Studies on the mechanisms of generation of microparticles and their role in the pathophysiology of coagulation

Microparticles are phospholipid vesicles shed by cells upon activation or during apoptosis. MP range in size from 50 nm to 1 µm [1]. Recent evidence have demonstrated that MP are involved in numerous physiological processes, including inflammation and blood coagulation. Because of the presence of negatively charged phospholipids on the outer leaflet of MP, these structures have been long attributed a role in blood coagulation, a process that requires the assembly of multimolecular complexes on the surface of negatively charged phospholipid membranes. More recently, however, it has become evident that MP also carry other components of the parental cell besides the phospholipids, which greatly broaden the spectrum of their potential effects as intercellular mediators [1]. For example, the presence on monocyte-derived MP of tissue factor, an essential cofactor for the initiation of blood coagulation [2], adds to their role in blood coagulation and thrombus formation [3]. The role of leukocyte- and endothelial cell-derived MP in coagulation processes involved both in physiological blood homeostasis both in vascular diseases has also been extensively investigated. MP can modulate cytokine expression in monocytes and endothelium; promote leukocyte-leukocyte aggregation and recruitment through P-selectin. Elevated platelet-derived MP associated with calpain activity have been documented in plasma of patients with thrombotic thrombocytopenic purpura [4]. Endothelial MP express the von Willebrand factor multimers that promote platelet aggregation and increase their stability, promoting the stability of the thrombus. Here we investigated the role of MP in thrombotic reaction associated to two widely widespread factors of risk to develop vascular diseases, like cigarette smoke and hypertension. In addition, we analyzed the role of MP bearing TF as mediators in the Instant blood-mediated inflammatory reaction, main responsible for the negative outcome of the pancreatic islets transplantation in type 1 diabetes

Chemochine e cancro: ruolo di MCP-1 e IL-8 nella proliferazione di cellule di tumore del polmone umano

RIASSUNTO Le chemochine sono piccole proteine basiche appartenenti alla famiglia delle citochine e caratterizzate da attività chemotattica. Esse regolano la migrazione direzionale di cellule leucocitarie e non (cellule endoteliali, fibroblasti, cellule epiteliali ecc.) sia in condizioni fisiologiche che patologiche. Negli ultimi anni si sono evidenziate proprietà delle chemochine che vanno al di là della capacità chemotattica, e che le rendono mediatori importanti in molti processi patologici, tra cui il cancro. Numerosi studi hanno dimostrano che le cellule trasformate secernono chemochine in grado di richiamare leucociti all’interno del tessuto tumorale. Questi infiltrati di cellule mononucleate non necessariamente rappresentano la risposta immunitaria dell’ospite contro il tumore, ma possono favorire la crescita, l’invasione e la metastatizzazione del tumore stesso. Nel presente lavoro di tesi si è studiato il ruolo delle chemochine Interleuchina-8 (IL-8/CXCL-8) e Monocyte Chemotactic Protein-1 (MCP-1/CCL2) nel tumore del polmone non a piccole cellule. IL-8 è un’importante fattore chemotattico per i neutrofili ed ha attività pro-angiogenica. MCP-1 è il principale responsabile del reclutamento dei mononucleati nel contesto dei tumori solidi. Nei nostri esperimenti sono state utilizzate tre linee cellulari di tumore del polmone (A549, Calu-6, e Calu-1). Con metodica immunoenzimatica ELISA abbiamo dimostrato la capacità di tutte le linee di produrre IL-8 ed MCP-1 solubili. La presenza dei recettori per queste chemochine sulle stesse cellule è stata valutata e confermata mediante citofluorimetria. Considerando i dati ottenuti, si è ipotizzato un effetto autocrino delle due chemochine in esame sulle cellule tumorali stesse. Per indagare questa ipotesi si è valutata in vitro, mediante test colorimetrici (XTT e MTT), l’effetto di MCP-1 e di IL-8 sulla proliferazione cellulare, sia in risposta a concentrazioni crescenti di chemochine esogene, sia dopo neutralizzazione anticorpo-mediata della chemochine prodotte dalle cellule stesse. Successivamente si è valutata l’induzione di morte cellulare per apoptosi con marcatura Annessina V/PI ed analisi al FACS, dopo neutralizzazione anticorpo-mediata di MCP-1 e IL-8 endogene. I risultati da noi ottenuti dimostrano, per la prima volta, la capacità di queste linee cellulari di produrre IL-8 ed MCP-1 e di esprimerne i recettori (CXCR1 e CCR2, rispettivamente), facendo ipotizzare la possibilità di un effetto autocrino di queste chemochine nel contesto del tumore del polmone. A corroborare questa teoria esiste la risposta dose-dipendente delle tre linee tumorali in risposta a concentrazioni crescenti di MCP-1 e di IL-8. Inoltre, la neutralizzazione di tali chemochine con gli opportuni anticorpi determina una significativa diminuzione dell’attività proliferativa delle cellule neoplastiche prese in esame. Dati preliminari ottenuti sulla linea cellulare A549 indicano che la neutralizzazione di MCP-1 e di IL-8 endogene possa indurre le cellule tumorali stesse in apoptosi. Concludendo, le tre linee cellulari di tumore del polmone producono e rilasciano nel terreno di coltura MCP-1 e IL-8 solubili e presentano sulla loro superficie cellulare i loro rispettivi recettori, CCR2 e CXCR1. Questo ha fatto pensare ad un possibile effetto autocrino di MCP-1 e di IL-8 sulle cellule tumorali stesse. I risultati ottenuti con i test di vitalità cellulare indicano che MCP-1 e IL-8 prodotte dalle tre linee cellulari hanno un’attività pro-proliferativa legata, almeno in parte alla loro capacità di inibire l’apoptosi nelle condizioni sperimentali da noi utilizzate. Tuttavia, sono necessari ulteriori esperimenti volti ad indagare dettagliatamente l’effetto delle due chemochine nel meccanismo di morte cellulare per apoptosi e/o nelle fasi del ciclo cellulare

Leptin induces the generation of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells

Obesity is linked to increased thrombotic risk. Circulating leptin concentration correlates with body mass index. Microparticles are small (.05-1μm) vesicles shed by activated and apoptotic cells, involved in numerous pathophysiologically relevant phenomena including blood coagulation and thrombosis. We tested the hypothesis that leptin induces the shedding of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells, and investigated the intracellular mechanisms leading to microparticle release upon incubation with leptin

C6: A monoclonal antibody specific for a fibronectin epitope situated at the interface between the oncofoetal extra-domain B and the repeat III8

Background Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN isoform that contains the \u201cextra-domain-B\u201d (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues in vivo. We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B. Methodology We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6. Conclusion The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity

Rheological properties, biocompatibility and in vivo performance of new hydrogel-based bone fillers

reserved7Three different heterologous substitutes for bone regeneration, manufactured with equine-derived cortical powder (CP), cancellous chips (CC) and demineralized bone matrix granules (DBM), were compared in in vitro and in vivo settings. We tested: a commercially available bone paste (Osteoplant-Activagen™, consisting of aqueous collagenous carrier, CP, DBM; named A); a second-generation injectable paste (20 kDa polyethylene glycol/hydroxypropyl-methyl cellulose-based hydrogel, CP, DBM; B); a pre-formed bone filler (400 kDa polyethylene oxide/hydroxypropyl-methyl cellulose-based hydrogel, CP, CC, DBM; C). Vitamin C acted as a visco-modulator during C and B β-rays sterilization, modifying graft injectability. For each filler, we examined dissolution in culture medium, gene expression of the substitute-exposed osteogenically-induced human bone marrow stromal cells (hBMSC), and performance in a rabbit bone defect model. A dissolved after 1 h, while fragmentation of B peaked after 8 h. C remained unaltered for 2 days, but affected the microenvironmental pH, slowing the proliferation of exposed cells. B-exposed hBMSC overexpressed bone sialoprotein, osteocalcin and RUNX2. For all fillers histological results evidenced bridged lesion margins, marrow replenishment and bone-remodeling. However, B-treated lesions displayed a metachromatic type II collagen-rich matrix with prehypertrophic-like cells, matching the in vitro expression of cartilage-specific markers, and suggesting a possible application of B/C double-layer monolithic osteochondral plugs for full-thickness articular defects.mixedGiannoni, Paolo; Villa, Federico; Cordazzo, Cinzia; Zardi, Luciano; Fattori, Paolo; Quarto, Rodolfo; Fiorini, MauroGiannoni, Paolo; Villa, Federico; Cordazzo, Cinzia; Zardi, Luciano; Fattori, Paolo; Quarto, Rodolfo; Fiorini, Maur

Interaction between C6 and the FN recombinant fragment formed by the type III repeats B and 8.

<p><b>A)</b> Schematic representation of the main interactions between the two proteins. The type III domains B and 8 are drawn as green ribbons, the scFv C6 is orange. Residues involved in the binding are reported as sticks, hydrogen bonds between amino acids are shown as dotted black lines. <b>B)</b> Residues of the scFv and of the type III repeats B and 8 that interact. The distances are displayed. <b>C)</b> Representation of the interaction between the type III domains B-8 (green surface) and the scFv C6 (orange surface).</p

Reactivity of the mAb C6 with FN recombinant fragments.

<p><b>A)</b> Different FN recombinant fragments tested with the mAb C6. <b>B)</b> Reactivity in ELISA of various concentrations of the mAb C6 with the FN recombinant fragment containing the type III repeats B and 8; it fragment B-8 with the mutation Glu1329Ala and fragment B-8 with the mutation Asp1385Glu.</p

Immunohistochemistry experiments using the mAb C6 on cryostat sections.

<p>A) Normal human lung; B) human lung adenocarcinoma; C) Normal human brain; D) Human mesothelioma; E) Human glioblastoma; F) Murine teratocarcinoma. Immunohistochemistry experiments using the recombinant antibody CGS1 on cryostat sections of human glioblastoma (G) and murine teratocarcinoma (H). CGS1 reacts directly with the ED-B (10) and thus, contrary of C6, it also reacts with murine tumours. C6 reacts, in an identical manner of CGS1, with normal and cancer human tissues but not with murine tumours (Modified from ref.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148103#pone.0148103.ref017" target="_blank">17</a>] under CC-BY license, with permission f from John Wiley and Sons, original copyright 2009.). </p

Structures of FN and of the scFv C6.

<p><b>A)</b> Model of the domain structure of a FN subunit. The three different types of repeats, the three sites of alternative splicing (ED-A, ED-B, IIICS) and the specificity of the mAb C6 for the interface between type III repeats B and 8 are shown. <b>B)</b> Sequence of the scFv C6; the linker between the VL and the VH is in red; the CDRs are framed. Amino acids involved in the interaction with C6 are in bold. <b>C)</b> Sequence of the FN type III repeats numbers 7 (blue), B (black) and 8 (red); the various loops between the beta sheets structures are framed. The amino acids involved in the interaction with C6 are in bold. Sequence from <a href="http://www.ncbi.nlm.gov.nuccore/47132556" target="_blank">http://www.ncbi.nlm.gov.nuccore/47132556</a>.</p