Hydroxychloroquine/Azithromycin Therapy and QT Prolongation in Hospitalized Patients With COVID-19.

OBJECTIVES: This study aimed to characterize corrected QT (QTc) prolongation in a cohort of hospitalized patients with coronavirus disease-2019 (COVID-19) who were treated with hydroxychloroquine and azithromycin (HCQ/AZM). BACKGROUND: HCQ/AZM is being widely used to treat COVID-19 despite the known risk of QT interval prolongation and the unknown risk of arrhythmogenesis in this population. METHODS: A retrospective cohort of COVID-19 hospitalized patients treated with HCQ/AZM was reviewed. The QTc interval was calculated before drug administration and for the first 5 days following initiation. The primary endpoint was the magnitude of QTc prolongation, and factors associated with QTc prolongation. Secondary endpoints were incidences of sustained ventricular tachycardia or ventricular fibrillation and all-cause mortality. RESULTS: Among 415 patients who received concomitant HCQ/AZM, the mean QTc increased from 443 ± 25 ms to a maximum of 473 ± 40 ms (87 [21%] patients had a QTc ≥500 ms). Factors associated with QTc prolongation ≥500 ms were age (p < 0.001), body mass index <30 kg/m2 (p = 0.005), heart failure (p < 0.001), elevated creatinine (p = 0.005), and peak troponin (p < 0.001). The change in QTc was not associated with death over the short period of the study in a population in which mortality was already high (hazard ratio: 0.998; p = 0.607). No primary high-grade ventricular arrhythmias were observed. CONCLUSIONS: An increase in QTc was seen in hospitalized patients with COVID-19 treated with HCQ/AZM. Several clinical factors were associated with greater QTc prolongation. Changes in QTc were not associated with increased risk of death.

Protocol driven periprocedural anticoagulation for left atrial ablation.

INTRODUCTION: A weight-based heparin dosing policy adjusted for preprocedural oral anticoagulation was implemented to reduce the likelihood of subtherapeutic dosing during left atrial catheter ablation procedures. We hypothesized that initiation of the protocol would result in a greater prevalence of therapeutic activated clotting time (ACT) values and decreased time to therapeutic ACT during left atrial ablation procedures. METHODS: A departmental protocol was initiated for which subjects received intravenous unfractionated heparin (UFH) to achieve and maintain a goal of ACT >300 s. Initial bolus dose was adjusted for pre-procedure oral anticoagulation and weight as follows: 50 units/kg for those receiving warfarin, 75 units/kg for those not anticoagulated, and 120 units/kg for those on direct oral anticoagulants (DOACs). A UFH infusion was initiated at 10% of the bolus per hour. One hundred consecutive left atrial ablation procedures treated with Protocol Guided heparin dosing were compared with a retrospective consecutive cohort of Usual Care heparin dosing. RESULTS: When the Usual Care and Protocol Guided cohorts were compared, significant findings were limited to those on pre-procedure DOAC. The initial UFH bolus increased from 99.3 ± 24.8 to 118.2 ± 22.8 units/kg (p < .001), the proportion of therapeutic ACT on the first draw after heparin administration increased from 57.7% to 76.6% (p = .010), and the time to therapeutic ACT after UFH administration decreased from 37.8 ± 19.8 to 30.2 ± 16.4 min (p = .032). CONCLUSION: A weight-based protocol for periprocedural UFH administration resulted in a higher proportion of therapeutic ACT values and decreased the time to therapeutic ACT for those on pre-procedure DOAC.

Pulsed field ablation for pulmonary vein isolation in the treatment of atrial fibrillation.

Pulsed-field ablation (PFA) is a promising new ablation modality for the treatment of atrial fibrillation. This energy form employs a train of microsecond duration high amplitude electrical pulses that ablate myocardium by electroporation of the sarcolemmal membrane without measurable tissue heating. The ablation pulse waveform has multiple variable components that can affect ablation efficacy, thus each proprietary system has unique properties that cannot be generalized to other systems. Success with PFA depends upon the proximity of the electrode to the target tissue, but not necessarily upon contact. A unique feature of PFA is tissue specificity. Myocardium is very susceptible to irreversible injury whereas the esophagus, phrenic nerves, pulmonary veins, and coronary arteries are relatively resistant to injury. The tissue specificity of PFA may result in a wide therapeutic range and improved safety profile during atrial fibrillation ablation. Vein isolation can be achieved very rapidly (seconds) promising that PFA may reduce procedure time to 1 hour or less. This attractive new technology promises to be a major advance in the field of atrial fibrillation ablation.

Synthesis, DNA-PK inhibition, anti-platelet activity studies of 2-(N-substituted-3-aminopyridine)-substituted-1,3-benzoxazines and DNA-PK and PI3K inhibition, homology modelling studies of 2-morpholino-(7,8-di and 8-substituted)-1,3-benzoxazines.

A number of new 2-(pyridin-3-ylamino)-4H-(substituted) benz[e]-1,3-oxazin-4-ones were synthesized 10a-g. These were then reacted with the hydro-halogen salt of 2, 3 and 4-(halo-methyl) pyridine in the presence of Cs(2)CO(3) to give eighteen new 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-benzoxazines (compounds 11a-i, 13a-c, and 15a-f). X-ray crystallography was used to confirm that the 2-N-substituted structures 11 and 13 were formed rather than the 3-N-substitution analogues 12 and 14. Eleven of the new compounds were tested for their effect on collagen induced platelet aggregation and it was found that the most active inhibitory compound was 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC(50) of 10 ± 2 µM. DNA-dependent protein kinase (DNA-PK) inhibition data for 12 previously prepared 2-morpholino substituted-1,3-benzoxazines (compounds 19-31) were measured and showed high to moderate activity where the most active compound was compound 27 with an IC(50) of 0.28 µM. Furthermore DNA-PK inhibition data for six newly prepared 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-benzoxazines (compounds 11b, 13a-b, 15a-b and 15e) and 8-methyl-7-(pyridin-3-ylmethoxy)-3-(pyridin-3-ylmethyl)-2H-benz[e]-1,3-oxazin-2,4(3H)-dione 17d were measured and moderate to low inhibitory activity was observed, with the most active of the compounds in this series being 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC(50) of 2.5 µM. PI3K inhibition studies revealed that compound 27 is highly potent (IC(50) for PI3Kα = 0.13 µM, PI3Kß = 0.14 µM, PI3Kγ = 0.72 µM, PI3Kδ = 2.02 µM). Compound 22 with 7-[2-(4-methylpiperazin-1-yl)ethoxy] group shows greater inhibition of DNA-PK over PI3K. Docking of some 2-morpholino-substituted-1,3-benzoxazine compounds 19-31 within the binding pocket and structure-activity relationships (SAR) analyses were performed with results agreeing well with observed activities.

Autophagic death of adult hippocampal neural stem cells following insulin withdrawal.

Novel therapeutic approaches using stem cell transplantation to treat neurodegenerative diseases have yielded promising results. However, survival of stem cells after transplantation has been very poor in animal models, and considerable efforts have been directed at increasing the viability of engrafted stem cells. Therefore, understanding the mechanisms that regulate survival and death of neural stem cells is critical to the development of stem cell-based therapies. Hippocampal neural (HCN) stem cells derived from the adult rat brain undergo cell death following insulin withdrawal, which is associated with downregulation of antiapoptotic Bcl-2 family members. To understand the type of cell death in HCN cells following insulin withdrawal, apoptosis markers were assessed. Of note, DNA fragmentation or caspase-3 activation was not observed, but rather dying cells displayed features of autophagy, including increased expression of Beclin 1 and the type II form of light chain 3. Electron micrographs showed the dramatically increased formation of autophagic vacuoles with cytoplasmic contents. Staurosporine induced robust activation of caspase-3 and nucleosomal DNA fragmentation, suggesting that the machinery of apoptosis is intact in HCN cells despite the apparent absence of apoptosis following insulin withdrawal. Autophagic cell death was suppressed by knockdown of autophagy-related gene 7, whereas promotion of autophagy by rapamycin increased cell death. Taken together, these data demonstrate that HCN cells undergo a caspase-independent, autophagic cell death following insulin withdrawal. Understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases. Disclosure of potential conflicts of interest is found at the end of this article.

IFN-gamma amplifies NFkappaB-dependent Neisseria meningitidis invasion of epithelial cells via specific upregulation of CEA-related cell adhesion molecule 1.

Temporal relationship between viral and bacterial infections has been observed, and may arise via the action of virus-induced inflammatory cytokines. These, by upregulating epithelial receptors targeted by bacteria, may encourage greater bacterial infiltration. In this study, human epithelial cells exposed to interferon-gamma but not tumour necrosis factor-alpha or interleukin 1-beta supported increased meningococcal adhesion and invasion. The increase was related to Opa but not Opc or pili adhesin expression. De novo synthesis of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a major Opa receptor, occurred in epithelial cells exposed to the cytokine, or when infected with Opa-expressing bacteria. Cell line-dependent differences in invasion that were observed could be correlated with CEACAM expression levels. There was also evidence for Opa/pili synergism leading to high levels of monolayer infiltration by capsulate bacteria. The use of nuclear factor-kappa B (NFkappaB) inhibitors, diferuloylmethane (curcumin) and SN50, abrogated bacterial infiltration of both untreated and interferon-gamma-treated cells. The studies demonstrate the importance of CEACAMs as mediators of increased cellular invasion under conditions of inflammation and bring to light the potential role of NFkappaB pathway in Opa-mediated invasion by meningococci. The data imply that cell-surface remodelling by virally induced cytokines could be one factor that increases host susceptibility to bacterial infection.

Critical determinants of the interactions of capsule-expressing Neisseria meningitidis with host cells: the role of receptor density in increased cellular targeting via the outer membrane Opa proteins.

Neisseria meningitidis capsule is an important virulence determinant required for survival in the blood but is reportedly involved in inhibiting cellular interactions mediated by meningococcal outer membrane adhesins. However, evidence from our previous studies suggested that target receptor density on host cells may determine whether or not capsulate bacteria can adhere via outer membrane proteins such as Opa. To confirm this and evaluate the impact of capsulation on bacterial interactions, we used Opa(+) and Opa(-) derivatives of capsulate and acapsulate meningococcal isolates and transfected cell lines expressing CEACAM1, a receptor targeted by Opa proteins. To assess the extent and rate of cell association, subpopulations of stably transfected Chinese hamster ovary cells with different receptor levels were derived. A quantitative correlation of CEACAM1 levels and Opa-dependent binding of both capsulate and acapsulate bacteria was demonstrated, which was accelerated at high receptor densities. However, it appears that invasion by Opa(+) capsulate bacteria only occurs when a threshold level of CEACAM density has been reached. Target cells expressing high levels of CEACAM1 (MFI c. 400) bound threefold more, but internalized 20-fold more Opa(+) capsulate bacteria than those with intermediate expression (MFI c. 100). No overall selection of acapsulate phenotype was observed in the internalized population. These observations confirm that capsule may not be an adequate barrier for cellular interactions and demonstrate the role of a host factor that may determine capsulate bacterial invasion potential. Upregulation of CEACAMs, which can occur in response to inflammatory cytokines, could lead to translocation of a small number of fully capsulate bacteria across mucosal epithelium into the bloodstream sufficient to cause a rapid onset of disseminated disease. Thus the data also suggest a novel rationale for the epidemiological observations that individuals with prior infectious/inflammatory conditions carry a high risk of invasive meningococcal disease.