ALBERT

Description: Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen–deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention.

Description: Patients with multiple myeloma (MM) are at increased risk of developing venous thromboembolism (VTE) compared to general individuals. With the introduction of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, substantially elevates the incidence of VTE. It was reported that patients with MM have significantly increased plasminogen activator inhibitor (PAI-1) level resulting in a decrease of fibrinolytic activity. To prevent VTE in MM patients receiving IMiDs, different prophylactic strategies such as low molecular weight heparin, aspirin and warfarin have been recommended. But most of those recommendations are based on limited evidence. Ginkgo biloba extract (GBE) is one of the most widely used herbal supplements, which has been used in thrombosis prevention. It prompts us to investigate the impact of thalidomide on fibrinolysis and whether GBE has affection on fibrinolysis. In this study, the human umbilical vein endothelial cells (HUVECs) were cultured alone or co-cultured with human multiple myeloma cell line (MMCs) in the medium with or without thalidomide and in the presence or absence of GBE. After 48 hours, culture supernatants were collected and protein concentration of t-PA and PAI-1 were analyzed by enzyme-linked immunosorbent assay. Results showed that MMCs and thalidomide can both altered the profibrinolytic potential of HUVECs by decreasing t-PA and increasing PAI-1 levels. When co-cultured with MMCs, thalidomide can further elevate PAI-1 levels but not further reduce t-PA. Whether HUVECs alone or co-cultured with MMCs, GBE was able to counteract these effects caused by thalidomide through increasing t-PA and decreasing PAI-1 levels. Results in this study indicate that GBE blunts the prothrombotic effect of thalidomide on HUVECs. It supports GBE clinic use for the prevention of VTE induced by IMiDs. Disclosures No relevant conflicts of interest to declare.

Description: Objective: Adult pure red cell aplasia (PRCA) is a syndrome characterized by a severe normocytic anemia, reticulocytopenia, and absence of erythroblasts from an otherwise normal bone marrow. Immunosuppressive therapy has been used as the initial treatment for acquired chronic PRCA. This article aims to evaluate the efficacy of cyclosporine A, and/or corticosteroids, and possible factors influencing it. Methods: 34 cases of PRCA were retrospectively analyzed at our institution.Clinical data of 23 inpatient cases and 11 outpatient cases from 2009 October to 2016 March were collected. These patients were treated by cyclosporine A (CsA), and/or corticosteroids (CS). Results: The causes of PRCA were as follows (table 1): idiopathic (15 cases, 44.1%), thymoma-associated (5 cases, 14.7%), and large granular lymphocyte (LGL) leukaemia-associated (7 cases, 20.6%), virus infection (4 cases, 11.7%), major ABO-mismatched allogeneic haematopoietic stem cell transplantation (1 case, 2.94%), rheumatoid arthritis (1 case, 2.94%), antierythropoietin antibody-mediated (1 case, 2.94%). One patient was lost to follow-up. Two patients were not evaluable for response due to short observation period (

Description: Hematopoietic microenvironment plays an important role in hematopoiesis, in which the bone marrow mesenchymal stem cells (BM-MSCs) can directly or indirectly affect the hematopoietic cells and regulate hematopoietic function. It has been reported that bone marrow hematopoietic progenitor cells were increased in p27 deficient mice, however, it is unclear whether BM-MSCs are mediated enhancing hematopoiesis resulting from p27 deficiency. To answer this question, the conditioned medium (CM) were harvested from BM-MSC cultures derived from WT and p27 knockout (KO) mice, respectively and added to the cultures of bone marrow cells derived from WT mice. After 4 days, the sca-1+ckit+Lin- cells (hematopoietic stem cells, HSC) and sca-1+ckit+Lin+ cells (hematopoietic progenitor cells, HPC) in the resulting cells were analyzed by flow cytometry, and the assay for colony forming cells (CFCs) was performed and the colony numbers of the CFC were counted. Results showed that the HPC fraction and the number of CFCs were increased significantly, but the HSC fraction was not increased in the bone marrow cell cultures with the CM from p27 KO mice compared with those with the CM from WT mice. To identify potential factors from the CM which enhance hematopoiesis caused by p27 deficiency, differences of protein expression profiles in the CM from the WT and p27 KO mice were examined by protein chip analysis. Five proteins were identified which were significantly increased in the CM from p27 KO mice, including interleukin-22 (IL-22), type I receptor of transforming growth factor-β, tumor necrosis factor- related apoptosis-inducing ligand, VE-cadherin and vascular endothelial growth factor B. To determine whether IL22, as a paracrine factor, plays a role in stimulating hematopoiesis by activating JAK-STAT signal pathway mediated by IL22 receptor A1 (IL22RA1), we examined the effect of IL22 on HPCs and the expression of signal molecules of JAK-STAT pathway. Results showed that the expression levels of IL22 at both mRNA and protein levels were up-regulated significantly in BM-MSCs derived from p27 deficient mice compared with those from WT mice. The HPC fraction and the number of CFCs were increased significantly in bone marrow cultures in the present of IL22, and were decreased significantly in the cultures with the CM from p27 knockout mice containing IL22 antibody. The percentages of IL22RA1, STAT3 and p-STAT3-S727 positive HPCs were increased significantly in the bone marrow from p27 KO mice compared with that from WT mice. Results in this study indicate that IL22, as a paracrine factor, might play an important role in stimulating hematopoiesis by activating JAK-STAT signal pathway mediated by IL22RA1. Disclosures No relevant conflicts of interest to declare.