IntroductionPhiladelphia chromosome-negative myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic disorders that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). 1,2 Recent studies have confirmed the pathogenetic involvement of an acquired, somatic, gain-offunction, activating, point mutation JAK2V617F in MPNs. [3][4][5][6] This represents a guanine to thymidine mutation in exon 14 resulting in a valine to phenylalanine substitution at codon 617 in the JH2 or pseudokinase domain of the JAK2 gene (a member of the Janus kinase [JAK] family of nonreceptor tyrosine kinases, JAK1, JAK2, JAK3, and TYK2). 2,6 Highly sensitive assays for JAK2 have determined that the JAK2V617F mutation is present in 90% of patients with PV and approximately 50% to 60% of patients with ET or PMF. 7 In addition, a subset of patients, most commonly with PV, are homozygous for the JAK2V617F allele, the result of copy-neutral loss of heterozygosity at the JAK2 locus, especially in patients with PV. 2,7,8 Mutations in exon 12 of JAK2 are present in almost all patients with PV who are JAK2V617F negative. 9,10 The JAK proteins function in the cytoplasm to relay signals initiated by membrane-bound cytokine receptors. Engagement of the receptor results in the phosphorylation of the receptor and JAK2, which recruits its substrate proteins such as signal transducers and activators of transcription (STATs). 11,12 STATs, especially STAT3 and STAT5, translocate to the nucleus and transactivate many genes involved in cell proliferation and survival (eg, Bcl-xL, cyclin D1, and PIM1). 8,11,12 The V617F mutation in JAK2 also activates the downstream signaling pathways through the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK). This contributes to diminished apoptosis of the hematopoietic progenitor cells (HPCs). 2,8 Overexpression of JAK2V617F in murine Ba/F3 cells with coexpression of the erythropoietin receptor (EpoR) confers in vitro cytokine-independent growth. 3,13 Recently, it was shown that enforced expression of JAK2V617F in human hematopoietic stem cells and myeloid progenitors directed differentiation toward the erythroid lineage, along with increased expression and phosphorylation of GATA-1 and decreased expression of PU.1. 14-16 JAK2V617F expression in retroviral models and in transgenic mice is sufficient to cause myeloproliferative disorders in the mice that recapitulate many clinicopathologic features observed in human PV, ET, and PMF. [17][18][19][20][21] 22,23 In vivo studies in mouse models have also shown that mutant JAK2V617F represents a novel target for therapeutic intervention with JAK2-selective tyrosine kinase inhibitors in MPNs. 21,24 For example, TG101348 inhibits myeloproliferation and myelofibrosis in a murine model of JAK2V617F-induced polycythemia. 21,22 Early clinical trials of several JAK2-selective kinase inhibitors (eg, XL019, TG101348, and INCB18424) are under way in JAK2-driven MPNs with poor prognosis (eg, PMF). ...

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Neutralizing antibodies (inhibitors) to replacement Factor-VIII impair the effective management of hemophilia-A1. Individuals with hemophilia-A due to major F8 gene disruptions lack antigenically cross-reactive material in their plasma (CRM-negative) and prevalence of inhibitors is >60%. Conversely, subjects with missense mutations are CRM-positive and the prevalence of inhibitors is <10%2. Individuals with the intron-22-inversion (~50% of individuals with severe hemophilia-A) should be in the former group based on the genetic defect. Although these individuals are CRM-negative, only 20% of them develop inhibitors3. Here we demonstrate the presence of comparable levels of F8 mRNA and intracellular Factor-VIII protein in B-lymphoblastoid cells and liver biopsies from healthy controls and subjects with the intron-22-inversion. These results support the hypothesis that most individuals with the intron-22-inversion are tolerized to Factor-VIII and thus do not develop inhibitors. Furthermore we developed a pharmacogenetic algorithm that permits the stratification of inhibitor risk for sub-populations by predicting immunogenicity using, as input, the number of putative T-cell epitopes in the infused FVIII and the competence of MHC-Class-II molecules to present such epitopes. The algorithm exhibited significant accuracy in predicting inhibitors in 25 unrelated individuals with the intron-22-inversion (AUC = 0.890; P = 0.001).

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Background and Purpose-The effectiveness of prothrombin complex concentrate (PCC) products available in the UnitedStates that contain low levels of factor VII (3-factor PCC) has not been tested. The purpose of this study was to review our experience with 3-factor PCC (Profilnine) in the setting of warfarin-associated intracranial hemorrhage (wICH). Methods-In November 2007, we implemented a protocol for reversal of anticoagulation in wICH using Profilnine.Additional treatment with fresh-frozen plasma was at the discretion of the treating physician.

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Splenectomy indications and outcome were evaluated in 124 adults with hemoglobin SC disease (Hb SC). Twelve patients (9.6%) required splenectomy. There was a significant difference between the splenectomy group and the non-splenectomy group, respectively, regarding Hb levels (median 7.2 g/dL vs. 12.5 g/dL, P < 0.0001), platelet counts (median 146 x 10(6)/L vs. 275 x 10(6)/L, P = 0.031), palpable spleen rate (66% vs. 16%, P = 0.0003%), acute chest syndrome frequency (75% vs. 12%P = 0.0004) and cholecystectomy rate (66% vs. 13%, P = 0.0004). No significant morbidity or mortality occurred postsplenectomy. There is a subgroup of Hb SC patients requiring splenectomy, in which splenectomy is effective. Although it appears to be safe regarding short-term complications of surgery, long-term adverse effects such as infections have to be evaluated cautiously.

The deleterious effects of hemolysis through its end product cell free hemoglobin(Hb) as a nitric oxide (NO) scavenger is well established and has been incriminated in the pathogenesis of some complications of SCD such as pulmonary hypertension, leg ulcers, renal dysfunction, and possibly stroke. These observations have led some investigators to hypothesize that these complications form a subphenotype of SCD related predominantly to hemolysis and endothelial dysfunction. In hemolytic states Hb released from RBC complexes with Haptoglobin (Hp) and is removed from the circulation by macrophages and monocytes through binding CD163, the Hb scavenger receptor expressed on these cells. When the binding capacity of Hp is exceeded, the concentration of free Hb rises in the plasma. Hp is a polymorphic protein encoded by a gene on chromosome 16q2.2; there are two allelic variants, Hp 1 and Hp 2. Hp 2 is believed to have resulted from an intragenic duplication event, leading to an elongated Hp a-chain. Individuals homozygous for the long a2 chain express large multimeric molecules (Hp 2-2). During the past decade, a considerable body of evidence has accumulated suggesting that Hp-2 allele is a major susceptibility gene for the development of vascular complications (coronary artery restenosis and development of cardiovascular disease) especially in diabetic patients. It has been hypothesized that the Hb-Hp2 complexes have a 10-fold greater affinity for the CD 163 receptor, and the binding of Hb-Hp2 complexes generates a more powerful inflammatory response with a more prominent cytokine release. Recently, we performed a preliminary analysis of the distribution of Hp1 and Hp2 alleles among pediatric and adult SCD patients and reported a significantly higher allele frequency for Hp2 among pediatric patients, suggesting a survival advantage for carriers of Hp1 allele (Yaun et al, Blood, 2005). We now report the results of an exploratory in vitro study of cytokine release from purified mononuclear cells obtained from a normal control and an SCD patient following exposure to Hb-Hp1-1 and Hb-Hp2-2 complexes. Mononuclear cells (106/well) isolated by Ficoll-Hypaque density gradient were incubated with Hb A-Hp1-1, Hb A-Hp2-2, Hb S Hp1-1, and Hb S-Hp2-2 complexes with a 1:1 ratio (wt/wt) at a final concentration of 1 mg/ml. After 24 hr incubation at 37°C, the supernatants collected after centrifugation were used for cytokine assays by a multiplex bead method. A blank (medium only), Hb A and Hb S without Hp were also incubated with mononuclear cells. Multiplex bead assays showed that cytokine release (GM-CSF, IL-1b, IL-6, IL-10, and TNFa) was much higher (3–12 fold) from both the control and SCD mononuclear cells upon exposure to Hb-Hp2-2 complexes, but much less or no effect by Hb-Hp1-1. The fold induction of TNFa and IL-1b was much higher in SCD cells than in control cells. There was no significant difference between Hb A and Hb S in terms of cytokine release when they were complexed with either Hp1-1 or Hp2-2, suggesting that the cytokine release was predominantly related to Hp type but not to Hb. Pure Hb A and Hb S increased cytokines over the control (blank) but to a significantly smaller extent than Hb (A or S) Hp-2-2 complexes. These preliminary results are confirmatory of a deleterious effect of the Hp2-2 genotype through a more pronounced inflammatory response and are suggestive of a potential novel mechanism whereby hemolysis could result in adverse outcomes related to Hp polymorphisms. If confirmed in larger studies and through phenotypic associations, attenuation of this response via anti-inflammatory modalities may provide a therapeutic strategy. Figure Figure