Monitoring the John Cunningham virus throughout natalizumab treatment in multiple sclerosis patients.

BACKGROUND AND PURPOSE: Progressive multifocal leukoencephalopathy (PML) cases have arisen amongst multiple sclerosis patients treated with natalizumab. Our objective was to gain a better understanding of the mechanisms that underlie the John Cunningham virus (JCV) infection which causes PML. METHODS: A study was made of (i) the quarterly JCV DNA levels in peripheral blood mononuclear cells (PBMCs), serum and urine samples in 100 multiple sclerosis patients during their natalizumab treatment (3-39 months), (ii) the association between human leukocyte antigen (HLA) class II and the previous viral detection and (iii) the identification of the JCV variants in those patients suspected of having PML. RESULTS: (i) JCV DNA in PBMCs and/or serum was detected in 23% of our cohort. Patients with an intermittent JCV excretion in urine had a significant increase of the viral load and prevalence in this compartment during natalizumab treatment. (ii) The frequency of the DRB1*07/DQA1*02:01/DQB1*02:02 haplotype tended to be higher in patients with detectable versus undetectable JCV DNA in PBMCs (P(corrected) = 0.108). (iii) The variants in PBMCs and serum of the non-PML patient matched the archetype. In the patient with non-fatal PML, the archetype and the same neurotropic variant in PBMCs, serum and cerebrospinal fluid was identified at the time PML was diagnosed, whereas in the patient with a worse PML prognosis, four neurotropic variants in the three previous compartments were found by the PML diagnosis. CONCLUSIONS: The detection of the neurotropic variant in blood during natalizumab treatment could be critical in the prevention of the development of severe PML, since this variant appears simultaneously with the clinical symptoms of PML and mutates quickly.

JC virus reactivation in patients with autoimmune rheumatic diseases treated with rituximab.

OBJECTIVES: To evaluate the presence of John Cunningham virus (JCV) DNA in patients with autoimmune rheumatic diseases (ARDs) treated with rituximab (RTX). METHOD: We assessed the JCV DNA levels in peripheral blood mononuclear cell (PBMC), serum, and urine samples by quantitative real-time polymerase chain reaction (qPCR) in a cohort of 42 ARD patients (20 of whom were being treated with RTX) and 42 healthy donors. Approximately 1 year later, we collected further samples from 32 of these 42 ARD patients, all of whom were being treated with RTX. We studied the association between these viral DNA prevalences and various clinical and demographic variables. RESULTS: The viral prevalence in PBMC, serum, and urine samples was 2.4% (1/42), 0%, and 50% (21/42), respectively, in the healthy donors, and 26% (8/31), 16% (5/31), and 86% (25/29), respectively, in the patients treated with RTX. The viral prevalences were not associated with any of the demographic or clinical variables included in the study. CONCLUSIONS: We detected a viral reactivation in PBMCs and serum during the RTX treatment that did not seem to be influenced by either use of immunosuppressive drugs or the length of treatment with the monoclonal antibody. Although this reactivation was asymptomatic, the viral presence in blood could increase the probability of the appearance of a neurotrophic variant of JCV.

Toll-like receptor-9 in Spanish multiple sclerosis patients: an association with the gender.

BACKGROUND AND PURPOSE: Toll-like receptor-9 (TLR9) is a potent inducer of innate immune system triggered by infection with viruses, some of them previously related to multiple sclerosis (MS). The aim of this study was to analyze the possible association of two TLR9 single nucleotide polymorphisms (SNPs; rs352162 and rs187084) with susceptibility to MS. METHODS: Two independent cohorts of MS patients and controls were included: 574 clinically definite relapsing-remitting MS patients (367 females) and 807 healthy controls (418 females) for the first cohort; and 366 relapsing-remitting MS patients (238 females) and 224 healthy controls (160 females) for the second cohort. Genotyping was performed by TaqMan assays. RESULTS: The AT haplotype was found to be significantly higher in women than in men (P = 0.013 and P = 0.044). CONCLUSIONS: Here two possible genetic markers are proposed that could be also associated with the differences observed in the clinical course of MS in both genders. Further studies with larger cohorts should be performed to confirm these results.

Expression of human endogenous retrovirus HERV-K18 is associated with clinical severity in osteoarthritis patients.

OBJECTIVES: The aim of this study was to evaluate the involvement of human endogenous retrovirus K18 (HERV-K18) in osteoarthritis (OA), by genotyping the HERV-K18 env locus in OA patients and controls, and analysing HERV-K18 RNA expression and its association with OA risk and clinical variables. METHOD: We recruited 558 patients with symptomatic OA and 600 controls. We performed the genotyping by TaqMan assays and the analysis of expression by quantitative real-time polymerase chain reaction (qRT-PCR). Scores on the Western Ontario and McMasters Universities Osteoarthritis Index (WOMAC), the Lequesne index, and the Stanford Health Assessment Questionnaire (HAQ) were analysed with regard to the expression levels of HERV-K18. RESULTS: The 18.3 haplotype tended towards an association with OA risk and concordantly this haplotype was associated with a higher HERV-K18 expression (p = 0.05). We found statistically significant differences when we compared the scores on the WOMAC, the Lequesne index for knee and hip, and the HAQ between OA patients with higher expression [normalization ratio (NR) > 10] and OA patients without HERV-K18 expression (p = 0.0003, 0.0005, 0.002, and 0.05, respectively), and also when the comparison was made between OA patients with higher expression (NR > 10) and OA patients with low expression of HERV-K18 (NR = 1) for the WOMAC and the Lequesne index for knee and hip (p = 0.002, 0.013, and 0.006, respectively). CONCLUSIONS: We found an association between health status measurement systems and severity index for OA and the levels of expression of HERV-K18. These results suggest the possible involvement of HERV-K18 in the aetiopathogenesis of the disease.

Human herpesvirus 6 and effectiveness of interferon ß1b in multiple sclerosis patients.

BACKGROUND: Human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) have been repeatedly associated with multiple sclerosis (MS) pathogenesis. Also, it has been speculated that, besides its immunomodulatory properties, the efficacy of interferon beta (IFN-beta) in treating the disease may be related to its antiviral properties. The objectives of this study were to evaluate the in vivo antiviral effect of IFN-beta-1b over HHV-6 and EBV and to analyze whether such effect could be involved in its effectiveness in MS. METHODS: A total of 54 patients with MS were included in an observational, multicentric, 24-month study. HHV-6 and EBV were detected by qPCR in blood and serum samples. IFN-beta-1b effectiveness was evaluated by presence, number and severity of relapses, reduction in the relapse rate, disability progression, and response to the treatment. RESULTS: Patients with HHV-6 in blood had a higher risk of severe relapses (P=0.01) and bad response (P=0.03). HHV-6 was detected more frequently during relapses than in remission in blood (P=0.024) and in serum (P=0.0002). Patients with HHV-6 in serum had more relapses (P=0.02), lesser reduction in the relapse rate (P=0.04), and a lower proportion of responders (P=0.02) than those without HHV-6 active replication. However, any association between EBV and clinical parameters could not be found. CONCLUSIONS: We concluded that presence of HHV-6 in blood and serum during IFN-beta treatment could be a good marker of poor response.

Inhibition of HERV-K (HML-2) in amyotrophic lateral sclerosis patients on antiretroviral therapy.

Reactivation of Human Endogenous Retrovirus K (HERV-K), subtype HML-2, has been associated with pathophysiology of amyotrophic lateral sclerosis (ALS). We aimed to assess the efficacy of antiretroviral therapy in inhibiting HML-2 in patients with ALS and a possible association between the change in HML-2 levels and clinical outcomes. We studied the effect of 24-weeks antiretroviral combination therapy with abacavir, lamivudine, and dolutegravir on HML-2 levels in 29 ALS patients. HML-2 levels decreased progressively over 24 weeks (P = 0.001) and rebounded within a week of stopping medications (P = 0.02). The majority of participants (82%), defined as "responders", experienced a decrease in HML-2 at week 24 of treatment compared to the pre-treatment levels. Differences in the evolution of some of the clinical outcomes could be seen between responders and non-responders: FVC decreased 23.69% (SE = 11.34) in non-responders and 12.71% (SE = 8.28) in responders. NPI score decreased 91.95% (SE = 6.32) in non-responders and 53.05% (SE = 10.06) in responders (P = 0.01). Thus, participants with a virological response to treatment showed a trend for slower progression of the illness. These findings further support the possible involvement of HML-2 in the clinical course of the disease.

Neutralizing antibodies, MxA expression and MMP-9/TIMP-1 ratio as markers of bioavailability of interferon-beta treatment in multiple sclerosis patients: a two-year follow-up study.

BACKGROUND: The objective of this study was to correlate the detection of neutralizing antibodies (NAbs) by the cytopathic effect (CPE) assay, with the expression of myxovirus resistance protein A (MxA), and the ratio between matrix metalloproteinase 9 (MMP-9) and its tissular inhibitor (TIMP-1), in order to evaluate their usefulness as markers of interferon beta (IFN-beta) bioavailability. METHODS: Pairs of blood and serum samples were collected from 50 patients with multiple sclerosis (MS) during 2 years of IFN-beta treatment. Expression of MxA, MMP-9 and TIMP-1 were analysed by quantitative PCR, and NAbs were measured by CPE assay. RESULTS: During the study, 60% of patients presented NAbs. The number of serum samples that were NAbs+ was significantly increased amongst patients with relapses (41/92 vs. 33/108, P = 0.04). With one serum sample and with a NAb titre >100 tenfold reduction unit (TRU), 66.7% of patients with MS suffered from relapses, 41.7% suffered from progression, and 75% was not an optimal clinical responder. We did not find any significant difference in MxA. We found that 62.5% of patients with MS patients whose ratio was increased twofold after 2 years suffered from relapses, 37.5% suffered from progression, and 68.7% was not an optimal clinical responder. CONCLUSION: The early detection of NAbs by CPE assay and the finding of only one serum sample with a NAb titre >100 TRU seem to be markers of low bioavailability of IFN-beta, whilst a twofold decrease in the MMP-9/TIMP-1 ratio by quantitative PCR assay seems to be a marker of high bioavailability of IFN-beta.

MHC2TA rs4774C and HHV-6A active replication in multiple sclerosis patients.

BACKGROUND AND PURPOSE: In a previous report, a strong gene-environment interaction between human herpesvirus 6A (HHV6A) active replication and MHC2TA rs4774C was demonstrated. The objectives of this study were: (i) to reappraise the association that was found in the previous study; (ii) to evaluate if MS patients with minor allele C and HHV-6A active infection had different clinical behavior; and (iii) to analyze the possible association of MHC2TA rs4774C with Epstein-Barr virus (EBV). METHODS: A total of 149 MS patients were analyzed both at the MHC2TA locus and by HHV-6A status in serum. We studied a G/C polymorphism (rs4774) by a TaqMan Assay-on-Demand. HHV-6A genomes in serum were evaluated by quantitative PCR. A control group of 562 healthy Spanish individuals was included for comparative purposes in the genetic analyses. A battery of clinical data was collected for all the MS patients included in the study. RESULTS: (i) MHC2TA/HHV-6A interaction: we found the same strong association of the rs4774C allele with HHV-6A active replication than in the previous study (P = 0.0001). (ii) CLINICAL DATA: the two main statistical significant differences for MS patients with HHV-6A active infection and minor allele C were: (a) a significant number of them were not free of progression (EDSS = 0) 2 years after the diagnosis (P = 0.01); (b) only a third of them responded to interferon beta treatment (P = 0.05). CONCLUSIONS: This study has verified previous results about the strong gene-environment interaction between HHV6A active replication and MHC2TA rs4774C. Furthermore, a different clinical behavior for MS patients with HHV-6A active infection and minor allele C was found.

CD46 expression and HHV-6 infection in patients with multiple sclerosis.

OBJECTIVES: The aim of this study was to investigate the possible association between the levels of the CD46 expression, and the presence and viral load of HHV-6 in patients with multiple sclerosis (MS). METHODS: We collected blood and serum samples of 103 patients with MS and the same number of healthy blood donors (HBD); total DNA and RNA was extracted from peripheral blood mononuclear cells (PBMCs) and serum, and then analyzed using quantitative real-time PCR for the detection of CD46 transcripts and HHV-6 genomes; the expression of rRNA18s was used for the calculation of the relative expression of CD46. RESULTS: Almost 80% of patients with MS had increased levels of CD46 in comparison with HBD, and a positive correlation was also found between the over-expression of CD46 in patients with MS and the HHV-6 DNA prevalence and viral load in the blood and serum. DISCUSSION: Therefore, the up-regulation of CD46 expression in patients with MS with HHV-6 infection could constitute an immunopathogenic factor that should be further investigated to elucidate its role in MS.

Interferon-beta treatment and active replication of the JC virus in relapsing-remitting multiple sclerosis patients.

We analyzed the effect of beta-interferon (beta-IFN) treatment over the active replication of JC virus (JCV) through the evaluation of JCV DNA prevalence and viral load in peripheral blood mononuclear cells (PBMCs) and serum samples, and mRNA prevalence and viral load, in relapsing-remitting multiple sclerosis (RRMS) patients. DNA extracted from PBMCs and serum, and mRNA extracted from PBMCs were analyzed in 146 RRMS patients (73 treated with beta-IFN, and 73 untreated patients), and 73 matched healthy blood donors for the presence of JCV genomes by quantitative real-time PCR assay. We found the same DNA prevalence in PBMC samples in RRMS patients treated with beta-IFN and in untreated ones: 6.8% (5/73). When we analyzed the viral active replication in both groups through the analysis of DNA prevalence in serum samples and the mRNA extracted from PBMCs, we did not find any positive sample. Regarding the viral load of those positive samples, we did not find any statistical significant difference between treated and untreated RRMS patients: 28.6 +/- 7.2 and 32.3 +/- 8.4 copies/microg of DNA, respectively. These results lead us to conclude that beta-IFN treatment in monotherapy has not any effect on JCV active replication.

Molecular characteristics of Human Endogenous Retrovirus type-W in schizophrenia and bipolar disorder.

Epidemiological and genome-wide association studies of severe psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD), suggest complex interactions between multiple genetic elements and environmental factors. The involvement of genetic elements such as Human Endogenous Retroviruses type 'W' family (HERV-W) has consistently been associated with SZ. HERV-W envelope gene (env) is activated by environmental factors and encodes a protein displaying inflammation and neurotoxicity. The present study addressed the molecular characteristics of HERV-W env in SZ and BD. Hundred and thirty-six patients, 91 with BD, 45 with SZ and 73 healthy controls (HC) were included. HERV-W env transcription was found to be elevated in BD (P<10-4) and in SZ (P=0.012) as compared with HC, but with higher values in BD than in SZ group (P<0.01). The corresponding DNA copy number was paradoxically lower in the genome of patients with BD (P=0.0016) or SZ (P<0.0003) than in HC. Differences in nucleotide sequence of HERV-W env were found between patients with SZ and BD as compared with HC, as well as between SZ and BD. The molecular characteristics of HERV-W env also differ from what was observed in Multiple Sclerosis (MS) and may represent distinct features of the genome of patients with BD and SZ. The seroprevalence for Toxoplasma gondii yielded low but significant association with HERV-W transcriptional level in a subgroup of BD and SZ, suggesting a potential role in particular patients. A global hypothesis of mechanisms inducing such major psychoses is discussed, placing HERV-W at the crossroads between environmental, genetic and immunological factors. Thus, particular infections would act as activators of HERV-W elements in earliest life, resulting in the production of an HERV-W envelope protein, which then stimulates pro-inflammatory and neurotoxic cascades. This hypothesis needs to be further explored as it may yield major changes in our understanding and treatment of severe psychotic disorders.

Influence of metyrapone treatment during pregnancy on the development and maturation of brain monoaminergic systems in the rat.

AIM: This study examines the effect of reducing the corticosterone levels of gestating rat dams on the postnatal development and maturation of monoaminergic systems in their offspring's brains. METHODS: Metyrapone, an inhibitor of CORT synthesis, was administered to pregnant rats from E0 to E17 of gestation. Monoamine concentrations were determined in male and female offspring at postnatal days (PN) 23 and 90 in the hippocampus, hypothalamus and striatum. RESULTS: Reducing maternal corticosterone (mCORT) during gestation led to alterations in dopamine and serotonin levels in all three brain areas studied at PN 23. Alterations persisted until at least PN 90 in the serotonergic systems; the dopamine content of the hippocampus also remained modified. Reduced mCORT during gestation also led to alterations in the development and maturation of the hypothalamic noradrenergic systems. Sexually dimorphic responses were observed in all these monoaminergic systems at different times. CONCLUSION: These results suggest that while they are still developing, brain monoaminergic systems are particularly sensitive to epigenetic influences. An adequate foetal level of CORT is required for the normal ontogeny of brain monoaminergic systems. The present data also provide that during the critical period of brain development, maternal CORT plays an important role in the sexual differentiation of monoaminergic systems, with particular influence on brain serotonergic neurones.

Herpesviruses and human endogenous retroviral sequences in the cerebrospinal fluid of multiple sclerosis patients.

OBJECTIVE: To analyze the possible role of human herpesvirus (HHVs) and human endogenous retroviruses (HERVs) infection in multiple sclerosis (MS) pathogenesis. METHODS: A total of 92 cerebrospinal fluid (CSF) samples were collected: 48 from MS patients at the first clinically evident demyelinating event, 23 from patients with other inflammatory neurological diseases (OINDs) and 21 from patients with other non-inflammatory neurological diseases (ONINDs). Total DNA and RNA were isolated, and the prevalences and viral loads of herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), HHV-6, HERV-H and HERV-W in the CSF of MS patients and controls were evaluated using a quantitative real-time polymerase chain reaction assay. RESULTS: (i) For HSV, 1/48 (2.1%, 86 copies/ml of CSF) MS patients and 1/23 (4.3%, 115.2 copies/ml of CSF) OIND patients (a myelitis case) had HSV sequences in the CSF; (ii) for EBV, only 1/48 (2.1%, 72 copies/ml of CSF) MS patients was positive for EBV; (iii) for HHV-6, only 5/48 (10.4%) MS patients had HHV-6 genomes in their CSF (128.1 copies/ml of CSF); (iv) we did not find any positive cases for VZV, CMV, HERV-H and HERV-W among MS patients or controls; (v) no cases of co-infections were found; (vi) the whole prevalence of HHVs was 7/48 (14.6%) for MS patients and 1/44 (2.3%) for controls (p = 0.038). CONCLUSION: The findings described here show that HHV infection is more frequent in the CSF of MS patients than in patients with other neurological diseases; however, only HHV-6 seems to be involved in the pathogenesis of MS in a subset of patients.