ALBERT

Description: Pruritus is a common symptom in patients with Philadelphia chromosome–negative myeloproliferative disorders (MPDs). The pathophysiology of MPD-associated pruritus is unclear. We have demonstrated that MPD mast cells (MCs) are involved by the malignant process. In the present study, we explored the hypothesis that MCs play an important role in the development of pruritogenesis in MPDs. We found that MPD MCs released significantly greater amounts of pruritogenic factors, including histamine, leukotrienes, and interleukin-31 (IL-31) than normal MCs. Elevated levels of IL-31 were also observed in MPD CD3+ cell-conditioned media. MPD MCs exhibited increased migratory behavior in response to stem cell factor or interleukin-8, which was associated with increased filamentous-actin content. Furthermore, the presence of pruritus in MPDs was statistically correlated with a greater number of MCs being generated by CD34+ cells, a greater number of MC colonies being formed by CD34+ cells, decreased apoptosis and prostaglandin D2 release by cultured MCs, and higher plasma levels of IL-31. These data demonstrate that functional abnormalities of MPD MCs probably lead to pruritogenesis in patients with MPDs. These studies provide cellular and molecular targets for the development of antipruritus drugs for patients with MPDs.

Description: Patients with HIV-1 immune-related thrombocytopenia (HIV-1–ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1–seropositive drug abusers are more prone to develop immune thrombocytopenia than non–drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non–drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti–GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 nonconserved peptides from HCV core envelope 1. Reactivity correlated inversely with platelet count (r2 = 0.7, P 〈 .01). Ab raised against peptide PHC09 in GPIIIa−/− mice induced thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as titer of anti–GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab versus PHC09 and significantly decreased their platelet count (P 〈 .001). Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66.

Description: Abstract 4432 Object This study was purposed to investigate the expression of HIF-1α and VEGF in leukemia cell lines and the effect of YC-1 on the regulation of HIF-1α and VEGF and the induction of apoptosis in U937 cell, exploring the mechanism of apoptosis after treatment of YC-1. Methods RT-PCR was used to determine the levels of HIF-1α mRNA and VEGF mRNA in K562,U937 and Jurkat cells. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour, 8 hours,16 hours and 24 hours respectively, the changes of morphologic features were observed by DAPI staining under fluorescent microscope and the apoptotic rates were assayed by flow cytometry with Annexin V-FITC/PI staining; the levels of HIF-1α mRNA and VEGF mRNA were measured with RT-PCR ; the protein expression of HIF-1α, VEGF, Bax,Bcl-2 and Caspase-3 were measured by Western blot. Results HIF-1α mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4μmol/L YC-1 for 0 hour,8 hours,16hours and 24hours respectively, the typical apoptotic morphologic features of U937 cells were observed under fluorescent microscope and the apoptotic rates were significantly increased in a time-dependent manner, they were (4.87±0.70)%, (27.27±2.00)%, (51.53±2.81) and (60.5±3.20)% respectively, the level of VEGF mRNA reduced, while the level of HIF-1α mRNA had no obviously changes. Furthermore, the expression of HIF-1α, VEGF and Bcl-2 decreased, while the expression of Bax and Caspase-3 increased in a time-dependent manner. Conclusion HIF-1α mRNA and VEGF mRNA are expressed in leukemia, YC-1 has significant effect of down-regulation the protein expression of HIF-1α and VEGF and induction of apoptosis in U937, its mechanisms may involve in up-regulating Bax/Bcl-2 ratio and expression of Caspase-3. Disclosures: No relevant conflicts of interest to declare.

Description: Protein Z is a vitamin K–dependent plasma glycoprotein that is involved in the regulation of blood coagulation. Plasma concentrations of protein Z vary widely between subjects and are greatly reduced during warfarin therapy. We developed a sensitive and quantitative assay for protein secretion using a secretory luciferase to explore the mode of secretion of protein Z compared with that of factor X. Protein Z secretion was much less efficient than factor X and was totally dependent upon added vitamin K, while factor X secretion was not. Protein Z secretion was highly sensitive to warfarin treatment of the synthesizing cells. In contrast, although factor X secretion was not precluded by warfarin, its γ-carboxylation was completely blocked. An exchange of the propeptide and/or γ-carboxyglutamic acid domain between protein Z and factor X reproduced the inefficient and warfarin-sensitive secretion pattern of protein Z, and vice versa. Joining of the propeptide and γ-carboxyglutamic acid domain to luciferase also demonstrated that the γ-carboxyglutamic acid domain of protein Z was responsible for its warfarin-sensitive secretion. Thus, it was concluded that the difference observed in secretion patterns of protein Z and factor X was mainly based on the structure of their γ-carboxyglutamic acid domains.

Description: Abstract 1910 Poster Board I-933 Primary myelofibrosis (PMF) is likely the consequence of both the acquisition of genetic mutations and epigenetic changes which silence critical genes influencing cell proliferation, differentiation, survival and trafficking. The abnormal trafficking of CD34+ cells in PMF has been attributed to reduced expression of CXCR4 (Shi et al, Cancer Res, 2007; Wang et al, Cancer Res, 2009). We examined the effects of treatment with chromatin modifying agents (CMA), including sequential treatment with a DNA methyltransferase inhibitor, decitabine “5-aza-2'-deoxycytidine (5azaD)”, followed by a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA) (5azaD/SAHA), or trichostatin A (5azaD/TSA) on the in vitro and in vivo behavior of PMF CD34+ cells (PCC). Sequential treatment with 5azaD/SAHA resulted in a reduction in the number of total cells, CD34+ cells and assayable hematopoietic progenitor cells (HPC) as compared with PCC treated with cytokines alone. By contrast, treatment of PCC with 5azaD/SAHA led to the generation of 2.4-fold greater numbers of CD34+CXCR4+ cells as compared to PCC exposed to cytokines alone and a 15.8-fold increase of the number of CD34+CXCR4+ cells when compared to input PCC (P

Description: Abstract 3916 Poster Board III-852 Polycythemia vera (PV) is a Philadelphia chromosome negative chronic myeloproliferative neoplasm (MPN) which is characterized by acquisition of a mutation in JAK2 (JAK2V617F). The administration of a pegylated form of interferon-alpha-2a (Peg IFNa-2a) to patients with PV has recently been reported to lead to hematological remissions and a reduction of the JAK2V617F allele burden in most patients receiving this modality of therapy. The mechanism underlying this profound clinical response of PV patients to Peg IFNa-2a has been the subject of a great deal of speculation. In order to evaluate the mechanism by which Peg IFNa-2a affects hematopoiesis in PV patients, CD34+ cells isolated from cord blood and the peripheral blood of patients with PV were cultured in semisolid media in the presence and absence of 200 and 500 U of Peg IFNa-2a. These relatively low doses of Peg IFNa-2a did not alter hematopoietic colony formation by CB CD34+ cells but inhibited PV CFU-GM colony formation by 35% and 50%, and BFU-E colony formation by 60% and 80%, respectively. Furthermore, the hematopietic colonies that formed in the presence of Peg IFNa-2a were composed of far fewer cells than those cultured in the presence of cytokines alone. In addition, individual hematopoietic colonies were plucked and the JAK2 genotype was assessed by nested allele-specific PCR assay. Exposure of PV CD34+ cells to Peg-IFNa-2a (500U) resulted in a reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells from 81.7±16.3% to 50.3±27.6% (p=0.004). Samples from 81.9% of the PV patients (9 of 11 samples) responded in this fashions to Peg IFNa 2a treatment. We then showed that incubation of PV CD34+ cells but not CB CD 34+ cells with 200 and 500U of Peg IFNa-2a resulted in increased rates of apoptosis by 4.3% and 15.3%, respectively. Erythroblasts and megakayoctes from patients with PV have been previously shown to be characterized by over-expression of the anti-apoptotic proteins Bcl-xL. We then examined if the effects of IFNa-2a could be enhanced by addition of the Bcl-xL inhibitor-ABT-737. After 2 days of treatment, Peg IFNa 2a plus ABT-737 induced significantly greater degree of apoptosis (∼50%) of a JAK2V617F positive erythroleukemia cell line (HEL cells) as compared to treatment with each agent alone, (Peg-IFNa-2a,

Description: Abstract 752 The Ph- myeloproliferative neoplasm (MPN) are associated with excessive production of red cells, platelets and granulocytes which largely determines their clinical manifestations. A mutation in the JAK2 tyrosine kinase (JAK2V617F) was identified in the majority of patients with MPNs. The JAK2V617F mutation has been shown to play a pivotal role in the pathogenesis of MPNs. We have reported that erlotinib (Tarceva), a kinase inhibitor which inhibits the epidermal growth factor induced kinase activity, is also a potent inhibitor of JAK2V617F activity. It has been shown that erythroblasts from patients with polycythemia vera (PV) express elevated levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL. In addition, we have recently documented that megakaryocytes derived from patients with primary myelofibrosis (PMF) undergo a delayed pattern of apoptosis in vitro which might be attributed to the over-expression of Bcl-xL. We hypothesize that a combination of a JAK2V617F inhibitor and a Bcl-xL inhibitor might be capable of selectively eliminating MPN cells while sparing normal cells, and therefore, providing an optimal treatment strategy for Ph− MPNs. We then evaluated the combinations of a JAK2V617 inhibitor (erlotinib or INCB018424) and a Bcl-xL inhibitor (ABT-737) for their ability to selectively eliminate MPN (JAK2V617F positive) cells while sparing normal cells in vitro using a variety of screening systems. We first tested the ability of each of the JAK2 inhibitors alone or in combination with ABT-737 to induce death of HEL cells, which harbors JAK2V617F. Among these treatments, ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1 nM) were shown to have similar capability of inducing HEL cell apoptosis (50-70%) which was significantly greater than that by each of the single agents (

Description: Abstract 4829 Object In many instances, Multidrug resistance (MDR) is mediated by increased expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe3O4 (MNP(Fe3O4)) and mdr1-shRNA Expression vetor.in K562/A02 leukemic cells. Methods To synthesis short hairpin RNA (shRNA)aiming divectly at the target sequence,we choice the 3491-3509,1539-1557and 3103-3121 nucleotide of mdr-1 mRNA as targets. Cloning in the plasmid vetor PGCSilencer-U6-neo-GFP, The recombinant plasmid vetors were called for PGY1-1,PGY1-2 and PGY1-3.The recombinant plasmid vetors were transfected into the cell 1ines K562/A02 by lipofection. After transfected 48 hours,the inhibition of mdr-1mRNA expression and the expression of P-gp was detected by realtime–PCR and Weston-blot, screening the recombinant plasmid vetor which has the most greatest mdr-1 gene inhibition ratio is PGY1-2.Analysis of the reveral ratio of multidrug resistance, the concentration of DNR and the content of mdr-1 gene and P-gp in K562/A02 cell line. Results The combination of daunorubicin (DNR) with either MNP(Fe3O4) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe3O4) and PGY1-2 cotreatment can synergistically down regulation the expression of mdr-1 gene and the expression of P-gp(P

Description: Abstract 3582 Poster Board III-519 Objective Cytochrome P450(CYP450-CYP1A2 /CYP2B6/CYP2C9) was transfected into human bone marrow-derived mesenchymal stem cells (hBMSCs), and the targed anti-tumor effect of BMSC-CYP450 cooperated with enzyme-prodrug(Dacarbazine (DTIC)/Cyclophosphamide (CPA)) was measured to provide laboratory data base for gene directed enzyme prodrug targeted anti-tumor therapy (GDEPT) which used BMSC as vehicles. Methods We respectively cloned CYP1A2/CYP2B6/CYP2C9 cDNA from human liver and constructed recombinant adenovirus vectors(pAd5CMV-NpA-CYP1A2/ pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9) which titer was 1×1012 pfu/mL. These hBMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction, and RT-PCR(A surface marker for the identification of MSCs-the neural ganglioside GD2 gene). The tropism of BMSCs for cancer cells was detected by Transwell inserts technique. These recombinant vectors were transferred into BMSCs and A375/K562 cells, and the expression of EGFP and CYP1A2/CYP2B6/CYP2C9 was detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI detected the anti-tumor effect of CYP450 recombinant adenovirus vectors combined with chemotherapeutic prodrug DTIC/CPA in vitro. A human melanoma(A375) BALB/c nude mice model and a human myelocytic leukemia(K562) BALB/c nude mice model was constructed and detected by immuno-histochemistry analysis. The CYP1A2 gene tranfected BMSCs were injected into the A375 BALB/c nude mice model in combination with DTIC through caudal vein, while CYP2B6/CYP2C9 gene tranfected BMSCs were injected into K562 BALB/c nude mice model in combination with CPA in the same way. The measurement of tumors size, fluorescence microscope and TUNEL were used to detect anti-tumor effect of BMSCs-CYP1A2 cooperating with DTIC and BMSCs-CYP2B6/CYP2C9 with CPA in vivo. Results We constructed the recombinant adenovirus vectors pAd5CMV-NpA-CYP1A2/pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9 and pAd5CMV-NpA-EGFP successfully. BMSCs was separated successfully, and it respectively showed that BMSCs can migrate through the polycarbonate filter toward K562 and A375 cells in the lower chamber in vitro. Fluorescence microscope detected the expression of EGFP, while both RT-PCR and Western blot detected high expression of CYP1A2/CYP2B6/CYP2C9 in gene-transfected group cells. Inverted microscope, MTT and Annexin V-FITC/PI confirmed that BMSCs transferred with CYP1A2/CYP2B6/CYP2C9 recombinant adenovirus vectors could activate DTIC/CPA and increase its anti-tumor effect(In the DTIC/CPA concentration(0.05 mmol/L/2.5 mmol/L) which BMSCs was relatively safe, the cell apoptosis was (38.38±2.27)% (P

Description: Abstract 4295 Syngeneic blood and marrow transplantation (BMT) has been applied in the treatment of many malignant or nonmalignant hematologic disorders with no or minimal and transient graft-versus-host disease (GVHD), much less transplant-related mortality (TRM) in contrast to allogeneic BMT, and lower relapse rate compared with autologous BMT. However, limited data in a single BMT center is not sufficient for statistical analysis. To evaluate the clinical outcomes of syngeneic BMT, CSBMT has performed a cooperative survey among BMT centers in mainland, Taiwan, and Hong Kong. From January 1964 to May 2009, 94 transplants from syngeneic donors have been performed in 32 BMT centers. The median age was 20 (1.5 to 51) years old. The diagnosis included AML (29 cases), SAA (26 cases), ALL (17 cases), CML (12 cases), lymphoma (3 cases), MDS (4 cases), neuroblastoma (2 cases), and large granular lymphocytosis (1 case). The main conditioning regimens were CYTBI or BUCY for malignant diseases, none or CY plus ATG for SAA. Bone marrow (BM, 34) or peripheral blood (PB, 49) or both BM and PB (11) as grafts were used. Five patients (SAA 2, AML 3) underwent the same donor's syngeneic BMT twice. One patient with large granular lymphocytosis and 1 case with SAA underwent the same donor's syngeneic BMT thrice. The median follow-up time was 28 months (1 month to 45 years). The median time for white blood cells 〉 1.0 × 109/L, and platelets 〉 20 × 109/L was 11 (2-30) days, 13 (0-122) days, respectively. Two patients (2.1%) had grade I acute GVHD (aGVHD), and 4 cases (4.3%) had grade II aGVHD. However, only one patient's specimen was consulted by pathologist. All aGVHD was controlled easily with low-dose steroid. No chronic GVHD was noted. Three-year disease-free survival (DFS) for the patients with nonmalignant disorders was 88.5%. Among them, the longest survivor was living and well for 45 years after transplant. Three-year DFS for the patients with malignant diseases was 62.9%. The overall survival rates at 3 years were 87.9%, and 69.5% for nonmalignant, and malignant diseases, respectively. 22 of 94 patients died after BMT (nonmalignant 3, malignant 19). The only cause of death for the patients with nonmalignant disorders was rejection. Relapse was the main cause of death in patients with malignancies (17/19). TRM was 2.1%. In conclusion, syngeneic BMT is a safe and effective therapeutic option for both nonmalignant and malignant hematologic disorders. Syngeneic donor, if available, should be the first choice in all cases of AA and hematological malignancies in general. The longest survivor of 45 years post-BMT is presented in this series. The good results and advantage of syngeneic BMT cast light on the potential utility of stored autologous placental-cord blood which is shared by the identical twin through the same placenta. Disclosures: No relevant conflicts of interest to declare.