Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing.

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina's TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.

Interleukin-10 producing-B cells and their association with responsiveness to rituximab in myasthenia gravis.

INTRODUCTION: A subset of regulatory B cells in humans and mice has been defined functionally by their ability to produce interleukin (IL)-10. We characterized IL-10-producing B (B10) cells in myasthenia gravis (MG) patients and correlated them with disease activity and responsiveness to rituximab therapy. METHODS: Frequencies of B10 cells from MG patients and healthy controls were monitored by fluorescence-activated cell sorting (FACS). RESULTS: MG patients had fewer B10 cells than controls, which was associated with more severe disease status. Moreover, patients who responded well to rituximab therapy exhibited rapid repopulation of B10 cells, whereas in patients who did not respond well to rituximab, B10 cell repopulation was delayed. The kinetics of B10 cells were related to the responsiveness to rituximab in MG. CONCLUSIONS: We have characterized a specific subset of B10 cells in MG patients which may serve as a marker for disease activity and responsiveness to immune therapy.

Head tremor secondary to MS resolved with rituximab.

We describe the case of a 33-year-old woman who presented with a 2-month history of worsening head tremor. The medical evaluation led to the new diagnosis of MS and the MRI of brain demonstrated prominently active disease. Intravenous rituximab was started according to the HERMES trial, and significant improvement was noted. She has received additional rituximab dosing approximately every 6 months, and at the 2-year follow-up the tremor has not recurred. The resolution of head tremor likely resulted from the complete suppression of MS disease activity, which must have allowed restoration of normal neural circuitry. In agreement with a growing body of evidence that supports early control of MS disease activity to prevent accumulation of fixed disability, this case advocates for aggressive immunological therapy at the onset of tremor in MS patients.

Cervical spondylosis is a risk factor for localized spinal cord lesions in multiple sclerosis.

OBJECTIVES: To answer the question whether cervical spondylosis would increase the incidence of cord lesions in MS patients, we investigated the statistical association between the two pathologies. METHODS: We extracted demographics, basic disease characteristics and MRI data of a cohort of 304 consecutive MS patients. For a subset of 176 patients, a detailed analysis independently assessed for each cervical level the co-existence of spinal canal narrowing from spondylosis and corresponding cord signal abnormalities. RESULTS: The cohort had typical demographics and in over 80 % of cases there was at least one cord lesion. EDSS correlated with age, disease duration, cerebral lesion burden and spinal cord lesions. After adjusting for either age, disease duration, central lesion burden, or EDSS, the presence of spinal spondylosis was not significantly associated with spinal cord lesions (p > 0.05). In the subset of 176 subjects with the level-by-level spine data, we found a highly statistically significant association (Pearson's χ2 = 23.7, p < 0.001) between canal narrowing and cord lesion at the level directly above or below. This association remained highly significant in both univariable and multivariable logistic regression models adjusting for age, disease duration, MS treatment, cerebral lesion burden and disability scores (p < 0.001). CONCLUSIONS: The data from our cohort of MS patients suggest an indirect contribution of cervical spondylosis to disability by increasing the risk of developing localized cord lesions. While further studies are needed to confirm the findings and clarify disease mechanisms, closer attention should be paid to worsening spondylosis in patients with MS.

Gene expression profiles reveal homeostatic dynamics during interferon-beta therapy in multiple sclerosis.

Understanding the mechanisms that sustain the effects of disease modifying drugs in multiple sclerosis (MS) may help refine current therapies and improve our knowledge of disease pathogenesis. By using cDNA microarrays, we investigated gene expression in the peripheral blood mononuclear cells (PBMC) of 7 MS patients, at baseline (T0) as well as after 1 (T1) and 3 months (T3) of interferon beta-1a (IFN-beta-1a; Rebif 44 microg) therapy. Gene expression changes involved genes of both immunological and non-immunological significance. We validated IL-10 up-regulation, which is in accordance with previous reports, and other novel changes that underscore the capacity of IFN-beta to impair antigen presentation and migration of inflammatory elements into the central nervous system (up-regulation of filamin B and down-regulation of IL-16 and rab7). Overall, gene expression changes became less pronounced after 3 months of therapy, suggesting a homeostatic response to IFN-beta. This may be of use for the design of new treatment schedules.

[DNA-microarray: new technological approaches on twin studies]. / DNA-microarray: nuovi approcci sperimentali per lo studio dei gemelli.

The completion of the Human Genome Project, and the innovations introduced in biotechnology are changing how to study twins. Here, we summarize some molecular studies performed on populations of discordant monozygotic twins (MZ) applying microarrays. Microarrays are an orderly arrangement of high numbers of probes (DNA, RNA or proteins), immobilized onto a matrix. The microarray approach allows a global analysis of gene expression, and therefore might point out the molecular mechanisms of MZ twins' discordance, such as epigenetic mechanisms. The application of microarray to twin studies will help better define, through bioinformatics, the role of genes and environment in the development of human diseases, thus suggesting new diagnostic and therapeutic approaches.

Dimethyl fumarate in the treatment of relapsing-remitting multiple sclerosis: an overview.

Multiple sclerosis (MS) shares an immune-mediated origin with psoriasis. Long-term safety and efficacy data generated in Europe from usage of fumaric acid formulations in the latter disease constituted grounds to investigate their effects in MS patients. Dimethyl fumarate (DMF) was found to be the active principle in those formulations and in vitro studies have demonstrated that DMF has immune-modulatory properties exerted through abilities to divert cytokine production toward a Th2 profile, both on lymphocytes and microglial cells. More importantly, DMF was discovered to impact the anti-oxidative stress cell machinery promoting the transcription of genes downstream to the activation of the nuclear factor (erythroid derived 2)-like2 (NRF2). DMF exposure increases the cytosol concentrations of NRF2, which besides immune regulatory effects, has the potential for cytoprotection on glial cells, oligodendrocytes and neurons. Extensive and rigorous clinical trials have assessed the efficacy and safety of DMF at the dose of 240 mg twice and three times a day in relapsing-remitting MS patients during one phase IIb and two phase III trials. Robust, positive results were obtained across a number of clinical and paraclinical parameters. In one study (DEFINE), the relative reductions of the adjusted annualized relapse rate of the low and high dose regimens in comparison with placebo were 53% and 48%, respectively (p < 0.001 for both comparisons). In the other trial (CONFIRM), DMF decreased the annualized relapse rate in comparison with placebo by 44% in the lower and by 51% in higher dosage group (also p < 0.001). The number and size of lesions as detected by magnetic resonance imaging were also significantly decreased in comparison with the patients receiving DMF at every dosage. Multiple post hoc and subgroup analyses corroborated the clinical data, rendering DMF an appealing medication whose potential for impacting the degenerative aspects of MS remains to be explored.

A whole genome screen for linkage disequilibrium in multiple sclerosis performed in a continental Italian population.

We have systematically screened the genome for evidence of linkage disequilibrium (LD) with multiple sclerosis (MS) by typing 6000 microsatellite markers in case-control and family based (AFBAC) cohorts from the Italian population. DNA pooling was used to reduce the genotyping effort involved. Four DNA pools were considered: cases (224 Italian MS patients), controls (231 healthy Italians), index (185 index cases from trio families) and parents (the 370 parents of the patient included in the Index pool), respectively. After refining analysis of the most promising 14 markers to emerge from this screening process, only marker D2S367 retained evidence for association.

Prolactin and prolactin receptor gene polymorphisms in multiple sclerosis and systemic lupus erythematosus.

Genes encoding for prolactin (PRL) and its receptor (PRLR) are possible candidates for multiple sclerosis (MS) and systemic lupus erythematosus (SLE) susceptibility. In fact: (1) a prolactin secretion dysfunction has been described in several autoimmune diseases including SLE and MS and their animal models; (2) both PRL and PRLR are structurally related to members of the cytokine/hematopoietin family and have a role in the regulation of the immune response; and (3) both PRL and PRLR genes map in genomic regions that showed linkage with autoimmunity. Prolactin maps on chromosome 6p, about 11-kb telomeric to HLA-DRB1 and PRLR in 5p12-13, which revealed evidence of linkage with MS in different populations. To evaluate a possible role of these two genes in SLE and MS we performed an association study of 19 PRL and PRLR single nucleotide polymorphisms (SNPs). These were directly searched by DHPLC in a panel of SLE and MS patients and selected from databases and the literature. The SNP allele frequencies were determined on patient and control DNA pools by primer-extension genotyping and HPLC analysis. Moreover a panel of HLA typed SLE and control individuals were individually genotyped for the PRL G-1149T polymorphism previously described to be associated with SLE. No statistically significant difference in the allele distribution was observed for any of the tested variations.

Extended interval dosing of natalizumab: a two-center, 7-year experience.

BACKGROUND: The enthusiasm for natalizumab, a highly efficacious agent in the treatment of multiple sclerosis (MS), has been tempered by the risks of progressive multifocal leukoencephalopathy associated with its use, and strategies to minimize those risks are of great interest. Extended interval dosing (EID) has been proposed as a way to maintain the efficacy of natalizumab while reducing exposure to it. We reviewed a cohort of patients who received natalizumab at 6-8-week intervals instead of the typical infusions every 4 weeks with the goal to assess if patients on EID had an increase in clinical relapses. METHODS: This is a retrospective review of all patients with MS treated with natalizumab at two MS centers where patients were offered the opportunity to switch to an EID every 6 or 8 weeks. RESULTS: A total of 361 patients received natalizumab for 22 ± 13 months (minimum duration 6 months). Of these, 96 patients received EID natalizumab at some point for 20 ± 11 months (minimum duration 6 months). Over the study period, there was no significant difference between the relapse rate in the monthly dosing (13%) and the EID (13%) groups of patients. CONCLUSION: Natalizumab is effective in controlling MS as very few clinical relapses were observed in our dataset. We found that EID did not compromise the treatment effect as measured by relapse rate and no significant breakthrough disease activity was observed. EID is an optional regimen for maintenance natalizumab therapy, but prospective studies are warranted to determine its efficacy.

Are randomized, blind clinical trials enough to guide individualized decisions for patients with neurologic diseases?

The practice of medicine relies on the patient-physician relationship, knowledge, and clinical judgment. Randomized controlled trials (RCTs) remain the least biased method for studying the effects of interventions in selected populations and are the only method to control adequately for unknown confounders. However, physicians face the limitations of RCTs on a daily basis as they treat relatively unselected populations and individual patients. We explore the benefits and limitations of RCTs for some neurologic disorders, and discuss the difficulties of predicting individualized outcomes and anticipating treatment responses in those heterogeneous conditions. Observational studies and advances in understanding neurologic diseases complement RCTs in decision-making. Considerable challenges remain for personalized medicine; for now, clinicians must rely on their ability to integrate evidence and clinical judgment.

From injection therapies to natalizumab: views on the treatment of multiple sclerosis.

Discoveries of the mechanisms that underlie the pathogenesis of multiple sclerosis have been acquired at an impressive rate over the last few decades and, as a consequence, a growing number of treatments are becoming available for this disease. This review first analyzes the experience from the early stages of the disease-modifying therapies, then, expanding on the concept of early treatment for improved outcomes, it focuses on natalizumab and its major complication, progressive multifocal leukoencephalopathy. We offer views on the risks associated with the use of natalizumab by underscoring the importance of the JC virus serology and by providing preliminary data on our experience with the extended interval dosing of natalizumab. This approach, which advocates individualized treatment plans, raises the question of the minimum effective natalizumab dose. Extended interval dosing suggests efficacy can be maintained while providing advantages of costs and convenience over regular monthly dosing. More data examining this strategy are necessary.

Antibody to α4 integrin suppresses natural killer cells infiltration in central nervous system in experimental autoimmune encephalomyelitis.

Natalizumab inhibits the influx of leukocytes into the central nervous system (CNS) via blockade of alpha-4 subunit of very late activation antigen (VLA)-4. The association of natalizumab therapy with progressive multifocal leukoencephalopathy (PML) suggests a disturbance of CNS immune surveillance in a small percentage of Multiple Sclerosis (MS) patients exposed to the medication. Natural killer (NK) cells are known to play an important role in modulating the evolution of different phases of this lymphocyte mediated disease, and we investigated the effects of natalizumab on the NK cell phenotype and infiltration in the CNS in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Our data show that both resting (from naïve mice) and activated (from EAE mice) NK cells express high levels of VLA-4, and anti-VLA-4 antibody treatment significantly decreases NK cells frequency in the CNS of EAE mice. Moreover, we find that anti-VLA-4 possibly impairs NK cells migratory potential, since unblocked VLA-4 expression levels were downregulated on those NK cells that penetrate the CNS. These data suggest that treatment with antibody to VLA-4 may alter immune surveillance of the CNS by impacting NK cell functions and might contribute to the understanding of the mechanisms leading to the development of PML in some MS patients.

A review of cases of neuromyelitis optica.

BACKGROUND: Under the therapeutic point of view, neuromyelitis optica (NMO) poses major challenges. Patients with NMO manifest severe disability from recurrent demyelinating lesions and the therapies are only partially effective. We performed a retrospective analysis of the records of patients followed at our institution and provide suggestions for management of acute relapses and preventive therapy. METHODS: We searched the electronic database for patients who met criteria for NMO spectrum between January 2003 and June 2009. Patient characteristics, clinical relapses, treatments, neurological status, and medical complications were recorded. RESULTS: In the 18 patients who met the criteria for NMO different regimens of chemotherapies seemed to be modestly effective in preventing clinical relapses. After the year 2006, when rituximab began to be used for NMO patients at our institution, a significant reduction of the relapse rate was observed. After the administration of rituximab, we have systematically been monitoring the percentage of the circulating B cells and we suggest that the clinical relapses occurring while on rituximab therapy correlate with the reconstitution of circulating B cells. CONCLUSIONS: The lack of response to therapies approved for multiple sclerosis demands prompt recognition of NMO patients and the NMO-antibody testing can be critically important for that purpose. We have observed remarkable variability of the disease course with long-lasting relapse-free intervals and clusters of severe, disabling attacks. The best effects in preventing and interrupting the high frequency of relapses is achieved with rituximab whose repeated dosing should be guided by monitoring the circulating B-cell counts.

Multiple sclerosis in twins from continental Italy and Sardinia: a nationwide study.

Knowledge about the balance between heritable and nonheritable risk in multiple sclerosis (MS) is based on twin studies in high-prevalence areas. In a study that avoided ascertainment limitations and directly compared continental Italy (medium-prevalence) and Sardinia (high-prevalence), we ascertained 216 pairs from 34,549 patients. This gives a twinning rate of 0.62% among MS patients, significantly less than that of the general population. In continental Italy, probandwise concordance was 14.5% (95% confidence interval, 5.1-23.8) for monozygotic and 4.0% (95% confidence interval, 0.8-7.1) for dizygotic twins. Results in Sardinia resemble those in northern populations but in limited numbers. Monozygotic concordance was 22.2% (95% confidence interval, 0-49.3) probandwise, but no concordant dizygotic pairs were identified. A questionnaire on 80 items possibly related to disease cause was administered to 70 twin pairs, 135 sporadic patients, and 135 healthy volunteers. Variables positively (7) or negatively (2) associated with predisposition and concordance in twins largely overlapped and were mainly linked to infection. If compared with previous studies, our data demonstrate that penetrance in twins appears to correlate with MS prevalence. They highlight the relevance of nonheritable variables in Mediterranean areas. The apparent underrepresentation of MS among Italian twins draws attention to protective factors, shared by twins, that may influence susceptibility.