ALBERT

Description: POEMS syndrome is a rare clonal plasma cell disorder without standard treatment. Based on the efficacy and low toxicity of a combination of melphalan and dexamethasone (MDex) for light chain amyloidosis, we conducted a prospective study of MDex treatment for patients with newly diagnosed POEMS syndrome. Thirty-one patients (19 men) were enrolled and the median age at the time of diagnosis was 44 years (range, 32-68 years). All patients received 12 cycles of MDex treatment. Twenty-five patients (80.6%) achieved hematologic response including 12 (38.7%) complete remission and 13 (41.9%) partial remission. Of all 31 patients, the neurologic response rate was 100%, assessed by overall neuropathy limitation scale (ONLS). The initial neurologic response was observed in 24 patients (77.4%) at 3 months after treatment and the median time to maximal neurologic response was 12 months (range, 3-15 months). Moreover, MDex substantially improved the level of serum vascular endothelial growth factor and relieved organomegaly, extravascular volume overload, and pulmonary hypertension. Only 6 patients (19.3%) suffered from grade 3 adverse events during treatment. All patients are alive and free of neurologic relapse after the median follow-up time of 21 months. Therefore, MDex is an effective and well-tolerated treatment option for patients with newly diagnosed POEMS syndrome.

Description: The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.

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: Key Points MGUS patients have significantly increased cortical bone porosity and reduced bone strength relative to matched controls.

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: Platelets play a supportive role in tumor metastasis. Impairment of platelet function within the tumor microenvironment may provide a clinically useful approach to inhibit metastasis. We developed a novel humanized single-chain antibody (scFv Ab) against integrin GPIIIa49-66 (named A11) capable of lysing activated platelets. In this study, we investigate the effect of A11 on the development of pulmonary metastases. In the Lewis lung carcinoma (LLC) metastatic model, A11 decreases the mean number of surface nodules and mean volume of pulmonary nodules. It protects against lung metastases in a time window that extended 4 hours before and 4 hours after the IV injection of LLCs. Coinjection of GPIIIa49-66 albumin reverses the antimetastatic activity of A11 in the B16 melanoma model, consistent with the pathophysiologic relevance of the platelet GPIIIa49-66 epitope. Significantly, A11 had no effect on angiogenesis using both in vitro and in vivo assays. The underlying molecular mechanisms are a combination of inhibition of each of the following interactions: between activated platelets and tumor cells, platelets and endothelial cells, and platelets and monocytes, as well as disaggregation of an existing platelet/tumor thrombus. Our observations may provide a novel antimetastatic strategy through lysing activated platelets in the tumor microenvironment using humanized anti–GPIIIa49-66 scFv Ab.

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: Abstract 4505 Background F Thrombotic and bleeding events are common and potentially fatal complications in patients receiving hematopoietic stem-cell transplantation (HSCT), which is profoundly associated with the survival of HSCT recipients. The hemostatic imbalance and endothelial injury are main manifestations in the course of HSCT, leading to thrombotic or bleeding disorders; however, there is still a lack of information on early differential diagnosis and prevention of those complications. In this study, we retrospectively investigated the outcomes and the risk factors of thrombotic and bleeding complications in HSCT recipients, and determined the clinical significance of hemostatic factors in thrombotic and bleeding events after HSCT. Methods F 423 hematologic patients receiving HSCT (113 auto-HSCT and 310 allo-HSCT recipients) were enrolled in the study, and their clinical manifestation and laboratory parameters were analyzed for evaluating the outcomes of hemostatic complications and related risk factors. Results: The overall incidence of bleeding disorders in 423 HSCT recipients was 65% (275 cases), in which 211 cases (76.8%) are allo-HSCT recipients. Bleeding was identified and evaluated based on a daily score of intensity. Minor bleeding was seen in 74.9% (206 cases), moderate bleeding was seen in 21.8% (60 cases), and severe bleeding was seen in 3.3% (9 cases) of all bleeding patients. The organs of hemorrhage involve skin or mucosa (36% in all HSCT recipients), gastrointestinal tract (36%), lung (1.2%), brain (0.4%), and urinary (39%). In regards to thrombotic complications, 12 recipients (2.8% in all HSCT recipients) developed thrombotic events, including 9 veno-occlusive diseases (VOD), 1 transplantation related thrombotic microangiopathy (TA-TMA), 1 pulmonary embolism (PE) and 1 deep vein thrombosis (DVT). The overall mortality after thrombotic events was 66.7% (8 cases) in all HSCT recipients with thrombotic complications. Both thrombotic and bleeding disorders were significantly correlated with age, disease category and pretreatment regimen (P0.05). No difference was found in the reconstruction time required for haematogenesis between recipients with or without bleeding disorders (P〉0.05), but the survival rate was correlated with the site and intensity of bleeding disorders (P