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.

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.

Design, synthesis and evaluation of an anthraquinone derivative conjugated to myelin basic protein immunodominant (MBP85-99) epitope: Towards selective immunosuppression.

Anthraquinone type compounds, especially di-substituted amino alkylamino anthraquinones have been widely studied as immunosuppressants. The anthraquinone ring is part of mitoxandrone that has been used for the treatment of multiple sclerosis (MS) and several types of tumors. A desired approach for the treatment of MS would be the immunosuppression and elimination of specific T cells that are responsible for the induction of the disease. Herein, the development of a peptide compound bearing an anthraquinone derivative with the potential to specifically destroy the encephalitogenic T cells responsible for the onset of MS is described. The compound consists of the myelin basic protein (MBP) 85-99 immunodominant epitope (MBP85-99) coupled to an anthraquinone type molecule (AQ) via a disulfide (S-S) and 6 amino hexanoic acid (Ahx) residues (AQ-S-S-(Ahx)6MBP85-99). AQ-S-S-(Ahx)6MBP85-99 could bind to HLA II DRB1*-1501 antigen with reasonable affinity (IC50 of 56 nM) The compound was localized to the nucleus of Jurkat cells (an immortalized line of human T lymphocytes) 10 min after its addition to the medium and resulted in lowered Bcl-2 levels (apoptosis). Entrance of the compound was abolished when cells were pre-treated with cisplatin, an inhibitor of thioredoxin reductase. Accordingly, levels of free thiols were elevated in the culture supernatants of Jurkat cells exposed to N-succinimidyl 3-(2-pyridyldithio) propionate coupled to (Ahx)6MBP85-99 via a disulphide (SPDP-S-S-(Ahx)6MBP85-99) but returned to normal after exposure to cisplatin. These results raise the possibility of AQ-S-S-(Ahx)6MBP85-99 being used as an eliminator of encephalitogenic T cells via implication of the thioredoxin system for the generation of the toxic, thiol-containing moiety (AQ-SH). Future experiments would ideally determine whether SPDP-S-S-(Ahx)6MBP85-99 could incorporate into HLA II DRB1*-1501 tetramers and neutralize encephalitogenic T cell lines sensitized to MBP85-99.

Treatment of surgical brain injury by immune tolerance induced by intrathymic and hepatic portal vein injection of brain antigens.

Surgical brain injury (SBI) defines complications induced by intracranial surgery, such as cerebral edema and other secondary injuries. In our study, intrathymic and hepatic portal vein injection of allogeneic myelin basic protein (MBP) or autogeneic brain cell suspensions were administered to a standard SBI model. Serum pro-inflammatory IL-2, anti-inflammatory IL-4 concentrations and the CD4(+)T/CD8(+)T ratio were measured at 1, 3, 7, 14 and 21 d after surgery to verify the establishment of immune tolerance. Furthermore, we confirmed neuroprotective effects by evaluating neurological scores at 1, 3, 7, 14 and 21 d after SBI. Anti-Fas ligand (FasL) immunohistochemistry and TUNEL assays of brain sections were tested at 21 d after surgery. Intrathymic injections of MBP or autogeneic brain cell suspensions functioned by both suppressing secondary inflammatory reactions and improving prognoses, whereas hepatic portal vein injections of autogeneic brain cell suspensions exerted a better effect than MBP. Intrathymic and hepatic portal vein injections of MBP had equal effects on reducing secondary inflammation and improving prognoses. Otherwise, hepatic portal vein injections of autogeneic brain cell suspensions had better outcomes than intrathymic injections of autogeneic brain cell suspensions. Moreover, the benefit of injecting antigens into the thymus was outweighed by hepatic portal vein injections.

Could Intrathymic Injection of Myelin Basic Protein Suppress Inflammatory Response After Co-culture of T Lymphocytes and BV-2 Microglia Cells?

BACKGROUND: The interaction between activated microglia and T lymphocytes can yield abundant pro-inflammatory cytokines. Our previous study proved that thymus immune tolerance could alleviate the inflammatory response. This study aimed to investigate whether intrathymic injection of myelin basic protein (MBP) in mice could suppress the inflammatory response after co-culture of T lymphocytes and BV-2 microglia cells. METHODS: Totally, 72 male C57BL/6 mice were randomly assigned to three groups (n = 24 in each): Group A: intrathymic injection of 100 µl MBP (1 mg/ml); Group B: intrathymic injection of 100 µl phosphate-buffered saline (PBS); and Group C: sham operation group. Every eight mice in each group were sacrificed to obtain the spleen at postoperative days 3, 7, and 14, respectively. T lymphocytes those were extracted and purified from the spleens were then co-cultured with activated BV-2 microglia cells at a proportion of 1:2 in the medium containing MBP for 3 days. After identified the T lymphocytes by CD3, surface antigens of T lymphocytes (CD4, CD8, CD152, and CD154) and BV-2 microglia cells (CD45 and CD54) were detected by flow cytometry. The expressions of pro-inflammatory factors of BV-2 microglia cells (interleukin [IL]-1ß, tumor necrosis factor-α [TNF-α], and inducible nitric oxide synthase [iNOS]) were detected by quantitative real-time polymerase chain reaction (PCR). One-way analysis of variance (ANOVA) and the least significant difference test were used for data analysis. RESULTS: The levels of CD152 in Group A showed an upward trend from the 3rd to 7th day, with a downward trend from the 7th to 14th day (20.12 ± 0.71%, 30.71 ± 1.14%, 13.50 ± 0.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The levels of CD154 in Group A showed a downward trend from the 3rd to 7th day, with an upward trend from the 7th to 14th day (10.00 ± 0.23%, 5.28 ± 0.69%, 14.67 ± 2.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The ratio of CD4+/CD8 + T in Group A showed a downward trend from the 3rd to 7th day, with the minimum at postoperative day 7, then an upward trend from the 7th to 14th day (P < 0.05). Meanwhile, the levels of CD45 and CD54 in Group A were found as the same trend as the ratio of CD4+/CD8 + T (CD45: 83.39 ± 2.56%, 82.74 ± 2.09%, 87.56 ± 2.11%; CD54: 3.80 ± 0.24%, 0.94 ± 0.40%, 3.41 ± 0.33% at postoperative days 3, 7, and 14, respectively, P < 0.05). The expressions of TNF-α, IL-1ß, and iNOS in Group A were significantly lower than those in Groups B and C, and the values at postoperative day 7 were the lowest compared with those at postoperative days 3 and 14 (P < 0.05). No significant difference was found between Groups B and C. CONCLUSIONS: Intrathymic injection of MBP could suppress the immune reaction that might reduce the secondary immune injury of brain tissue induced by an inflammatory response.

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.

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.

Therapeutic window for combination therapy of A91 peptide and glutathione allows delayed treatment after spinal cord injury.

Immunisation with neural-derived peptides is a promising strategy in models of spinal cord (SC) injury. Recent studies have also demonstrated that the addition of glutathione monoethyl ester (GHSE) to this strategy further improves motor recovery, tissue protection and neuronal survival after SC injury. As it is realistic to envision that this combination therapy could be tested in clinical trials, the therapeutic window should be experimentally explored before implementing its use in SC-injured human beings. For this purpose, 50 rats (10 per group) were subjected to a moderate SC contusion. The combined therapy was initiated at 10 min., 24, 72 or 120 hr after injury. Motor recovery and the survival of rubrospinal (RS) and ventral horn (VH) neurones were evaluated 60 days after injury. Results showed a significant motor improvement even if the combined therapy was initiated up to 72 hr after injury. BBB scores were as follows: 10 min.: 10.5 ± 0.7, 24 hr: 10.7 ± 0.5, 72 hr: 11.0 ± 1.3 and PBS: 6.7 ± 1 (mean ± S.D.). Initiation of combined therapy 120 hr after injury had no beneficial effect on motor recovery. Survival of RS and VH neurones was significantly higher in animals treated during the first 72 hr than those treated only with PBS. In this case again, animals treated with combined therapy 120 hr after injury did not present significant survival of neurones. Treatment with this combined strategy has a clinically feasible therapeutic window. This therapy provides enough time to transport and diagnose the patient and allows the concomitant use of other neuroprotective therapies.

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.

TCR affinity for self-ligands influences the development and function of encephalitogenic T cells.

The specificity and affinity of self-reactive T cells is likely to impact the development of autoimmune-disease causing T cells in the thymus as well as their function in the periphery. We identified a naturally occurring, low affinity variant of an MBP Ac1-11/I-A(u) specific TCR that is known to induce EAE. Thymocytes in mice carrying the transgenes for this low affinity TCR were poorly positively selected, as compared to their high affinity TCR expressing counterparts. Nonetheless, CD4 T cells bearing the low affinity TCR accumulated in the periphery of the mice. Unlike mice expressing the high affinity TCR, these mice very rarely developed disease. However, if endogenous TCR expression was eliminated by breeding to RAG1 deficient mice, 100% of the mice carrying either the high or the low affinity versions of the TCR developed EAE. Intriguingly, while the incidence of EAE increased, the age of onset of disease in both mice was identical. These data suggest disease onset occurs during a short window of mouse development.

Inhibition of the kynurenine-NAD+ pathway leads to energy failure and exacerbates apoptosis in pneumococcal meningitis.

Pneumococcal meningitis causes neurological sequelae, including learning and memory deficits in up to half of the survivors. In both humans and in animal models of the disease, there is apoptotic cell death in the hippocampus, a brain region involved in learning and memory function. We previously demonstrated that in an infant rat model of pneumococcal meningitis, there is activation of the kynurenine (KYN) pathway in the hippocampus, and that there was a positive correlation between the concentration of 3-hydroxykynurenine and the extent of hippocampal apoptosis. To clarify the role of the KYN pathway in the pathogenesis of hippocampal apoptosis in pneumococcal meningitis, we specifically inhibited 2 key enzymes of the KYN pathway and assessed hippocampal apoptosis, KYN pathway metabolites, and nicotinamide adenine dinucleotide (NAD) concentrations by high-performance liquid chromatography. Pharmacological inhibition of kynurenine 3-hydroxylase and kynureninase led to decreased cellular NAD levels and increased apoptosis in the hippocampus. The cerebrospinal fluid levels of tumor necrosis factor and interleukin-1α and -ß were not affected. Our data suggest that activation of the KYN pathway in pneumococcal meningitis is neuroprotective by compensating for an increased NAD demand caused by infection and inflammation;this mechanism may prevent energy failure and apoptosis in the hippocampus.

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.

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.

Autoantibodies to myelin basic protein (MBP) in healthy individuals and in patients with multiple sclerosis: a role in regulating cytokine responses to MBP.

Anti-myelin basic protein (-MBP) autoantibodies have generally been considered to be absent from sera from healthy individuals, but to be detectable in sera from some patients with multiple sclerosis (MS). However, their pathogenic role is uncertain. We demonstrate the presence of MBP-reactive autoantibodies in sera from 17 healthy individuals and 17 MS patients. The addition of MBP to the sera caused a dose-dependent deposition of MBP and co-deposition of immunoglobulin M (IgM) and fragments of complement component 3 (C3) on allogeneic monocytes. Calcium chelation abrogated the immunoglobulin deposition, indicating that formation of complement-activating immune complexes played a role in the binding process. Furthermore, MBP elicited tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 production by normal mononuclear cells in the presence of serum from both patients and controls. Mononuclear cells from MS patients responded to MBP with the production of interferon (IFN)-gamma, IL-4 and IL-5, in addition to TNF-alpha and IL-10. The production of IFN-gamma and IL-5 was increased when MS serum was added rather than normal serum. Denaturation of MBP strongly inhibited MBP deposition and the MBP-induced IgM deposition and cytokine production, indicating that these events were facilitated by autoantibodies recognizing conformational epitopes on MBP. We infer that MBP-elicited TNF-alpha and IL-10 responses are promoted to equal extents by naturally occurring MBP autoantibodies and autoantibodies contained in MS sera. However, the latter seem to be more efficient in facilitating the production of IFN-gamma and IL-5.

[Inhibiton of experimental autoimmune encephalomyelitis in Lewis rats by nasal administration of encephalitogenic MBP peptides: synergistic effects of MBP 68-86 and 87-99].

AIM: To explore the synergistic effect of MBP 68-86 and 87-99, on the inhibition of experimental autoimmune encephalomyelitis (EAE) in Lewis rat by nasal administration. METHODS: Three different MBP peptides(MBP 68-86, 87-99, and the non-encephalitogenic peptide 110-128) were synthesized and administrated nasally to Lewis rat on day-11, -10, -9, -8 and -7 prior to immunization with the guinea pig MBP (gp-MBP)+CFA, which was used to induce EAE. The protective effect on Lewis rat from EAE by the MBP peptides was evaluated. RESULTS: Protection was achieved with the encephalitogenic peptides MBP 68-86 and 87-99, MBP 68-86 being more potent, but not with MBP 110-128. Neither MBP 68-86 nor 87-99 used alone conferred complete protection to gp-MBP-induced EAE. In contrast, nasal administration of a mixture of MBP 68-86 and 87-99 completely blocked gp-MBP-induced EAE even at lower dosage than being used alone. Rats tolerized with MBP 68-86+87-99 nasally showed decreased T cell responses to MBP, reflected by lymphocyte proliferation and IFN-gamma ELISPOT assays. Rats tolerized with MBP 68-86+87-99 also had abrogated MBP-reactive IFN-gamma and TNF-alpha mRNA expression in lymph node cells compared to rats receiving MBP 110-128 nasally, while similar low levels of MBP-reactive TGF-beta and IL-4 mRNA expressing cells were observed in the two groups. CONCLUSION: Nasal administration of encephalitogenic MBP peptides can induce antigen-specific T cell tolerance and confer incomplete protection to gp-MBP-induced EAE, and MBP 68-86 and 87-99 have synergistic effects. Non-regulatory mechanisms are proposed to be responsible for tolerance development after nasal peptide administration.

Downregulation of transforming growth factor-beta2 facilitates inflammation in the central nervous system by reciprocal astrocyte/microglia interactions.

The central nervous system is an immune privileged organ in which inflammatory reactions are normally downregulated by mechanisms that are not completely understood. Transforming growth factor (TGF)-beta2 is constitutively expressed in the adult central nervous system and little is known about its regulation and modulatory role during neuroinflammation. In this study, we show that TGFbeta2 mRNA and protein are downregulated in the acute phase of chronic relapsing experimental autoimmune encephalomyelitis, whereas the homologous cytokine TGFbeta1 is upregulated. To further characterize regulatory mechanisms, we resorted to an in vitro glial cell culture system. The proinflammatory cytokines IFNgamma and TNFalpha suppressed TGFbeta2 secretion by astrocytes, the major intracerebral producers of TGFbeta2. On the cellular level, activated microglia inhibited TGFbeta2 secretion but induced TGFbeta1 through soluble factors. On the other hand, TGFbeta2 influenced antigen-presenting cell functions of microglia by downregulating major histocompatibility complex class II expression and costimulatory/adhesion molecules, and thereby inhibited myelin basic protein-specific T cell proliferation. These data suggest that TGFbeta2 plays a central role in maintenance of the immune privilege of the central nervous system. Downregulation of astrocytic TGFbeta2 by T cell- and microglia-secreted cytokines appears to be a critical step in providing the grounds for acute and chronic neuroinflammation.

Involvement of regulatory T cells in the experimental autoimmune encephalomyelitis-preventive effect of dendritic cells expressing myelin oligodendrocyte glycoprotein plus TRAIL.

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4(+)CD25(+) regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4(+)CD25(+) T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3(+) cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4(+)CD25(+) T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4(+)CD25(+) Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.

A role for CXCL12 (SDF-1alpha) in the pathogenesis of multiple sclerosis: regulation of CXCL12 expression in astrocytes by soluble myelin basic protein.

The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1alpha), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1beta and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1beta. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.