Figures
Abstract
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.
Citation: Horakova D, Zivadinov R, Weinstock-Guttman B, Havrdova E, Qu J, Tamaño-Blanco M, et al. (2013) Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study. PLoS ONE 8(1): e53996. https://doi.org/10.1371/journal.pone.0053996
Editor: Robert Shin Fujinami, University of Utah School of Medicine, United States of America
Received: October 4, 2012; Accepted: December 7, 2012; Published: January 8, 2013
Copyright: © 2013 Horakova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by Czech Ministries of Education and Health (NT13237-4/2012, MSM 0021620849, PRVOUK-P26/LF1/4, RVO-VFN64165/2012). The SET study is an investigator-initiated study supported by Biogen Idec Inc. Support from the National Multiple Sclerosis Society (RG3743 and RG4836), the Department of Defense Multiple Sclerosis Program (MS090122) and a Pediatric MS Center of Excellence Center Grant) is gratefully acknowledged. The funders had no role in data analysis, decision to publish, or preparation of the manuscript.
Competing interests: Dr. Dana Horakova received speaker honoraria and consultant fees from Biogen Idec, Novartis, Merck Serono, Teva and Bayer Schering and financial support for research activities from Biogen Idec. The SET study is an investigator-initiated study supported by Biogen Idec Inc. Dr. Zivadinov has received speaker honoraria and consultant fees from Teva Neurosciences, Biogen Idec, Questcor, Genzyme-Sanofi, Novartis, Bracco, Bayer and EMD Serono. He has received research support from the National Multiple Sclerosis Society, Department of Defense, Biogen Idec, Teva Neuroscience, Teva Pharmaceuticals, EMD Serono, Genzyme-Sanofi, Questcor, Bracco and Greatbatch. Dr. Weinstock-Guttman received honoraria for serving in advisory boards and educational programs from Teva Neuroscience, Biogen Idec, Novartis, Accorda EMD Serono, Pfizer, Novartis, Genzyme and Sanofi. She also received support for research activities from the National Institutes of Health, National Multiple Sclerosis Society, National Science Foundation, Department of Defense, EMD Serono, Biogen Idec, Teva Neuroscience, Cyberonics, Novartis, Acorda and the Jog for the Jake Foundation. Dr. Eva Havrdova received honoraria and consulting fees from Genzyme, Biogen Idec, Sanofi-Aventis, Merck Serono, Roche, Teva and Novartis for consulting services, speaking and serving on a scientific advisory boards. Dr. Jun Qu received research funding from the Department of Defense, the National Science Foundation, the National Institutes of Health, the American Heart Association, Kinex, CH3, Pfizer, Novartis, Abbott and Amgen. These are unrelated to the research presented in this report. Drs. Michaela Tyblova, Jan Krasensky, Manuela Vaneckova, and Zdenek Seidl have received financial support for research activities from Biogen Idec. Dr. Petra Lelkova has no conflicts to disclose. Dr. Tomas Kalincik received compensation for travel and honoraria from Novartis, Biogen Idec, Sanofi Aventis, Teva and Merck Serono. Dr. Murali Ramanathan received research funding or consulting fees from EMD Serono, Biogen Idec, Allergan, Netezza, Pfizer, Novartis, Monsanto the National Multiple Sclerosis Society, the Department of Defense, Jog for the Jake Foundation, the National Institutes of Health and National Science Foundation. He received compensation for serving as an Editor from the American Association of Pharmaceutical Scientists. These are unrelated to the research presented in this report. Miriam Tamaño-Blanco, Darlene Badgett, Niels Bergsland, Sara Hussein, Laura Willis, Michael G. Dwyer, Haoying Yu, Ming Zhang and Xiaotao Duan have no conflicts to disclose. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Introduction
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the brain and spinal cord that causes physical and cognitive disability. Disease progression in MS is mediated by inflammatory and neurodegenerative processes that cause injury to the brain and spinal cord.
Epstein-Barr virus (EBV) exposure, decreased vitamin D levels and smoking are the best-established environmental risk factors for MS [1]–[3]. Efforts to integrate environmental risk factors with the risk contributions of genetic variations such HLA DRB1*1501 have been reported [3], [4].
Cross-sectional studies suggest that exposure to EBV, cigarette smoking and low vitamin D are also associated with MS disease progression. Higher levels of EBV antibodies are associated with greater brain atrophy [4]–[6]. Smoking increases lesion volume and the risk of developing secondary-progressive MS [7]–[9] whereas increased vitamin D levels have been associated with fewer relapses [10]–[13].
The first clinical demyelinating event, often known as clinically isolated syndrome (CIS), represents a crucial opportunity for understanding the factors involved in conversion to MS. The environmental factors in CIS have not been extensively investigated prospectively and their role in MS disease course and progression is not well characterized. Anti-EBV nuclear antigen (EBNA-1) antibodies were associated with progression to clinically definite MS (CDMS) and with the formation of new lesions [14] and smoking has been linked to increased risk of developing CDMS and to decreased time to first relapse [15]. Here, we examine the relationship between multiple environmental risk factors for MS and disease progression as assessed by clinical and MRI measures obtained longitudinally in a cohort of CIS patients after the initial demyelinating event in a controlled, multi-center, observational study. Drawing on the body of prior research, pre-treatment serum from this CIS cohort was assessed for anti-EBV EBNA-1, anti-EBV viral capsid antigen (VCA), anti-cytomegalovirus (CMV) antibodies, vitamin D levels and active smoking status via cotinine levels. All patients were initiated on intramuscular interferon beta-1a (AVONEX®) therapy at time of entry.
Methods
Study Population
Clinical Study Design.
The Observational Study of Early Interferon beta 1-a Treatment in High Risk Subjects after CIS (SET study) is a prospective observational clinical study coordinated by the Charles University in Prague. It involves eight centers from the Czech Republic.
The objective of the SET study is to determine clinical and MRI predictors of response to interferon beta 1-a therapy in CIS. All patients are treated with 30 µg, intramuscular interferon beta 1-a (AVONEX®). The trial includes clinical visits every 3-months for 4 years and subsequent long term follow up in routine clinical practice. A range of clinical and MRI outcomes (including time to CDMS, disability progression, quality of life measures and yearly volumetric MRI scans) were obtained longitudinally.
Study Population.
The study population were CIS patients with the following characteristics: 18–55 years of age, enrolled within 4 months from the clinical event, EDSS ≤3.5, presence of ≥2 T2-hyperintense lesions on diagnostic MRI, and presence of ≥2 oligoclonal bands in CSF obtained at the screening visit prior to steroid treatment. All patients were treated with 3–5 g of methylprednisolone for the first symptom and baseline MRI was performed at least 30 days after steroid administration.
Of the 220 CIS patients enrolled in the SET study, 216 CIS patients had available clinical follow-up and MRI data. This analysis was limited to 211 subjects with environmental factor biomarkers or HLA DRB1*1501 genotyping available (Results S1).
Clinical and MRI assessments were obtained at baseline, 6, 12 and 24 months. Clinical assessments were performed using the Kurtzke Expanded Disability Status Scale (EDSS). In case of relapses, patients were evaluated within 4 days from onset of the new symptoms.
Ethics Statement.
The Medical Ethics Committees of the General University Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech Republic, approved the study protocol and the informed consent procedure. In addition, approvals were obtained from local medical ethics committees of all other participating centers (KZ Hospital, Teplice; University Hospitals in Brno, Plzen and Olomouc; St. Anne's University Hospital, Brno; Motol University Hospital, Prague and Kralovske Vinohrady University Hospital, Prague). Written informed consent was obtained from all patients at enrolment.
Environmental Factors
The technicians conducting analyses of anti-EBV and anti-CMV antibodies, smoking status, vitamin D and HLA DRB1*1501 status were blinded to the patients' clinical status. All analyses were conducted in serum samples obtained at the screening visit prior to any corticosteroid or interferon beta-1a treatment.
Anti-EBV and Anti-CMV antibodies.
Enzyme-linked immunosorbent assay (ELISA) kits from Diamedix Corporation (Miami, FL) were used to quantify anti-CMV IgG, anti-EBV VCA, EBNA-1 and early antigen (EA-D) IgG antibodies. Serial dilutions of positive control samples provided with each kit were used to generate standard curves. The anti-CMV, EBNA-1 and VCA IgG levels were normalized to the manufacturer's cut-off calibrator standard, which represents a sample that is just positive. Anti-CMV and anti-EBV antibody levels were available for 193 patients. The anti-EBNA-1 and anti-VCA relative concentrations were categorized into quartiles.
Smoking Status.
Cotinine levels were measured using a validated liquid chromatography-mass spectrometry method. A cotinine level threshold of 10 ng/ml was used to categorize subject as active smokers [16], [17]. Cotinine levels were available for 194 patients.
Vitamin D Levels.
The vitamin D metabolite 25 hydroxy vitamin D3 (25(OH)VD3) was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods with stable-isotope-labeled internal standards as previously described [18].
Raw 25(OH)VD3 levels were deseasonalized using sinusoidal regression [19] and dichotomized based on the clinical threshold for vitamin D deficiency (25(OH)VD3 level <20 ng/ml) [20]. Levels of 25(OH)VD3 were available for 185 patients. The missing subjects lacked serum or had insufficient serum.
Genotyping
HLA DRB1*1501 status was obtained by genotyping DNA from peripheral blood for rs3135005, a SNP strongly correlated with HLA DRB1*1501 status, using allele discrimination (Applied Biosystems, Redwood City, CA) [21]. HLA DRB1*1501 status was available for 198 patients. The missing genotypes were due to non-availability of DNA samples or ambiguous genotypes.
MRI Acquisition and Analysis
Image Acquisition.
MRI was performed on all patients using a 1.5 T magnet (Philips Gyroscan NT 15, Best, the Netherlands). Acquisition details are in Results S1.
Image Analysis.
The scans were collected centrally at the Department of Radiology at Charles University (Prague, Czech Republic). All MRI scans were transferred analyzed by the Buffalo Neuroimaging Analysis Center (Buffalo, New York, USA).
Lesion Measures.
The T2- and contrast enhancing lesion (CEL) number and lesion volumes (LVs) were measured as previously described [22]. For each time point, the identification of new and enlarging T2-lesions was performed via a “subtraction image” methodology (See Results S1).
Global and Tissue-Specific Atrophy Measures.
For baseline analyses, SIENAX software was used (version 2.6). Normalized whole brain volume (WBV), normalized gray matter volume (NGMV) and normalized white matter volume (NWMV) were measured as previously described [23]. For longitudinal changes of the WBV, we used the SIENA method [24] to calculate the percentage brain volume change (PBVC). To quantify longitudinal GM and WM volume changes, we used a modified hybrid of FMRIB's SIENA and SIENAX tools (See Results S1).
Data Analysis
SPSS (IBM Inc., Armonk, NY, version 19.0) statistical program was used for all statistical analyses. In view of the multiple testing, a conservative p-value of ≤0.01 was used to assess significance; p-values ≤0.05 were considered to be trends. The clinical variables were analyzed with appropriate regression analyses that included age, sex and the genetic or environmental variable of interest as predictors. The regression models for MRI progression variables additionally included the corresponding baseline value of the MRI variable as a predictor. Additional details of the statistical analysis are in Results S1.
Results
Overview of Study Cohort
Tables 1, 2, 3 show the clinical, demographic and MRI features and environmental factor distributions of the cohort.
During the 2-year period, 43% of patients (90 of 211) experienced relapses; the median time to relapse was 5.6 months. Figure 1A shows the cumulative hazard for time to the first relapse.
Figure 1A shows the cumulative hazard function for time to first relapse in all subjects. Figures 1B-D show the cumulative hazard functions for the age <35 years (red line) vs. age ≥35 years (green line), CEL present as baseline (red line) vs. CEL not present at baseline (green line) and CMV positive (red line) vs. CMV negative sub-groups, respectively. The corresponding covariate p-values from Cox regression are also shown.
Based on EDSS change, 25 of 211 (12%) showed EDSS progression and 36 of 211 (17%) showed EDSS improvement (defined as ≥1 point decrease in EDSS at Month 24 compared to EDSS at baseline). Four patients (1.9%) showed EDSS progression but did not experience relapses during the 2-year period.
Anti-CMV Positivity is Associated with Relapses
Associations of Anti-CMV Positivity with Relapses.
Anti-CMV antibody positivity was associated as a trend with an increase in the number of relapses during the 2-year period (p = 0.014). The marginal mean for the number of relapses during the 2-year period was 0.52± SE 0.096 in the anti-CMV negative group compared to 0.90± SE 0.14 in the anti-CMV positive group.
Anti-CMV positivity was associated with progression to CDMS over the 2-year period (p = 0.004, Odds ratio = 2.51, 95% confidence interval = 1.35–4.68). In the anti-CMV antibody positive group, 51% (55 out of 107) experienced relapses compared to 31% (27 out of 86) in the anti-CMV antibody negative group.
Anti-CMV antibody positivity was associated with time to first relapse (Figure 1, p = 0.008, hazard ratio = 1.9).
Associations of Other Factors with Relapses.
Anti-EBV VCA highest quartile status, anti-EBV EBNA-1 highest quartile status, HLA DRB1*1501 positivity, active smoking status and vitamin D deficiency status were not associated with the number of relapses (all p>0.26), progression to CDMS (all p>0.44) or time to first relapse (all p-values >0.41).
However, younger age and female sex were associated with increased number of relapses over the 2-year period (p<0.001 for age and p = 0.021 for sex). Increased age (p = 0.009) and age ≥35 years status (p = 0.045, a trend, Figure 1) were associated with increased time to first relapse.
The presence (p<0.001) and number of CEL (p = 0.004) at baseline were also associated with the number of relapses. The presence of CEL at baseline was associated with decreased time to relapse (Figure 1, p<0.001). The number of T2 lesions at baseline was also associated with a trend toward decreased time to first relapse (p = 0.013).
Anti-EBV VCA Antibodies are Associated with Disability Progression
Anti-EBV VCA in the highest quartile (p = 0.023, OR = 2.84, 95% CI = 1.16–6.96) was associated as a trend with EDSS progression. EDSS progression occurred for 11 of 49 (25%) patients in the highest quartile of anti-EBV VCA compared to 13 of 145 (9%) patients in the lower quartiles of anti-EBV VCA.
Anti-CMV positivity, anti-EBV EBNA-1 in the highest quartile, HLA DRB1*1501 positivity, active smoking status and vitamin D deficiency were not associated with EDSS progression (p = 0.063 for CMV positivity; all other p>0.46).
Anti-EBV VCA Antibodies are Associated with MRI Progression
Contrast Enhancing Lesions.
Anti-EBV-VCA highest quartile status was associated as a trend with increased CEL number (p = 0.031) but not CE-LV between baseline and 2 years (p = 0.31). The marginal mean ± SE for the highest anti-EBV VCA quartile was 0.49±0.15 lesions compared to 0.24±0.048 for the lower quartiles. The cumulative number of CEL over 2-years was associated as a trend with anti-EBV VCA highest quartile (p = 0.041, marginal mean ± SE = 0.90±0.22 for the highest quartile vs. 0.51±0.077 for the lower quartiles).
Anti-EBV-EBNA-1 in the highest quartile was associated with increased CEL number (p = 0.003) but not CE-LV between baseline and 2-years (p = 0.21). The marginal mean ± SE for the highest anti-EBV EBNA-1 quartile was 0.52±0.14 lesions compared to 0.20±0.045 for the lower quartiles.
New and Newly Enlarging T2 Lesions.
Anti-EBV VCA highest quartile status was associated with a trend toward increased number of new and newly enlarging T2 lesions (Figure 2C, p = 0.018). The marginal mean for the highest anti-EBV VCA quartile was 4.53±0.83 lesions vs. 2.80±0.29 lesions for the lower quartiles.
Figures 2A and 2B show the dependence of change in CEL number and change in CE-LV, respectively, on smoking status. Figures 2C shows the dependence of number of new and newly enlarging T2-lesions on anti-EBV VCA highest quartile status. Figure 2D shows the dependence of number of new T2-lesions on anti-EBV VCA highest quartile status. The bars represent mean values and the error bars are standard errors. The red colors denote the positive group and the green bars the negative group. The corresponding covariate p-values from regression are also shown.
The effect of anti-EBV VCA highest quartile status on the number of new and newly enlarging T2 lesions was explained by its effect on the number of new T2 lesions (p = 0.006). The marginal mean for the number of new lesions in the patients in the highest quartile of anti-EBV VCA levels was 3.56±0.67 lesions compared to 2.01±0.22 lesions for those in the lower quartiles.
Smoking is Associated with Contrast Enhancing Lesions
Active smoker status was associated with increase in CEL number (p = 0.002, Figure 2A) and a trend toward increased in CE-LV between baseline and 2-year (p = 0.014, Figure 2B). The marginal mean number of CEL at 2 years in the active smoker group was 0.51±0.13 lesions compared 0.19±0.044 lesions in the non-active smoker group.
Active smoking status was not associated with the number of new and newly enlarging T2 lesions (p = 0.86) or brain atrophy as assessed by PBVC (p = 0.64).
Vitamin D, Anti-CMV Positivity and HLA DRB1*1501 and MRI Progression
Contrast Enhancing Lesions.
Anti-CMV positivity, HLA DRB1*1501 positivity and vitamin D deficiency were not associated with cumulative CEL number over 2-years, CEL number or volume at 2 years or with new or newly enlarging T2 lesions (all p-values >0.35).
New and Newly Enlarging T2 Lesions.
A trend was found for increased number of new T2 lesions and vitamin D deficiency (p = 0.019, marginal means ± SE = 2.54±0.25 new lesions in vitamin D deficient vs. 1.49±0.32 in the vitamin D insufficient or sufficient group).
Brain Atrophy.
HLA DRB1*1501 positivity (p = 0.046, rp = −0.15) was associated with trend of decreased PBVC over the 2-year period. A weak trend was found for anti-CMV positivity (p = 0.054, rp = −0.14). We did not obtain evidence for significant associations of PBVC with anti-EBV EBNA-1 (p = 0.43) and vitamin D deficiency (p = 0.69).
Effects of Risk Factor Combinations
These results are summarized in Results S1. Based on these analyses, we surmise that the anti-CMV positivity-anti-EBV VCA highest quartile combination is a parsimonious explanatory predictor because it exhibits stronger associations with more clinical and MRI variables.
Discussion
In this study of CIS patients with oligoclonal bands in CSF and 2 or more lesions on the brain MRI as entry criteria, we examined the associations of environmental factors on disease progression. Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA highest quartile status was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of whole brain atrophy. We also investigated combinations of risk factors and found that the anti-CMV positivity-anti-EBV VCA highest quartile status combination was additively associated with both clinical and MRI outcomes.
Our findings of associations of anti-EBV VCA antibodies with brain atrophy are consistent with a previous study in an Italian patient group with MS [6]. In contrast, Lunemann et al. reported that only anti-EBV EBNA-1 antibodies were associated with conversion to CDMS [14] and found associations for new T2-lesions with both anti-EBV EBNA-1 and anti-EBV VCA antibodies. Our prospective study was conducted in cohort of high-risk patients on interferon-beta-1a treatment, whereas only 65.5% of the subjects in the Lunemann et al. study had oligoclonal bands [14]. Ingram et al. did not find associations between anti-EBV EBNA-1 IgG levels and disease activity in MS [25].
We did not obtain evidence that smoking was associated with decreased time to relapse as reported by Di Pauli et al. [15]. The discordance may be attributable to methodological differences: Di Pauli et al. [15] had 3-year follow-up and used a smoking questionnaire. We did not obtain patient smoking history but measured cotinine to objectively assesses active smoking status.
The genetics models for MS etiology implicate a network of immune related processes with HLA loci at the hub [26]. Although HLA DRB*1501 has been associated with low N-acetyl-aspartate (NAA) concentration in WM and WBV [27], numerous studies have reported lack of associations with MS disease severity [28]–[31]. Our finding of a trend between HLA DRB1*1501 positivity and decreased PBVC must be interpreted conservatively given its borderline p-value and the lack of associations with GM or WM atrophy and with CEL or T2-lesions, which are related to inflammatory activity.
Although vitamin D deficiency exhibited a trend with increased new T2 lesions, we did not find the associations with relapse rate that have been reported for MS [10]–[13]. We attribute the lack of evidence for vitamin D effects on relapse rate to the low frequency of vitamin D sufficiency in our study sample. Vitamin D supplementation, which is frequent among MS patients, is less likely in the younger CIS population.
The immediate early 1 (IE1) protein of CMV is an antagonist of Type 1 interferon but stimulates interferon-gamma responses via signal transducer and activator of transcription 1 (STAT1) [32]. This may provide a mechanistic framework for understanding our CMV findings in our interferon-beta treated cohort. We used the anti-CMV positivity anti-VCA highest quartile status combination risk factor score to assess the effects of the combination because a risk factor score is easily interpreted. EBV and CMV are both herpesviruses and evidence from transplant patients and immunological studies suggest that CMV infection can cause reactivation of EBV and alter cytokine production from immune cell populations [33]–[36].
We now discuss the strengths and weaknesses of our study. Although our study has a small sample size compared to genomewide association studies, it was conducted in the framework of a prospective, longitudinal, multi-center study and we had available clinical and MRI measures over a 2-year period. The relative homogeneity of the Czech patient sample and the single interferon-beta 1a treatment study design increases the power to detect effects. However, the homogeneity may potentially limit the ability to extrapolate the findings to other populations and to other treatments. Additionally, the results represent the contributions of environmental factors to the combined effects of both treatment and disease progression. The study inclusion criteria limited our ability to assess conversion according to the 2010 Polman-McDonald criteria [37].
In conclusion, our results suggest that environmental factors such as cigarette smoking, CMV exposure and responses to EBV may contribute to disease progression in interferon beta treated CIS patients. Smoking cessation and therapeutic and preventive interventions directed against CMV and EBV may potentially be useful in further slowing disease progression in this patient population.
Acknowledgments
We thank the other Clinical Centers and investigators who participated in the SET study:
KZ a.s. Hospital, Teplice: M. Vachova, S. Machalicka, J. Kotalova.
University Hospital, Brno, Bohunice: Y.Benesova, P. Praksova, P. Stourac.
St. Anne's University Hospital, Brno: M. Dufek.
Charles University in Prague, 2nd Faculty of Medicine, Motol: E. Meluzinova, J. Pikova, E. Houzvickova.
Charles University in Prague, 3rd Faculty of Medicine, Kralovske Vinohrady: D. Zimova.
University Hospital, Plzen: J. Sucha.
University Hospital, Olomouc: V. Sladkova, J. Mares.
The authors thank the subjects who participated in this study.
Author Contributions
Conceived and designed the experiments: DH EH ZS MV BWG JQ RZ MR. Performed the experiments: MTB DB MT PL NB SH LW JK MGD MZ HY XD. Analyzed the data: MR RZ TK. Wrote the paper: MR BWG DH EH TK RZ.
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