The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells.

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef's dileucine motif LL164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif-dependent manner. Treating HIV-1-infected CD4+ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4+ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4+ T cells.

Inhibiteurs de Nef du VIH-1 : applications et développements pour une guérison efficace.

Résumé La thérapie anti-rétrovirale peut contrôler la réplication du virus de l'immunodéficience humaine de type 1 (VIH-1) chez les individus vivant avec le VIH. Par contre, ces traitements ne constituent pas une guérison et aucune approche pour une guérison du VIH-1 n'a encore montré de succès lors des études cliniques. Les approches de guérison sont souvent contrées in vivo par des barrières développées par le VIH-1. L'inhibition pharmacologique de la protéine accessoire Nef du VIH-1 représente une approche ambitieuse et prometteuse pour développer une nouvelle stratégie de guérison. Des petites molécules inhibitrices de Nef peuvent inverser les défauts reliés à l'infection par le VIH dans la signalisation des récepteurs des cellules T et les kinases, l'apoptose, l'autophagie et surtout, la présentation d'antigène. Ensemble, ces activités démontrent la grande capacité des inhibiteurs de Nef à être appliqués comme agents thérapeutiques dans un traitement contre le VIH-1. Dans cette revue, nous présentons les motifs pour lesquels Nef constitue une cible thérapeutique et nous soulignons les progrès effectués dans l'identification et le développement d'inhibiteurs de Nef.

Inhibitors of HIV-1 Nef: applications and developments for a practical cure.

Antiretroviral therapy can control human immunodeficiency virus type 1 (HIV-1) replication in people living with HIV; however, these treatments are not curative and no practical approach for an HIV-1 cure has yet shown success in clinical trials. Counteracting the multiple barriers HIV-1 presents against a practical cure is a direct means to functionalize these curative approaches in vivo. Pharmacological inhibition of the HIV-1 accessory protein, Nef, represents a particularly promising and ambitious approach, with Nef inhibitors holding the potential to reverse HIV-1-related defects in T cell receptor and kinase signaling, apoptosis, autophagy and most importantly, antigen presentation. Together, the capacity for Nef inhibitors to restore these activities underscores their potential as supportive agents in a practical HIV-1 cure. In this review, we outline a rationale for pharmacologically targeting Nef and review the progress made in the identification and development of Nef inhibitors.

PACS-1 Interacts with TRPC3 and ESyt1 to Mediate Protein Trafficking while Promoting SOCE and Cooperatively Regulating Hormone Secretion.

Corticotropic cells of the anterior pituitary gland release adrenocorticotropic hormone (ACTH) in a regulated manner to promote the production of cortisol and androgens. The process of ACTH secretion is partly mediated by the phosphofurin acidic cluster sorting protein 1 (PACS-1); however, the underlying mechanisms behind this regulation remain unclear. Herein, we demonstrated PACS-1 interactions with the short transient receptor potential channel 3 (TRPC3) calcium transporter and the extended synaptotagmin-1 (ESyt1) endoplasmic reticulum-plasma membrane tethering protein. Importantly, PACS-1 promoted interactions between TRPC3 and ESyt1 and regulated their plasma membrane localization. Lastly, we demonstrated that PACS-1 is required for a proper store-operated calcium entry (SOCE) response and that ESyt1 regulates ACTH secretion through an unknown mechanism regulated by PACS-1. Overall, our study provides new insights into the physiological role PACS-1 plays in modulating intracellular calcium levels and regulating ACTH secretion in corticotropic cells.

PACS-1 contains distinct motifs for nuclear-cytoplasmic transport and interacts with the RNA-binding protein PTBP1 in the nucleus and cytosol.

Phosphofurin acidic cluster sorting protein 1 (PACS-1) is canonically a cytosolic trafficking protein, yet recent reports have described nuclear roles for PACS-1. Herein, we sought to define the nuclear transport mechanism of PACS-1. We demonstrate that PACS-1 nucleocytoplasmic trafficking is dependent on its interaction with the nuclear transport receptors importin alpha 5 and exportin 1. PACS-1 nuclear entry and exit are defined by a nuclear localization signal (NLS, residues 311-318) and nuclear export signal (NES3, residues 366-375). Mutation of the PACS-1 NLS and NES3 altered the localization of a complex formed between PACS-1 and an RNA-binding protein, polypyrimidine tract-binding protein 1. Overall, we identify the nuclear localization mechanism of PACS-1 and highlight a potential role for PACS-1 in RNA-binding protein trafficking.

Outcomes and Safety of Fluoroless Catheter Ablation for Atrial Fibrillation.

BACKGROUND: Intracardiac echocardiography and 3D mapping systems allow catheter ablation for atrial fibrillation (AF) to be conducted without fluoroscopy; however, the safety and effectiveness of fluoroless AF ablation are not well defined. METHODS: We examined consecutive radiofrequency AF catheter ablations at a large academic teaching hospital from November 2017 to July 2019. Outcomes for fluoroscopy-guided (N = 176) and fluoroless (N = 147) ablations were compared. Cases were designated as fluoroless at the outset of the procedure. RESULTS: Mean age was 59.5 ± 10 years, 66.9% were male, 71.8% had paroxysmal AF, and the mean CHA2DS2-VASc score was 1.7 ± 1.4. There were no differences in patient baseline characteristics. In the fluoroless group, minimal fluoroscopy was used in 17 patients (median, 3 seconds; interquartile range, 1.2-4.8). Mean procedure time, fluoroscopy time, and radiation dose (± standard deviation) were greater in the fluoroscopy group compared with the fluoroless group (194 ± 56 vs 176 ± 46 minutes, P = 0.0021; 10.7 ± 6.6 vs 0.008 ± 0.03 minutes, P < 0.0001; 2759.2 ± 1911 vs 5.4 ± 24 µGy m2, P < 0.0001). In multivariable linear regression models, fluoroless AF ablation was independently associated with reduced procedure times (ß = -16.5 minutes, P = 0.01). Acute procedural success (95.5% vs 98.6%, P = 0.1), complication rates (4.5% vs 2.0%, P = 0.24), and 1-year AF recurrence rates (28.7% vs 27.1%, log-rank P = 0.69) were similar between fluoroscopy and fluoroless groups. Excluding the 17 patients receiving fluoroscopy in the fluoroless group did not impact our results (P = 0.013). After exclusion of redo cases, fluoroless AF ablation was no longer associated with reduced procedure times (ß = -11.4 minutes, P = 0.106). CONCLUSIONS: Fluoroless radiofrequency AF ablation had similar effectiveness and safety compared with conventional fluoroscopy-guided AF ablation.

CONTEXTE: L'échocardiographie intracardiaque et les systèmes de cartographie 3D permettent l'ablation par cathéter de la fibrillation auriculaire (FA) sans fluoroscopie; l'innocuité et l'efficacité d'une telle approche ne sont toutefois pas bien connues. MÉTHODOLOGIE: Nous avons examiné les résultats d'ablations par cathéter de la FA par radiofréquences menées de façon consécutive dans un hôpital universitaire d'envergure entre novembre 2017 et juillet 2019. Les résultats des ablations par fluoroscopie (n = 176) et des ablations sans fluoroscopie (n = 147) ont été comparés. Les cas étaient désignés comme n'ayant pas utilisé la fluoroscopie à la fin de l'intervention, le cas échéant. RÉSULTATS: L'âge moyen était de 59,5 ± 10 ans, 66,9 % des patients étaient des hommes, 71,8 % étaient atteints de FA paroxystique, et le score CHA2DS2-VASc moyen était de 1,7 ± 1,4. Il n'y avait pas de différences entre les caractéristiques des patients au départ. Dans le groupe ayant subi une ablation sans fluoroscopie, une fluoroscopie minimale a été utilisée chez 17 patients (médiane : 3 secondes; intervalle interquartile : 1,2-4,8). La durée moyenne de l'intervention, la durée de la fluoroscopie, et la dose de rayonnements (± écart type) ont été plus élevées dans le groupe avec fluoroscopie que dans le groupe sans fluoroscopie (194 ± 56 vs 176 ± 46 minutes, p = 0,0021; 10,7 ± 6,6 vs 0,008 ± 0,03 minute, p < 0,0001; 2759,2 ± 1911 vs 5,4 ± 24 µGy m2, p < 0,0001). Dans des modèles de régression linéaire multivariables, l'ablation de la FA sans fluoroscopie a été associée de façon indépendante à des interventions de plus courte durée (ß = −16,5 minutes, p = 0,01). Le succès immédiat de l'intervention (95,5 % vs 98,6 %, p = 0,1), le taux de complications (4,5 % vs 2,0 %, p = 0,24), et le taux de récidive de la FA après 1 an (28,7 % vs 27,1 %, p (test du log-rank = 0,69) ont été comparables dans les groupes avec et sans fluoroscopie. L'exclusion des 17 patients chez qui la fluoroscopie avait été utilisée dans le groupe sans fluoroscopie n'a pas modifié ces résultats (p = 0,013). Après l'exclusion des cas où l'intervention était une reprise, l'ablation de la FA sans fluoroscopie n'était plus associée à une réduction des durées d'intervention (ß = −11,4 minutes, p = 0,106). CONCLUSIONS: L'ablation de la FA par radiofréquences sans fluoroscopie est associée à une efficacité et à une innocuité comparables à celles de l'ablation de la FA classique guidée par fluoroscopie.

Acetaminophen in late pregnancy and potential for in utero closure of the ductus arteriosus-a pharmacokinetic evaluation and critical review of the literature.

Acetaminophen has become a novel treatment option for patent ductus arteriosus closure in premature infants. This raises concerns about whether acetaminophen should be avoided in late pregnancy, similar to nonsteroidal anti-inflammatory drugs, because of the risk of in utero ductus arteriosus closure. This article critically evaluated the literature reporting an association between acetaminophen use and in utero ductus arteriosus closure and provided a comparative pharmacokinetic analysis of fetal acetaminophen exposure in pregnancy vs drug levels in neonates, with the goal of making an expert recommendation regarding its safety. Here, 1 prospective cohort study and 12 case reports and series evaluating the risk of premature ductus arteriosus closure with prenatal acetaminophen use were reported and overall do not suggest causation. Pharmacokinetic studies showed that acetaminophen fetal transplacental exposures are well below the levels shown to close the ductus arteriosus in neonates. Short-term use of acetaminophen in the third trimester of pregnancy poses a negligible risk of premature ductus arteriosus closure and can still be considered safe in the third trimester of pregnancy at recommended doses.

Prevalence of Left Atrial Thrombus in Anticoagulated Patients With Atrial Fibrillation.

BACKGROUND: The prevalence of left atrial (LA) thrombus in patients with atrial fibrillation (AF) or atrial flutter (AFL) on guideline-directed anticoagulation is not well known, yet this may inform transesophageal echocardiogram (TEE) use before cardioversion or catheter ablation. OBJECTIVES: The purpose of this study was to quantify LA thrombus prevalence among patients with AF/AFL on guideline-directed anticoagulation and to identify high-risk subgroups. METHODS: EMBASE, MEDLINE, and CENTRAL were systematically searched from inception to July 2020 for studies reporting on LA thrombus prevalence among patients with AF/AFL undergoing TEE following at least 3 weeks of continuous therapeutic oral anticoagulation with vitamin K antagonists (VKAs) or direct oral anticoagulants (DOACs). Meta-analysis was performed using random effects models. RESULTS: Thirty-five studies describing 14,653 patients were identified. The mean-weighted LA thrombus prevalence was 2.73% (95% confidence interval [CI]: 1.95% to 3.80%). LA thrombus prevalence was similar for VKA- and DOAC-treated patients (2.80%; 95% CI: 1.86% to 4.21% vs. 3.12%; 95% CI: 1.92% to 5.03%; p = 0.674). Patients with nonparoxysmal AF/AFL had a 4-fold higher LA thrombus prevalence compared with paroxysmal patients (4.81%; 95% CI: 3.35% to 6.86% vs. 1.03%; 95% CI: 0.52% to 2.03%; p < 0.001). LA thrombus prevalence was higher among patients undergoing cardioversion versus ablation (5.55%; 95% CI: 3.15% to 9.58% vs. 1.65%; 95% CI: 1.07% to 2.53%; p < 0.001). Patients with CHA2DS2-VASc scores ≥3 had a higher LA thrombus prevalence compared with patients with scores ≤2 (6.31%; 95% CI: 3.72% to 10.49% vs. 1.06%; 95% CI: 0.45% to 2.49%; p < 0.001). CONCLUSIONS: LA thrombus prevalence is high in subgroups of anticoagulated patients with AF/AFL, who may benefit from routine pre-procedural TEE use before cardioversion or catheter ablation.