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The L-type calcium current (ICaL) is the first step in cardiac excitation–contraction-coupling and plays an important role in regulating contractility, but also in electrical and mechanical remodeling. Primary culture of cardiomyocytes, a widely used tool in cardiac ion channel research, is associated with substantial morphological, functional and electrical changes some of which may be prevented by electrical pacing. We therefore investigated ICaL directly after cell isolation and after 24 h of primary culture with and without regular pacing at 1 and 3 Hz in rat left ventricular myocytes. Moreover, we analyzed total mRNA expression of the pore forming subunit of the L-type Ca2+ channel (cacna1c) as well as the expression of splice variants of its exon 1 that contribute to specificity of ICaL in different tissue such as cardiac myocytes or smooth muscle. 24 h incubation without pacing decreased ICaL density by ~ 10% only. Consistent with this decrease we observed a decrease in the expression of total cacna1c and of exon 1a, the dominant variant of cardiomyocytes, while expression of exon 1b and 1c increased. Pacing for 24 h at 1 and 3 Hz led to a substantial decrease in ICaL density by 30%, mildly slowed ICaL inactivation and shifted steady-state inactivation to more negative potentials. Total cacna1c mRNA expression was substantially decreased by pacing, as was the expression of exon 1b and 1c. Taken together, electrical silence introduces fewer alterations in ICaL density and cacna1c mRNA expression than pacing for 24 h and should therefore be the preferred approach for primary culture of cardiomyocytes. [ABSTRACT FROM AUTHOR]

Lengthy scales or testlets pose certain challenges for structural equation modeling (SEM) if all the items are included as indicators of a latent construct. Three general approaches to modeling lengthy scales in SEM (parceling, latent scoring, and shortening) have been reviewed and evaluated. A hypothetical population model is simulated containing two exogenous constructs with 14 indicators each and an endogenous construct with four indicators. The simulation generates data sets with varying numbers of response options, two types of distributions, factor loadings ranging from low to high, and sample sizes ranging from small to moderate. The population model is varied to incorporate one of the following: (a) single parcels, (b) various parcels as indicators of two exogenous constructs, (c) latent scores as observed exogenous variables, and (d) four and six individual items as indicators of two exogenous constructs. The dependent variables evaluated are biases in the covariance and partial covariance population parameters. Biases in these parameters are found to be minimal under the following conditions: (a) when parcels of indicators of five response options are used as indicators of two latent exogenous constructs, (b) when latent scores are used as observed variables at sample sizes above 100 and with indicators that are relatively less skewed in the case of dichotomous indicators, and (c) when four or six individual items with high or diverse factor loadings are used as indicators of two exogenous constructs. These findings provide guidelines for resolving the inconsistency of findings from applying various approaches to empirical data. (Contains 4 tables and 1 figure.)

Importance: Progressive multifocal leukoencephalopathy (PML) is a life-threatening viral infection with no approved antiviral treatment., Objective: To determine whether restoring the compromised immune system of patients with PML with directly isolated allogeneic virus-specific (DIAVIS) T cells is a promising therapeutic strategy, especially if other curative options are absent., Design, Setting, and Participants: A retrospective case series of patients with PML who were treated with DIAVIS T cells was conducted between March 2020 and February 2022. T cells were isolated from healthy donors within 24 hours and targeted against the BK polyomavirus. Patients with PML were treated monocentrically. Eligibility for treatment with DIAVIS T cells was assessed for patients with confirmed PML, and exclusion criteria included stable PML disease and previous treatment with natalizumab., Exposure: Fresh DIAVIS T cells were administered with a maximum dose of 2 × 104 CD3+ cells/kg body weight. Remaining T cells were cryopreserved in divided doses and administered in additional treatments approximately 2 and 6 weeks later., Main Outcomes and Measures: Primary outcome measures were clinical response and survival of patients, compared with the outcomes of a historical reference group of PML cases receiving best supportive treatment (BST) and with recently published real-world data of patients with PML who were treated with immune checkpoint inhibition., Results: The study cohort consisted of 28 patients (median [IQR] age, 60 [51-72] years; 20 male [71.4%]). Twenty-two patients (79%) treated with DIAVIS T cells showed response, resulting in significant clinical stabilization or improvement and a reduction in viral load. Six individuals (21%) were classified as nonresponders, deteriorated rapidly, and died, as did 2 other patients during a 12-month follow-up. Older age was the only predictor of a poor treatment response. Survival analysis revealed better 12-month survival rates (hazard ratio, 0.42; 95% CI, 0.24-0.73; P =.02) from diagnosis for patients treated with DIAVIS T cells (18 of 26 [69%]; 12-mo survival rate, 69%) compared with historical controls with BST (57 of 113 [50%]; 12-mo survival rate, including censored data, 45%)., Conclusion and Relevance: This case series of DIAVIS T-cell therapy in PML provides first class IV evidence suggesting efficacy to reduce mortality and improve functional outcome. Further prospective studies are required to confirm these results.