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Background Allergen-specific IL-4+ and IL-13+ CD4+ cells (type 2 cells) are essential for helping B cells to class-switch to IgE and establishing an allergic milieu in the gastrointestinal tract. The role of T cells in established food allergy is less clear. Objective We examined the food allergen–specific T-cell response in participants of 2 food allergen immunotherapy trials to assess the relationship of the T-cell response to clinical phenotypes, including response to immunotherapy. Methods Blood was obtained from 84 participants with peanut allergy and 142 participants with egg allergy who underwent double-blind placebo-controlled food challenges. Peanut- and egg-responsive T cells were identified by CD154 upregulation after stimulation with the respective extract. Intracellular cytokines and chemokine receptors were also detected. The response to peanut epicutaneous immunotherapy (Peanut Epicutaneous Phase II Immunotherapy Clinical Trial [CoFAR6]; 49 participants receiving epicutaneous immunotherapy) and egg oral immunotherapy or a baked egg diet (Baked Egg or Egg Oral Immunotherapy for Children With Egg Allergy [CoFAR7]; 92 participants) was monitored over time. Results Peanut-specific type 2 and CCR6+ T cells were negatively correlated with each other and differently associated with immune parameters, including specific IgE level and basophil activation test result. At baseline, type 2 cells, but not CCR6+ cells, were predictive of clinical parameters, including a successfully consumed dose of peanut and baked egg tolerance. Exposure to peanut or egg immunotherapy was associated with a decrease in type 2 cell frequency. At baseline, high egg-specific type 2 cell frequency was the immune feature most predictive of oral immunotherapy failure. Conclusion Food-specific type 2 T cells at baseline are informative of threshold of reactivity and response to immunotherapy.

Introduction: Mutations in the lamin A/C ( LMNA ) gene have been causally linked to atrial arrhythmias and cardiac conduction disease (CCD) in young adults. However, the mechanism by which the laminopathy leads to perturbed cardiac electrophysiology has not been fully elucidated. Hypothesis: We hypothesize that protein-truncating variants in LMNA will impair the mechano-protection force in the nuclear envelope and will lead to early-onset cardiomyocyte degeneration and CCD. Methods: In a multi-regional registry of early-onset CCD and atrial fibrillation in Japan, we performed whole-exome DNA sequencing of 23 probands. Using CRISPR/Cas9, we generated indels of candidate gene homologues in zebrafish and characterized cardiac physiology using optical mapping technology and immunohistochemistry. Results: Among all probands, our study revealed four rare nonsense variants in the nuclear protein-coding genes. We focused on a LMNA protein-truncating variant, c.339dupT (p.K114X fsX1), and created a variant in the zebrafish lmna that produced a similar truncation. Lmna -/- zebrafish larvae showed shortened atrial action potential duration (APD) (msec.) compared to wild-type controls (162 ± 19 vs 227 ± 79, p lmna -/- embryos exhibited prolonged APD (399 ± 51 vs 322 ± 47, p lmna -/- zebrafish displayed significantly decreased cell numbers and smaller cell size compared to those of controls, which in turn developed into abnormal nuclear structures in adult. Conclusions: These findings suggest that lamin A is a prerequisite for proper atrial cardiomyocyte morphology in embryonic zebrafish, and is indispensable for correct cardiac electrophysiology and -conduction.

Background: Enhanced diastolic calcium (Ca 2+ ) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca 2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca 2+ /calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca 2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca 2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Background: Racial inequities for patients with heart failure (HF) have been widely documented. HF patients who receive cardiology care during a hospital admission have better outcomes. It is unknown whether there are differences in admission to a cardiology or general medicine service by race. This study examined the relationship between race and admission service, and its effect on 30-day readmission and mortality Methods: We performed a retrospective cohort study from September 2008 to November 2017 at a single large urban academic referral center of all patients self-referred to the emergency department and admitted to either the cardiology or general medicine service with a principal diagnosis of HF, who self-identified as white, black, or Latinx. We used multivariable generalized estimating equation models to assess the relationship between race and admission to the cardiology service. We used Cox regression to assess the association between race, admission service, and 30-day readmission and mortality. Results: Among 1967 unique patients (66.7% white, 23.6% black, and 9.7% Latinx), black and Latinx patients had lower rates of admission to the cardiology service than white patients (adjusted rate ratio, 0.91; 95% CI, 0.84–0.98, for black; adjusted rate ratio, 0.83; 95% CI, 0.72–0.97 for Latinx). Female sex and age >75 years were also independently associated with lower rates of admission to the cardiology service. Admission to the cardiology service was independently associated with decreased readmission within 30 days, independent of race. Conclusions: Black and Latinx patients were less likely to be admitted to cardiology for HF care. This inequity may, in part, drive racial inequities in HF outcomes.

// Juan Wang 1,2 , Liang Peng 2 , Ruihua Zhang 2 , Zihan Zheng 2,3 , Chun Chen 4,5 , Ka Lung Cheung 6 , Miao Cui 7 , Guanglin Bian 2 , Feihong Xu 2 , David Chiang 2 , Yuan Hu 2 , Ye Chen 1 , Geming Lu 2 , Jianjun Yang 2 , Hui Zhang 2 , Jianfei Yang 8 , Hongfa Zhu 7 , Shu-hsia Chen 2 , Kebin Liu 9 , Ming-Ming Zhou 6 , Andrew G. Sikora 2 , Liwu Li 5 , Bo Jiang 1 and Huabao Xiong 2 1 Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, China 2 Department of Medicine, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 3 Department of Biology, College of Arts and Sciences, University of North Carolina at Chapel Hill, NC, USA 4 Amgen Inc., Thousand Oaks, CA, USA 5 Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA 6 Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 7 Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 8 Tempero Pharmaceuticals, GlasxoSmithKline, Cambridge, MA, USA 9 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USA Correspondence to: Huabao Xiong, email: // Bo Jiang, email: // Keywords : 5-FU, TH17, TS, Immunology and Microbiology Section, Immune response, Immunity Received : November 24, 2015 Accepted : March 07, 2016 Published : March 24, 2016 Abstract While it is well established that treatment of cancer patients with 5-Fluorouracil (5-FU) can result in immune suppression, the exact function of 5-FU in the modulation of immune cells has not been fully established. We found that low dose 5-FU selectively suppresses T H 17 and T H 1 cell differentiation without apparent effect on Treg, T H 2, and significantly suppresses thymidylate synthase (TS) expression in T H 17 and T H 1 cells but has a lesser effect in tumor cells and macrophages. Interestingly, the basal expression of TS varies significantly between T helper phenotypes and knockdown of TS significantly impairs T H 17 and T H 1 cell differentiation without affecting the differentiation of either Treg or T H 2 cells. Finally, low dose 5-FU is effective in ameliorating colitis development by suppressing T H 17 and T H 1 cell development in a T cell transfer colitis model. Taken together, the results highlight the importance of the anti-inflammatory functions of low dose 5-FU by selectively suppressing T H 17 and T H 1 immune responses.

Mutations in the lamin A/C ( LMNA ) gene have been causally linked to dilated cardiomyopathy with conduction disease. However, the mechanism by which the laminopathy leads to electrophysiological disorders has not been fully elucidated. In a multi-regional registry of early-onset cardiac conduction

Background: Duchenne muscular dystrophy patients are prone to ventricular arrhythmias, which may be caused by abnormal calcium (Ca 2+ ) homeostasis and elevated reactive oxygen species. CaMKII (Ca 2+ /calmodulin-dependent protein kinase II) is vital for normal Ca 2+ homeostasis, but excessive CaMKII activity contributes to abnormal Ca 2+ homeostasis and arrhythmias in cardiomyocytes. Reactive oxygen species induce CaMKII to become autonomously active. We hypothesized that genetic inhibition of CaMKII oxidation (ox-CaMKII) in a mouse model of Duchenne muscular dystrophy can alleviate abnormal Ca 2+ homeostasis, thus, preventing ventricular arrhythmia. The objective of this study was to test if selective loss of ox-CaMKII affects ventricular arrhythmias in the mdx mouse model of Duchenne muscular dystrophy. Methods and Results: 5-(6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate staining revealed increased reactive oxygen species production in ventricular myocytes isolated from mdx mice, which coincides with elevated ventricular ox-CaMKII demonstrated by Western blotting. Genetic inhibition of ox-CaMKII by knockin replacement of the regulatory domain methionines with valines (MM-VV [CaMKII M281/282V]) prevented ventricular tachycardia in mdx mice. Confocal calcium imaging of ventricular myocytes isolated from mdx :MM-VV mice revealed normalization of intracellular Ca 2+ release events compared with cardiomyocytes from mdx mice. Abnormal action potentials assessed by optical mapping in mdx mice were also alleviated by genetic inhibition of ox-CaMKII. Knockout of the NADPH oxidase regulatory subunit p47 phox normalized elevated ox-CaMKII, repaired intracellular Ca 2+ homeostasis, and rescued inducible ventricular arrhythmias in mdx mice. Conclusions: Inhibition of reactive oxygen species or ox-CaMKII protects against proarrhythmic intracellular Ca 2+ handling and prevents ventricular arrhythmia in a mouse model of Duchenne muscular dystrophy.

Background: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors. Methods: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%–40%), and severe HF (ejection fraction Results: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum. Conclusions: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular “sponge” of PP1c.

The molecular mechanisms underlying the early development of atrial fibrillation (AF) remain poorly understood. Emerging evidence suggests that abnormal epigenetic modulation via microRNAs (miRNAs) might be involved in the pathogenesis of paroxysmal AF (pAF).To identify key molecular changes associated with pAF, we conducted state-of-the-art transcriptomic studies to identify the abnormal miRNA-mRNA interactions potentially driving AF development.High-quality total RNA including miRNA was isolated from atrial biopsies of age-matched and sex-matched pAF patients and control patients in sinus rhythm (SR; n=4 per group) and used for RNA-sequencing and miRNA microarray. Results were analyzed bioinformatically and validated using quantitative real-time (qRT)-PCR and 3'UTR luciferase reporter assays.113 genes and 49 miRNAs were differentially expressed (DE) in pAF versus SR patients. Gene ontology analysis revealed that most of the DE genes were involved in the "gonadotropin releasing hormone receptor pathway" and "p53 pathway". Of these DE genes, bioinformatic analyses identified 23 pairs of putative miRNA-mRNA interactions that were altered in pAF (involving 15 miRNAs and 17 mRNAs). Using qRT-PCR and 3'UTR luciferase reporter assays, the interaction between upregulation of miR-199a-5p and downregulation of FKBP5 was confirmed in samples from pAF patients.Our combined transcriptomic analysis and miRNA microarray study of atrial samples from pAF patients revealed novel pathways and miRNA-mRNA regulations that may be relevant in the development of pAF. Future studies are required to investigate the potential involvement of the gonadotropin releasing hormone receptor and p53 pathways in AF pathogenesis.

BACKGROUND Cardiovascular disease is a leading cause of death worldwide. Arrhythmias are associated with significant morbidity and mortality related to cardiovascular disease. Recent work illustrates that many cardiac arrhythmias are initiated by a pathologic imbalance between kinase and phosphatase activities in excitable cardiomyocytes. OBJECTIVE To test the relationship between myocyte kinase/phosphatase imbalance and cellular and whole animal arrhythmia phenotypes associated with ankyrin-B cardiac syndrome. METHODS By using a combination of biochemical, electrophysiological, and in vivo approaches, we tested the ability of calcium/calmodulin-dependent kinase (CaMKII) inhibition to rescue imbalance in kinase/phosphatase pathways associated with human ankyrin-B-associated cardiac arrhythmia. RESULTS The cardiac ryanodine receptor (RyR 2 ), a validated target of kinase/phosphatase regulation in myocytes, displays abnormal CaMKII-dependent phosphorylation (pS2814 hyperphosphorylation) in ankyrin-B +/− heart. Notably, RyR 2 dysregulation is rescued in myocytes from ankyrin-B +/− mice overexpressing a potent CaMKII-inhibitory peptide (AC3I), and aberrant RyR 2 open probability observed in ankyrin-B +/− hearts is normalized by treatment with the CaMKII inhibitor KN-93. CaMKII inhibition is sufficient to rescue abnormalities in ankyrin-B +/− myocyte electrical dysfunction including cellular afterdepolarizations, and significantly blunts whole animal cardiac arrhythmias and sudden death in response to elevated sympathetic tone. CONCLUSIONS These findings illustrate the complexity of the molecular components involved in human arrhythmia and define regulatory elements of the ankyrin-B pathway in pathophysiology. Furthermore, the findings illustrate the potential impact of CaMKII inhibition in the treatment of a congenital form of human cardiac arrhythmia.

Food allergy has rapidly increased in prevalence, suggesting an important role for environmental factors in disease susceptibility. The immune response of food allergy is characterized by IgE production, and new findings from mouse and human studies indicate an important role of the cytokine IL-9, which is derived from both T cells and mast cells, in disease manifestations. Emerging evidence suggests that route of exposure to food, particularly peanut, is important. Exposure through the skin promotes sensitization while early exposure through the gastrointestinal tract promotes tolerance. Evidence from mouse studies indicate a role of the microbiome in development of food allergy, which is supported by correlative human studies showing a dysbiosis in food allergy. There is no approved treatment for food allergy, but emerging therapies are focused on allergen immunotherapy to provide desensitization, while pre-clinical studies are focused on using adjuvants or novel delivery approaches to improve efficacy and safety of immunotherapy., Highlights • Emerging evidence suggests that route of exposure to food allergens in early life determines sensitization versus tolerance. • The microbiota and dietary factors appear to play a key role in susceptibility to food allergy. • Immunotherapy applied via different routes is currently the most promising form of experimental treatment for food allergy.

The association between systolic cardiac dysfunction and arrhythmia development in patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) is generally assumed but has not been extensively studied. The purpose of this study was to describe arrhythmias in patients with DMD and BMD in the present era and determine whether arrhythmia development is associated with cardiac dysfunction. This is a single-center retrospective review of 237 Holters from 91 patients with DMD (mean = 17 ± 4 years, range 3 to 27 years) and 64 Holters from 21 patients with BMD (mean = 18 ± 7 years, range 4 to 31 years) with corresponding echocardiography. Holters were stratified by age of patient at the time of study and ejection fraction: normal (≥55%), mild (55% and ≥45%), moderate (45% and ≥30%), and severe (30%). Arrhythmias included frequent atrial and ventricular premature complexes (10/hr), couplets, and runs of supraventricular and ventricular tachycardias. Arrhythmias occurred in 44% of DMD and 57% of BMD patients and were significantly associated with decrease in cardiac function. Clinically significant arrhythmias (supraventricular tachycardia and ventricular tachycardia) occurred in 10% of all Holters obtained in patients with DMD and 25% of all Holters obtained in patients with BMD. Subgroup analysis of Holters from patients with DMD demonstrated that arrhythmias increased with decreasing ejection fraction regardless of age, but that age was also a significant predictor of arrhythmia development. In conclusion, among patients with DMD or BMD, arrhythmias increase with development of cardiac dysfunction.

Allergen immunotherapy for the treatment of food allergy has been a subject of intensive study within the last 10 years. After an unsuccessful attempt with subcutaneous immunotherapy for peanut allergy, other routes with varying degrees of safety and efficacy have been tested for peanut, milk, and egg allergies. In this review, we summarize the results to date with oral immunotherapy, sublingual immunotherapy, and epicutaneous immunotherapy for the treatment of food allergy. While results of immunotherapy trials are promising, increases in efficacy are commonly associated with an increased side effect profile. There is a need for additional research beginning at the preclinical level to develop safe and effective treatments for food allergy.

Background— Cardiac device implantation can be complicated by inability to adequately place leads because of significant lead capture issues. This study sought to determine whether there are genetic bases that underlie poor lead capture. Methods and Results— Retrospective review of all patients with structurally normal hearts who underwent new device implantation at Texas Children’s Hospital between 2009 and 2014 was performed. Patients with inability to capture at 10 V or a final capture threshold ≥3 V at 0.4 ms during implant were analyzed. Among a total of 136 patients (median age, 13 years; range, 3 days to 46 years), 11 patients (8.1%) who underwent dual chamber device implantation had elevated thresholds in the atria (4), ventricle (3), or both chambers (4; atrial-lead threshold, 4.7±4.3 versus 0.7±0.3 V; ventricular-lead, 3.0±3.3 versus 0.7±0.3 V). All 11 patients presented with sinus node dysfunction and 10 had atrial arrhythmias. At implant, inability to find atrial capture was seen in 4 patients. Three demonstrated intermittent complete loss of ventricular capture after implantation: 1 has recurrent syncope, 2 eventually died. Genetic testing performed in 10 demonstrated 7 patients with 6 distinct SCN5A mutations, all predicted to be severe loss-of-function mutations by bioinformatic analyses. In the remaining patients, although putative pathogenic mutations were not found, multiple SCN5A polymorphisms were identified in 2 and a desmin mutation in 1. Conclusions— This study suggests that significant capture issues at implant may be because of loss-of-function SCN5A mutations, providing new insights into SCN5A function. Recognition of this association may be critical for planning device implantation strategies and patient follow-up.

Background The molecular mechanisms underlying the early development of atrial fibrillation (AF) remain poorly understood. Emerging evidence suggests that abnormal epigenetic modulation via microRNAs (miRNAs) might be involved in the pathogenesis of paroxysmal AF (pAF).

Background— Enhanced sarcoplasmic reticulum Ca 2+ -leak via ryanodine receptor type-2 (RyR2) contributes to the pathogenesis of atrial fibrillation (AF). Recent studies have shown that the level of RyR2 protein is elevated in atria of patients with paroxysmal AF, suggesting that microRNA-mediated post-transcriptional regulation of RyR2 might be an underlying mechanism. Bioinformatic analysis suggests that miR-106b and miR-93, members of the miR-106b-25 cluster, could bind to RyR2-3′-untranslated region and suppress its translation. Thus, we tested the hypothesis that loss of the miR-106b-25 cluster promotes AF via enhanced RyR2-mediated sarcoplasmic reticulum Ca 2+ -leak. Methods and Results— Quantitative real-time polymerase chain reaction showed that the levels of mature miR-106b, miR-93, and miR-25 were lower in atria of patients with paroxysmal AF when compared with patients in sinus rhythm. In vitro assay showed that miR-93 reduced RyR2-3′-untranslated region luciferase activity. Total RyR2 protein in atrial tissue of miR-106b-25 −/− mice was increased by 42% when compared with wild-type littermates but still maintained a normal subcellular distribution. Ca 2+ -spark frequency and total sarcoplasmic reticulum Ca 2+ -leak were increased in atrial myocytes of miR-106b-25 −/− mice. Telemetry ECG recordings revealed that miR-106b-25 −/− mice exhibited more frequent atrial ectopy and were also more susceptible to pacing-induced AF than wild-type littermates. Increased sarcoplasmic reticulum Ca 2+ -release and AF susceptibility in miR-106b-25 −/− mice were abolished by the RyR2 blocker K201. Conclusions— These results suggest that miR-106b-25 cluster–mediated post-transcriptional regulation of RyR2 is a potential molecular mechanism involved in paroxysmal AF pathogenesis. As such, the miR-106b-25 cluster could be a novel gene-therapy target in AF associated with enhanced RyR2 expression.

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in the clinical setting [(1)][1]. In the general population, the prevalence of AF increases with age, ranging from 0.5% in patients younger than 40 years to 5% in patients older than 65 years [(2)][2]. Less is known about the

Background Peanut allergy is common, life-threatening, and without therapeutic options. We evaluated peanut epicutaneous immunotherapy (EPIT) by using Viaskin Peanut for peanut allergy treatment. Objective We sought to evaluate the clinical, safety, and immunologic effects of EPIT for the treatment of peanut allergy. Methods In this multicenter, double-blind, randomized, placebo-controlled study, 74 participants with peanut allergy (ages 4-25 years) were treated with placebo (n = 25), Viaskin Peanut 100 μg (VP100; n = 24) or Viaskin Peanut 250 μg (VP250; n = 25; DBV Technologies, Montrouge, France). The primary outcome was treatment success after 52 weeks, which was defined as passing a 5044-mg protein oral food challenge or achieving a 10-fold or greater increase in successfully consumed dose from baseline to week 52. Adverse reactions and mechanistic changes were assessed. Results At week 52, treatment success was achieved in 3 (12%) placebo-treated participants, 11 (46%) VP100 participants, and 12 (48%) VP250 participants ( P = .005 and P = .003, respectively, compared with placebo; VP100 vs VP250, P = .48). Median change in successfully consumed doses were 0, 43, and 130 mg of protein in the placebo, VP100, and VP250 groups, respectively (placebo vs VP100, P = .014; placebo vs VP250, P = .003). Treatment success was higher among younger children ( P = .03; age, 4-11 vs >11 years). Overall, 14.4% of placebo doses and 79.8% of VP100 and VP250 doses resulted in reactions, predominantly local patch-site and mild reactions ( P = .003). Increases in peanut-specific IgG 4 levels and IgG 4 /IgE ratios were observed in peanut EPIT-treated participants, along with trends toward reduced basophil activation and peanut-specific T H 2 cytokines. Conclusions Peanut EPIT administration was safe and associated with a modest treatment response after 52 weeks, with the highest responses among younger children. This, when coupled with a high adherence and retention rate and significant changes in immune pathways, supports further investigation of this novel therapy.

BackgroundAtrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown.ObjectivesThe purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits.MethodsCardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry–based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit–PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation.ResultsA total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone.ConclusionsSubcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c–R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+ remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.

Background— The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca 2+ leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca 2+ release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms. Methods and Results— CREM-IbΔC-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca 2+ release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca 2+ /calmodulin-dependent protein kinase II–mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca 2+ release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca 2+ -dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. Conclusions— RyR2-mediated SR Ca 2+ leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca 2+ release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.

Background— Electrical, structural, and Ca 2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca 2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca 2+ -release events in paroxysmal AF (pAF) are unknown. Method and Results— Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10–20 days preoperatively) were analyzed with simultaneous measurements of [Ca 2+ ] i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca 2+ current, and Na + /Ca 2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca 2+ leak and incidence of delayed after-depolarizations in pAF. Ca 2+ -transient amplitude and sarcoplasmic reticulum Ca 2+ load (caffeine-induced Ca 2+ -transient amplitude, integrated Na + /Ca 2+ -exchange current) were larger in pAF. Ca 2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca 2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca 2+ leak and sarcoplasmic reticulum Ca 2+ -release events, causing delayed after-depolarizations/triggered activity in pAF. Conclusions— Increased diastolic sarcoplasmic reticulum Ca 2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca 2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Purpose: Atrial fibrillation (AF) is the most common cardiac arrhythmia with a growing prevalence yet current pharmaceutical treatments are ineffective. Previous studies suggest that protein phosphatase 1 (PP1) is dysregulated globally in AF patients but its local regulation on key Ca2+-handling proteins such as ryanodine receptor type-2 (RyR2) is not known. In this study we hypothesize that decreased PP1 regulation of RyR2 promotes AF through increasing Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2. Methods: To study the local regulation of RyR2 by PP1, we used spinophilin knockout mice (KO), since PP1 targets RyR2 via spinophilin. The interaction between RyR2 and PP1 was measured using co-immunoprecipitation while RyR2 phosphorylation level was quantified using Western blot. RyR2 activity was determined by measuring 1) single RyR2 channel activity in a lipid bilayer system and 2) Ca2+ sparks frequency in atrial myocytes loaded with Fluo4-AM. Baseline cardiac characteristics were determined using echocardiography and histological staining while AF inducibility was assessed by intracardiac electrophysiology study. Results: The interaction of PP1 with RyR2 was reduced by 55% in the KO hearts compared to WT (P

Atrial fibrillation (AF) is the most common sustained arrhythmia yet current pharmacological treatments are only moderately effective. Enhanced sarcoplasmic reticulum (SR) Ca2+ leak via ryanodine receptor type-2 (RyR2) contributes to AF pathogenesis and preliminary data in atrial samples from