No evidence of JC virus reactivation in natalizumab treated multiple sclerosis patients: an 18 month follow-up study.

BACKGROUND AND AIM: Natalizumab, used as therapy for multiple sclerosis (MS), has been associated with progressive multifocal leucoencephalopathy (PML), a potentially fatal disease caused by JC virus (JCV), which is not predictable by specific markers. This study evaluated whether JCV reactivation occurred in the urine and/or plasma in 42 MS patients treated with natalizumab over 18 months, and followed by a thorough monitoring programme. METHODS: 42 patients (F/M: 24/18, mean age 34.4±8.9 years) were followed-up by: urine and plasma JCV-DNA PCR assay, immune cell subsets analysis, clinical and MRI evaluation, quality of life, fatigue and mood assessment. RESULTS: JCV data. At baseline, 11/42 (26%) patients had JCV viruria, persistent at serial controls. One patient acquired viruria at month 1 and one patient at month 12. No patient had JCV viraemia at baseline; three patients acquired viraemic (one at month 6, one at month 13 (both transiently) and one at month 12 (persistently viraemic)). The prevalence of JCV in both urine and plasma did not change significantly from baseline to months 12 and 18. No patient had clinical or MRI evidence of PML. Immunological data. Circulating B cells showed greater expansion (300% increase in absolute number) since the first infusion. NK cell count doubled with no change in percentage while T cell count increased with a reduced percentage, reflecting a clear redistribution in the lymphocyte compartment. CD4+ and CD8+ T cells increased proportionally, with no change in their percentage. Clinical data. 27 patients (64%) were disease free after 1 year. A marked improvement in quality of life was reported by 72% of patients. CONCLUSIONS: No evidence of subclinical JCV reactivation was found in our natalizumab treated MS patients up to 18 months of therapy, notwithstanding the marked increase in circulating B cells observed. Moreover, the efficacy of natalizumab, its tolerability and the positive impact on quality of life were confirmed in this study.

Magnetic resonance evidence of cerebellar cortical pathology in multiple sclerosis.

BACKGROUND: Although neuropathological observations suggest that cerebellar cortex is a major site of demyelination in multiple sclerosis (MS), only a few MRI studies on cerebellar cortical pathology in MS are available. OBJECTIVE: To analyse cerebellar cortical volume (CCV) and leucocortical lesions (CL) in MS, and their impact on clinical disability. METHODS: The authors studied 125 patients divided into 38 Clinical Isolated Syndrome (CIS), 35 relapsing remitting multiple sclerosis (RRMS), 27 secondary progressive multiple sclerosis (SPMS) and 25 primary progressive multiple sclerosis, and 32 normal controls (NC). CCV and cerebellar CL number and volume were evaluated by means of Freesurfer software and Double Inversion Recovery, respectively. RESULTS: Compared with NC (mean 113.2 + or - 2.6 cm(3)), the CCV was significantly reduced in CIS (105.4 + or - 2.2 cm(3), p=0.018), RRMS (104.0 + or - 2.0 cm(3), p=0.012), SPMS (98.8 + or - 2.0 cm(3), p<0.001) and PPMS (100.6 + or - 2.2 cm(3), p<0.001), even after age, gender and mean cortical volume correction. CL were observed in all patient groups and were an independent predictor of CCV and cerebellar dysfunction. DISCUSSION: The authors confirm that the cerebellar cortex is severely and early affected by MS pathology. The monitoring of cerebellar cortical atrophy and CL may help to understand the mechanism underlying disability progression in MS.

Cyclophosphamide as second-line therapy in multiple sclerosis: benefits and risks.

Cyclophosphamide (Cy) is an alkylating agent used over the past 40 years to halt rapidly progressive forms of multiple sclerosis (MS). High doses of Cy produce marked immunosuppression and an anti-inflammatory immune deviation. Cy is most effective in young patients, with very active MS (frequent relapses, rapid accumulation of disability, and gad+ lesions on brain MRI). Monthly intravenous pulses of Cy for 1 year, followed by bimonthly pulses for the second year are a well-tolerated protocol in MS. Most side effects (mild alopecia, nausea and vomiting, and cystitis) are transient, dose dependent, and reversible. Permanent amenorrhoea and bladder cancer have rarely been described. As second-line therapy, Cy can be used in non-responders to IFN-beta or glatiramer acetate. As induction therapy, a short course (6-12 months) of Cy can precede immunomodulatory drugs in selected patients with an aggressive MS onset.

Severe relapses after the first infusion of natalizumab in active relapsing-remitting multiple sclerosis.

We describe three patients suffering from a very active form of relapsing-remitting multiple sclerosis (MS), who experienced severe disease worsening, associated with a marked increase in brain inflammation, a few days after the first administration of natalizumab. In line with preclinical studies, our observations suggest that natalizumab, when administered during active disease phases, may worsen disease evolution possibly by modifying the regulatory network in the brain. We suggest that relapsing-remitting MS patients having had a recent relapse should be treated with natalizumab only after achieving complete clinical and radiological remission.

Cortical lesions and atrophy associated with cognitive impairment in relapsing-remitting multiple sclerosis.

BACKGROUND: Neuropsychological deficits in patients with multiple sclerosis (MS) have been shown to be associated with the major pathological substrates of the disease, ie, inflammatory demyelination and neurodegeneration. Double inversion recovery sequences allow cortical lesions (CLs) to be detected in the brain of patients with MS. Modern postprocessing techniques allow cortical atrophy to be assessed reliably. OBJECTIVE: To investigate the contribution of cortical gray matter lesions and tissue loss to cognitive impairment in patients with relapsing-remitting MS. DESIGN: Cross-sectional survey. SETTING: Referral, hospital-based MS clinic. Patients Seventy patients with relapsing-remitting MS. MAIN OUTCOME MEASURES: Neuropsychological performance was tested using the Rao Brief Repeatable Battery of Neuropsychological Tests, version A. Patients who scored 2 SDs below the mean normative values on at least 1 test of the Rao Brief Repeatable Battery of Neuropsychological Tests, version A, were considered to be cognitively impaired. A composite cognitive score (the cognitive impairment index) was computed. T2 hyperintense white matter lesion volume, contrast-enhancing lesion number, CL number and volume, normalized brain volume, and normalized neocortical gray matter volume were also assessed. RESULTS: Twenty-four patients with relapsing-remitting MS (34.3%) were classified as cognitively impaired. T2 hyperintense white matter lesion volume and contrast-enhancing lesion number were not different between cognitively impaired and cognitively unimpaired patients. Cognitively impaired patients had a higher CL number (P = .01) and volume (P < .001) and decreased normalized brain volume (P = .02) and normalized neocortical gray matter volume (P = .002) when compared with cognitively unimpaired patients. Multivariate analysis revealed that age (beta = 0.228; P = .02), CL volume (beta = 0.452; P < .001), and normalized neocortical gray matter volume (beta = 0.349; P < .001) were independent predictors of the cognitive impairment index (r(2) = 0.55; F = 23.903; P < .001). CONCLUSION: The burden of CLs and tissue loss are among the major structural changes associated with cognitive impairment in relapsing-remitting MS.

Cyclophosphamide-based combination therapies for autoimmunity.

Immunomodulatory agent (IMA)-unresponsive multiple sclerosis (MS) can quickly evolve to a dramatic and irreversible disability. Treating these patients with appropriate immunosuppressive therapies can be a chance to arrest disease activity and progression. Cyclophosphamide (Cyc)-based intense immunosuppression has been successfully used to treat rapidly deteriorating, IMA-refractory MS patients. Therapeutic protocols combining Cyc and interferon beta (IFNbeta) have also been successfully applied to treat IFNbeta-unresponsive MS. The association of Cyc with other immunomodulatory drugs or monoclonal antibodies is currently being investigated in clinical trials aimed at treating severe forms of autoimmune diseases.

Morphology and evolution of cortical lesions in multiple sclerosis. A longitudinal MRI study.

Cortical lesions (CLs) can be detected in the majority of patients with established multiple sclerosis (MS), but little is known about their evolution over time. This study was performed to investigate the short-term MRI evolution of CLs, with the ultimate aim to achieve a better in vivo understanding of their nature. Seven hundred and sixty-eight CLs from 107 MS patients (76 with relapsing-remitting [RR] and 31 with secondary progressive [SP] MS) were followed with brain MR examinations, including a double inversion recovery (DIR) sequence, every 6 months for 1 year. CLs' number, volume and morphological features were assessed at each time-point. Six hundred and eighty CLs (88.5%) remained morphologically unchanged during the follow-up period, while 74 (9.6%) showed an increase in size. Only 6 (0.8%) CLs seen at baseline (all in RRMS patients) disappeared at follow-up MRI scans. No enlarging CLs spread into the subcortical white matter. No CLs ever showed gadolinium enhancement. At baseline, the mean number of CLs was higher in SPMS than in RRMS patients (p<0.001), whereas the mean number of new CLs per patient after 1 year did not differ between the two groups. Over a one-year period, CLs can increase their number and size in a relevant proportion of MS patients, without spreading into the subcortical white matter or showing inflammatory features similar to those of white matter lesions. The short-term rate of CLs accumulation does not seem to vary according to the clinical stage of MS.

Extensive cortical inflammation is associated with epilepsy in multiple sclerosis.

INTRODUCTION: Epilepsy is three to six times more frequent in MS than in the general population. Previous studies based on conventional magnetic resonance (MR) imaging have suggested a possible correlation between cortical inflammatory pathology and epileptic seizures. However, pure intracortical lesions (ICLs) are unlikely to be demonstrated with conventional MR. We applied the double inversion recovery (DIR) sequence in relapsing remitting MS (RRMS) patients with or without epileptic seizures in order to clarify the relationship between ICLs and epilepsy in MS in vivo. METHODS: Twenty RRMS patients who had epileptic seizures (RRMS/E) during the course of the disease were studied for the presence of ICLs. A group of 80 RRMS patients with no history of seizures and matched for gender, age, disease duration, Expanded Disability Status Scale (EDSS) grading, and T2 lesion volume (T2-WMLV) was selected as reference population. ICLs were detected by applying the DIR sequence. RESULTS: ICLs were observed in 18/20 (90%) RRMS/E and in 39/80 (48%) RRMS (p = 0.001). RRMS/E showed five times more ICLs (7.2 +/- 8.4) than RRMS (1.5 +/- 2.4; p = 0.015). The total ICLs volume was 6 times larger in RRMS/E than in RRMS (1.2 +/- 1.7 cm3 versus 0.2 +/- 0.2 cm3, p = 0.016). No significant difference was observed between RRMS and RRMS/E with regard to the number and volume of juxtacortical lesions and T2-WMLV. DISCUSSION: Our findings indicate that RRMS/E have more extensive cortical inflammation than RRMS patients with no history of epilepsy. Inflammatory ICLs may be responsible for epilepsy in MS.

Detection of cortical inflammatory lesions by double inversion recovery magnetic resonance imaging in patients with multiple sclerosis.

BACKGROUND: A significant inflammatory pathologic disorder in the cortex of patients with multiple sclerosis (MS) has been demonstrated by ex vivo studies. OBJECTIVE: To determine the frequency, time of appearance, and clinical relevance of intracortical lesions (ICLs) in MS in vivo. DESIGN: Double inversion recovery sequence study. SETTING: Multiple Sclerosis Centre of the Veneto Region. Patients We enrolled 380 patients (116 with clinically isolated syndrome [CIS], 163 with relapsing-remitting MS [RRMS], and 101 with secondary progressive MS [SPMS]) and 40 age- and sex-matched healthy volunteers between May 1, 2005, and December 31, 2006. MAIN OUTCOME MEASURES: We assessed the frequency and number of ICLs and brain parenchyma fraction, white matter T2 lesion volume, and clinical disability. RESULTS: Although never observed in healthy volunteers, ICLs were detected in 58% of patients (36% of patients with CIS, 64% of patients with RRMS, and 73% of patients with SPMS). The number of ICLs was higher in patients with SPMS than in those with CIS or RRMS (P <.001), and patients with ICLs had a higher Expanded Disability Status Scale score (P = .004), a higher white matter T2 lesion volume (P = .008), a lower brain parenchyma fraction (P = .009), and a higher frequency of IgG oligoclonal bands (IgGOBs) (P <.001) than patients without ICLs. Patients positive for IgGOBs had more ICLs than patients negative for IgGOBs (P = .02). The number of ICLs correlated with the Expanded Disability Status Scale score (r = 0.48, P <.001), white matter T2 lesion volume (r = 0.38, P = .001), and brain parenchyma fraction (r = -0.47, P = .001). A significant association between ICLs and male sex was observed. CONCLUSIONS: Although more frequent in patients with SPMS, ICLs were observed from the early disease stages. The ICLs were more frequently detected in patients with IgGOBs and were associated with a higher clinical disability score and male sex. The ICLs may help to define MS clinical heterogeneity and prognosis in clinical settings.

The safety profile of cyclophosphamide in multiple sclerosis therapy.

Cyclophosphamide (Cyc) is an alkylating agent used to treat malignancies and autoimmune diseases, such as lupus nephritis, rheumatoid arthritis and immune-mediated neuropathies. Over the past 40 years, Cyc has also been applied to treat multiple sclerosis (MS) and the effective stabilisation of rapidly progressive forms of MS has been demonstrated in several studies. Cyc has a dose-dependent bimodal effect on the immune system. High doses have been demonstrated to induce an anti-inflammatory immune deviation (i.e., suppression of T helper 1 and enhancement of T helper 2 activity), affect CD4CD25(high) regulatory T cells and establish a state of marked immunosuppression. Data from the literature suggest that Cyc is particularly indicated in the treatment of young MS patients, suffering from a very active inflammatory disease characterised by frequent relapses and rapid accumulation of disability and displaying gadolinium-enhancing lesions on brain magnetic resonance. The most common Cyc-based therapeutic protocol applied in MS consists of monthly intravenous pulses for 1 year followed by bimonthly pulses for the second year, with or without prior infusion of corticosteroids. This protocol is usually well tolerated by the patients. Indeed, most of the side effects (mild alopecia, nausea and vomiting, cystitis) are dose dependent, transient and completely reversible. Definitive amenorrhoea is observed only in older female patients (aged > 40 years). Cyc has a safety and efficacy profile similar to that of mitoxantrone and can be used in patients whose disease is not controlled by IFN-beta or glatiramer acetate. Short course (6-12 months) of Cyc therapy can precede the initiation of immunomodulatory treatment in selected patients with an aggressive MS onset.

Cortical atrophy is relevant in multiple sclerosis at clinical onset.

INTRODUCTION: Increasing evidence suggests relevant cortical gray matter pathology in patients with Multiple Sclerosis (MS), but how early this pathology begins; its impact on clinical disability and which cortical areas are primarily affected needs to be further elucidated. METHODS: 115 consecutive patients (10 Clinically Isolated Syndrome (CIS), 32 possible MS (p-MS), 42 Relapsing Remitting MS (RR-MS), 31 Secondary Progressive MS (SP-MS)), and 40 age/gender-matched healthy volunteers (HV) underwent a neurological examination and a 1.5 T MRI. Global and regional Cortical Thickness (CTh) measurements, brain parenchyma fraction and T2 lesion load were analyzed. RESULTS: We found a significant global cortical thinning in p-MS (2.22 +/- 0.09 mm), RR-MS (2.16 +/- 0.10 mm) and SP-MS (1.98 +/- 0.11 mm) compared to CIS (2.51 +/- 0.11 mm) and HV (2.48 +/- 0.08 mm). The correlations between mean CTh and white matter (WM) lesion load was only moderate in MS (r = -0.393, p = 0.03) and absent in p-MS (r = -0.147, p = 0.422). Analysis of regional CTh revealed that the majority of cortical areas were involved not only in MS, but also in p-MS. The type of clinical picture at onset (in particular, pyramidal signs/symptoms and optic neuritis) correlated with atrophy in the corresponding cortical areas. DISCUSSION: Cortical thinning is a diffuse and early phenomenon in MS already detectable at clinical onset. It correlates with clinical disability and is partially independent from WM inflammatory pathology.

Cannabidiol, unlike synthetic cannabinoids, triggers activation of RBL-2H3 mast cells.

Cannabidiol (CBD), a prominent psychoinactive component of cannabis with negligible affinity for known cannabinoid receptors, exerts numerous pharmacological actions, including anti-inflammatory and immunosuppressive effects, the underlying mechanisms of which remain unclear. In the current study, we questioned whether CBD modulates activation of mast cells, key players in inflammation. By using the rat basophilic leukemia mast cell line (RBL-2H3), we demonstrate that CBD (3-10 muM) augments beta-hexosaminidase release, a marker of cell activation, from antigen-stimulated and unstimulated cells via a mechanism, which is not mediated by G(i)/G(o) protein-coupled receptors but rather is associated with a robust rise in intracellular calcium ([Ca(2+)](i)) levels sensitive to clotrimazole and nitrendipine (10-30 muM). This action, although mimicked by Delta(9)-tetrahydrocannabinol (THC), is opposite to that inhibitory, exerted by the synthetic cannabinoids WIN 55,212-2 and CP 55,940. Moreover, the vanilloid capsaicin, a full agonist of transient receptor potential channel VR1, did not affect [Ca(2+)](i)levels in the RBL-2H3 cells, thus excluding the involvement of this receptor in the CBD-mediated effects. Together, these results support existence of yet-to-be identified sites of interaction, i.e., receptors and/or ion channels associated with Ca(2+) influx of natural cannabinoids such as CBD and THC, the identification of which has the potential to provide for novel strategies and agents of therapeutic interest.

Longitudinal analysis of immune cell phenotypes in early stage multiple sclerosis: distinctive patterns characterize MRI-active patients.

To investigate whether peripheral immune abnormalities are associated with brain inflammation in multiple sclerosis, and whether differences in MRI activity are paralleled by changes in leukocyte composition, we conducted a prospective longitudinal study in patients at their clinical onset. Twenty patients presenting a first inflammatory event in the central nervous system suggestive of multiple sclerosis underwent, every 45 days for one year, immunophenotyping of 98 blood cell subsets together with brain MRI and clinical evaluation. Six patients showed intense MRI activity, six patients did not display MRI activity, while the remaining 8 patients had low (i.e. intermediate) MRI activity during the follow-up. Our results show that MRI-active and MRI-inactive patients display significant differences in ten lymphocyte subsets. Among these, there are both effector (CCR7-CD45RA-CD4+ alphabeta T cells, CCR5+ gammadelta T cells) and regulatory (DN CD28+ alphabeta T cells and CD25+CD8+ alphabeta T cells) lymphocytes pertaining to the innate and the acquired arms of the immune system. Moreover, these differences were, upon employment of a class prediction procedure based on "support vector machines" algorithm utilizing leave-one-out cross validation procedures, able to correctly assign patients to their respective MRI activity group. All 6 MRI-active and 6 MRI-inactive patients were correctly classified, and, upon application of a class prediction model in an unsupervised manner to the 8 patients with intermediate MRI activity, 6 were predicted as MRI-active and 2 as MRI-inactive patients. Also, when the mean values of the first three time points (T0, T1 and T2) were used for the prediction of all patients, the selected lymphocyte subsets correctly classified 90% of patients. Sensitivity was 91.7% and specificity was 87.5%. These results provide evidence showing that brain inflammation in multiple sclerosis is associated with distinct changes in peripheral lymphocyte subsets, and raise the possibility that the identified subsets may, after adequate validation, assist in the prediction of MRI activity in the early stages of multiple sclerosis.

HPLC electrochemical detection of trace amines in human plasma and platelets and expression of mRNA transcripts of trace amine receptors in circulating leukocytes.

We evaluated, using a multi-channel electrochemical HPLC system, whether trace amines are detectable in plasma and platelets of healthy control subjects. To this end, levels of tyramine, octopamine and synephrine were assessed in samples obtained from eight males and eight females, age matched and free from drugs. In plasma, octopamine was detectable in all subjects, synephrine in 15 and tyramine in six out of 16 subjects. Likewise, detectable levels of octopamine together with synephrine were, in contrast to tyramine, found within platelets of most individuals. Intracellular levels of the amines significantly diminished following platelet activation (ADP or collagen). In addition, circulating leukocytes from these same subjects are herein shown to express mRNA transcripts for the recently discovered 'trace amine receptors' (TAR-1, -3, -4 and -5). Thus, although baseline plasma levels of octopamine tyramine or synephrine may vary among healthy individuals, the observation that platelets store and actively release these trace amines suggests that they may be effectors involved in platelet-mediated signaling events in the bloodstream.