Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction
Background: Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2 WT CMs or CCND2 OE CMs, respectively) and injected into infarcted pig hearts. Methods: Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2 WT CMs or CCND2 OE CMs (3×10 7 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. Results: The number of the engrafted CCND2 OE CMs exceeded that of the engrafted CCND2 WT CMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2 WT CMs or the delivery vehicle, the administration of CCND2 OE CM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2 OE CM-treated than from the CCND2 WT CM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2 OE CMs (CCND2 OE Exos) than from the CCND2 WT CMs (CCND2 WT Exos). As demonstrated by our study, CCND2 OE Exos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2 OE Exos versus CCND2 WT Exos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. Conclusions: We demonstrated that enhanced potency of CCND2 OE CMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2 OE CMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.
The Inter- and Intra- Observer Agreement in Confocal Endomicroscopy Guided Diagnosis Of Pancreatic Cysts and Evaluation of Diagnostic Accuracy Compared to Histopathology
AACC Practical Recommendations for Implementing and Interpreting SARS-CoV-2 Emergency Use Authorization and Laboratory-Developed Test Serologic Testing in Clinical Laboratories
Abstract Background The clinical laboratory continues to play a critical role in managing the coronavirus pandemic. Numerous US Food and Drug Administration emergency use authorization (EUA) and laboratory-developed test (LDT) serologic assays have become available. The performance characteristics of these assays and their clinical utility continue to be defined in real time during this pandemic. The AACC convened a panel of experts from clinical chemistry, microbiology, and immunology laboratories; the in vitro diagnostics industry; and regulatory agencies to provide practical recommendations for implementation and interpretation of these serologic tests in clinical laboratories. Content The currently available EUA serologic tests and platforms, information on assay design, antibody classes including neutralizing antibodies, and the humoral immune responses to SARS-CoV-2 are discussed. Verification and validation of EUA and LDT assays are described, along with a quality management approach. Four indications for serologic testing are outlined. Recommendations for result interpretation, reporting comments, and the role of orthogonal testing are also presented. Summary This document aims to provide a comprehensive reference for laboratory professionals and healthcare workers to appropriately implement SARS-CoV-2 serologic assays in the clinical laboratory and to interpret test results during this pandemic. Given the more frequent occurrence of outbreaks associated with either vector-borne or respiratory pathogens, this document will be a useful resource in planning for similar scenarios in the future.
Impact of surgeon and hospital factors on surgical decision-making for grade 1 degenerative lumbar spondylolisthesis: a Quality Outcomes Database analysis
OBJECTIVE Surgical treatment for degenerative spondylolisthesis has been proven to be clinically challenging and cost-effective. However, there is a range of thresholds that surgeons utilize for incorporating fusion in addition to decompressive laminectomy in these cases. This study investigates these surgeon- and site-specific factors by using the Quality Outcomes Database (QOD). METHODS The QOD was queried for all cases that had undergone surgery for grade 1 spondylolisthesis from database inception to February 2019. In addition to patient-specific covariates, surgeon-specific covariates included age, sex, race, years in practice (0–10, 11–20, 21–30, > 30 years), and fellowship training. Site-specific variables included hospital location (rural, suburban, urban), teaching versus nonteaching status, and hospital type (government, nonfederal; private, nonprofit; private, investor owned). Multivariable regression and predictor importance analyses were performed to identify predictors of the treatment performed (decompression alone vs decompression and fusion). The model was clustered by site to account for site-specific heterogeneity in treatment selection. RESULTS A total of 12,322 cases were included with 1988 (16.1%) that had undergone decompression alone. On multivariable regression analysis clustered by site, adjusting for patient-level clinical covariates, no surgeon-specific factors were found to be significantly associated with the odds of selecting decompression alone as the surgery performed. However, sites located in suburban areas (OR 2.32, 95% CI 1.09–4.84, p = 0.03) were more likely to perform decompression alone (reference = urban). Sites located in rural areas had higher odds of performing decompression alone than hospitals located in urban areas, although the results were not statistically significant (OR 1.33, 95% CI 0.59–2.61, p = 0.49). Nonteaching status was independently associated with lower odds of performing decompression alone (OR 0.40, 95% CI 0.19–0.97, p = 0.04). Predictor importance analysis revealed that the most important determinants of treatment selection were dominant symptom (Wald χ2 = 34.7, accounting for 13.6% of total χ2) and concurrent diagnosis of disc herniation (Wald χ2 = 31.7, accounting for 12.4% of total χ2). Hospital teaching status was also found to be relatively important (Wald χ2 = 4.2, accounting for 1.6% of total χ2) but less important than other patient-level predictors. CONCLUSIONS Nonteaching centers were more likely to perform decompressive laminectomy with supplemental fusion for spondylolisthesis. Suburban hospitals were more likely to perform decompression only. Surgeon characteristics were not found to influence treatment selection after adjustment for clinical covariates. Further large database registry experience from surgeons at high-volume academic centers at which surgically and medically complex patients are treated may provide additional insight into factors associated with treatment preference for degenerative spondylolisthesis.
Automated Analysis of Multiparametric Magnetic Resonance Imaging/Magnetic Resonance Elastography Exams for Prediction of Nonalcoholic Steatohepatitis
BackgroundNonalcoholic fatty liver disease (NAFLD) affects 25% of the global population. The standard of diagnosis, biopsy, is invasive and affected by sampling error and inter‐reader variability. We hypothesized that widely available rapid MRI techniques could be used to predict nonalcoholic steatohepatitis (NASH) noninvasively by measuring liver stiffness, with magnetic resonance elastography (MRE), and liver fat, with chemical shift‐encoded (CSE) MRI. Besides, we validate an automated image analysis technique to maximize the utility of these methods.PurposeTo implement and test an automated system for analyzing CSE‐MRI and MRE data coupled with model‐based prediction of NASH.Study TypeProspective.SubjectsEighty‐three patients with suspected NAFLD.Field Strength/SequenceA 1.5 T using a flow‐compensated motion‐encoded gradient echo MRE sequence and a multiecho CSE‐MRI sequence.AssessmentsThe MRE and CSE‐MRI data were analyzed by two readers (5+ and 1 years of experience) and an automated algorithm. A logistic regression model to predict pathology‐diagnosed NASH was trained based on stiffness and proton density fat fraction, and the area under the receiver operating characteristic curve (AUROC) was calculated using 10‐fold cross validation for models based on both automated and manual measurements. A separate model was trained to predict the NASH severity score (NAS).Statistical TestsPearson's correlation, Bland–Altman, AUROC, C‐statistic.ResultsThe agreement between automated measurements and the more experienced reader (R2 = 0.87 for stiffness and R2 = 0.99 for proton density fat fraction [PDFF]) was slightly better than the agreement between readers (R2 = 0.85 and 0.98). The model for predicting biopsy‐diagnosed NASH had an AUROC of 0.87. The NAS‐prediction model had a C‐statistic of 0.85.Data ConclusionWe demonstrated a workflow that used a limited MRI acquisition protocol and fully automated analysis to predict NASH with high accuracy. These methods show promise to provide a reliable noninvasive alternative to biopsy for NASH‐screening in populations with NAFLD.Level of Evidence2Technical Efficacy Stage2
Eosinophils attenuate hepatic ischemia-reperfusion injury in mice through ST2-dependent IL-13 production
Abstract Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.
Midterm results of different treatment methods for myocardial bridging in patients after septal myectomy