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We present a parameter sensitivity analysis method that is appropriate for stochastic models, and we demonstrate how this analysis generates experimentally testable predictions about the factors that influence local Ca(2+) release in heart cells. The method involves randomly varying all parameters, running a single simulation with each set of parameters, running simulations with hundreds of model variants, then statistically relating the parameters to the simulation results using regression methods. We tested this method on a stochastic model, containing 18 parameters, of the cardiac Ca(2+) spark. Results show that multivariable linear regression can successfully relate parameters to continuous model outputs such as Ca(2+) spark amplitude and duration, and multivariable logistic regression can provide insight into how parameters affect Ca(2+) spark triggering (a probabilistic process that is all-or-none in a single simulation). Benchmark studies demonstrate that this method is less computationally intensive than standard methods by a factor of 16. Importantly, predictions were tested experimentally by measuring Ca(2+) sparks in mice with knockout of the sarcoplasmic reticulum protein triadin. These mice exhibit multiple changes in Ca(2+) release unit structures, and the regression model both accurately predicts changes in Ca(2+) spark amplitude (30% decrease in model, 29% decrease in experiments) and provides an intuitive and quantitative understanding of how much each alteration contributes to the result. This approach is therefore an effective, efficient, and predictive method for analyzing stochastic mathematical models to gain biological insight.

BackgroundAlthough rotors have been considered among the drivers of atrial fibrillation (AF), the rotor definition is inconsistent. We evaluated the nature of rotors in 2D and 3D in- silico models of persistent AF (PeAF) by analyzing phase singularity (PS), dominant frequency (DF), Shannon entropy (ShEn), and complex fractionated atrial electrogram cycle length (CFAE-CL) and their ablation.MethodsMother rotor was spatiotemporally defined as stationary reentries with a meandering tip remaining within half the wavelength and lasting longer than 5 s. We generated 2D- and 3D-maps of the PS, DF, ShEn, and CFAE-CL during AF. The spatial correlations and ablation outcomes targeting each parameter were analyzed.Results1. In the 2D PeAF model, we observed a mother rotor that matched relatively well with DF (>9 Hz, 71.0%, p<0.001), ShEn (upper 2.5%, 33.2%, p<0.001), and CFAE-CL (lower 2.5%, 23.7%, p<0.001). 2. The 3D-PeAF model also showed mother rotors that had spatial correlations with DF (>5.5 Hz, 39.7%, p<0.001), ShEn (upper 8.5%, 15.1%, p <0.001), and CFAE (lower 8.5%, 8.0%, p = 0.002). 3. In both the 2D and 3D models, virtual ablation targeting the upper 5% of the DF terminated AF within 20 s, but not the ablations based on long-lasting PS, high ShEn area, or lower CFAE-CL area.ConclusionMother rotors were observed in both 2D and 3D human AF models. Rotor locations were well represented by DF, and their virtual ablation altered wave dynamics and terminated AF.

Adoptive transfer of regulatory T (Treg) cells could be an alternative to chronic immunosuppression for prevention of allogeneic graft rejection. While polyspecific Treg cells can prevent immune responses under lymphopenic conditions, Ag-specific Treg cells are needed to treat autoimmunity and graft rejection. Yet, reliable markers for Ag-specific Treg cells are missing. We report that latency-associated peptide (LAP) and glycoprotein A repetitions predominant (GARP) can identify human Ag-specific Treg cells. In addition,we show that the depletion of CD154 + cells from LAP + or GARP + Treg cells increases the Treg-cell purity to over 90%, as assessed by epigenetic analysis. These Ag-specific Treg cells can be isolated magnetically and might contribute to the development of GMP-based protocols. In addition, Ag-specific Treg cells are functionally far superior to CD4 + CD25 high or CD4 + CD25 high CD127 low Treg cells in vitro and in preventing strong alloreactions in humanized mice. They could, therefore, have a high therapeutic potential for the control of alloimmune, autoimmune, and allergic immune responses in patients.Keywords: Antigen-specific r CD154 r GARP r LAP r Regulatory T (Treg) cells Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionIn organ transplantation, acute rejection can be prevented by a combination of nonspecific immunosuppressive drugs and immunomodulatory induction therapies, yet chronic rejection and chronic graft dysfunction remain a clinical problem [1]. In addition, chronic nonspecific immunosuppression causes renal dysfunction and predisposes patients to infections and increased Correspondence: Dr. Elmar Jaeckel e-mail: Jaeckel.Elmar@mh-hannover.de risk of malignancies [1]. Innate and adaptive immune responses can be controlled by Treg cells, thereby offering new therapeutic options after organ transplantation [2][3][4][5][6]. CD4 + CD25 high FOXP3 + Treg cells have been shown to be of major importance in allospecific tolerance in animal models and to influence donor-specific immune responses in transplant patients [7][8][9][10]. After allogeneic hematopoietic stem cell transplantation, polyspecific Treg cells can prevent GVH disease without influencing * These authors contributed equally to this work.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 2592-2602 Cellular immune response 2593 graft-versus-leukemia responses [11,12]. Therefore, polyspecific CD4 + CD25 high cells are currently being evaluated after stem cell transplantation [13]. However, under nonlymphopenic conditions as seen in patients with autoimmune diseases or in patients after organ transplantation, polyspecific Treg cells have so far been largely ineffective in controlling immune responses [5,14,15] [24], and CD137 (4-1BB) [25] were described as Treg-cell activation markers after polyspecific Treg-cell activation.In this manuscript, we demonstrated for the first time that LAP and GARP can be use...

Background: Magnesium (Mg)-based alloys are considered to be promising materials for implant application due to their excellent biocompatibility, biodegradability, and mechanical properties close to bone. However, low corrosion resistance and fast degradation are limiting their application. Mg-Ca alloys have huge potential owing to a similar density to bone, good corrosion resistance, and as Mg is essential for Ca incorporation into bone. The objective of the present work is to determine the in vitro degradation and in vivo performance of binary Mg-xCa alloy (x = 0.5 or 5.0 wt%) to assess its usability for degradable implant applications. Methods: Microstructural evolutions for Mg-xCa alloys were characterized by optical, SEM, EDX, and XRD. In vitro degradation tests were conducted via immersion test in phosphate buffer saline solution. In vivo performance in terms of interface, biocompatibility, and biodegradability of Mg-xCa alloys was examined by implanting samples into rabbit femoral condyle for 2 and 4 weeks. Results: Microstructural results showed the enhancement in intermetallic Mg 2 Ca phase with increase in Ca content. Immersion tests revealed that the dissolution rate varies linearly, with Ca content exhibiting more hydrogen gas evolution, increased pH, and higher degradation for Mg-5.0Ca alloy. In vivo studies showed good biocompatibility with enhanced bone formation for Mg-0.5Ca after 4 weeks of implantation compared with Mg-5.0Ca alloy. Higher initial corrosion rate with prolonged inflammation and rapid degradation was noticed in Mg-5.0Ca compared with Mg-0.5Ca alloy. Conclusions: The results suggest that Mg-0.5Ca alloy could be used as a temporary biodegradable implant material for clinical applications owing to its controlled in vivo degradation, reduced inflammation, and high bone-formation capability.