Dynamic modeling and simulation of leukocyte integrin activation through an electronic design automation framework

Model development and analysis of biological systems is recognized as a key requirement for integrating in-vitro and in-vivo experimental data. In-silico simulations of a biochemical model allows one to test different experimental conditions, helping in the discovery of the dynamics that regulate the system. Several characteristics and issues of biological system modeling are common to the electronics system modeling, such as concurrency, reactivity, abstraction levels, as well as state space explosion during verification. This paper proposes a modeling and simulation framework for discrete event-based execution of biochemical systems based on SystemC. SystemC is the reference language in the electronic design automation (EDA) field for modeling and verifying complex systems at different abstraction levels. SystemC-based verification is the de-facto an alternative to model checking when such a formal verification technique cannot deal with the state space complexity of the model. The paper presents how the framework has been applied to model the intracellular signalling network controlling integrin activation mediating leukocyte recruitment from the blood into the tissues, by handling the solution space complexity through different levels of simulation accuracy

Urokinase Plasminogen Activator Inhibits HIV Virion Release from Macrophage-Differentiated Chronically Infected Cells via Activation of RhoA and PKCε

HIV replication in mononuclear phagocytes is a multi-step process regulated by viral and cellular proteins with the peculiar feature of virion budding and accumulation in intra-cytoplasmic vesicles. Interaction of urokinase-type plasminogen activator (uPA) with its cell surface receptor (uPAR) has been shown to favor virion accumulation in such sub-cellular compartment in primary monocyte-derived macrophages and chronically infected promonocytic U1 cells differentiated into macrophage-like cells by stimulation with phorbol myristate acetate (PMA). By adopting this latter model system, we have here investigated which intracellular signaling pathways were triggered by uPA/uPAR interaction leading the redirection of virion accumulation in intra-cytoplasmic vesicles.uPA induced activation of RhoA, PKCδ and PKCε in PMA-differentiated U1 cells. In the same conditions, RhoA, PKCδ and PKCε modulated uPA-induced cell adhesion and polarization, whereas only RhoA and PKCε were also responsible for the redirection of virions in intracellular vesicles. Distribution of G and F actin revealed that uPA reorganized the cytoskeleton in both adherent and polarized cells. The role of G and F actin isoforms was unveiled by the use of cytochalasin D, a cell-permeable fungal toxin that prevents F actin polymerization. Receptor-independent cytoskeleton remodeling by Cytochalasin D resulted in cell adhesion, polarization and intracellular accumulation of HIV virions similar to the effects gained with uPA.These findings illustrate the potential contribution of the uPA/uPAR system in the generation and/or maintenance of intra-cytoplasmic vesicles that actively accumulate virions, thus sustaining the presence of HIV reservoirs of macrophage origin. In addition, our observations also provide evidences that pathways controlling cytoskeleton remodeling and activation of PKCε bear relevance for the design of new antiviral strategies aimed at interfering with the partitioning of virion budding between intra-cytoplasmic vesicles and plasma membrane in infected human macrophages

Nanovesicles from adipose-derived mesenchymal stem cells inhibit T lymphocyte trafficking and ameliorate chronic experimental autoimmune encephalomyelitis

Cell based-therapies represent promising strategies for the treatment of neurological diseases. We have previously shown that adipose stem cells (ASC) ameliorate chronic experimental autoimmune encephalomyelitis (EAE). Recent evidence indicates that most ASC paracrine effects are mediated by extracellular vesicles, i.e. micro- and nanovesicles (MVs and NVs). We show that preventive intravenous administration of NVs isolated from ASC (ASC-NVs) before disease onset significantly reduces the severity of EAE and decreases spinal cord inflammation and demyelination, whereas therapeutic treatment with ASC-NVs does not ameliorate established EAE. This treatment marginally inhibits antigen-specific T cell activation, while reducing microglial activation and demyelination in the spinal cord. Importantly, ASC-NVs inhibited integrin-dependent adhesion of encephalitogenic T cells in vitro, with no effect on adhesion molecule expression. In addition, intravital microscopy showed that encephalitogenic T cells treated with ASC NVs display a significantly reduced rolling and firm adhesion in inflamed spinal cord vessels compared to untreated cells. Our results show that ASC-NVs ameliorate EAE pathogenesis mainly by inhibiting T cell extravasation in the inflamed CNS, suggesting that NVs may represent a novel therapeutic approach in neuro-inflammatory diseases, enabling the safe administration of ASC effector factors

Spatiotemporal organization and mechanosensory function of podosomes

Podosomes are small, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. They comprise a protrusive actin core module and an adhesive ring module composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Furthermore, podosomes are associated with an actin network and often organize into large clusters. Recent results from our laboratory and others have shed new light on podosome structure and dynamics, suggesting a revision of the classical "core-ring" model. Also, these studies demonstrate that the adhesive and protrusive module are functionally linked by the actin network likely facilitating mechanotransduction as well as providing feedback between these two modules. In this commentary, we briefly summarize these recent advances with respect to the knowledge on podosome structure and discuss force distribution mechanisms within podosomes and their emerging role in mechanotransduction

Substrate stiffness influences phenotype and function of human antigen-presenting dendritic cells

Contains fulltext : 181654.pdf (publisher's version ) (Open Access

Selective Rac1 mutants promote retina ganglion cells survival and regeneration by ERK1/2 and PAK activation after optic nerve injury.

In adult mammalian central nervous system (CNS) neurons fail to regenerate after injury and most of them die by apoptosis within few days. The neuronal pathways underlying such events are far from being fully understood. Rac1 is a rho-related small GTPase that regulates cytoskeletal dynamics critically involved in neuronal development, axon growth and cell survival. Its constitutively active (CA) mutant is able to promote the axonal growth through an inhibitory environment both in vitro and in vivo and also to promote cell survival in vitro. However Rac1 CA mutant simultaneously activates many downstream concurrent signaling pathways, because Rac1 can potentially bind several downstream effectors. Thus, in order to investigate the effect of Rac1 activation on neuronal survival and axonal regeneration after injury, we used two Rac1 double mutants in which a first L61 mutation leads to constitutive activation state whereas a second mutation (F37A or Y40C) prevents the specific interaction with some downstream effectors, conferring selectivity for the delivery of downstream signaling. A TAT Trojan nanovector sequence allows overstepping of the cell plasma membrane leading to accumulation into the cell. We injected these two mutants intravitreally and we investigated, by means of immunofluorescence and imaging techniques, the retina ganglion cell (RGCs) survival and the axonal regeneration at 15 and 30 days after optic nerve crush. These mutants have been previously characterized, but to our knowledge they have never been used in vivo in the CNS. Our results show that both Rac1-mutants were able to improve cell survival, however with different mechanisms: the F37A increased p21 activated kinase (PAK) and extracellular signal regulated kinases (ERK1/2) activation directly in RGCs, displaying a dose-dependent effect on survival, whereas the Y40C mutant increased the ERK1/2 phosphorylation selectively in Muller glial cells, giving an indirect effect on RGCs. Between the two mutants only the F37A was able to improve axonal regeneration until 15 days post-injury, suggesting that the manipulation of a single pathway is insufficient to obtain a massive regeneration. However, our data suggest also that long-term repetitive treatments could counteract the normal axonal degeneration occurring after crush. Our study clarifies the role of Rac1 as a pro-survival signaling molecule in neurons in vivo and supports the possibility to use Trojan nanovector-based approaches in the treatment of neuronal damages

PLD-dependent phosphatidic acid microdomains are signaling platforms for podosome formation

Contains fulltext : 202101.pdf (publisher's version ) (Open Access