Nicotinamide nucleotide transhydrogenase expression analysis in multiple sclerosis patients.

Background: Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial redox-induced proton pump that links NADPH synthesis to the mitochondrial metabolic pathway. It also participates in the regulation of immune responses. A long non-coding RNA namely NNT-antisense 1 (NNT-AS1) has been shown to be transcribed from the same locus and exert anti-proliferative effects in some tissues. Methods: In the current study, we evaluated expression of NNT and NNT-AS1 in peripheral blood of 50 relapsing-remitting multiple sclerosis patients compared with healthy subjects. The difference in NNT expression was significant in only in male subjects aged over 50 when compared with the corresponding control subgroup. Results: For NNT-AS1, based on the results of Quantile regression and adjustment of the effects of age and sex as well as the interaction between sex and disease status, no significant difference was found between cases and controls. Moreover, NNT and NNT-AS1 expressions were correlated with age in controls and in female subjects respectively. Conclusion: Finally, we assessed correlations between expressions of these genes and detected significant pairwise correlations between transcript levels of NNT and NNT-AS1 genes in both cases and controls. The current study highlights a gender-specific role for NNT in the pathogenesis of MS.

Phosphorylated SIRT1 as a biomarker of relapse and response to treatment with glatiramer acetate in multiple sclerosis.

We have previously shown that SIRT1 mRNA expression was significantly lower in relapsing MS patients compared to those in remission. Our goal was to longitudinally investigate the role of active, phosphorylated SIRT1 (p-SIRT1) as a potential biomarker of relapse and predictor for response to glatiramer acetate (GA) treatment in patients with relapsing remitting multiple sclerosis (MS). We also want to investigate the downstream effects of SIRT1 activation by measuring the trimethylation of histone 3 at lysine 9 (H3K9me3). A cohort of 15 GA-treated patients was clinically monitored using the Expanded Disability Status Scale (EDSS) and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 6, and 12 months after initiation of the therapy. P-SIRT1 and H3K9me3 levels were assayed by Western blotting using specific antibodies. Statistically significant lower levels of p-SIRT1 protein (p < 0.0001) and H3K9me3 (p = 0.001) were found during relapses when compared to stable MS patients. Non-responders to GA treatment were defined as patients who exhibited at least two relapses following initiation of GA treatment. Statistically significant lower levels of p-SIRT1 protein (p = 0.02) and H3K9me3 (p = 0.004) were found in GA non-responders compared to responders. Using receiver operating characteristic analysis, area under the curve (AUC) for p-SIRT1 was 77% (p = 0.007) and for H3K9me3 was 81% (p = 0.002) for prediction of relapse. For predicting responsiveness to GA treatment, AUC was 75% (P = 0.01) for H3K9me3. Our data suggest that p-SIRT1 and H3K9me3 could serve as potential biomarkers for MS relapse. In addition, H3K9me3 could serve as possible biomarker to predict response to GA treatment.

Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients.

Multiple sclerosis (MS) is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh) contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS) patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD), is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis.

The p38 mitogen-activated protein kinase cascade modulates T helper type 17 differentiation and functionality in multiple sclerosis.

The p38 mitogen-activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) -mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4(+)  CD27(+)  CD45RA(+) naive T cells, both in healthy donors and in patients affected by the relapsing-remitting form of MS. Our data also indicate that p38 activation is essential for interleukin-17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4(+) T cells isolated from individuals with relapsing-remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17-mediated autoimmune diseases.

TIMP-1 resistant matrix metalloproteinase-9 is the predominant serum active isoform associated with MRI activity in patients with multiple sclerosis.

BACKGROUND: The activity of matrix metalloproteinase-9 (MMP-9) depends on two isoforms, an 82 kDa active MMP-9 modulated by its specific tissue inhibitor (TIMP-1), and a 65 kDa TIMP-1 resistant active MMP-9. The relevance of these two enzymatic isoforms in multiple sclerosis (MS) is still unknown. OBJECTIVE: To investigate the contribution of the TIMP-1 modulated and resistant active MMP-9 isoforms to MS pathogenesis. METHODS: We measured the serum levels of the 82 kDa and TIMP-1 resistant active MMP-9 isoforms by activity assay systems in 86 relapsing-remitting MS (RRMS) patients, categorized according to clinical and magnetic resonance imaging (MRI) evidence of disease activity, and in 70 inflammatory (OIND) and 69 non-inflammatory (NIND) controls. RESULTS: Serum levels of TIMP-1 resistant MMP-9 were more elevated in MS patients than in OIND and NIND (p < 0.05, p < 0.02, respectively). Conversely, 82 kDa active MMP-9 was higher in NIND than in the OIND and MS patients (p < 0.01 and p < 0.00001, respectively). MRI-active patients had higher levels of TIMP-1 resistant MMP-9 and 82 kDa active MMP-9, than did those with MRI inactive MS (p < 0.01 and p < 0.05, respectively). CONCLUSION: Our findings suggested that the TIMP-1 resistant MMP-9 seem to be the predominantly active isoform contributing to MS disease activity.

Roles of the ubiquitin peptidase USP18 in multiple sclerosis and the response to interferon-ß treatment.

BACKGROUND AND PURPOSE: Ubiquitin specific peptidase 18 (USP18) is a deubiquitinating enzyme that functions as a negative regulator of the type I interferon (IFN) signalling pathway and is specifically induced by type I IFNs. In the present study, previous observations by our group were expanded suggesting an implication of USP18 in multiple sclerosis (MS) based on the finding of a deficient expression of the gene in peripheral blood mononuclear cells from MS patients compared with healthy controls. METHODS: Two polymorphisms, rs2542109 (intronic) and rs9618216 (promoter), were genotyped in a cohort of 691 relapse-onset MS patients and 1028 healthy controls and in 225 MS patients treated with IFNß and classified into responders and non-responders after 2 years of treatment according to clinical criteria. Correlations between genotypes and expression levels for USP18 and its target ISG15 were performed by real-time polymerase chain reaction. RESULTS: Two USP18 haplotypes were significantly associated with MS, TG and CG. Additional experiments revealed that CG carriers were characterized by lower USP18 gene expression levels in peripheral blood mononuclear cells and higher clinical disease activity. Finally, AA homozygosis for the intronic polymorphism rs2542109 was associated with the responder phenotype; however, USP18 expression levels induced by IFNß did not differ amongst MS patients carrying different rs2542109 genotypes. CONCLUSIONS: Altogether, these results point to a role of USP18 in MS pathogenesis and the therapeutic response to IFNß.

Immunogenic effects of recombinant interferon-beta therapy disrupt the JAK/STAT pathway in primary immune cells from patients with multiple sclerosis.

BACKGROUND: Immunogenicity of recombinant interferon-ß (IFN-ß) is a known complication in the therapy of relapsing-remitting multiple sclerosis (RRMS). Neutralizing antibodies (NAbs) that can interfere with efficacy are quantified using in vitro bioassays; however, these assays do not reveal the immunogenic state of the patient and are not predictive of treatment outcome. OBJECTIVE: Assessment of the impact of NAbs on IFN-ß responsive cells and signalling pathways in peripheral blood mononuclear cells (PBMCs) with phospho-specific flow cytometry. METHOD: PBMCs from 10 IFN-ß-treated patients with RRMS, two untreated patients, and two healthy controls were re-stimulated in autologous sera and media with a serial dilution of IFN-ß (0-8000 U/ml) and levels of phosphorylation of STAT1/3/4/5/6 transcription factors were quantified in PBMC subtypes (NAb titres 0 to > 6000 neutralizing units). Data was subjected to principal component analysis, Hotelling's T (2), and partial least squares analysis. RESULTS: Three significantly distinct clusters of individuals were revealed in autologous sera: therapy-naïve and healthy, treated NAb-negative, and treated NAb-positive. Compared with controls STATs signalling patterns were modulated in treated NAb-negative patients and inhibited in all treated NAb-positive patients independently of NAb titres. In media no clustering of patients could be found. The predictability of NAb titres based on the phospho-flow data was 74%. CONCLUSION: Phospho-specific flow cytometry can delineate subset-specific cell responses that can act as surrogates for NAb exposure in blood. Immunogenic effects alter the response in primary cells even at low NAb levels. Cell line-based immunogenicity testing is not readily transferable to the immunogenic response in patients.

Role of the Bruton tyrosine kinase pathway in multiple sclerosis.

Multiple sclerosis (MS) is a chronic, immune-mediated, neurodegenerative condition that results in progressive accumulation of disability over the course of the disease. MS presents heterogeneously, and, as the disease progresses, patients develop a range of physical and neurologic problems that include reduced mobility, cognitive impairment, weakness, fatigue, pain, and defects in speech or vision. Economically, MS is costly, including both direct costs stemming from clinical care and medications and the indirect costs of productivity losses. These costs pose a substantial burden to patients, families, caregivers, employers, and society. There are 21 approved disease-modifying therapies for MS across several drug classes. The importance of early MS treatment has been confirmed, and progress has been made in the treatment of relapsing-remitting MS, although this progress has not been replicated for progressive presentations of the disease. Ongoing research continues to elucidate the exact mechanisms of disease in MS as well as potential new treatment strategies that may better address current gaps, such as disability progression in secondary progressive MS without activity. One of the novel pathways under investigation is the inhibition of Bruton tyrosine kinase, a cytoplasmic tyrosine kinase, which is expressed in B cells and other potentially targetable hematopoietic lineage cells. This review examines emerging hypotheses that targeting both B cells and myeloid cells within the periphery and central nervous system could yield clinical effects in key areas of MS pathophysiology that are currently unaddressed.

Characterization of immune cell subsets during the active phase of multiple sclerosis reveals disease and c-Jun N-terminal kinase pathway biomarkers.

BACKGROUND: Autoimmune activation and deregulated apoptosis of T lymphocytes are involved in multiple sclerosis (MS). c-Jun N-terminal kinase (JNK) plays a role in T-cell survival and apoptosis. OBJECTIVES: The aim of this work was to investigate the role of the JNK-dependent apoptosis pathway in relapsing-remitting MS (RRMS). METHODS: The immunomodulatory effect of AS602801, a JNK inhibitor, was firstly evaluated on activated peripheral blood mononuclear cells (PBMCs) from healthy volunteers (HVs) and secondly in unstimulated purified CD4+, CD8+ and CD11b+ cells from RRMS patients and HVs. Moreover JNK/inflammation/apoptosis related genes were investigated in RRMS and HV samples. RESULTS: In activated PBMCs from HVs, we showed that AS602801 blocked T-lymphocyte proliferation and induced apoptosis. In RRMS CD4+ and CD8+ cells, AS602801 induced apoptosis genes and expression of surface markers, while in RRMS CD11b+ cells it induced expression of innate immunity receptors and co-stimulatory molecules. Untreated cells from RRMS active-phase patients significantly released interleukin-23 (IL-23) and interferon-gamma (IFN-γ) and expressed less apoptosis markers compared to the cells of HVs. Moreover, gene expression was significantly different in cells from RRMS active-phase patients vs. HVs. By comparing RRMS PBMCs in the active and stable phases, a specific genomic signature for RRMS was indentified. Additionally, CASP8AP2, CD36, ITGAL, NUMB, OLR1, PIAS-1, RNASEL, RTN4RL2 and THBS1 were identified for the first time as being associated to the active phase of RRMS. CONCLUSIONS: The analysis of the JNK-dependent apoptosis pathway can provide biomarkers for activated lymphocytes in the active phase of RRMS and a gene expression signature for disease status. The reported results might be useful to stratify patients, thereby supporting the development of novel therapies.

Chitinase effects on immune cell response in neuromyelitis optica and multiple sclerosis.

BACKGROUND: Recent studies conducted in arthritis, asthma, and inflammatory bowel disease suggest that chitinases are important in inflammatory processes and tissue remodeling. OBJECTIVE: To investigate the role of chitinases in multiple sclerosis (MS) and neuromyelitis optica (NMO). METHODS: Levels of chitotriosidase, acid mammalian chitinase (AMCase), and chitinase 3-like-1 (CHI3L1) were measured using ELISA, in cerebrospinal fluid (CSF) and in serum from 24 patients with relapsing remitting (RR) MS, 24 patients with secondary progressive (SP) MS, 12 patients with NMO, 24 patients with other inflammatory neurological diseases (OIND), and 24 healthy controls (HCs). The number of anti-MOG cytokine-secreting cells was studied using ELISPOT. Eotaxins, MCP-1, RANTES, and IL-8 were assessed using ELISA. Cell transmigration was determined using an in vitro blood-brain barrier (BBB) model, in the presence and absence of chitinases. RESULTS: CSF chitinase levels were significantly increased in patients with RRMS and NMO compared with HCs and patients with SPMS and OIND. In contrast, no significant differences were detected in serum chitinase levels between groups. Chitinase CSF levels showed correlation with anti-MOG IL-13-producing cells, and eotaxin levels. In vitro experiments showed macrophage chitinase secretion was significantly increased by IL-13, but not by IL-5, IL-6, IL-12, or IFN-γ. Moreover, chitinases enhanced IL-8, RANTES, MCP-1, and eotaxin production, increasing migratory capacity in eosinophils, T cells, and macrophages across an in vitro BBB model. CONCLUSIONS: Chitinases increased in the CSF from patients with NMO in response to IL-13. These enhanced levels could contribute to central nervous system inflammation by increasing immune cell migration across the BBB.

PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity.

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5's link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.

Teriflunomide Treatment of Multiple Sclerosis Selectively Modulates CD8 Memory T Cells.

Background and Objectives: Inhibition of de novo pyrimidine synthesis in proliferating T and B lymphocytes by teriflunomide, a pharmacological inhibitor of dihydroorotate dehydrogenase (DHODH), has been shown to be an effective therapy to treat patients with MS in placebo-controlled phase 3 trials. Nevertheless, the underlying mechanism contributing to the efficacy of DHODH inhibition has been only partially elucidated. Here, we aimed to determine the impact of teriflunomide on the immune compartment in a longitudinal high-dimensional follow-up of patients with relapse-remitting MS (RRMS) treated with teriflunomide. Methods: High-dimensional spectral flow cytometry was used to analyze the phenotype and the function of innate and adaptive immune system of patients with RRMS before and 12 months after teriflunomide treatment. In addition, we assessed the impact of teriflunomide on the migration of memory CD8 T cells in patients with RRMS, and we defined patient immune metabolic profiles. Results: We found that 12 months of treatment with teriflunomide in patients with RRMS does not affect the B cell or CD4 T cell compartments, including regulatory TREG follicular helper TFH cell and helper TH cell subsets. In contrast, we observed a specific impact of teriflunomide on the CD8 T cell compartment, which was characterized by decreased homeostatic proliferation and reduced production of TNFα and IFNγ. Furthermore, we showed that DHODH inhibition also had a negative impact on the migratory velocity of memory CD8 T cells in patients with RRMS. Finally, we showed that the susceptibility of memory CD8 T cells to DHODH inhibition was not related to impaired metabolism. Discussion: Overall, these findings demonstrate that the clinical efficacy of teriflunomide results partially in the specific susceptibility of memory CD8 T cells to DHODH inhibition in patients with RRMS and strengthens active roles for these T cells in the pathophysiological process of MS.

Prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a complex, inflammatory and neurodegenerative disease of the central nervous system leading to long-term disability. Recent studies indicate a close association between inflammation and neurodegeneration in all lesions and disease stages of MS. Prolyl oligopeptidase (POP) is a proline-specific serine protease that cleaves several neuroactive peptides. This peptidase has been implicated in neurodegeneration, as well as in the modulation of the inflammatory response. METHODS: We examined plasma POP and the levels of an endogenous POP inhibitor from relapsing remitting MS patients and compared these with healthy controls, by monitoring the fluorescent changes due to standard fluorescently labelled substrate cleavage. We analysed the data in relationship to patient age and disease disability status. RESULTS: We observed a significant decrease in POP activity in plasma of relapsing remitting MS patients relative to healthy controls, coupled with an increase of POP endogenous inhibitor. The POP activity was also correlated with patient age and disability status. The lowered POP activity from plasma of MS patients could be rescued by reductants CONCLUSIONS: The decrease in circulating POP activity measured in MS is reverted by reductants. This suggests that POP inactivation in MS might be a result of the oxidative conditions prevailing in the plasma of the diseased patients. Plasma levels of POP activity as well as those of their endogenous inhibitor are suggested as biomarkers of inflammation and oxidative stress in MS.

Alterations in serum MMP-8, MMP-9, IL-12p40 and IL-23 in multiple sclerosis patients treated with interferon-beta1b.

BACKGROUND: Interferon-beta1b (IFN-beta1b), an effective treatment for multiple sclerosis (MS), lessens disease severity in MS patients. However, the mechanisms of its immunoregulatory and anti-inflammatory effects in MS remain only partially understood. Matrix metalloproteinases (MMP) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are involved in blood brain barrier disruption and formation of MS lesions. Th1/Th17 cytokines e.g. interleukins IL-12p40, IL-17, and IL-23, are associated with MS disease activity and are significant players in pathogenesis of MS. OBJECTIVE: During a 1-year prospective study, we serially measured serum MMP-8, MMP-9, TIMP-1, IL-12p40, IL-17, and IL-23 in 24 patients with relapsing-remitting MS. We compared the results to clinical course and to brain magnetic resonance imaging. IFN-beta1b decreased serum MMP-8 and MMP-9 (not TIMP-1). RESULTS: The sustained treatment with IFN-beta1b attenuated the pro-inflammatory environment by significantly reducing the serum IL-12p40, IL-23, and showed a trend for decreasing IL-17. Decreased serum MMP-8, MMP-9, IL-12 and IL-23 levels were correlated with a decrease in the number of contrast-enhanced T2-weighted lesions. CONCLUSION: Early treatment of MS with IFN-beta1b may stabilize clinical disease by attenuating levels of inflammatory cytokines and MMPs. Serial measurement of inflammatory mediators may serve as sensitive markers to gauge therapeutic responses to IFN-beta1b during the first year of treatment.

Increased cerebrospinal fluid chitotriosidase index in patients with multiple sclerosis.

OBJECTIVE: To investigate chitotriosidase (CTTS) activity in serum and cerebrospinal fluid (CSF) in multiple sclerosis (MS) patients in relation to disease course and CSF markers for immune activation or inflammation. MATERIALS AND METHODS: We studied 80 patients with relapsing-remitting MS (RRMS), 24 with secondary progressive MS (SPMS), 20 with primary progressive MS (PPMS) and 29 patients with other neurological disorders (OND). We measured CTTS activity and studied the correlation with CSF mononuclear cell count (MNC) and intrathecal IgG production. RESULTS: CTTS activity was significantly higher in CSF, but not in serum, from the total MS group compared with OND and controls. In RRMS and SPMS CTTS, index was increased compared with controls (RRMS, 0.10 +/- 0.21; SPMS, 0.10 +/- 0.15; controls, 0.021 +/- 0.020), but not in PPMS (0.061 +/- 0.052). CTTS index was higher in MS patients with elevated MNC or CSF-restricted oligoclonal IgG bands than in MS patients without these CSF findings. CONCLUSIONS: CTTS index is elevated in RRMS and SPMS. The CTTS index is related to CSF markers of inflammation or immune activation.

Macrophages of multiple sclerosis patients display deficient SHP-1 expression and enhanced inflammatory phenotype.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-kappaB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-kappaB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-kappaB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-kappaB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.

Plasma chitotriosidase activity in multiple sclerosis.

A recent study has shown that chitotriosidase (Chit) may play a role in the pathogenesis of multiple sclerosis (MS). Plasma Chit activity was investigated in 219 untreated MS patients and 160 healthy controls (HC) by means of a fluorometric enzyme activity assay. Chit activity was also measured in a subgroup of 46 patients following treatment with interferon-beta (IFNbeta). Overall, plasma Chit activity was significantly increased in MS patients compared with HC, but no differences were observed between relapsing and progressive clinical forms. In addition, Chit activity was similar between patients during relapse and patients during clinical remission. Treatment with IFNbeta was associated with a significant increase in Chit activity compared with untreated patients in both responders and non-responders to treatment. Although these findings suggest a role of Chit in MS, our data do not support an association between plasma Chit activity and MS clinical course and clinical response to IFNbeta treatment.

Serum NADPH oxidase concentrations and the associations with iron metabolism in relapsing remitting multiple sclerosis.

BACKGROUND: Overproduction of reactive oxygen species (ROS) and impaired iron metabolism are considered to be possible factors in the pathogenesis of Multiple sclerosis (MS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the primary sources of regulated ROS production. The NADPH oxidase (NOX) family consists of seven catalytic homologues, NOX1-5 and two dual oxidases. NOX1 and NOX5 are associated with endothelial dysfunction and inflammation but NOX4 has a protective effect on vascular function. The aims of this study were to investigate the status of NOX1, NOX4 and NOX5 and its relationship with serum iron metabolism biomarkers in relapsing-remitting MS patients. METHODS: The study included 53 RRMS patients and 45 control subjects. Serum NOX1,4,5, ferritin, iron, unbound-iron binding capacity, C-reactive protein (CRP), white blood count (WBC) and erythrocyte sedimentation rate (ESR) levels were measured in all the study subjects. RESULTS: Higher serum NOX5 (p < 0.0001), CRP (p = 0.014), ferritin (p = 0.040) and lower serum NOX4 (p < 0.0001) and iron (p = 0.013) concentrations were found in the patients than in controls. No correlation was found between NOXs, CRP, WBC, ESR and iron metabolism biomarkers in patients. CONCLUSION: Our data suggest that increased NOX5 expression and decreased levels of NOX4 might be related with oxidative stress related vascular changes in MS patients. These findings provide future opportunities to combat MS with separately target individual NOX isoforms.

The value of anti-JCV antibody index assessment in multiple sclerosis patients treated with natalizumab with respect to demographic, clinical and radiological findings.

BACKGROUND: Natalizumab-related progressive multifocal leukoencephalopathy (PML) is associated with the presence of anti-John Cunningham virus (JCV) antibodies. The aim of this investigation was to evaluate the long-term stability of anti-JCV antibody serum levels and their relation to various demographic, clinical and radiological characteristics in patients suffering from multiple sclerosis (MS). METHODS: Seventy-eight relapsing-remitting MS patients treated with natalizumab and evaluated for the presence of serum anti-JCV antibodies over a time period of 1-6 years (3-11 samples) were included in the study. Anti-JCV antibody levels and their changes were correlated with various demographic, clinical and radiological findings. RESULTS: Median follow-up time was 43.5 months, with a median of 5.3 samples available per patient. At baseline, 46 (59%) of the patients were seropositive. During follow-up, anti-JCV antibody status changed from negative to positive or vice versa in 23% of patients. Baseline anti-JCV antibody index correlated positively with age (p = 0.03). PATIENTS: with stable positive anti-JCV antibody index had more T2 hyperintensities (20.2 vs. 13.1; p < 0.007) on cerebral magnetic resonance imaging (MRI) and were also older than the stable negative anti-JCV antibody index group of patients (45.2.vs. 40.3 years; p < 0.01). No significant increase in T2 hyperintensities after seroconversion was revealed. Average duration from beginning of natalizumab therapy to seroconversion (n = 13) was 33 months. Annual seroconversion rate of anti-JCV antibody status was 6.5%. A baseline anti-JCV antibody index of >0.90 (n = 33) predicted stable seropositivity (100%), while baseline anti-JCV antibody index <0.20 did not predicted stable seronegativity (59%). PML was not diagnosed in any of the patients studied during the follow-up. CONCLUSIONS: A positive baseline anti-JCV antibody index of >0.90 predicts stable positive JCV serostatus, in contrast with a baseline anti-JCV antibody index of <0.20, which remained negative in 59% of cases. Stable positive anti-JCV index patients have more MRI T2 hyperintensities and are older compared with stable negative anti-JCV index patients.

Combination therapy with interferon beta-1a and doxycycline in multiple sclerosis: an open-label trial.

OBJECTIVE: To evaluate the efficacy, safety, and tolerability of combination therapy with intramuscular interferon beta-1a and oral doxycycline, a potent inhibitor of matrix metalloproteinases, in patients with relapsing-remitting multiple sclerosis (RRMS) having breakthrough disease activity. DESIGN: Open-label, 7-month trial. SETTING: Louisiana State University Health Sciences Center, Shreveport. PATIENTS: Fifteen patients with RRMS taking interferon beta-1a with breakthrough disease activity took doxycycline for 4 months. Patients underwent monthly neurologic examination, magnetic resonance imaging of the brain using triple-dose gadolinium, and safety blood work. INTERVENTIONS: Ongoing treatment with intramuscular interferon beta-1a plus oral doxycycline, 100 mg daily, for 4 months. MAIN OUTCOME MEASURES: The primary end point was gadolinium-enhancing lesion number change, and the secondary end points were relapse rates, safety and tolerability of the combination of interferon beta-1a and doxycycline in patients with MS, Expanded Disability Status Scale score, serum matrix metalloproteinase-9 levels, and transendothelial migration of monocytes exposed to serum from patients with RRMS. RESULTS: Combination of doxycycline and interferon beta-1a treatment resulted in reductions in contrast-enhancing lesion numbers and posttreatment Expanded Disability Status Scale values (P < .001 for both). Only 1 patient relapsed. Multivariate analyses indicated correlations between decreased serum matrix metalloproteinase-9 levels and enhancing lesion activity reduction. Transendothelial migration of monocytes incubated with serum from patients with RRMS undergoing combination therapy was suppressed. Adverse effects were mild; no adverse synergistic effects of combination therapy or unexpected adverse events were reported. CONCLUSIONS: Combination of intramuscular interferon beta-1a and oral doxycycline treatment was effective, safe, and well tolerated. Controlled clinical trials in larger cohorts of patients with MS are needed to evaluate the efficacy and tolerability of this combination. Trial Registration clinicaltrials.gov Identifier: NCT00246324