Anna Repice

Autologous haematopoietic stem cell transplantation with an intermediate intensity conditioning regimen in multiple sclerosis: the Italian multi-centre experience

Background: Over recent years numerous patients with severe forms of multiple sclerosis (MS) refractory to conventional therapies have been treated with intense immunosuppression followed by autologous haematopoietic stem cell transplantation (AHSCT). The clinical outcome and the toxicity of AHSCT can be diverse, depending on the various types of conditioning protocols and on the disease phase. Objectives: To report the Italian experience on all the consecutive patients with MS treated with AHSCT with an intermediate intensity conditioning regimen, named BEAM/ATG, in the period from 1996 to 2008. Methods: Clinical and magnetic resonance imaging outcomes of 74 patients were collected after a median follow-up period of 48.3 (range = 0.8–126) months. Results: Two patients (2.7%) died from transplant-related causes. After 5 years, 66% of patients remained stable or improved. Among patients with a follow-up longer than 1 year, eight out of 25 subjects with a relapsing–remitting course (31%) had a 6–12 months confirmed Expanded Disability Status Scale improvement > 1 point after AHSCT as compared with one out of 36 (3%) patients with a secondary progressive disease course (p = 0.009). Among the 18 cases with a follow-up longer than 7 years, eight (44%) remained stable or had a sustained improvement while 10 (56%), after an initial period of stabilization or improvement with median duration of 3.5 years, showed a slow disability progression. Conclusions: This study shows that AHSCT with a BEAM/ATG conditioning regimen has a sustained effect in suppressing disease progression in aggressive MS cases unresponsive to conventional therapies. It can also cause a sustained clinical improvement, especially if treated subjects are still in the relapsing–remitting phase of the disease.

Azathioprine versus Beta Interferons for Relapsing-Remitting Multiple Sclerosis: A Multicentre Randomized Non-Inferiority Trial

For almost three decades in many countries azathioprine has been used to treat relapsing-remitting multiple sclerosis. However its efficacy was usually considered marginal and following approval of β interferons for this indication it was no longer recommended as first line treatment, even if presently no conclusive direct β interferon-azathioprine comparison exists. To compare azathioprine efficacy versus the currently available β interferons in relapsing-remitting multiple sclerosis, a multicenter, randomized, controlled, single-blinded, non-inferiority trial was conducted in 30 Italian multiple sclerosis centers. Eligible patients (relapsing-remitting course; ≥2 relapses in the last 2 years) were randomly assigned to azathioprine or β interferons. The primary outcome was annualized relapse rate ratio (RR) over 2 years. Key secondary outcome was number of new brain MRI lesions. Patients (n = 150) were randomized in 2 groups (77 azathioprine, 73 β interferons). At 2 years, clinical evaluation was completed in 127 patients (62 azathioprine, 65 β interferons). Annualized relapse rate was 0.26 (95% Confidence Interval, CI, 0.19–0.37) in the azathioprine and 0.39 (95% CI 0.30–0.51) in the interferon group. Non-inferiority analysis showed that azathioprine was at least as effective as β interferons (relapse RRAZA/IFN 0.67, one-sided 95% CI 0.96; p<0.01). MRI outcomes were analyzed in 97 patients (50 azathioprine and 47 β interferons). Annualized new T2 lesion rate was 0.76 (95% CI 0.61–0.95) in the azathioprine and 0.69 (95% CI 0.54–0.88) in the interferon group. Treatment discontinuations due to adverse events were higher (20.3% vs. 7.8%, p = 0.03) in the azathioprine than in the interferon group, and concentrated within the first months of treatment, whereas in the interferon group discontinuations occurred mainly during the second year. The results of this study indicate that efficacy of azathioprine is not inferior to that of β interferons for patients with relapsing-remitting multiple sclerosis. Considering also the convenience of the oral administration, and the low cost for health service providers, azathioprine may represent an alternative to interferon treatment, while the different side effect profiles of both medications have to be taken into account. Trial Registration EudraCT 2006-004937-13

Modulating dendritic cells (DC) from immunogenic to tolerogenic responses: A novel mechanism of AZA/6-MP

Azathioprine (Aza), 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are thiopurine drugs widely used as immunosuppressants/anti-inflammatory agents in organ transplantation and chemotherapy. Aza is well tolerated and effective in modifying the course of MS. Here we investigated the action of 6-MP on human dendritic cells (DCs). We described for the first time that 6-MP impairs in vitro differentiation of DCs, has an inhibitory effect during DC activation processes inducing a functionally less immunogenic phenotype. Moreover, 6-MP significantly reduces DC IL-23 production and CCR7 expression, at the same time induces IL-10 augmentation. All these findings add a novel action mechanism in Aza immune modulation.

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

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

Identification of single nucleotide variations in the coding and regulatory regions of the myelin-associated glycoprotein gene and study of their association with multiple sclerosis

The myelin-associated glycoprotein (MAG) gene is an appealing candidate in the 19q13 Multiple Sclerosis (MS) candidate region. Using denaturing high performance liquid chromatography (DHPLC), we identified 14 single nucleotide polymorphisms (SNPs) in MAG coding and regulatory regions, and we tested their possible association with MS in Italian patient and control DNA pools. Eight variations had a frequency <0.05, i.e. below the detection limit in the pools. Of these, Arg537Cys was further studied with individually genotyped individuals and was detected in 1/189 patients and 0/85 controls. The frequency of the six remaining SNPs were not significantly different in pools including a total of 1266 patient and 1612 control chromosomes. Considering the statistical power of the experimental design, these results exclude the MAG gene as an MS susceptibility factor with an odds ratio (OR) equal or higher than 1.3.

Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis

BackgroundGene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status.MethodsThirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches.ResultsSeveral immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only.ConclusionsAnalysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification.

Detection of JCPyV microRNA in blood and urine samples of multiple sclerosis patients under natalizumab therapy.

Polyomavirus JC (JCPyV) reactivation and development of progressive multifocal leukoencephalopathy is a health concern in multiple sclerosis patients under natalizumab therapy. Here, the JCPyV microRNA-J1-3p and microRNA-J1-5p expressions and genomic variability were investigated in blood and urine samples of multiple sclerosis patients before and under natalizumab therapy and in healthy controls. The two JCPyV microRNAs were detected in the JCPyV-DNA-positive peripheral blood mononuclear cell samples and in the exosomes derived from plasma and urine obtained from JCPyV-DNA-positive and JCPyV-DNA-negative patients. In particular, the increased JCPyV microRNA expression in samples of multiple sclerosis patients under natalizumab therapy was consistent with the high JCPyV-DNA positivity observed in these samples. Moreover, JCPyV microRNA genomic region showed few nucleotide differences in samples obtained from blood and urine of multiple sclerosis patients and healthy controls. Overall, these data suggest a potential role of the JCPyV microRNA expression in counteracting the viral reactivation to maintain JCPyV asymptomatic persistence in the host.

Disappearance of motor tics after Wernicke's encephalopathy in a patient with Tourette's syndrome

Article abstract-Tourettes syndrome (TS) is a disease characterized by multiple motor and vocal tics as well as behavioral abnormalities. The anatomic substrates of this syndrome are not defined. We report a 48-year-old man with TS in whom motor tics disappeared after the onset of a midbrain syndrome related to thiamine deficiency (Wernickes encephalopathy). MRI study showed a lesion in the dorsal area of the midbrain. This case suggests that loops located in the midbrain tegmentum may influence the presence of motor tics in patients with TS. NEUROLOGY 1997;48: 381-383

No proinflammatory signature in CD34+ hematopoietic progenitor cells in multiple sclerosis patients.

Autologous hematopoietic stem cell transplantation (aHSCT) has been used as a therapeutic approach in multiple sclerosis (MS). However, it is still unclear if the immune system that emerges from autologous CD34+ hematopoietic progenitor cells (HPC) of MS patients is pre-conditioned to re-develop the proinflammatory phenotype. The objective of this article is to compare the whole genome gene and microRNA expression signature in CD34+ HPC of MS patients and healthy donors (HD). CD34+ HPC were isolated from peripheral blood of eight MS patients and five HD and analyzed by whole genome gene expression and microRNA expression microarray. Among the differentially expressed genes (DEGs) only TNNT1 reached statistical significance (logFC=3.1, p<0.01). The microRNA expression was not significantly different between MS patients and HD. We did not find significant alterations of gene expression or microRNA profiles in CD34+ HPCs of MS patients. Our results support the use of aHSCT for treatment of MS.

Markers of JC virus infection in patients with multiple sclerosis under natalizumab therapy

Objective: To evaluate the frequency of JC polyomavirus (JCPyV) infection and anti-JCPyV antibodies in patients with multiple sclerosis under natalizumab therapy. Methods: Presence of anti-JCPyV antibodies and JCPyV DNA was analyzed in 39 patients with relapsing-remitting multiple sclerosis undergoing natalizumab therapy. Anti-JCPyV antibodies were evaluated in serum by a 2-step virus-like particle-based ELISA assay (Stratify), and JCPyV DNA was evaluated in peripheral blood mononuclear cells, plasma, and urine by quantitative PCR. The anti-JCPyV antibodies were evaluated in serum samples collected at the same time or later than those collected for DNA analysis. Results: JCPyV DNA was detected in 59% of patients, and anti-JCPyV antibodies were present in 67%. JCPyV DNA occurred more often in blood than in urine. Anti-JCPyV antibodies were observed in 70% of the JCPyV-infected patients, and JCPyV DNA was detected in 50% of the patients without anti-JCPyV antibodies. When JCPyV DNA was investigated in blood and urine the frequency of infection was higher than previously described. Conclusion: Under these experimental conditions, with respect to the observed frequency of JCPyV infection, the sensitivity of the anti-JCPyV antibody assay was lower than expected.