The bifunctional microRNA miR-9/miR-9* regulates REST and CoREST and is downregulated in Huntington's disease.

The transcription factor REST silences neuronal gene expression in non-neuronal cells. In neurons, the protein is sequestered in the cytoplasm in part through binding to huntingtin. Polyglutamine expansions in huntingtin, which causes Huntington's disease (HD), abrogates REST-huntingtin binding. Consequently, REST translocates to the nucleus, occupies RE1 repressor sequences and decreases neuronal gene expression. In this work, we found that levels of several microRNAs (miRNAs) with upstream RE1 sites are decreased in HD patient cortices relative to healthy controls. Interestingly, one of these, the bifunctional brain enriched miR-9/miR-9*, targets two components of the REST complex: miR-9 targets REST and miR-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates.

Treatment with the phosphodiesterase type-4 inhibitor rolipram fails to inhibit blood--brain barrier disruption in multiple sclerosis.

Rolipram, a prototypic phosphodiesterase-4 inhibitor, is highly effective in suppressing Th1 autoimmunity in multiple animal models, including experimental autoimmune encephalomyelitis. In addition, rolipram has been extensively studied as a potential neuroprotective agent. Based on its anti-inflammatory activity, we tested the efficacy of rolipram in suppressing inflammatory disease activity in multiple sclerosis in a proof-of-principle phase I/II open-label clinical trial. Enrolled MS patients were evaluated by monthly MRI and clinical examinations during 3 months (four MRIs) of pretreatment baseline and 8 months of rolipram therapy. The primary outcome was a change in contrast-enhanced lesions between baseline and the last 4 months of rolipram therapy. Previously defined biomarkers of rolipram-mediated immunomodulation were evaluated during the study. The trial was stopped prematurely because the drug was poorly tolerated and because of safety concerns: we observed an increase, rather than decrease, in the brain inflammatory activity measured by contrast-enhanced lesions on brain MRI. At the administered doses rolipram was active in vivo as documented by immunological assays. We conclude that the reasons underlying the discrepancy between the therapeutic efficacy of rolipram in experimental autoimmune encephalomyelitis versus multiple sclerosis are at present not clear.

Optimized clonotypic analysis of T-cell receptor repertoire in immune reconstitution.

OBJECTIVE: In recent years, T-cell receptor (TCR) sequencing analysis has proven an effective technique for the identification of T-cell populations of interest in cancer and autoimmunity, as well as for the characterization of peripheral immune repertoire reconstitution after hematopoietic stem cell transplantation (HSCT). However, despite its increased utilization, to our knowledge no group has investigated the minimum number of sequences necessary to accurately and efficiently describe the composition of TCR repertoire. The primary aim of this study was to optimize a procedure for clonotypic analysis of the TCR repertoire in patients undergoing autologous HSCT. MATERIALS AND METHODS: TCR beta-chain diversity was analyzed by DNA sequencing and CDR3 spectratyping CD8(+) T cells isolated from three patients with multiple sclerosis undergoing autologous HSCT. Samples were collected at baseline and 1 or 2 years post-HSCT. RESULTS: Using DNA cloning and high throughput sequencing, we analyzed over 1500 in-frame TCR sequences, allowing us to evaluate how our measures of TCR repertoire diversity change with increasing numbers of sequences included in the analysis. Our findings show that by analyzing 75 to 100 in-frame sequences, we are able to estimate TCR diversity within 5.0% to 7.4% of the values obtained at endpoint analysis (213-312 sequences per sample). CONCLUSIONS: This study confirms the use of TCR sequencing as an effective technique for the characterization of immune renewal after autologous HSCT. In addition, we demonstrate for the first time convincing evidence to support the use of moderate sample sizes to accurately and efficiently evaluate TCR repertoire diversity.

Clonotypic analysis of cerebrospinal fluid T cells during disease exacerbation and remission in a patient with multiple sclerosis.

Migration of autoreactive T cells into the central nervous system (CNS) compartment is thought to be an important step in the pathogenesis of multiple sclerosis (MS). To follow the evolution of T cell repertoire in the CNS of a patient with relapsing-remitting MS, we analyzed cerebrospinal fluid (CSF) cells obtained during an acute clinical exacerbation, and subsequent disease remission after 13 months of immunomodulatory therapy. T cell receptor CDR3 region length distribution was significantly altered during the relapse, demonstrating the presence of clonally expanded T cells in the CSF. CDR3 spectratyping is a valuable approach to identify disease-associated T cells in the CNS.

Effect of anti-CD25 antibody daclizumab in the inhibition of inflammation and stabilization of disease progression in multiple sclerosis.

BACKGROUND: Several questions arise concerning the use of the anti-CD25 antibody daclizumab to treat multiple sclerosis (MS). OBJECTIVES: To answer the following 3 questions related to the efficacy of daclizumab therapy in patients with MS: Is the therapeutic effect of daclizumab dependent on combination with interferon beta? Is a higher dosage of daclizumab more efficacious in patients with persistent disease activity? Can biomarkers predict full vs partial therapeutic response to daclizumab? DESIGN: An open-label baseline vs treatment phase II clinical trial of daclizumab in patients having MS with inadequate response to interferon beta. Three months of interferon beta treatment at baseline were followed by 5.5 months of interferon beta-daclizumab combination therapy. If patients experienced more than 75% reduction of contrast-enhancing lesions (CELs) on brain magnetic resonance imaging at month 5.5 compared with baseline, daclizumab was continued as monotherapy for 10 months. Otherwise, the dosage of daclizumab was doubled. SETTING: Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland. PATIENTS: Fifteen patients with MS receiving standard preparations of interferon beta who experienced more than 1 MS exacerbation or whose clinical disability increased in the preceding 12 months and who had at least 2 CELs on baseline brain magnetic resonance images. INTERVENTION: Daclizumab (1 mg/kg) as an intravenous infusion every 4 weeks in combination with interferon beta (months 0-5.5) and as monotherapy (months 6.5-15.5). MAIN OUTCOME MEASURES: The primary outcome was the reduction of CELs among interferon beta monotherapy, interferon beta-daclizumab combination therapy, and daclizumab monotherapy. The secondary outcomes included immunologic biomarkers and changes in clinical disability. RESULTS: Overall, 5 of 15 patients (33%) experienced adverse effects of therapy. Two patients developed systemic adverse effects, and daclizumab therapy was discontinued. Although daclizumab monotherapy was efficacious in 9 of 13 patients with MS, interferon beta-daclizumab combination therapy was necessary to stabilize disease activity in the other 4 patients. Daclizumab therapy led to 72% inhibition of new CELs and significant improvement in clinical disability. Pilot biomarkers (increase in CD56bright natural killer cells and decrease in CD8+ T cells) were identified that can differentiate between full and partial daclizumab responders. CONCLUSIONS: Daclizumab monotherapy is effective in most patients who experienced persistent MS disease activity with interferon beta therapy. Interferon beta-daclizumab combination therapy or higher dosages of daclizumab may be necessary to achieve optimal therapeutic response in all patients. Biomarkers may identify patients with suboptimal response to daclizumab monotherapy. Administration among a large patient sample during a longer period is needed to fully define the safety and long-term efficacy of daclizumab as treatment for high-inflammatory MS. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00001934.