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Abstract Background Interferon-beta (IFNB) therapy for multiple sclerosis can lead to the induction of neutralizing antibodies (NAbs) against IFNB. Various methods are used for detection and quantification of NAbs. Methods Blood samples from 125 IFNB-1b–treated patients, which were tested NAb negative or NAb positive after conclusion of a clinical study, were retested three years after first being assessed in four different laboratories that offer routine NAb testing to practicing neurologists. The myxovirus protein A (MxA) induction assay, the cytopathic effect (CPE) assay (two laboratories), or the luciferase assay were used. Intra- and inter-laboratory agreement between assays with respect to NAb detection and NAb titer quantification were evaluated. Results High agreement for NAb detection (kappa coefficient, 0.86) and for titer levels was observed for the intra-laboratory comparison in the laboratory using the MxA induction assay performed three years ago and now. A similarly high agreement for NAb detection (kappa coefficient, 0.87) and for titer quantification was noted for the MxA assay of this laboratory with one of two laboratories using the CPE assay. All other inter-laboratory comparisons showed kappa values between 0.57 and 0.68 and remarkable differences in individual titer levels. Conclusions There are considerable differences in the detection and quantification of IFNB-induced NAbs among laboratories offering NAb testing for clinical practice using different assay methods. It is important that these differences are considered when interpreting NAb results for clinical decision-making and when developing general recommendations for potentially clinically meaningful NAb titer levels.

Keller's systemic model of youth mentoring posits there are multiple pathways through which all stakeholders in the youth mentoring process, including the program staff who support the match (or case managers), influence youth outcomes. This study examines case managers' direct and indirect contributions to match outcomes and tests how transitive interactions facilitate a theorized sequence of mentoring interactions to effect greater closeness and length, specifically in nontargeted mentoring programs. A structural equations model of case manager contributions to match outcomes was tested using data from 758 mentor-mentee matches, supported by 73 case managers across seven mentoring agencies. Results reveal direct effects of mentor-reported match support quality on match length and indirect influences on match length through increasing youth-centeredness, goal-focused orientation, and closeness. The findings confirm the presence of multiple pathways of influence, including indirect effects on outcomes via transitive interactions in match support that scaffold youth-centeredness and goal-focused interactions in the match. Findings also suggest supervisors' evaluations of case managers may provide little information about how match support influences the nature of mentor-mentee interactions., (© 2023 Wiley Periodicals LLC.)

This paper describes a multi-phase effort to develop a web-based training for adults serving as mentors in school-based programs for youth with a parent in the military. In Phase 1, we conducted focus groups with military parents to: gauge their receptivity to this type of supportive intervention, identify program features that would make the option of mentoring for their children more or less appealing, and identify specific training needs for adult volunteers preparing for the role of mentor to youth in this population. In Phase 2, we used an iterative process to develop the training protocol, including cycling through multiple drafts, creating a web-based platform, reviewing and incorporating feedback from various stakeholders, and then pilot testing the training with two groups of mentor volunteers as part of a school-based mentoring program for military-connected students. We report on what we learned from the military parent focus groups, including parent skepticism about the need for such a program, concerns about potential stigma, and the need for mentors to have some understanding of military culture. We describe how we used that information to develop a practical and accessible training module for volunteer mentors, especially those without a military background, who could be matched with military-connected youth.

Background: Urothelial carcinoma (UC) is the fifth most common cancer in the developed world. Delineation of differentiation subtypes in UC highlighted the importance of aberrant differentiation. Understanding underlying mechanisms may facilitate diagnosis and development of efficient therapy strategies. It is well accepted that epigenetic mechanisms are involved. Long noncoding RNAs (lncRNAs), a new class of epigenetic factors, are thought to mediate molecular differences between cell types to control cellular identity. The present study focuses on the lncRNA HOTAIR, originating from the HOXC locus. Its overexpression induces an aggressive phenotype in many cancers and aberrant expression of homeotic HOX transcription factors, especially HOXD10, that regulate differentiation and tissue homeostasis. The aim of the present study was to determine the functional role of HOTAIR in UC with regard to aggressive phenotype, regulation of aberrant differentiation and altered HOX gene expression., Methods: We determined RNA expression levels of HOTAIR and HOX genes in UC tissues and cell lines. Knockdown of HOTAIR and ectopic overexpression was performed to determine the effect on reported target genes in UC. Cell lines were stably transfected with HOTAIR to investigate changes in phenotype and HOX gene expression., Results: HOTAIR was overexpressed in approximately half of UC tissues and cell lines. Effects of HOTAIR overexpression differed between cell lines. Whereas VM-CUB1 cells acquired the expected phenotype with increased proliferation, clonogenicity, anchorage independent growth, migratory activity and epithelial-to-mesenchymal transition, 5637 cells grew more slowly displaying induction of senescence and related immune response genes. Other UC lines showed intermediate effects. Expression profiling revealed divergent effects on HOX genes, cell cycle regulators and differentiation according with the phenotypic differences between HOTAIR-overexpressing VM-CUB1 and 5637 cells., Conclusions: Our data indicate that HOTAIR overexpression may affect differentiation state and aggressiveness of UC cells, but in a cell-type dependent manner. Our functional studies and the comparison of our expression data sets with those from other cancer cell types, which revealed minimal overlaps, indicate that effects of HOTAIR are strongly tissue-dependent and can even differ within one cancer type. Thus, HOTAIR functions and target genes cannot simply be transferred from one cancer type to the other.

To simulate the cardiac niche, a bioreactor system was designed and constructed to incorporate cyclic stretch, rhythmic electrical stimulation, and constant perfusion. The homogeneity of surface strain distribution across the cell culture substrate was confirmed with ARAMIS deformation analysis. The proliferation marker, Ki-67, detected in human umbilical vein endothelial cells and 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide cytotoxicity assay performed on human atrial fibroblasts confirmed biocompatibility of this novel device. Cyclic stretch treatment for 24 h resulted in the perpendicular alignment of human atrial fibroblasts. An electrical stimulation system containing carbon electrodes was characterized by electrochemical impedance spectroscopy and charge injection/recovery studies, which indicated that increased corrosive reactions were associated with a higher input voltage and prolonged pulse duration. Field stimulation delivered through this system could induce rhythmic contractions in adult rat ventricular myocytes, with contractile characteristics similar to those paced in a standard field stimulation chamber. In conclusion, this bioreactor provides a novel tool to study the interaction between physical stimulation and cardiac cell physiology.

Previous investigations had suggested that signaling from the overexpressed beta(2) adrenergic in the heart of transgenic TG4 mice was dampened in the atria. Using an RT-PCR based strategy, we have identified Regulator of G-protein Signaling 5 (RGS5) as being up-regulated in the atria of TG4 mice. Northern blot analysis demonstrated that RGS5 levels were 3 fold higher in the atria of TG4 mice. Western blot analysis of a panel of rat tissues demonstrated that basal expression of RGS5 protein was confined to the heart and skeletal muscle. Furthermore, RGS5 protein was detected in skeletal muscle C2C12 and cardiomyocyte HL-1 cultured cell lines. As observed for RGS5 mRNA levels in TG4 mice, RGS5 protein levels were increased in the atria of rats that were administered the beta adrenergic agonist isoproterenol during a 14 day period. Taken together, these results indicate that RGS5 is a housekeeping RGS in the heart and in skeletal muscle while its beta adrenergic-mediated induction in the atrium suggests that it also has a highly specialized function.

Recent progress in implantations of differentiated cardiac and non-cardiac cells as well as adult stem cells into the heart suggests that the irreversible loss of viable cardiac myocytes that occurs during myocardial infarction can be at least partly substituted. We evaluated an alternative approach by reconstituting cardiac tissue grafts in vitro and implanting them as spontaneously and coherently contracting tissues. For this purpose we have optimized a method to generate ring-shaped three-dimensional engineered heart tissue (EHT) in vitro from neonatal rat cardiac myocytes. When subjected to isometric force measurements in organ baths, electrically stimulated EHTs exhibit a Frank-Starling behavior, a positive inotropic response to increases in extracellular calcium, a positive inotropic and lusitropic response to isoprenaline, and a negative inotropic response to the muscarinic agonist carbachol ('accentuated antagonism'). Twitch tension under maximal calcium amounts to 1-2 mN/ mm2. Importantly, passive (resting) tension is low, yielding a ratio of active/passive tension of approximately 1.5 under basal and 14 under maximal calcium. Morphologically, EHTs represent a highly interconnected three-dimensional network of cardiac myocytes resembling loose cardiac tissue with a high fraction of binucleated cardiac myocytes, strong eosin staining and elongated centrally located nuclei. Electron microscopy demonstrated well developed sarcomeric structures, T-tubules, SR vesicles, T-tubule-SR-junctions, all types of intercellular connective structures, and a basement membrane. Thus, EHTs comprise functional and morphological properties of intact, ventricular myocardium. First implantation experiments of EHTs in the peritoneum of Fischer 344 rats showed that EHTs survived for at least 14 days, maintained a network of differentiated cardiac myocytes, and were strongly vascularized. Thus, EHTs may serve as material for a novel tissue replacement approach.

In transgenic mice (TG4) overexpressing the human beta2-adrenoceptor (beta2-AR), unoccupied receptors are supposed to activate spontaneously the signalling cascade, leading to enhanced levels of cAMP. This second messenger shifts activation curves of the hyperpolarization-activated current If towards less negative potentials. Here, we characterize If of ventricular myocytes from non-transgenic littermate (LM) and TG4 mice and investigate whether If is modulated by spontaneous beta2-AR signalling. If was activated in whole-cell voltage-clamp experiments during test steps ranging from -65 mV to -135 mV (holding potential: -55 mV; 36 degrees C). In TG4 the maximum amplitude was fivefold larger than in LM myocytes (-1.10 +/- 0.11 pA/pF vs. -0.22 +/- 0.04 pA/pF at -135 mV), and the potential for half-maximum If current (VI0.5) was less negative (-100.5 +/- 1.0 mV in TG4 vs. -108.4 +/- 2.6 mV in LM). (-)-Isoproterenol (1 microM) shifted VI0.5 of LM myocytes by 10.4 mV towards less negative potentials but had no significant effect in TG4. However, the inverse beta2-AR agonist ICI 118,551 (300 nM) shifted VI0.5 of TG4 myocytes to values observed in LM under control conditions, suggesting a relation to spontaneously active beta2-ARs. Enhanced expression of hyperpolarization-activated and cyclic nucleotide gated channels (HCN) could contribute to increased maximum If amplitude in TG4 myocytes. Semi-quantitative RT-PCR analysis demonstrated a 1.8-fold elevation of HCN4 mRNA and no significant change for HCN2 mRNA in TG4 ventricle. Cardiac hypertrophy was not detected in TG4 mice investigated here. We conclude that spontaneous beta2-AR signalling in hearts of TG4 mice shifts If current-voltage relation towards less negative potentials. Increased maximum If amplitude in TG4 myocytes is in line with enhanced expression of HCN channels. Both mechanisms could contribute to larger inward current at physiological diastolic potentials.

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

The atria and ventricles of transgenic mice (TGbeta(2)) with cardiac overexpression of the human beta(2)-adrenoceptor (beta(2)AR) were initially reported to show maximum contractility in the absence of beta-AR stimulation. However, we have previously observed a different phenotype in these mice, with myocytes showing normal contractility but reduced betaAR responses. We have investigated the roles of cyclic AMP and Gi in basal and betaAR function in these myocytes. ICI 118,551 at inverse agonist concentrations decreased contraction by 32%. However, the cyclic AMP antagonist Rp-cAMPS had no effect on contraction in TGbeta(2) myocytes, indicating that there was no tonic influence of raised cyclic AMP. These findings cannot be explained by the proposed model for inverse agonism, where the activated receptor (R*) raises cyclic AMP levels and so increases contraction in the absence of agonist. After pertussis toxin (PTX) pretreatment to produce inactivation of Gi, the basal contraction in 1 mM Ca(2+) was increased in TGbeta(2) mice (7.82+/-0.47%, n=23) compared to LM mice (3.60+/-0.59%, n=11) (P<0.001). The contraction amplitude of myocytes to the maximal concentration of isoprenaline was also increased significantly by PTX in TGbeta(2) mice (9.40+/-1.22%, n=8) and was no longer reduced compared to LM mice (8.93+/-1.50%, n=11). Both beta(1)- and beta(2)AR subtypes were affected both by the original desensitization and by the resensitization with PTX. PTX treatment has therefore restored the original phenotype, with high basal contractility and little further effect of isoprenaline. We suggest that both beta-AR desensitization and lack of increased basal contraction in ventricular myocytes from our colony of TGbeta(2) mice were due to increased activity of PTX-sensitive G-proteins.

Under Na(+)-free conditions, low-voltage-activated Ca(2+) currents in cardiomyocytes from various species have been described either as Ni(2+)-sensitive T-type Ca(2+) current (I(Ca(T))) or as tetrodotoxin (TTX)-sensitive Ca(2+) current (I(Ca(TTX))). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which I(Ca(T)) and I(Ca(TTX)) can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na(+)-free solutions. Whereas rat myocytes lack I(Ca(T)) and exhibit I(Ca(TTX)) only, guinea pig myocytes possess both of these low-voltage-activated Ca(2+) currents, which are separated pharmacologically by superfusion with TTX or Ni(2+). I(Ca(T)) and I(Ca(TTX)) were of similar amplitude but significantly differed in their electrophysiological properties: I(Ca(TTX)) activated at more negative potentials than did I(Ca(T)), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. I(Ca(TTX)) but not I(Ca(T)) increased after membrane rupture ("run-up"). Isolation of I(Ca(TTX)) by application of the bivalent cation Ni(2+) is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca(2+) channel blocker mibefradil (10 micromol/L) is a suitable tool for the study of I(Ca(TTX)). However, mibefradil not only blocked I(Ca(T)) by 85+/-2% but also decreased I(Ca(TTX)) by 48+/-8%. We conclude that under Na(+)-free conditions I(Ca(T)) and I(Ca(TTX)) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of I(Ca(T)) will have to consider the TTX-sensitive current component to avoid possible interference.

Putative beta4-adrenoceptors mediate cardiostimulation and arrhythmias in mammalian heart. Both beta1- and putative beta4-adrenoceptors mediate arrhythmias but through different mechanisms. To elucidate further the mechanisms of cardiostimulation and arrhythmias we measured Ca2+ transients and L-type Ca2+ currents in mouse ventricular myocytes. We used (-)-CGP 12177, an antagonist of beta1- and beta2-adrenoceptors with agonist properties at the putative beta4-adrenoceptor, and (-)-isoprenaline as an agonist for beta1- and beta2-adrenoceptors. (-)-CGP 12177 increased Ca2+ transients in electrically stimulated cells loaded with Indo-1. The maximum increase of Ca2+ transients caused by (-)-CGP 12177 amounted to approximately one-third of that caused by maximally effective (-)-isoprenaline concentrations. Both (-)-CGP 12177 and (-)-isoprenaline caused concentration-dependent arrhythmic Ca2+ transients. The arrhythmias appeared at paced Ca2+ transients and between paced Ca2+ transients. The arrhythmic potency of (-)-CGP 12177 (-logEC50=9.4) was approximately 40 times greater than that of (-)-isoprenaline (-logEC50=7.8). L-type Ca2+ current was measured in the whole cell configuration of the patch clamp technique. In the presence of both 3-isobutyl 1-methylxanthine (6 micromol/l) and (-)-propranolol (500 nmol/l), (-)-CGP 12177 (100 nmol/l) increased significantly L-type Ca2+ current by 19% of the effect of (-)-isoprenaline. The (-)-CGP 12177-evoked increase of Ca2+ transients contrasts with the smaller effects on L-type Ca2+ current, suggesting that activation of the putative beta4-adrenoceptor causes a more efficient Ca2+-induced Ca2+ release than activation of the beta1-adrenoceptor. Beta4-Adrenoceptors mediate arrhythmias with smaller Ca2+ transients and smaller increases of L-type Ca2+ current than beta1-adrenoceptors, in line with different but still unknown mechanisms as previously suggested for the intact heart.

Objectives: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human beta 2-adrenoceptor (beta 2-AR) was explained by spontaneously active beta 2-ARs that stimulate the beta-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls., Methods: Cardiac beta-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria., Results: Overexpression of beta 2-ARs was confirmed by an almost 400-fold increase in beta-AR density. The beta 1:beta 2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o -4.82 +/- 0.48 vs. -6.56 +/- 0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 microM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice., Conclusions: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating beta 2-ARs in left atrium and ventricle. A contractile deficit unrelated to the beta-adrenoceptor pathway is evident in TG4 myocytes and left atria.

The antiarrhythmic action of 6-benzoylheteratisine (6-bh), a C(19) diterpenoid alkaloid from Aconitum tanguticum (Maxim.) Stapf was investigated in left and right guinea pig isolated atria. Furthermore, possible effects on transmembrane action potential of isolated papillary muscles were studied using microelectrode techniques. At concentrations of more than 6 × 10(-8) mol/1, preincubation with 6-bh suppressed arrhythmias induced by aconitine, veratridine and ouabain. Bradycardia of the right atria as a sign of toxicity occurred at 1 × 10(-6) mol/1. The alkaloid significantly reduced the maximum rate of rise of the action potential as well as the action potential amplitude, indicating inhibition of voltage-dependent sodium channels as a functional principle. Additionally, a use-dependent mode of drug-action could be demonstrated. We conclude therefore, that 6-bh is a naturally occurring class-I antiarrhythmic substance. The compound is a main alkaloid of Aconitum tanguticum, a plant used to prepare a poison antidote in Chinese and Tibetan folk medicine. It may be speculated that the poison antidote effect is at least partially based on the antiarrhythmic properties of 6-bh., (Copyright © 1997 Gustav Fischer Verlag. Published by Elsevier GmbH.. All rights reserved.)

Several regions within the 35-kDa COOH-terminal portion of caldesmon have been implicated in the ability of caldesmon to inhibit actin-activated myosin ATPase activity. To further define the functional regions of caldesmon, we have studied the effects of three chymotryptic fragments, one fragment produced by CNBr digestion and two fragments produced by digestion with submaxillaris arginase C protease, on the relaxed stiffness and active force of rabbit psoas fibers. Each of the regions of caldesmon studied had either direct or indirect effects on single-fiber mechanics. The 35-kDa and 20-kDa fragments of caldesmon, like intact caldesmon, were effective inhibitors of fiber stiffness, a measure of cross-bridge attachment. The 7.3-kDa and 10-kDa fragments, which constitute the NH2 and COOH halves of the 20-kDa fragment, inhibited both relaxed fiber stiffness and active force production, but with a reduced efficacy compared to the 20-kDa fragment. These results suggest that several regions within the 35-kDa COOH-terminal region of caldesmon are required for optimum function of caldesmon and that function includes inhibition of weak cross-bridge attachment and force production.

The physical environment can be an important contributor to occupational stress. Factors that contribute to stress and other negative outcomes include: lack of control over the environment, distractions from coworkers, lack of privacy, noise, crowding, and environmental deprivations (such as lack of windows and aesthetic impoverishment). The design of "salutogenic" environments requires not only the elimination of negative stress inducing features, but also the addition of environmental enhancements, including such factors as increased personal control, contact with nature and daylight, aesthetically pleasing spaces, and spaces for relaxation alone or with others. Salutogenic environments also take into consideration positive psychosocial "fit," as well as functional fit between people and environments. At the heart of the current interest in the work environment are two major concerns: organizational productivity and employee well being.