Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes.

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

Confirmation and refutation of very luminous galaxies in the early Universe.

During the first 500 million years of cosmic history, the first stars and galaxies formed, seeding the Universe with heavy elements and eventually reionizing the intergalactic medium1-3. Observations with the James Webb Space Telescope (JWST) have uncovered a surprisingly high abundance of candidates for early star-forming galaxies, with distances (redshifts, z), estimated from multiband photometry, as large as z ≈ 16, far beyond pre-JWST limits4-9. Although such photometric redshifts are generally robust, they can suffer from degeneracies and occasionally catastrophic errors. Spectroscopic measurements are required to validate these sources and to reliably quantify physical properties that can constrain galaxy formation models and cosmology10. Here we present JWST spectroscopy that confirms redshifts for two very luminous galaxies with z > 11, and also demonstrates that another candidate with suggested z ≈ 16 instead has z = 4.9, with an unusual combination of nebular line emission and dust reddening that mimics the colours expected for much more distant objects. These results reinforce evidence for the early, rapid formation of remarkably luminous galaxies while also highlighting the necessity of spectroscopic verification. The large abundance of bright, early galaxies may indicate shortcomings in current galaxy formation models or deviations from physical properties (such as the stellar initial mass function) that are generally believed to hold at later times.

Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

IMPORTANCE: HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

Safety of catheter ablation in patients with recently implanted cardiac implantable electronic device: A 5-year experience.

INTRODUCTION: Catheter ablation (CA) can interfere with cardiac implantable electronic device (CIED) function. The safety of CA in the 1st year after CIED implantation/lead revision is uncertain. METHODS: This single center, retrospective cohort included patients who underwent CA between 2012 and 2017 and had a CIED implant/lead revision within the preceding year. We assessed the frequency of device/lead malfunctions in this population. RESULTS: We identified 1810 CAs in patients between 2012 and 2017, with 170 CAs in 163 patients within a year of a CIED implant/lead revision. Mean age 68 ± 12 years (68% men). Time between the CIED procedure and CA was 158 ± 99 days. The CA procedures included AF ablation (n = 57, 34%), AV node ablation (n = 40, 24%), SVT ablation (n = 37, 22%), and PVC/VT ablations (n = 36, 21%). The cumulative frequency of lead dislodgement, significant CIED dysfunction, and/or CIED-related infection following CA was (n = 1/170, 0.6%). There was a single atrial lead dislodgement (0.6%). There were no instances of power-on-reset or CIED-related infection. Following CA, there was no significant difference in RA or RV lead sensing (p = 0.52 and 0.84 respectively) or thresholds (p = 0.94 and 0.17 respectively). The RA impedance slightly decreased post-CA from 474 ± 80 Ohms to 460 ± 73 Ohms (p = 0.002), as did the RV impedance (from 515 ± 111 Ohms to 497 ± 98 Ohms, p < 0.0001). CONCLUSIONS: CA can be performed within 1 year following CIED implantation/lead revision with a low risk of CIED/lead malfunction or lead dislodgement. The ideal time to perform CA after a CIED remains uncertain.

Cardiac resynchronization therapy for pacing induced cardiomyopathy: Role of baseline right ventricular pacing burden.

BACKGROUND: Cardiac resynchronization therapy (CRT) is indicated for patients with heart failure with reduced left ventricular ejection fraction (LVEF) and chronic right ventricular (RV) pacing burden ≥40% (pacing-induced cardiomyopathy, PICM). It is uncertain whether baseline RV pacing burden impacts response to CRT. METHODS: We conducted a retrospective study of all CRT upgrades for PICM at our hospital from January 2017 to December 2018. Univariate and multivariable-adjusted changes in LVEF, and echocardiographic response (≥10% improvement in LVEF) at 3-12 months post-CRT upgrade were compared in those with RV pacing burden ≥90% versus <90%. RESULTS: We included 75 patients (age 74 ± 11 years, 71% male) who underwent CRT upgrade for PICM. The baseline RV pacing burden was ≥90% in 56 patients (median 99% [IQR 98%-99%]), and <90% in 19 patients (median 79% [IQR 73%-87%]). Improvement in LVEF was greater in those with baseline RV pacing burden ≥90% versus <90% (15.7 ± 9.3% vs. 7.5 ± 9.6%, p = .003). Baseline RV pacing burden ≥90% was a strong predictor of an improvement in LVEF ≥10% after CRT upgrade both in univariate and multivariate-adjusted models (p = .005 and .02, respectively). CONCLUSION: A higher baseline RV pacing burden predicts a greater improvement in LVEF after CRT upgrade for PICM.

Electrocardiographic Z-axis QRS-T voltage-time-integral in patients with typical right bundle branch block - Correlation with echocardiographic right ventricular size and function.

BACKGROUND: Right bundle branch block (RBBB) can be benign or associated with right ventricular (RV) functional and structural abnormalities. Our aim was to evaluate QRS-T voltage-time-integral (VTI) compared to QRS duration and lead V1 R' as markers for RV abnormalities. METHODS: We included adults with an ECG demonstrating RBBB and echocardiogram obtained within 3 months of each other, between 2010 and 2020. VTIQRS and VTIQRST were obtained for 12 standard ECG leads, reconstructed vectorcardiographic X, Y, Z leads and root-mean-squared (3D) ECG. Age, sex and BSA-adjusted linear regressions were used to assess associations of QRS duration, amplitudes, VTIs and lead V1 R' duration/VTI with echocardiographic tricuspid annular plane systolic excursion (TAPSE), RV tissue Doppler imaging S', basal and mid diameter, and systolic pressure (RVSP). RESULTS: Among 782 patients (33% women, age 71 ± 14 years) with RBBB, R' duration in lead V1 was modestly associated with RV S', RV diameters and RVSP (all p ≤ 0.03). QRS duration was more strongly associated with RV diameters (both p < 0.0001). AmplitudeQRS-Z was modestly correlated with all 5 RV echocardiographic variables (all p ≤ 0.02). VTIR'-V1 was more strongly associated with TAPSE, RV S' and RVSP (all p ≤ 0.0003). VTIQRS-Z and VTIQRST-Z were among the strongest correlates of the 5 RV variables (all p < 0.0001). VTIQRST-Z.√BSA cutoff of ≥62 µVsm had sensitivity 62.7% and specificity 65.7% for predicting ≥3 of 5 abnormal RV variables (AUC 0.66; men 0.71, women 0.60). CONCLUSION: In patients with RBBB, VTIQRST-Z is a stronger predictor of RV dysfunction and adverse remodeling than QRS duration and lead V1 R'.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Emergent activity, heterogeneity, and robustness in a calcium feedback model of the sinoatrial node.

The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN activity emerges at an early point in life and maintains a steady rhythm for the lifetime of the organism. The ion channel composition and currents of SAN cells can be influenced by a variety of factors. Therefore, the emergent activity and long-term stability imply some form of dynamical feedback control of SAN activity. We adapt a recent feedback model-previously utilized to describe control of ion conductances in neurons-to a model of SAN cells and tissue. The model describes a minimal regulatory mechanism of ion channel conductances via feedback between intracellular calcium and an intrinsic target calcium level. By coupling a SAN cell to the calcium feedback model, we show that spontaneous electrical activity emerges from quiescence and is maintained at steady state. In a 2D SAN tissue model, spatial variability in intracellular calcium targets lead to significant, self-organized heterogeneous ion channel expression and calcium transients throughout the tissue. Furthermore, multiple pacemaking regions appear, which interact and lead to time-varying cycle length, demonstrating that variability in heart rate is an emergent property of the feedback model. Finally, we demonstrate that the SAN tissue is robust to the silencing of leading cells or ion channel knockouts. Thus, the calcium feedback model can reproduce and explain many fundamental emergent properties of activity in the SAN that have been observed experimentally based on a minimal description of intracellular calcium and ion channel regulatory networks.

Modeling incomplete penetrance in long QT syndrome type 3 through ion channel heterogeneity: an in silico population study.

Many cardiac diseases are characterized by an increased late sodium current, including heart failure, hypertrophic cardiomyopathy, and inherited long QT syndrome type 3 (LQT3). The late sodium current in LQT3 is caused by a gain-of-function mutation in the voltage-gated sodium channel Nav1.5. Despite a well-defined genetic cause of LQT3, treatment remains inconsistent because of incomplete penetrance of the mutation and variability of antiarrhythmic efficacy. Here, we investigate the relationship between LQT3-associated mutation incomplete penetrance and variability in ion channel expression, simulating a population of 1,000 individuals with the O'Hara-Rudy model of the human ventricular myocyte. We first simulate healthy electrical activity (i.e., in the absence of a mutation) and then incorporate heterozygous expression for three LQT3-associated mutations (Y1795C, I1768V, and ΔKPQ), to directly compare the effects of each mutation on individuals across a diverse population. For all mutations, we find that susceptibility, defined by either the presence of an early afterdepolarization (EAD) or prolonged action potential duration (APD), primarily depends on the balance between the conductance of IKr and INa, for which individuals with a higher IKr-to-INa ratio are less susceptible. Furthermore, we find distinct differences across the population, observing individuals susceptible to zero, one, two, or all three mutations. Individuals tend to be less susceptible with an appropriate balance of repolarizing currents, typically via increased IKs or IK1. Interestingly, the more critical repolarizing current is mutation specific. We conclude that the balance between key currents plays a significant role in mutant-specific presentation of the disease phenotype in LQT3.NEW & NOTEWORTHY An in silico population approach investigates the relationship between variability in ion channel expression and gain-of-function mutations in the voltage-gated sodium channel associated with the congenital disorder long QT syndrome type 3 (LQT3). We find that ion channel variability can contribute to incomplete penetrance of the mutation, with mutant-specific differences in ion channel conductances leading to susceptibility to proarrhythmic action potential duration prolongation or early afterdepolarizations.

Long-term changes in heart rate and electrical remodeling contribute to alternans formation in heart failure: a patient-specific in silico study.

Individuals with chronic heart failure (CHF) have an increased risk of ventricular arrhythmias, which has been linked to pathological cellular remodeling and may also be mediated by changes in heart rate. Heart rate typically fluctuates on a timescale ranging from seconds to hours, termed heart rate variability (HRV). This variability is reduced in CHF, and this HRV reduction is associated with a greater risk for arrhythmias. Furthermore, variations in heart rate influence the formation of proarrhythmic alternans, a beat-to-beat alternation in the action potential duration (APD), or intracellular calcium (Ca). In this study, we investigate how long-term changes in heart rate and electrical remodeling associated with CHF influence alternans formation. We measure key statistical properties of the RR-interval sequences from ECGs of individuals with normal sinus rhythm (NSR) and CHF. Patient-specific RR-interval sequences and synthetic sequences (randomly generated to mimicking these statistical properties) are used as the pacing protocol for a discrete time-coupled map model that governs APD and intracellular Ca handling of a single cardiac myocyte, modified to account for pathological electrical remodeling in CHF. Patient-specific simulations show that beat-to-beat differences in APD vary temporally in both populations, with alternans formation more prevalent in CHF. Parameter studies using synthetic sequences demonstrate that increasing the autocorrelation time or mean RR-interval reduces APD alternations, whereas increasing the RR-interval standard deviation leads to higher alternans magnitudes. Importantly, we find that although both the CHF-associated changes in heart rate and electrical remodeling influence alternans formation, variations in heart rate may be more influential.NEW & NOTEWORTHY Using patient-specific data, we show that both the changes in heart rate and electrical remodeling associated with chronic heart failure influence the formation of proarrhythmic alternans in the heart.

Ephaptic Coupling as a Resolution to the Paradox of Action Potential Wave Speed and Discordant Alternans Spatial Scales in the Heart.

Previous computer simulations have suggested that existing models of action potential wave propagation in the heart are not consistent with observed wave propagation behavior. Specifically, computer models cannot simultaneously reproduce the rapid wave speeds and small spatial scales of discordant alternans patterns measured experimentally in the same simulation. The discrepancy is important, because discordant alternans can be a key precursor to the development of abnormal and dangerous rapid rhythms in the heart. In this Letter, we show that this paradox can be resolved by allowing so-called ephaptic coupling to play a primary role in wave front propagation in place of conventional gap-junction coupling. With this modification, physiological wave speeds and small discordant alternans spatial scales both occur with gap-junction resistance values that are more in line with those observed in experiments. Our theory thus also provides support to the hypothesis that ephaptic coupling plays an important role in normal wave propagation.

Multicenter assessment of the outcomes of subcutaneous ICD implantation in patients with prior or future sternotomy.

BACKGROUND: The subcutaneous ICD (S-ICD) is a viable alternative to transvenous ICD and avoids intravascular complications in patients without a pacing indication. The outcomes of S-ICD implantation are uncertain in patients with prior sternotomy. OBJECTIVE: We aim to compare the implant techniques and outcomes with S-ICD implantation in patients with and without prior sternotomy. METHODS: Multicenter retrospective cohort study including adult patients with an S-ICD implanted between January 2014 and June 2020. Outcomes were compared between patients with and without prior sternotomy. RESULTS: Among the 212 patients (49 ± 15 years old, 43% women, BMI 30 ± 8 kg/m2 , 68% primary prevention, 30% ischemic cardiomyopathy, LVEF median 30% IQR 25%-45%) who underwent S-ICD implantation, 47 (22%) had a prior sternotomy. There was no difference in the sensing vector (57% vs. 53% primary, p = 0.55), laterality of the S-ICD lead to the sternum (94% vs. 96% leftward, p = 0.54), or the defibrillation threshold (65 ± 1.4 J vs. 65 ± 0.8 J, p = 0.76) with versus without prior sternotomy. The frequency of 30-day complications was similar with and without prior sternotomy (n = 3/47 vs. n = 15/165, 6% vs. 9%, p = 0.56). Over a median follow-up of 28 months (IQR 10-49 months), the frequency of inappropriate shocks was similar between those with and without prior sternotomy (n = 3/47 and n = 16/165, 6% vs. 10%, p = 0.58). CONCLUSION: Implantation of an S-ICD in patients with prior sternotomy is safe with a similar risk of 30-day complications and inappropriate ICD shocks as patients without prior sternotomy.

Electrocardiographic prediction of left ventricular hypertrophy in women and men with left bundle branch block - Comparison of QRS duration, amplitude and voltage-time-integral.

BACKGROUND: Standard ECG criteria for left ventricular (LV) hypertrophy rely on QRS amplitudes. However, in the setting of left bundle branch block (LBBB), ECG correlates of LV hypertrophy are not well established. We sought to evaluate quantitative ECG predictors of LV hypertrophy in the presence of LBBB. METHODS: We included adult patients with typical LBBB having ECG and transthoracic echocardiogram performed within 3 months of each other in 2010-2020. Orthogonal X, Y, Z leads were reconstructed from digital 12­lead ECGs using Kors's matrix. In addition to QRS duration, we evaluated QRS amplitudes and voltage-time-integrals (VTIs) from all 12 leads, X, Y, Z leads and 3D (root-mean-squared) ECG. We used age, sex and BSA-adjusted linear regressions to predict echocardiographic LV calculations (mass, end-diastolic and end-systolic volumes, ejection fraction) from ECG, and separately generated ROC curves for predicting echocardiographic abnormalities. RESULTS: We included 413 patients (53% women, age 73 ± 12 years). All 4 echocardiographic LV calculations were most strongly correlated with QRS duration (all p < 0.00001). In women, QRS duration ≥ 150 ms had sensitivity/specificity 56.3%/64.4% for increased LV mass and 62.7%/67.8% for increased LV end-diastolic volume. In men, QRS duration ≥ 160 ms had a sensitivity/specificity 63.1%/72.1% for increased LV mass and 58.3%/74.5% for increased LV end-diastolic volume. QRS duration was best able to discriminate eccentric hypertrophy (area under ROC curve 0.701) and increased LV end-diastolic volume (0.681). CONCLUSIONS: In patients with LBBB, QRS duration (≥ 150 in women and ≥ 160 in men) is a superior predictor of LV remodeling esp. eccentric hypertrophy and dilation.

A data-assimilation approach to predict population dynamics during epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a biological process that plays a central role in embryonic development, tissue regeneration, and cancer metastasis. Transforming growth factor-ß (TGFß) is a potent inducer of this cellular transition, comprising transitions from an epithelial state to partial or hybrid EMT state(s), to a mesenchymal state. Recent experimental studies have shown that, within a population of epithelial cells, heterogeneous phenotypical profiles arise in response to different time- and TGFß dose-dependent stimuli. This offers a challenge for computational models, as most model parameters are generally obtained to represent typical cell responses, not necessarily specific responses nor to capture population variability. In this study, we applied a data-assimilation approach that combines limited noisy observations with predictions from a computational model, paired with parameter estimation. Synthetic experiments mimic the biological heterogeneity in cell states that is observed in epithelial cell populations by generating a large population of model parameter sets. Analysis of the parameters for virtual epithelial cells with biologically significant characteristics (e.g., EMT prone or resistant) illustrates that these sub-populations have identifiable critical model parameters. We perform a series of in silico experiments in which a forecasting system reconstructs the EMT dynamics of each virtual cell within a heterogeneous population exposed to time-dependent exogenous TGFß dose and either an EMT-suppressing or EMT-promoting perturbation. We find that estimating population-specific critical parameters significantly improved the prediction accuracy of cell responses. Thus, with appropriate protocol design, we demonstrate that a data-assimilation approach successfully reconstructs and predicts the dynamics of a heterogeneous virtual epithelial cell population in the presence of physiological model error and parameter uncertainty.

Computational modeling of aberrant electrical activity following remuscularization with intramyocardially injected pluripotent stem cell-derived cardiomyocytes.

Acute engraftment arrhythmias (EAs) remain a serious complication of remuscularization therapy. Preliminary evidence suggests that a focal source underlies these EAs stemming from the automaticity of immature pluripotent stem cell-derived cardiomyocytes (PSC-CMs) in nascent myocardial grafts. How these EAs arise though during early engraftment remains unclear. In a series of in silico experiments, we probed the origin of EAs-exploring aspects of altered impulse formation and altered impulse propagation within nascent PSC-CM grafts and at the host-graft interface. To account for poor gap junctional coupling during early PSC-CM engraftment, the voltage dependence of gap junctions and the possibility of ephaptic coupling were incorporated. Inspired by cardiac development, we also studied the contributions of another feature of immature PSC-CMs, circumferential sodium channel (NaCh) distribution in PSC-CMs. Ectopic propagations emerged from nascent grafts of immature PSC-CMs at a rate of <96 bpm. Source-sink effects dictated this rate and contributed to intermittent capture between host and graft. Moreover, ectopic beats emerged from dynamically changing sites along the host-graft interface. The latter arose in part because circumferential NaCh distribution in PSC-CMs contributed to preferential conduction slowing and block of electrical impulses from host to graft myocardium. We conclude that additional mechanisms, in addition to focal ones, contribute to EAs and recognize that their relative contributions are dynamic across the engraftment process.

Electromagnetic interference from left ventricular assist devices detected in patients with implantable cardioverter-defibrillators.

INTRODUCTION: Electromagnetic interference (EMI) from left ventricular assist devices (LVADs) can cause implantable cardioverter-defibrillator (ICD) oversensing. We sought to assess the frequency of inappropriate shocks/oversensing due to LVAD-related EMI and prospectively compare integrated (IB) versus dedicated bipolar (DB) sensing in patients with LVADs. METHODS: Single-center study in LVAD patients with Medtronic or Abbott ICDs between September 2017 and March 2020. We excluded patients that were pacemaker dependent. Measurements were obtained of IB and DB sensing and noise to calculate a signal-to-noise ratio (SNR). Device checks were reviewed to assess appropriate and inappropriate sensing events. RESULTS: Forty patients (age 52 ± 14 years, 75% men, 38% ischemic cardiomyopathy) were included with the median time between LVAD implantation and enrollment of 6.7 months (2.3, 11.4 months). LVAD subtypes included: HeartWare (n = 22, 55%), Heartmate II (n = 10, 25%), and Heartmate III (n = 8, 20%). Over a follow-up duration of 21.6 ± 12.9 months after LVAD implantation, 5% of patients (n = 2) had oversensing of EMI from the LVAD (both with HeartWare LVADs and Abbott ICDs) at 4 days and 10.8 months after LVAD implantation. Both patients underwent adjustment of ventricular sensing with resolution of oversensing and no further events over 5 and 15 months of further follow-up. The SNR was similar between IB and DB sensing (50 [29-67] and 57 [41-69], p = 0.89). CONCLUSION: ICD oversensing of EMI from LVADs is infrequent and can be managed with reprogramming the sensitivity. There was no significant difference in the R-wave SNR with IB versus DB ICD leads.

Emerging Implantable-Device Technology for Patients at the Intersection of Electrophysiology and Heart Failure Interdisciplinary Care.

Cardiac implantable electronic devices, including implantable cardioverter-defibrillators and therapy, are part of guideline-indicated treatment for a subset of patients with heart failure with reduced ejection fraction. Current technological advancements in cardiac implantable electronic devices have allowed the detection of specific physiological parameters that are used to forecast clinical decompensation through algorithmic, multiparameter remote monitoring. Other recent emerging technologies, including cardiac contractility modulation and baroreflex activation therapy, may provide symptomatic or physiological benefits in patients without indications for cardiac resynchronization. Our goal in this state-of-the-art review is to describe the new commercially available technologies, their purported mechanisms of action, and the evidence surrounding their clinical roles, limitations and future directions. Finally, we underline the need for standardized workflow and close interdisciplinary management of this population to ensure the delivery of high-quality care.

Metabolomics and cytokine profiling of mesenchymal stromal cells identify markers predictive of T-cell suppression.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have shown great promise in the field of regenerative medicine, as many studies have shown that MSCs possess immunomodulatory function. Despite this promise, no MSC therapies have been licensed by the Food and Drug Administration. This lack of successful clinical translation is due in part to MSC heterogeneity and a lack of critical quality attributes. Although MSC indoleamine 2,3-dioxygnease (IDO) activity has been shown to correlate with MSC function, multiple predictive markers may be needed to better predict MSC function. METHODS: Three MSC lines (two bone marrow-derived, one induced pluripotent stem cell-derived) were expanded to three passages. At the time of harvest for each passage, cell pellets were collected for nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography mass spectrometry (MS), and media were collected for cytokine profiling. Harvested cells were also cryopreserved for assessing function using T-cell proliferation and IDO activity assays. Linear regression was performed on functional data against NMR, MS and cytokines to reduce the number of important features, and partial least squares regression (PLSR) was used to obtain predictive markers of T-cell suppression based on variable importance in projection scores. RESULTS: Significant functional heterogeneity (in terms of T-cell suppression and IDO activity) was observed between the three MSC lines, as were donor-dependent differences based on passage. Omics characterization revealed distinct differences between cell lines using principal component analysis. Cell lines separated along principal component one based on tissue source (bone marrow-derived versus induced pluripotent stem cell-derived) for NMR, MS and cytokine profiles. PLSR modeling of important features predicted MSC functional capacity with NMR (R2 = 0.86), MS (R2 = 0.83), cytokines (R2 = 0.70) and a combination of all features (R2 = 0.88). CONCLUSIONS: The work described here provides a platform for identifying markers for predicting MSC functional capacity using PLSR modeling that could be used as release criteria and guide future manufacturing strategies for MSCs and other cell therapies.

Morphological landscapes from high content imaging reveal cytokine priming strategies that enhance mesenchymal stromal cell immunosuppression.

Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify "priming" conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e., priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-γ) and tumor necrosis factor-⍺ (TNF-⍺) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T-cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-γ/TNF-⍺ priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors, for example, PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.

UltraSound Axillary Vein Access (USAA): Learning curve and randomized comparison to traditional venous access for cardiac device implantation.

BACKGROUND: Many techniques exist for venous access (VA) during cardiac implantable electronic device (CIED) implantation. OBJECTIVE: We sought to evaluate the learning curve with ultrasound (US) guided axillary vein access (USAA). METHODS: Single-center prospective randomized controlled trial of patients undergoing CIED implantation. Patients were randomized in a 2:1 fashion to USAA versus conventional VA techniques. The primary outcomes were the success rates, VA times and 30-day complication rates. RESULTS: The study included 100 patients (age 68 ± 14 years, BMI 27 ± 4 kg/m2 ). USAA was successful in 66/70 implants (94%). Initial attempts at conventional VA included 47% axillary (n = 14), 30% (n = 9) cephalic, and 23% (n = 7) subclavian. The median access time was longer for USAA than conventional access (8.3 IQR 4.2-15.3 min vs. 5.2 IQR 3.4-8.6 min, p = .009). Among the five inexperienced USAA implanters, there was a significant improvement in median access time from first to last tertile of USAA implants (17.0 IQR 7.0-21.0 min to 8.6 IQR 4.5-10.8 min, p = .038). The experienced USAA implanter had similar access times with USAA compared with conventional access (4.0 IQR 3.3-4.7 min vs. 5.2 IQR 3.4-8.6 min, p = .15). Venograms were less common with USAA than conventional access (2% vs. 33%, p < .0001). The 30-day complication rate was similar with USAA (n = 4/70, 6%) versus conventional (n = 3/30, 10%, p = .44). CONCLUSION: Although the success rate with USAA was high, there was a significant learning curve. Once experienced with the USAA technique, there is the potential for reduced complications without adding to the procedure duration.