Targeting the adenosinergic system in restless legs syndrome: A pilot, "proof-of-concept" placebo-controlled TMS-based protocol.

Restless Legs Syndrome (RLS) is a common sleep disorder characterized by an urge to move the legs that is responsive to movement (particularly during rest), periodic leg movements during sleep, and hyperarousal. Recent evidence suggests that the involvement of the adenosine system may establish a connection between dopamine and glutamate dysfunction in RLS. Transcranial magnetic stimulation (TMS) is a non-invasive electrophysiological technique widely applied to explore brain electrophysiology and neurochemistry under different experimental conditions. In this pilot study protocol, we aim to investigate the effects of dipyridamole (a well-known enhancer of adenosinergic transmission) and caffeine (an adenosine receptor antagonist) on measures of cortical excitation and inhibition in response to TMS in patients with primary RLS. Initially, we will assess cortical excitability using both single- and paired-pulse TMS in patients with RLS. Then, based on the measures obtained, we will explore the effects of dipyridamole and caffeine, in comparison to placebo, on various TMS parameters related to cortical excitation and inhibition. Finally, we will evaluate the psycho-cognitive performance of RLS patients to screen them for cognitive impairment and/or mood-behavioral dysfunction, thus aiming to correlate psycho-cognitive findings with TMS data. Overall, this study protocol will be the first to shed lights on the neurophysiological mechanisms of RLS involving the modulation of the adenosine system, thus potentially providing a foundation for innovative "pharmaco-TMS"-based treatments. The distinctive TMS profile observed in RLS holds indeed the potential utility for both diagnosis and treatment, as well as for patient monitoring. As such, it can be considered a target for both novel pharmacological (i.e., drug) and non-pharmacological (e.g., neuromodulatory), "TMS-guided", interventions.

A Next-Generation Sequencing Study in a Cohort of Sicilian Patients with Parkinson's Disease.

Parkinson's disease (PD) is a multisystem and multifactorial disorder and, therefore, the application of modern genetic techniques may assist in unraveling its complex pathophysiology. We conducted a clinical-demographic evaluation of 126 patients with PD, all of whom were Caucasian and of Sicilian ancestry. DNA was extracted from the peripheral blood for each patient, followed by sequencing using a Next-Generation Sequencing system. This system was based on a custom gene panel comprising 162 genes. The sample underwent further filtering, taking into account the allele frequencies of genetic variants, their presence in the Human Gene Mutation Database, and their association in the literature with PD or other movement/neurodegenerative disorders. The largest number of variants was identified in the leucine-rich repeat kinase 2 (LRRK2) gene. However, variants in other genes, such as acid beta-glucosidase (GBA), DNA polymerase gamma catalytic subunit (POLG), and parkin RBR E3 ubiquitin protein ligase (PRKN), were also discovered. Interestingly, some of these variants had not been previously associated with PD. Enhancing our understanding of the genetic basis of PD and identifying new variants possibly linked to the disease will contribute to improved diagnostic accuracy, therapeutic developments, and prognostic insights for affected individuals.

H-magnetic resonance spectroscopy: diagnostic tool in recurrent headache in systemic lupus erythematosus. A case report.

We describe serial MR-spectroscopy studies in a patient with systemic lupus erythematosus and headache. We used MR-spectroscopy to monitor disease activity during periods with and without headache. MR-spectroscopy investigates metabolic alterations and was used to explore the pathophysiological mechanism involved in the complications of systemic lupus erythematosus. Our patient underwent serial conventional MRI and MR-spectroscopy at times of controlled and uncontrolled headache, with or without visual aura. MR-spectroscopy showed an increase in the choline/creatine ratio in thalamus and posterior white matter only during periods of uncontrolled headache with visual aura. Conventional MRI scans were normal at all times. MR-spectroscopy should be used in the diagnosis and follow-up of headache in patients with systemic lupus erythematosus.

Disruption of neurite morphology parallels MS progression.

OBJECTIVES: To apply advanced diffusion MRI methods to the study of normal-appearing brain tissue in MS and examine their correlation with measures of clinical disability. METHODS: A multi-compartment model of diffusion MRI called neurite orientation dispersion and density imaging (NODDI) was used to study 20 patients with relapsing-remitting MS (RRMS), 15 with secondary progressive MS (SPMS), and 20 healthy controls. Maps of NODDI were analyzed voxel-wise to assess the presence of abnormalities within the normal-appearing brain tissue and the association with disease severity. Standard diffusion tensor imaging (DTI) parameters were also computed for comparing the 2 techniques. RESULTS: Patients with MS showed reduced neurite density index (NDI) and increased orientation dispersion index (ODI) compared with controls in several brain areas (p < 0.05), with patients with SPMS having more widespread abnormalities. DTI indices were also sensitive to some changes. In addition, patients with SPMS showed reduced ODI in the thalamus and caudate nucleus. These abnormalities were associated with scores of disease severity (p < 0.05). The association with the MS functional composite score was higher in patients with SPMS compared with patients with RRMS. CONCLUSIONS: NODDI and DTI findings are largely overlapping. Nevertheless, NODDI helps interpret previous findings of increased anisotropy in the thalamus of patients with MS and are consistent with the degeneration of selective axon populations.

JC Virus-DNA Detection Is Associated with CD8 Effector Accumulation in Peripheral Blood of Patients with Multiple Sclerosis under Natalizumab Treatment, Independently from JC Virus Serostatus.

Although natalizumab (anti-α4 integrin) represents an effective therapy for relapsing remitting multiple sclerosis (RRMS), it is associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). The aim of this study was to explore natalizumab-induced phenotypic changes in peripheral blood T-lymphocytes and their relationship with JCV reactivation. Forty-four patients affected by RRMS were enrolled. Blood and urine samples were classified according to natalizumab infusion number: 0 (N0), 1-12 (N12), 13-24 (N24), 25-36 (N36), and over 36 (N > 36) infusions. JCV-DNA was detected in plasma and urine. T-lymphocyte phenotype was evaluated with flow cytometry. JCV serostatus was assessed. Ten healthy donors (HD), whose ages and sexes matched with the RRMS patients of the N0 group, were enrolled. CD8 effector (CD8 E) percentages were increased in natalizumab treated patients with detectable JCV-DNA in plasma or urine compared to JCV-DNA negative patients (JCV-) (p < 0.01 and p < 0.001, resp.). Patients with CD8 E percentages above 10.4% tended to show detectable JCV-DNA in plasma and/or urine (ROC curve p = 0.001). The CD8 E was increased when JCV-DNA was detectable in plasma or urine, independently from JCV serology, for N12 and N24 groups (p < 0.01). As long as PML can affect RRMS patients under natalizumab treatment with a negative JCV serology, the assessment of CD8 E could help in the evaluation of JCV reactivation.

NMR-based metabolomic approach to study urine samples of chronic inflammatory rheumatic disease patients.

The nuclear magnetic resonance (NMR)-based metabolomic approach was used as analytical methodology to study the urine samples of chronic inflammatory rheumatic disease (CIRD) patients. The urine samples of CIRD patients were compared to the ones of both healthy subjects and patients with multiple sclerosis (MS), another immuno-mediated disease. Urine samples collected from 39 CIRD patients, 25 healthy subjects, and 26 MS patients were analyzed using 1H NMR spectroscopy, and the NMR spectra were examined using partial least squares-discriminant analysis (PLS-DA). PLS-DA models were validated by a double cross-validation procedure and randomization tests. Clear discriminations between CIRD patients and healthy controls (average diagnostic accuracy 83.5 ± 1.9%) as well as between CIRD patients and MS patients (diagnostic accuracy 81.1 ± 1.9%) were obtained. Leucine, alanine, 3-hydroxyisobutyric acid, hippuric acid, citric acid, 3-hydroxyisovaleric acid, and creatinine contributed to the discrimination; all of them being in a lower concentration in CIRD patients as compared to controls or to MS patients. The application of NMR metabolomics to study these still poorly understood diseases can be useful to better clarify the pathologic mechanisms; moreover, as a holistic approach, it allowed the detection of, by means of anomalous metabolic traits, the presence of other pathologies or pharmaceutical treatments not directly connected to CIRDs, giving comprehensive information on the general health state of individuals. Graphical abstract NMR-based metabolomic approach as a tool to study urine samples in CIRD patients with respect to MS patients and healthy controls.

Natalizumab Affects T-Cell Phenotype in Multiple Sclerosis: Implications for JCV Reactivation.

The anti-CD49d monoclonal antibody natalizumab is currently an effective therapy against the relapsing-remitting form of multiple sclerosis (RRMS). Natalizumab therapeutic efficacy is limited by the reactivation of the John Cunningham polyomavirus (JCV) and development of progressive multifocal leukoencephalopathy (PML). To correlate natalizumab-induced phenotypic modifications of peripheral blood T-lymphocytes with JCV reactivation, JCV-specific antibodies (serum), JCV-DNA (blood and urine), CD49d expression and relative abundance of peripheral blood T-lymphocyte subsets were longitudinally assessed in 26 natalizumab-treated RRMS patients. Statistical analyses were performed using GraphPad Prism and R. Natalizumab treatment reduced CD49d expression on memory and effector subsets of peripheral blood T-lymphocytes. Moreover, accumulation of peripheral blood CD8+ memory and effector cells was observed after 12 and 24 months of treatment. CD4+ and CD8+ T-lymphocyte immune-activation was increased after 24 months of treatment. Higher percentages of CD8+ effectors were observed in subjects with detectable JCV-DNA. Natalizumab reduces CD49d expression on CD8+ T-lymphocyte memory and effector subsets, limiting their migration to the central nervous system and determining their accumulation in peripheral blood. Impairment of central nervous system immune surveillance and reactivation of latent JCV, can explain the increased risk of PML development in natalizumab-treated RRMS subjects.

Early versus delayed rehabilitation treatment in hemiplegic patients with ischemic stroke: proprioceptive or cognitive approach?

BACKGROUND: Early/intensive mobilization may improve functional recovery after stroke but it is not clear which kind of "mobilization" is more effective. Proprioceptive neuromuscular facilitation (PNF) and cognitive therapeutic exercise (CTE) are widespread applied in post-stroke rehabilitation but their efficacy and safety have not been systematically investigated. AIM: To compare PNF and CTE methods in a two different time setting (early versus standard approach) in order to evaluate different role of time and techniques in functional recovery after acute ischemic stroke. DESIGN: We designed a prospectical multicenter blinded interventional study of early versus standard approach with two different methods by means of both PNF and CTE. SETTING: A discrete stroke-dedicated area for out-of-thrombolysis patients, connected with two different comprehensive stroke centres in two different catchment areas. POPULATION: Three hundred and forty consecutive stroke patient with first ever sub-cortical ischemic stroke in the mean cerebral artery (MCA) territory and contralateral hemiplegia admitted within 6 and 24 hours from symptoms onset. METHODS: All patients were randomly assigned by means of a computer generated randomization sequence in blocks of 4 to one to the 4 interventional groups: early versus delayed rehabilitation programs with Kabat's schemes or Perfetti's technique. Patients in both delayed group underwent to a standard protocol in the acute phase. PRIMARY OUTCOME: disability at 3-12 months. Disability measures: modified Rankin Score and Barthel Index. Safety outcome: immobility-related adverse events. SECONDARY OUTCOME MEASURES: Six-Minute Walking Test, Motricity Index, Mini-Mental State Examination, Beck Depression Inventory. RESULTS: Disability was not different between groups at 3 months but Barthel Index significantly changed between early versus delayed groups at 12 months (P=0.01). Six-Minute Walking Test (P=0.01) and Motricity Index in both upper (P=0.01) and lower limbs (P=0.001) increased in early versus delayed groups regardless rehabilitation schedule. CONCLUSIONS: A time-dependent effect of rehabilitation on post stroke motor recovery was observed, particularly in lower limb improvement. According to our results, rehabilitation technique seems not to affect long term motor recovery. CLINICAL REHABILITATION IMPACT: These results show a significant effect of time but not of technique that may impact the decision making in the acute phase of care.

Human polyomavirus JC replication and non-coding control region analysis in multiple sclerosis patients under natalizumab treatment.

In the last years, the treatment of multiple sclerosis (MS) patients with natalizumab has been associated with the occurrence of progressive multifocal leukoencephalopathy (PML) caused by human polyomavirus JC (JCV). Here, we have shown a significant correlation between patients with JC viruria and positive JC-specific antibody response and patients without JCV-specific antibodies after 1 year of natalizumab (p = 0.0006). Furthermore, JCV-specific quantitative PCR on urine and plasma samples, collected at the enrollment (t0) and every 4 months (t1, t2, t3) in the first year and at two time points (t4 and t5) in the second year of natalizumab treatment, indicated the prevalence of JC viremia rather than JC viruria only in the second year of treatment (p = 0.04). Moreover, the analysis of JCV non-coding control region (NCCR) sequences in peripheral blood mononuclear cells of patients with JC-specific antibodies after 12 natalizumab infusions (t3) revealed the presence of rearranged sequences, whereas the prevalence of genotypes 1A, 1B, and 4 was detected in these patients by VP1 sequence analysis. In summary, JC viruria evaluation seems to be useful to identify early those patients who do not already develop a humoral immune response against JCV. It may also be interesting to study the JCV NCCR rearrangements since they could give us new insights on the onset of neuro-invasive viral variants.

Intracranial hemodynamic changes in primary Sjögren syndrome: a transcranial Doppler case-control study.

The aim of this case-control study is to evaluate the cerebral hemodynamic parameters in primary Sjögren syndrome patients by means of transcranial Doppler and the possible relationship with neuroimaging structural alteration, immunological markers and subclinical neurological involvement. 87 consecutive treatment-naïve outpatients with primary Sjögren syndrome and 86 age- and sex-matched healthy controls underwent transcranial Doppler for bilateral measurement of mean flow velocities, pulsatility index and systolic-diastolic ratio, brain magnetic resonance imaging, clinical evaluation with neuropsychological test and serological assessment. 28 patients and 4 controls (32 vs. 4 %, p .001) had executive function disorders at neuropsychological tests. Mean pulsatility index and systolic-diastolic ratio were significantly higher in both mean cerebral arteries of the patients than in controls (1.3 ± 0.6 vs. 0.9 ± 0.6, p .01 and 3.4 ± 1.7 vs. 1.6 ± 0.7, p .001, respectively). White matter hyperintensities were present in 21 patients and 18 controls. Only age was significantly associated with WMHs in both groups (p < .0001). The increase in systolic-diastolic ratio significantly correlates with neuropsychological impairment. Anti-SSA autoantibodies positively correlate with impaired systolic-diastolic ratio and with neuropsychiatric symptoms. The correlation between haemodynamic changes and anti-SSA autoantibodies suggests a role for the autoimmune response in determining early cerebral hemodynamic dysfunctions. The functional impairment of the endothelium may play a pivotal role in vasomotor dysfunction before any organic damage. The subsequent structural damage of the arterial wall may be responsible for the increase in resistances in small cerebral arteries and sustained hypoperfusion.

Atorvastatin treatment and carotid plaque morphology in first-ever atherosclerotic transient ischemic attack/stroke: a case-control study.

BACKGROUND: A relationship between echolucency of carotid plaques and the consequent risk of ipsilateral ischemic stroke has been observed. An aggressive lipid-lowering therapy may increase the echogenicity of carotid plaque in patients with elevated low-density lipoprotein cholesterol levels. The aim of this study is to prospectively evaluate the long-term effect of high-dose atorvastatin on carotid plaque morphology in patients with first-ever transient ischemic attack or stroke. METHODS: All patients with symptomatic first ischemic atherosclerotic cerebrovascular event occurred within the previous 10 days were enrolled. Carotid Doppler ultrasound of the neck vessels with 7-11 MHz probe for the definition of the atherosclerotic carotid framework was performed. The analysis of the gray-scale median (GSM) of each plate was carried out with image processing software. RESULTS: A total of 240 symptomatic plaques were included and divided into 3 groups: 80 in group A (atorvastatin 80 mg), 80 in group B (atorvastatin 40 mg), and 80 to group C (no atorvastatin). GSM score increases significantly more extensive in group A than in group B (+48.65 vs. +39.46, P < .02) and group C (+48.65 vs. 19.3, P = .0002). An inverse association between reduction of low-density lipoprotein and the increase in the GSM score (r = -.456, P = .007) has been observed. Moreover, the reduction of high-sensitive C-reactive protein correlates inversely with the increase of the GSM (r = -.398, P = .021). CONCLUSIONS: Dose-dependent effect of atorvastatin on symptomatic carotid plaque morphology may suggest a specific role of this drug in the atherosclerotic stroke prevention.

Ultrasonographic evaluation of cerebral arterial and venous haemodynamics in multiple sclerosis: a case-control study.

OBJECTIVE: Although recent studies excluded an association between Chronic Cerebrospinal Venous Insufficiency and Multiple Sclerosis (MS), controversial results account for some cerebrovascular haemodynamic impairment suggesting a dysfunction of cerebral autoregulation mechanisms. The aim of this cross-sectional, case-control study is to evaluate cerebral arterial inflow and venous outflow by means of a non-invasive ultrasound procedure in Relapsing Remitting (RR), Primary Progressive (PP) Multiple Sclerosis and age and sex-matched controls subjects. MATERIAL AND METHODS: All subjects underwent a complete extra-intracranial arterial and venous ultrasound assessment with a color-coded duplex sonography scanner and a transcranial doppler equipment, in both supine and sitting position by means of a tilting chair. Basal arterial and venous morphology and flow velocities, postural changes in mean flow velocities (MFV) of middle cerebral arteries (MCA), differences between cerebral venous outflow (CVF) in clinostatism and in the seated position (ΔCVF) and non-invasive cerebral perfusion pressure (CPP) were evaluated. RESULTS: 85 RR-MS, 83 PP-MS and 82 healthy controls were included. ΔCVF was negative in 45/85 (52.9%) RR-MS, 63/83 (75.9%) PP-MS (p = 0.01) and 11/82 (13.4%) controls (p<0.001), while MFVs on both MCAs in sitting position were significantly reduced in RR-MS and PP-MS patients than in control, particularly in EDSS ≥ 5 subgroup (respectively, 42/50, 84% vs. 66/131, 50.3%, p<0.01 and 48.3 ± 2 cm/s vs. 54.6 ± 3 cm/s, p = 0.01). No significant differences in CPP were observed within and between groups. CONCLUSIONS: The quantitative evaluation of cerebral blood flow (CBF) and CVF and their postural dependency may be related to a dysfunction of autonomic nervous system that seems to characterize more disabled MS patients. It's not clear whether the altered postural control of arterial inflow and venous outflow is a specific MS condition or simply an "epiphenomenon" of neurodegenerative events.

MR venography in patients with multiple sclerosis and correlation with clinical and MRI parameters.

BACKGROUND AND PURPOSE: Multiple sclerosis (MS) has been associated with chronic cerebrospinal venous insufficiency. We aim to evaluate the correlation between extracranial veins stenosis evaluated with MR venography (MRV) and clinical/MR parameters of MS. METHODS: In 29 consecutive MS patients we performed a standard brain MRI protocol, completed by the evaluation of extra-cerebral venous system using a phase-contrast and a Volumetric Interpolated Breath Hold Examination (VIBE) sequence before and after gadolinium. The T2-proton density images were used to calculate the lesion volume. The jugular veins were evaluated qualitatively (in terms of presence and severity of stenoses) and quantitatively (degree of stenosis). The phase-contrast images were analyzed to calculate the average and peak velocity in the internal jugular veins. RESULTS: Postcontrast VIBE successfully showed the jugular veins in all the subjects. T2-lesion-volume was 8.2 [4.6] cm³. A stenosis of the internal jugular veins > of 50% was observed in 10/29(33%) patients. No significant correlation was observed between T2-lesion-volume and degree-of-stenosis (r = .362, P = .302). No different flow parameters were found in the subgroups of patients with and without stenosis (P = .54). CONCLUSIONS: In MS the presence/severity of jugular vein stenosis identified with 3T-MRV is not related to MR-visible tissue damage. Moreover no abnormal flow parameters were found in stenosed veins.

Neurological involvement in primary Sjögren syndrome: a focus on central nervous system.

OBJECTIVES: Sjögren syndrome is an autoimmune disease involving mainly salivary and lacrimal glands. Beyond widely described PNS involvement, high variable prevalence of CNS manifestations ranging from 2.5 and 60% of all pSS patients has been reported, without specific syndrome definition. The aim of this cohort study was to evaluate the prevalence of CNS signs and symptoms in pSS patients and to identify possible biomarkers of CNS damage. METHODS: 120 patients with pSS diagnosis according to the 2002 American-European Consensus Group criteria were enrolled after exclusion of secondary causes. All patients underwent to a wide neurological, neuropsychological, psychiatric, neuroradiological and ultrasonographic evaluation. RESULTS: Central and peripheral nervous system involvement was observed in 81 patients with a prevalence of 67.5%. The prevalence of CNS involvement was significantly higher than PNS disease (p 0.001). 68 patients (84%) shown non-focal CNS symptoms and 64 (79%) focal CNS deficits with headache as the most common feature (46.9%), followed by cognitive (44.4%) and mood disorders (38.3%). Particularly, we observed a high prevalence of migraine without aura, subcortical frontal executive functions and verbal memory impairment and apathy/alexythimia. MR spectroscopy revealed a reduction of NAA levels or NAA/Cr ratio decrease in subcortical frontal and basal ganglia white matter, while ultrasonography showed an impairment of microvasculature response. At multivariate analysis, headache, cognitive disorders and psychiatric symptoms was significantly associated to serological markers (anti-SSA), MRS and ultrasonographic features. CONCLUSIONS: The higher prevalence of MWO-mimic headache, cognitive dys-executive syndrome and mood disorders observed in this series confirmed previous evidences of a higher diffused CNS compromission rather than focal involvement such as SM-like clinical course or NMO-like syndrome. The association with immunological biomarkers, metabolic cerebral dysfunction and microvascular damage suggests a possible endothelial dysfunction of the cerebral microcirculation or a potential inflammation-mediated shift of the neurovascular coupling.

Human Polyomavirus JC monitoring and noncoding control region analysis in dynamic cohorts of individuals affected by immune-mediated diseases under treatment with biologics: an observational study.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) onset, caused by Polyomavirus JC (JCPyV) in patients affected by immune-mediated diseases during biological treatment, raised concerns about the safety profile of these agents. Therefore, the aims of this study were the JCPyV reactivation monitoring and the noncoding control region (NCCR) and viral protein 1 (VP1) analysis in patients affected by different immune-mediated diseases and treated with biologics. METHODS: We performed JCPyV-specific quantitative PCR of biological samples collected at moment of recruitment (t0) and every 4 months (t1, t2, t3, t4). Subsequently, rearrangements' analysis of NCCR and VP1 was carried out. Data were analyzed using χ2 test. RESULTS: Results showed that at t0 patients with chronic inflammatory rheumatic diseases presented a JCPyV load in the urine significantly higher (p≤0.05) than in patients with multiple sclerosis (MS) and Crohn's disease (CD). It can also be observed a significant association between JC viruria and JCPyV antibodies after 1 year of natalizumab (p=0.04) in MS patients. Finally, NCCR analysis showed the presence of an archetype-like sequence in all urine samples, whereas a rearranged NCCR Type IR was found in colon-rectal biopsies collected from 2 CD patients after 16 months of infliximab. Furthermore, sequences isolated from peripheral blood mononuclear cells (PBMCs) of 2 MS patients with JCPyV antibody at t0 and t3, showed a NCCR Type IIR with a duplication of a 98 bp unit and a 66 bp insert, resulting in a boxB deletion and 37 T to G transversion into the Spi-B binding site. In all patients, a prevalence of genotypes 1A and 1B, the predominant JCPyV genotypes in Europe, was observed. CONCLUSIONS: It has been important to understand whether the specific inflammatory scenario in different immune-mediated diseases could affect JCPyV reactivation from latency, in particular from kidneys. Moreover, for a more accurate PML risk stratification, testing JC viruria seems to be useful to identify patients who harbor JCPyV but with an undetectable JCPyV-specific humoral immune response. In these patients, it may also be important to study the JCPyV NCCR rearrangement: in particular, Spi-B expression in PBMCs could play a crucial role in JCPyV replication and NCCR rearrangement.