The Effects of Japanese Encephalitis Vaccine and Accelerated Dosing Scheduling on the Immunogenicity of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine: A Phase II, Randomized, Open-Label, Single-Center Trial in Adults Aged 18 to 45 Years in the United States.

BACKGROUND: Dengue is a global health problem requiring an effective, safe dengue vaccine. METHODS: We report the results of a phase II, randomized, open-label, single-center trial in adults aged 18 to 45 years in the United States designed to explore the effects of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine (CYD-TDV, Dengvaxia) when administered on its designated schedule (months 0, 6, and 12) or on an accelerated dosing schedule (months 0, 2, and 6) and/or given before, or concomitantly with, a vaccine against Japanese encephalitis (JE). RESULTS: Based on dengue virus serotype-specific neutralizing antibody (NAb), the accelerated dosing schedule was comparable to the 0, 6, and 12-month schedule. Giving JE vaccine concurrently with CYD-TDV did not result in an increase in overall NAb titers. Immunophenotyping of peripheral blood mononuclear cells revealed an increase in activated CD8+ T cells after CYD-TDV vaccination, a phenomenon that was greatest for the JE vaccine primed. CONCLUSIONS: We conclude that an accelerated dosing schedule of CYD-TDV results in essentially equivalent dengue serotype-specific NAb titers as the currently used schedule, and there may be an early benefit in antibody titers and activated CD8+ T cells by the administration of the JE vaccine before CYD-TDV vaccination.

Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

Tpeak-Tend interval during therapeutic hypothermia can predict upcoming ventricular fibrillation in subjects with aborted arrhythmic sudden cardiac death: 3-years follow-up results.

AIMS: Prolonged Tpeak-Tend interval has been shown to be markers of arrhythmogenesis in various cardiac disorders. However, its dynamicity is one of the obstacles to predict fatal ventricular arrhythmia. This study investigated whether Tpeak-Tend interval during therapeutic hypothermia (TH) is associated with ventricular fibrillation (VF) inducibility and clinical arrhythmia in subjects with aborted arrhythmic sudden cardiac death (SCD). METHODS AND RESULTS: The study group included 31 patients (24 males, age 39.1 ± 17.6 years) presenting with arrhythmic SCD in whom Tpeak-Tend interval and J-wave amplitude were measured in electrocardiogram (ECG) of the earliest medical contact and during TH; these patients underwent programmed ventricular stimulation. The summation of J-wave amplitude and QTc interval increased during TH. However, it was not associated with VF inducibility. Patients with inducible VF showed a small Tpeak-Tend interval dispersion in the baseline 12-lead ECG (68.8 ± 24.7 vs. 94.0 ± 55.6 ms, P = 0.044) and a marked increase of the dispersion during the TH (36.2 ± 51.2 vs. -6.1 ± 45.5 ms, P = 0.039). Twenty-four patients underwent implantable cardioverter defibrillator (ICD) implantation. Among them, the patients with long QTc, Tpeak-Tend, and precordial Tpeak-Tend during the TH developed VF more frequently (QTc, 511.9 ± 53.71 ms vs. 566.5 ± 56.08 ms, P = 0.038; Tpeak-Tend interval, 145.6 ± 38.4 ms vs. 185.7 ± 49.95 ms, P = 0.048; precordial Tpeak-Tend interval, 139.3 ± 35.11 ms vs. 185.7 ± 49.95 ms, P = 0.018). The initial VF inducibility was not related with the VF development in follow-up. CONCLUSION: In patients with aborted arrhythmic SCD, long Tpeak-Tend interval and QTc interval during TH could predict VF development in their follow-up.

Connexins and pannexins in the immune system and lymphatic organs.

Connexin43 and pannexin1 are found in immune cells. While gap junctional communication has been demonstrated between immune cells, hemichannels have been implicated in many cellular functions. Among the functions involved as being connexin dependent and pannexin dependent are cell migration, phagocytosis, antigen presentation, T-cell reactivity and B-cell responses. Surprisingly, many of these connexin-related and pannexin-related functions are not recapitulated in in vivo models. This is leading to a reevaluation of the role of these proteins in immune function.

Connexin43 is dispensable for phagocytosis.

Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

Performance of Inaugural Student Cohorts in 1-Year Special Master's Program.

SUNY Upstate Medical University (Upstate) recently revised two 1-year special master's programs (SMPs), designed to enhance academics in preparation for medical school through rigorous graduate science coursework. Upstate SMP program success was measured by graduate performance during the first year of medical school. First-year performance measures included academic deficiencies, exam performance, and participation grades in a longitudinal case-based course on ethics and public health. Upstate SMP students with MCAT scores below the 50th percentile or who self-report classified as underrepresented in medicine outperformed controls on several metrics. Upstate SMP students consistently bested controls in the longitudinal course. Overall, Upstate SMP graduates performed commensurately with classmates.

Connexin40 and connexin43 determine gating properties of atrial gap junction channels.

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and

Transseptal dispersion of repolarization and its role in the development of Torsade de Pointes arrhythmias.

OBJECTIVE: This study was designed to quantitate transseptal dispersion of repolarization (DR) and delineate its role in arrhythmogenesis using the calcium agonist BayK 8644 to mimic the gain of function of calcium channel current responsible for Timothy syndrome. BACKGROUND: Amplification of transmural dispersion of repolarization (TDR) has been shown to contribute to development of Torsade de Pointes (TdP) arrhythmias under long-QT conditions. METHODS: An arterially perfused septal wedge preparation was developed via cannulation of the septal artery. Action potentials (APs) were recorded using floating microelectrodes together with a transseptal electrocardiogram (ECG). These data were compared to those recorded from arterially perfused canine left ventricular (LV) wedge preparations. RESULTS: Under control conditions, the shortest AP duration measured at 90% repolarization (APD(90)) was observed in right ventricular (RV) endocardium (181.8 +/- 15 ms), APD(90) peaked close to midseptum (278.0 +/- 32 ms), and abbreviated again as LV endocardium was approached (207.3 +/- 9 ms). Transseptal DR averaged 106 +/- 24 ms and T(peak)-T(end) 84 +/- 7 ms (n = 6). TDR and T(peak)-T(end) recorded from LV wedge were 36 +/- 9 ms and 34 +/- 19 ms, respectively (n = 30). BayK 8644 increased transseptal DR to 123.2 +/- 35 ms (n = 5) and induced early and delayed afterdepolarizations (3/5), rate-dependent ST-T-wave alternans (5/5), and TdP arrhythmias (3/5). CONCLUSIONS: Our data indicate that dispersion of repolarization across the interventricular septum is twice that of the LV free wall, predisposing to development of TdP under long-QT conditions. Our findings suggest that the coronary-perfused ventricular septal preparation may be a sensitive model in which to assess the potential arrhythmogenic effects of drugs and pathophysiological conditions.

Cellular basis for the electrocardiographic and arrhythmic manifestations of Timothy syndrome: effects of ranolazine.

BACKGROUND: Timothy syndrome is a multisystem disorder associated with QT interval prolongation and ventricular cardiac arrhythmias. The syndrome has been linked to mutations in Ca(V)1.2 resulting in gain of function of the L-type calcium current (I(Ca,L)). Ranolazine is an antianginal agent shown to exert an antiarrhythmic effect in experimental models of long QT syndrome. OBJECTIVE: The purpose of this study was to develop and characterize an experimental model of Timothy syndrome by using BayK8644 to mimic the gain of function of I(Ca,L) and to examine the effects of ranolazine. METHODS: Action potentials from epicardial and M regions and a pseudo-electrocardiogram (ECG) were simultaneously recorded from coronary-perfused left ventricular wedge preparations, before and after addition of BayK8644 (1 microM). RESULTS: BayK8644 preferentially prolonged action potential duration of the M cell, leading to prolongation of the QT interval and an increase in transmural dispersion of repolarization (from 44.3 +/- 7 ms to 86.5 +/- 25 ms). Stimulation at cycle lengths of 250-500 ms led to ST-T wave alternans due to alternation of the plateau voltage of the M cell action potential as well as development of delayed afterdepolarizations in epicardial and M cell action potentials. Ventricular extrasystoles and tachycardia (monomorphic, bidirectional, or torsades de pointes) developed spontaneously or after rapid pacing. Peak and late I(Na) were unaffected by BayK8644. Clinically relevant concentrations of ranolazine (10 microM) suppressed all actions of BayK8644. CONCLUSION: A left ventricular wedge model of long QT syndrome created by augmentation of I(Ca,L) recapitulates the ECG and arrhythmic manifestations of Timothy syndrome, which can be suppressed by ranolazine.

Development of a coronary-perfused interventricular septal preparation as a model for studying the role of the septum in arrhythmogenesis.

BACKGROUND: Coronary-perfused ventricular wedge preparations have proven valuable in the elucidation of the mechanisms of arrhythmias. This study was undertaken to develop an arterially perfused model of the interventricular (IV) septum. METHODS: A canine septal preparation was developed via cannulation of the septal artery. Action potentials were recorded from ventricular endocardial surfaces and locations within the septum using floating microelectrodes; a transseptal electrocardiogram was simultaneously recorded. In some experiments, the calcium agonist BayK 8644 was used to enhance transseptal heterogeneity of action potential (AP) duration. RESULTS: Distinctive electrocardiographic waveforms and dissimilar AP morphologies and durations were observed across the IV septum. The range of AP durations observed exceeded that found in the left ventricular wedge. BayK 8644 further accentuated these differences and induced torsades de pointes arrhythmias. CONCLUSIONS: The arterially perfused septal preparation is a sensitive model for the study of arrhythmias that may arise from the IV septum.

Spontaneous cardiac calcinosis in BALB/cByJ mice.

BALB/c mice are predisposed to dystrophic cardiac calcinosis-the mineralization of cardiac tissues, especially the right ventricular epicardium. In previous reports, the disease appeared in aged animals and had an unknown etiology. In the current study, we report a substrain of BALB/c mice (BALB/cByJ) that develops disease early and with high frequency. Here we analyzed hearts grossly to identify the presence and measure the severity of disease and to compare BALB/c substrains. Histologic analysis and fluorescent and immunofluorescent microscopy were used to characterize the calcinotic lesions. BALB/cByJ mice exhibited more frequent and severe calcium deposition than did BALB/c mice of other substrains (90% compared with 3% at 5 wk). At this age, lesions covered an average of 30% of the total ventricular surface area in BALB/cByJ mice, compared with less than 1% in other strains. In bone-marrow-chimeric mice, green fluorescent protein was used as a marker to show that the lesions contain an infiltration of cells of bone marrow origin. Lesion histology showed that calcium deposits were surrounded by fibrosis with interspersed immune cells. Lymphocytes, macrophages, and granulocytes were all present. Internalization of the gap-junction protein connexin 43 was observed in myocytes adjacent to lesions. In conclusion, BALB/cByJ mice exhibit more frequent and severe dystrophic cardiac calcinosis than do other BALB/c substrains. Our findings suggest that immune cells are actively recruited to lesions and that myocyte gap junctions are altered near lesions.

Antiarrhythmic effects of ranolazine in canine pulmonary vein sleeve preparations.

BACKGROUND: Ectopic activity arising from the pulmonary veins (PV) plays a prominent role in the development of atrial fibrillation (AF). OBJECTIVE: This study sought to determine the electrophysiological effects of ranolazine in canine PV sleeve preparations. METHODS: Transmembrane action potentials were recorded from canine superfused left superior or inferior PV sleeves using standard microelectrode techniques. Acetylcholine (ACh, 1 microM), isoproterenol (1 microM), high calcium ([Ca(2+)](o) = 5.4 mM) or a combination was used to induce early or delayed afterdepolarizations (EADs or DADs) and triggered activity. RESULTS: Ranolazine (10 microM) significantly accentuated use-dependent depression of maximal rate of increase of action potential upstroke (V(max)). Reducing basic cycle length (BCL) from 2000 to 200 ms resulted in a decrease of V(max) from 279 +/- 58 to 146 +/- 23 V/s (47.7%) in control subjects and from 241 +/- 71 to 72 +/- 63 V/s (70.2%) after 10 microM ranolazine (n = 4, P <.05). Ranolazine slightly abbreviated action potential duration, but induced significant rate-dependent prolongation of effective refractory period due to development of postrepolarization refractoriness (n = 6, P <.05). Ranolazine (10 microM) caused loss of excitability resulting in 2:1 activation failure at BCLs