ALBERT

Description: Author(s): Isao Ishii, Hitoshi Muneshige, Shuhei Kamikawa, Takahiro K. Fujita, Takahiro Onimaru, Naohiro Nagasawa, Toshiro Takabatake, Takashi Suzuki, Genki Ano, Mitsuhiro Akatsu, Yuichi Nemoto, and Terutaka Goto To investigate the origin of a phase transition at T Q =0.06 K simultaneously occurring with a superconducting transition in a cage compound PrRh 2 Zn 20 , we carried out ultrasonic measurements on a single-crystalline sample. The transverse modulus ( C 11 − C 12 )/2 is intimately coupled to the non-Kramers gro... [Phys. Rev. B 87, 205106] Published Tue May 07, 2013

Description: Erythropoietin (Epo) gene expression is under the control of hypoxia-inducible factor 1 (HIF-1), and is negatively regulated by GATA. Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α), which increase the binding activity of GATA and inhibit Epo promoter activity, are increased in patients with anemia of chronic disease (ACD). We previously demonstrated the ability of K-7174 (a GATA-specific inhibitor), when injected intraperitoneally, to improve Epo production that had been inhibited by IL-1β or TNF-α treatment. In the present study, we examined the ability of both K-11706, which inhibits GATA and enhances HIF-1 binding activity, and K-13144, which has no effect on GATA or HIF-1 binding activity, to improve Epo production following inhibition by IL-1β or TNF-α in Hep3B cells in vitro and in an in vivo mouse assay. Oral administration of K-11706 reversed the decreases in hemoglobin and serum Epo concentrations, reticulocyte counts, and numbers of erythroid colony-forming units (CFU-Es) induced by IL-1β or TNF-α. These results raise the possibility of using orally administered K-11706 for treating patients with ACD.

Description: The most characteristic clinical feature of PNH is intravascular hemolysis of PNH-affected RBC due to deficiency in the expression of GPI-anchored membrane proteins with complement-regulatory activity, CD55 and CD59. PNH-affected (CD55- and CD59-negative) RBC with complement sensitivity should have a shortened life span due to intravascular hemolysis, although life span of PNH-RBC separately from normal RBC could not be measured, at least clinically in PNH patients. We recently developed a sensitive flow cytometric method to analyse the PNH-phenotype (CD59-negative) in reticulocyte and whole RBC (Sato S et al: Reticulocyte-gated flow cytometric analysis of red blood cells in paroxysmal nocturnal hemogobinuria. Laboratory Hematology12: 82–85, 2006). This 2-color (RNA/CD59) flow cytometry could analyse the phenotype of reticulocyte fraction in detail, that enables to assess PNH phenotype of reticulocytes in several hematological conditions such as an emergence of RBC with PNH phenotype in aplastic anemia and a minimal residual production of PNH-RBC after hematopoietic stem cell transplantation for PNH. Analyses of PNH phenotype consecutively in total 7 patients with PNH showed differences in ratios between reticulocytes and whole RBC, where % CD59-negative population in the former was always higher than the latter due to hemolysis of CD59-negative mature RBC in the circulation. On an assumption that CD59-negative reticulocytes are not destroyed in the reticulocyte maturation time and CD59-positive RBC has a mean life span (MLS) of 120 days, we proposed a formula to estimate PNH-RBC’s MLS: W/100 = R x M / [ (100-R) x 120 + R x M ], where W, % CD59-negative whole RBC; R, % CD59-negative reticulocytes; M, MLS (day) of PNH-RBC. By this formula, PNH-RBC’s MLS was estimated at 16–45 days in the patients. It showed a poor correlation with absolute reticulocyte count or hemolysis-related laboratory data (lactate dehydrogenase (LD) etc), suggesting erythropoietic ability in response to anemia was different among PNH patients due to their various hematological backgrounds underlying PNH. Comparable degree of anemia did not induce same degree of reticulocytosis in these patients. Hence, we are proposing to use an erythropoietic ability-adjusted index, red cell turnover index (RCTI), calculated from the following formula to assess the total body hemolysis of PNH-RBC: PNH-RCTI = [(absolute reticulocyte count) x (% CD59-negative retiuclocyte) / 100] / (PNH-RBC’s MLS). PNH-RCTI is assumed to correlate with turnover rate of PNH-RBC by complement-mediated lysis in the circulation. PNH-RCTI ranged 0.88–15.3 x 103 reticulocytes/μl/day (reference range, normal RCTI 0.4–0.8). Serum LD level is a non-specifc parameter being increased by in vivo hemolysis, especially intravascular hemolysis. While PNH-RBC’s MLS did not show any significant correlation with serum LD levels (r=0.306), PNH-RCTI correlated positively with serum LD levels (r=0.704, 15 points) in 7 PNH patients. These data indicate that shortened PNH-RBC’s MLS estimated from the reticulocyte-gated flow cytometry could be a reliable parameter reflecting in vivo hemolysis in PNH.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is clinically characterized by the complement (C’)-mediated intravascular lysis of PNH-affected erythrocytes and thrombotic diathesis. Treatment methods of intravascular hemolysis, especially on the hemolytic crisis, have not been established yet. Although several reagents have been used for the treatment of PNH hemolysis, the value of prednisone/prednisolone in treating the hemolysis of PNH is controversial; anti-C5 antibody is under clinical study. We previously demonstrated that heparin and low-molecular weight heparin (LMWH) inhibit C’-mediated hemolysis of PNH erythrocytes in vitro, by inhibiting insertion of C5b-7 into the erythrocyte membranes (Ninomiya et al: Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparon. Brit J Haematol109,875–881,2000). However, that the concentration of heparin (or even that of LMWH) required to inhibit C’-mediated lysis of PNH erythrocytes efficiently induces a profound prolongation of APTT (activated partial thromboplastin time) of plasma unables their clinical application. In the current study, we examined the inhibitory effects of heparin, LMWH (dalteparin), and N-desulfated heparin on C’-mediated lysis of PNH erythrocytes (sucrose hemolytic assay) and their anticoagulant effects on normal plasma coagulation system (APTT, TT (thrombin time), and AT-III (antithrombin)-enhancing activity). N-desulfated heparin inhibited C’-mediated lysis of PNH erythrocytes, equivalently to heparin or LMWH, on the basis of their uronic acid (UA) contents; IC50 values of heparin, LMWH, and N-desulfated heparin, was 0.8 U/ml (UA 2.9 μg/ml), 0.5 IU/ml (UA 2.3μg/ml), and 3.0μg/ml (UA 0.9μg/ml), respectively. Whereas the concentrations of heparin and LMWH, that exerted their anti-C’ activity efficiently, induced prolongations of APTT profoundly to clinically unapplicable extents, N-desulfated heparin (up to 50 μg/ml) did not show any anticoagulant activity. These data show a potential value of N-desulfated heparin for the treatment of C’-mediated intravascular lysis of PNH erythrocytes without bleeding complications due to its administration. Because anticoagulant therapy may be beneficial and indicated for the treatment of hemolysis in PNH, especially in hemolytic crisis increasing the risk of thrombotic complications, cocktail therapy of heparin (or LMWH) with N-desulfated heparin may be proposed.

Description: Alloantigen targeting adoptive immunotherapy is a powerful therapeutic approach for the treatment of hematologic malignancies. A compelling example is represented by donor lymphocyte infusions after allogeneic hematopoietic stem cell transplantation. The clinical efficacy of T-cell based therapy relies not only on the ability of T cells to mediate a direct anti-tumor effect (GvT) and a protective immune response against pathogens, but also in their capacity to persist and expand in vivo, providing a long-term protection from disease relapse. Despite undeniable efficacy, the extensive exploitation of donor lymphocytes is limited by the risk of a severe and potentially life-threatening complication: Graft-versus-host disease (GvHD). To solve this double bind, we investigated the therapeutic potential of donor lymphocytes retrovirally transduced to express the suicide gene thymidine kinase of Herpes Simplex virus (TK) in patients affected by hematologic malignancies, and showed that the suicide machinery controls severe GvHD. To maximize the extent and persistence of GvT activity mediated by TK cells, we exploited the positive effects of IL-7 in maintaining the homeostasis of memory lymphocytes. We observed that while stimulation with anti-CD3 antibodies and culture in the presence of high doses of IL-2 generates mainly CD45RA−CD62L− effector memory (EM) TK cells with a limited ability to engraft and persist in vivo, stimulation with anti-CD3/CD28 conjugated cell sized beads and culture with low doses of IL-7 result in the generation of CD45RA−/CD62L+ TK cells with central memory (CM) functional phenotype, producing high levels of IL-2 upon stimulation, and expressing persistent high levels of IL-2R alpha and IL-7R alpha, a molecule associated to activation and long term survival memory T-cells, respectively. In mixed lymphocytes cultures (MLR), CM TK cells showed higher proliferative potential and lower sensitivity to apoptosis than EM TK cells, and such alloreactive CM TK cells kept CCR7 and IL-7R alpha expression even after 2ndary MLR. Accordingly, CM cells were more potent than EM cells in mediating allogeneic GvHD, after infusion in NOD/Scid mice, previously transplanted with allogeneic human skin. Newly developed homeostatic CM TK cells combine a high alloreactive potential with the selective sensitivity to GCV-mediated cell death, providing a tool for maximal anti-tumor activity with control of GvHD.

Description: Idiopathic thrombocytopenic purpura (ITP) is one of the major causes of thrombocytopenia. Currently, the diagnosis of ITP is principally based on the exclusion of other possible concurrent causes of thrombocytopenia. In the guidelines proposed by the American Society of Hematology, the panel recommended that no specific laboratory tests are considered necessary for the diagnosis. However, availability of reliable laboratory assays should be helpful in supporting the diagnosis of ITP. Recently, 2 of us (MK and YI) reported that erythrocyte count, leukocyte count, anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, reticulated platelets and thrombopoietin measured at first visit were useful to predict a future diagnosis of chronic ITP (manuscript submitted). To confirm this, we conducted a multicenter prospective study involving 113 patients with thrombocytopenia and a normal peripheral blood film at first visit. Patients with clinically apparent associated conditions that can cause thrombocytopenia were excluded. Each patient underwent physical examination and routine laboratory tests, and was prospectively followed for 〉6 months. Anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, reticulated platelets and plasma thrombopoietin were also examined at first visit. Clinical diagnosis was made in a blinded fashion based on bone marrow findings and the clinical course. Eighty-nine patients were diagnosed as having chronic ITP, and 24 had a non-ITP disorder, including 11 with aplastic anemia (AA), 10 with myelodysplastic syndrome (MDS), and one each with Fanconi anemia, May-Hegglin anomaly and myelofibrosis. Six laboratory findings were identified as initial parameters that discriminated future diagnosis of chronic ITP from non-ITP. These included the absence of anemia, absence of leukopenia, increased anti-GPIIb/IIIa antibody-producing B cell frequency, increased platelet-associated anti-GPIIb/IIIa antibodies, elevated reticulated platelet percentage and normal or slight increase of thrombopoietin (all for P 〈 0.001). Stepwise multiple regression analysis revealed that anemia, anti-GPIIb/IIIa antibody-producing B cell frequency, reticulated platelet percentage and thrombopoietin were factors that independently contributed to the later diagnosis of chronic ITP. Three or more of 6 ITP-associated laboratory findings were present at presentation in 78 (93%) patients later diagnosed as chronic ITP, compared with 6 (25%) patients whose disorder was non-ITP (P 〈 10−5). All 6 “false-positive” patients were diagnosed as AA or MDS, but 4 of them probably had overlapping immune thrombocytopenia. In summary, this multicenter prospective study confirms usefulness of 6 laboratory tests for the diagnosis of chronic ITP. The identification of ITP-associated laboratory findings encourages the future development of reliable diagnostic criteria for chronic ITP.

Description: To elucidate the contributions of GATA-1 to definitive hematopoiesis in vivo, we have examined adult mice that were rendered genetically defective in GATA-1 synthesis (Takahashi et al, J Biol Chem272:12611, 1997). Because the GATA-1 gene is located on the X chromosome, which is randomly inactivated in every cell, heterozygous females can bear either an active wild-type or mutant (referred to asGATA-1.05) GATA-1 allele, consequently leading to variable anemic severity. These heterozygous mutant mice usually developed normally, but they began to die after 5 months. These affected animals displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells) was expressed profusely in the abnormally abundant cells generated in the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally differentiate, this leads to progenitor proliferation in the spleen and consequently death. Thus, GATA-1 plays important in vivo roles for directing definitive hematopoietic progenitors to differentiate along both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human myelodysplastic syndrome and thus may serve as a useful model for this disorder.

Description: We studied the role of adenosine (Ado), which is generated from adenine nucleotides via the activity of ecto-5′-nucleotidase (ecto-5′-NT), in the inhibition of platelet aggregation by endothelial cells (ECs). The enzymatic activity of nucleotidases on human umbilical vein endothelial cells (HUVECs) was examined with regard to (1) the inhibition of adenosine diphosphate (ADP)–induced platelet aggregation and (2) the liberation of inorganic phosphate from adenine nucleotides. Adenosine 5′-monophosphate (AMP) preincubated with HUVECs significantly inhibited ADP-induced platelet aggregation. This was completely blocked by the treatment of HUVECs with a specific inhibitor of ecto-5′-NT, 5′-[αβ-methylene] diphosphate (APCP), or by the addition of an A2a receptor antagonist. Neither nitric oxide nor prostacyclin was involved in this inhibitory activity, suggesting that Ado generated in the incubation medium by the activity of 5′-NT on HUVECs inhibited platelet aggregation. When ADP was incubated on HUVECs, it lost most of its agonistic activity for platelets. Pretreatment of HUVECs with APCP at a concentration that abolished ecto-5′-NT activity partially restored ADP-induced platelet aggregation. Ecto-5′-NT contributes to EC function by inhibiting platelet aggregation in cooperation with ATP diphosphohydrolase, which degrades ADP to AMP.