ALBERT

Description: Introduction Hepatic veno-occlusive disease (VOD) and thrombotic microangiopathy (TMA) are important and serious thrombotic complications after hematopoietic stem cell transplantation (HSCT), but the early diagnosis remains difficult since their clinical manifestations are similar to those of other transplantation-related complications. Our aim in this study is to illustrate the early alteration of hemostatic parameters in recipients of hematopoietic stem cell transplantation and then determine its value in transplantation-related thrombotic complications and other post-HSCT clinical settings, such as acute graft-versus-host disease (aGVHD) and infection. Methods Plasma from 95 patients undergoing HSCT was collected prior to conditioning therapy and then weekly until five weeks after HSCT. Hemostatic parameters were evaluated prospectively in our institution. 1. Plasminogen activator inhibitor-1(PAI-1), tissue-plasminogen activator(t-PA), protein C(PC), von Willebrand factor(vWF)and thrombomodulin(TM)were investigated by enzymimmunoassay. Other hemostatic parameters such as activated partial thromboplastin time(APTT), prothrombin time(PT), fibrinogen (Fg), antithrombin III(AT III) and D-dimer(D-Di) were measured with hemagglutinin equipment in the same time. 2. According to the different settings after transplantation, three groups of transplant associated complications were classified as thrombus group (VOD n=5, TMA n=1), aGVHD group (n=29) and infection group (n=19). Systemic analyses were carried out for the hemostatic parameters and transplantation-related thrombotic complications or other clinical settings. Results Significant increase was observed in the levels of fibrinogen, t-PA and PAI-1 after transplant, while Protein C and ATIII decreased significantly(P0.05). All the patients with three different complications presented with significantly increased PAI-1 and lower level of Protein C compared with those who had no complication (P0.05). However, 6 patients with thrombotic complications (VOD5, TMA1) extremely showed elevated PAI-1 levels after the clinical onset of thrombotic complications by comparison with highest post-HSCT values in the aGVHD patients or infection patients (P

Description: Osteoporosis is a very common problem among adolescent and adult patients with thalassaemia major (TM). The pathogenesis of osteoporosis in TM is related to several factors including iron overload. Bone is derived from osteoblasts. And osteoblasts are differentiated from mesenchymal stem cells (MSC). Therefore, iron-overload induced MSC damage may contribute to osteopenia and osteoporosis. The effect of iron-overload on MSC has not been investigated previously. We hypothesize that iron-overload may induce apoptosis in MSC by caspase-dependent pathway, and haematopoietic growth factor thrombopoietin (TPO) and calcium channel blocker amlodipine may have a protective effect on iron-induced apoptosis in these cells. We have shown that iron (FeCl3) reduced hMSCs viability in a dose-dependent manner (0–0.6 mM) (n=5). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM) for 72 hrs (n=4). The expression of active caspase-3 was significantly increased in iron-treated cells (0.15mM, 0.3mM) (n=5). Iron treatment also increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential. TPO exerted protective effect on iron-induced apoptosis in hMSCs. Human MSCs were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/ml) for 72 hrs (n=4). The cell viability was significantly increased with the treatment of TPO. Dot-plot analysis of annexin V/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells. Incubation with TPO also decreased the iron-induced caspase-3 expression. Flow cytometric dot-plot analysis of hMSCs also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO. The population of phospho-Erk1/2 was also significantly increased in TPO-treatment, and the increased phospho-Erk was significantly reversed by the upstream signaling inhibitor PD098059. Calcium channel blocker amlodipine (10−9M) also had a protective effect on iron-induced apoptosis in these cells. Our findings suggest that iron-overload induces apoptosis in hMSCs via the caspase-dependent pathway and that TPO and amlodipine might exert a protective effect on iron-induced apoptosis via the activation of Erk1/2 signaling. The use of either haematopoietic growth factor or calcium channel blocker for the protection of hMSCs from iron induced toxicity is a novel concept. Our study has the potential in minimizing the bone damage induced by iron-overload in patients with thalassaemia major.

Description: Patients with myeloproliferative disorders (MPD) are at a high risk of developing thrombotic events. We hypothesize that one of the contributory factors to this thrombotic tendency is the involvement of vascular endothelial cells (EC) by the malignant process. In vitro and in vivo assays were used to determine the involvement of EC in patients with MPD. Endothelial progenitor cells (EPC) were assayed from the peripheral blood (PB) mononuclear cells (MNCs) of 3 normal controls (NC) and 16 patients with MPD (12 polycythemia vera (PV), 4 primary myelofibrosis (PMF). MNC were cultured for 2 days in EC growth media on fibronectin(FN)-coated plates. The non-adherent cells were then harvested and transferred to a secondary FN-coated plate for additional 5–14 days. EC colonies were identified by their morphological appearance. The colonies were plucked and analyzed for PECAM-1(CD31), VE-Cadherin(CD144), VEGFR-2, vWF, Endoglin(CD105), ULEX-1, CD45, CD14 by flow cytometry and acetylated LDL(Ac-ADL) uptake. EC colonies were CD31+CD144+VEGFR2+ULEX-1+vWF+CD105+CD45+CD14+ and capable of taking up Ac-LDL and when exposed to TNF-α and IL-1β, expressing ICAM(CD54) and E-selectin(CD62e). MPD MNC formed fewer numbers of EC colonies than normal MNC (31.1±34.2 vs 78.8±28.9; p

Description: Primary myelofibrosis (PMF) is a myeloproliferative disorder characterized by abnormal trafficking of CD34+ cells, resulting in their constitutive mobilization. This abnormal trafficking of PMF CD34+ cells has been related to the reduced expression of the chemokine receptor CXCR4 (Shi et al, Cancer Research67: 6417, 2007). We further tested this hypothesis by studying the homing of PMF CD34+ cells to the marrow and the spleens of sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and determining if this defect could be corrected by treatment with chromatin modifying agents. Following the infusion of PMF (n=10) or G-CSF mobilized peripheral blood (mPB) (n=4) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and assayable human CFU-GM were detected in the marrow of these mice (139±47 PMF CD34+ cells/106 BMCs vs. 1493±946 mPB CD34+ cells/106 BMCs, 2.0±0.8% of input PMF CFU-GM vs. 12.6±6.6% of input mPB CFU-GM, P

Description: HIV-ITP patients have a unique Ab against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell106: 551, 2001; JCI113: 973, 2004). Since HCV-ITP, like HIV-ITP, is associated with circulating immune complexes, we asked whether the complexes could contain platelet fragments induced by oxidative platelet fragmentation and whether HCV-ITP could be induced by molecular mimickry with an HCV peptide. The incidence of Hepatitis-C related ITP varies from 10–40% increasing with severity of liver disease. HIV-ITP is more frequent in drug abusers compared to non-drug abusers (37% vs 16%); and more severe in HIV-drug abusers than non-drug abusers (platelets

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: The Philadelphia chromosome negative (Ph−) myeloproliferative disorders (MPD), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are thought to result from the transformation of a multipotent hematopoietic stem cell (HSC). These Ph− MPDs are characterized by trilineage bone marrow hyperplasia with increased production of red cells, granulocytes and platelets which largely determines their clinical manifestations. Pruritus is common in Ph− MPDs occurring in approximately 50% of cases. The itching occurs spontaneously or appears when taking a hot shower or following other sudden environmental changes. In the present study, we explored the hypothesis that mast cells (MCs) play an important role in pruritogenesis in Ph− MPDs. PB CD34+ cells from 18 PV, 11 PMF, and 7 G-CSF mobilized (Gmob) volunteers were cultured in the presence of SCF (100ng/ml) and IL-6 (50ng/ml) for 7–8 weeks. After 49 days of culture, Gmob PB CD34+ cells generated significantly greater numbers of MCs (range: 5.6–20.1×106; mean±SD: 11.3±5.2×106) than PV (0.5–3.8×106; 1.2±0.9×106; p

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: Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)- mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Restricting transgene expression to late erythroblasts and red blood cells (RBC) may reduce the risk of activating oncogenes in HSC and its offspring in all lineages. In this study, we sought to evaluate the feasibility of redirecting a fraction of robust protein-synthesis machinery in maturing erythroid cells for production of alpha-L-iduronidase (IDUA), the lysosomal enzyme missing in Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). We first utilized a murine erythroid leukemia (MEL) cell line to compare IDUA expression and release from LVs using human elongation factor 1α promoter (EF), LTR of SFFV (SF), or a erythroid specific hybrid promoter (IHK) containing human ALAS2 intron 8 erythroid specific enhancer, HS40 core element from human alpha LCR and human ankyrin-1 promoter. We have previously shown highly erythroid-specific expression in vivo using this hybrid promoter, which is sustained in secondary transplantation recipient mice and resists proviral silencing (Moreau-Gaudry et al. Blood, 2001; Mohamedali et al, Mol Ther 2004). MEL cells were introduced to differentiation with hexamethylene bis-acetamide, and progressive erythroid differentiation was confirmed by morphologic evaluation in cytospins. Relatively low levels of expression from IHK (5% of SF, and 8% of EF) were observed in non-induced MEL cells; however, it increased steadily during induction and reached a similarly high expression level as those from the SF promoter. Expression from EF promoter reduced significantly, and the levels from SF promoter remained unchanged with erythroid differentiation. A similar pattern was found in IDUA activities released from stably transduced MEL cells during induction. We then evaluated in vivo the systemic IDUA production in an enzyme-deficient MPS I mouse model using erythroid-specific LV. Lineage-negative bone marrow cells (Lin−) were isolated with 92–97% purity by lineage depletion using immunomagnetic cell sorting. After a short pre-stimulation period, Lin− cells were transduced twice with LV-IHK-IDUA-ires-GFP for a total multiplicity of infection (MOI) at 2–20, resulting in up to 71% transduction efficiency. Four-months after transplantation of Lin- cells transduced at high MOI, sustained and higher-than normal plasma IDUA levels were achieved in all treated MPS I mice (1.5 to 8-fold of normal levels) with GFP transgene expression detected in up to 7% of Ter119+ blood cells. To evaluate transgene expression pattern during erythroid differentiation in bone marrow, we defined erythroblast subpopulations by immunostaining with CD71 and Ter119 for enrichment in proerythroblasts (I), basophilic erythroblasts (II), polychromatophilic erythroblasts (III), and orthrochromatic erythroblasts and reticulocytes (IV). GFP expression was predominantly detected in population III (9–17.3%), followed by the most differentiated population IV. To determine transduction efficiency in HSC, spleen colony-forming-cell assay was performed that showed 15–25% GFP+ spleen colonies in 2o BMT recipients. Moreover, long-term systemic metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in liver and spleen, with relatively moderate improvement in brain. These results demonstrate for the first time that a lysosomal enzyme can be produced and secreted successfully and steadily at superphysiological levels in circulation by erythroid cells using tissue-specific LV, with phenotypic correction of Hurler Syndrome in mice. This data warrants further evaluation for the potential application of erythroid-specific vectors in treating non-RBC related diseases.