Induced pluripotent stem cells (iPSCs) are an essential tool for modeling how causal genetic variants impact cellular function in disease, as well as an emerging source of tissue for regenerative medicine. The preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes limiting the scale and reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming enabling automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. We demonstrate that automated reprogramming and the pooled selection of polyclonal pluripotent cells results in high-quality, stable iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single-colony subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines.

The ␤ 2 integrin CD11b plays a central role in inflammation and the systemic inflammatory response syndrome (SIRS). The CD11b molecule activates in two ways: the density of membranebound CD11b up-regulates and the molecule undergoes a conformational change that confers adhesiveness to counter-receptors. We studied the kinetics of CD11b activation in patients with SIRS. We found a significantly diminished CD11b activation in response to tumor necrosis factor ␣ (TNF-␣). This affected all circulating polymorphonuclear neutrophils (PMN) and was an intrinsic property of the cells and not due to antagonism by soluble TNF-␣ receptors or loss of cellular receptors for TNF-␣. Diminished responsiveness correlated with the severity of organ failure and lasted for months in some patients but had no impact on mortality. We speculate that reduced CD11b responsiveness in SIRS contributes to the high risk of recurrent infection, but that it may also be protective against excessive PMN activation within the vascular space.

To determine the effect atherosclerosis has on myocardial contractility, we studied the contractile properties of right ventricular papillary muscles from 34 atherosclerotic and 17 control rabbits. We produced atherosclerosis by feeding for 2 to 8 months a diet of 5% lard, 5% peanut oil, 0.5% cholesterol, and 89.5% rabbit pellets. The controls received only rabbit pellets during the same time interval. Contracting isometrically 12 times per minute at 25 degrees C, muscles from the atherosclerotic rabbits developed tension at a lower maximum rate (max dT/dt), had a longer latency, and required longer to develop tension at the maximum rate and to develop peak tension. In isotonic contractions, they shortened with lower maximum velocities and required longer to accelerate to maximum velocity and to shorten maximally. We found no evidence that developed tension or distance shortened differed between the two groups of muscles. Raising the contraction frequency to 24 contractions per minute between the two groups of muscles. Raising the contraction frequency to 24 contractions per minute brought performance of the two groups of muscles closer in both types of contraction. Norepinephrine (1.5 x 10-5 M) nearly abolished differences between performance of the two groups. The loss of contractility correlates poorly with coronary and aortic atherosclerosis. It occurred early in the feeding of the atherogenic diet. We think it was due to a lipid-induced defect in the cardiac cell's handling of calcium.

Background Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a potentially fatal genetic arrhythmia syndrome and, despite optimal beta-blocker (BB) therapy, patients often experience life-threatening arrhythmic events (LAE). The role of implantable cardioverter defibrillators (ICD) on reducing mortality in patients with CPVT is debated. Purpose 1) To assess the survival benefit of ICD in the overall CPVT population; 2) to investigate the benefit-to-harm ratio in the population of CPVT patients carriers of ICD. Methods We followed-up consecutive patients with genetically-proven CPVT, treated with optimal BB mono-therapy. The probability of surviving at the occurrence of a first LAE (defined as: sudden cardiac death, aborted cardiac arrest or hemodynamically non-tolerated ventricular tachycardia) during BB mono-therapy was compared between ICD carriers and non-ICD carriers. In the cohort of ICD carriers, we assessed the rate of a first major complication (i.e., a complication requiring surgical intervention) and we calculated the benefit-to-harm ratio of ICD (defined as rate of appropriate shocks on LAE over rate of major complications). Results We enrolled 228 CPVT patients with a pathogenic RYR2 (n=216) or CASQ2 (n=12) mutation, of whom 87 (38%) had an ICD implanted. Overall, during 1,558 person-years of follow-up, 31/228 (14%) patients experienced a first LAE on BB mono-therapy (annual LAE rate 2.0%, 95% CI: 1.4–2.8%). Of the 31 individuals who experienced an LAE, 21 patients had an ICD when the LAE occurred, and all survived after ICD intervention. Of the remaining 10 patients who were not carriers of an ICD, 6/10 (60%) survived after external defibrillation or spontaneous arrhythmia termination, while 4/10 (40%) died suddenly. The probability of surviving at the occurrence of a first LAE in BB mono-therapy was thirty-fold higher (Odds Ratio: 30.0, 95% CI: 1.4–629.0; p=0.029) in patients with an ICD, as compared to patients without an ICD. Overall, 15/87 (17%) patients with an ICD experienced a first major complication over 602 person-years of follow-up (2.5% per year, 95% CI: 1.4%-4.1%). Importantly, no patient died for a pro-arrhythmic effect or complications related to ICD. Comparing the rate of major complications to the rate of appropriate ICD shocks on LAE (LAE rate 4.7% per year, 95% CI: 3.1–7.0%), the benefit-to-harm ratio was 1.9, thus favouring the benefit of ICD therapy. Conclusions In our population, ICD therapy conferred a significant survival benefit in patients with CPVT at the occurrence of a first LAE. Overall, the benefit of ICD implant outweighed the harm, as shown by almost two times greater benefit-to-harm ratio. This study provides evidence supporting ICD implantation in patients with high-risk CPVT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca Corrente Funding Scheme of the Italian Ministry of Health

Background. Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a highly lethal recessive arrhythmogenic disease associated with mutations in the calsequestrin 2 ( CASQ2 ) gene. We previously demonstrated that CASQ2 gene delivery was able to rescue the arrhythmic phenotype in CASQ2 knock-out (KO) mice 5 months after the viral infection. The aim of the present study is to investigate the long-term effects of viral gene delivery in CASQ2-R33Q missense mutation mouse model. Methods. Newborn CPVT mice were infected with adeno-associated viral vector 9 (AAV9) containing the coding sequence of the wild type CASQ2 co-expressed with green fluorescent protein (GFP) gene. Furthermore, we evaluated the effect at 7 and 12 months after viral infection by in vivo ECG analysis, in vitro electrophysiological and molecular assays. Results. Telemeter recording of ECG demonstrated in vivo ventricular tachycardias (VT) after epinephrine (2 mg/Kg) administration in only 17% (2 out of 12) of the infected mice, while 87% (7/8) of the control CASQ2-R33Q homozygous mice presented a clear arrhythmic phenotype (p<0.005). Additionally, electrophysiological analysis showed that delayed after depolarization (DADs) and triggered activity (TA) were almost abolished in GFP-positive cardiomyocytes (7 months: DADs: 0%, TA 0%, n=18; 12 months: DADs: 8%, TA: 8%, n=12) in comparison with their internal control (GFP-negative cells, 7 months: DADs:100%, TA: 83%, n=6; 12 months: DADs: 100%, TA: 71%, n=7; p<0.001). Finally, western blot revealed increased CASQ2 expression in the infected mice and immunofluorescence assay indicated its correct localization along the z-lines. Conclusions. In the present work we show that the viral expression of CASQ2 is effective for a long period of time and is able to revert the functional abnormalities of the mutant endogenous protein and to prevent life-threatening arrhythmias in the knock-in CASQ2-R33Q mice. These data suggest that despite R33Q mice, at variance with KO mice, express abnormal CASQ2, the gene replacement therapy is still able to prevent arrhythmogenic mechanisms in vitro and VTs in vivo thus suggesting that CASQ2-gene transfer may become a novel therapy for recessive CPVT.