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Pathogenesis – Immunology Poster Presentation

P0936 - Active MS patients revealed a higher expression of EBV-DNA in their CD19+ cells in comparison to stable MS patients and controls (ID 1832)

Speakers
Authors
Presentation Number
P0936
Presentation Topic
Pathogenesis – Immunology

Abstract

Background

Multiple sclerosis (MS) is a chronic, inflammatory neurodegenerative disease affecting more than 2.3 million people worldwide and for which, there is no cure currently. The etiology of the disease is multifactorial including genetic predisposition, abnormal immune responses and environmental factors. One environmental factor in particular, exposure to EBV, has been associated with MS through increased antibody titers to EBV viral proteins in the blood and the cerebrospinal fluid. EBV is a ubiquitous human herpesvirus latent in B-cells where 95% of the population by young adulthood are known to be positive by seropositivity rate. EBV causes infectious mononucleosis (IM) and individuals who have had IM have a higher risk of developing MS.

Objectives

Compare the frequency and magnitude of detection of EBV-DNA by digital droplet PCR (ddPCR) in normal donors (ND) and stable and active MS patients.

Methods

25 stable and 25 active MS patients and 26 ND were analyzed in this study. Active patients were defined as patients with ≥ 1 cerebral enhancing lesions (CEL) at the sample date. PBMCs and CD19+-B cells sorted by flow cytometry and magnetic beads were analyzed. DNA was extracted and used as templates to evaluate the presence of EBV-DNA by ddPCR. EBV copy number per 106 cells was assessed for the BAMHI region of EBV compared to the housekeeping gene, RPP30.

Results

Our data show that the frequency of EBV positive CD19+ cells in active MS patients was significantly elevated (20/25, 80%) compared to stable MS patients (7/25, 28%; p=0.0002) and normal controls (11/26, 42%; p=0.006). To evaluate if the increase in the frequency of EBV positivity in the active MS patients was specific for EBV, cells from these cohorts were used to amplify another ubiquitous B cell tropic virus, JCV, that also establishes latency in B cells. JCV was not detected in any of the active MS patients CD19+ cells. These data demonstrate that the increase in the frequency of EBV DNA detection in active MS patients in comparison to the stable and the ND controls was specific to EBV. Significant difference was also observed in the frequency of detection of EBV DNA between the active (9/25, 36%) versus the stable MS patients (3/25, 12%; p=0.047) when PBMCs were used as template to amplify EBV-DNA. However, the magnitude of the EBV viral load was not significantly different between the three cohorts as determined by ANOVA test.

Conclusions

Our experiments indicate that in active MS patients the percentage of CD19+-B cells positive for EBV-DNA is doubled in contrast to stable MS and normal donor controls supporting a role for EBV in the pathogenesis of MS. Our data indicate that the use of sorted CD19+ B cells versus total PBMCs may be a more sensitive method for EBV-DNA detection.

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Presenter Of 1 Presentation

Pathogenesis – Immunology Poster Presentation

P0936 - Active MS patients revealed a higher expression of EBV-DNA in their CD19+ cells in comparison to stable MS patients and controls (ID 1832)

Speakers
Authors
Presentation Number
P0936
Presentation Topic
Pathogenesis – Immunology

Abstract

Background

Multiple sclerosis (MS) is a chronic, inflammatory neurodegenerative disease affecting more than 2.3 million people worldwide and for which, there is no cure currently. The etiology of the disease is multifactorial including genetic predisposition, abnormal immune responses and environmental factors. One environmental factor in particular, exposure to EBV, has been associated with MS through increased antibody titers to EBV viral proteins in the blood and the cerebrospinal fluid. EBV is a ubiquitous human herpesvirus latent in B-cells where 95% of the population by young adulthood are known to be positive by seropositivity rate. EBV causes infectious mononucleosis (IM) and individuals who have had IM have a higher risk of developing MS.

Objectives

Compare the frequency and magnitude of detection of EBV-DNA by digital droplet PCR (ddPCR) in normal donors (ND) and stable and active MS patients.

Methods

25 stable and 25 active MS patients and 26 ND were analyzed in this study. Active patients were defined as patients with ≥ 1 cerebral enhancing lesions (CEL) at the sample date. PBMCs and CD19+-B cells sorted by flow cytometry and magnetic beads were analyzed. DNA was extracted and used as templates to evaluate the presence of EBV-DNA by ddPCR. EBV copy number per 106 cells was assessed for the BAMHI region of EBV compared to the housekeeping gene, RPP30.

Results

Our data show that the frequency of EBV positive CD19+ cells in active MS patients was significantly elevated (20/25, 80%) compared to stable MS patients (7/25, 28%; p=0.0002) and normal controls (11/26, 42%; p=0.006). To evaluate if the increase in the frequency of EBV positivity in the active MS patients was specific for EBV, cells from these cohorts were used to amplify another ubiquitous B cell tropic virus, JCV, that also establishes latency in B cells. JCV was not detected in any of the active MS patients CD19+ cells. These data demonstrate that the increase in the frequency of EBV DNA detection in active MS patients in comparison to the stable and the ND controls was specific to EBV. Significant difference was also observed in the frequency of detection of EBV DNA between the active (9/25, 36%) versus the stable MS patients (3/25, 12%; p=0.047) when PBMCs were used as template to amplify EBV-DNA. However, the magnitude of the EBV viral load was not significantly different between the three cohorts as determined by ANOVA test.

Conclusions

Our experiments indicate that in active MS patients the percentage of CD19+-B cells positive for EBV-DNA is doubled in contrast to stable MS and normal donor controls supporting a role for EBV in the pathogenesis of MS. Our data indicate that the use of sorted CD19+ B cells versus total PBMCs may be a more sensitive method for EBV-DNA detection.

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