In vitro and in vivo model of ebv-positive activated diffuse large b cell lymphoma with plasmacytic differentiation

Le malattie linfoproliferative croniche possono evolvere istologicamente in linfomi ad alto grado di malignit\ue0. E\u2019 noto come il virus di Epstein-Barr giochi un ruolo importante nella patogenesi di alcuni linfomi aggressivi e come sia associato alla trasformazione istologica di forme indolenti. E\u2019 nota inoltre la sua capacit\ue0 trasformante di cellule in vitro. Le linee cellulari rappresentano un importante strumento di ricerca poich\ue9 facilitano lo studio della biologia di molte malattie e l\u2019applicazione di nuove terapie mirate. VR09 \ue9 una nuova linea cellulare di linfoma B diffuso a grandi cellule (DLBCL) con differenziazione plasmacitica realizzata nel nostro laboratorio e derivante da un caso di malattia linfoproliferativa B. Abbiamo dettagliatamente studiato la linea cellulare attraverso la caratterizzazione immunofenotipica, immunoistochimica, molecolare, citogenetica e mediante FISH. Abbiamo inoltre testato il potenziale tumorigenico di VR09 in vivo. Le cellule in sospensione hanno mostrato capacit\ue0 proliferativa in vitro dopo alcuni mesi e capacit\ue0 di sviluppare una massa sottocutanea quando inoculate sottocute in topi immunodeficienti Rag2-/- \u3b3-chain-/- . Sia le cellule in vitro che quelle derivate dalla massa sottocutanea hanno mostrato un profilo immunoistochimico corrispondente ad uno stadio \u201cattivato\u201d della maturazione B con aspetti di differenziazione plasmacitica (CD19+, CD20+, CD79a+, CD79b+/-, CD138+/-, cyclina D1-, Ki67 80%, IgM+, IgD+, MUM1+, MDNA+, CD10-, CD22+, CD23+, CD43+, K+, \u3bb-, Bcl2+, Bcl6-) e la presenza di EBV in forma episomica. E\u2019 stata inoltre identificata la trisomia del cromosoma 12, la presenza di ipermutazioni somatiche nella regione VH, la mutazione dei geni Card11 e CD79B, e la presenza di p53 in forma wild-type. Questa nuova linea cellulare potrebbe essere utile per caratterizzare in modo pi\uf9 approfondito le forme di DLBCL con differenziazione plasmacitica e testare in esse nuove terapie mirate.\u2003Background: B-cell lymphoproliferative diseases can show plasmacytic differentiation and may potentially progress to diffuse large B cell lymphoma (DLBCL). Epstein-Barr virus infection may cause the transformation of malignant cells in vitro. Design and Method: we established VR09 cell line, a DLBCL cell line with plasmacytic differentiation, obtained from a case of atypical B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization to characterize this cell line. We also assessed whether VR09 has tumorigenic potential in vivo. Results: cells in suspension revealed plasmacytic features and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2-/- \u3b3-chain-/- mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/- CD138+/- cycline D1- Ki67 80% IgM+ IgD+ MUM1+ MDNA+ CD10- CD22+ CD23+ CD43+ K+, \u3bb- Bcl2+ Bcl6-); in addition they displayed episomal EBV genome, chromosome 12 trisomy, absence of c-MYC rearrangement, presence of somatic hypermutation in the VH region, mutations of Card 11 and CD79B genes, and wild-type p53. Conclusion: This new EBV-positive cell line may be useful to further characterize activated DLBCL with plasmacytic features. \u200

Thrombotic and hemorrhagic complications in idiopathic erythrocytosis

We report clinical features of a large cohort of patients with IE compared to a cohort of patients with PV, focusing on the thrombotic and hemorrhagic risk

A proliferation-inducing ligand (APRIL) serum levels predict time to first treatment in patients affected by B-cell chronic lymphocytic leukemia

Purpose: A proliferation-inducing ligand (APRIL), a tumor necrosis factor superfamily member involved inB-lymphocytes differentiation and survival, plays a role in protecting B-Cell Chronic lymphocytic leukemia(B-CLL) cells from apoptosis. Having observed that APRIL serum (sAPRIL) levels were higher in B-CLLpatients with CLL at diagnosis as compared to healthy donors (14.61 \ub1 32.65 vs. 4.19 \ub1 3.42 ng \u2044 mL;P < 0.001), we tested the correlation existing in these patients between sAPRIL, clinical\u2013biological parametersand disease progression. Experimental design: sAPRIL levels were measured by ELISA in 130patients with B-CLL at diagnosis and in 25 healthy donors. Results: sAPRIL levels did not correlate withgender, age, clinical stage, blood cell counts, b2-microglobulin (b2M) levels, ZAP-70 and CD38 expression.Using median sAPRIL natural logarithm (ln) as cutoff, we distinguished two groups of patients (APRILLOWand APRILHIGH) who were comparable with regard to clinical\u2013biological parameters and overall survival, butdifferent with regard to time to the first treatment (TTFT; P = 0.035). According to univariate analysis, highlymphocyte count, high b2M, Binet stage B\u2013C, ZAP-70 expression and ln(sAPRIL) above median wereassociated with earlier TTFT. Advanced clinical stage, high b2M, ZAP-70 expression and ln(sAPRIL) abovemedian remained independently predictive of shorter TTFT at multivariate analysis. Moreover, sAPRILincreased its prognostic significance when patients were stratified according to independent favorable clinical\u2013biological characteristics (low b2M, stage A and lack of ZAP-70 expression). Conclusions: sAPRIL is anovel indicator of shorter TTFT in B-CLL and a predictor of progression especially in patients otherwiseconsidered at low risk according to validated prognostic factors

Evaluation of Coaguchek®Pro II coagulation testing device performance to assess direct oral anticoagulant action. The DOAC-CHECK study

Direct oral anticoagulants (DOAC) measurement is recommended in specific conditions. A point-of-care testing should be used in emergency to qualitatively rule out relevant DOAC concentrations. The DOAC-CHECK Study aims to evaluate whether the use of CoaguChek® Pro II (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) coagulation testing device can provide reliable information in patients treated with DOAC. The study was carried out in two FCSA (Italian Federation of Thrombosis Centers) centers. We choose 3 different concentration thresholds for our analysis (30, 50 and 100 ng/mL) and by ROC curves the ideal cut-off point was selected to be the one that yielded a sensitivity of at least 95% associated with the highest possible specificity. 512 patients were enrolled. For Edoxaban and Rivaroxaban, both CoaguChek® Pro II prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests showed a sensitivity >95% corresponding to satisfying specificity values; negative predictive values resulted in the range 90-100%. At variance, CoaguChek® Pro II PT and aPTT tests did not seem to be useful for identifying Apixaban and Dabigatran concentrations higher than the pre-defined thresholds. Our results suggest that CoaguChek® Pro II coagulation testing device can be used to qualitatively identify relevant concentrations of Edoxaban or Rivaroxaban, but not of Apixaban or Dabigatran

Analysis of 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia

65Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L.openopenPatrizia Noris; Nicole Schlegel; Catherine Klersy; Paula G. Heller; Elisa Civaschi; Nuria Pujol-Moix; Fabrizio Fabris; Remi Favier; Paolo Gresele; Véronique Latger-Cannard; Adam Cuker; Paquita Nurden; Andreas Greinacher; Marco Cattaneo; Erica De Candia; Alessandro Pecci; Marie-Françoise Hurtaud-Roux; Ana C. Glembotsky; Eduardo Muñiz-Diaz; Maria Luigia Randi; Nathalie Trillot; Loredana Bury; Thomas Lecompte; Caterina Marconi; Anna Savoia; Carlo L. Balduini; Sophie Bayart; Anne Bauters; Schéhérazade Benabdallah-Guedira; Françoise Boehlen; Jeanne-Yvonne Borg; Roberta Bottega; James Bussel; Daniela De Rocco; Emmanuel de Maistre; Michela Faleschini; Emanuela Falcinelli; Silvia Ferrari; Alina Ferster; Tiziana Fierro; Dominique Fleury; Pierre Fontana; Chloé James; Francois Lanza; Véronique Le Cam Duchez; Giuseppe Loffredo; Pamela Magini; Dominique Martin-Coignard; Fanny Menard; Sandra Mercier; Annamaria Mezzasoma; Pietro Minuz; Ilaria Nichele; Lucia D. Notarangelo; Tommaso Pippucci; Gian Marco Podda; Catherine Pouymayou; Agnes Rigouzzo; Bruno Royer; Pierre Sie; Virginie Siguret; Catherine Trichet; Alessandra Tucci; Béatrice Saposnik; Dino VeneriPatrizia, Noris; Nicole, Schlegel; Catherine, Klersy; Paula G., Heller; Elisa, Civaschi; Nuria Pujol, Moix; Fabrizio, Fabris; Remi, Favier; Paolo, Gresele; Véronique Latger, Cannard; Adam, Cuker; Paquita, Nurden; Andreas, Greinacher; Marco, Cattaneo; Erica De, Candia; Alessandro, Pecci; Marie Françoise Hurtaud, Roux; Ana C., Glembotsky; Eduardo Muñiz, Diaz; Maria Luigia, Randi; Nathalie, Trillot; Loredana, Bury; Thomas, Lecompte; Caterina, Marconi; Savoia, Anna; Carlo L., Balduini; Sophie, Bayart; Anne, Bauters; Schéhérazade Benabdallah, Guedira; Françoise, Boehlen; Jeanne Yvonne, Borg; Bottega, Roberta; James, Bussel; DE ROCCO, Daniela; Emmanuel de, Maistre; Faleschini, Michela; Emanuela, Falcinelli; Silvia, Ferrari; Alina, Ferster; Tiziana, Fierro; Dominique, Fleury; Pierre, Fontana; Chloé, James; Francois, Lanza; Véronique Le Cam, Duchez; Giuseppe, Loffredo; Pamela, Magini; Dominique Martin, Coignard; Fanny, Menard; Sandra, Mercier; Annamaria, Mezzasoma; Pietro, Minuz; Ilaria, Nichele; Lucia D., Notarangelo; Tommaso, Pippucci; Gian Marco, Podda; Catherine, Pouymayou; Agnes, Rigouzzo; Bruno, Royer; Pierre, Sie; Virginie, Siguret; Catherine, Trichet; Alessandra, Tucci; Béatrice, Saposnik; Dino, Vener

La gentilezza nelle attivit\ue0 di counselling

Oggi giorno si sente spesso parlare di Networking Skills. Queste riguardano quell\u2019insieme di abilit\ue0 che permettono all\u2019individuo di crearsi una propria rete sociale a cui rivolgersi per ricevere diversi aiuti e supporti al fine di poter raggiungere i propri obiettivi (Castells, 2007). L\u2019essere in grado di saper instaurare relazioni positive con l\u2019altro, a nostro avviso, \ue8 importante in vari ambiti della vita umana. Harris e Williams (2014), a questo riguardo, ritengono che nella nostra societ\ue0 e nell\u2019attuale mercato del lavoro sia importante riuscire a creare una propria \u2018rete\u2019, dalla quale poter ricavare informazioni che ci permettono di aumentare le probabilit\ue0 di trovare un posto di lavoro, ma anche che ci stimoli a generare idee nuove e creative. Scrimin e Axia (2004) ritengono che il supporto sociale sia importante anche per poter fronteggiare le difficolt\ue0 ascrivibili all\u2019ambito della salute. Si potrebbero riportare molti altri esempi, ma \ue8 ormai abbastanza consolidato da molti anni in letteratura che senza l\u2019Altro non ci sarebbe un S\ue9 (Gallup, 1997; Aronson, Wilson, e Akert, 2006) In tutto questo, a nostro avviso, un ruolo importante pu\uf2 essere giocato dalla gentilezza, un punto di forza della persona che pu\uf2 portare dei vantaggi a s\ue9 e agli altri in quanto il praticare atti di gentilezza pu\uf2 aumentare il benessere e la felicit\ue0 non solo in chi mette in atto i comportamenti gentili ma anche in chi li riceve. Ci piace ricordare le parole del Dalai Lama (Gyatso Tenzin), che nel suo libro \u201cLa felicit\ue0 \ue8 negli altri\u201d (Dalai Lama - Gyatso Tenzin e Victor Chan, 2014) mette in evidenza, attraverso il racconto di alcuni incontri significativi della sua vita, come l\u2019essere gentili e compassionevoli verso gli altri pu\uf2 portare alla felicit\ue0. Secondo il Dalai Lama, imparare ad amare e aiutare gli altri non \ue8 sempre facile, ma la ricompensa \ue8 grande e duplice: la felicit\ue0 interiore e la possibilit\ue0 di costruire un mondo migliore. Considerando che con sempre maggiore frequenza nell\u2019ambito del counseling si ritiene essenziale dare enfasi ai punti di forza delle persone per poter migliorare la qualit\ue0 della vita degli stessi (Soresi e Polo, in questo volume), abbiamo voluto approfondire il costrutto della gentilezza. Con questo capitolo ci proponiamo, quindi, di sintetizzare una serie di riflessioni presenti in letteratura sulla gentilezza, al fine di poter fornire delle definizioni, presentare strumenti di indagine e descrivere alcune azioni che possono essere considerate utili a sostenere la gentilezza anche nell\u2019ambito del counseling

Thrombocytopenias: a clinical point of view

The finding of thrombocytopenia may sometimes be a warning sign of high-risk situations requiring swift therapeutic decisions. The diagnosis must start with the patient\u2019s history and data, with a careful evaluation of all blood counts and confirmation of the thrombocytopenia, followed by a morphological evaluation of the peripheral blood smear which, together with objective clinical findings, such as bleeding, lymphadenopathy and splenomegaly, will indicate the need for immediate therapy or appropriate further diagnostic investigations. Thrombocytopenia occurring in surgical patients can basically be attributed to DIC or HIT: the differential diagnosis is made on the basis of the correct laboratory investigations. In our opinion, the possibility of an ongoing pregnancy should always be evaluated in thrombocytopenic women of childbearing age, since a confirmed pregnancy implies that clinical forms of strictly obstetric pertinence are also possible. In conclusion, the overall picture of thrombocytopenias is extremely variegated, leading to numerous diagnostic and therapeutic problems whose solutions require close collaboration between clinicians and laboratory specialists

In Vitro and In Vivo Model of EBV-Positive Non-Hodgkin Plasmablastic Lymphoma with Focal Plasmacytic Differentiation

Assessment of EBV-positive large B-cell lympoma cell line with plasmacytic differentiatio

B-cell chronic lymphocytic leukemia cells espress functional death receptor 3 following stimulation of the B cell receptor

Background. In the microenvironments where B-CLL develops, complex molecular networks propagate signals that confer growth advantage to CLL. Within this context, interplaying between TNF superfamily members and their cognate receptors has been shown to play a relevant role in controlling B-CLL growth and survival. TNF-like protein 1A (TL1A) is a recently discovered member of the TNF superfamily expressed on various cell types, including macrophages, dendritic cells, and endothelial cells. The TL1A cognate receptor, death receptor 3 (DR3), is a member of the TNF receptor superfamily expressed on various cell types, including activated T cells and macrophages. TL1A-DR3 interactions have been shown to modulate immune and inflammatory functions. So far, the expression of DR3 on B-lineage cells as well as possible roles of TL1A-DR3 interplaying in CLL biology has not been explored. Aims. The objective of the study was to investigate DR3 expression and function(s) in B-CLL. Methods. B cells were purified from PBMC of 23 B-CLL patients and 13 healthy donors by negative selection with magnetic beads. DR3 surface expression was measured by flow cytometry at baseline and various time points following stimulation with F(ab’)2 anti-human IgM conjugated to latex microspheres. DR3 mRNA was detected by quantitative RT-PCR. Phosphorylation states of NF-kappaB, Erk1/2, and TBK1 were measured by using phospho-specific antibodies and flow cytometry in basal condition and following DR stimulation of cells with agonistic antibody at different time points (15, 30, 60, 180 minutes). Results. Although both healthy and CLL B cells did not express DR3 in basal conditions, stimulation of B cell receptor (BCR) induced a statistically significant increase of DR surface expression in healthy as well as malignant B cells (p<0.001 for both cells). Time course analysis showed that DR3 expression peaked at 24 hour after stimulation. DR3 expression was confirmed also at the mRNA level. Time course analysis showed that DR3 mRNA in B-CLL cells peaked at 2.5 hour following anti-IgM stimulation (4-fold change with respect to basal conditions). Anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells if compared with healthy B cells (p<0.05). Interestingly, when B-CLL patients were stratified by IGVH mutational status, anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells harboring unmutated IGVH genes compared with cells with mutated IGVH genes. To assess whether the anti-IgM-induced DR3 molecule was functionally active in B-CLL cells, we examined the ability of DR3 to trigger phosphorylation events in biologically relevant signaling nodes (i.e. Erk1/2, NF-kappaB, and TBK1) by stimulating cells with anti-DR3 agonistic antibodies. Phospho-specific flow cytometry analysis showed that DR3 engagement induced phosphorylation of Erk1/2 but not NF-kappaB or TBK1. Conclusions. We described for the first time the expression of functional DR3 molecules in B-lineage cells activated by BCR stimulation in healthy and pathological cells (i.e. B-CLL cells). The findings that anti-IgM-induced DR3 expression was higher in B-CLL cells if compared with healthy B cells and that, among B-CLL patients, this expression was higher in cells with unmutated IGVH genes, suggest that DR3 stimulation may have a role in B-CLL pathogenesis