Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7

Loss of ATP-Sensitive Potassium Channel Surface Expression in Heart Failure Underlies Dysregulation of Action Potential Duration and Myocardial Vulnerability to Injury.

The search for new approaches to treatment and prevention of heart failure is a major challenge in medicine. The adenosine triphosphate-sensitive potassium (KATP) channel has been long associated with the ability to preserve myocardial function and viability under stress. High surface expression of membrane KATP channels ensures a rapid energy-sparing reduction in action potential duration (APD) in response to metabolic challenges, while cellular signaling that reduces surface KATP channel expression blunts APD shortening, thus sacrificing energetic efficiency in exchange for greater cellular calcium entry and increased contractile force. In healthy hearts, calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the Kir6.2 KATP channel subunit initiating a cascade responsible for KATP channel endocytosis. Here, activation of CaMKII in a transaortic banding (TAB) model of heart failure is coupled with a 35-40% reduction in surface expression of KATP channels compared to hearts from sham-operated mice. Linkage between KATP channel expression and CaMKII is verified in isolated cardiomyocytes in which activation of CaMKII results in downregulation of KATP channel current. Accordingly, shortening of monophasic APD is slowed in response to hypoxia or heart rate acceleration in failing compared to non-failing hearts, a phenomenon previously shown to result in significant increases in oxygen consumption. Even in the absence of coronary artery disease, failing myocardium can be further injured by ischemia due to a mismatch between metabolic supply and demand. Ischemia-reperfusion injury, following ischemic preconditioning, is diminished in hearts with CaMKII inhibition compared to wild-type hearts and this advantage is largely eliminated when myocardial KATP channel expression is absent, supporting that the myocardial protective benefit of CaMKII inhibition in heart failure may be substantially mediated by KATP channels. Recognition of CaMKII-dependent downregulation of KATP channel expression as a mechanism for vulnerability to injury in failing hearts points to strategies targeting this interaction for potential preventives or treatments

Alcohol and HIV-Derived Hepatocyte Apoptotic Bodies Induce Hepatic Stellate Cell Activation

Recently, we found that both HIV and acetaldehyde, an alcohol metabolite, induce hepatocyte apoptosis, resulting in the release of large extracellular vesicles called apoptotic bodies (ABs). The engulfment of these hepatocyte ABs by hepatic stellate cells (HSC) leads to their profibrotic activation. This study aims to establish the mechanisms of HSC activation after engulfment of ABs from acetaldehyde and HIV-exposed hepatocytes (ABAGS+HIV). In vitro experiments were performed on Huh7.5-CYP (RLW) cells to generate hepatocyte ABs and LX2 cells were used as HSC. To generate ABs, RLW cells were pretreated for 24 h with acetaldehyde, then exposed overnight to HIV1ADA and to acetaldehyde for 96 h. Thereafter, ABs were isolated from cell suspension by a differential centrifugation method and incubated with LX2 cells (3:1 ratio) for profibrotic genes and protein analyses. We found that HSC internalized ABs via the tyrosine kinase receptor, Axl. While the HIV gag RNA/HIV proteins accumulated in ABs elicited no productive infection in LX2 and immune cells, they triggered ROS and IL6 generation, which, in turn, activated profibrotic genes via the JNK-ERK1/2 and JAK-STAT3 pathways. Similarly, ongoing profibrotic activation was observed in immunodeficient NSG mice fed ethanol and injected with HIV-derived RLW ABs. We conclude that HSC activation by hepatocyte ABAGS+HIV engulfment is mediated by ROS-dependent JNK-ERK1/2 and IL6 triggering of JAK-STAT3 pathways. This can partially explain the mechanisms of liver fibrosis development frequently observed among alcohol abusing PLWH

STAT3 imparts BRCAness by impairing homologous recombination repair in Epstein-Barr virus-transformed B lymphocytes.

Epstein-Barr virus (EBV) causes lymphomas and epithelial cell cancers. Though generally silent in B lymphocytes, this widely prevalent virus can cause endemic Burkitt lymphoma and post-transplant lymphoproliferative disorders/lymphomas in immunocompromised hosts. By learning how EBV breaches barriers to cell proliferation, we hope to undermine those strategies to treat EBV lymphomas and potentially other cancers. We had previously found that EBV, through activation of cellular STAT3 prevents phosphorylation of Chk1, and thereby, suppresses activation of the intra-S phase cell-cycle checkpoint, a potent barrier to oncogene-driven proliferation. This observation prompted us to examine the consequences on DNA repair since homologous recombination repair, the most error-free form, requires phosphoChk1. We now report that the defect in Chk1 phosphorylation also curtails RAD51 nucleation, and thereby, homologous recombination repair of DNA double strand breaks. The resulting reliance on error-prone microhomology-mediated end-joining (MMEJ) repair makes EBV-transformed cells susceptible to PARP inhibition and simultaneous accrual of genome-wide deletions and insertions resulting from synthesis-dependent MMEJ. Analysis of transcriptomic and drug susceptibility data from hundreds of cancer lines reveals a STAT3-dependent gene-set predictive of susceptibility of cancers to synthetic lethal PARP inhibition. These findings i) demonstrate how the tumor virus EBV re-shapes cellular DNA repair, ii) provide the first genome-wide evidence for insertions resulting from MMEJ in human cells, and iii) expand the range of cancers (EBV-related and -unrelated) that are likely to respond to synthetic lethal inhibitors given the high prevalence of cancers with constitutively active STAT3

Monophasic action potential duration shortening is blunted in failing hearts.

<p>Monophasic action potentials were measured from a single left ventricular epicardial position in isolated hearts from TAB and sham mice before and during exposure to hypoxia or abrupt heart rate acceleration from 400 beats per minute (bpm) to 667 bpm (150 msec cycle length to 90 msec cycle length) driven by pacing. <b>A)</b> Representative normalized monophasic action potentials at baseline (solid lines) and in response to hypoxia (dotted lines) from hearts of sham (left panel) and TAB (right panel) mice. A horizontal line designates the point at which monophasic action potential duration at 90% repolarization (MAPD₉₀) was measured. <b>B)</b> Summary of MAPD₉₀ shortening (left), half-time (t_1/2) of MAPD₉₀ shortening (middle), and rate of MAPD₉₀ shortening (right) in hearts from sham and TAB mice in response to hypoxia (*p < .05). <b>C)</b> Summary of MAPD₉₀ shortening (left), half-time (t_1/2) of MAPD₉₀ shortening (middle), and rate of MAPD₉₀ shortening (right) in hearts from sham and TAB mice in response to heart rate acceleration (*p < .05).</p

Morphological and electrophysiological changes in transverse aortic banding (TAB) <i>vs</i>. sham operated mice.

<p><b>A)</b> Representative parasternal long axis echocardiographic images of the ventricles of sham and TAB operated mice. <b>B)</b> Summary data of left ventricular ejection fraction (**p<0.01 <i>vs</i>. sham). <b>C)</b> Representative action potentials recorded in isolated ventricular cardiomyocytes from sham and TAB mice. <b>D)</b> Summary data of action potential duration in isolated ventricular myocytes of sham and TAB mice (**p<0.01 <i>vs</i>. sham). APD₅₀, APD₇₅, APD₉₀: action potential duration at 50%, 75% and 90% repolarization, respectively. E_m: membrane potential.</p

K_ATP channel surface expression is down-regulated in ventricles of hearts after transverse aortic banding.

<p>A) Representative western blots of the K_ATP Kir6.2 subunit and the sodium-potassium pump (Na⁺-K⁺ pump) from the biotinylated membrane fraction of ventricular tissue from isolated hearts of sham and TAB mice. <b>B)</b> Summary of ventricular membrane Kir6.2 expression normalized to Na⁺-K⁺ pump expression in hearts of sham and TAB mice (*p<0.05 <i>vs</i>. sham). <b>C)</b> Representative current profiles of isolated left ventricular myocytes before and after application of the K_ATP channel activators, pinacidil (PIN, 100 μM) and 2,4-dinitrophenol (DNP, 200 μM). <b>D)</b> Summary of K_ATP channel current density (after-before K_ATP channel activations) from isolated ventricular cardiomyocytes of sham and TAB mice (**p<0.01 <i>vs</i>. sham).</p

Dynamic downregulation of K_ATP channel current density is blunted in ventricular cardiomyocytes of TAB mice.

<p><b>A)</b> Representative tracings of pinacidil (PIN, 100 μM)- and 2,4-dinitrophenol (DNP, 200 μM)-stimulated K_ATP channel current density in response to isoproterenol (ISO, 1 μM). <b>B)</b> Summary of percent inhibition of pinacidil and DNP-stimulated K_ATP channel current density by isoproterenol (1 μM) in isolated cardiomyocytes of sham and TAB mice (**p<0.01 <i>vs</i>. sham).</p

CaMKII is activated in ventricles of hearts following transverse aortic banding.

<p><b>A)</b> Representative western blots of phosphorylated CaMKII (P-CaMKII), oxidized CaMKII (Ox-CaMKII), total CaMKII (T-CaMKII) and GAPDH in ventricles of sham and TAB mice as labelled by whole-heart biotinylation. <b>B)</b> Summary of P-CaMKII, Ox-CaMKII and T-CaMKII quantification from western blots in ventricles of sham and TAB mice (*p<0.05, **p < .01 <i>vs</i>. sham).</p