Haematologica 2004;89: supplement no. 8 - Supplements ...

24Oral Communicationsshowed that the proportion of PMN expressing TFwas increased by 185±10 and 241±50% of basal valuesafter P-selectin and fMLP stimulation, respectively.Conclusions: our data show that the procoagulantactivity observed in stimulated PMN wasassociated to the expression of functional TF.CO-010SRC-DEPENDENT SIGNALING IS NECESSARY FOR β2-INTEGRINFUNCTION AND PMN FIRM ADHESION IN FLOWTotani L, Piccoli A, Barone S, Pecce R, Federico L,Manarini S, Evangelista VLaboratory of Vascular Biology and Pharmacology,Mario Negri Sud, Santa Maria Imbaro, ItalyNeutrophil (PMN) tethering by P-or E-selectin promotesthe adhesive function of β2-integrins responsiblefor firm adhesion in flow. SRC kinases may regulateβ2-integrin function in PMN. We investigatedthe role of SRC kinases on PMN adhesion to surfaceadherentplatelet, HUVEC or E-selectin expressingcells (CHO-E), in flow. In our model after an initialrecruitment phase, the shear was stepped up to 5dynes/cm 2 and adherent cells perfused with freshmedium. Firmly adhered and rolling PMN were quantifiedin the high shear phase. Results. Up to 95% ofPMN recruited on adherent platelets at 2 dynes/cm 2remained firmly adhered when shear stress wasstepped up to 5 dynes/cm 2 . P-selectin was requiredfor PMN recruitment. Similarly to β2-integrin blockadeby monoclonal antibodies the SRC inhibitors PP1and PP2 but not their inactive analog PP3 barelyaffected the total number of interacting cells butdose-dependently reduced the number of firmlyadhered PMN and increased the rolling fraction.After initial recruitment at 0.25 dynes/cm 2 , P-selectin, β2-integrins and SRC kinase activity werealso necessary for PMN to firmly adhere to thrombin-activatedHUVEC. Like P-selectin, E-selectin mayalso trigger β2-integrin function. β2-integrin blockadedecreased to about 20±7% of the control firmadhesion of PMN to CHO-E. PP2 dose-dependentlyreduced both total PMN and firmly adhered fraction.PMN adhesion to CHO-E, was accompanied by β2integrin or SRC dependent tyrosine phosphorylationof the 110 kDa, focal adhesion kinase PYK-2. Finally,confocal microscopy study showed that two distinctactivation-dependent epitopes of β2-integrins, wereup regulated at the sites of CHO-E-PMN adhesion.These epitopes were practically absent in mixed cellconjugates formed by PMN treated with SRCinhibitors. SRC, allowing and/or stabilizing clusteringof activated β2-integrins, translate short-lived membraneto membrane contacts to firm PMN adhesionin flow. This process involves PYK2.CO-011GENE EXPRESSION ANALYSIS OF HUMAN LPS-STIMULATEDAND UN-STIMULATED HUMAN LEUKOCYTES BY MICROARRAYTECHNOLOGY: DEVELOPMENT OF A NEW BAYESIAN MODELGiusti B, Poggi F, Rossi L, Biggeri A,° Blangiardo M,°Magi A, # Sestini I, Cesari F, Ferrini S, Gensini F,Abbate R, Gensini GF, Pepe GDipartimento di Area Critica Medico Chirurgica,sezione di Clinica Medica Generale e ClinicheSpecialistiche, University of Florence, Florence;°Dipartimento di Statistica “G. Parenti”, Universityof Florence; # Unita’ Operativa di Genetica eCitogenetica, Azienda Ospedaliera Careggi,Florence, ItalyMicroarray technology (MT) allows monitoring ofexpression levels for thousands of genes in a singleassay. The basic strategy is to isolate RNA from twosources (control and test). mRNAs from the twostates are differentially labeled with two fluorescentmolecules (Cy3 and Cy5) and co-hybridized to themicroarray. The arrays are scanned and independentimages are generated for each pair of samples. Forimage quantitation, there are many commercial andfreely available softwares and developed methods.A challenging issue in MT is the cutoff value used todistinguish differential expression from natural variability.One of the most used method of analysis wasintroduced by Newton (1999). Aims of this studywere: to evaluate and validate the experimental andanalysis approach of MT by using an experimentalmodel of LPS-stimulated and un-stimulated humanleukocytes. Mononuclear cells were isolated fromperipheral blood of 20 controls. For experiments ofself-self hybridization, RNA was extracted from thepellets and pooled. For gene expression analysis ofLPS-stimulated and un-stimulated leukocytes, cellswere incubated at 37°C for 3 hours in presence orabsence of LPS (10 µg/mL) and then RNA wasextracted and pooled. Data analysis was performedby Newton method and two Bayesian models, onedeveloped by Tseng (2001) and one developed by us.We observed 14 differentially expressed genesaccording to Newton analysis, 40 genes according toTseng and 34 according to our model. Ten out of the14 Newton genes were identified by the three model;25/34 genes identified by our model were alsoidentified by Tseng model. For 12/25 genes identifiedby the two Bayesian models there were well establishedevidences in the literature confirming the differentialexpression after LPS stimulation (e.g. IL-1β,-RA; PAI-2; MIP-1A, -1B); for the other 13/25 therewere indirect evidences of involvement or new identifiedgenes up-or down-regulated after LPS stimulation.Bayesian models and in particular our modelseems to be more efficient in identifying differen-haematologica vol. 89(suppl. n. 8):september 2004