Determination of biomechanical and architectural muscle properties : from single muscle fibre to whole muscle mechanics

The work presented in this thesis aims to provide a more detailed insight in the complex physiology of certain muscle tissue types. This thesis builds upon the results of in vitro contractile and ex vivo architectural experiments with muscle tissue preparations from rats (Rattus norvegicus), rabbits (Oryctolagus cuniculus) and pigs (Sus scrofa domesticus) - investigated by experimental and modelling approaches. During the course of this work the chapters are intended to determine, describe and interprete the distinct properties of muscle tissue samples of striated skeletal and smooth musculature. These species-specific properties have not been observed before, but are needed for modelling approaches and a better understanding of contractile mechanics and muscle growth. Despite the numerous studies on skeletal and smooth muscle tissue, there are still fundamental questions about the physiology and force generation of the muscle. Hence, the determination of specific biomechanical and architectural muscle properties allows a quantitative understanding of the mechanisms involved in force development. Moreover, this is a crucial step towards reliable, realistic muscle models and thus also to increased predictive quality of muscle-driven multi-body models.Die in dieser Thesis vorgestellte Arbeit ist dahingehend ausgerichtet, einen detaillierten Einblick in die komplexe Physiologie bestimmter Muskelgewebearten zu geben. Die Arbeit baut auf den Ergebnissen von kontraktilen in vitro und architektonischen ex vivo Experimenten mit Muskelgewebepräparaten von Ratten (Rattus norvegicus), Kaninchen (Oryctolagus cuniculus) und Schweinen (Sus scrofa domesticus) auf. Im Laufe dieser Arbeit werden die charakteristischen Eigenschaften von Muskelgewebeproben, sowohl von quergestreifter skelettaler als auch glatter Muskulatur, untersucht. Die erstmalige Erfassung dieser artspezifischen Eigenschaften wird sowohl für Modellierungsansätze als auch für ein besseres Verständnis der kontraktilen Mechanik und des Muskelwachstums benötigt. Trotz der zahlreichen Studien an quergestreifter Skelett- und glatter Muskulatur gibt es noch grundlegende Fragen zur Physiologie und Krafterzeugung des Muskels. Die Bestimmung spezifischer biomechanischer- und architektonischer Muskeleigenschaften erlaubt mitunter ein quantitatives Verständnis der an der Kraftentwicklung beteiligten Mechanismen. Des Weiteren stellt dies einen entscheidenden Schritt auf dem Weg zu verlässlichen, realistischen Muskelmodellen und damit auch zu einer verbesserten Vorhersagekraft von muskelgetriebenen Mehrkörpermodellen dar

Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel–deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel–deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4^+ and CD8^+ T cells from c-Rel–deficient NOD mice showed significantly decreased T-cell receptor–induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel–competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel–deficient NOD mice. The results suggest that c-Rel–dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel–dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell–dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity

Power amplification increases with contraction velocity during stretch-shortening cycles of skinned muscle fibers

Muscle force, work, and power output during concentric contractions (active muscle shortening) are increased immediately following an eccentric contraction (active muscle lengthening). This increase in performance is known as the stretch-shortening cycle (SSC)-effect. Recent findings demonstrate that the SSC-effect is present in the sarcomere itself. More recently, it has been suggested that cross-bridge (XB) kinetics and non-cross-bridge (non-XB) structures (e.g., titin and nebulin) contribute to the SSC-effect. As XBs and non-XB structures are characterized by a velocity dependence, we investigated the impact of stretch-shortening velocity on the SSC-effect. Accordingly, we performed in vitro isovelocity ramp experiments with varying ramp velocities (30, 60, and 85% of maximum contraction velocity for both stretch and shortening) and constant stretch-shortening magnitudes (17% of the optimum sarcomere length) using single skinned fibers of rat soleus muscles. The different contributions of XB and non-XB structures to force production were identified using the XB-inhibitor Blebbistatin. We show that (i) the SSC-effect is velocity-dependent - since the power output increases with increasing SSC-velocity. (ii) The energy recovery (ratio of elastic energy storage and release in the SSC) is higher in the Blebbistatin condition compared with the control condition. The stored and released energy in the Blebbistatin condition can be explained by the viscoelastic properties of the non-XB structure titin. Consequently, our experimental findings suggest that the energy stored in titin during the eccentric phase contributes to the SSC-effect in a velocity-dependent manner

In vitro Evidence That Combination Therapy With CD16-Bearing NK-92 Cells and FDA-Approved Alefacept Can Selectively Target the Latent HIV Reservoir in CD4+ CD2hi Memory T Cells

Elimination of the latent HIV reservoir remains the biggest hurdle to achieve HIV cure. In order to specifically eliminate HIV infected cells they must be distinguishable from uninfected cells. CD2 was recently identified as a potential marker enriched in the HIV-1 reservoir on CD4+ T cells, the largest, longest-lived and best-characterized constituent of the HIV reservoir. We previously proposed to repurpose FDA-approved alefacept, a humanized α-CD2 fusion protein, to reduce the HIV reservoir in CD2hi CD4+ memory T cells. Here, we show the first evidence that alefacept can specifically target and reduce CD2hi HIV infected cells in vitro. We explore a variety of natural killer (NK) cells as mediators of antibody-dependent cell-mediated cytotoxicity (ADCC) including primary NK cells, expanded NK cells as well as the CD16 transduced NK-92 cell line which is currently under study in clinical trials as a treatment for cancer. We demonstrate that CD16.NK-92 has a natural preference to kill CD2hi CD45RA– memory T cells, specifically CD45RA– CD27+ central memory/transitional memory (TCM/TM) subset in both healthy and HIV+ patient samples as well as to reduce HIV DNA from HIV+ samples from donors well controlled on antiretroviral therapy. Lastly, alefacept can combine with CD16.NK-92 to decrease HIV DNA in some patient samples and thus may yield value as part of a strategy toward sustained HIV remission

Locational and Directional Dependencies of Smooth Muscle Properties in Pig Urinary Bladder

The urinary bladder is a distensible hollow muscular organ, which allows huge changes in size during absorption, storage and micturition. Pathological alterations of biomechanical properties can lead to bladder dysfunction and loss in quality of life. To understand and treat bladder diseases, the mechanisms of the healthy urinary bladder need to be determined. Thus, a series of studies focused on the detrusor muscle, a layer of urinary bladder made of smooth muscle fibers arranged in longitudinal and circumferential orientation. However, little is known about whether its active muscle properties differ depending on location and direction. This study aimed to investigate the porcine bladder for heterogeneous (six different locations) and anisotropic (longitudinal vs. circumferential) contractile properties including the force-length-(FLR) and force-velocity-relationship (FVR). Therefore, smooth muscle tissue strips with longitudinal and circumferential direction have been prepared from different bladder locations (apex dorsal, apex ventral, body dorsal, body ventral, trigone dorsal, trigone ventral). FLR and FVR have been determined by a series of isometric and isotonic contractions. Additionally, histological analyses were conducted to determine smooth muscle content and fiber orientation. Mechanical and histological examinations were carried out on 94 and 36 samples, respectively. The results showed that maximum active stress (pact) of the bladder strips was higher in the longitudinal compared to the circumferential direction. This is in line with our histological investigation showing a higher smooth muscle content in the bladder strips in the longitudinal direction. However, normalization of maximum strip force by the cross-sectional area (CSA) of smooth muscle fibers yielded similar smooth muscle maximum stresses (165.4 ± 29.6 kPa), independent of strip direction. Active muscle properties (FLR, FVR) showed no locational differences. The trigone exhibited higher passive stress (ppass) than the body. Moreover, the bladder exhibited greater ppass in the longitudinal than circumferential direction which might be attributed to its microstructure (more longitudinal arrangement of muscle fibers). This study provides a valuable dataset for the development of constitutive computational models of the healthy urinary bladder. These models are relevant from a medical standpoint, as they contribute to the basic understanding of the function of the bladder in health and disease

Deficiency of Nuclear Factor-κB c-Rel Accelerates the Development of Autoimmune Diabetes in NOD Mice

The nuclear factor-κB protein c-Rel plays a critical role in controlling autoimmunity. c-Rel–deficient mice are resistant to streptozotocin-induced diabetes, a drug-induced model of autoimmune diabetes. We generated c-Rel–deficient NOD mice to examine the role of c-Rel in the development of spontaneous autoimmune diabetes. We found that both CD4^+ and CD8^+ T cells from c-Rel–deficient NOD mice showed significantly decreased T-cell receptor–induced IL-2, IFN-γ, and GM-CSF expression. Despite compromised T-cell function, c-Rel deficiency dramatically accelerated insulitis and hyperglycemia in NOD mice along with a substantial reduction in T-regulatory (Treg) cell numbers. Supplementation of isogenic c-Rel–competent Treg cells from prediabetic NOD mice reversed the accelerated diabetes development in c-Rel–deficient NOD mice. The results suggest that c-Rel–dependent Treg cell function is critical in suppressing early-onset autoimmune diabetogenesis in NOD mice. This study provides a novel natural system to study autoimmune diabetes pathogenesis and reveals a previously unknown c-Rel–dependent mechanistic difference between chemically induced and spontaneous diabetogenesis. The study also reveals a unique protective role of c-Rel in autoimmune diabetes, which is distinct from other T-cell–dependent autoimmune diseases such as arthritis and experimental autoimmune encephalomyelitis, where c-Rel promotes autoimmunity

The need for evidence-based climate risk and adaptation assessments:Lessons learned from the AGRICA project

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Emerging concepts about NAIP/NLIRC4 inflammasomes

Neuronal apoptosis inhibitory protein (NAIP)/NOD-like receptor (NLR) containing a caspase activating and recruitment domain (CARD) 4 (NLRC4) inflammasome complexes are activated in response to proteins from virulent bacteria that reach the cell cytosol. Specific NAIP proteins bind to the agonists and then physically associate with NLRC4 to form an inflammasome complex able to recruit and activate pro-caspase-1. NAIP5 and NAIP6 sense flagellin, component of flagella from motile bacteria, whereas NAIP1 and NAIP2 detect needle and rod components from bacterial type III secretion systems, respectively. Active caspase-1 mediates the maturation and secretion of the pro-inflammatory cytokines, 11,113 and 11,18, and is responsible for the induction of pyroptosis, a pro-inflammatory form of cell death. in addition to these well-known effector mechanisms, novel roles have been described for NAIP/NLRC4 inflammasomes, such as phagosomal maturation, activation of inducible nitric oxide synthase, regulation of autophagy, secretion of inflammatory mediators, antibody production, activation of T cells, among others. These effector mechanisms mediated by NAIP/NLRC4 inflammasomes have been extensively studied in the context of resistance of infections and the potential of their agonists has been exploited in therapeutic strategies to non-infectious pathologies, such as tumor protection. Thus, this review will discuss current knowledge about the activation of NAIP/NLRC4 inflammasomes and their effector mechanisms.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCTVUniversidade Federal de São Paulo, Ctr Terapia Celulare & Mol CTC Mol, BR-04044010 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-04044010 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celulare & Mol CTC Mol, BR-04044010 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, BR-04044010 São Paulo, SP, BrazilFAPESP: 2013/16010-5Web of Scienc