Toll-like receptor 4-mediated microglial inflammation exacerbates early white matter injury following experimental subarachnoid hemorrhage.

Neuroinflammation has been reported to be associated with white matter injury (WMI) after subarachnoid hemorrhage (SAH). As the main resident immune cells of the brain, microglia can be activated into proinflammatory and anti-inflammatory phenotypes. Toll-like receptor 4 (TLR4), expressed on the surface of the microglia, plays a key role in microglial inflammation. However, the relationship between TLR4, microglial polarization, and WMI following SAH remains unclear. In this study, a total of 121 male adult C57BL/6 wild-type (WT) mice, 20 WT mice at postnatal day 1 (P1), and 41 male adult TLR4 gene knockout (TLR4-/-) mice were used to investigate the potential role of TLR4-induced microglial polarization in early WMI after SAH by radiological, histological, microstructural, transcriptional, and cytological evidence. The results indicated that microglial inflammation was associated with myelin loss and axon damage, shown as a decrease in myelin basic protein (MBP), as well as increase in degraded myelin basic protein (dMBP) and amyloid precursor protein (APP). Gene knockout of TLR4 revised microglial polarization toward the anti-inflammatory phenotype and protected the white matter at an early phase after SAH (24 h), as shown through reduction of toxic metabolites, preservation of myelin, reductions in APP accumulation, reductions in white matter T2 hyperintensity, and increases in FA values. Cocultures of microglia and oligodendrocytes, the cells responsible for myelin production and maintenance, were established to further elucidate the relationship between microglial polarization and WMI. In vitro, TLR4 inhibition decreased the expression of microglial MyD88 and phosphorylated NF-κB, thereby inhibiting M1 polarization and mitigating inflammation. Decrease in TLR4 in the microglia increased preservation of neighboring oligodendrocytes. In conclusion, microglial inflammation has dual effects on early WMI after experimental SAH. Future explorations on more clinically relevant methods for modulating neuroinflammation are warranted to combat stroke with both WMI and gray matter destruction.

Noradrenaline through ß-adrenoceptor contributes to sexual dimorphism in primary CD4+ T-cell response in DA rat EAE model?

Males exhibit stronger sympathetic nervous system (SNS) activity, but weaker primary CD4+ T-cell (auto)immune responses. To test the role of catecholamines, major end-point SNS mediators, in this dimorphism, influence of propranolol (ß-adrenoceptor blocker) on mitogen/neuroantigen-stimulated CD4+ T cells from female and male EAE rat draining lymph node (dLN) cell cultures was examined. Male rat dLNs exhibited higher noradrenaline concentration and frequency of ß2-adrenoceptor-expressing CD4+ T lymphocytes and antigen presenting cells. Propranolol, irrespective of exogenous noradrenaline presence, more prominently augmented IL-2 production and proliferation of CD4+ lymphocytes in male than female rat dLN cell cultures. In neuroantigen-stimulated dLN cells of both sexes propranolol increased IL-1ß and IL-23/p19 expression and IL-17+ CD4+ cell frequency, but enhanced IL-17 production only in male rat CD4+ lymphocytes, thereby abrogating sexual dimorphism in IL-17 concentration observed in propranolol-free cultures. Thus, ß-adrenoceptor-mediated signalling may contribute to sex bias in rat IL-17-producing cell secretory capacity.

Inclusion of non-target antigen in vaccination favors generation of OVA specific CD4 memory T cells.

Inducing long-lived memory T cells by sub-unit vaccines has been a challenge. Subunit vaccines containing single immunogenic target antigen from a given pathogen have been designed with the presumption of mimicking the condition associated with natural infection, but fail to induce quality memory responses. In this study, we have included non-target antigens with vaccine candidate, OVA, in the inoculum containing TLR ligands to suffice the minimal condition of pathogen to provoke immune response. We found that inclusion of immunogenic HEL (hen egg lysozyme) or poorly immunogenic MBP (Myelin Basic protein) non-target antigen enhances the OVA specific CD4 T cell responses. Interestingly, poorly immunogenic MBP was found to strongly favor the generation of OVA specific memory CD4 T cells. MBP not only improves magnitude of T cell response but also promotes the T cells to undergo higher cycles of division, one of the characteristic of central memory T cells. Inclusion of MBP with vaccine targets was also found to promote multiple cytokine producing CD4 T cells. We also found that challenge of host with non-target antigen MBP favors generation of central Memory T cells.

Interaction of the cryptic fragment of myelin basic protein with mitochondrial voltage-dependent anion-selective channel-1 affects cell energy metabolism.

In demyelinating nervous system disorders, myelin basic protein (MBP), a major component of the myelin sheath, is proteolyzed and its fragments are released in the neural environment. Here, we demonstrated that, in contrast with MBP, the cellular uptake of the cryptic 84-104 epitope (MBP84-104) did not involve the low-density lipoprotein receptor-related protein-1, a scavenger receptor. Our pull-down assay, mass spectrometry and molecular modeling studies suggested that, similar with many other unfolded and aberrant proteins and peptides, the internalized MBP84-104 was capable of binding to the voltage-dependent anion-selective channel-1 (VDAC-1), a mitochondrial porin. Molecular modeling suggested that MBP84-104 directly binds to the N-terminal α-helix located midway inside the 19 ß-blade barrel of VDAC-1. These interactions may have affected the mitochondrial functions and energy metabolism in multiple cell types. Notably, MBP84-104 caused neither cell apoptosis nor affected the total cellular ATP levels, but repressed the aerobic glycolysis (lactic acid fermentation) and decreased the l-lactate/d-glucose ratio (also termed as the Warburg effect) in normal and cancer cells. Overall, our findings implied that because of its interactions with VDAC-1, the cryptic MBP84-104 peptide invoked reprogramming of the cellular energy metabolism that favored enhanced cellular activity, rather than apoptotic cell death. We concluded that the released MBP84-104 peptide, internalized by the cells, contributes to the reprogramming of the energy-generating pathways in multiple cell types.

Application of N-Dodecyl l-Peptide to Enhance Serum Stability while Maintaining Inhibitory Effects on Myometrial Contractions Ex Vivo.

N-Alkylation and N-acylation of the prostaglandin-F2α allosteric modulator l-PDC31 were performed to install various alkyl, PEG and isoprenoid groups onto the l-enantiomer of the peptide. Among the different bio-conjugates studied, the N-dodecyl analog reduced prostaglandin-F2α-induced mouse myometrium contractions ex vivo. Furthermore, N-dodecyl-l-PDC31 exhibited improved stability in a mouse serum assay, likely due to protection from protease degradation by the lipid chain.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP72-85-induced EAE in Lewis rats. The Lys91 and Pro96 of MBP87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala96]MBP87-99, cyclo(87-99)[Ala91,96]MBP87-99 and cyclo(87-99)[Arg91, Ala96]MBP87-99, but not wild-type linear MBP87-99, strongly inhibited MBP72-85-induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg91, Ala96]MBP87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Spinal activity of interleukin 6 mediates myelin basic protein-induced allodynia.

Mechanosensory fibers are enveloped by myelin, a unique multilamellar membrane permitting saltatory neuronal conduction. Damage to myelin is thought to contribute to severe pain evoked by innocuous tactile stimulation (i.e., mechanical allodynia). Our earlier (Liu et al., 2012) and present data demonstrate that a single injection of a myelin basic protein-derived peptide (MBP84-104) into an intact sciatic nerve produces a robust and long-lasting (>30days) mechanical allodynia in female rats. The MBP84-104 peptide represents the immunodominant epitope and requires T cells to maintain allodynia. Surprisingly, only systemic gabapentin (a ligand of voltage-gated calcium channel α2δ1), but not ketorolac (COX inhibitor), lidocaine (sodium channel blocker) or MK801 (NMDA antagonist) reverse allodynia induced by the intrasciatic MBP84-104. The genome-wide transcriptional profiling of the sciatic nerve followed by the bioinformatics analyses of the expression changes identified interleukin (IL)-6 as the major cytokine induced by MBP84-104 in both the control and athymic T cell-deficient nude rats. The intrasciatic MBP84-104 injection resulted in both unilateral allodynia and unilateral IL-6 increase the segmental spinal cord (neurons and astrocytes). An intrathecal delivery of a function-blocking IL-6 antibody reduced the allodynia in part by the transcriptional effects in large-diameter primary afferents in DRG. Our data suggest that MBP regulates IL-6 expression in the nervous system and that the spinal IL-6 activity mediates nociceptive processing stimulated by the MBP epitopes released after damage or disease of the somatosensory nervous system.

MOG-induced experimental autoimmune encephalomyelitis in the rat species triggers anti-neurofascin antibody response that is genetically regulated.

BACKGROUND: Ιn multiple sclerosis (MS), axonal damage leads to permanent neurological disabilities and the spreading of the autoimmune response to axonal antigens is implicated in disease progression. Experimental autoimmune encephalomyelitis (EAE) provides an animal model that mimics MS. Using different EAE models, we investigated the pathophysiological basis of epitope spreading to neurofascin, a protein localized at the node of Ranvier and its regulation by non-MHC genes. METHODS: We used two different EAE models in DA rat; one which is induced with myelin oligodendrocyte glycoprotein (MOG) which leads to disease characterized by profound demyelination, and the second which is induced with myelin basic protein (MBP) peptide 63-88 which results in severe central nervous system (CNS) inflammation but little or no demyelination. We determined anti-neurofascin antibody levels during the course of disease. Furthermore, the anti-neurofascin IgG response was correlated with clinical parameters in 333 (DAxPVG.1AV1) x DA rats on which we performed linkage analysis to determine if epitope spreading to neurofascin was affected by non-MHC genes. RESULTS: Spreading of the antibody response to neurofascin occurred in demyelinating MOG-induced EAE but not in EAE induced with MBP peptide 63-88. Anti-neurofascin IgG levels correlated with disease severity in (DAxPVG.1AV1) x DA rats, and a genomic region on chromosome 3 was found to influence this response. CONCLUSIONS: Inter-molecular epitope spreading to neurofascin correlates with disease severity in MOG-EAE is dependent on extensive demyelination and is influenced by non-MHC genes. The findings presented here may shed light on factors involved in the severity of MS and its genetics.

Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats.

BACKGROUND: Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins - myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) - results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. MATERIAL AND METHODS: Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. RESULTS: Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. CONCLUSIONS: The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction.

Myelin basic protein induces inflammatory mediators from primary human endothelial cells and blood-brain barrier disruption: implications for the pathogenesis of multiple sclerosis.

AIM: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, characterized by demyelination of white matter, loss of myelin forming oligodendrocytes, changes in the blood-brain barrier (BBB) and leucocyte infiltration. Myelin basic protein (MBP) is a component of the myelin sheath. Degradation of myelin is believed to be an important step that leads to MS pathology. Transmigration of leucocytes across the vasculature, and a compromised BBB participate in the neuroinflammation of MS. We examined the expression and regulation of the chemokine (C-C motif) ligand 2 (CCL2) and the cytokine interleukin-6 (IL-6) in human endothelial cells (EC), a component of the BBB, after treatment with MBP. METHODS: EC were treated with full-length MBP. CCL2 and IL-6 protein were determined by ELISA. Western blot analysis was used to determine signalling pathways. A BBB model was treated with MBP and permeability was assayed using albumin conjugated to Evan's blue dye. The levels of the tight junction proteins occludin and claudin-1, and matrix metalloprotease (MMP)-2 were assayed by Western blot. RESULTS: MBP significantly induced CCL2 and IL-6 protein from EC. This induction was partially mediated by the p38 MAPK pathway as there was phosphorylation after MBP treatment. MBP treatment of a BBB model caused an increase in permeability that correlated with a decrease in occludin and claudin-1, and an induction of MMP2. CONCLUSION: These data demonstrate that MBP induces chemotactic and inflammatory mediators. MBP also alters BBB permeability and tight junction expression, indicating additional factors that may contribute to the BBB breakdown characteristic of MS.

TLR-4 ligation of dendritic cells is sufficient to drive pathogenic T cell function in experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) depends on the initial activation of CD4(+) T cells responsive to myelin autoantigens. The key antigen presenting cell (APC) population that drives the activation of naïve T cells most efficiently is the dendritic cell (DC). As such, we should be able to trigger EAE by transfer of DC that can present the relevant autoantigen(s). Despite some sporadic reports, however, models of DC-driven EAE have not been widely adopted. We sought to test the feasibility of this approach and whether activation of the DC by toll-like receptor (TLR)-4 ligation was a sufficient stimulus to drive EAE. FINDINGS: Host mice were seeded with myelin basic protein (MBP)-reactive CD4+ T cells and then were injected with DC that could present the relevant MBP peptide which had been exposed to lipopolysaccharide as a TLR-4 agonist. We found that this approach induced robust clinical signs of EAE. CONCLUSIONS: DC are sufficient as APC to effectively drive the differentiation of naïve myelin-responsive T cells into autoaggressive effector T cells. TLR-4-stimulation can activate the DC sufficiently to deliver the signals required to drive the pathogenic function of the T cell. These models will allow the dissection of the molecular requirements of the initial DC-T cell interaction in the lymphoid organs that ultimately leads to autoimmune pathology in the central nervous system.

Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury.

Immunization with neurally derived peptides (INDP) boosts the action of an autoreactive immune response that has been shown to induce neuroprotection in several neurodegenerative diseases, especially after spinal cord (SC) injury. This strategy provides an environment that promotes neuronal survival and tissue preservation. The mechanisms by which this autoreactive response exerts its protective effects is not totally understood at the moment. A recent study showed that INDP reduces lipid peroxidation. Lipid peroxidation is a neurodegenerative phenomenon caused by the increased production of reactive nitrogen species such as nitric oxide (NO). It is possible that INDP could be interfering with NO production. To test this hypothesis, we examined the effect of INDP on the amount of NO produced by glial cells when cocultured with autoreactive T cells. We also evaluated the amount of NO and the expression of the inducible form of nitric oxide synthase (iNOS) at the injury site of SC-injured animals. The neural-derived peptides A91 and Cop-1 were used to immunize mice and rats with SC injury. In vitro studies showed that INDP significantly reduces the production of NO by glial cells. This observation was substantiated by in vivo experiments demonstrating that INDP decreases the amount of NO and iNOS gene expression at the site of injury. The present study provides substantial evidence on the inhibitory effect of INDP on NO production, helpingour understanding of the mechanisms through which protective autoimmunity promotes neuroprotection.

Adhesive properties and inflammatory potential of citrullinated myelin basic protein peptide 45-89.

Deimination of arginyl residue of myelin basic protein (MBP) reduces cationicity of MBP and impedes the normal myelin membrane assembly. Less ordered structure of MBP is more susceptible to proteolytic attack that may lead to the release of highly immunogenic deiminated peptides into extracellular milieu. We have studied the association of peptides 45-89 derived from citrullinated MBP (C8 isomer) and phosphorylated MBP (C3 isomer) with the myelin lipids in a model membrane system using optical waveguide lightmode spectrometry. The analysis of association/dissociation kinetics to planar lipids under controlled hydrodynamic conditions has shown that MBP 45-89 peptide from citrullinated C8 isomer is less effectively adsorbed on the lipid membrane, than peptide from phosphorylated C3 isomer and packing densities for phosphorylated 45-89 MBP peptide is higher than for citrullinated forms. On the other hand, our results shown that continuous (24 h) exposure of mixed oligodendrocyte/microglial cells to peptides 45-89 from MBP-C8 induces apoptosis via mitochondrial pathway. In addition, peptides 45-89 stimulated the secretion of nitric oxide from microglial cells via induction of iNOS and decreased the level of the inhibitory protein IkB, indicating involvement of the transcription factor NF-kB in these processes. Our results suggest that some citrullinated peptides, initially released from oligodendrocytes, might activate microglia, which produces reactive nitrogen species and generates in turn fatal feedbacks that kill oligodendrocytes.

Dexmedetomidine Increases MMP-12 and MBP Concentrations after Coronary Artery Bypass Graft Surgery with Extracorporeal Circulation Anaesthesia without Impacting Cognitive Function: A Randomised Control Trial.

Postoperative neurological deficits remain a concern for patients undergoing cardiac surgeries. Even minor injuries can lead to neurocognitive decline (i.e., postoperative cognitive dysfunction). Dexmedetomidine may be beneficial given its reported neuroprotective effect. We aimed to investigate the effects of dexmedetomidine on brain injury during cardiac surgery anaesthesia. This prospective observational study analysed data for 46 patients who underwent coronary artery bypass graft surgery with extracorporeal circulation between August 2018 and March 2019. The patients were divided into two groups: control (CON) with typical anaesthesia and dexmedetomidine (DEX) with dexmedetomidine infusion. Concentrations of the biomarkers matrix metalloproteinase-12 (MMP-12) and myelin basic protein (MBP) were measured preoperatively and at 24 and 72 h postoperatively. Cognitive evaluations were performed preoperatively, at discharge, and 3 months after discharge using Addenbrooke's Cognitive Examination version III (ACE-III). The primary endpoint was the ACE-III score at discharge. Increased MMP-12 and MBP concentrations were observed in the DEX group 24 and 72 h postoperatively. No significant differences in ACE-III scores were observed between the groups at discharge; however, the values were increased when compared with initial values after 3 months (p = 0.000). The current results indicate that the administration of dexmedetomidine as an adjuvant to anaesthesia can increase MMP-12 and MBP levels without effects on neurocognitive outcomes at discharge and 3 months postoperatively.

MicroRNA-21-5p agomir inhibits apoptosis of oligodendrocyte precursor cell and attenuates white matter injury in neonatal rats.

BACKGROUND AND RESEARCH QUESTION/HYPOTHESIS: Excessive oligodendrocyte precursor cell (OPC) apoptosis occurs during intrauterine infection-induced white matter injury (WMI) in premature infants, preventing excessive apoptosis of OPCs is one of the mechanisms protecting WMI. Micro-RNA-21-5p (miR-21-5p) mediating anti-apoptotic activity was observed in other diseases. Therefore, the aim of this study was to determine whether miR-21-5p protects against WMI by modulating phosphatase and tensin homologue deleted on chromosome 10/phosphatidylinositol-3-kinase/protein kinase B (PTEN/PI3K/Akt) signalling pathway. METHODS: A lipopolysaccharide (LPS)-induced neonatal Sprague-Dawley (SD) rat model of preterm WMI was established. To explore the effect of miR-21-5p on WMI, we intraventricularly injected miR-21-5p agomir and miR-21-5p antagomir to activate or inhibit endogenous miR-21-5p. Immunofluorescent labelling of myelin basic protein, immunohistochemical labelling of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and terminal deoxynucleotidyl transferase dUTP nick end labelling assays were conducted to observe pathological white matter changes. The antibody of anti-oligodendrocyte marker 4 (O4) was used to specifically recognise OPCs. The expressions of miR-21-5p and PTEN mRNA in the brain were detected with quantitative real-time polymerase chain reaction (qRT-PCR). PTEN, Akt, and phosphorylated Akt (p-Akt) protein levels were assayed with western blotting, and apoptotic proteins associated with PI3K/Akt signalling were quantified. RESULTS: Intense white matter dysplasia and excessive OPC apoptosis were observed in the brains of rats with WMI. When the miR-21-5p agonist miR-21-5p agomir was used in the WMI group, apoptosis of OPCs was significantly reduced, and myelin maturation increased. MiR-21-5p agomir relieved WMI. MiR-21-5p agomir inhibited the mRNA and protein expression of PTEN, increased p-Akt phosphorylation, and decreased the expression and activation of related apoptotic proteins.On the other hand, the administration of miR-21-5p specific blocker, miR-21-5p antagomir, reduced the level of p-AKT, increased OPC apoptosis, and worsened WMI. INTERPRETATION: Our findings revealed that miR-21-5p agomir had anti-OPC over-apoptotic effects and enhanced myelin development in WMI by modulating the PTEN/Akt signalling pathway.

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog.

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.

Silencing Nogo-A promotes functional recovery in demyelinating disease.

OBJECTIVE: To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS). METHODS: A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in 2 models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. Enzyme-linked immunosorbent assay, flow cytometry, and (3)H-thymidine incorporation were used to determine immunological changes in myelin-specific T cells in mice with EAE. RESULTS: The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair, as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T-cell proliferation and cytokine production were unchanged in the siRNA-NogoA-treated mice. INTERPRETATION: Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.

Both MHC and non-MHC genes regulate inflammation and T-cell response after traumatic brain injury.

Genetic regulation of autoimmune neuroinflammation is a well known phenomenon, but genetic influences on inflammation following traumatic nerve injuries have received little attention. In this study we examined the inflammatory response in a rat traumatic brain injury (TBI) model, with a particular focus on major histocompatibility class II (MHC II) presentation, in two inbred rat strains that have been extensively characterized in experimental autoimmune encephalomyelitis (EAE); DA and PVG. In addition, MHC and Vra4 congenic strains on these backgrounds were studied to give information on MHC and non-MHC gene contribution. Thus, allelic differences in Vra4, harboring the Ciita gene, was found to regulate expression of the invariant chain at the mRNA level, with a much smaller effect exerted by the MHC locus itself. Notably, however, at the protein level the MHC congenic PVG-RT1(av1) strain displayed much stronger MHCII(+) presentation, as shown both by immunolabeling and flow cytometry, than the PVG strain, dwarfing the effect of Ciita. The PVG-RT1(av1) strain had significantly more T-cell influx than both DA and PVG, suggesting regulation both by MHC and non-MHC genes. Finally, in terms of outcome, the EAE susceptible DA strain displayed a significantly smaller resulting lesion volume than the resistant PVG-RT1(av1) strain. These results provide additional support for a role of adaptive immune response after neurotrauma and demonstrate that outcome is significantly affected by host genetic factors.

Cytokine secretion pattern in treatment of lymphocytes of multiple sclerosis patients with fumaric acid esters.

The present study was performed to investigate the effects of dimethylfumarate (DMF) and methylhydrogen fumarate (MHF) on the cytokine pattern of peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients. The PBMCs from patients and healthy controls were stimulated with myelin basic protein (MBP) or phytohemagglutinin (PHA) and cultured in the presence of DMF and MHF. The percentage of CD4+IL-4+ and CD4+IFN-γ+ cells was determined by means of intracellular cytokine staining. CD4+IL-4+ cells were significantly increased in the presence of DMF and MHF when PBMCs were stimulated by MBP (P < 0.003). The same significant result was obtained by PHA stimulation (P < 0.049). In terms of CD4+IFN-γ+ cells, the percentage of cells did not significantly differ between the cultures stimulated with MBP or PHA in the presence and absence of the drugs. Results of MBP stimulation in control group also showed a significant increase in CD4+IL-4+ cells in the presence of DMF and MHF. In comparison between patient and control groups, no statistically significant changes were observed. In conclusion, both DMF and MHF effectively increased IL-4 production, whereas they did not significantly change IFN-γ level, indicating the role of these drugs in increasing the production of beneficial cytokines such as IL-4.

Oral administration of L-mR18L, a single domain cationic amphipathic helical peptide, inhibits lesion formation in ApoE null mice.

We have shown that Ac-hE18A-NH2, a dual-domain cationic apolipoprotein-mimetic peptide, reduces plasma cholesterol levels in dyslipidemic mice. Two single-domain cationic peptides based on the lytic class L peptide 18L were developed to test the hypothesis that a single-domain cationic amphipathic peptide can reduce atherosclerosis in apolipoprotein (apo)E null mice when orally administered. To incorporate anti-inflammatory properties, aromatic residues were clustered in the nonpolar face similar to peptide 4F, resulting in modified 18L (m18L). To reduce lytic properties, the Lys residues of 18L were replaced with Arg with the resulting peptide called modified R18L (mR18L). Biophysical studies showed that mR18L had stronger interactions with lipids than did m18L. Peptide mR18L was also more effective than m18L in promoting LDL uptake by HepG2 cells. ApoE null mice received normal chow or chow containing m18L or mR18L for six weeks. A significant reduction in plasma cholesterol and aortic sinus lesion area was seen only in the mR18L group. Plasma from mice administered mR18L, unlike those from the control and m18L groups, did not enhance monocyte adhesion to endothelial cells. Thus oral administration of mR18L reduces plasma cholesterol and lesion formation and inhibits monocyte adhesion.