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

Studio dei meccanismi trasduzione del segnale che controllano l'attivazione integrinica nei leucociti

Il reclutamento leucocitario \ue8 un processo finemente modulato e svolge un ruolo fondamentale nel controllo della risposta immune. Il processo avviene attraverso una sequenza di pi\uf9 tappe controllate sia da molecole d\u2019adesione sia da fattori attivanti. Il riconoscimento dell\u2019endotelio vascolare da parte dei leucociti \ue8 tradizionalmente descritto come una sucessione di almeno tre eventi mediati da distinte famiglie di proteine (Fig1){1}. Le Selectine controllano il contatto iniziale (tethering) e il rotolamento (rolling) dei globuli bianchi circolanti sui carboidrati presentati dall\u2019endotelio{2}. Il thetering consiste in una iniziale e transiente adesione dei leucociti sull\u2019endotelio vasale, su leucociti gi\ue0 adesi o su piastrine; successivamente, l\u2019adesione si fa pi\uf9 stabile ed i leucociti cominciamo a rotolare. Il lento movimento della fase di rolling permette ai leucociti di interagire con i chemoattrattanti esposti sulla membrana delle cellule endoteliale. Successivamente, i chemoattrattanti (come, ad esempio, le chemochine) inducono un segnale intracellulare, attraverso recettori a sette domini transmembrana accoppiati a proteina trimeriche G (GPCRs), che causa l\u2019aumento dell\u2019avidit\ue0 integrinica (e quindi dell\u2019adesivit\ue0 dei leucociti) per il controligando endoteliale appartenente alla famiglia delle proteine con domini immunoglobulinici. Quest\u2019ultimo evento, che promuove l\u2019adesione stabile (firm adhesion) e la migrazione transendoteliale dei leucociti, \ue8 ora conosciuto come segnale \u201cinside-out\u201d{3}. Partendo da questo semplice schema, recentemente sono stati fatti numerosi progressi nella comprensione e nella revisione del fenomeno. Inanzitutto, sono state meglio caratterizzate le fasi di rolling e di tethering durante le quali si sono viste partecipare, oltre alle Selectine, anche le integrine. Cos\uec, il dogma secondo cui il rolling doveva essere un processo indipendente dell\u2019attivazione (integrinica) \ue8 stato inficiato dalla scoperta che le integrine \u3b14 (e in certe condizioni anche le \u3b22) potevano supportare il rolling prima dell\u2019attivazione indotta da chemochine {4,5}.Il rolling quindi \ue8 stato nuovamente distinto in lento e veloce includento la possibilit\ue0 che segnali intracellulari attivati da Selectine, e non solo da chemochine, possano giocare un ruolo nell\u2019attivazione integrinica sotto flusso{6}. Queste osservazioni portarono a pensare che, durante il rolling, i segnali attivati da Selectine, 6 sebbene non in grado di attivare completamente le integrine, partecipino alla loro \u201cpreattivazione\u201d (priming) per la sucessiva totale attivazione indotta da chemochine. Inoltre, negli ultimi anni \ue8 stata definitivamente identificata la modalit\ue0 di attivazione integrinica indotta da chemochine e responsabile dell\u2019arresto sotto flusso dei leucociti e sono stati ottenuti significativi avanzamenti nella comprensione degli eventi intracellulari che controllano l\u2019intero processo. Dal momento in cui Selectine, Integrine, chemoattrattanti e i loro recettori sono stati identificati e si sono trovati possedere profili di espressione leucocita-specifici, si \ue8 sviluppato il concetto di \u201carea-code\u201d (letteralmente \u201ccodice postale\u201d) tessuto-specifico {7-9}. In tale modello, le Selectine, le chemochine e le integrine generano una grande diversit\ue0 combinatoriale in base alle diverse coppie di selettina-carboidrato, chemochinarecettore e integrina-ligando imunoglobulinico presentate rispettivamente sul leucocita o sulla cellula endoteliale. Il leucocita per migrare in un certo tessuto o organo deve riconoscere il \u201ccodice\u201d espresso dal distretto vascolare del tessuto stesso. In assenza di \u201ccodice\u201d il leucocita rimane nel circolo sanguigno. Un leucocita che \ue8 in grado di rotolare e di aderire, ma non di migrare, non si accumula nel tessuto e torna nel circolo sanguigno senza svolgere la propria funzione. La superficie del vaso sanguigno \ue8 campionata in cerca dei corretti elementi che compongano un giusto codice. Se il codice \ue8 giusto il leucocita completer\ue0 la sequenza e migrer\ue0 nel tessuto, altrimenti ritorner\ue0 nel circolo. Queste scoperte hanno fornito di recente spunto per importanti applicazioni in campo biomedico. Negli organi linfoidi, le molecole d\u2019adesione PNAd e MAdCAM-1, insieme con i loro ligandi linfocitari e la coppia chemochina-recettore, creano un \u201ccodice\u201d specifico per la migrazione dei linfociti nativi (na\uefve) {2}. In ogni caso, ad oggi, nessun \u201ccodice assoluto\u201d di molecole specifiche per la migrazione in un esclusivo sito di migrazione \ue8 mai stato caratterizzato. Ad esempio, le mucine, Selectine, ed integrine come VLA-4 e LFA-1, sono state viste essere implicate nella migrazione di leucociti in diversi organi infiammati. Lo steso dicasi per le chemochine. Di fatto, esiste un considerevole livello di ridondanza e sovrapposizione, il che suggerisce che altri meccanimsi rendano il processo coerente e non abiguo. In realta\u2019 dati recenti suggeriscono che paramteri quantitativi come le caratteristiche emodinamiche del vaso sanguigno (cio\ue8 flusso lento contro flusso veloce), il livello di densit\ue0 (alto o basso) di molecole d\u2019adesione, l\u2019espressione di recettori per chemochine e, in fine, il momento di inizio del processo infiammatorio (presto Vs ritardato) possono specificatamente selezionare una sottopopolazione linfocitaria rispetto ad un\u2019altra durante l\u2019intero processo infiammatorio.Non disponibil

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

Comparative analysis of normal versus CLL B-lymphocytes reveals patient-specific variability in signaling mechanisms controlling LFA-1 activation by chemokines

Activation of lymphocyte function-associated antigen-1 (LFA-1) by chemokines is fine-tuned by inside-out signaling mechanisms responsible for integrin-mediated adhesion modulation. In the present study, we investigated the possibility of qualitative variability of signaling mechanisms controlling LFA-1 activation in chronic lymphocytic leukemia (CLL) cells. We pursued a multiplexed comparative analysis of the role of the recently described chemokine-triggered rho-signaling module in human normal versus CLL B-lymphocytes. We found that the rho-module of LFA-1 affinity triggering is functionally conserved in normal B-lymphocytes. In contrast, in malignant B-lymphocytes isolated from patients with B-CLL, the role of the rho-module was not maintained, showing remarkable differences and variability. Specifically, RhoA and phospholipase D1 were crucially involved in LFA-1 affinity triggering by CXCL12 in all analyzed patients. In contrast, Rac1 and CDC42 involvement displayed a consistent patient-by-patient variability, with a group of patients showing LFA-1 affinity modulation totally independent of Rac1 and CDC42 signaling activity. Finally, phosphatidylinositol-4-phosphate 5-kinase isoform 1gamma (PIP5KC) was found without any regulatory role in all patients. The data imply that the neoplastic progression may completely bypass the regulatory role of Rac1, CDC42, and PIP5KC, and show a profound divergence in the signaling mechanisms controlling integrin activation in normal versus neoplastic lymphocytes, suggesting that patients with CLL can be more accurately evaluated on the basis of the analysis of signaling mechanisms controlling integrin activation. Our findings could potentially affect the diagnosis, prognosis, and therapy of CLL disorder