Pharmacometabolomics applied to low-dose interleukin-2 treatment in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The immunosuppressive functions of regulatory T lymphocytes (Tregs) are impaired in ALS, and correlate to disease progression. The phase 2a IMODALS trial reported an increase in Treg number in ALS patients following the administration of low-dose (ld) interleukin-2 (IL-2). We propose a pharmacometabolomics approach to decipher metabolic modifications occurring in patients treated with ld-IL-2 and its relationship with Treg response. Blood metabolomic profiles were determined on days D1, D64, and D85 from patients receiving 2 MIU of IL-2 (n = 12) and patients receiving a placebo (n = 12). We discriminated the three time points for the treatment group (average error rate of 42%). Among the important metabolites, kynurenine increased between D1 and D64, followed by a reduction at D85. The percentage increase of Treg number from D1 to D64, as predicted by the metabolome at D1, was highly correlated with the observed value. This study provided a proof of concept for metabolic characterization of the effect of ld-IL-2 in ALS. These data could present advances toward a personalized medicine approach and present pharmacometabolomics as a key tool to complement genomic and transcriptional data for drug characterization, leading to systems pharmacology.

Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote liver allograft tolerance in humans.

BACKGROUND & AIMS: CD4+CD25+Foxp3+ regulatory T cells (Tregs) are essential to maintain immunological tolerance and have been shown to promote liver allograft tolerance in both rodents and humans. Low-dose IL-2 (LDIL-2) can expand human endogenous circulating Tregs in vivo, but its role in suppressing antigen-specific responses and promoting Treg trafficking to the sites of inflammation is unknown. Likewise, whether LDIL-2 facilitates the induction of allograft tolerance has not been investigated in humans. METHODS: We conducted a clinical trial in stable liver transplant recipients 2-6 years post-transplant to determine the capacity of LDIL-2 to suppress allospecific immune responses and allow for the complete discontinuation of maintenance immunosuppression (ClinicalTrials.gov NCT02949492). One month after LDIL-2 was initiated, those exhibiting at least a 2-fold increase in circulating Tregs gradually discontinued immunosuppression over a 4-month period while continuing LDIL-2 for a total treatment duration of 6 months. RESULTS: All participants achieved a marked and sustained increase in circulating Tregs. However, this was not associated with the preferential expansion of donor-reactive Tregs and did not promote the accumulation of intrahepatic Tregs. Furthermore, LDIL-2 induced a marked IFNγ-orchestrated transcriptional response in the liver even before immunosuppression weaning was initiated. The trial was terminated after the first 6 participants failed to reach the primary endpoint owing to rejection requiring reinstitution of immunosuppression. CONCLUSIONS: The expansion of circulating Tregs in response to LDIL-2 is not sufficient to control alloimmunity and to promote liver allograft tolerance, due, at least in part, to off-target effects that increase liver immunogenicity. Our trial provides unique insight into the mechanisms of action of immunomodulatory therapies such as LDIL-2 and their limitations in promoting alloantigen-specific effects and immunological tolerance. CLINICAL TRIALS REGISTRATION: The study is registered at ClinicalTrials.gov (NCT02949492). IMPACT AND IMPLICATIONS: The administration of low-dose IL-2 is an effective way of increasing the number of circulating regulatory T cells (Tregs), an immunosuppressive lymphocyte subset that is key for the establishment of immunological tolerance, but its use to promote allograft tolerance in the setting of clinical liver transplantation had not been explored before. In liver transplant recipients on tacrolimus monotherapy, low-dose IL-2 effectively expanded circulating Tregs but did not increase the number of Tregs with donor specificity, nor did it promote their trafficking to the transplanted liver. Low-dose IL-2 did not facilitate the discontinuation of tacrolimus and elicited, as an off-target effect, an IFNγ-orchestrated inflammatory response in the liver that resembled T cell-mediated rejection. These results, supporting an unexpected role for IL-2 in regulating the immunogenicity of the liver, highlight the need to carefully evaluate systemic immunoregulatory strategies with investigations that are not restricted to the blood compartment and involve target tissues such as the liver.

Amyotrophic lateral sclerosis transcriptomics reveals immunological effects of low-dose interleukin-2.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis.

Repeated 5-day cycles of low dose aldesleukin in amyotrophic lateral sclerosis (IMODALS): A phase 2a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Low-dose interleukin-2 (ld-IL-2) enhances regulatory T-cell (Treg) function in auto-inflammatory conditions. Neuroinflammation being a pathogenic feature of amyotrophic lateral sclerosis (ALS), we evaluated the pharmacodynamics and safety of ld-IL-2 in ALS subjects. METHODS: We performed a single centre, parallel three-arm, randomised, double-blind, placebo-controlled study. Eligibility criteria included age < 75 years, disease duration < 5 years, riluzole treatment > 3 months, and a slow vital capacity ≥ 70% of normal. Patients were randomised (1:1:1) to aldesleukin 2 MIU, 1 MIU, or placebo once daily for 5 days every 4 weeks for 3 cycles. Primary outcome was change from baseline in Treg percentage of CD4+ T cells (%Tregs) following a first cycle. Secondary laboratory outcomes included: %Treg and Treg number following repeated cycles, and plasma CCL2 and neurofilament light chain protein (NFL) concentrations as surrogate markers of efficacy. Safety outcomes included motor-function (ALSFRS-R), slow vital capacity (SVC), and adverse event reports. This trial is registered with ClinicalTrials.gov, NCT02059759. FINDINGS: All randomised patients (12 per group), recruited from October 2015 to December 2015, were alive at the end of follow-up and included in the intent-to-treat (ITT) analysis. No drug-related serious adverse event was observed. Non-serious adverse events occurred more frequently with the 1 and 2 MIU IL-2 doses compared to placebo, including injection site reactions and flu-like symptoms. Primary outcome analysis showed a significant increase (p < 0·0001) in %Tregs in the 2 MIU and 1 MIU arms (mean [SD]: 2 MIU: +6·2% [2·2]; 1 MIU: +3·9% [1·2]) as compared to placebo (mean [SD]: -0·49% [1·3]). Effect sizes (ES) were large in treated groups: 2 MIU ES=3·7 (IC95%: 2·3-4·9) and 1 MIU ES=3·5 (IC95%: 2·1-4·6). Secondary outcomes showed a significant increase in %Tregs following repeated cycles (p < 0·0001) as compared to placebo, and a dose-dependent decrease in plasma CCL2 (p = 0·0049). There were no significant differences amongst the three groups on plasma NFL levels. INTERPRETATION: Ld-IL-2 is well tolerated and immunologically effective in subjects with ALS. These results warrant further investigation into their eventual therapeutic impact on slowing ALS disease progression. FUNDING: The French Health Ministry (PHRC-I-14-056), EU H2020 (grant #633413), and the Association pour la Recherche sur la SLA (ARSLA).

Genome-wide survey of copy number variants finds MAPT duplications in progressive supranuclear palsy.

BACKGROUND: Progressive supranuclear palsy is a neurodegenerative tauopathy manifesting clinically as a progressive akinetic-rigid syndrome. In this study, we sought to identify genetic variants influencing PSP susceptibility through a genome-wide association analysis of a cohort of well-characterized patients who had participated in the Neuroprotection and Natural History in Parkinson Plus Syndromes and Blood Brain Barrier in Parkinson Plus Syndromes studies. METHODS: We genotyped single-nucleotide polymorphisms in 283 PSP cases from the United Kingdom, Germany, and France and compared these with genotypes from 4472 controls. Copy number variants were identified from genotyping data. RESULTS: We observed associations on chromosome 17 within or close to the MAPT gene and explored the genetic architecture at this locus. We confirmed the previously reported association of rs1768208 in the MOBP gene (P = 3.29 × 10-13 ) and rs1411478 in STX6 (P = 3.45 × 10-10 ). The population-attributable risk from the MAPT, MOBP, and STX6 single-nucleotide polymorphisms was found to be 0.37, 0.26, and 0.08, respectively. In addition, we found 2 instances of copy number variants spanning the MAPT gene in patients with PSP. These copy number variants include tau but few other genes within the chromosome 17 haplotype region, providing additional support for the direct pathogenicity of MAPT in PSP. CONCLUSIONS: Clinicians should also be aware of MAPT duplication as a possible genetic cause of PSP, especially in patients presenting with young age at onset. © 2019 International Parkinson and Movement Disorder Society.

Joint genome-wide association study of progressive supranuclear palsy identifies novel susceptibility loci and genetic correlation to neurodegenerative diseases.

BACKGROUND: Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease for which the genetic contribution is incompletely understood. METHODS: We conducted a joint analysis of 5,523,934 imputed SNPs in two newly-genotyped progressive supranuclear palsy cohorts, primarily derived from two clinical trials (Allon davunetide and NNIPPS riluzole trials in PSP) and a previously published genome-wide association study (GWAS), in total comprising 1646 cases and 10,662 controls of European ancestry. RESULTS: We identified 5 associated loci at a genome-wide significance threshold P < 5 × 10- 8, including replication of 3 loci from previous studies and 2 novel loci at 6p21.1 and 12p12.1 (near RUNX2 and SLCO1A2, respectively). At the 17q21.31 locus, stepwise regression analysis confirmed the presence of multiple independent loci (localized near MAPT and KANSL1). An additional 4 loci were highly suggestive of association (P < 1 × 10- 6). We analyzed the genetic correlation with multiple neurodegenerative diseases, and found that PSP had shared polygenic heritability with Parkinson's disease and amyotrophic lateral sclerosis. CONCLUSIONS: In total, we identified 6 additional significant or suggestive SNP associations with PSP, and discovered genetic overlap with other neurodegenerative diseases. These findings clarify the pathogenesis and genetic architecture of PSP.

Stage at which riluzole treatment prolongs survival in patients with amyotrophic lateral sclerosis: a retrospective analysis of data from a dose-ranging study.

BACKGROUND: Riluzole is the only drug to prolong survival for amyotrophic lateral sclerosis (ALS) and, at a dose of 100 mg, was associated with a 35% reduction in mortality in a clinical trial. A key question is whether the survival benefit occurs at an early stage of disease, late stage, or is spread throughout the course of the disease. To address this question, we used the King's clinical staging system to do a retrospective analysis of data from the original dose-ranging clinical trial of riluzole. METHODS: In the original dose-ranging trial, patients were enrolled between December, 1992, and November, 1993, in Belgium, France, Germany, Spain, Canada, the USA, and the UK if they had probable or definite ALS as defined by the El Escorial criteria. The censor date for the riluzole survival data was set as the original study end date of Dec 31, 1994. For this analysis, King's clinical ALS stage was estimated from the electronic case record data of the modified Norris scale, UK Medical Research Council score for muscle strength, El Escorial category, vital capacity, and gastrostomy insertion data. The lowest allocated stage was 2 because the original trial only included patients with probable or definite ALS. We used a χ2 test to assess the independence of stage at trial enrolment and treatment group, Kaplan-Meier product limit distribution to test the transition from each stage to subsequent stages, and Cox regression to confirm an effect of treatment group on time in stage, controlling for covariates. We did sensitivity analyses by combining treatment groups, using alternative strategies to stage, stratifying by stage at trial enrolment, and using multistate outcome analysis of treatments (MOAT). FINDINGS: We analysed the case records of all 959 participants from the original dose-ranging trial, 237 assigned to 50 mg/day riluzole, 236 to 100 mg/day, 244 to 200 mg/day, and 242 to daily placebo. Clinical stage at enrolment did not significantly differ between treatment groups (p=0·22). Time in stage 4 was longer for patients receiving 100 mg/day riluzole than for those receiving placebo (hazard ratio [HR] 0·55, 95% CI 0·36-0·83; log-rank p=0·037). Combining treatment groups and stratifying by stage at enrolment showed a similar result (HR 0·638, 95% CI 0·464-0·878; p=0·006), as did analysis with MOAT where the mean number of days spent in stage 4 was numerically higher for patients given riluzole at higher doses compared with patients receiving placebo. Time from stages 2 or 3 to subsequent stages or death did not differ between riluzole treatment groups and placebo (p=0·83 for stage 2 and 0·88 for stage 3). INTERPRETATION: We showed that riluzole prolongs survival in the last clinical stage of ALS; this finding needs to be confirmed in a prospective study, and treatment effects at stage 1 still need to be analysed. The ALS stage at which benefit occurs is important for counselling of patients before starting treatment. Staging should be used in future ALS clinical trials to assess the stage at which survival benefit occurs, and a similar approach could be used for other neurodegenerative diseases. FUNDING: NIHR Maudsley Biomedical Research Centre, The European Union Joint Programme on Neurodegeneration, and the King's Summer Undergraduate Studentship.

Value of percutaneous radiofrequency ablation with or without percutaneous vertebroplasty for pain relief and functional recovery in painful bone metastases.

OBJECTIVE: To evaluate the effectiveness of percutaneous radiofrequency (RF) ablation with or without percutaneous vertebroplasty (PV) on pain relief, functional recovery and local recurrence at 6 months' follow-up (FU), in patients with painful osseous metastases. MATERIALS AND METHODS: Thirty RF ablations were performed in 24 patients (mean age: 61 years) with bone metastases. Half of the patients had an additional PV. The primary end point was pain relief evaluated by a visual analogue scale (VAS) before treatment, and at 1 and 6 months' FU. Functional outcome was assessed according to the evolution of their ability to walk at 6 months' FU. Imaging FU was available in 20 out of 24 patients with a mean delay of 4.7 months. RESULTS: Reduction of pain was obtained at 6 months FU in 81% of cases (15 out of 18). Mean pretreatment VAS was 6.4 (±2.7). Mean VAS was 1.9 (±2.4) at 1 month FU, and 2.3 (±2.9) at 6 months' FU. Pain was significantly reduced at 6 months FU (mean VAS reduction = 4.1; P < 0.00001). Functional improvement was obtained in 74% of the cases. Major complications rate was 12.5 % (3 out of 24) with 2 skin burns, and 1 case of myelopathy. Local tumour recurrence or progression was recorded in 5 cases. CONCLUSION: Radiofrequency ablation is an effective technique in terms of pain relief and functional recovery for the treatment of bone metastases, which provides a relatively low rate of local recurrence.

Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial.

BACKGROUND: An improper balance of regulatory/effector T (Treg/Teff) cells is central to the development of autoimmune diseases, including type 1 diabetes. We previously showed that low-dose interleukin 2 (IL2) induced Treg cell expansion and activation and clinical improvement in patients with hepatitis-C-virus-induced vasculitis. We aimed to establish which low doses of IL2 would be safe and induce Treg cells in patients with type 1 diabetes, considering that: (1) type 1 diabetes might be linked to alteration of the IL2/IL2R activation pathway; (2) activation of pathogenic Teff cells by IL2 could exacerbate disease; and (3) the safety of low-dose IL2 is not known in type 1 diabetes. METHODS: This was a single-centre phase 1/2 study. 24 adult patients (18-55 years) with established insulin-dependent type 1 diabetes and at least one diabetes-related autoantibody were enrolled and randomly assigned (in a 1:1:1:1 ratio, by computer-generated randomisation list, with block size four) to placebo or IL2 at 0.33 MIU/day, 1 MIU/day, or 3 MIU/day for a 5-day course and were followed up for 60 days. All investigators and participants were masked to assignment. The primary outcome was change in Treg cells, measured by flow cytometry, and expressed as a percentage of CD4+ T cells, from day 1 to day 60. This trial is registered with ClinicalTrials.gov, number NCT01353833. FINDINGS: Six patients were assigned to each group between June 1, 2011, and Feb 3, 2012. IL2 was well tolerated at all doses, with no serious adverse events. However, there was a dose-response association for non-serious adverse events during the treatment phase (days 1-6); one patient in the placebo group, three patients in the 0.33 MIU group, five patients in the 1 MIU group, and six patients in the 3 MIU group had non-serious adverse events. The most common adverse events in the treatment phase were injection-site reaction (no patients with placebo vs three patients with 0.33 MIU and 1 MIU vs two patients with 3 MIU) and influenza-like syndrome (no patients with placebo vs one patient with 0.33 MIU and 1 MIU vs four patients with 3 MIU). After the treatment phase, adverse events did not differ between groups. IL2 did not induce deleterious changes in glucose-metabolism variables. IL2 induced a dose-dependent increase in the proportion of Treg cells, significant at all doses compared with placebo (placebo mean increase 0.5% [SD 0.4]; 0.33 MIU 2.8% [1.2], p=0.0039; 1 MIU 3.9% [1.8], p=0.0039; 3 MIU 4.8% [1.9] p=0.0039). INTERPRETATION: We have defined a well-tolerated and immunologically effective dose range of IL2 for application to type 1 diabetes therapy and prevention, which could be relevant to other disorders in which a Treg cell increase would be desirable.

Elevated serum ferritin is associated with reduced survival in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the loss of motor neurons. Its etiology remains unknown, but several hypothesis have been raised to explain motor neuron death, including oxidative stress. Dysregulation of cellular iron metabolism can lead to increased oxidative stress, and existing data argue for a role of iron metabolism in ALS pathophysiology. METHODS: We performed a retrospective analysis of iron metabolism (IM) variables (serum levels of iron, transferrin, ferritin, and TSC for Transferrin Saturation Coefficient) in a cohort of 694 ALS patients and 297 healthy controls. RESULTS: Serum ferritin levels and TSC were higher, whereas serum transferrin levels were lower in ALS patients than controls. In addition, patients with a high level serum ferritin had a shorter survival time compared to those with low level serum ferritin (618 days versus 921 days for men subgroup; p = .007). Site of onset and ALS-FRS score were not associated with IM variables. CONCLUSION: This study suggests that ALS patients may have increased iron storage, as measured by increased serum ferritin and TSC. Elevated serum ferritin may also have a deleterious impact on survival in ALS.

The economic costs of progressive supranuclear palsy and multiple system atrophy in France, Germany and the United Kingdom.

Progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) are progressive disabling neurological conditions usually fatal within 10 years of onset. Little is known about the economic costs of these conditions. This paper reports service use and costs from France, Germany and the UK and identifies patient characteristics that are associated with cost. 767 patients were recruited, and 760 included in the study, from 44 centres as part of the NNIPPS trial. Service use during the previous six months was measured at entry to the study and costs calculated. Mean six-month costs were calculated for 742 patients. Data on patient sociodemographic and clinical characteristics were recorded and used in regression models to identify predictors of service costs and unpaid care costs (i.e., care from family and friends). The mean six-month service costs of PSP were €24,491 in France, €30,643 in Germany and €25,655 in the UK. The costs for MSA were €28,924, €25,645 and €19,103 respectively. Unpaid care accounted for 68-76%. Formal and unpaid costs were significantly higher the more severe the illness, as indicated by the Parkinson's Plus Symptom scale. There was a significant inverse relationship between service and unpaid care costs.

Disease severity and progression in progressive supranuclear palsy and multiple system atrophy: validation of the NNIPPS--Parkinson Plus Scale.

BACKGROUND: The Natural History and Neuroprotection in Parkinson Plus Syndromes (NNIPPS) study was a large phase III randomized placebo-controlled trial of riluzole in Progressive Supranuclear Palsy (PSP, n = 362) and Multiple System Atrophy (MSA, n = 398). To assess disease severity and progression, we constructed and validated a new clinical rating scale as an ancillary study. METHODS AND FINDINGS: Patients were assessed at entry and 6-montly for up to 3 years. Evaluation of the scale's psychometric properties included reliability (n = 116), validity (n = 760), and responsiveness (n = 642). Among the 85 items of the initial scale, factor analysis revealed 83 items contributing to 15 clinically relevant dimensions, including Activity of daily Living/Mobility, Axial bradykinesia, Limb bradykinesia, Rigidity, Oculomotor, Cerebellar, Bulbar/Pseudo-bulbar, Mental, Orthostatic, Urinary, Limb dystonia, Axial dystonia, Pyramidal, Myoclonus and Tremor. All but the Pyramidal dimension demonstrated good internal consistency (Cronbach α ≥ 0.70). Inter-rater reliability was high for the total score (Intra-class coefficient = 0.94) and 9 dimensions (Intra-class coefficient = 0.80-0.93), and moderate (Intra-class coefficient = 0.54-0.77) for 6. Correlations of the total score with other clinical measures of severity were good (rho ≥ 0.70). The total score was significantly and linearly related to survival (p<0.0001). Responsiveness expressed as the Standardized Response Mean was high for the total score slope of change (SRM = 1.10), though higher in PSP (SRM = 1.25) than in MSA (SRM = 1.0), indicating a more rapid progression of PSP. The slope of change was constant with increasing disease severity demonstrating good linearity of the scale throughout disease stages. Although MSA and PSP differed quantitatively on the total score at entry and on rate of progression, the relative contribution of clinical dimensions to overall severity and progression was similar. CONCLUSIONS: The NNIPPS-PPS has suitable validity, is reliable and sensitive, and therefore is appropriate for use in clinical studies with PSP or MSA. TRIAL REGISTRATION: ClinicalTrials.gov NCT00211224.

A new MRI rating scale for progressive supranuclear palsy and multiple system atrophy: validity and reliability.

AIM: To evaluate a standardised MRI acquisition protocol and a new image rating scale for disease severity in patients with progressive supranuclear palsy (PSP) and multiple systems atrophy (MSA) in a large multicentre study. METHODS: The MRI protocol consisted of two-dimensional sagittal and axial T1, axial PD, and axial and coronal T2 weighted acquisitions. The 32 item ordinal scale evaluated abnormalities within the basal ganglia and posterior fossa, blind to diagnosis. Among 760 patients in the study population (PSP = 362, MSA = 398), 627 had per protocol images (PSP = 297, MSA = 330). Intra-rater (n = 60) and inter-rater (n = 555) reliability were assessed through Cohen's statistic, and scale structure through principal component analysis (PCA) (n = 441). Internal consistency and reliability were checked. Discriminant and predictive validity of extracted factors and total scores were tested for disease severity as per clinical diagnosis. RESULTS: Intra-rater and inter-rater reliability were acceptable for 25 (78%) of the items scored (≥ 0.41). PCA revealed four meaningful clusters of covarying parameters (factor (F) F1: brainstem and cerebellum; F2: midbrain; F3: putamen; F4: other basal ganglia) with good to excellent internal consistency (Cronbach α 0.75-0.93) and moderate to excellent reliability (intraclass coefficient: F1: 0.92; F2: 0.79; F3: 0.71; F4: 0.49). The total score significantly discriminated for disease severity or diagnosis; factorial scores differentially discriminated for disease severity according to diagnosis (PSP: F1-F2; MSA: F2-F3). The total score was significantly related to survival in PSP (p<0.0007) or MSA (p<0.0005), indicating good predictive validity. CONCLUSIONS: The scale is suitable for use in the context of multicentre studies and can reliably and consistently measure MRI abnormalities in PSP and MSA. Clinical Trial Registration Number The study protocol was filed in the open clinical trial registry (http://www.clinicaltrials.gov) with ID No NCT00211224.

Regulatory T cell content in the bone marrow graft does not predict the occurrence of acute GVHD.

The subpopulation of regulatory T cells (Treg) was shown to play a key role in alloreactive responses. In allogeneic hematopoietic stem cell transplantation, several groups tested whether Treg content in transplants correlates with graft-versus-host disease (GVHD) with controversial results. In a retrospective study of 49 consecutive HLA-matched sibling transplantations, we studied the relationship between Treg content in bone marrow transplants and acute GVHD (aGVHD) occurrence. We observed a large variability in Treg in bone marrow grafts. However, contrary to previous observations in peripheral blood stem cells transplantation, we report that the Treg content of allogeneic bone marrow transplantation did not predict the occurrence of aGVHD.

Platelet serotonin level predicts survival in amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease involving upper and lower motor neurons loss. Clinical features are highly variable among patients and there are currently few known disease-modifying factors underlying this heterogeneity. Serotonin is involved in a range of functions altered in ALS, including motor neuron excitability and energy metabolism. However, whether serotoninergic activity represents a disease modifier of ALS natural history remains unknown. METHODOLOGY: Platelet and plasma unconjugated concentrations of serotonin and plasma 5-HIAA, the major serotonin metabolite, levels were measured using HPLC with coulometric detection in a cohort of 85 patients with ALS all followed-up until death and compared to a control group of 29 subjects. PRINCIPAL FINDINGS: Platelet serotonin levels were significantly decreased in ALS patients. Platelet serotonin levels did not correlate with disease duration but were positively correlated with survival of the patients. Univariate Cox model analysis showed a 57% decreased risk of death for patients with platelet serotonin levels in the normal range relative to patients with abnormally low platelet serotonin (p = 0.0195). This protective effect remained significant after adjustment with age, gender or site of onset in multivariate analysis. Plasma unconjugated serotonin and 5-HIAA levels were unchanged in ALS patients compared to controls and did not correlate with clinical parameters. CONCLUSIONS/SIGNIFICANCE: The positive correlation between platelet serotonin levels and survival strongly suggests that serotonin influences the course of ALS disease.

Cognitive impairment in patients with multiple system atrophy and progressive supranuclear palsy.

This article reports the severity and profile of neuropsychological impairment on a prevalent cohort of patients with a clinical diagnosis of either multiple system atrophy (n=372) or progressive supranuclear palsy (n=311) from the Neuroprotection and Natural History in Parkinson Plus Syndromes cohort. The Dementia Rating Scale and Frontal Assessment Battery were used to assess global cognition and executive dysfunction. For the Dementia Rating Scale impairment was observed in approximately 57% of the progressive supranuclear palsy group and 20% of the multiple system atrophy group. In the former, impairment in a single cognitive domain was observed in 40%, with the same number showing impairment in multiple domains, while in the latter the figures were 28.6 and 13.5%, respectively. On the Frontal Assessment Battery, impairment was observed in 62.0% of patients with progressive supranuclear palsy and 31.8% of those with multiple system atrophy. Although the progressive supranuclear palsy group performed worse overall, the cognitive profiles of the two groups on the Dementia Rating Scale subscales were identical, with the main impairment of the Initiation and Perseveration subscale. The impaired patients in the two groups were largely indistinguishable, qualitatively and quantitatively. Impairment was associated with greater age and clinical disability in both groups and was evident even in the early stages (22% in multiple system atrophy and 50% in progressive supranuclear palsy). Where a pathological diagnosis was available, the original clinical diagnosis was confirmed in the majority of cases, including those with significant cognitive impairment. The rate of impairment in those with a confirmed pathological diagnosis was comparable to that of the sample as a whole. These results demonstrate, in the largest prospectively recruited cohort of patients with progressive supranuclear palsy and multiple system atrophy studied to date, the existence of a cognitive profile similar to that previously reported in idiopathic Parkinson's disease. The results indicate a high level of cognitive impairment associated with progressive supranuclear palsy, but also point to comparable dysfunction in a substantial proportion of the patients with multiple system atrophy. Significant cognitive impairment appears consistent with a diagnosis of multiple system atrophy, even early in the disease, with important implications for diagnosis, research and management.

P-glycoprotein expression and function are increased in an animal model of amyotrophic lateral sclerosis.

The efflux pumps located at the blood-brain barrier (BBB) prevent drugs entering the brain. As such, efflux pumps are a major obstacle to drug brain distribution. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with little therapeutics available: riluzole is the only drug approved in its treatment. The lack of response to treatment in ALS may be, at least in part, due to increased activities of efflux pumps in relation to disease, leading to subtherapeutic brain concentrations of drugs. In the present study, we used a transgenic mouse model of ALS (G86R mSOD1 mice) to test this hypothesis. Expression and functionality of P-glycoprotein (ABCB1, P-gp) and Breast Cancer Resistance Protein (ABCG2, BCRP), two major efflux pumps, were studied. We observed an increased P-gp expression (1.5-fold) in presymptomatic mSOD1 mice compared to wild-type controls. Consistent with this, P-gp function was also increased by 1.5-fold and riluzole brain disposition was decreased by 1.7-fold in mSOD1 mice. Contrasting with this, BCRP expression and function were unaltered by the pathology. These results demonstrate that BBB transport proteins are modified in G86R mSOD1 mice ALS model. Such findings underline potential problems in extrapolating the results of animal studies to humans and developing clinical trials, especially for drugs transported by P-gp.

Genetic variants of the alpha-synuclein gene SNCA are associated with multiple system atrophy.

BACKGROUND: Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism, cerebellar ataxia and autonomic dysfunction. Pathogenic mechanisms remain obscure but the neuropathological hallmark is the presence of alpha-synuclein-immunoreactive glial cytoplasmic inclusions. Genetic variants of the alpha-synuclein gene, SNCA, are thus strong candidates for genetic association with MSA. One follow-up to a genome-wide association of Parkinson's disease has identified association of a SNP in SNCA with MSA. METHODOLOGY/FINDINGS: We evaluated 32 SNPs in the SNCA gene in a European population of 239 cases and 617 controls recruited as part of the Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) study. We used 161 independently collected samples for replication. Two SNCA SNPs showed association with MSA: rs3822086 (P = 0.0044), and rs3775444 (P = 0.012), although only the first survived correction for multiple testing. In the MSA-C subgroup the association strengthened despite more than halving the number of cases: rs3822086 P = 0.0024, OR 2.153, (95% CI 1.3-3.6); rs3775444 P = 0.0017, OR 4.386 (95% CI 1.6-11.7). A 7-SNP haplotype incorporating three SNPs either side of rs3822086 strengthened the association with MSA-C further (best haplotype, P = 8.7 x 10(-4)). The association with rs3822086 was replicated in the independent samples (P = 0.035). CONCLUSIONS/SIGNIFICANCE: We report a genetic association between MSA and alpha-synuclein which has replicated in independent samples. The strongest association is with the cerebellar subtype of MSA. TRIAL REGISTRATION: ClinicalTrials.gov NCT00211224.

Brain and plasma riluzole pharmacokinetics: effect of minocycline combination.

PURPOSE: amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by the loss of motorneurons. The only drug approved is riluzole. Minocycline is an antibiotic with numerous neuroprotective properties. riluzole and minocycline were given to an animal model of ALS and had beneficial effect on the disease. The combination was then tested in humans in phase II and phase III studies with less beneficial effects and a faster decline of the disease in the group treated with minocycline. In a previous study, we showed that riluzole is transported out of the brain by the P-glycoprotein at the blood-brain barrier level. METHODS: in this work, we studied in CF1 mice, the plasmatic and cerebral pharmacokinetics of riluzole combined or not with minocycline. RESULTS: our results showed that the kinetics of riluzole are not linear with dose, but that cerebral AUC0-infinity increase proportionally with plasmatic AUC0-infinity. At the dose of 10 mg/kg, the cerebral AUC0-infinity /plasmatic AUC0-infinity ratio was 4.6 in mdr1a (-/-) mice and 2.4 in mdr1a (+/+) mice. The combination of minocycline (170 mg/kg) and riluzole (10 mg/kg) induced a 2 fold increase in the cerebral AUC0-infinity of riluzole and induced a neuromuscular toxicity in mice. This effect of minocycline was not found at low concentration (10 mg/kg of minocycline). CONCLUSIONS: if our results are confirmed in humans, riluzole cerebral concentrations could be predicted by plasmatic concentrations. Furthermore, the combination of high doses of minocycline with riluzole could induce neurological toxicity that lead to deceiving results in ALS clinical studies.