EBNA1 Inhibitors Block Proliferation of Spontaneous Lymphoblastoid Cell Lines From Patients With Multiple Sclerosis and Healthy Controls.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes lifelong latency in memory B cells and has been identified as a major risk factor of multiple sclerosis (MS). B cell depletion therapies have disease-modifying benefit in MS. However, it is unclear whether this benefit is partly attributable to the elimination of EBV+ B cells. Currently, there are no EBV-specific antiviral therapies available for targeting EBV latent infection in MS and limited experimental models to study EBV in MS. METHODS: In this study, we describe the establishment of spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls and treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells. RESULTS: We showed that a small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV+ cells, while having no discernible effects on EBV- cells. We validate that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR. DISCUSSION: This study shows that patient-derived SLCLs provide a useful tool for interrogating the role of EBV+ B cells in MS and suggests that a clinical trial testing the effect of EBNA1 inhibitors in MS may be warranted.

Energetic Radiation From Galactic Cosmic Ray Interactions With Saturn's Main Rings.

Saturn's main rings have an energetic particle and gamma ray photon radiation environment produced by ring interactions of galactic cosmic ray (GCR) protons and heavier ions penetrating the planetary dipolar magnetic field. Accurate models of this radiation environment are important for interpretation of Pioneer 11 and Cassini in situ measurements near the rings and for constraints on radiolytic contributions to neutral gas production and ice chemistry. A GEANT (GEometry ANd Tracking) based simulation is used to model flux spectra of protons, electrons, positrons, charged pions, neutrons, and gamma ray photons emitted from GCR interactions with H2O ice spheres approximating the ring material. Dependent on location in the A to D rings within the planetary magnetic field of Saturn, only GCR protons above respective energies of 20 to 72 GeV can reach the rings without being deflected away by the magnetic field. Calculated differential and integral fluxes from our simulations have good agreement with in situ Pioneer-11 measurements in selected energy channels. The charged particle and neutral radiation measurements are sensitive, respectively, to the sizes and areal mass densities of ring bodies. Computed gamma ray emission fluxes are 8% of our calculated limit for detection from the Earth by the Fermi Large Area Telescope. Addition of charged particle sensors and neutron-photon imaging spectrometers to a future Saturn Ring Observer mission would provide valuable information on the ring mass structure. The present paper provides a foundation for modeling of Pioneer 11 and Cassini radiation measurements across the main rings and future measurements of radiation from the rings.

Differential α4(+)/(-)ß2 Agonist-binding Site Contributions to α4ß2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

Two α4ß2 nicotinic acetylcholine receptor (α4ß2-nAChR) isoforms exist with (α4)2(ß2)3 and (α4)3(ß2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)ß2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)ß2 sites. However, the relative roles of the conserved α4(+)/(-)ß2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4ß2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of ß2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (ß2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)ß2 sites contribute equally to function, making identical ß2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)ß2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.