Molecular identification of Sicilian (deltaß)º-thalassemia associated with ß-thalassemia and hemoglobin S in Brazil

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (deltaß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (deltaß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(deltaß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (deltaß)º-thalassemia in association with ß-thalassemia documented at the molecular level.87387

Variant rh alleles and rh immunisation in patients with sickle cell disease

Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced. RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined. We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC. In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered.Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corr1317277FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Mechanical Properties Of Stored Red Blood Cells Using Optical Tweezers

We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.593016Ashkin, A., Dziedzic, J.M., Optical trapping and manipulation of viruses and bacteria (1987) Science, 235, pp. 1517-1520Barjas-Castro, M.L., Brandão, M.M., Fontes, A., Costa, F.F., Cesar, C.L., Saad, S.T.O., Elastic properties of irradiated red blood cell units measured by optical tweezer (2002) Transfusion, 42, pp. 1196-1199Brandão, M.M., Fontes, A., Barjas-Castro, M.L., Barbosa, L.C., Costa, F.F., Cesar, C.L., Saad, S.T.O., Optical tweezers for measuring red blood cell elasticity: Application to the study of drug response in sickle cell disease (2003) European Journal of Haematology, 70, pp. 207-211Williamson, L.M., Warwick, R.M., Transfusion-associated graft-versus-host disease and its prevention (1995) Blood Rev., 9, pp. 251-261Button, L.N., Dewolf, W.C., Newburger, P.E., The effecr of irradiation on blood components (1981) Transfusion, 21, pp. 419-426Platt, O.S., The sickle syndrome (1995) Blood: Principles and Practice of Hematology, , R. I Hadlin, S. E. Lux, T. P. Stossel, J. B. Lippincott, PhiladelphiaBallas, S.K., Dover, G.J., Charache, S., Effect of hydroxyurea on the rheological properties of sickle erythrocytes in vivo (1989) Am. J. Hematol, 32, pp. 104-111Groner, W., Mohandas, N., Bessis, M., New optical technique for measuring erythrocyte deformability with the ektacytometer (1980) Clin. Chem., 26, pp. 1435-1442De Franceschi, L., Bachir, D., Galacteros, F., Tchernia, G., Cynober, T., Alper, S., Platt, O., Brugnara, C., Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease (1997) J Clin Invest, 100, pp. 1847-1852Hochmuth, R.M., Worthy, P.R., Evans, E.A., Red cell extensional recovery and the determination of membrane viscosity (1979) Biophys. J., 26, pp. 101-114Evans, E.A., La Celle, P.L., Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation (1975) Blood, 45, pp. 29-43Itoh, T., Chien, S., Usami, S., Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation (1995) Blood, 85, pp. 2245-2253Evans, E.A., Mohandas, N., Membrane-associated sickle hemoglobin: A major determinant of sickle erythrocyte rigidity (1987) Blood, 70, pp. 1443-1449Dong, C., Chadwick, R.S., Schechter, A.N., Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation (1992) Biophys. J., 63, pp. 774-783Suzuki, Y., Tateishi, N., Cicha, I., Decreased deformability of the X-ray irradiated red blood cells stored in manitol-adenine-phosphate medium (2000) Clin. Hemorheol. Micro-cire., 22, pp. 131-14

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

As hypoxia-induced inflammatory angiogenesis may contribute to sickle cell disease manifestations, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from steady-state sickle cell anemia patients presented elevated concentrations of pro-angiogenic factors (Angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly augmenting endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice, compared with non-sickle cell disease mice, consistent with an upregulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy displayed a pro-angiogenic profile and had more significant effects on endothelial cell proliferation and capillary formation than plasma of patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factor profile, in association with an inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, sickle cell anemia and retinopathic hemoglobin SC individuals present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy for preventing progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.As hypoxia-induced inflammatory angiogenesis may contribute to sickle cell disease manifestations, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated an1006730739FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2008/57441-0; 2009/16334-0565036/201

Abnormal hedgehog pathway in myelodysplastic syndrome and its impact on patients' outcome

sem informação10012e491e493CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçãosem informaçã

IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in jak2v617f-positive myeloproliferative neoplasms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)The recurrent V617F mutation in JAK2 (JAK2(V617F)) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2(V617F) HEL cells, but not in the JAK2(WT) U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2(V617F)-positive but not JAK2(WT) specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34(+) cells from essential thrombocythemia patients compared to healthy donors, and in JAK2(V617F) MPN patients when compared to JAK2(WT). Our data indicate that IRS2 is a binding partner of JAK2(V617F) in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN.The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indi7669486959sem informaçãoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)sem informaçã