Darlene Badgett

Genomic Effects of IFN-β in Multiple Sclerosis Patients

The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN-β-1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing-remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 μg of IFN-β-1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN-β-1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1–4 h of IFN-β treatment. The mRNAs for several genes involved in IFN-β signaling, such as IFN-α/β receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9–27), IFN-induced transmembrane protein 2 (1–8D), β2-microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN-β treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy.

In vivo gene expression revealed by cDNA arrays: the pattern in relapsing–remitting multiple sclerosis patients compared with normal subjects

OBJECTIVES To use DNA arrays to identify differences in gene expression associated with relapsing-remitting (RR) MS. METHODS Total RNA was isolated from monocyte depleted peripheral blood mononuclear cells of 15 RR MS patients and 15 age- and sex-matched controls. The RNA was reverse transcribed to radiolabeled cDNA and the resultant cDNA was used to probe a DNA array containing over 4000 named human genes. The binding of radiolabeled cDNA to the probes on the array was measured by phosphorimager. RESULTS Of more than 4000 genes tested, only 34 were significantly different in RR-MS patients from controls. Of these, 25 were significantly increased and 9 significantly decreased in the RR MS patients. Twelve of these genes have inflammatory and/or immunological functions that could be relevant to the MS disease process. The potentially relevant genes that were elevated (15% to 28%) were P protein, LCK, cAMP responsive element modulator, IL-7 receptor, matrix metalloproteinase-19, M130 antigen, and peptidyl-prolyl isomerase. Those that were significantly decreased (15% to 35%) were SAS transmembrane 4 superfamily protein, STRL22 (C-C chemokine receptor 6), AFX protein, DNA fragmentation factor-45 and immunoglobulin gamma 3 (Gm marker). CONCLUSIONS The RR-MS disease effect was relatively restricted and most of the mRNAs tested were not different from the normal controls. However, there were significant differences identified in the expression of a subset of mRNAs, including 13 with inflammatory/immune functions that could be relevant to MS. The systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in MS pathogenesis and its phenotypic heterogeneity.

Lipid profiles are associated with lesion formation over 24 months in interferon-β treated patients following the first demyelinating event

Objectives To investigate the associations of serum lipid profile with disease progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event. Methods High density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were obtained in pretreatment serum from 135 high risk patients with CIS (≥2 brain MRI lesions and ≥2 oligoclonal bands) enrolled in the Observational Study of Early Interferon β-1a Treatment in High Risk Subjects after CIS study (SET study), which prospectively evaluated the effect of intramuscular interferon β-1a treatment following the first demyelinating event. Thyroid stimulating hormone, free thyroxine, 25-hydroxy vitamin D3, active smoking status and body mass index were also obtained. Clinical and MRI assessments were obtained within 4 months of the initial demyelinating event and at 6, 12 and 24 months. Results The time to first relapse and number of relapses were not associated with any of the lipid profile variables. Higher LDL-C (p=0.006) and TC (p=0.001) levels were associated with increased cumulative number of new T2 lesions over 2 years. Higher free thyroxine levels were associated with lower cumulative number of contrast-enhancing lesions (p=0.008). Higher TC was associated as a trend with lower baseline whole brain volume (p=0.020). Higher high density lipoprotein was associated with higher deseasonalised 1,25-dihydroxy vitamin D3 (p=0.003) levels and a trend was found for deseasonalised 25-hydroxy vitamin D3 (p=0.014). Conclusions In early multiple sclerosis, lipid profile variables particularly LDL-C and TC levels are associated with inflammatory MRI activity measures.

Environmental factors associated with disease progression after the first demyelinating event: results from the multi-center SET study.

Objectives To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event. Methods We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months. Results The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies. Conclusions Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.

Interferon-β modulates bone-associated cytokines and osteoclast precursor activity in multiple sclerosis patients

Purpose Multiple sclerosis (MS) patients have a high risk of low bone density. The purpose of this study was to examine the molecular mechanisms potentially capable of modulating bone home-ostasis in response to interferon-β-1a (IFN-β-1a) treatment and the focus was the bone-modulating system comprised of receptor activator of nuclear factor-κB (RANK), its ligand RANKL and its decoy receptor, osteoprotegerin (OPG). Methods In this open-label pharmacodynamic study, peripheral blood was obtained from relapsing remitting MS patients just prior to and at multiple time points after intramuscular injection of 30 μg IFN-β-1a. Samples were analysed for RANKL, tumour necrosis factor related apoptosis-inducing ligand (TRAIL), OPG and macrophage inflammatory protein-1α/β expression. Osteoclast precursor differentiation from peripheral blood cells of MS patients in the presence of exogenously added IFN-β-1a was also assessed. Additionally, the changes in plasma levels of osteocalcin and the C-telopeptides after 1 year of treatment were measured as surrogate markers of bone formation and degradation, respectively. Results IFN-β-1a treatment modulated RANKL and OPG in a selective, time-dependent manner. The levels of OPG protein decreased 25% at the 8-h time point, then increased 43% at the 24-h time point. The levels of free RANKL reached a maximum at the 8-h time point. Increases in the levels of macrophage inflammatory protein-1β (MIP-1β), a chemokine that increases osteolysis, were observed. The levels of the bone formation marker, osteocalcin, were lower in MS patients compared to controls and increased after one year of treatment. Ex vivo treatment of peripheral blood lymphocytes with IFN-β resulted in a marked reduction of osteoclast-like cells in the presence of RANKL and macrophage colony stimulating factor. Conclusions IFN-β treatment induces complex, specific and time-dependent changes in multiple proteins and mRNAs related to bone homeostasis in MS patients.

Genomic effects of once-weekly, intramuscular interferon-β1a treatment after the first dose and on chronic dosing : Relationships to 5-year clinical outcomes in multiple sclerosis patients

PURPOSE To characterize gene expression in multiple sclerosis (MS) patients after the first dose and chronic dosing of 30 microg, once weekly, intramuscular interferon-beta1a (IFN-beta) and to delineate the pharmacogenomic differences between Good Responders and Partial Responders to IFN-beta therapy. METHODS The treatment responses after the first IFN-beta dose and chronic IFN-beta dosing were assessed in 22 relapsing MS patients (17 females, 5 males; average age: 41.5+/-SD 10.4 years). Gene expression profiles in peripheral blood mononuclear cells were obtained prior to treatment and at 1, 2, 4, 8, 24, 48, 120, 168 h after the first IFN-beta dose and at 1, 6 and 12 months after chronic dosing with once-weekly 30 microg IFN-beta-1a intramuscularly. Repeated measures statistics with false discovery rate control were used. The functional characteristics, biological pathways and transcription factor sites were analyzed. RESULTS Of the 1000 genes modulated following the first dose and upon chronic dosing of IFN-beta in MS patients, approximately 35% were up-regulated and 65% were down- regulated; the percentage of modulated genes in common was approximately 50%. The expression of the pharmacodynamic mRNA markers of IFN-beta effect showed differences in time profiles for the Good Responder and Partial Responders to IFN-beta therapy and the Jak-STAT, TNFRSF10B, IL6, TGFbeta, retinoic acid and CDC42 pathways were differentially modulated. The patients with side effects to therapy showed differences in the TGFbeta1, IFNG/STAT3 and TNF pathways. CONCLUSIONS Gene expression is a valuable tool for understanding the molecular mechanisms of IFN-beta action in MS patients.

Dynamics of immune cell trafficking in interferon-β treated multiple sclerosis patients

PURPOSE To investigate the effects of interferon-beta-1a (IFN-beta-1a) on the trafficking of cell populations in peripheral blood cells of multiple sclerosis (MS) patients. METHODS In this open-label pharmacodynamic study, peripheral blood was obtained from 10 relapsing-remitting (RR) MS patients just prior to and at 1, 2, 4, 8, 24, 48, 120, and 168 h after intramuscular injection of 30-microg IFN-beta-1a. Timed samples were also obtained from five controls at 0, 8, 24, 48 and 168 h. The blood cells were analyzed using four-color flow cytometry with antibody conjugates directed against cell surface proteins specific for T cells, B cells, NK cells, and the activation marker, CD69. RESULTS IFN-beta-1a treatment resulted in selective, time-dependent effects on many cell populations in peripheral blood. The trafficking of T-helper and T-suppressor/cytotoxic subsets of T cells were qualitatively different. The most prominent effects were on the trafficking of natural killer cells, the levels of which decreased to 23.5% of pretreatment values at 8 h after treatment. The levels of CD69-positive NK cells increased to a peak value of 606% of pretreatment levels at the 24-h time point. In untreated controls, these characteristic trafficking effects were not observed. There was inter-patient heterogeneity in the levels of activated NK cells at the 6-month time point that may potentially be relevant for individualizing IFN-beta therapy. CONCLUSIONS IFN-beta treatment can induce specific, selective, and time-dependent trafficking of cells and its effects on different subsets of a given cell type are not qualitatively similar. The dynamics indicate that the activation of NK cells by IFN-beta is possibly dependent on the trafficking of NK cells. The activated NK cell levels after prolonged therapy may potentially provide a surrogate marker for IFN-beta exposure.

Associations of moderate alcohol consumption with clinical and MRI measures in multiple sclerosis

OBJECTIVE To examine the associations of alcohol consumption patterns with disability and brain injury in multiple sclerosis (MS) patients. DESIGN This study included 423 subjects (272 MS patients, 151 healthy controls) participating in a study of clinical, environmental and genetic risk factors in MS. Disability was assessed with the Expanded Disability Status Scale (EDSS) and the MS Severity Scale (MSSS). Brain injury was assessed using the quantitative MRI measures of T2-lesion volume (T2-LV), T1-LV, normalized volumes of brain parenchyma (NBV), gray matter (NGMV) and lateral ventricle (NLVV). Information related to alcohol-consumption patterns was obtained with standardized questionnaire during an in-person interview. The associations of alcohol consumption variables with disability and MRI measures were assessed in regression analyses. RESULTS The frequency of MS patients who did not consume alcohol after MS (19.4%) was higher than the frequency before MS (p<0.001). The EDSS, NGMV and NLVV exhibited a non-linear dependence on duration of alcohol consumption after MS onset: non-linear regression analyses indicated that EDSS and NLVV were lower and the NGMV was greater in MS patients who had consumed for a period of 15years or less after MS onset compared those who did not consume alcohol or consumed it for more than 15years. CONCLUSION The duration of alcohol consumption is associated with disability and MRI measures in MS. Prospective, longitudinal studies of the role of alcohol in MS disease progression are warranted.

Chronic cerebrospinal vascular insufficiency is not associated with HLA DRB1*1501 status in multiple sclerosis patients.

Background Chronic cerebrospinal venous insufficiency (CCSVI) was described as a vascular condition characterized by anomalies of veins outside the skull was reported to be associated with multiple sclerosis (MS). The objective was to assess the associations between HLA DRB1*1501 status and the occurrence of CCSVI in MS patients. Methodology/Principal Findings This study included 423 of 499 subjects enrolled in the Combined Transcranial and Extracranial Venous Doppler Evaluation (CTEVD) study. The HLA DRB1*1501 status was obtained in 268 MS patients and 155 controls by genotyping rs3135005, a SNP associated with DRB1*1501 status. All subjects underwent a clinical examination and Doppler scan of the head and neck. The frequency of CCSVI was higher (OR = 4.52, p<0.001) in the MS group 56.0% vs. 21.9% in the controls group and also higher in the progressive MS group 69.8% vs. 49.5% in the non-progressive MS group. The 51.9% frequency of HLA DRB1*1501 positivity (HLA+) in MS was higher compared (OR = 2.33, p<0.001) to 31.6% to controls. The HLA+ frequency in the non-progressive (51.6%) and progressive MS groups (52.3%) was similar. The frequency of HLA+ CCSVI+ was 40.7% in progressive MS, 27.5% in non-progressive MS and 8.4% in controls. The presence of CCSVI was independent of HLA DRB1*1501 status in MS patients. Conclusions/Significance The lack of strong associations of CCSVI with HLA DRB1*1501 suggests that the role of the underlying associations of CCSVI in MS should be interpreted with caution. Further longitudinal studies should determine whether interactions between these factors can contribute to disease progression in MS.

Humoral response to EBV is associated with cortical atrophy and lesion burden in patients with MS

Objective: Because dysregulated Epstein-Barr virus (EBV)-infected B cells may induce meningeal inflammation, which contributes to cortical pathology in multiple sclerosis (MS), we investigated associations between antibody responses to EBV and development of cortical pathology in MS. Methods: We included 539 patients with MS (369 with relapsing-remitting MS, 135 with secondary progressive MS, and 35 with primary progressive MS), 66 patients with clinically isolated syndrome (CIS), 63 patients with other neurologic diseases (OND), and 178 age- and sex-matched healthy controls (HC). All participants were scanned on 3T MRI. Serum samples were analyzed for IgG antibodies against EBV viral capsid antigen (VCA) and EBV nuclear antigen-1 (EBNA-1), and their quartiles were determined on the whole study sample. Differences between the study groups were assessed using analysis of covariance adjusted for multiple comparisons. Results: More than 30% of patients with MS and CIS presented with the highest quartile of anti-EBV-VCA and -EBNA-1 status compared to ≤10% of HC (p < 0.001). The figures were 9 (14.3%) and 7 (12.3%) for patients with OND. Patients with MS with the highest quartile of anti-EBV-VCA showed significantly increased T2 lesion volume (p = 0.001), T1 lesion number (p = 0.002), and T1 lesion volume (p = 0.04) and decreased gray matter (p = 0.041) and cortical (p = 0.043) volumes compared to patients with MS with lower quartiles. No significant differences of MRI outcomes in patients with CIS, patients with OND, and HC with lower or highest quartiles of anti-EBV-VCA and -EBNA-1 were detected. Conclusions: Humoral response to anti-EBV-VCA and -EBNA-1 is associated with more advanced cortical atrophy, accumulation of chronic T1 black holes, and focal white matter lesions in patients with MS.