matrix. Notably, the resistivity-temperature profile is very reproducible and with a negative temperature coefficient of -0.002 K-1, which suggests the potential application of the composite as a temperature sensor. Overall, the industrial scale by which A-PPy-OH can be produced offers a straightforward alternative for the scale-up production of suitable polymers to generate multifunctional nanocomposites

Passive experimental autoimmune encephalomyelitis in C57BL/6 with MOG: evidence of involvement of B cells

Experimental autoimmune encephalomyelitis (EAE) is the most relevant animal model to study demyelinating diseases such as multiple sclerosis. EAE can be induced by active (active EAE) or passive (at-EAE) transfer of activated T cells in several species and strains of rodents. However, histological features of at-EAE model in C57BL/6 are poorly described. The aim of this study was to characterize the neuroinflammatory and neurodegenerative responses of at-EAE in C57BL/6 mice by histological techniques and compare them with that observed in the active EAE model. To develop the at-EAE, splenocytes from active EAE female mice were harvested and cultured in presence of MOG 35-55 and IL-12, and then injected intraperitoneally in recipient female C57BL6/J mice. In both models, the development of EAE was similar except for starting before the onset of symptoms and presenting a higher EAE cumulative score in the at-EAE model. Spinal cord histological examination revealed an increased glial activation as well as more extensive demyelinating areas in the at-EAE than in the active EAE model. Although inflammatory infiltrates composed by macrophages and T lymphocytes were found in the spinal cord and brain of both models, B lymphocytes were significantly increased in the at-EAE model. The co-localization of these B cells with IgG and their predominant distribution in areas of demyelination would suggest that IgG-secreting B cells are involved in the neurodegenerative processes associated with at-EAE

Oxidative stress and proinflammatory cytokines contribute to demyelination and axonal damage in a cerebellar culture model of neuroinflammation

Background: Demyelination 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 Findings: To 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. Conclusion: The 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

A Network Analysis of the Human T-Cell Activation Gene Network Identifies Jagged1 as a Therapeutic Target for Autoimmune Diseases

Understanding complex diseases will benefit the recognition of the properties of the gene networks that control biological functions. Here, we set out to model the gene network that controls T-cell activation in humans, which is critical for the development of autoimmune diseases such as Multiple Sclerosis (MS). The network was established on the basis of the quantitative expression from 104 individuals of 20 genes of the immune system, as well as on biological information from the Ingenuity database and Bayesian inference. Of the 31 links (gene interactions) identified in the network, 18 were identified in the Ingenuity database and 13 were new and we validated 7 of 8 interactions experimentally. In the MS patients network, we found an increase in the weight of gene interactions related to Th1 function and a decrease in those related to Treg and Th2 function. Indeed, we found that IFN-ß therapy induces changes in gene interactions related to T cell proliferation and adhesion, although these gene interactions were not restored to levels similar to controls. Finally, we identify JAG1 as a new therapeutic target whose differential behaviour in the MS network was not modified by immunomodulatory therapy. In vitro treatment with a Jagged1 agonist peptide modulated the T-cell activation network in PBMCs from patients with MS. Moreover, treatment of mice with experimental autoimmune encephalomyelitis with the Jagged1 agonist ameliorated the disease course, and modulated Th2, Th1 and Treg function. This study illustrates how network analysis can predict therapeutic targets for immune intervention and identified the immunomodulatory properties of Jagged1 making it a new therapeutic target for MS and other autoimmune diseases