BLOOD First Edition Papers

Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal-center B cells

Thu, 03 Dec 2009 13:46:14 PST

BCL6 is a transcriptional repressor required for mature B-cell germinal center (GC) formation and implicated in lymphomagenesis. BCL6 physiologic function is only partially known since the complete set of its targets in GC B-cell has not been identified. To address this issue, we used an integrated biochemical-computational-functional approach to identify BCL6 direct targets in normal GC B-cells. This approach includes: i) identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation, ii) inference of transcriptional relationships using a regulatory network reverse engineering approach (ARACNe), and iii) validation of physiologic relevance of the candidate targets down-regulated in GC B-cells. Our approach showed that a large set of promoters (>4000) is physically bound by BCL6, but that only a fraction of them is subjected to transcriptional repression in GC B-cells. This set of 1,207 targets identifies a number of cellular functions directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine signaling, toll-like receptor signaling and differentiation. These results define a broad role of BCL6 in preventing centroblasts from responding to signals leading to exit from the GC before they complete the phase of proliferative expansion and immunoglobulin gene remodeling necessary for their selection based on antibody affinity.

Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion

Thu, 03 Dec 2009 13:46:05 PST

Human plasmacytoid dendritic cells (pDC) are crucially involved in the modulation of adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. In several of these diseases elevated extracellular levels of the serine protease granzyme B (GrB) are observed. Here we demonstrate that human pDC can be an abundant source of GrB and that such GrB⁺ pDC potently suppress T cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. Moreover, we show that GrB expression is strictly regulated on a transcriptional level involving JAK1, STAT3 and STAT5 and that IL-3, a cytokine secreted by activated T cells, plays a central role for GrB induction. Moreover, we find that the immunosuppressive cytokine IL-10 enhances, while toll-like-receptor agonists and CD40 ligand strongly inhibit GrB secretion by pDC. GrB-secreting pDC may play a regulatory role for immune evasion of tumors, anti-viral immune responses, and autoimmune processes. Our results provide novel information about the complex network of pDC-T cell interactions and may contribute to an improvement of prophylactic and therapeutic vaccinations.

Suppression of B cell lymphomagenesis by theBH3-only proteins Bmf and Bad

Thu, 03 Dec 2009 13:45:58 PST

Oncogenic c-Myc is known to balance excessive proliferation by apoptosis that can be triggered by p53-dependent and p53-independent signaling networks. Here, we provide evidence that the "BH3-only" pro-apoptotic Bcl-2 family members Bmf and Bad are potent antagonists of c-Myc-driven B cell lymphomagenesis. Tumor formation was preceded by accumulation of preneoplastic pre-B and immature IgM⁺ B cells in hematopoietic organs of Eµ-myc/bmf^-/- mice, whereas Eµ-myc/bad^-/- mice showed an increase of pre-B cells limited to the spleen. While loss of Bad had no impact on the tumor immunophenotype, Bmf-deficiency favored the development of IgM⁺ B cell over pre-B cell tumors. This phenomenon was due to a strong protection of immature IgM⁺ B cells from oncogene-driven apoptosis caused by loss of bmf and c-Myc-induced repression of Bmf expression in premalignant pre-B cells. Steady-state levels of B cell apoptosis were also reduced in the absence of Bad, in support of its role as a sentinel for trophic factor-deprivation. Loss of Bmf reduced the pressure to inactivate p53, whereas Bad-deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc.

CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain engineered monoclonal antibody

Wed, 02 Dec 2009 14:16:16 PST

CD19 is a B-cell specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified Fc-domain designed to enhance binding of FcRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity and antibody dependent cellular phagocytosis but not complement mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC as compared to both the humanized anti-CD19 non-engineered antibody it is derived from and also Rituximab, a therapeutic antibody widely utilized in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a Granzyme B dependent mechanism. The NK-cell mediated cytolytic and secretory function with XmAb5574 as compared to the non-engineered antibody is associated with enhanced NK cell activation, interferon production, Erk1/2 phosphorylation downstream of Fc receptor and no increased NK cell apoptosis. Notably, enhanced NK-cell mediated ADCC with Xm5574 was enhanced further by Lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with Lenalidomide for the therapy of CLL and related CD19⁺ B-cell malignancies.

SNP array analysis of tyrosine kinase inhibitor (TKI) resistant chronic myeloid leukemia (CML) identifies heterogeneous secondary genomic alterations

Wed, 02 Dec 2009 14:16:05 PST

To elucidate whether tyrosine kinase inhibitor (TKI) resistance in CML is associated with characteristic genomic alterations, we analyzed DNA samples from 45 TKI resistant CML patients with 250K single nucleotide polymorphism (SNP) arrays. From 20 patients, matched serial samples of pre-treatment and TKI resistance time points were available. 11 of the 45 TKI resistant patients had mutations of BCR-ABL1, including two T315I mutations. Besides known TKI resistance associated genomic lesions such as duplication of the BCR-ABL1 gene (n=8) and trisomy 8 (n=3), recurrent submicroscopic alterations including acquired uniparental disomy were detectable on chromosomes 1, 8, 9, 17, 19 and 22. On chromosome 22, newly acquired and recurrent deletions of the IGLC1 locus were detected in three patients, who had previously presented with lymphoid or myeloid blast crisis. This may support a hypothesis of TKI induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity.

Alterations of the systemic environment are the primary cause of impaired B- and T-lymphopoiesis in telomere dysfunctional mice

Wed, 02 Dec 2009 14:15:51 PST

There is growing evidence that telomere dysfunction can contribute to human aging. Telomere dysfunction limits lymphopoiesis in aging telomerase knockout (mTerc^-/-) mice primarily by the induction of stem cell extrinsic alterations. The relative contribution of alterations in the stem cell niche and the systemic environment to the impairment of lymphopoiesis in response to telomere dysfunction is currently unknown. This study revealed a minor impact of stem cell intrinsic defects on the impairment of B- and T- lymphopoiesis in response to telomere dysfunction. The impairment in B- and T-lymphopoiesis in aging telomere dysfunctional mice was mainly due to alterations of the systemic environment. Telomere dysfunction had no significant cell-autonomous effects impairing the function of thymic or bone marrow niches in supporting B- and T-lymphopoiesis. Moreover, age-related alterations in the cellular composition of the thymic epithelium in telomere dysfunctional mice were rescued by transplantation of the thymus into a wildtype environment and these rejuvenated thymi supported normal T-lymphopoiesis in recipient mice. Together, these data place alterations in the systemic environment on top of the hierarchy of events limiting lymphopoiesis in response to telomere dysfunction.

CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus

Tue, 01 Dec 2009 13:44:15 PST

T lymphopoiesis requires settling of the thymus by bone marrow derived precursors throughout adult life. Progenitor entry into the thymus is selective, but the molecular basis of this selectivity is incompletely understood. The chemokine receptor CCR9 has been demonstrated to be important in this process. However, progenitors lacking CCR9 can still enter the thymus, suggesting a role for additional molecules. Here we report that the chemokine receptor CCR7 is also required for efficient thymic settling. CCR7 is selectively expressed on bone marrow progenitors previously shown to have the capacity to settle the thymus, and CCR7^-/- progenitors are defective in settling the thymus. We further demonstrate that CCR7 sustains thymic settling in the absence of CCR9. Mice deficient for both CCR7 and CCR9 have severe reductions in the number of early thymic progenitors, and in competitive assays CCR7^-/-CCR9^-/- double knock-out progenitors are almost completely restricted from thymic settling. However, these mice possess near-normal thymic cellularity. Compensatory expansion of intrathymic populations can account for at least a part of this recovery. Together our results illustrate the critical role of chemokine receptor signaling in thymic settling and help to clarify the cellular identity of the physiologic thymic settling progenitors.

Regulation of human NK cell cytokine and chemokine productionby target cell recognition

Fauriat, C., Long, E. O., Ljunggren, H.-G., Bryceson, Y. T. — Tue, 01 Dec 2009 13:44:07 PST

NK cell recognition of infected or neoplastic cells can induce cytotoxicity and cytokine secretion. So far, it has been difficult to assess the relative contribution of multiple NK cell activation receptors to cytokine and chemokine production upon target cell recognition. Using Drosophila cells expressing ligands for the NK cell receptors LFA-1, NKG2D, DNAM-1, 2B4, and CD16, we studied the minimal requirements for secretion by freshly isolated, human NK cells. Target cell stimulation induced secretion of predominately pro-inflammatory cytokines and chemokines. Release of chemokines MIP-1, MIP-1β, and RANTES was induced within one hour of stimulation, whereas release of TNF- and IFN- occurred later. Engagement of CD16, 2B4, or NKG2D sufficed for chemokine release, whereas induction of TNF- and IFN- required engagement of additional receptors. Remarkably, our results revealed that, upon target cell recognition, CD56^dim NK cells were more prominent cytokine and chemokine producers than CD56^bright NK cells. The present data demonstrate how specific target cell ligands dictate qualitative and temporal aspects of NK cell cytokine and chemokine responses. Conceptually, the results point to CD56^dim NK cells as an important source of cytokines and chemokines upon recognition of aberrant cells, producing graded responses depending on the multiplicity of activating receptors engaged.

Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long term correction of oxidase activity in peripheral blood neutrophils

Tue, 01 Dec 2009 13:43:58 PST

Chronic granulomatous disease (CGD) is associated with significant morbidity and mortality from infection. The first CGD gene therapy trial¹ resulted in only short term marking of 0.01-0.1% of neutrophils. Recently, Ott et al.², using busulfan conditioning and an SFFV retrovirus vector achieved >20% marking in two patients with X-linked CGD. However, oxidase correction per marked neutrophil was less than normal and not sustained. Despite this, patients clearly benefited in that severe infections resolved. As such, we initiated a gene therapy trial for X-CGD to treat severe infections unresponsive to conventional therapy. We treated three adults using busulfan conditioning and an MFGS retroviral vector encoding gp91^phox, achieving early marking of 26%, 5% and 4% of neutrophils, respectively, with sustained long term marking of 1.1% and 0.03% of neutrophils in two of the patients. Gene marked neutrophils have sustained full correction of oxidase activity for 34 and 11 months, respectively, with full or partial resolution of infection in those two patients. Gene marking is polyclonal with no clonal dominance. We conclude that busulfan conditioning together with an MFGS vector is capable of achieving long term correction of neutrophil oxidase function sufficient to provide benefit in management of severe infections. This study is registered at http://clinicaltrials.gov as NCT00394316.

FOXO transcription factor activity is partially retained in quiescent CML stem cells and induced by tyrosine kinase inhibitors in CML progenitor cells

Tue, 01 Dec 2009 13:43:49 PST

Chronic Myeloid Leukaemia (CML) is initiated and maintained by the tyrosine kinase BCR-ABL. ABL-specific tyrosine kinase inhibitors (TKIs), whilst effective against mature CML cells, induce little apoptosis in stem/progenitor cells. However, in stem/progenitor cells TKIs exert potent anti-proliferative effects through a poorly understood mechanism. We showed that in CD34⁺ CML cells FOXO1, 3a and 4 (FOXOs) were phosphorylated, predominantly cytoplasmic and inactive, consequent to BCR-ABL expression. TKIs decreased phosphorylation of FOXOs, leading to their re-localisation from cytoplasm (inactive) to nucleus (active), thus inducing G1 arrest. Of key importance, despite BCR-ABL activity, primitive quiescent CML stem cells showed low levels of FOXO phosphorylation and predominant nuclear localisation, resembling the pattern in normal stem cells. These results demonstrate for the first time that TKI-induced G1 arrest in CML progenitor cells is mediated by re-activation of FOXOs, whilst quiescence of CML stem cells is regulated by sustained FOXO activity. These data contribute to our understanding of CML stem cell quiescence and TKI activity, suggesting new strategies to target CML stem/progenitor cells by preventing or reversing this effect.

Melphalan 200 mg/m2 versus Melphalan 100 mg/m2 in newly diagnosed myeloma patients: a prospective, multi-center phase III study

Tue, 01 Dec 2009 13:43:41 PST

High-dose (200 mg/m², MEL200) and intermediate-dose melphalan (100 mg/m², MEL100) showed significant activity in myeloma. Differences in toxicity and efficacy between them have never been evaluated. In a phase III study, 298 patients were randomly assigned to receive two autologous transplantations after conditioning with MEL200 or MEL100. Ninety-six/149 (64%) completed the MEL200 arm, 103/149 (69%) the MEL100 arm. Best response to MEL200 was: complete remission (CR) 22/149 (15%); partial remission (PR) 95/149 (64%), for an overall response rate of 79%. Best response to MEL100 was: CR 12/149 (8%); PR 95/149 (64%), for an overall response rate of 72%. Overall-survival did not differ (p=0.13); median progression-free-survival (31.4 vs 26.2 months, p=0.01), median time-to-progression (34.4 vs 27.0 months, p=0.014) were longer in the MEL200. Treatment-related mortality was 3.1% in the MEL200 and 2.9% in the MEL100 group. The incidence of severe neutropenia and infections were marginally superior; whereas severe thrombocytopenia, mucositis, gastrointestinal adverse events and the overall occurrence of at least one non-hematological grade 3-4 adverse event were significantly higher in the MEL200 cohort. We conclude that MEL200 leads to longer remission duration and should be considered the standard conditioning regimen for autologous transplantation. This study is registered at http://clinicaltrials.gov as NCT00950768.

The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome

Mon, 30 Nov 2009 14:36:53 PST

Multiple Myeloma (MM) is a plasma cell (PC) neoplasm that proceeds through a pre-malignant state of monoclonal gammopathy of unknown significance (MGUS), however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared to normal PCs and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1 and the Sumo-inducer Arf5 were elevated in MM patient samples and cell lines. Survival correlated with expression since 80% of patients with low UBE2I and PIAS1 were living 6 years after transplant, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I (UBE2I-DN) mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell (BMSC) and decreased BMSC-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

p38 mitogen-activated protein kinase activation during platelet storage: consequences for platelet recovery and hemostatic function in vivo

Mon, 30 Nov 2009 14:36:42 PST

Platelets undergo several modifications during storage which reduce their post transfusion survival and functionality. One important feature of these changes, which are known as platelet storage lesion, is the shedding of the surface glycoproteins GPIb and GPV. We recently demonstrated that TNF-alpha converting enzyme (TACE/ADAM17) mediates mitochondrial injury-induced shedding of adhesion receptors and that TACE activity correlates with reduced post transfusion survival of these cells. We now confirm that TACE mediates receptor shedding and clearance of platelets stored for 16 hours at 37°C or 22°C. We further demonstrate that both storage and mitochondrial injury lead to the phosphorylation of p38 MAPK in platelets and that TACE-mediated receptor shedding from mouse and human platelets requires p38 MAP kinase signaling. Protein kinase C, ERK MAPK, and caspases were not involved in TACE activation. Both inhibition of p38 MAPK and inactivation of TACE during platelet storage led to a markedly improved post transfusion recovery and hemostatic function of platelets in mice. p38 MAPK inhibitors had only minor effects on the aggregation of fresh platelets under static or flow conditions in vitro. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Gene expression profiling identifies emerging oncogenic pathways operating in extranodal NK/T-cell lymphoma, nasal-type

Mon, 30 Nov 2009 14:36:32 PST

Biopsies and cell lines of NK/T-cell lymphoma, nasal-type (NKTCL) were subject to combined gene expression profiling and array-based comparative genomic hybridization analyses. Compared to PTCL, NOS, NKTCL had higher transcript levels for NK-cell markers and cytotoxic molecules, especially granzyme H, a novel sensitive biomarker of NKTCL. Compared to normal NK cells, tumors were closer to activated than resting cells and overexpressed several genes related to vascular biology, EBV-induced genes and PDGFRA. Notably, PDGFR and its phosphorylated form were confirmed at the protein level, and in vitro the MEC04 NKTCL-cell line was sensitive to imatinib. Deregulation of the AKT, JAK-STAT and NF-B pathways suggested by bioinformatical analysis, was corroborated by nuclear expression of phosphorylated AKT, STAT3 and RelA in NKTCL, and several deregulated genes in these pathways mapped to regions of recurrent copy number aberrations (AKT3 (1q44), IL6R (1q21.3), CCL2 (17q12), TNFRSF21 (6p12.3)). Several features of NKTCL uncovered by this analysis (overexpression of VEGFA and its receptor KDR by the tumor cells, overexpression of MET-HGF) suggest perturbation of angiogenic pathways. Integrative analysis also evidenced deregulation of the tumor suppressor HACE1 in the frequently deleted 6q21 region. This study highlights emerging oncogenic pathways in NKTCL and identifies novel diagnostic and therapeutic targets.

Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug

Mon, 30 Nov 2009 14:36:22 PST

Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled-AnxA5 and also imaged with atomic force microscopy (AFM). IgG levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells (HUVECs) and a syncytialized trophoblast cell line (STCs). AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by AFM. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of HUVECs and STCs, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs, cultured cells and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

Liprin {beta}1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity

Mon, 30 Nov 2009 14:36:11 PST

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response and lipid absorption, and the development of in vitro models should allow for better understanding of the mechanisms regulating lymphatic vascular growth, repair and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. We furthermore identify liprin β1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.

Hereditary fibrinogen A {alpha}-chain amyloidosis: phenotypic characterization of a systemic disease and the role of liver transplantation

Mon, 30 Nov 2009 14:36:01 PST

Variants of fibrinogen A -chain (AFib) cause the commonest type of hereditary renal amyloidosis in in Europe, and, possibly in the USA as well. Variant fibrinogen is produced in the liver, and solitary renal allografts fail within 1-7 years with recurrent amyloidosis. We assessed 22 patients with AFib for combined liver and kidney transplantation (LKT) and report the clinical features and outcome. Twenty-one had E526V, and one the R554L variant. Coronary atherosclerosis was identified in 68% of cases, and systemic atheromatosis in 55%. Vascular atheroma excised at endarterectomy and endomyocardial biopsies contained purely variant fibrinogen amyloid. Half of cases had autonomic neuropathy. Six of the nine patients who received LKT are alive (cumulative survival 67%), with good allograft function and no amyloidosis at median 67 (33-155) months' follow-up. Serial ^99mTc-DMSA renal scintigraphy in 2 cases of pre-emptive LKT before haemodialysis demonstrates preserved native kidney residual function at 5 years follow-up. Four explanted livers were used successfully for domino transplantation. Fibrinogen amyloidosis is a systemic amyloid disease with visceral, vascular, cardiac and neurological involvement. LKT is curative, however, cardiovascular amyloidosis may preclude this option. Our data encourage evaluation of pre-emptive solitary liver transplantation early in the course of amyloid nephropathy to prevent haemodialysis and kidney transplantation.

Immunomodulatory derivatives (IMiDs) induce PU.1 downregulation, myeloid maturation arrest, and neutropenia

Wed, 25 Nov 2009 12:16:47 PST

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma (MM) but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, Cathepsin G (CG) stored in the azurophilic granules of promyelocytes. High levels of CG subsequently may increase the risk of VTE. To our knowledge this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in MM.

TNF-related apoptosis-inducing ligand1 (TRAIL1) enhances the transition of red blood cells from the larval to adult type during metamorphosis in Xenopus

Tamura, K., Mawaribuchi, S., Yoshimoto, S., Shiba, T., Takamatsu, N., Ito, M. — Wed, 25 Nov 2009 12:16:38 PST

The transition of red blood cells (RBCs) from primitive to definitive erythropoiesis is conserved across vertebrates. In anuran amphibians, the larval RBCs from primitive erythropoiesis are replaced by adult RBCs from definitive erythropoiesis during metamorphosis. The molecular mechanisms by which the primitive (larval) blood cells are specifically removed from circulation are not yet understood. In this study, we identified Xenopus TNF-related apoptosis-inducing ligand1 (xTRAIL1) and xTRAIL2 as ligands of Xenopus death receptor-Ms (xDR-Ms), and investigated whether TRAIL signaling could be involved in this transition. The xTRAIL and xDR-M genes were highly expressed in the liver and RBCs, respectively, during metamorphosis. Interestingly, xTRAIL1 enhanced the transition of the RBCs, and a dominant-negative form of the xTRAIL1 receptor attenuated it, when injected into tadpoles. Moreover, xTRAIL1 induced apoptosis in larval RBCs, but had little effect on adult RBCs in vitro. We also found that adult RBCs treated with staurosporine, a PKC inhibitor, were sensitized to xTRAIL1. The mRNAs for PKC isoforms were up-regulated in RBCs during metamorphosis. These results suggest that xTRAIL1 can cause apoptosis, probably mediated through xDR-Ms, in larval RBCs, but may not kill adult RBCs, presumably owing to PKC activation, as part of the mechanism for RBC switching.

DNA methylation for subtype classification and prediction of treatment outcome in patients with childhood acute lymphoblastic leukemia

Wed, 25 Nov 2009 12:16:31 PST

Despite improvements in the prognosis of childhood acute lymphoblastic leukemia (ALL), subgroups of patients would benefit from alternative treatment approaches. Our aim was to identify genes with DNA methylation profiles that could identify such groups. We determined the methylation levels of 1,320 CpG sites in regulatory regions of 416 genes in cells from 401 children diagnosed with ALL. Hierarchical clustering of 300 CpG sites distinguished between T-ALL and B-cell precursor (BCP) ALL and between the main cytogenetic subtypes of BCP ALL. It also stratified patients with high hyperdiploidy and t(12;21) ALL into two subgroups with different probability of relapse. Using supervised learning we constructed multivariate classifiers by external cross-validation procedures. We identified 40 genes that consistently contributed to accurate discrimination between the main subtypes of BCP ALL and gene sets that discriminated between subtypes of ALL and between ALL and controls in pair-wise classification analyses. We also identified 20 individual genes with DNA methylation levels that predicted relapse of leukemia. Thus, methylation analysis should be explored as a method to improve stratification of ALL patients. The genes highlighted in our study are not enriched to specific pathways, but the gene expression levels are inversely correlated to the methylation levels.

The t(14;18)(q32;q21)/IGH-MALT1 translocation in MALT lymphomas contains templated nucleotide insertions and a major breakpoint region similar to follicular and mantle cell lymphoma

Wed, 25 Nov 2009 12:16:19 PST

The t(14;18)(q32;q21) involving the immunoglobulin heavy chain locus (IGH) and the MALT1 gene is a recurrent abnormality in MALT lymphomas. However, the nucleotide sequence of only one t(14;18)-positive MALT lymphoma has been reported so far. We here report the molecular characterization of the IGH-MALT1 fusion products in 5 new cases of t(14;18)-positive MALT lymphomas. Similar to the IGH-associated translocations in follicular and mantle cell lymphomas, the IGH-MALT1 junctions in MALT lymphoma showed all features of a recombination signal sequence (RSS)-guided V(D)J-mediated translocation at the IGH locus. Furthermore, analogous to follicular and mantle cell lymphoma, templated T-nucleotides were identified at the t(14;18)/IGH-MALT1 breakpoint junctions. On chromosome 18, we identified a novel major breakpoint region (MBR) in MALT1 upstream of its coding region. Moreover, the presence of duplications of MALT1 nucleotides in one case suggests an underlying staggered DNA-break process not consistent with V(D)J-mediated recombination. The molecular characteristics of the t(14;18)/IGH-MALT1 resemble those found in the t(14;18)/IGH-BCL2 in FL and t(11;14)/CCND1-IGH in MCL suggesting that these translocations could be generated by common pathomechanisms involving illegitime V(D)J-mediated recombination on IGH as well as new synthesis of T-nucleotides and non-homologous end joining (NHEJ) or alternative NHEJ repair pathways on the IGH-translocation partner.

Post-translational stability of the heme biosynthetic enzyme ferrochelatase is dependent on iron availability and intact iron-sulfur cluster assembly machinery

Crooks, D. R., Ghosh, M. C., Haller, R. G., Tong, W.-H., Rouault, T. A. — Wed, 25 Nov 2009 12:16:10 PST

Mammalian ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, possesses an iron-sulfur [2Fe-2S] cluster that does not participate in catalysis. We investigated ferrochelatase expression in iron-deficient erythropoietic tissues of mice lacking iron regulatory protein 2 (IRP2), in iron-deficient MEL cells, and in human patients with ISCU myopathy. Ferrochelatase activity and protein levels were dramatically decreased in Irp2^-/- spleens, whereas ferrochelatase mRNA levels were increased, demonstrating post-transcriptional regulation of ferrochelatase in vivo. Translation of ferrochelatase mRNA was unchanged in iron-depleted MEL cells, and the stability of mature ferrochelatase protein was also unaffected. However, the stability of newly formed ferrochelatase protein was dramatically decreased during iron deficiency. Ferrochelatase was also severely depleted in muscle biopsies and cultured myoblasts from patients with ISCU myopathy, a disease caused by deficiency of a scaffold protein required for Fe-S cluster assembly. Together, these data suggest that decreased Fe-S cluster availability due to cellular iron depletion or impaired Fe-S cluster assembly causes reduced maturation and stabilization of apo-ferrochelatase, providing a direct link between Fe-S biogenesis and completion of heme biosynthesis. We propose that decreased heme biosynthesis due to impaired Fe-S cluster assembly can contribute to the pathogenesis of diseases caused by defective Fe-S cluster biogenesis.

Rap1 controls lymphocyte adhesion cascades and interstitial migration within lymph nodes in RAPL-dependent and -independent manners

Wed, 25 Nov 2009 12:16:02 PST

The small GTPase Rap1 and its effector RAPL regulate lymphocyte adhesion and motility. However, their precise regulatory roles in the adhesion cascade preceding entry into lymph nodes and during interstitial migration are unclear. Here, we show that Rap1 is indispensably required for the chemokine-triggered initial arrest step of rolling lymphocytes through LFA-1, whereas RAPL is not involved in rapid arrest. RAPL and talin play a critical role in stabilizing lymphocyte arrest to the endothelium of blood vessels under flow or to the high endothelial venules of peripheral lymph nodes in vivo. Further, mutagenesis and peptide studies suggest that release of a trans-acting restraint from the β2 cytoplasmic region of LFA-1 is critical for Rap1-dependent initial arrest. Rap1 or RAPL deficiency severely impaired lymphocyte motility over lymph node stromal cells in vitro, and RAPL deficiency impaired high-velocity directional movement within lymph nodes. These findings reveal the several critical steps of Rap1, which are RAPL-dependent and -independent, in lymphocyte trafficking.

Thrombotic risk assessment in the antiphospholipid syndrome requires more than the quantification of lupus anticoagulants

Devreese, K., Peerlinck, K., Hoylaerts, M. F. — Wed, 25 Nov 2009 12:15:50 PST

Lupus anticoagulants (LAC) are associated with thromboembolic complications (TEC). LAC can be detected via their anticoagulant properties in coagulation tests and thrombin generation assays, via the peak height (PH) and lag time (LT). To assess the thrombotic risk in LAC-positive patients, we have presently expressed the LAC-activity quantitatively via PH/LT calibration curves, constructed for mixtures of monoclonal antibodies with LAC-activity against β2-glycoprotein I (β2GPI) and prothrombin, spiked in normal plasma. PH/LT was determined in plasma of LAC patients, with (n=38) and without (n=21) TEC and converted into arbitrary LAC-units on parallel calibration lines. Patient LAC-titers ranged from 0-200 AU/ml, with 5/59 patients being negative. In the positive LAC-titer population (54/59), LAC-titers and anti-β2GPI IgG titers correlated with TEC, with odds ratios of 3.54 (CI: 1.07-11.7) and 10.0 (CI: 1.98-50.6), respectively. In patients with single or combined low titers, useful predictions on thrombosis could be made only after additional measurements of the prothrombotic plasma markers soluble P-selectin and FVII. This layered strategy yielded PPV, NPV, sensitivity and specificity values around 90% in this subgroup. Hence, LAC- and anti-β2GPI IgG titers, when combined with selected markers of the hypercoagulable state, allow a relevant thrombotic risk assessment in nearly all LAC patients.

KSHV induced notch components render endothelial and mural cell characteristics and cell survival

Tue, 24 Nov 2009 13:43:01 PST

Kaposi's sarcoma-associated herpesvirus (KSHV) infection is essential to the development of Kaposi's sarcoma. Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine if Kaposi's sarcoma-associated herpesvirus (KSHV) regulates Notch components. KSHV infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, vIL-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes) and endothelial precursor CD133. Targeting Notch pathway with -secretase inhibitor (GSI) and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.

Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively-spliced transcripts

Tue, 24 Nov 2009 13:42:54 PST

Splicing mutations account for ~10% of lesions causing genetic diseases, but few branchpoint sequence (BPS) lesions have been reported. In three families with autosomal recessive congenital erythropoietic porphyria (CEP) due to uroporphyrinogen III synthase (URO-synthase) deficiency, sequencing the promoter, all 10 exons and the intron/exon boundaries did not detect a mutation. Northern analyses of lymphoblast mRNAs from two patients and RT/PCR of lymphoblast mRNAs from all three patients revealed multiple longer transcripts involving intron 9 and low levels of wild-type message. Sequencing intron 9 RT/PCR products and genomic DNA in each case revealed homozygosity for a novel BPS mutation (c.661-31T>G) and alternatively-spliced transcripts containing 81, 246, 358, and 523 nucleotides (nt) from intron 9. Real-time PCR revealed aberrant transcripts in both wild-type and CEP lymphoblasts, while BPS mutation reduced the wild-type transcript and enzyme activity in CEP lymphoblasts to ~10% and 15% of normal, respectively. Though the +81 nt alternative transcript was in-frame, it only contributed ~0.2% of the lymphoblast URO-synthase activity. Thus, the BPS mutation markedly reduced the wild-type transcript and enzyme activity, thereby causing the disease. This is the first BPS mutation in the last intron, presumably accounting for the observed 100% intron retention without exon skipping.

High Mobility Group protein HMGB2 regulates human erythroid differentiation through trans-activation of Gfi-1B transcription

Tue, 24 Nov 2009 13:42:44 PST

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the Gfi-1B gene in mice leads to embryonic death due to failure of producing differentiated red cells. Accordingly, Gfi-1B expression is tightly regulated during erythropoiesis but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2, a high mobility HMG protein, as a key regulator of Gfi-1B transcription. HMGB2 binds to the Gfi-1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and, to a lesser extent, of GATA-1 and NF-Y to the Gfi-1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of Gf1-1B transcription. Importantly, knock down of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of Gfi-1B by Oct-1 and thereby controls erythroid differentiation.

Expression of terminal {alpha}2-6 linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13

Tue, 24 Nov 2009 13:42:37 PST

Von Willebrand factor (VWF) platelet tethering function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by 2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was only reduced to 50±14% by ADAMTS13, compared to 11±7% for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O ≥ B > A ≥ AB). However, ABO blood group regulation of ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. Sialic acid-mediated enhancement of VWF proteolysis by ADAMTS13 was significantly attenuated at high urea concentration (≥2M), suggesting that VWF sialylation enhances ADAMTS13 proteolysis by either promoting an ADAMTS13-specific permissive conformation, or by improving the VWF-ADAMTS13 interaction. These novel data demonstrate that sialic acid protects VWF against proteolysis by serine and cysteine proteases, but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key regulator of VWF multimeric composition, and as such, may be of pathophysiological significance.

Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells

Tue, 24 Nov 2009 13:42:26 PST

Abstract (200)Complex molecular mechanisms control B-cell fate to become a memory or a plasma-cell. IL-24 is a class-II family cytokine of poorly understood immune function which regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27+ memory B cells and CD5 expressing B cells whereas it is low to undetectable in centroblasts and plasma cells. Addition of rIL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and IgG production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma-cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; rIL-24 increased CD40-induced B cell proliferation and modulated the transcription of key transcription factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription- (STAT)-3, and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-Dependent Ag-driven B-cell differentiation and suggest its physiological role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

Down syndrome acute lymphoblastic leukemia: a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the iBFM Study Group

Tue, 24 Nov 2009 13:42:14 PST

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down Syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either IgH@ translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients a novel activating somatic mutation, F232C in CRLF2 was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and interestingly all 10 had high CRLF2 expression. CRLF2 and mutated Jak2 cooperated in conferring cytokine independent growth to BaF3 pro-B-cells. Intriguingly the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Lenalidomide treatment promotes CD154 expression on CLL cells and enhances production of antibodies by normal B Cells through a PI3-kinase dependent pathway

Tue, 24 Nov 2009 13:42:07 PST

Chronic lymphocytic leukemia (CLL) has a profound humoral immune defect and tumor-specific humoral tolerance that contributes directly to the disease morbidity and mortality. While CD154 gene therapy has demonstrated the ability reverse this immune defect, attempts to do this pharmacologically have been unsuccessful. Lenalidomide is an immune modulating agent with clinical activity in CLL whose mechanism of action is not clearly understood. We demonstrate that clinically relevant lenalidomide concentrations induce surface expression of functional CD154 antigen on CLL cells, via enhanced transcription mediated by a NFATc1/NF-B complex to the CD154 promoter and also through down-stream mRNA stabilization that is dependent upon activation of the PI3-kinase pathway. Importantly, CD154-positive CLL cells promote co-stimulatory activation of normal B cells to produce antibodies, up-regulate BID, DR5, and p73, and become sensitized to TRAIL-mediated apoptosis. Similar evidence of CD154 activation is observed in vivo among patients receiving lenalidomide, including the induction of BID, DR5, and p73 in vivo and also development of a ROR1 anti-tumor directed antibody. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells and may reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.

Rho GTPases in hematopoiesis and hemopathies

Mulloy, J. C., Cancelas, J. A., Filippi, M.-D., Kalfa, T. A., Guo, F., Zheng, Y. — Tue, 24 Nov 2009 13:41:58 PST

Rho family GTPases are intracellular signaling proteins regulating multiple pathways involved in cell actomyosin organization, adhesion and proliferation. Our knowledge of their cellular functions comes mostly from previous biochemical studies using mutant overexpression approaches in various clonal cell lines. Recent progress in understanding Rho GTPase functions in blood cell development and regulation by gene targeting of individual Rho GTPases in mice has allowed a genetic understanding of their physiologic roles in hematopoietic progenitors and mature lineages. In particular, mouse gene targeting studies have provided convincing evidence that individual members of the Rho GTPase family are essential regulators of cell type-specific functions and stimuli-specific pathways in regulating hematopoietic stem cell interaction with bone marrow niche, erythropoiesis and red blood cell actin dynamics, phagocyte migration and killing, and T- and B-cell maturation. In addition, deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as neutrophil dysfunction, leukemia, and Fanconi anemia, raising the possibility that Rho GTPases and downstream signaling pathways are of therapeutic value. In this review we discuss recent genetic studies of Rho GTPases in hematopoiesis and several blood lineages and the implications of Rho GTPase signaling in hematologic malignancies, immune pathology and anemia.

A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma

Mon, 23 Nov 2009 13:16:15 PST

The serine/threonine Pim kinases are up regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by c-Myc, N-Myc, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2,4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre T-LBL/T-ALL) being highly sensitive. Incubation of pre T-LBL cells with SMI-4a induced G1 phase cell cycle arrest secondary to a dose dependent induction of p27^Kip1, apoptosis through the mitochondrial pathway, and inhibition of the mTORC1 pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, two substrates of this enzyme. Additionally, treatment of these cells with SMI-4a was found to induce phosphorylation of ERK1/2, and the combination of SMI-4a and a MEK1/2 inhibitor was highly synergistic in killing pre T-LBL cells. In immuno-deficient mice carrying subcutaneous pre T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre T- LBL.

Vascular endothelial growth factor activation of endothelial cells is mediated by early growth response-3

Suehiro, J.-i., Hamakubo, T., Kodama, T., Aird, W. C., Minami, T. — Mon, 23 Nov 2009 13:16:05 PST

Endothelial cell activation and dysfunction underlie many vascular disorders including atherosclerosis, tumor growth and sepsis. Endothelial cell activation, in turn, is mediated primarily at the level of gene transcription. Here, we show that in response to a number of activation agonists, including vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-, and thrombin, endothelial cells demonstrate rapid and profound induction of the early growth response genes, Egr-1 and Egr-3. In VEGF-treated endothelial cells, induction of Egr-3 was far greater and more prolonged compared with Egr-1. VEGF-mediated stimulation of Egr-3 involved the inducible binding of NFATc, serum response factor (SRF) and CREB to their respective consensus motifs in the upstream promoter region of Egr-3. Knockdown of Egr-3 markedly impaired VEGF-mediated proliferation, migration, and tube formation of endothelial cells, and blocked VEGF-induced monocyte adhesion. Egr-3 knockdown abrogated VEGF-mediated vascular outgrowth from ex vivo aortic rings, and attenuated Matrigel plug vascularization and melanoma tumor growth in vivo. Together, these findings suggest that Egr-3 is a critical determinant of VEGF signaling in activated endothelial cells. Thus, Egr-3 represents a potential therapeutic target in VEGF-mediated vasculopathic diseases.

Novel IL-21 signaling pathway upregulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas

Fri, 20 Nov 2009 10:40:03 PST

IL-21, a member of the IL-2 cytokine family, has diverse regulatory effects on NK, T and B cells. In contrast to other cytokines that are usually immunostimulatory, IL-21 can induce apoptosis of murine B cells at specific activation-differentiation stages. This effect may be employed for treatment of B cell malignancies. Herein we report that diffuse large B cell lymphoma (DLBCL) cell lines exhibit widespread expression of the IL-21R and that IL-21 stimulation leads to cell cycle arrest and caspase-dependent apoptosis. IL-21 also induces apoptosis in de novo DLBCL primary tumors but does not affect viability of human healthy B cells. Furthermore, IL-21 promotes tumor regression and prolongs survival of mice harboring xenograft DLBCL tumors. The anti-lymphoma effects of this cytokine are dependent on a mechanism involving IL-21-activated STAT3 upregulating expression of c-Myc. This upregulation promotes a decrease in expression of anti-apoptotic Bcl-2 and Bcl-X_L proteins triggering cell death. Our results represent one of the first examples in which the STAT3 - c-Myc signaling pathway, which can promote survival and oncogenesis, can induce apoptosis in neoplastic cells. Moreover, based on IL-21's potency in vitro and in animal models, our findings indicate that this cytokine should be examined in clinical studies of DLBCL.

Malignant cells fuel tumor growth by educating infiltrating leukocytes to produce the mitogen Gas6

Fri, 20 Nov 2009 10:39:52 PST

The transforming and tumor growth promoting properties of Axl, a member of the Tyro3, Axl and Mer family of Receptor tyrosine kinases (TAMR), are well recognized. In contrast, little is known about the role of the TAMR ligand Gas6 in tumor biology. By using Gas6 deficient (Gas6^-/-) mice, we show that bone marrow-derived Gas6 promotes growth and metastasis in different experimental cancer models, including one resistant to VEGF inhibitors. Mechanistic studies reveal that circulating leukocytes produce minimal Gas6. However, once infiltrated in the tumor, leukocytes upregulate Gas6, which is mitogenic for tumor cells. Consistent herewith, impaired tumor growth in Gas6^-/- mice is rescued by transplantation of wild type (WT) bone marrow and, conversely, mimicked by transplantation of Gas6^-/- bone marrow into WT hosts. These findings highlight a novel role for Gas6 in a positive amplification loop, whereby tumors promote their growth by educating infiltrating leukocytes to upregulate the production of the mitogen Gas6. Hence, inhibition of Gas6 might offer novel opportunities for the treatment of cancer.

Thrombosis in primary myelofibrosis: incidence and risk factors

Fri, 20 Nov 2009 10:39:43 PST

We assessed frequency and predictive factors for major cardiovascular (CV) events in 707 patients with primary myelofibrosis (PMF) followed in 4 European Institutions. A total of 236 deaths (33%) was recorded for an overall mortality of 7.7% patient-years (pt-yr). Fatal and non-fatal thrombosis were registered in 51 (7.2%) patients, with a rate of 1.75% pt-yr. If deaths from non-CV causes were considered as competing events, we estimated that the adjusted rate of major thrombotic events would have been 2.2% pt-yr. In a multivariable model, age >60 years (HR 2.34, 95% CI 1.24-4.39, p=0.01) and JAK2 mutational status (HR 1.92, 95%CI 1.10-3.34, p=0.02) were significantly associated with thrombosis while the strength of the association between leukocyte count > 15x10⁹/L and CV events was of borderline significance (HR 1.72, 95% CI 0.97-2.72, p=0.06). The highest incidence of fatal and non-fatal thrombosis was observed when the mutation was present along with leukocytosis (3.9% pt-yr, HR 3.13, 95% CI 1.26-7.81). This study is the largest hitherto carried out in this setting and shows that the rate of major CV events in PMF is comparable to that reported in essential thrombocythemia and it is increased in aged patients and those with JAK2V617F mutation and leukocytosis.

Induction of nitric oxide by erythropoietin is mediated by the {beta} common receptor and requires interaction with VEGF receptor 2

Fri, 20 Nov 2009 10:39:34 PST

Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) have profound effects on the endothelium and endothelial progenitor cells (EPCs), which originate from the bone marrow and differentiate into endothelial cells. Both EPO and VEGF have demonstrated an ability to increase the number and performance properties of EPCs. EPC behavior is highly dependent on nitric oxide (NO) and both VEGF and EPO can stimulate intracellular NO. EPO can bind to the homodimeric EPO receptor (EPO-R) and the heterodimeric receptor, EPO-R and the common β receptor (βC-R). While VEGF has a number of receptors, VEGF-R2 appears most critical to EPC function. We demonstrate that EPO induction of NO is dependent on the βC-R and VEGF-R2, that VEGF induction of NO is dependent on the expression of the βC-R, and that the βC-R and VEGF-R2 interact. This is the first definitive functional and structural evidence of an interaction between the two receptors and has implications for EPO's side effects.

The severity of trauma determines the immune response to PF4/heparin and the frequency of heparin-induced thrombocytopenia

Fri, 20 Nov 2009 10:39:26 PST

Heparin can induce heparin-induced thrombocytopenia (HIT). The combined effect of type of surgery (major vs minor) and heparin on this prothrombotic immune reaction to PF4/heparin was analyzed. In a randomized, double-blind, single center study, trauma patients receiving low-molecular-weight (LMWH) or unfractionated heparin (UFH) for thrombosis prophylaxis were assessed for PF4/heparin-antibody seroconversion, clinical HIT, and thrombosis according to type of surgery. The risk for seroconversion increased with the magnitude of surgical trauma: major vs minor surgery OR 7.98 [95%CI 2.06-31.00; p=0.003, controlled for potential confounders] as did the risk for HIT (2.2% [95%CI 0.3-4.1%] vs 0.0%, p=0.010). During LMWH compared to UFH thromboprophylaxis, HIT (1/298 vs 4/316; p=0.370) and PF4/heparin seroconversion (1.7% vs.6.6%; p=0.002) were less frequent, driven by differences in patients undergoing major surgery: incidence of HIT (LMWH 0.8% vs UFH 4.0%; p=0.180); seroconversion rates (4.0% vs 17.0%; p=0.001). After minor surgery no case of HIT occurred. The severity of trauma and the need for major surgery strongly influence the risk of forming an anti-PF4/heparin immune response, which is then increased by UFH. In major trauma certoparin may be safer than UFH as it induces HIT-antibody seroconversion - and the corresponding risk of HIT - less frequently. This study is registered at http://clinicaltrials.gov as NCT00196417.

Cyclosporine-induced immune suppression alters establishment of HTLV-1 infection in a rabbit model

Fri, 20 Nov 2009 10:39:18 PST

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia (ATL) and a number of lymphocyte-mediated inflammatory diseases. Persistent HTLV-1 infection is determined by a balance between host immune responses and virus spread. Immunomodulatory therapy involving HTLV-1 infected patients occurs in a variety of clinical settings. Knowledge of how these treatments influence host-virus relationships is not understood. Herein, we examined the effects of Cyclosporine A (CsA)-induced immune suppression during early infection of HTLV-1. Twenty four New Zealand white rabbits were split into 4 groups. Three groups were treated with either 10 or 20 mg/kg CsA or saline before infection. The fourth group was treated with 20 mg/kg CsA 1 week post infection. Immune suppression, plasma CsA concentration, ex vivo lymphocyte HTLV-1 p19 production, anti-HTLV-1 serologic responses and proviral load levels were measured during infection. Our data indicated that CsA-treatment prior to HTLV-1 infection enhanced early viral expression compared to untreated HTLV-1-infected rabbits, and altered long term viral expression parameters. However, CsA treatment 1 week post infection diminished HTLV-1 expression throughout the 10 week study course. Collectively, these data indicates immunologic control is a key determinant of early HTLV-1 spread and has important implications for therapeutic intervention during HTLV-1-associated diseases.

Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow

Schaff, U. Y., Dixit, N., Procyk, E., Yamayoshi, I., Tse, T., Simon, S. I. — Fri, 20 Nov 2009 10:39:09 PST

Orai1 was reported to function as a calcium channel subunit that facilitates store operated calcium entry (SOCE) in T-cells and is necessary for formation of the immune synapse. We reasoned that SOCE via Orai1 might regulate PMN activation during recruitment to inflamed endothelium. Orai1 function was assessed by real-time imaging of calcium transients as PMNs were stimulated to roll, arrest, and migrate on E-selectin and ICAM-1 in shear flow. Calcium entry was significantly reduced when Orai1 function was impaired by heterozygous knockout in a mouse model or by siRNA knockdown in HL-60 cells. Reduced Orai-1 expression correlated with the delayed onset of arrest and reduced ability to transition to a polarized migratory phenotype. Inhibition of SOCE by treatment with 2-APB, or blocking phospholipase-C (PLC) mediated calcium store release with U73122, abrogated formyl peptide induced calcium elevation and delayed subsequent cell arrest and polarization. These results suggest that calcium entry via Orai1 is the predominant SOCE that cooperates with cytoplasmic calcium store release in coordinating integrin dependent PMN arrest and migration in the acute response to inflammation.

Dual targeting of the PI3K/Akt/mTOR pathway as an anti-tumor strategy in Waldenstrom's macroglobulinemia

Thu, 19 Nov 2009 12:59:53 PST

We have previously shown clinical activity of a TORC1 inhibitor in Waldenstrom's Macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the PI3K/Akt/mTOR in WM, and mechanisms of overcoming resistance to therapy. We first demonstrated that primary WM cells show constitutive activation of the PI3K/Akt pathway, supported by decreased expression of PTEN at the gene and protein levels, together with constitutive activation of Akt and mTOR. We illustrated that dual targeting of the PI3K/mTOR pathway by the novel inhibitor NVP-BEZ235 showed higher cytotoxicity on WM cells compared to inhibition of the PI3K or mTOR pathways alone. In addition, NVP-BEZ235 inhibited both Rictor and Raptor, thus abrogating the Rictor-induced Akt phosphorylation. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the forkhead box transcription factors. In addition, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment leading to significant inhibition of migration, adhesion in vitro and homing in vivo. These studies therefore show that dual targeting of the PI3K/mTOR pathway is a better modality of targeted therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade such as WM.

VEGF/VEGFR2 interaction downregulates matrix metalloproteinase-9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration

Thu, 19 Nov 2009 12:59:43 PST

B-cell chronic lymphocytic leukemia (B-CLL) migration involves several molecules including matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF). We have studied whether VEGF regulates MMP-9. VEGF significantly reduced MMP-9 protein expression in a dose-dependent manner, measured by gelatin zymography. Blocking the VEGFR2 receptor restored MMP-9 levels, implicating this receptor in the observed effect. Downregulation of MMP-9 by VEGF resulted in significant inhibition of B-CLL cell migration through Matrigel or HUVEC, confirming the crucial role of MMP-9 in these processes. RT-PCR analyses revealed that VEGF regulated MMP-9 at the transcriptional level. Indeed, VEGF induced STAT1 tyrosine phosphorylation and this was blocked by inhibiting VEGFR2. STAT1 was responsible for MMP-9 downregulation, as STAT1 gene silencing restored MMP-9 production and B-CLL cell migration in the presence of VEGF. Thus, the levels of VEGF and MMP-9 influence B-CLL cell expansion and both molecules could constitute therapeutic targets for this disease.

A phase II pilot study of tacrolimus/sirolimus GVHD prophylaxis for sibling donor hematopoietic stem cell transplant using three conditioning regimens

Thu, 19 Nov 2009 12:59:22 PST

Combination tacrolimus and sirolimus GVHD prophylaxis for allogeneic transplant in patients conditioned with a fractionated total body irradiation (TBI)-based regimen has shown encouraging results. We studied this prophylaxis combination in 85 patients receiving a matched-sibling transplant conditioned with 3 different regimens: fludarabine-melphalan (n=46); TBI-etoposide (n=28), and busulfan-cyclophosphamide (n=11). The conditioning regimens were completed on day -4. Sirolimus and tacrolimus were started on day -3 to avoid overlap with conditioning therapy. All patients engrafted, with a median time to neutrophil engraftment of 15 days. The cumulative incidence of acute GVHD grade II-IV and III-IV was 43% and 19%, respectively, with no significant difference by conditioning regimen. The two-year cumulative incidence of chronic GVHD was 46%. With a median follow-up of 26 months, DFS was 58% and OS, 66%. The day 100 and two year non-relapse mortality (NRM) was 4.8% and 10.2%, respectively. The overall incidence of thrombotic microangiopathy (TMA) was 19%, and was significantly higher with Bu/Cy (55%, p=0.005). Tacrolimus plus sirolimus is an effective combination for acute GVHD prophylaxis and is associated with very low NRM. TMA is a significant complication with this regimen, particularly in patients receiving Bu/Cy.

Results of a phase II study of bortezomib in patients with relapsed or refractory indolent lymphoma

Thu, 19 Nov 2009 12:59:15 PST

This study evaluated the efficacy and safety of single-agent bortezomib in indolent B-cell lymphoma that had relapsed from/was refractory to rituximab. Sixty patients enrolled; 59 were treated with bortezomib 1.3 mg/m² Days 1, 4, 8, and 11 for up to eight 21-day cycles; responders could receive 4 additional cycles; maintenance was optional. Fifty-three evaluable patients completed >2 cycles. Median age 70 years, 53% female, Ann Arbor stage III-IIIE (28%) and IV (65%); 43 patients (72%) had >2 prior regimens. Six patients went on to maintenance. Overall responses: 1 CR (1.9%), 3 CRu (5.7%), 3 PR (5.7%), 34 SD (64.2%), and 12 PD (22.6%). Median time to response=2.2 months (range 1.2-5.3); duration of response=7.9 months (2.8-21.3); 1-yr survival was 73% and 2-yr survival was 58%; median survival= 27.7 months (range, 1.4-30.9); median PFS=5.1 months (range, 0.2-27.7), median TTP=5.1 months (range, 0.2-27.7), and median event-free survival=1.8 months (range, 0.2-27.7). Treatment-related Grade 3-4 AEs included: thrombocytopenia (20%), fatigue (10%), neutropenia (8.5%), and neuropathy and diarrhea (6.8%, each). This study demonstrates that bortezomib has modest activity against marginal zone and follicular lymphoma; it has the potential for combination with other agents in low-grade lymphomas. Maintenance therapy should be explored further.

DNA vaccination with all-trans retinoic acid treatment induces long term survival and elicits specific immune responses requiring CD4+ and CD8+ T-cell activation in an acute promyelocytic leukemia mouse model

Thu, 19 Nov 2009 12:59:33 PST

DNA vaccination and all-trans retinoic acid (ATRA) results in a survival advantage in a mouse model of acute promyelocytic leukemia (APL). Depletion of CD4+ or CD8+ cells abolished this effect. CD4+ depletions of long term survivors (LTS) resulted in relapse and death within 3 months, thus demonstrating the need of both CD4+ and CD8+ subsets for the generation of DNA-driven anti-leukemic immune responses and underscores a crucial role of CD4+ cells in the maintenance of durable remissions. Degranulation and cytotoxic CFSE-based assays showed MHC-restricted APL-specific T-cell mediated immune responses. Sorted APL-specific CD8+CD107a+ T-cells showed an increase of anti-leukemic activity. Effectors from ATRA+DNA-treated mice were shown to secrete interferon-gamma (IFN-) when stimulated with either APL cells or peptides from the PML-RAR vaccine derived sequences as detected by ELISPOT assays. Our results demonstrate that DNA vaccination with ATRA confers the effective boosting of IFN- producing and cytotoxic T-cells in the leukemic mice.

Allogeneic hematopoietic stem cell transplantation (alloSCT) for chronic myeloid leukemia in the imatinib era; evaluation of its impact within a subgroup of the randomized German CML Study IV

Wed, 18 Nov 2009 13:49:07 PST

The role of allogeneic stem cell transplantation in chronic myeloid leukemia is being reevaluated. Whereas drug treatment has been shown to be superior in first line treatment, data on allo SCT as second line therapy after imatinib failure are scarce. Using an interim safety analysis of the German randomized CML-Study IV designed to optimize imatinib therapy by combination, dose-escalation and transplantation we here report on 84 patients consecutively transplanted according to predefined criteria (low EBMT score, imatinib failure, and advanced disease). Three year survival after transplantation of 56 patients in chronic phase was 91% (median follow-up 30 months). Transplantation related mortality was 8%. In a matched pair comparison of transplanted and non-transplanted patients, survival was not different. Three year survival after transplantation of 28 patients in advanced phase was 59%. 88% of transplanted patients achieved complete molecular remissions. We conclude that allo SCT could become the preferred second line option after imatinib failure for suitable patients with a donor. The study is registered at the National Institute of Health, ClinicalTrials.gov: NCT00055874.

PTEN is a tumor suppressor in CML stem cells and BCR-ABL induced leukemias in mice

Peng, C., Chen, Y., Yang, Z., Zhang, H., Osterby, L., Rosmarin, A. G., Li, S. — Wed, 18 Nov 2009 13:48:58 PST

The tumor suppressor gene PTEN is inactivated in many human cancers. However, it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome positive (Ph⁺) leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. Using our mouse model of BCR-ABL induced leukemias, we show that PTEN is down-regulated by BCR-ABL in leukemia stem cells (LSCs) in CML, and that PTEN deletion causes acceleration of CML development. In addition, overexpression of PTEN delays the development of CML and B-ALL, and prolongs survival of leukemia mice. PTEN suppresses LSCs and induces cell cycle arrest of leukemia cells. Moreover, PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL induced leukemias and suggest a potential strategy for the treatment of Ph⁺ leukemia.

Impaired fibrinolysis as a risk factor for Budd-Chiari syndrome

Wed, 18 Nov 2009 13:48:47 PST

In Budd-Chiari syndrome (BCS) thrombosis develops in the hepatic veins or inferior vena cava. To study the relationship between impaired fibrinolysis and BCS, we measured plasma levels of fibrinolysis proteins in 101 BCS-patients and 101 healthy controls and performed a plasma-based clot lysis assay. In BCS-patients, plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in controls (median 6.3 vs. 1.4 IU/ml, p<0.001). Thrombin-activatable fibrinolysis inhibitor and plasmin inhibitor levels were lower than in controls (13.8 vs. 16.9 µg/ml and 0.91 vs. 1.02 U/l, both p<0.001). Median plasma clot lysis time (CLT) was 73.9 min in BCS-patients and 73.0 min in controls (p=0.329). A subgroup of cases displayed clearly elevated CLTs. A CLT above the 90^th (93.1 min) or 95^th (98.1 min) percentile of the controls was associated with an increased risk of BCS, with odds ratios of 2.4 (95%CI 1.1-5.5) and 3.4 (95%CI 1.2-9.7), respectively. In controls, only PAI-1 activity was significantly associated with CLT. Analysis of single nucleotide polymorphisms of fibrinolysis proteins revealed no significant differences between cases and controls. This case-control study provides the first evidence that an impaired fibrinolytic potential, at least partially caused by elevated PAI-1 levels, is related to the presence of BCS.

Immunization with host type CD8{alpha}+ dendritic cells reduces experimental acute GVHD in an IL-10 dependent manner

Wed, 18 Nov 2009 13:48:37 PST

Little is known about the active role of immunization in suppressing undesirable immune responses. Because CD8⁺ dendritic cells (DCs) suppress certain immune responses, we tested the hypothesis that immunization of donors with host-derived CD8⁺ DCs will reduce host-specific donor T cell responses. BALB/c T cells from the animals that were immunized with B6 CD8⁺ DCs demonstrated, in vitro and in vivo, significantly reduced proliferation and secretion of inflammatory cytokines, but showed enhanced secretion of IL-10. The responses against third party and model antigens were preserved demonstrating antigen specificity. The in vivo relevance was further demonstrated by the reduction on GVHD in both a MHC mismatched clinically relevant BALB/c->B6 model and MHC-matched, minor-mismatched C3H.SW-> B6 model of GVHD. Immunization of the donors that were deficient in IL-10 (IL-10^-/-) or with CD8⁺ DCs from B6 class II (class II^-/-) failed to reduce T cell responses demonstrating (a) critical role for secretion of IL-10 by donor T cells and (b) for a direct contact between the T cells and the CD8⁺ DCs. Together these data may represent a novel strategy for reducing GVHD and suggest a broader counter intuitive role for vaccination strategies in mitigating undesirable immune responses in an antigen specific manner.

Molecular signatures to improve diagnosis in peripheral T-cell lymphoma and prognostication in angioimmunoblastic T-cell lymphoma

Wed, 18 Nov 2009 13:48:26 PST

Peripheral T-cell lymphoma (PTCL) is often challenging to diagnose and classify. Since most patients also have a poor outcome with standard chemotherapy, a better understanding of PTCL may lead to more effective diagnosis and therapy. Gene expression profiling (GEP) was performed on 144 cases of PTCL and natural killer (NK)-cell lymphoma and robust molecular classifiers were constructed for angioimmunoblastic T-cell lymphoma (AITL), ALK positive anaplastic large cell lymphoma (ALK+ALCL), and adult T-cell leukemia/lymphoma (ATLL). PTCL-Unclassifiable (PTCL-U) was molecularly heterogeneous, but we were able to identify a molecular subgroup with features of cytotoxic T- lymphocytes and a poor survival compared to the remaining PTCL-U cases. Many of the pathological features and substantial components of the molecular signature of AITL are contributed by the follicular dendritic cells, B-cell and other stromal components. The expression of Th17 associated molecules in ALK(+)ALCL was noted and may represent aberrant activation of Th17 cell differentiation by abnormal cytokine secretion. ATLL has a homogeneous molecular signature showing high expression of HTLV-1 induced genes. These classifiers reflect the biology of the tumor cells as well as their microenvironment. We also constructed a molecular prognosticator for AITL which appears to be largely related to the microenvironmental signature and the high expression of two immunosuppressive signatures are associated with poor outcome. Oncogenic pathways and tumor-host interactions were also identified and these findings may lead to better therapies and outcome in the future.

Leukemic transformation by the APL fusion protein PRKAR1A-RAR{alpha} critically depends on recruitment of RXR{alpha}

Tue, 17 Nov 2009 13:25:49 PST

PRKAR1A (R1A)-retinoic acid receptor (R1A-RAR) is the sixth RAR-containing fusion protein in acute promyelocytic leukemia (APL). Using the murine bone-marrow retroviral transduction/transformation assay (RTTA), we showed that R1A-RAR fusion protein could transform bone-marrow progenitor/stem cells. In gel-shift assays, R1A-RAR was able to bind to a panel of retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXR, and demonstrated distinct DNA-binding characteristics when compared to wild-type RAR/RXR or other X-RAR chimeric proteins. The ratio of R1A-RAR to RXR proteins affected the RARE interaction pattern of R1A-RAR/RXR complexes. Studies comparing R1A-RAR with R1A-RAR(RIIa) demonstrated that the RIIa protein interaction domain located within R1A was responsible for R1A-RAR homodimeric DNA binding and interaction with wild-type R1A protein. However, the RIIa domain was not required for R1A-RAR-mediated transformation, since its deletion in R1A-RAR(RIIa) did not compromise its transformation capability. In contrast, introduction of point mutations within the RAR portion of either R1A-RAR or R1A-RAR(RIIa) previously demonstrated to eliminate RXR interaction or treatment of transduced cells with RXR shRNA or a RXR agonist reduced transformation capability. Thus, leukemic transformation by APL fusion protein PRKAR1A-RAR is critically dependent on RXR which urges RXR as a promising target for APL.

Gastric MALT lymphoma B cells express polyreactive, somatically mutated immunoglobulins

Tue, 17 Nov 2009 13:25:40 PST

Gastric B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) arises against a background of chronic inflammation caused by persistent Helicobacter pylori infection. The clinical and histopathological features of the human tumor can be reproduced by Helicobacter infection of BALB/c mice. In this study, we have analyzed the antibody sequences and antigen specificity of a panel of murine and human MALT lymphoma-derived antibodies. We find that a majority of tumors in patients as well as experimentally infected mice are monoclonal. The tumor immunoglobulin heavy chain genes have undergone somatic hypermutation, and approximately half of all tumors show evidence of intraclonal variation and positive and/or negative selective pressure. Recombinantly expressed MALT lymphoma antibodies bind with intermediate affinity to various unrelated self and foreign antigens, including Helicobacter sonicate, IgG, DNA and stomach extract; antigen binding is blocked in a dose-dependent manner in competitive ELISAs. A strong bias towards the use of V_H gene segments previously linked to auto- and/or polyreactive antibodies in B-cell malignancies or autoimmune pathologies supports the experimental finding of polyreactivity. Our results suggest that MALT lymphoma development may be facilitated by an array of local self and foreign antigens providing direct antigenic stimulation of the tumor cells via their B-cell receptor.

Inhibition of Syk with fostamatinib disodium has significant clinical activity in non Hodgkin's lymphoma and chronic lymphocytic leukemia

Tue, 17 Nov 2009 13:25:31 PST

Certain malignant B-cells rely upon B-cell receptor (BCR)-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the BCR signal. In in vivo analyses of B-cell lymphoma cell lines and primary tumors, Syk inhibition induces apoptosis. These data prompted a phase I/II clinical trial of fostamatinib disodium, the first clinically available oral Syk inhibitor, in patients with recurrent B-cell NHL. Dose-limiting toxicity in the phase I portion was neutropenia, diarrhea and thrombocytopenia and 200 mg bid was chosen for Phase II testing. 68 patients with recurrent B-NHL were then enrolled in 3 cohorts: DLBCL; FL, and other NHL, including MCL; marginal zone/MALT; lymphoplasmacytic; and SLL/CLL. Common toxicities included diarrhea, fatigue, cytopenias, hypertension and nausea. Response rates were 22% for (5/23) DLBCL, 10% (2/21) for follicular lymphoma, 55% (6/11) for SLL/CLL and 11% (1/9) for mantle cell lymphoma. Stable disease was observed in an additional 22 patients, including 11 with FL, 4 with DLBCL, 4 with MCL, 2 with SLL/CLL and 1 with marginal zone lymphoma. Median progression-free survival was 4.2 months, and median response duration exceeded 4 months. Disrupting BCR-induced signaling by inhibiting Syk represents a novel therapeutic approach to NHL and SLL/CLL. This study is registered at http://clinicaltrials.gov as NCT00446095.

Induction of immune tolerance to FIX by intramuscular AAV gene transfer is independent of the activation status of dendritic cells

Bharadwaj, A. S., Kelly, M., Kim, D., Chao, H. — Tue, 17 Nov 2009 13:25:23 PST

The nature of viral vectors is suggested to be a significant contributor to undesirable immune responses subsequent to gene transfer. Such viral vectors recognized as "danger signals" by the host immune system, activate dendritic cells (DCs), causing unwanted anti-vector and/or transgene product immunity. We recently reported efficient induction of immune tolerance to FIX by direct intramuscular injection of AAV1-FIX. AAV vectors are non-pathogenic and elicit minimal inflammatory response. We hypothesized that the non-pathogenic nature of AAV plays a critical role in induction of tolerance following AAV gene transfer. We observed inefficient recruitment and activation of DCs subsequent to intramuscular injection of AAV. To further validate our hypothesis, we examined immune responses to FIX following intramuscular injection of AAV with simultaneous activation of DCs. We were able to achieve phenotypic and functional activation of DCs following administration of LPS and anti-CD40 antibody. However, we observed efficient induction of FIX tolerance irrespective of DC activation in mice with different genetic and MHC backgrounds. Furthermore, activation of DCs did not exaggerate the immune response induced following intramuscular injection of AAV2. Our results demonstrate that induction of FIX tolerance following AAV gene transfer is independent of DC activation status.

Rituximab inhibits B-cell receptor signaling

Tue, 17 Nov 2009 13:25:15 PST

Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B cell derived lymphoid neoplasias and of antibody-mediated auto-immune diseases. Beside of cell- and complement-mediated B cell depletion, RTX is thought to inhibit B cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mTOR. However, surprisingly, although B cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here for the first time that pre-treatment with RTX results in a time-dependent inhibition of the BCR signaling cascade involving Lyn, Syk, PLC2, Akt and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics and signaling might contribute to the immunosuppressive activity of the drug.

Myeloid progenitor cells lacking p53 exhibit delayed upregulation of Puma and prolonged survival after cytokine deprivation

Tue, 17 Nov 2009 13:25:07 PST

Loss of p53-dependent apoptosis contributes to the development of haematological malignancies and failure to respond to treatment. Pro-apoptotic Bcl-2 family member, Puma, is essential for apoptosis in HoxB8-immortalised IL-3 dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated WT, p53^-/- and FoxO3a^-/- FDM cells and found that p53^-/- but not FoxO3a^-/- cells were protected against IL-3 withdrawal. Loss of p21^cip/waf, which is critical for p53 mediated cell cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53^-/- hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53^-/- cells was substantially delayed when compared to WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally upregulates Puma, which initiates apoptosis.

Differential expression of CD21 identifies developmentally and functionally distinct subsets of human transitional B cells

Tue, 17 Nov 2009 13:24:55 PST

The transitional stage of B-cell development represents an important step where autoreactive cells are deleted, allowing the generation of a mature functional B-cell repertoire. In mice, three subsets of transitional B cells have been identified. In contrast, most studies of human transitional B cells have focussed on a single subset defined as CD24^hiCD38^hi B cells. Here, we have identified two subsets of human transitional B cells based on the differential expression of CD21. CD21^hi transitional B cells displayed higher expression of CD23, CD44, IgD, and BAFF-R and exhibited greater proliferation and Ig secretion in vitro than CD21^lo transitional B cells. In contrast, the CD21^lo subset expressed elevated levels of Lymphoid enhancing binding factor-1 (LEF-1), a transcription factor highly expressed by immature lymphocytes, and produced higher amounts of autoreactive Ab. These phenotypic, functional and molecular features suggest that CD21^lo transitional B cells are less mature than the CD21^hi subset. This was confirmed by analysing B-cell deficient patients with X-linked agammaglobulinaemia and the kinetics of B-cell reconstitution following stem cell transplant, which revealed that the development of CD21^lo transitional B cells preceded that of CD21^hi transitional B cells. These findings provide important insights into the process of human B cell development and have implications for understanding the processes underlying perturbed B-cell maturation in autoimmune and immunodeficient conditions.

SETBP1 overexpression is a novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia

Mon, 16 Nov 2009 13:08:03 PST

Acute myeloid leukemias (AML) result from multiple genetic alterations in hematopoietic stem cells. We describe a novel t(12;18)(p13;q12) involving ETV6 in a patient with AML. The translocation resulted in overexpression of SETBP1 (18q12), located close to the breakpoint. Overexpression of SETBP1 through retroviral insertion has been reported to confer growth advantage in hematopoietic progenitor cells. We show that SETBP1 overexpression protects SET from protease cleavage, increasing the amount of full-length SET protein, and leading to the formation of a SETBP1-SET-PP2A complex that results in PP2A inhibition, promoting proliferation of the leukemic cells. The prevalence of SETBP1 overexpression in AML at diagnosis (n=192) was 27.6%, and was associated with unfavorable cytogenetic prognostic group, monosomy 7, and EVI1 overexpression (p<0.01). Patients with SETBP1 overexpression had a significantly shorter overall survival (OS), and the prognosis impact was remarkably poor remarkable in patients older than 60 years in both OS (p=0.015) and event free survival (p=0.015). In summary, our data show a novel leukemogenic mechanism through SETBP1 overexpression; moreover, multivariate analysis confirms the negative prognostic impact of SETBP1 overexpression in AML, especially in elderly patients, where it could be used as a predictive factor in any future clinical trials with PP2A activators.

Usefulness of repeated D-dimer testing after stopping anticoagulation for a first episode of unprovoked venous thromboembolism: the PROLONG II prospective study

Mon, 16 Nov 2009 13:07:53 PST

The PROLONG randomized trial showed that a normal D-dimer (D-d) at 1 month after anticoagulation suspension for unprovoked venous thromboembolism (VTE) was associated with a low risk of late recurrences (4.4 percent patient-years). However it is unknown whether D-d changes subsequently. The aim of this prospective multi-center study was to assess D-d time course and its relation with late recurrences in patients with normal D-d at 1 month after anticoagulation suspension for a first episode of unprovoked VTE. D-d was measured with a qualitative method (Clearview Simplify D-dimer, Inverness Medical Professional Diagnostics, Bedford, UK). Patients with a normal D-d at 1 month after stopping anticoagulation repeated D-d testing every two months for a year. D-d was normal in 68% (243/355) patients at 1 month after anticoagulation suspension. Patients in whom D-d became abnormal at the 3^rd month and remained abnormal afterwards had a higher risk of recurrence (7/31; 27 percent patient-years; 95% CI:12-48) than patients in whom D-d remained normal at the 3r^d month and afterwards (4/149; 2.9 percent patient-years; 95% CI:1-7) (adjusted hazard ratio: 7.9; 95% CI:2.1-30; p=0.002). Repeated D-d testing after anticoagulation suspension for a first episode of unprovoked VTE could help tailor the duration of treatment. This trial is registered at http://clinicaltrials.gov as NCT00266045.

gp96, an endoplasmic reticulum master chaperone for integrins and Toll-like receptors, selectively regulates early T and B lymphopoiesis

Fri, 13 Nov 2009 12:28:30 PST

Integrins contribute to lymphopoiesis, whereas Toll-like receptors (TLRs) facilitate the myeloid replenishment during inflammation. The combined role of TLRs and integrin on hematopoiesis remains unclear. gp96 (grp94, HSP90b1) is an endoplasmic reticulum (ER) master chaperone for multiple TLRs. We report herein that gp96 is also essential for expression of 14 hematopoietic system-specific integrins. Genetic deletion of gp96 thus enables us to determine the collective roles of gp96, integrins and TLRs in hematopoiesis. We found that gp96-null hematopoietic stem cells could support long-term myelopoiesis. B and T cell development, however, was severely compromised with transitional block from pro-B to pre-B cells and inability of thymocytes to develop beyond CD4^-CD8^- stage. These defects were cell-intrinsic and could be recapitulated on bone marrow (BM) stromal cell culture. Furthermore, defective lymphopoiesis correlated strongly with failure of hematopoietic progenitors to form close contact with stromal cell niche and was not due to defect in the assembly of antigen receptor or IL-7 signaling. These findings define gp96 as the only known molecular chaperone to specifically regulate T and B cell development.

Indirect inhibition of in vivo and in vitro T cell responses by intravenous immunoglobulins due to impaired antigen presentation

Aubin, E., Lemieux, R., Bazin, R. — Fri, 13 Nov 2009 12:28:23 PST

Several clinical studies done with intravenous immunoglobulin (IVIg)-treated autoimmune patients as well as a number of in vitro studies have revealed that IVIg can reduce polyclonal T cell activation and modify their cytokine secretion pattern. However, their effect on (auto)antigen-specific T cell responses has never been addressed directly. In the present work, we used an in vivo model of induction of antigen-specific T cell responses and an in vitro antigen presentation system to study the effects of IVIg on T cell responses. The results obtained showed that IVIg inhibited both the in vivo and in vitro antigen-specific T cell responses but that this effect was the indirect consequence of a reduction in the antigen presentation ability of antigen presenting cells (APC). The inhibitory effect of IVIg was FcRIIb-independent, suggesting that IVIg must interfere with activating FcRs expressed on APC to reduce their ability to present antigens. Such inhibition of T cell responses by reducing antigen presentation may therefore contribute to the well-known anti-inflammatory effects of IVIg in autoimmune diseases.

Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma

Fri, 13 Nov 2009 12:28:16 PST

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade^TM), and importantly, triggers apoptosis in MM cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid^TM) induces synergistic anti-MM activity in vitro using MM cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with: 1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and PARP; 2) activation of BH-3 protein BIM; 3) translocation of BIM to endoplasmic reticulum; 4) inhibition of migration of MM cells and angiogenesis; and 5) suppression of chymotrypsin-like, caspase-like and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

Granzyme B is not required for regulatory T cell-mediated suppression of graft-versus-host disease

Cai, S. F., Cao, X., Hassan, A., Fehniger, T. A., Ley, T. J. — Fri, 13 Nov 2009 12:28:09 PST

Regulatory T (T_reg) cells can suppress a wide variety of immune responses, including anti-tumor and alloimmune responses. The mechanisms by which T_reg cells mediate their suppressive effects depend on the context of their activation. We previously reported that granzyme B is important for T_reg cell-mediated suppression of anti-tumor immune responses. We therefore hypothesized that granzyme B may likewise be important for suppression of graft-versus-host disease (GvHD). We found that allogeneic mismatch induces the expression of granzyme B in mixed lymphocyte reactions, and in a model of graft-versus-host disease (GvHD). However, wild-type and granzyme B-deficient T_reg cells were equally able to suppress effector T (T_eff) cell proliferation driven by multiple stimuli, including allogeneic antigen-presenting cells. Surprisingly, adoptive transfer of granzyme B-deficient T_reg cells prevented GvHD lethality, suppressed serum cytokine production in vivo, and prevented target organ damage. These data contrast strikingly with our previous study, demonstrating that granzyme B plays a non-redundant role in T_reg cell-mediated suppression of anti-tumor responses. Taken together, these findings suggest that targeting specific T_reg cell suppressive mechanisms, such as granzyme B, may be therapeutically beneficial for segregating GvHD and graft-versus-tumor immune responses.

ADAMTS13 gene deletion aggravates ischemic brain damage: a possible neuroprotective role of ADAMTS13 by ameliorating post-ischemic hypoperfusion

Fri, 13 Nov 2009 12:28:01 PST

Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, that are dependent on the platelet GPIb-von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion (MCAO) model in Adamts13-/- and wild-type mice. After reperfusion for 0.5 hour or 23.5 hours, the regional cerebral blood flow (rCBF) in the ischemic cortex was decreased markedly in Adamts13-/- mice compared to wild-type mice (P<0.05), which also resulted in a larger infarct volume after 24 hours for Adamts13-/- compared to wild-type mice (P<0.01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke.

Targeting EXT-1 reveals a crucial role for heparan sulfate in the growth of multiple myeloma

Fri, 13 Nov 2009 12:27:53 PST

Expression of the heparan sulfate proteoglycan (HSPG) syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side-chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT-1, a co-polymerase critical for HS-chain biosynthesis, had similar effects. By employing an innovative myeloma xenotransplant model in Rag-2^-/-_c^-/- mice, we demonstrate that induction of EXT-1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS-chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM.

The impact on outcome of the addition of all-trans retinoic acid to intensive chemotherapy in younger patients with non-acute promyelocytic acute myeloid leukemia: overall results and results in genotypic subgroups defined by mutations in NPM1, FLT3 and CEBPA

Thu, 12 Nov 2009 12:14:00 PST

We investigated the benefit of adding All-trans retinoic acid (ATRA) to chemotherapy for younger patients with non-Acute Promyelocytic AML and high risk MDS and to consider interactions between treatment and molecular markers. Overall, 1075 patients under 60 years were randomized to receive or not ATRA in addition to DAT (Daunorubicin/Ara-C/Thioguanine) chemotherapy with Ara-C at standard or double standard dose. There were data on FLT3 internal tandem duplications (FLT3/ITD) and NPM1 mutations (n=592), CEBPA mutations (n=423) and MN1 expression (n=195). The complete remission (CR) rate was 68% with CRi in an additional 16%; 8-year overall survival was 32%. There was no significant treatment effect for any outcome, with no significant interactions between treatment and demographics, or cytarabine randomization. Importantly, there were no interactions by FLT3/ITD, NPM1, or CEBPA mutation. There was a suggestion that ATRA reduced relapse in patients with lower MN1 levels, but no significant effect on overall survival. Results were consistent when restricted to patients with normal karyotype. ATRA has no overall effect on treatment outcomes in this group of patients. The study did not identify any subgroup of patients likely to derive a significant survival benefit from the addition of ATRA to chemotherapy. This study is registered at http://www.controlled-trials.com under ISRCTN17833622.

Regulating human Th17 cells via differential expression of IL-1 receptor

Thu, 12 Nov 2009 12:13:51 PST

In humans, IL-1β has been suggested as an essential cytokine for developing interleukin 17 (IL-17 or IL-17A) producing CD4⁺ T helper cells (Th17 cells). However, little is known about the relationship of IL-1 receptor expression and Th17 cell differentiation. We report here the presence of two distinct CD4⁺ T cell populations with and without expression of IL-1 receptor I (IL-1RI) that correlates with the capacity to produce IL-17 in naive and memory CD4⁺ T cells of human peripheral blood. IL-1RI⁺ memory CD4⁺ T cells had increased gene expression of IL17, RORC and IRF4 even before T cell receptor (TCR) triggering, indicating that the effect of IL-1β is programmed in these cells via IL-1RI. While CD4⁺ T cells from umbilical cord blood did not express IL-1RI, the cytokines IL-7, IL-15 and TGF-β up-regulated IL-1RI expression on naive CD4⁺ T cells, suggesting that IL-1RI⁺ naive CD4⁺ T cells develop in periphery. Furthermore, IL-17 production from the cytokine-treated naive CD4⁺ T cells was induced by IL-1β and this induction was blocked by IL-1R antagonist. These results indicate that human Th17 cell differentiation is regulated via differential expression of IL-1RI which is controlled by IL-7 and IL-15.

Comparison of gene expression profiles between human and mouse monocyte subsets

Thu, 12 Nov 2009 12:13:39 PST

Human and mouse blood each contain two main monocyte subsets. Here, we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in human and 550 genes in mouse were differentially expressed between subsets by ≥ 2.0-fold. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation, some molecules were conversely expressed between the two species' subsets, including CD36, CD9, and TREM-1. Other differences included a prominent PPAR signature in mouse monocytes, which is absent in man, and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus, whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized, important differences between the species deserve consideration when models of human disease are studied in the mouse.

An emerging player in the adaptive immune response: microRNA-146a is a modulator of IL-2 expression and AICD in T lymphocytes

Thu, 12 Nov 2009 12:13:20 PST

MicroRNAs (miRNAs) are a class of small noncoding RNAs that act as key post-transcriptional regulators of gene expression. Activation of the T cell-mediated immune response has been associated with changes in the expression of specific miRNAs. However, the role of miRNAs in the development of an effective immune response is just beginning to be explored. This study focuses on the functional role of miR-146a in T lymphocyte-mediated immune response and provides interesting clues on the transcriptional regulation of miR-146a during T cell activation. We show that miR-146a is low in human naive T cells and is abundantly expressed in human memory T cells; consistently, miR-146a is induced in human primary T lymphocytes upon TCR stimulation. Moreover, we identified NF-kB and c-ETS binding sites as required for the induction of miR-146a transcription upon TCR engagement. Our results demonstrate that several signalling pathways, other than inflammation, are influenced by miR-146a. In particular, we provide experimental evidence that miR-146a modulates AICD, acting as an anti-apoptotic factor, and that FADD is a target of miR-146a. Furthermore, miR-146a enforced expression impairs both AP-1 activity and IL-2 production induced by TCR engagement, thus suggesting a role of this miRNA in the modulation of adaptive immunity.

A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy

Wang, H., Liu, Y., Li, Z.-Y., Fan, X., Hemminki, A., Lieber, A. — Thu, 12 Nov 2009 12:13:09 PST

Many tumors, including lymphomas, upregulate expression of CD46 to escape destruction by complement. Tumor cells are therefore relatively resistant to therapy by monoclonal antibodies, which act through complement-dependent cytotoxicity (CDC). From an E.coli expression library of adenovirus type 35 fiber knob mutants, we selected a variant (Ad35K++) that had a higher affinity to CD46 than the natural Ad35 fiber knob. We demonstrated that incubation of lymphoma cells with recombinant Ad35K++ protein resulted in transient removal of CD46 from the cell surface. Pre-incubation of lymphoma cells with Ad35K++ sensitized cells to CDC triggered by the CD20-specific monoclonal antibody rituximab. In xenograft models with human lymphoma cells, pre-injection of Ad35K++ dramatically increased the therapeutic effect of rituximab. Blood cell counts and organ histology were normal after intravenous injection of Ad35K++ into mice that express human CD46. The presence of polyclonal anti-Ad35K++ antibodies did not affect the ability of Ad35K++ to enhance rituximab-mediated CDC in in vitro assays. The Ad35K++-based approach has potential implications in monoclonal antibody therapy of maligancies beyond the combination with rituximab.

Annexin A2 tetramer activates human and murine macrophages through TLR4

Swisher, J. F. A., Burton, N., Bacot, S., Vogel, S. N., Feldman, G. M. — Thu, 12 Nov 2009 12:12:59 PST

Annexins are an abundant family of intracellular phospholipid-binding proteins, yet several extracellular roles have been identified. Specifically, annexin A2 found in a heterotetrameric complex with S100A10 not only serves as a key extracellular binding partner for pathogens and host proteins alike, but also can also be shed or secreted. We reported previously that soluble annexin A2 tetramer (A2t) activates human monocyte-derived macrophages (MDM), resulting in secretion of inflammatory mediators and enhanced phagocytosis. Although a receptor for A2t has been cloned from bone marrow stromal cells, data contained herein demonstrate that it is dispensable for A2t-dependent activation of MDM. Furthermore, A2t activates wild-type (WT) murine bone marrow-derived macrophages (BMDM), whereas macrophages from myeloid differentiation factor 88 deficient (MyD88^-/-) mice display a blunted response, suggesting a role for Toll-like Receptor (TLR) signaling. siRNA knockdown of TLR4 in human MDM reduced the response to A2t, blocking antibodies against TLR4 (but not TLR2) blocked activation altogether, and BMDM from TLR4^-/- mice were refractory to A2t. These data demonstrate that the modulation of macrophage function by A2t is mediated through TLR4, suggesting a previously unknown but important role for this stress-sensitive protein in the detection of danger to the host, whether from injury or invasion.

Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development

Thu, 12 Nov 2009 12:12:50 PST

Proper thymocyte development is required to establish T cell central tolerance and to generate naive T cells, both of which are essential for T cell homeostasis and a functional immune system. Here we demonstrate that the loss of transcription factor Foxp1 results in the abnormal development of T cells. Instead of generating naive T cells, Foxp1-deficient single-positive thymocytes acquire an activated phenotype prematurely in the thymus and lead to the generation of peripheral CD4⁺ T and CD8⁺ T cells that exhibit an activated phenotype, increased apoptosis and readily produce cytokines upon T cell receptor engagement. These results identify Foxp1 as an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development.

Src tyrosine kinase pre-activation is associated with platelet hypersensitivity in essential thrombocythemia and polycythemia vera

Thu, 12 Nov 2009 12:13:30 PST

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders characterized by an increased incidence of thrombotic complications and tendency to evolve into myelofibrosis or acute myeloid leukemia. The acquired somatic JAK2 V617F-mutation is present in the majority of PV and ET patients. Since aberrant protein Tyr-phosphorylation has been associated with hematopoietic malignancies, the activity of the tyrosine kinases Src and JAK2 was analyzed in resting and thrombin-stimulated platelets from 13 PV and 42 ET patients. JAK2 was found inactive in healthy and pathological resting cells regardless of the presence of the V617F-mutation. Also Src was inactive in all resting platelets, but in the pathological specimens it was present in a pre-activated conformation as a consequence of anomalous dephosphorylation of its inhibitory phospho-Tyr527 residue, likely mediated by SHP-2 Tyr-phosphatase, whose constitutive activity correlated with its recruitment to Src. Low thrombin concentration triggered a more rapid Src-signaling activation, higher [Ca2⁺]_c increase and aggregation in pathological platelets compared to control ones. Thrombin-induced Src-activation preceded JAK2-activation, which occurred simultaneously in normal and pathological platelets. Our results indicate that a constitutive Src-kinase pre-activation is implicated in platelet hypersensitivity and likely involved, at least partially, in the functional abnormalities of PV and ET platelets.

Memories that last forever: strategies for optimizing vaccine T cell memory

Ahlers, J. D., Belyakov, I. M. — Tue, 10 Nov 2009 12:46:05 PST

For acute self limiting infections a vaccine is successful if it elicits memory at least as good as the natural experience; however, for persistent and chronic infections such as HIV, HCV, HPV, and human Herpes Viruses, this paradigm is not applicable. At best, during persistent virus infection the individual must be able to maintain the integrity of the immune system in equilibrium with controlling replicating virus. New vaccine strategies are required that elicit both potent high avidity CD8⁺ T cell effector/memory and central memory responses that can clear the nidus of initial virus infected cells at mucosal surfaces in order to prevent mucosal transmission or significantly curtail development of disease. The objective of an HIV-1 T cell vaccine is to generate functional CD8⁺ effector memory cells at mucosal portals of virus entry to prevent viral transmission. In addition, long-lived CD8⁺ and CD4⁺ central memory cells circulating through secondary lymphoid organs and resident in bone marrow respectively are needed to provide a concerted second wave of defense that can contain virus at mucosal surfaces and prevent systemic dissemination. Further understanding of factors which can influence long-lived effector and central memory cell differentiation will significantly contribute to development of effective T cell vaccines. In this review we will focus on discussing mechanisms involved in T cell memory and provide promising new approaches toward expanding current vaccine strategies to enhance antiviral memory.

ALK1 signaling regulates early postnatal lymphatic vessel development

Niessen, K., Zhang, G., Ridgway, J. B., Chen, H., Yan, M. — Tue, 10 Nov 2009 12:45:54 PST

In vertebrates, endothelial cells form two hierarchical tubular networks, the blood vessels and the lymphatic vessels. Despite the difference in their structure and function, and genetic programs that dictate their morphogenesis, common signaling pathways have been recognized that regulate both vascular systems. ALK1 is a member of the TGFβ type I family of receptors, and compelling genetic evidence suggests its essential role in regulating blood vascular development. Here we report that ALK1 signaling is intimately involved in lymphatic development. Lymphatic endothelial cells express key components of ALK1 pathway and respond robustly to ALK1 ligand stimulation in vitro. Blockade of ALK1 signaling results in defective lymphatic development in multiple organs of neonatal mice. We found that ALK1 signaling regulates the differentiation of lymphatic endothelial cells to influence the lymphatic vascular development and remodeling. Furthermore, simultaneous inhibition of ALK1 pathway increases apoptosis in lymphatic vessels caused by blockade of VEGFR3 signaling. Thus, our study reveals a novel aspect of ALK1 signaling in regulating lymphatic development, and suggests that targeting ALK1 pathway might provide additional control of lymphangiogenesis in human diseases.

The effect of prolonged administration of hydroxyurea on morbidity and mortality in adult patients with sickle-cell syndromes: results of a 17-year, single center trial (LaSHS)

Tue, 10 Nov 2009 12:45:44 PST

The aim of this prospective study was to evaluate the long-term efficacy and safety of hydroxyurea (HU) in sickle-cell disease (SCD). Thirty-four patients with sickle-cell anemia (HbS/HbS), 131 with HbS/β⁰-thal and 165 with HbS/β⁺-thal were participated into this trial. HU was given to 131 patients, while 199 patients were conventionally treated. The median follow-up period was 8 years for HU patients and 5 years for non-HU patients. HU produced a dramatic reduction of the frequency of severe painful crises, transfusion requirements, hospital admissions, and incidence of acute chest syndrome. The probability of 10-year survival was 86% and 65% for HU and non-HU patients, respectively (p=0.001), although HU patients had more severe form of SCD. The 10-year probability of survival for HbS/HbS, HbS/β⁰-thal, and HbS/IVSI-110 patients was 100%, 87% and 82%, respectively for HU patients and 10%, 54% and 66%, for non-HU patients. The multivariate analysis showed that HbF values at baseline and percentage change of LDH between baseline and 6-month were independently predicted for survival in the HU group. These results highlight the beneficial effect of HU which seems to modify the natural history of SCD and raise the issue of expanding its use in all SCD patients.

Quantitative DNA-methylation predicts survival in adult acute myeloid leukemia

Tue, 10 Nov 2009 12:45:27 PST

Acute myeloid leukemia (AML) is characterized by molecular heterogeneity that is not fully reflected in the current classification system. Recent insights point towards a significant role of aberrant DNA-methylation in leukemogenesis. Therefore, we investigated the prognostic impact of DNA-methylation in AML. In order to screen for promoter methylation in AML we applied a combination of base-specific cleavage biochemistry and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS), a powerful methodology allowing for quantitatively investigating DNA-methylation status to a large series of both promoter regions and leukemia samples. We analyzed 92 genomic regions in 182 patient samples, correlated findings with clinical and molecular data, and validated the results in an independent cohort of 74 AML samples. Using this approach, we were able to identify novel leukemia subgroups based on distinct DNA-methylation patterns. Furthermore, we defined a methylation-based outcome predictor for patient survival (P<0.01) that in multivariable analysis provided independent prognostic information (hazard ratio 1.52, 95%-CI 1.06 – 2.16). Here, we report the first large scale methylation-based outcome predictor in AML, and thereby our findings support the use of genomic methylation markers for improved molecular classification and prognostication in adult AML.

Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages

Tue, 10 Nov 2009 12:45:18 PST

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. Additionally, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin, and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.

Education of human natural killer cells by activating killer cell immunoglobulin-like receptors

Fauriat, C., Ivarsson, M. A., Ljunggren, H.-G., Malmberg, K.-J., Michaelsson, J. — Tue, 10 Nov 2009 12:45:09 PST

Expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-MHC class I molecules provides an educational signal that generates functional NK cells. However, the effects of activating KIRs specific for self-MHC class I on NK cell education remain elusive. Here, we provide evidence that the activating receptor KIR2DS1 tunes down the responsiveness of freshly isolated human NK cells to target cell stimulation in donors homozygous for HLA-C2, the ligand of KIR2DS1. The tuning was apparent in KIR2DS1⁺ NK cells lacking expression of inhibitory KIRs and CD94/NKG2A, as well as in KIR2DS1⁺ NK cells co-expressing the inhibitory MHC class I-specific receptors CD94/NKG2A and KIR2DL3, but not KIR2DL1. However, the tuning of responsiveness was restricted to target cell recognition, since KIR2DS1⁺ NK cells responded well to exogenous cytokines. Our results provide the first example of human NK cell education by an activating KIR and suggest that the education of NK cells via activating KIRs is a mechanism to secure tolerance that complements education via inhibitory KIRs.

The role of the Th1 transcription factor T-bet in a mouse model of immune-mediated bone marrow failure

Tang, Y., Desierto, M. J., Chen, J., Young, N. S. — Tue, 10 Nov 2009 12:44:58 PST

The transcription factor T-bet is a key regulator of type 1 immune responses. We examined the role of T-bet in an animal model of immune-mediated bone marrow (BM) failure, using mice carrying a germ-line T-bet gene deletion (T-bet^-/-). In comparison to normal C57BL6 (B6) controls, T-bet^-/- mice had normal cellular composition in lymphohematopoietic tissues, but T-bet^-/- lymphocytes were functionally defective. Infusion of 5 x 10⁶ T-bet^-/- lymph node (LN) cells into sublethally-irradiated, major histocompatibility complex mismatched, CByB6F1 (F1) recipients failed to induce the severe marrow hypoplasia and fatal pancytopenia which is produced by injection of similar numbers of B6 LN cells. Increasing T-bet^-/- LN cell dose to 10-23 x 10⁶ per recipient led to only mild hematopoietic deficiency. Recipients of T-bet^-/- LN cells had no expansion in T cells or gamma interferon (IFN-)-producing T cells, but showed a significant increase in Lin^-Sca1⁺CD117⁺CD34^- BM cells. Plasma transforming growth factor β and interleukin 17 concentrations were increased in T-bet^-/- LN cell recipients, possibly a compensatory up-regulation of the Th17 immune response. Continuous infusion of IFN- resulted in hematopoietic suppression but did not cause T-bet^-/- LN cell expansion or BM destruction. Our data provided fresh evidence demonstrating a critical role of T-bet in immune mediated BM failure.

In vivo biotinylation of the vasculature in B cell lymphomaidentifies BST-2 as a target for antibody-based therapy

Tue, 10 Nov 2009 12:44:43 PST

The discovery of accessible markers of lymphoma may facilitate the development of antibody-based therapeutic strategies. Here, we describe the results of a chemical proteomic study, based on the in vivo biotinylation of vascular proteins in lymphoma-bearing mice followed by mass spectrometric and bioinformatic analysis, to discover proteins expressed at the tissue-blood border of disseminated B cell lymphoma. From a list of 58 proteins which were more than 10-fold up-regulated in nodal and extranodal lymphoma lesions compared to their levels in the corresponding normal host organs, we validated BST-2 as a novel vascular marker of B cell lymphoma, using immunochemical techniques and in vivo biodistribution studies. Furthermore, targeting BST-2 with two independent monoclonal antibodies delayed lymphoma growth in a syngeneic mouse model of the disease. The results of this study delineate a strategy for the treatment of systemic B cell lymphoma in humans and suggest that anti-BST-2 antibodies may facilitate pharmacodelivery approaches which target the tumor-stroma interface.

Autoimmune B cell lymphopenia following successful adoptive therapy with telomerase-specific T lymphocytes

Tue, 10 Nov 2009 12:44:31 PST

Telomerase (TERT) is a good candidate for cancer immunotherapy since it is overexpressed in 85% of all human tumors and implicated in maintenance of the transformed phenotype. TERT-based cancer vaccines have been shown to be safe, not inducing any immune-related pathology, but their impact on tumor progression is modest. Here we show that adoptive cell therapy (ACT) utilizing high avidity T lymphocytes reactive against telomerase can control growth of different established tumors. Moreover, in transgenic adenocarcinoma mouse prostate (TRAMP) mice, which develop prostate cancer, TERT-based ACT halted progression to more aggressive and poorly differentiated tumors, significantly prolonging mouse survival. We also demonstrated that human tumors, including a Burkitt's lymphoma, and human cancer stem cells, are targeted in vivo by TERT-specific CTLs. Effective therapy with T cells against telomerase, differently from active vaccination, however led to autoimmunity marked by a consistent, although transient, B cell depletion in primary and secondary lymphoid organs, associated with alteration of the spleen cytoarchitecture. These results indicate B cells as an in vivo target of TERT-specific CTLs during successful immunotherapy.

The architectural pattern of FOXP3-positive T cells in follicular lymphoma is an independent predictor of survival and histologic transformation

Farinha, P., Al-Tourah, A., Gill, K., Klasa, R., Connors, J. M, Gascoyne, R. D — Mon, 09 Nov 2009 13:18:51 PST

Previous studies of FL patients treated heterogeneously have suggested that decreased numbers of regulatory T-cells (Tregs) correlates with improved survival. We studied advanced-stage FL patients from a single institution phase II trial. All patients were treated uniformly with multi-agent chemotherapy and radiation. Tissue microarrays were constructed using diagnostic biopsies available in 105 patients and stained with CD4, CD8, CD25 and FOXP3 antibodies. Both cell content and cell distribution were evaluated. For all antibodies, there were cases with a predominant intrafollicular or perifollicular localization of cells ("follicular" pattern) while others displayed a diffuse ("diffuse") pattern. The median follow-up of living patients was 17.1 years. The International Prognostic Index (IPI) score predicted overall survival (OS) (p=0.004) but not risk of transformation (RT). Cell content did not impact survival while immunoarchitectural patterns of CD4/CD8 were significant for progression-free survival (PFS) (p=0.056), CD25 for both PFS and OS (p=0.0019 and p=0.024, respectively) and FOXP3⁺ predicted PFS, OS and RT (p=0.0003, p<0.0001 and p=0.002, respectively). A Cox multivariate model showed both IPI and FOXP3⁺ pattern were independent predictors of OS (p=0.008 and p<0.001, respectively), while only FOXP3⁺ pattern predicted RT (p=0.004). We conclude that FOXP3⁺ cell distribution significantly predicts survival and RT in FL.

Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11 acute myeloid leukemias

Mon, 09 Nov 2009 13:18:43 PST

Early relapse detection in AML is possible using standardized real time quantitative polymerase chain reaction (RQ-PCR) protocols. However, optimal sampling intervals have not been defined and are likely to vary according to the underlying molecular lesion. In 74 patients experiencing hematological relapse (HR) and harboring aberrations amenable to RQ-PCR (mutated NPM1 [designated NPM1c], PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11), we observed strikingly different relapse kinetics. The median doubling time of the CBFB-MYH11 leukemic clone was significantly longer (36 days) than that of clones harboring other markers (RUNX1-RUNX1T1 14 days, PML-RARA, 12 days and NPM1c, 11 days, P<0.001). Furthermore, we employed a mathematical model to determine frequency of relapse detection (RDF) and median time from detection of minimal residual disease (MRD) to HR (t_m) as a function of sampling interval length. For example, to obtain an RDF of 90% and a t_m of 60 days, blood sampling every 6^th month should be performed for CBFB-MYH11 leukemias. By contrast, in NPM1c+/FLT3-ITD-, NPM1c+/FLT3-ITD+, RUNX1-RUNX1T1 and PML-RARA leukemias bone marrow sampling is necessary every 6^th, 4^th, 4^th and 2^nd month, respectively. These data carry important implications for the development of optimal RQ-PCR monitoring schedules suitable for evaluation of MRD-directed therapies in future clinical trials.

International Prognostic Scoring System-independent cytogenetic risk categorization in primary myelofibrosis

Hussein, K., Pardanani, A. D., van Dyke, D. L., Hanson, C. A., Tefferi, A. — Mon, 09 Nov 2009 13:18:17 PST

Among 200 patients with primary myelofibrosis, karyotype at diagnosis was abnormal in 83 (42%). In order to assess their individual prognostic impact, specific cytogenetic abnormalities with ≥5 informative cases were identified and the rest grouped separately as "other abnormalities". Median survival in patients with sole +9 (n=6), sole 20q- (n=21), sole 13q- (n=8), normal karyotype (n=117), "other abnormalities" (n=28), complex karyotype (n=13), and sole +8 (n=7) were "not reached", 112, 105, 80, 46, 34 and 28 months, respectively (p=0.01). Accordingly, four cytogenetic risk groups were considered: i) favorable (sole +9, 20q- or 13q-), ii) normal, iii) unfavorable (complex karyotype or sole +8), and iv) "other abnormalities". Multivariable analysis confirmed the International Prognostic Scoring System (IPSS)-independent prognostic value of both 4-way and 2-way (i.e. favorable/normal vs. unfavorable/other abnormalities; IPSS-adjusted HR 0.37, 95% CI 0.24-0.58) cytogenetic risk categorization (p<0.01). The ability to prognostically dissect a specific IPSS category has major therapeutic implications.

Malaria in patients with sickle cell anemia: burden, risk factors and outcome at outpatient clinic and during hospitalization

Mon, 09 Nov 2009 13:18:07 PST

Approximately 280,000 children are born with sickle cell anemia (SCA) in Africa annually yet few survive beyond childhood. Falciparum malaria is considered a significant cause of this mortality. We conducted a 5-year prospective surveillance study for malaria parasitemia, clinical malaria and severe malarial anemia (SMA) in Dar-es-Salaam, Tanzania between 2004 and 2009. We recorded 10,491 visits to the outpatient clinic among 1,808 patients with SCA and 773 visits among 679 patients without SCA. Similarly, we recorded 691 hospital admissions among 497 patients with SCA and 2,017 in non-SCA patients. Overall, the prevalence of parasitemia was lower in SCA than in non-SCA patients both at clinic (0.7% versus 1.6%; OR 0.53, 95% confidence interval 0.32-0.86; p=0.008) and during hospitalization (3.0% versus 5.6%; 0.46; 0.25-0.94; 0.01). Furthermore, SCA patients had higher rates of malaria during hospitalization than at clinic, the ORs being 4.29 (2.63-7.01; p<0.001) for parasitemia, 17.66 (5.92-52.71; p<0.001) for clinical malaria, and 21.11 (8.46-52.67; p<0.001) for SMA respectively. Although malaria was rare among patients with SCA, parasitemia during hospitalization was associated with both severe anemia and death. Effective treatment for malaria during severe illness episodes and further studies to determine the role chemoprophylaxis are required.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation

Mon, 09 Nov 2009 13:17:58 PST

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, while Nrf2 is a key activator of stress-responsive genes. Since p45 and Nrf2 have similar DNA binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently utilized as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that two characteristic gene clusters are defined within p45 target genes; platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

MT1-MMP controls human mesenchymal stem cell trafficking and differentiation

Lu, C., Li, X.-Y., Hu, Y., Rowe, R. G., Weiss, S. J. — Mon, 09 Nov 2009 13:17:42 PST

Human mesenchymal stem cells (hMSCs) localized to bone marrow, non-hematopoietic organs as well as perivascular niches are postulated to traffic through type I collagen-rich stromal tissues in order to first infiltrate sites of tissue damage, inflammation or neoplasia and then, differentiate. Nevertheless, the molecular mechanisms supporting the ability of hMSCs to remodel 3-dimensional (3-D) collagenous barriers during trafficking or differentiation remain undefined. Herein, we demonstrate that hMSCs degrade and penetrate type I collagen networks in tandem with the expression of a five-member set of collagenolytic matrix metalloproteinases (MMPs). Specific silencing of each of these proteases reveals that only a single membrane-tethered metalloenzyme, termed MT1-MMP, plays a required role in hMSC-mediated collagenolysis, 3-D invasion and intravasation. Further, once confined within type I collagen-rich tissue, MT1-MMP also controls hMSC differentiation in a 3-D-specific fashion. Together, these data demonstrate that hMSC invasion and differentiation programs fall under the control of the pericellular collagenase, MT1-MMP.

Inflammation induces lymphangiogenesis through upregulation of VEGFR-3 mediated by NF-{kappa}B and Prox1

Mon, 09 Nov 2009 13:18:34 PST

The concept of inflammation-induced lymphangiogenesis (i.e., formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The two primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the NF-B family; however, the role of NF-B in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-B pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-B followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-B by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-B to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-B pathway by inflammatory stimuli activates Prox1, and both NF-B and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

Congenic interval of CD45 / Ly-5 congenic mice contains multiple genes that may influence hematopoietic stem cell engraftment

Waterstrat, A., Liang, Y., Swiderski, C. F., Shelton, B. J., Van Zant, G. — Mon, 09 Nov 2009 13:18:24 PST

The B6.SJL-Ptprc(d)Pep3(b)/BoyJ (B6.SJL) congenic mouse strain, a valuable and widely used tool in murine bone marrow transplantation studies, has long been considered equivalent to the parental C57B/L6 (B6) strain with the exception of a small congenic interval on chromosome 1 harboring an alternative CD45/Ly-5 alloantigen (Ly-5.1). In this study we compared functional properties of stem and stromal cells between the strains, and delineated the boundary of the B6.SJL congenic interval. We identified a 25% reduction in homing efficiency, 3.8-fold reduction in transplantable long-term hematopoietic stem cells (LT-HSCs), a 5-fold reduction in LT-HSCs capable of 24-hour homing, and a cell-intrinsic engraftment defect of approximately 30-50% in B6.SJL-derived bone marrow cells relative to B6-derived cells. These functional differences were independent of stem cell number, cycling, or apoptosis. Genotypic analysis revealed a 42.1 mbp congenic interval in B6.SJL including 306 genes, and at least 124 genetic polymorphisms. Moreover, expression profiling revealed 288 genes differentially expressed between non-hematopoietic stromal cells of the two strains. These results indicate that polymorphisms between the B6 and SJL genotype within the B6.SJL congenic interval influence HSC engraftment and result in transcriptional variation within bone marrow stroma.

Purified T-depleted, CD34+ peripheral blood and bone marrow cell transplantation from haploidentical mother to child with thalassemia

Fri, 06 Nov 2009 12:50:36 PST

Feto-maternal microchimerism suggests immunological tolerance between mother and fetus. Thus, we performed primary hematopoietic stem-cell transplantation (HSCT) from mismatched mother to thalassemic patient without an HLA-identical donor. Twenty-two patients with thalassemia major were conditioned with 60 mg/kg hydroxyurea and 3 mg/kg azathioprine from day -59 to -11, 30 mg/m² fludarabine from day -17 to -11, 14 mg/kg busulfan starting on day -10, and 200 mg/kg cyclophosphamide, 10 mg/kg Thiotepa, and 12.5 mg/kg anti-thymocyte globulin daily from day -5 to -2. Fourteen patients received CD34⁺ mobilized peripheral and bone marrow progenitor cells; eight patients received marrow graft selected PBSC CD34⁺ and BM CD3/CD19 depleted . T-cell dose was adjusted to 2 x 10⁵/kg by fresh marrow cell addback at the time of transplant. Both groups received cyclosporine for graft versus host disease (GVHD) prophylaxis for two months post transplant. Two patients died (cerebral EBV lymphoma or CMV pneumonia), six patients reject their grafts, and 14 showed full chimerism with functioning grafts at a median follow-up of 40 months. None of the 14 patients who showed full chimerism developed acute or chronic GVHD. These results suggest that maternal haploidentical HSCT is feasible for patients with thalassemia who lack a matched related donor.

Differential genome-wide array-based methylation profiles in prognostic subsets of chronic lymphocytic leukemia

Fri, 06 Nov 2009 12:50:29 PST

Global hypomethylation and regional hypermethylation are well-known epigenetic features of cancer, however, in chronic lymphocytic leukemia (CLL), studies on genome-wide epigenetic modifications are limited. Here we analyzed the global methylation profiles in CLL, by applying high-resolution methylation micro-arrays (27,578 CpG sites) to 23 CLL samples, belonging to the immunoglobulin heavy-chain variable (IGHV) mutated (favorable) and IGHV unmutated/IGHV3-21 (poor-prognostic) subsets. Overall, results demonstrated significant differences in methylation patterns between these subgroups. Specifically, in IGHV unmutated CLL, we identified methylation of 7 known or candidate tumor suppressor genes (e.g. VHL, ABI3 and IGSF4) as well as 8 unmethylated genes involved in cell proliferation and tumor progression (e.g. ADORA3 and PRF1 enhancing the NF-B and MAPKinase pathways, respectively). In contrast, these latter genes were silenced by methylation in IGHV mutated patients. The array data was validated for selected genes using methylation-specific PCR, quantitative RT-PCR and bi-sulfite sequencing. Finally, the significance of DNA methylation in regulating gene promoters was shown by re-inducing 4 methylated tumor suppressor genes (e.g. VHL and ABI3) in IGHV unmutated samples using the methyl-inhibitor 5-aza-2'-deoxycytidine. Taken together, our data for the first time reveal differences in global methylation profiles between prognostic subsets of CLL, which may unfold epigenetic silencing mechanisms involved in CLL pathogenesis.

Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant V{alpha}14-J{alpha}18 TCR{alpha} gene

Fri, 06 Nov 2009 12:50:22 PST

Establishment of a system with efficient generation of NKT cells from embryonic stem (ES) cells would enable us to identify the cells with NKT cell potential and obtain NKT cells with desired function. Here, by using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system, which preferentially developed functional NKT cells, and also identified early NKT progenitors which first appeared on day 11 as a c-kit⁺ population in the co-cultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kit^lo/- on day 14. Interestingly, in the presence of Notch signals NKT-ES cells differentiated only to thymic CD44^lo CD24^hi NKT cells producing mainly IL-4, while NKT cells resemble to CD44^hi CD24^lo liver NKT cells producing mainly IFN- and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT cell development and for the establishment of NKT cell therapy.

Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis

Fri, 06 Nov 2009 12:50:14 PST

Tissue infiltration of phagocytes exacerbates several human pathologies including chronic inflammations or cancers. However, the mechanisms involved in macrophage migration through interstitial tissues are poorly understood. We investigated the role of Hck, a Src-family kinase involved in the organization of matrix adhesion and degradation structures called podosomes. In hck^-/- mice submitted to peritonitis, we found that macrophages accumulated in interstitial tissues and poorly reached the peritoneal cavity. In vitro, three-dimensional (3D)-migration and matrix degradation abilities, two protease-dependent properties of bone marrow-derived macrophages (BMDMs), were affected in hck^-/- BMDMs. These macrophages formed few and undersized podosome rosettes and, consequently, had reduced matrix proteolysis operating underneath despite normal expression and activity of Matrix Metalloproteases. Finally, in fibroblasts unable to infiltrate matrix, ectopic expression of Hck provided the gain of 3D-migration function which correlated positively with formation of podosome rosettes. In conclusion, spatial organization of podosomes as large rosettes, proteolytic degradation of extracellular matrix and 3D-migration appeared to be functionally linked and regulated by Hck in macrophages. Hck being the first protein combining a phagocyte-limited expression to a role in 3D-migration, it could be a target for new anti-inflammatory and anti-tumour molecules.

CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK cell subsets

Fri, 06 Nov 2009 12:50:06 PST

Human CD56^bright natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIR), high IFN- production, but little cytotoxicity. CD56^dim NK cells have high KIR expression, produce little IFN-, yet display high cytotoxicity. We hypothesized that if human NK maturation progresses from a CD56^bright to a CD56^dim phenotype, an intermediary NK cell must exist which demonstrates more functional overlap than these two subsets, and we utilized CD94 expression to test our hypothesis. CD94^highCD56^dim NK cells express CD62L, CD2, and KIR at levels between CD56^bright and CD94^lowCD56^dim NK cells. CD94^highCD56^dim NK cells produce less monokine-induced IFN- than CD56^bright NK cells but much more than CD94^lowCD56^dim NK cells due to differential IL-12-mediated STAT4 phosphorylation. CD94^highCD56^dim NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56^bright NK cells but lower than CD94^lowCD56^dim NK cells. Collectively, our data suggest that density of CD94 surface expression on CD56^dim NK cells identifies a functional and likely developmental intermediary between CD56^bright and CD94^lowCD56^dim NK cells. This supports the notion that in vivo, human CD56^bright NK cells progress through a continuum of differentiation that ends with a CD94^lowCD56^dim phenotype.

Combination immunosuppressant therapy for patients with chronic refractory immune thrombocytopenic purpura

Fri, 06 Nov 2009 06:15:10 PST

Treatment options for patients with chronic refractory immune thrombocytopenic purpura (ITP) are limited. Because combination immunosuppressant therapy had proved effective in ITP and other disorders, we used this approach in patients with particularly severe and refractory ITP. In this retrospective, observational study, we determined the response (platelet count above 30x10⁹/L and doubling of baseline) among 19 refractory ITP patients. Treatment consisted of azathioprine, mycophenolate mofetil and cyclosporine. The patients had failed a median of 6 prior treatments, including splenectomy (in all except one). Of 19 patients, 14 (73.7%) achieved a response lasting a median of 24.0 months after which time 8 (57.1%) relapsed. Of the 8 relapsing patients, 6 responded to additional treatments. Of the 14 patients who achieved an initial response, 2 (14.3%) remained in remission after eventually stopping all medications. Severe adverse events did not occur. Combination immunosuppressant therapy can produce a rise in the platelet count, sometimes sustained in refractory ITP patients.

Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin

Fri, 06 Nov 2009 06:15:00 PST

Cobalamin (Cbl, vitamin B12) deficiency in humans is a common cause of hematological and neurological disorders. We show here that the cellular export of Cbl, in contrast to the carrier- and receptor-dependent cellular import of Cbl, occurs by transmembrane transport of 'free' Cbl. Screening of candidate transporters by cellular gene silencing revealed a role in cellular cobalamin efflux of the ATP-binding cassette (ABC)-drug transporter, ABCC1, alias Multidrug Resistance Protein 1 (MRP1), which is present in the basolateral membrane of intestinal epithelium and in other cells. The ability of MRP1 to mediate ATP-dependent Cbl transport was confirmed by vesicular transport experiments, and a physiological role of MRP1 in mammalian Cbl homeostasis is indicated by the phenotype of knockout mice with targeted disruption of MRP1. These animals have a reduced concentration of Cbl in plasma and in the storage organs liver and kidney. In contrast, Cbl accumulates in the terminal part of the intestine of these mice suggesting a functional malabsorbtion due to a lower epithelial basolateral Cbl efflux. The identification of this Cbl export mechanism now allows the delineation of a coherent pathway for Cbl trafficking from food to the body cells.

Rapid activation of endothelial cells enables P. falciparum adhesion to platelet decorated von Willebrand factor strings

Fri, 06 Nov 2009 06:14:45 PST

During Plasmodium falciparum malaria infections, von Willebrand factor (VWF) levels are elevated, post-mortem studies show platelets co-localised with sequestered infected erythrocytes (IE) at brain microvascular sites, while in vitro studies have demonstrated platelet-mediated IE adhesion to TNF-activated brain endothelium via a bridging mechanism. This current study demonstrates how all these observations could be linked through a completely novel mechanism whereby IE adhere via platelet decorated ultra-large VWF strings on activated endothelium. Using an in vitro laminar flow model, we have demonstrated tethering and firm adhesion of IE to the endothelium specifically at sites of platelet accumulation. We also show that an IE pro-adhesive state, capable of supporting high levels of binding within minutes of induction can be removed through the action of the VWF protease ADAMTS-13. We propose that this new mechanism contributes to sequestration both independently of and in concert with current adhesion mechanisms.

Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML

Fri, 06 Nov 2009 06:14:37 PST

Activation of p53 by MDM2 antagonist nutlin-3a or inhibition of XIAP induces apoptosis in AML cells. We demonstrate here that concomitant inhibition of MDM2 by nutlin-3a and of XIAP by small molecule antagonists synergistically induced apoptosis in p53 wild-type OCI-AML3 and Molm13 cells. Knockdown of p53 by shRNA blunted the synergy, and downregulation of XIAP by antisense oligonucleotide (ASO) enhanced nutlin-3a-induced apoptosis, suggesting that the synergy was mediated by p53 activation and XIAP inhibition. This is supported by data showing that inhibition of both MDM2 and XIAP by their respective ASOs induced significantly more cell death than either ASO alone. Importantly, p53 activation and XIAP inhibition enhanced apoptosis in blasts from patients with primary AML, even when the cells were protected by stromal cells. Mechanistic studies demonstrated that XIAP inhibition potentiates p53-induced apoptosis by decreasing p53-induced p21 and that p53 activation enhances XIAP inhibition-induced cell death by promoting mitochondrial release of SMAC and by inducing the expression of caspase-6. Since both XIAP and p53 are presently being targeted in ongoing clinical trials in leukemia, the combination strategy holds promise for expedited translation into the clinic.

A population based cytogenetic study of adults with acute lymphoblastic leukaemia (ALL)

Moorman, A. V, Chilton, L., Wilkinson, J., Ensor, H. M, Bown, N., Proctor, S. J — Fri, 06 Nov 2009 06:14:26 PST

Chromosomal abnormalities are increasingly used to risk stratify adults with ALL. Published data describing the age-specific incidence of chromosomal abnormalities and their prognostic relevance is largely derived from clinical trials. Trials frequently have age restrictions and low recruitment rates. Thus we investigated these factors in a population-based cohort of 349 patients diagnosed over 19 years in the North of the UK. The incidence of most chromosomal abnormalities varied significantly with age. The incidence of t(9;22)(q34;q11) increased in each successive decade, up to 24% among 40-49 year olds. Thereafter the incidence reached a plateau. t(4;11)(q21;q23) and t(1;19)(q23;p13) were rare among patients aged over 60 years. In contrast, t(8;14)(q24;q32) and t(14;18)(q32;q21) increased with age. High hyperdiploidy occurred in 13% of patients <20 years but in only 5% of older patients. The incidence of low hypodiploidy / near-triploidy (HoTr) and complex karyotype increased with age from 4% (15-29 years) to 15% (60+ years). Overall survival varied significantly by age and cytogenetics. Older patients and those with t(9;22), t(4;11), Ho-Tr or complex karyotype had a significantly inferior outcome. These population-based results demonstrate the cytogenetic heterogeneity of adult ALL. These data will inform service planning and the design of new age-focussed clinical trials.

Force-induced cleavage of single VWF A1A2A3-tridomains by ADAMTS-13

Wu, T., Lin, J., Cruz, M. A., Dong, J.-F., Zhu, C. — Fri, 06 Nov 2009 06:14:19 PST

A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs 13 (ADAMTS-13) regulates hemostasis by cleaving a cryptic peptide bond inside the folded A2 domain of von Willebrand factor (VWF). This cleavage is regulated mechanically by hemodynamic forces as it occurs in flowing blood. We tested the hypothesis that force-induced A2 domain unfolding facilitates cleavage using atomic force microscopy to pull single VWF A1A2A3-tridomain polypeptides by platelet glycoprotein (GP) Ib or antibodies to measure time, distance, and force. Structural destabilization of A1A2A3 was induced by 5-80 pN forces, manifesting as an abrupt molecular length increase distributed around 20 and 50 nm, likely due to uncoupling A1A2A3 (or partially unfolding A2) and fully unfolding A2, respectively. Time required to destabilize A1A2A3 first increased (catch), reaching a maximum of 0.2 s at 20 pN, then decreased (slip) with increasing force, independent of ADAMTS-13. Time required to rupture A1A2A3 exhibited similar catch-slip behavior when pulled by GPIb but only slip behavior when pulled by antibody, which was progressively shortened by increasing concentration of ADAMTS-13 and by decreasing force after (but not before) structural destabilization, indicating that cleavage of A2 requires the force-induced A2 unfolding. Analysis with a model for single-substrate trimolecular enzymatic kinetics estimated a cleavage rate k_cat of 2.9 ± 0.59 s^-1 and a K_d of 5.6 ± 3.4 nM for ADAMTS-13/A1A2A3 binding. These findings quantify the mechanical regulation of VWF cleavage by ADAMTS-13 at the level of single A1A2A3-tridomain.

The AC133+CD38-, but not the rhodamine-low phenotype, tracks LTC-IC and SRC function in human cord blood ex vivo expansion cultures

Fri, 06 Nov 2009 06:14:11 PST

Phenotypic markers associated with human hematopoietic stem cells (HSC) were developed and validated using uncultured cells. Since phenotype and function can be dissociated during culture, better markers to prospectively track and isolate HSC in ex vivo cultures would be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRC), we demonstrated that the Rhodamine-low phenotype was lost, while AC133 expression was retained throughout culture. Furthermore, the AC133⁺CD38^- subpopulation was significantly enriched in long term culture-initiating cells (LTC-IC) and SRC post-culture. Pre- and post-culture analysis of total nucleated cell number, LTC-IC and limiting dilution analysis in NOD/SCID mice, showed a 43-fold expansion of the AC133⁺CD38^- subpopulation that corresponded with a 7.3 and 4.4-fold expansion of LTC-IC and SRC in this subpopulation, respectively. Thus, AC133⁺CD38^- is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures.

Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells

Han, J., Koh, Y. J., Moon, H. R., Ryoo, H. G., Cho, C.-H., Kim, I., Koh, G. Y. — Fri, 06 Nov 2009 06:13:58 PST

The stromal vascular fraction (SVF) in adipose tissue contains a pool of various stem and progenitor cells, but the existence of hematopoietic stem and progenitor cells (HSPCs) in the SVF has not been seriously considered. We detected the presence of HSPCs in the SVF by phenotypically probing with Lin^-Sca-1⁺c-kit⁺ (LSK) and functionally confirming the presence using colony-forming cell assay and assessing the long-term multilineage reconstitution ability after SVF transplantation. The LSK population in the SVF was 0.004±0.001%, and 5x10⁵ freshly isolated SVF cells gave rise to 13±4 multilineage colonies. Also, 0.15±0.03% of SVF cells was home to bone marrow (BM), especially near vascular and endosteal regions, 24 hours after blood transplantation. SVF transplantation was capable of generating a long-term (>16 weeks), but variable extent (2.1-32.1%) multilineage reconstitution in primary recipients, which was subsequently transferred to the secondary recipients by BM transplantation. All HSPCs within the SVF originated from the BM. Furthermore, the G-CSF mobilization of HSPCs from BM markedly elevated the number of phenotypic and functional HSPCs in the SVF, which induced a high efficiency long-term reconstitution in multilineage hematopoiesis in vivo. Our results provide compelling evidence that adipose tissue is a novel extramedullary tissue possessing phenotypic and functional HSPCs.

Proteomics-based discovery of a novel, structurally unique, and developmentally regulated plasminogen receptor, Plg-RKT, a major regulator of cell surface plasminogen activation

Fri, 06 Nov 2009 06:14:53 PST

Activation of plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiological and pathological processes. Carboxypeptidase B-sensitive plasminogen binding sites promote plasminogen activation on eukaryotic cells. However, no integral membrane plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we utilize the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-R_KT (C9orf46 homolog). Plg-R_KT was highly co-localized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-R_KT also interacts directly with tissue plasminogen activator. Furthermore, Plg-R_KT markedly promoted cell surface plasminogen activation. Database searching revealed that Plg-R_KT mRNA is broadly expressed by migratory cell types, including leukocytes, breast cancer, leukemic and neuronal cells. This structurally unique plasminogen receptor, represents a novel control point for regulating cell surface proteolysis.

Evidence for a cross-talk between human neutrophils and Th17 cells

Wed, 04 Nov 2009 13:36:26 PST

Interleukin (IL)-17A and IL-17F are two of several cytokines produced by T helper 17 cells (Th17) which are able to indirectly induce the recruitment of neutrophils. Recently, human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors, including CCR2 and CCR6. Herein, we show that highly purified neutrophils cultured with IFN plus LPS produce the CCL2 and CCL20 chemokines, the known ligands of CCR2 and CCR6, respectively. Accordingly, supernatants from activated neutrophils induced chemotaxis of Th17 cells, which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment, neutrophils and Th17 cells were found in gut tissue from Crohn's disease and synovial fluid from rheumatoid arthritis patients. Finally, we report that, although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via GM-CSF, TNF and IFN release, these latter cells cannot be activated by IL-17A and IL-17F, due to their lack of IL-17RC expression. Collectively, our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells, which may represent a useful target for the treatment of chronic inflammatory diseases.

Functional characterization of alloreactive T-cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy

Wed, 04 Nov 2009 13:36:11 PST

Immunotherapy with allodepleted donor T-cells (ADTs) improves immunity after T-cell depleted SCT, but infection/relapse remain problematic. To refine this approach, we characterized the expression of surface markers/cytokines on proliferating alloreactive T-cells (ATs). CD25 was expressed on 83 % of CFSE^-dim ATs, confirming this as an excellent target for allodepletion. 70 % of CD25^-ve ATs expressed CD71 (transferrin receptor), identifying this as a novel marker to target ATs persisting after CD25 depletion. Comparison of residual alloreactivity after combined CD25/71 vs. CD25 immunomagnetic depletion showed enhanced depletion of alloreactivity to host with CD25/71 depletion in both 2^o MLRs (p < 0.01) and IFN- ELISPOT assays (p < 0.05) with no effect on 3rd party responses. In pentamer/IFN- ELISPOT assays, anti-viral responses to CMV, EBV and adenovirus were preserved after CD25/71 allodepletion. CD25/71 ADTs can be redirected to recognize leukemic targets through lentiviral transfer of a chimeric CD19 TCR. Finally, we have established conditions for clinically applicable CD25/71 allodepletion under Good Manufacturing Practice conditions, resulting in highly effective, reproducible and selective depletion of ATs (median residual alloreactivity to host in 2^o MLR 0.39% vs to 3rd party 62%, n=5). This strategy enables further clinical studies of adoptive immunotherapy with larger doses of ADTs to enhance immune reconstitution after T-cell depleted SCT.

Induction of heme oxygenase-1 in factor VIII-deficient mice reduces the immune response to therapeutic factor VIII

Wed, 04 Nov 2009 13:35:57 PST

Replacement therapy with exogenous factor VIII (FVIII) to treat hemorrhages induces anti-FVIII inhibitory IgG in up to 30% of patients with hemophilia A. Chronic inflammation associated with recurrent bleedings is a proposed risk factor for FVIII inhibitor development. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory activity. Here, we demonstrate that induction of HO-1 prior to FVIII administration drastically reduces the onset of the anti-FVIII humoral immune response. The protective effect was specific for HO-1 since it was reproduced upon administration of the end products of HO-1 activity, carbon monoxide (CO) and bilirubin, and prevented by the pharmacological inhibition of HO-1 using tin mesoporphyrin IX. HO-1 induction was associated with decreased MHC class II expression by splenic antigen-presenting cells and reduced T-cell proliferation. Triggering the endogenous anti-inflammatory machinery before FVIII administration may represent a novel therapeutic option for preventing the development of FVIII inhibitors in hemophilia A patients.

Dysregulation of bone remodelling by imatinib mesylate

Vandyke, K., Fitter, S., Dewar, A. L., Hughes, T. P., Zannettino, A. C.W. — Wed, 04 Nov 2009 13:35:44 PST

Imatinib mesylate is a rationally designed tyrosine kinase inhibitor that has revolutionised the treatment of chronic myeloid leukaemia and gastro-intestinal stromal tumours. Although the efficacy and tolerability of imatinib is a vast improvement over conventional chemotherapies, the drug exhibits off-target effects. An unanticipated side-effect of imatinib therapy is hypophosphataemia and hypocalcaemia, which in part has been attributed to drug-mediated changes to renal and gastro-intestinal handling of phosphate and calcium. However, emerging data suggests that imatinib also targets cells of the skeleton, stimulating the retention and sequestration of calcium and phosphate to bone, leading to decreased circulating levels of these minerals. The aim of this review is to highlight our current understanding of the mechanisms surrounding the effects of imatinib on the skeleton. In particular, it examines recent studies which suggest that imatinib has direct effects on bone-resorbing osteoclasts and bone-forming osteoblasts through inhibition of c-fms, c-kit, carbonic anhydrase II and the platelet-derived growth factor receptor. The potential application of imatinib in the treatment of cancer induced osteolysis will also be discussed.

Heterozygous deletion of the PU.1 locus in human AML

Bonadies, N., Pabst, T., Mueller, B. U. — Wed, 04 Nov 2009 13:35:30 PST

The transcription factor PU.1 is essential for myeloid development. Targeted disruption of an upstream regulatory element (URE) decreases PU.1 expression by 80% and leads to AML in mice. Here, we sequenced the URE sequences of PU.1 in 120 AML patients. Four polymorphisms (SNP) in the URE were observed, with homozygosity in all SNPs in 37 patients. Among them, we compared samples at diagnosis and remission, and one patient with cytogenetically normal AML-M2 was identified with heterozygosity in three of the SNPs in the URE at remission. Loss of heterozygosity (LOH) was further found in this patient at two polymorphic sites in the 5' promoter region and in two intronic sites flanking exon 4, thus suggesting LOH covering at least 40kb of the PU.1-locus. Consistently, PU.1 expression in this patient was markedly reduced. Our study suggests that heterozygous deletion of the PU.1 locus can be associated with human AML.

Comparative gene expression analysis of zebrafish and mammals identifies common regulators in hematopoietic stem cells

Kobayashi, I., Ono, H., Moritomo, T., Kano, K., Nakanishi, T., Suda, T. — Wed, 04 Nov 2009 13:35:14 PST

Hematopoiesis in teleost fish is maintained in the kidney. We previously reported that Hoechst dye efflux activity of hematopoietic stem cells (HSCs) is highly conserved in vertebrates, and that Hoechst can be used to purify HSCs from teleost kidneys. Regulatory molecules that are strongly associated with HSC activity may also be conserved in vertebrates. Here, we identified evolutionarily conserved molecular components in HSCs by comparing the gene expression profiles of zebrafish, murine, and human HSCs. Microarray data of zebrafish kidney side population cells (zSPs) showed that genes involved in "cell junction" and "signal transduction" tended to be up-regulated in zSPs, while genes involved in "DNA replication" tended to be down-regulated. These properties of zSPs were similar to those of mammalian HSCs. Overlapping gene expression analysis showed that 40 genes were commonly up-regulated in these three HSCs. Some of these genes, such as egr1, gata2, and id1, have been previously implicated in the regulation of HSCs. In situ hybridization in zebrafish kidney revealed that expression domains of egr1, gata2, and id1 overlapped with that of abcg2a, a marker for zSPs. These results suggest that the overlapping genes identified here are regulated in HSCs and play important roles in their functions.

Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting

Tue, 03 Nov 2009 13:30:19 PST

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferases (DNMT) inhibitor which induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in G0-G1 phase and decreasing the production of proinflammatory cytokines such as TNF and IFN. This effect was not due to a pro-apoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes which induce cell growth arrest, such as DCUN1D2, U2AF2, GADD45B or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, over-expression of FOXP3 and expansion of regulatory T cells. Finally, the administration of 5-azaC post-transplant prevented the development of GVHD leading to a significant increase in survival in a fully mismatched BMT mouse model. In conclusion, the current study shows the effect of 5-azaC in T-lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways which must be explored in order to prevent graft-versus-host disease.

The contribution of cyclooxygenase-1 and -2 to persistent thromboxane biosynthesis in aspirin-treated essential thrombocythemia: implications for antiplatelet therapy

Tue, 03 Nov 2009 13:30:11 PST

We tested whether cyclooxygenase (COX)-2 expression and unacetylated COX-1 in newly-formed platelets, might contribute to persistent thromboxane (TX) biosynthesis in aspirin-treated essential thrombocythemia (ET). Forty-one patients on chronic aspirin (100 mg/day) and 24 healthy subjects were studied. Platelet COX-2 expression was significantly increased in patients and correlated with tiazole orange-positive platelets (r=0.71, p<0.001). The rate of TXA₂ biosynthesis in vivo, as reflected by urinary 11-dehydro TXB₂ (TXM) excretion, and the maximal biosynthetic capacity of platelets, as reflected by serum TXB₂ were higher in patients as compared to aspirin-treated healthy volunteers. Serum TXB₂ was significantly reduced by the selective COX-2 inhibitor, NS-398, added in vitro. Patients were randomized to adding the selective COX-2 inhibitor, etoricoxib, or continuing aspirin for 7 days. Etoricoxib significantly reduced by approximately 25% TXM excretion and serum TXB₂. Fourteen of the 41 patients were studied again 21±7 months after first visit. Serum TXB₂ was consistently reduced by approximately 30% by adding NS398 in vitro, while it was completely suppressed 50µM aspirin. Accelerated platelet regeneration in the majority of aspirin-treated ET patients may explain aspirin-persistent TXA₂ biosynthesis through enhanced COX-2 activity and faster renewal of unacetylated COX-1. These findings may help reassessing the optimal antiplatelet strategy in ET.

Osteoclasts are important for bone angiogenesis

Tue, 03 Nov 2009 13:30:03 PST

Increased osteoclastogenesis and angiogenesis occur in physiological and pathological conditions. However, it is unclear if or how these two processes are linked. Therefore, to test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with PTHrP increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with RANKL or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined if MMP-9, an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo respectively from MMP-9^-/- mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo, but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most likely by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.

Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells

Weishaupt, H., Sigvardsson, M., Attema, J. L. — Tue, 03 Nov 2009 13:29:54 PST

Heritable epigenetic signatures are proposed to serve as an important regulatory mechanism in lineage fate determination. To investigate this, we profiled chromatin modifications in murine hematopoietic stem cells, lineage restricted progenitors and CD4⁺ T cells using modified genome-scale miniChIP technology. We show that genes involved in mature hematopoietic cell function associate with distinct chromatin states in stem and progenitor cells, prior to their activation or silencing upon cellular maturation. Many lineage-restricted promoters are associated with bivalent histone methylation and highly combinatorial histone modification patterns, which may determine their selective priming of gene expression during lineage commitment. These bivalent chromatin states are conserved in mammalian evolution, with a particular over-representation of promoters encoding key regulators of hematopoiesis. Following differentiation into progenitors and T cells, activating histone modifications persist at transcriptionally repressed promoters, suggesting that these transcriptional programs might be reactivated after lineage restriction. Collectively, our data reveal the epigenetic framework that underlies the cell fate options of hematopoietic stem cells.

The normal IGHV1-69-derived B-cell repertoire contains "stereotypic" patterns characteristic of unmutated CLL

Tue, 03 Nov 2009 13:29:44 PST

The cell of origin of CLL has long been sought and immunoglobulin gene analysis provides new clues. In the unmutated subset (U-CLL), there is strikingly increased usage of the 51p1-related alleles of the IGHV1-69 gene, often combined with selected IGHD genes and with IGHJ6. Shared sequence "stereotypic" characteristics of the HCDR3 result, and suggest antigen selection of the leukemic clones. We have now analyzed 51p1/IGHJ6 combinations in normal blood B cells from 3 healthy individuals (>51yr) for parallel sequence patterns. A high proportion (33.3% of sequences) revealed stereotypic patterns, several (15.0%) being similar to those described in U-CLL. Additional CLL-associated stereotypes, not yet reported, were detected in 4.8%. Stereotypes (13.6%) not detected in CLL were also found. The HCDR2-IGHJ6 sequences were essentially unmutated. Junctional amino acids in normal B cells were heterogeneous, as in cases of stereotyped CLL. Phenotypically, normal B cells expressing 51p1-derived IgM were naive. This snapshot of the naive B-cell repertoire reveals subsets of B cells closely related to those characteristic of CLL. Conserved patterns in the 51p1-encoded IgM of normal B cells suggest a restricted sequence repertoire shaped by evolution to recognize common pathogens. Proliferative pressure on these cells is the likely route to U-CLL.

Bortezomib and high dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with de novo multiple myeloma: a phase II study of the Intergroupe Francophone du Myelome (IFM)

Mon, 02 Nov 2009 14:22:50 PST

Autologous stem cell transplantation (ASCT) is recommended for younger newly diagnosed multiple myeloma (MM) patients. Achieving complete response (CR) or at least very good partial response (VGPR) is a major prognostic factor for survival with 20-30% of patients achieving CR after ASCT. Bortezomib has demonstrated synergistic effects with Melphalan and no prolonged hematological toxicity. In this IFM phase II study, 54 untreated patients were enrolled between July and December 2007 to receive bortezomib (1mg/m²x4) and melphalan (200mg/m²) as conditioning regimen (Bor-HDM). Overall, 70% of patients achieved at least VGPR including 17 patients with CR (32%) after ASCT. No toxic deaths were observed. Bortezomib did not increase hematological toxicity. Only 1 grade 3-4 peripheral neuropathy was reported. A matched control analysis was conducted comparing our cohort to patients from the IFM 2005-01 trial (HDM alone). Patients were matched for response to induction therapy and type of induction: CR was higher in the Bor-HDM group (35% vs. 11%) (P =.001), regardless of induction therapy. These results suggest that Bor-HDM is a safe and promising conditioning regimen. Randomized studies are needed to assess whether this conditioning regimen is superior to HDM alone. The original trial is registered at www.clinicaltrials.gov as NCT00642395.

Evidence for direct involvement of epirubicin in the formation of chromosomal translocations in t(15;17) therapy-related acute promyelocytic leukemia

Mon, 02 Nov 2009 14:22:30 PST

Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17)(q22;q12~21) involving the PML and RARA genes, is associated with exposure to agents targeting topoisomerase II (topoII), particularly mitoxantrone and epirubicin. We previously have shown that mitoxantrone preferentially induces topoII-mediated DNA damage in a "hotspot region" within PML intron 6. To investigate mechanisms underlying epirubicin associated t-APL, t(15;17) genomic breakpoints were characterized in 6 cases with prior breast cancer. Significant breakpoint clustering was observed in PML and RARA loci (p=0.009 and p=0.017, respectively), with PML breakpoints lying outside the mitoxantrone-associated hotspot region. Recurrent breakpoints identified in the PML and RARA loci in epirubicin-related t-APL were shown to be preferential sites of topoII-induced DNA damage, enhanced by epirubicin. Although site preferences for DNA damage differed between mitoxantrone and epirubicin, the observation that particular regions of the PML and RARA loci are susceptible to these agents may underlie their respective propensities to induce t-APL.

Antibody mediated B cell depletion prior to adoptive immunotherapy with T cells expressing CD20-specific chimeric T cell receptors facilitates eradication of leukemia in immunocompetent mice

Fri, 30 Oct 2009 10:14:14 PDT

We have established a model of leukemia immunotherapy using T cells expressing chimeric T cell receptors (cTCR) targeting the CD20 molecule expressed on normal and neoplastic B cells. Following transfer into human CD20 (hCD20) transgenic mice, cTCR⁺ T cells demonstrated antigen-specific delayed egress from the lungs, concomitant with T cell deletion. Few cTCR⁺ T cells reached the bone marrow (BM) in hCD20 transgenic mice, precluding effectiveness against leukemia. Anti-hCD20 antibody-mediated B cell depletion prior to adoptive T cell therapy permitted egress of mouse CD20-specific cTCR⁺ T cells from the lungs, enhanced T cell survival, and promoted cTCR⁺ T cell-dependent elimination of established mouse CD20⁺ leukemia. Furthermore, CD20-specific cTCR⁺ T cells eliminated residual B cells refractory to depletion with monoclonal antibodies. These findings suggest that combination of antibody therapy that depletes antigen-expressing normal tissues with adoptive T cell immunotherapy enhances the ability of cTCR⁺ T cells to survive and control tumors.

A genome-wide association analysis of serum iron concentrations

Fri, 30 Oct 2009 10:14:04 PDT

To investigate genetic variants that affect iron plasma concentrations in persons not affected by overt genetic disorders of iron metabolism, a genome-wide association study was conducted in the InCHIANTI Study (N=1206) and the Baltimore Longitudinal Study of Aging (BLSA, N=713). The top two SNPs were examined for replication in the Women's Health and Aging Study (WHAS I & II, N=569). The SNP most strongly associated with lower serum iron concentration was rs4820268 (p=5.12x10^-9) located in exon13 of the transmembrane protease serine 6 (TMPRSS6) gene, an enzyme that promotes iron absorption and recycling by inhibiting hepcidin antimicrobial peptide transcription. The allele associated with lower iron concentrations was also associated with lower hemoglobin levels, smaller red cells, and more variability in red cell size (high red blood cell distribution width). Our results confirm the association of TMPRSS6 variants with iron level and provide further evidence of association with other anemia-related phenotypes.

The new oral anticoagulants

Garcia, D., Libby, E., Crowther, M. A. — Fri, 30 Oct 2009 10:13:55 PDT

Although their first application in clinical practice occurred in the 1940s, vitamin K antagonists (VKAs) remain the only form of oral anticoagulant medication approved for long-term use. While the available VKAs are highly effective for the prevention and/or treatment of most thrombotic disease, the significant inter- and intra-patient variability in dose-response, the narrow therapeutic index, and the numerous drug and dietary interactions associated with these agents have led clinicians, patients, and investigators to search for alternative agents. Three new orally administered anticoagulants (apixaban, dabigatran, and rivaroxaban) are in the late stages of development and several others are just entering (or moving through) earlier phases of investigation. These novel anticoagulant medications are being studied for the prevention and treatment of venous thromboembolism, the treatment of acute coronary syndromes and the prevention of stroke in patients with atrial fibrillation. This review will summarize published clinical trial data pertinent to apixaban, dabigatran and rivaroxaban.

A prototype nonpeptidyl, hydrazone class, thrombopoietin receptor agonist, SB-559457, is toxic to primary human myeloid leukemia cells

Kalota, A., Gewirtz, A. M. — Fri, 30 Oct 2009 10:13:45 PDT

Biologic characterization of SB-559457 (SB), a non-peptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity towards human leukemia cells. Anti-proliferative effects followed by significant, non-apoptotic, cell death within 72 hours occurred in 24/26 AML, 0/6 ALL, and 3/6 CML patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB, but not in rhTpo, treated cells. Expression profiling of cells exposed to SB vs rhTpo revealed statistically significant, >2-fold changes in GAPDH and REDD1 gene expression, confirmed by QRT-PCR. These genes, induced in energy or hypoxia stressed cells, have been implicated in cell death pathways, and may provide important clues to the mechanism of SB induced, leukemic cell death. These results suggest that nonpeptidyl, hydrazone class Mpl agonists may be clinically useful anti-leukemic agents by virtue of their combined thrombopoietic and anti-leukemic effects.

Alternative activation of macrophages by IL-4 impairs phagocytosis of pathogens but potentiates microbial-induced signalling and cytokine secretion

Varin, A., Mukhopadhyay, S., Herbein, G., Gordon, S. — Fri, 30 Oct 2009 10:13:38 PDT

Alternatively activated macrophages play an important role in host defense in the context of a Th2 microenvironment such as parasitic infection. However, the role of these macrophages during secondary challenge with Th1 type pathogens is poorly defined. In this study, thioglycollate-elicited mouse peritoneal macrophages were treated with IL-4 or IL-13 in vitro and challenged with Neisseria meningiditis (N.m.). After 8-12 hours IL-4 pretreatment, the non-opsonic phagocytic uptake of N.m. was markedly reduced, depending on the common IL-4R chain, but independent of Scavenger receptor A and MARCO, two known receptors for N.m. Inhibition of phagocytosis extended to several other microbial particles, zymosan and other bacteria. Concomitantly, IL-4 potentiated secretion of proinflammatory cytokines, after additional bacterial stimulation, which depended on the MyD88 signalling pathway. Similar results were obtained after intraperitoneal stimulation of IL-4 and N.m. in vivo. Further in vitro studies revealed a striking correlation with inhibition of Akt phosphorylation and stimulation of the MAP kinase pathway; inhibition of phagocytosis was associated with inhibition of phagosome formation. These findings are relevant to host defense in mixed infections within a Th2 microenvironment, and shed light on immunological functions associated with alternative priming and full activation of macrophages.

Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway

Fri, 30 Oct 2009 10:13:29 PDT

LGL leukemia results from chronic expansion of cytotoxic T cells or NK cells. Apoptotic resistance resulting from constitutive activation of survival signaling pathways is a fundamental pathogenic mechanism. Recent network modeling analyses identified PDGF as a key master switch in controlling these survival pathways in T cell LGL leukemia. Here we show that an autocrine PDGF regulatory loop mediates survival of leukemic LGL of both T and NK cell origin. We found high levels of circulating PDGF-BB in platelet-poor plasma samples from LGL leukemia patients. Production of PDGF-BB by leukemic LGL was demonstrated by immunocytochemical staining. Leukemic cells expressed much higher levels of PDGFR-β transcripts than purified normal CD8⁺ T cells or NK cells. We observed that Phosphatidylinositol-3-kinase (PI3 kinase), Src family kinase (SFK), and downstream PKB/AKT pathways were constitutively activated in both T- and NK-LGL leukemia. Pharmacologic blockade of these pathways led to apoptosis of leukemic LGL. Neutralizing antibody to PDGF-BB inhibited PKB/AKT phosphorylation induced by LGL leukemia sera. These results suggest that targeting of PDGF-BB, a pivotal regulator for the long term survival of leukemic LGL, may be an important therapeutic strategy.

Long term outcome of EBV specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients

Fri, 30 Oct 2009 10:13:21 PDT

T-cell immunotherapy that takes advantage of Epstein-Barr virus (EBV)-stimulated immunity has the potential to fill an important niche in targeted therapy for EBV-related cancers. To address questions of long-term efficacy, safety and practicality, we studied 114 patients who had received infusions of EBV-specific cytotoxic T lymphocytes (CTLs) at three different centers to prevent or treat EBV-positive lymphoproliferative disease (LPD) arising after hematopoietic stem cell transplantation. Toxicity was minimal, consisting mainly of localized swelling at sites of responsive disease. None of the 101 patients who received CTL prophylaxis developed EBV-positive LPD while 11 of the 13 patients treated with CTLs for biopsy-proven or probable LPD achieved sustained complete remissions. The gene-marking component of this study enabled us to demonstrate the persistence of functional CTLs for up to 9 years. A preliminary analysis indicated that a patient-specific CTL line can be manufactured, tested and infused for $6,095, a cost that compares favorably with other modalities used in the treatment of LPD. We conclude that the CTL lines described here provide safe and effective prophylaxis or treatment for lymphoproliferative disease in transplant recipients and the manufacturing methodology is robust and can be transferred readily from one institution to another without loss of reproducibility. These trials were registered at clinicaltrials.gov under NCT00058812.

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Saur, S. J., Sangkhae, V., Geddis, A. E., Kaushansky, K., Hitchcock, I. S. — Fri, 30 Oct 2009 10:13:10 PDT

Regulation of growth factor and cytokine signaling is essential for maintaining physiological numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, following Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K₅₅₃ and K₅₇₃). By mutating these residues to arginine, ubiquitination and degradation was significantly reduced and caused hyperproliforation in cell lines expressing these mutated receptors. Using siRNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.

Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel,on behalf of the European LeukemiaNet

Fri, 30 Oct 2009 05:46:32 PDT

In 2003, an international working group last reported on recommendations for diagnosis, response assessment, and treatment outcomes in acute myeloid leukemia (AML). Since that time, considerable progress has been made in elucidating the molecular pathogenesis of the disease that has resulted in the identification of new diagnostic and prognostic markers. Furthermore, therapies are now being developed that target disease-associated molecular defects. Recent developments prompted an international expert panel to provide updated evidence- and expert opinion-based recommendations for the diagnosis and management of AML, that contain both minimal requirements for general practice as well as standards for clinical trials. A new standardized reporting system for correlation of cytogenetic and molecular genetic data with clinical data is proposed.

Gene expression classifiers for relapse-free survival and minimal residual disease improve risk classification and outcome prediction in pediatric B-precursor acute lymphoblastic leukemia

Fri, 30 Oct 2009 05:46:24 PDT

To determine if gene expression profiling could improve outcome prediction in children with acute lymphoblastic leukemia (ALL) at high risk for relapse, we profiled pre-treatment leukemic cells in 207 uniformly-treated children with high-risk B-precursor ALL. A 38-gene expression classifier predictive of relapse-free survival (RFS) could distinguish two groups with differing relapse risks: low (4 yr RFS: 81%, n=109) vs. high (4 yr RFS: 50%, n=98) (P< 0.0001). In multivariate analysis, the gene expression classifier (P=0.001) and flow cytometric measures of minimal residual disease (MRD) (P=0.001) each provided independent prognostic information. Together, they could be used to classify children with high-risk ALL into low (87% RFS), intermediate (62% RFS), or high risk (29% RFS) groups (P<0.0001). A 21-gene expression classifier predictive of end-induction MRD effectively substituted for flow MRD, yielding a combined classifier that could distinguish these three risk groups at diagnosis (P< 0.0001). These classifiers were further validated on an independent high-risk ALL cohort (P=0.006) and retained independent prognostic significance (P<0.0001) in the presence of other recently described poor prognostic factors (IKAROS/IKZF1 deletions, JAK mutations, and kinase expression signatures). Thus, gene expression classifiers improve ALL risk classification and allow prospective identification of children who respond or fail current treatment regimens. These trials were registered at http://clinicaltrials.gov under NCT00005603.

Estrogen receptor signaling promotes dendritic cell differentiation by increasing expression of the transcription factor IRF4

Fri, 30 Oct 2009 05:46:12 PDT

During inflammation, elevated GM-CSF directs the development of new dendritic cells (DC). This pathway is influenced by environmental factors, and we previously showed that physiological levels of estradiol, acting through estrogen receptor alpha (ER), promote the GM-CSF-mediated differentiation of a CD11b⁺ DC subset from myeloid progenitors (MP). We now have identified interferon regulatory factor 4 (IRF4), a transcription factor induced by GM-CSF and critical for CD11b⁺ DC development in vivo, as a target of ER signaling during this process. In MP, ER potentiates and sustains GM-CSF-induction of IRF4. Furthermore, retroviral delivery of the Irf4 cDNA to undifferentiated ER-/- bone marrow cells restored the development of the estradiol/ER-dependent DC population, indicating that an elevated amount of IRF4 protein substitutes for ER signaling. Thus at an early stage in the MP response to GM-CSF, ER signaling induces an elevated amount of IRF4, which leads to a developmental program underlying CD11b⁺ DC differentiation.

A non-coding antisense RNA in tie-1 locus regulates tie-1 function in vivo

Fri, 30 Oct 2009 05:46:00 PDT

Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear & brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 vs. tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate non-coding RNA mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiological process such as vascular development.

A randomized phase III study on the effect of thalidomide combined with Adriamycin, dexamethasone (TAD), and high-dose melphalan, followed by thalidomide maintenance in patients with multiple myeloma

Fri, 30 Oct 2009 05:45:53 PDT

The phase III trial HOVON-50 was designed to evaluate the effect of thalidomide during induction treatment and as maintenance in patients with Multiple Myeloma (MM) who were transplant candidates. 556 patients were randomly assigned to arm A: 3 cycles of Vincristine, Doxorubicin, Dexamethasone (VAD) or to arm B, Thalidomide 200 orally, days 1-28 + AD (TAD). After induction therapy and stem cell mobilization patients were to receive High Dose Melphalan 200mg/m² (HDM) followed by maintenance with - Interferon (Arm A) or Thalidomide 50 mg daily (Arm B). Thalidomide significantly improved overall response rate (ORR) as well as quality of the response before and after HDM. Best ORR on protocol was 88% and 79 % (p=0.005), at least Very Good Partial Remission (VGPR) 66 % and 54% (p=0.005), Complete remission (CR) 31% and 23 % (p=0.04), respectively in favour of the Thalidomide arm. Thalidomide also significantly improved Event Free Survival (EFS) from median 22 months to 34 months (p<0.001) and PFS from median 25 to 34 months (p<0.001). Median survival was longer in the thalidomide arm, 73 versus 60 months, however this difference was not significant (P=0.77). Patients randomized to thalidomide had strongly reduced survival after relapse. This trial was registered on www.controlled-trials.com as ISRCTN06413384.

Selecting optimal second-line tyrosine kinase inhibitor therapy for chronic myeloid leukemia patients after imatinib failure - does the BCR-ABL mutation status really matter?

Branford, S., Melo, J. V, Hughes, T. P. — Fri, 30 Oct 2009 05:45:37 PDT

Pre-clinical studies of BCR-ABL mutation sensitivity to nilotinib or dasatinib suggested the majority would be sensitive. Correspondingly, the initial clinical trials demonstrated similar response rates for CML patients after imatinib failure, irrespective of the mutation status. However, upon closer examination clinical evidence now indicates that some mutations are less sensitive to nilotinib (Y253H, E255K/V and F359V/C) or dasatinib (F317L and V299L). T315I is insensitive to both. Novel mutations (F317I/V/C and T315A) are less sensitive/insensitive to dasatinib. We refer to these collectively as second-generation inhibitor (SGI) clinically-relevant mutations. By in-vitro analysis, other mutations confer a degree of insensitivity; however, clinical evidence is currently insufficient to define them as SGI clinically-relevant. Here we examine the mutations that are clearly SGI clinically-relevant, those with minimal impact on response, and those for which more data are needed. In our series of patients with mutations upon imatinib cessation and/or at nilotinib or dasatinib commencement, 43% had SGI clinically-relevant mutations, including 14% with T315I. The frequency of SGI clinically-relevant mutations was dependent on the disease phase upon imatinib failure. The clinical data suggest that a mutation will often be detectable after imatinib failure for which there is compelling clinical evidence that one SGI should be preferred.

Alpha-defensins secreted by dysplastic granulocytes inhibit the differentiation of monocytes in chronic myelomonocytic leukemia

Wed, 28 Oct 2009 13:45:22 PDT

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that occurs in elderly patients. One of the main diagnostic criteria is the accumulation of heterogeneous monocytes in the peripheral blood. We further explored this cellular heterogeneity and observed that part of the leukemic clone in the peripheral blood was made of immature dysplastic granulocytes with a CD14^-/CD24⁺ phenotype. The proteome profile of these cells is dramatically distinct from that of CD14⁺/CD24^- monocytes from CMML patients or healthy donors. More specifically, CD14^-/CD24⁺ CMML cells synthesize and secrete large amounts of alpha-defensin 1-3 (HNP1-3). Recombinant HNPs inhibit M-CSF driven differentiation of human peripheral blood monocytes into macrophages. Using transwell, antibody-mediated depletion, suramin inhibition of purinergic receptors and competitive experiments with UDP/UTP, we demonstrate that HNP1-3 secreted by CD14^-/CD24⁺ cells inhibit M-CSF-induced differentiation of CD14⁺/CD24^- cells at least in part through P2Y6, a receptor involved in macrophage differentiation. Altogether, these observations suggest that a population of immature dysplastic granulocytes contributes to the CMML phenotype through production of alpha-defensins HNP1-3 that suppress the differentiation capabilities of monocytes.

Mta3-NuRD complex is a master regulator for initiation of primitive hematopoiesis in vertebrate embryos

Li, X., Jia, S., Wang, S., Wang, Y., Meng, A. — Wed, 28 Oct 2009 13:45:12 PDT

Metastasis-associated antigen (Mta) 1/2/3 are components of NuRD complexes and have been found to play roles in embryonic development and homeostasis. However, their functions in primitive hematopoiesis are unknown. In this study, we demonstrate that knockdown of mta3 by antisense morpholinos abolishes primitive hematopoietic lineages and causes abnormal angiogenesis in zebrafish embryos. However, the expression of the pronephric duct and paraxial mesoderm markers is unaltered and the specification of angioblasts is unaffected in mta3 morphants. The results suggest that mta3 is specifically required for primitive hematopoiesis. Furthermore, inhibition of deacetylase activity with the inhibitors VPA or TSA in zebrafish embryos completely blocks primitive hematopoiesis, resulting in hematopoietic defects almost identical to those seen in mta3 morphants. Importantly, overexpression of scl or scl and lmo2, two master genes for primitive hematopoiesis, is able to overturn effects of mta3 knockdown or VPA/TSA treatment; and overexpression of mta3, human MBD3 or HDAC1, two other components of NuRD complex, enhances the expression of scl and lmo2 in the posterior lateral plate mesoderm during early primitive hematopoiesis. We conclude that Mta3-NuRD complex is essential for the initiation of primitive hematopoiesis. Thus, our findings provide new insight into the regulatory hierarchy of primitive hematopoiesis in vertebrates.

W41/W41 blastocyst complementation: a system for genetic modeling of hematopoiesis

Jansson, L., Larsson, J. — Wed, 28 Oct 2009 13:45:04 PDT

We report a rapid and highly efficient approach to generate mice in which the hematopoietic system is derived from embryonic stem (ES) cells. We show that ES cells injected into blastocysts from the c-kit deficient W41/W41 mouse strain have a clear advantage over the W41/W41 blastocyst-derived inner cell mass cells in founding the hematopoietic system. Fetal liver HSCs from W41/W41 blastocyst complementation embryos can be transplanted to establish large cohorts of bone marrow chimeras with hematopoiesis of practically pure ES cell origin. Using ES cells with site-directed modifications we show how this system can be employed to drive inducible transgene expression in hematopoietic cells in a robust and reliable manner both in vitro and in vivo. The approach avoids the cost and time constraints associated with the creation of standard transgenic mouse strains while taking advantage of the sophisticated site-directed manipulations that are possible in ES cells.

Epigenetic regulation of dendritic cell differentiation and function by oxidized phospholipids

Wed, 28 Oct 2009 13:44:51 PDT

Dendritic cells (DCs) are the key cell type in the regulation of an adaptive immune response. Under inflammatory conditions monocytes can give rise to immunostimulatory DCs, depending on microenvironmental stimuli. Here we show that oxidized phospholipids (Ox-Pls), which are generated during inflammatory reactions, dysregulate the differentiation of DCs. DCs generated in the presence of Ox-Pls (Ox-DCs) upregulated the typical DC marker DC-SIGN, but did not express CD1a, CD1b and CD1c. These Ox-DCs had a substantially diminished T cell stimulating capacity after stimulation with TLR-ligands. TLR-ligand-induced production of IL-12 was also strongly diminished, whereas induction of CD83 was not altered. In addition, we found that Ox-Pls strongly inhibit inflammatory stimuli-induced phosphorylation of histone H3, a key step of IL-12 production, yet leaving activation of NF-B unaltered. Taken together, Ox-Pls present during differentiation yielded DCs with a reduced capacity to become immunostimulatory mature DCs. Furthermore, the presence of Ox-Pls blocked histone modifications required for full activation of DCs. Therefore, inflammation-derived Ox-Pls control DC functions in part by epigenetic mechanisms.

Loss-of-function Additional sex combs-like1 mutations disrupt hematopoiesis but do not cause severe myelodysplasia or leukemia

Tue, 27 Oct 2009 13:41:35 PDT

The Additional sex combs-like 1 (Asxl1) gene is one of 3 mammalian homologs of the Additional sex combs (Asx) gene of Drosophila. Asx is unusual because it is required to maintain both activation and silencing of Hox genes in flies and mice. Asxl proteins are characterized by an amino terminal homology domain, by interaction domains for nuclear receptors, and by a C terminal PHD protein-protein interaction domain. A recent study of patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) revealed a high incidence of truncation mutations that would delete the PHD domain of ASXL1. Here, we show that Asxl1 is expressed in all hematopoietic cell fractions analyzed. Asxl1 knockout mice exhibit defects in frequency of differentiation of lymphoid and myeloid progenitors, but not in multipotent progenitors. We do not detect effects on hematopoietic stem cells, or in peripheral blood. Notably, we do not detect severe myelodysplastic phenotypes, or leukemia in this loss of function model. We conclude that Asxl1 is needed for normal hematopoiesis. The mild phenotypes observed may be because Asx-like genes have redundant function with Asxl1, or alternatively, MDS or oncogenic phenotypes may result from gain-of-function Asxl mutations caused by genomic amplification, gene fusion, or truncation of Asxl1.

The small 11kDa non-structural protein of human parvovirus B19 plays a key role in inducing apoptosis during B19 virus infection of primary erythroid progenitor cells

Tue, 27 Oct 2009 13:41:25 PDT

Human parvovirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leads to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the three B19V nonstructural proteins, 7.5kDa, 11kDa and NS1, for their function in inducing apoptosis in transfection of primary ex vivo expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11kDa is a more significant inducer of apoptosis than NS1, while 7.5kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11kDa is expressed at least 100 times more than nucleus-localized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11kDa expression using anti-sense oligos targeting specifically to the 11kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11kDa protein contributes to erythroid progenitor cell death during B19V infection.

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

Tue, 27 Oct 2009 13:41:13 PDT

Coagulation factor V (FV), present in plasma and platelets, is indispensable to thrombin formation, yet patients with undetectable plasma FV seldom experience major bleeding. We used thrombin generation assays to explore the role of platelet FV in four patients with severe congenital FV deficiency (three with plasma FV:C <1%). When triggered with tissue factor (TF) concentrations up to 50 pM, platelet-poor plasma (PPP) from the patients with undetectable plasma FV showed no thrombin generation, whereas platelet-rich plasma (PRP) formed thrombin already at 1-5 pM TF. Thrombin generation in PRP from the FV-deficient patients was enhanced to near-normal levels by platelet activators (collagen or Ca²⁺-ionophore) and could be completely suppressed by specific FV inhibitors, suggesting FV-dependence. Accordingly, platelet FV antigen and activity were measurable in all FV-deficient patients and platelet FVa could be visualized by Western blotting. Normalization of the tissue factor pathway inhibitor (TFPI) level, which is physiologically low in FV-deficient plasma, almost completely abolished thrombin generation in PRP from the FV-deficient patients. In conclusion, patients with undetectable plasma FV may contain functional FV in their platelets. In combination with the low TFPI level, residual platelet FV allows sufficient thrombin generation to rescue these patients from fatal bleeding.

Human Siglec-10 can bind to vascular adhesion protein-1 (VAP-1) and generate enzymatic products

Tue, 27 Oct 2009 13:40:50 PDT

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular Adhesion Protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counter-receptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production indicating that Siglec-10 serves as a substrate for VAP-1. Thus, Siglec-10 -- VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

How I treat multiple myeloma in younger patients

Stewart, A. K., Richardson, P. G., San-Miguel, J. F. — Tue, 27 Oct 2009 13:40:37 PDT

No abstract

Hes1 immortalizes committed progenitors and plays a role in blast crisis transition in chronic myelogenous leukemia

Tue, 27 Oct 2009 13:40:26 PDT

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3, conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered into irradiated mice, the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML), resulting in rapid death of the recipient mice. On the other hand, BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive, Sca-1-positive, and lineage-negative hematopoietic stem cells (KSLs), but not committed progenitors CMPs or GMPs, as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly, Hes1 was highly expressed in eight of 20 patients with CML in blast crisis, but not in the chronic phase, and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML.

Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4 and with mutations in CFH, CFI, CD46, and C3 in patients with atypical haemolytic uraemic syndrome

Tue, 27 Oct 2009 13:40:11 PDT

Factor H autoantibodies have been reported in ~10% of aHUS patients and are associated with deficiency of factor H related proteins 1 and 3. In this study we have examined the prevalence of factor H autoantibodies in the Newcastle cohort of aHUS patients, determined whether the presence of such autoantibodies is always associated with deficiency of factor H related proteins 1 and 3, and examined whether such patients have additional susceptibility factors and/or mutations in the genes encoding complement regulator/activators. We screened 142 aHUS patients and found factor H autoantibodies in 13 individuals (aged 1-11yrs). The presence of the autoantibodies was confirmed by Western blotting. Using MLPA we measured CFHR1 and CFHR3 copy number. In 10 of the 13 patients there were zero copies of CFHR1 and in three there were two. In three of the patients with zero copies of CFHR1 there were was one copy of CFHR3, and these individuals exhibited a novel deletion incorporating CFHR1 and CFHR4. In five patients mutations were identified: one in CFH, one in CFI, one in CD46 and two in C3. The latter observation emphasizes that multiple concurrent factors may be necessary in individual patients for disease manifestation.

The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis

Sroczynska, P., Lancrin, C., Kouskoff, V., Lacaud, G. — Mon, 26 Oct 2009 13:22:38 PDT

The transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatio-temporal expression is tightly regulated during embryonic development and is under the control of two alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these two promoters remain largely uncharacterized. To investigate these activities, we introduced two reporter genes under the control of the proximal and distal promoters in ES cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knock-out embryos appears normal. Altogether, our data demonstrate that the differential activities of the two Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.

Specific promoter methylation identifies different subgroups of MLL-rearranged infant acute lymphoblastic leukemia, influences clinical outcome, and provides therapeutic options

Fri, 23 Oct 2009 13:16:18 PDT

MLL-rearranged infant Acute Lymphoblastic Leukemia (ALL) remains the most aggressive type of childhood leukemia, displaying a unique gene-expression profile. Here we hypothesized that this characteristic gene-expression signature may have been established by potentially reversible epigenetic modifications. To test this hypothesis, we used Differential Methylation Hybridization (DMH) to explore the DNA methylation patterns underlying MLL-rearranged ALL in infants. The obtained results were correlated with gene-expression data to confirm gene silencing as a result of promoter hypermethylation. Distinct promoter CpG island methylation patterns separated different genetic subtypes of MLL-rearranged ALL in infants. MLL translocations t(4;11) and t(11;19) characterized extensively hypermethylated leukemias, whereas t(9;11)-positive infant ALL and infant ALL carrying wild-type MLL genes epigenetically resembled normal bone marrow. Furthermore, the degree of promoter hypermethylation among infant ALL patients carrying t(4;11) or t(11;19) appeared to influence relapse-free survival, with patients displaying accentuated methylation being at high relapse risk. Finally, we show that the demethylating agent zebularine reverses aberrant DNA methylation, and effectively induces apoptosis in MLL-rearranged ALL cells. Collectively these data suggest that aberrant DNA methylation occurs in the majority of MLL-rearranged infant ALL cases and guides clinical outcome. Therefore inhibition of aberrant DNA methylation may be an important novel therapeutic strategy for MLL-rearranged ALL in infants.

Comprehensive genomic screens identify a role for PLZF-RAR{alpha} as a positive regulator of cell proliferation via direct regulation of c-MYC

Fri, 23 Oct 2009 13:16:07 PDT

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of APL, involving the production of reciprocal fusion proteins, PLZF-RAR and RAR-PLZF. PLZF-RAR mediates transformation partly by acting as a dominant negative retinoic acid receptor, binding to and dysregulating RAR/RXR target genes. Using a combination of ChIP-chip and gene expression arrays, we identify novel, direct target genes of PLZF-RAR that tend to be repressed in APL as compared to other myeloid leukemias, supporting the role of PLZF-RAR as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RAR promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RAR binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RAR may act as a dominant negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RAR-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc downregulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RAR.

BCL6 regulates tonic BCR signaling in diffuse large B-cell lymphomas by repressing the SYK phosphatase, PTPROt

Fri, 23 Oct 2009 13:15:45 PDT

Tonic B-cell receptor (BCR) signaling is an important checkpoint during B-cell ontogenesis and a key survival pathway in a subset of diffuse large B-cell lymphomas (DLBCLs). We previously demonstrated that BCR-dependent DLBCL cell lines and primary tumors underwent apoptosis following treatment with an ATP-competitive inhibitor of the BCR-associated spleen tyrosine kinase (SYK). These "BCR-type" tumors also have more abundant expression of the transcriptional repressor, BCL6, and increased sensitivity to BCL6 inhibition. Herein, we evaluated potential connections between BCL6-mediated transcriptional repression and SYK-dependent BCR signaling. In transcriptionally profiled normal B-cell subsets (naive, germinal center [GC], and memory B cells) and in primary DLBCLs, there were reciprocal patterns of expression of BCL6 and the SYK tyrosine phosphatase, PTPROt. BCL6 repressed PTPROt transcription via a direct interaction with functional BCL6 binding sites in PTPROt promoter. Enforced expression of BCL6 in normal naive B cells and RNAi-mediated depletion of BCL6 in GC B-cells directly modulated PTPROt expression. In "BCR-type" DLBCLs, BCL6 depletion increased PTPROt expression and decreased phosphorylation of SYK and the downstream adaptor protein, BLNK. Since BCL6 augments BCR signaling and BCL6 and SYK are both promising therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.

Immunoregulatory functions of KLRG1 cadherin interactions are dependent on forward and reverse signaling

Banh, C., Fugere, C., Brossay, L. — Fri, 23 Oct 2009 13:15:34 PDT

KLRG1 is an inhibitory receptor expressed on a subset of mature T and NK cells. Recently, E-, N-, and R-cadherin, have been identified as ligands for KLRG1. Cadherins are a large family of transmembrane or membrane-associated glycoproteins that were thought to only bind specifically to other cadherins to mediate specific cell-cell adhesion in a Ca²⁺-dependent manner. The consequences of cadherin KLRG1 molecular interactions are not well characterized. Here, we report that the first two extracellular domains of cadherin are sufficient to initiate a KLRG1 dependent signaling. We also demonstrate that KLRG1 engagement inhibits cadherin dependent cellular adhesion and influences dendritic cell secretion of inflammatory cytokines, thereby exerting immunosuppressive effects. Consistent with this, engagement of cadherin by KLRG1 molecule induces cadherin tyrosine phosphorylation. Therefore, KLRG1/cadherin interaction leads to the generation of a bidirectional signal, in which both KLRG1 and cadherin activate downstream signaling cascades simultaneously. Taken together, our results provide novel insights on how KLRG1 and E-cadherin interactions are integrated to differentially regulate not only KLRG1 positive cells, but also E-cadherin-expressing cells, such as dendritic cells.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Fri, 23 Oct 2009 13:15:23 PDT

Protease nexin-1 (PN-1) is a serpin which inhibits plasminogen activators, plasmin and thrombin. PN-1 is barely detectable in plasma, but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in alpha-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody, and using platelets from PN-1-deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator (uPA). Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1-deficient mice respond to sub-threshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl₃-induced injury, is significantly increased in PN-1-deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

In vitro differentiated T/NK cell progenitors derived from human CD34+ cells mature in the thymus

Fri, 23 Oct 2009 13:15:14 PDT

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a treatment option for patients suffering from hematopoietic malignancies. Haplo-HSCT is hampered by treatment-related morbidity and mortality, in part due to opportunistic infections; a direct consequence of delayed T cell recovery. Thymic output can be improved by facilitation of thymic immigration, known to require pre-commitment of CD34⁺ cells. We demonstrate that Delta-like ligand (DLL)-mediated pre-differentiation of mobilized CD34⁺ (mCD34⁺) cells in vitro results in a population of thymocyte-like cells arrested at a T/NK cell progenitor stage. Upon intrahepatic transfer to Rag2^-/-c^-/- mice, these cells selectively home to the thymus and differentiate towards surface TCRβ⁺ mature T cells considerably faster than animals transplanted with non-cultured CD34⁺ cells. This finding creates the opportunity to develop an early T cell reconstitution therapy to combine with HSCT.

Molecular and functional analysis of the stem cell compartment of chronic myelogenous leukemia reveals the presence of a CD34- cell population with intrinsic resistance to imatinib

Fri, 23 Oct 2009 13:15:57 PDT

We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin^-CD34^-) hematopoietic stem cells from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular karyotyping and quantitative analysis of BCR-ABL transcript demonstrated that about one third of CD34^- cells are leukemic. CML Lin^-CD34^- cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures induced CD34 expression on some cells, cell cycling, increased clonogenic activity and expression of BCR-ABL transcript. Lin^-CD34^- cells showed hematopoietic cell engraftment rate in two immunodeficient mouse strains similar to Lin^-CD34⁺ cells whereas endothelial cell engraftment was significantly higher. Gene expression profiling revealed the down-regulation of cell cycle arrest genes, genes involved in antigen presentation and processing, while the expression of genes related to tumor progression, such as angiogenic factors, was strongly up-regulated when compared to normal counterparts. Phenotypic analysis confirmed the significant down-regulation of HLA class I and II molecules in CML Lin^-CD34^-cells. Imatinib mesylate did not reduce fusion transcript levels, BCR-ABL kinase activity and clonogenic efficiency of CML Lin^-CD34^- cells in vitro. Moreover, leukemic CD34^- cells survived exposure to BCR-ABL inhibitors in vivo. Thus, we identified a novel CD34^- leukemic stem cell subset in CML with peculiar molecular and functional characteristics.

AML1/RUNX1 point mutation possibly promotes leukemic transformation in myeloproliferative neoplasms

Ding, Y., Harada, Y., Imagawa, J., Kimura, A., Harada, H. — Thu, 22 Oct 2009 14:04:20 PDT

Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages. Some patients exhibit leukemic transformation (LT) by unknown mechanisms, and chemotherapy may increase the risk of LT. To clarify the molecular mechanisms of LT, gene alterations involved in LT from patients in the chronic phase (CP) of MPN were identified. Among 18 patients who progressed to leukemia, AML1/RUNX1 mutations were detected in five patients at the LT but in none at the CP. To investigate the leukemogenic effect of AML1/RUNX1 mutants, the AML1D171N mutant was transduced into CD34⁺ cells from patients in the CP of MPN. The D171N transduction resulted in proliferation of immature myeloid cells, enhanced self-renewal capacity and proliferation of primitive progenitors. Taken together, these results indicate that AML1/RUNX1 point mutations may have a leukemogenic potential in MPN stem cells, and they may promote leukemic transformation in MPN.

How I treat CLL upfront

Gribben, J. G — Thu, 22 Oct 2009 14:04:10 PDT

Chronic lymphocytic leukemia (CLL) remains incurable, with patients having a clinical course of disease progression to requirement for treatment followed by an unremitting course of relapse of disease. Over the past decade there have been major advances in understanding of the pathophysiology of CLL and in the treatment of this disease. This has led to greatly increased response rates and durations of response, but until recently little evidence that this had led to improvement in survival. Advances in the use of prognostic factors that identify patients at high risk of progression have led us to the question that if there still a role for a "watch and wait" approach in asymptomatic high risk patients or if they should be treated earlier in their disease course. Questions remain including; what is the optimal first line treatment and its timing and is there any role of maintenance therapy or stem cell transplantation in this disease? CLL is a disease of the elderly and not all of these patients are eligible for aggressive upfront chemo-immunotherapy regimens, so what is the optimal treatment approach for more frail elderly patients? It is highly likely that our treatment approaches will continue to evolve as the results of ongoing clinical trials are released and that further improvements in the outcome of this disease will result from identification of therapies that target the underlying pathophysiology of CLL.

Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival and response to DNA damage

Thu, 22 Oct 2009 14:03:49 PDT

Deletion of TP53 gene, under routine assessment by FISH analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We have used methodologies with similar sensitivity for the detection of deletions (I-FISH) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; subset of p53-wt cases (n=132) was screened repeatedly during disease course. The most common type of TP53 inactivation, i.e. mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5 %). Among additional defects, the frequency of the isolated TP53 mutation (n=20; 5 %) and the combination of two or more mutations on separate alleles (n=5; 1.3 %) greatly exceeded the sole deletion (n=3; 0.8 %). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation since novel defects may be selected by previous therapies.

MicroRNA 29b functions in acute myeloid leukemia

Thu, 22 Oct 2009 14:03:40 PDT

MicroRNAs (miRNAs) are associated with cytogenetics and molecular subtypes of acute myelogeneous leukemia (AML), but their impact on AML pathogenesis is poorly understood. We have previously shown that miR-29b expression is deregulated in primary AML blasts. In this work, we investigated the functional role of miR-29b in leukemogenesis. Restoration of miR-29b in AML cell lines and primary samples induces apoptosis and dramatically reduces tumorigenicity in a xenograft leukemia model. Transcriptome analysis after ectopic transfection of synthetic miR-29b into leukemia cells indicates that miR-29b target apoptosis, cell cycle and proliferation pathways. A significant enrichment for apoptosis genes, including MCL-1, was found among the mRNAs inversely correlated with miR-29b expression in 45 primary AML samples. Together, the data support a tumor suppressor role for miR-29b and provide a rationale for the use of synthetic miR-29b oligonucleotides as a novel strategy to improve treatment response in AML.

Scl regulates the quiescence and the long-term competence of hematopoietic stem cells

Thu, 22 Oct 2009 14:03:32 PDT

The majority of long term reconstituting hematopoietic stem cells (LT-HSC) in the adult is in G0 while a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSC when compared to short term reconstituting HSCs (ST-HSCs) and progenitors, and that SCL negatively regulates the G0-G1 transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at ~1 CRU was two-fold decreased when Scl gene dosage was decreased. Second, Scl^+/- HSCs were at a marked competitive disadvantage with Scl^+/+ cells when transplanted at 4 CRU equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared to controls. At the molecular level, we found that SCL occupies the Cdkn1a and Id1 loci in primary hematopoietic cells and that the expression levels of these two regulators of HSC cell cycle and long term functions is sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long term competence.

TLR8-dependent TNF{alpha} over-expression in Fanconi anemia group C cells

Thu, 22 Oct 2009 14:03:24 PDT

A Fanconi anemia (FA) protein complex facilitates ubiquitinylation of FANCI and FANCD2. Whether FA sub complexes influence ubiquitinylation of other substrates is unknown. Here we show, using gene expression microarray and proteomics methods, that genes encoding proteins directly involved in ubiquitinylation are over-represented in the signature of FA bone marrow cells and that while some proteins are uniquely ubiquitinylated in complemented cells, others are ubiquitinylated only in mutant cells. One in the latter category was toll-like receptor 8 (TLR8). Because TNF production is abnormally high in FA-C cells and contributes to the pathogenesis of the marrow failure phenotype, we tested the hypothesis that TNF gene over-expression in FA-C cells is TLR8-dependent. We confirmed that TLR8 (or a TLR8-associated protein) is ubiquitinylated in mutant FA-C cells, and that high level TNF synthesis in mutant cells depended upon TLR8 and the canonical downstream signaling intermediates IRAK-1 and IKK-alpha/beta. FANCC deficient THP-1 cells over-expressed TNF in response to TLR8 agonists but not other TLR agonists, and primary splenic macrophages from Fancc^-/- mice were also hypersensitive to the TLR8 agonist R848. TNF production in FA-C cells was suppressed by inhibitors of TLR8, p38 MAPK, IRAK, and IKK. Engineered point mutations of FANCC were capable of complementing the mitomycin C hypersensitivity phenotype of FANCC mutant cells but did not suppress TNF overproduction. In conclusion, FANCC suppresses production of TNF in mononuclear phagocytes by suppressing TLR8 activity and this function of FANCC is independent of its function in protecting the genome from cross-linking agent-induced damage.

Identification of pro-apoptotic Bim as a tumor suppressorin neoplastic mast cells: role of KIT D816V and effects of various targeted drugs

Thu, 22 Oct 2009 14:03:57 PDT

Systemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MC) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MC. Recent data suggest that the pro-apoptotic BH3-only death regulator Bim plays a role as a tumor-suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816-induced downregulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and to promote expression of Bim in the MC leukemia cell lines HMC-1.1 (D816V-negative) and HMC-1.2 (D816V-positive). Both drugs were also found to counteract growth of primary neoplastic MC. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth-inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Together, our data show that KIT D816V suppresses expression of pro-apoptotic Bim in neoplastic MC. Targeting of Bcl-2-family members by drugs promoting Bim-(re)expression or by BH3-mimetics like obatoclax, may be an attractive therapeutic approach in SM.

An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization

Wed, 21 Oct 2009 14:03:53 PDT

Human plasma cells (PCs) and their precursors play an essential role in humoral immune response, but are rare and difficult to harvest. We report here i) the generation of human syndecan-1⁺ and immunoglobulin secreting PCs starting from memory B cells (MBCs) in a 3-step- and 10-day (D) culture, including a 6-fold cell amplification. ii) We report the detailed phenotypic and Affymetrix gene expression profiles of these in vitro PCs as well as of intermediate cells - activated B cells (actBCs) and plasmablasts (PBs) - compared to MBCs and bone marrow PCs, which is accessible through an open web ATLAS (http://amazonia.transcriptome.eu/). iii) We show this B cell to PC differentiation to involve IRF4 and AICDA expressions in D4 actBCs, decrease of PAX5 and BCL6 expressions and increase in PRDM1 and XBP1 expressions in D7 PBs and D10 PCs. It involves downregulation of genes controlled by Pax5, induction of genes controlled by Blimp-1 and XBP1 (unfold protein response). iv) The phenotype of D10 PCs resembles that of peripheral blood PCs detected after immunization of healthy donors. This in vitro model will facilitate further studies in PC biology. It will likewise be helpful to study plasma-cell dyscrasias, including multiple myeloma.

The mouse mutation "thrombocytopenia and cardiomyopathy" (trac) disrupts Abcg5: a spontaneous single gene model for human hereditary phytosterolemia/sitosterolemia

Wed, 21 Oct 2009 14:03:44 PDT

The spontaneous mouse mutation "thrombocytopenia and cardiomyopathy" (trac) causes macrothrombocytopenia, prolonged bleeding times, anemia, leukopenia, infertility, cardiomyopathy, and shortened life span. Homozygotes show a 20-fold decrease in platelet numbers and a 3-fold increase in platelet size with structural alterations and functional impairments in activation and aggregation. Megakaryocytes in trac/trac mice are present in increased numbers, have poorly developed demarcation membrane systems, and decreased polyploidy. The thrombocytopenia is not intrinsic to defects at the level of hematopoietic progenitor cells but is associated with a microenvironmental abnormality. The trac mutation maps to mouse chromosome 17, synteneic with human chromosome 2p21-22. A G to A mutation in exon 10 of the ATP-binding cassette sub-family G, member 5 (Abcg5) gene, alters a tryptophan codon (UGG) to a premature stop codon (UAG). Crosses with mice doubly transgenic for the human ABCG5 and ABCG8 genes rescued platelet counts and volumes. ABCG5 and ABCG8 form a functional complex that limits dietary phytosterol accumulation. Phytosterolemia in trac/trac mice confirmed a functional defect in the ABCG5/ABCG8 transport system. The trac mutation provides a new clinically significant animal model for human phytosterolemia and provides a new means for studying the role of phytosterols in hematological diseases and testing therapeutic interventions.

Identification of microRNA expression patterns and definition of a microRNA/mRNA regulatory network in distinct molecular groups of multiple myeloma

Wed, 21 Oct 2009 14:03:36 PDT

To date, little evidence of miRNA expression/deregulation in multiple myeloma (MM) has been reported. To characterize miRNA in the context of the major MM molecular types, we generated miRNA expression profiles of highly-purified malignant plasma cells from 40 primary tumors. Furthermore, transcriptional profiles, available for all patients, were used to investigate the occurrence of miRNA/predicted target mRNA pairs anti-correlations, and the miRNA and genome-wide DNA data were integrated in a subset of patients to evaluate the influence of allelic imbalances on miRNA expression. Differential miRNA expression patterns were identified which were mainly associated with the major IGH translocations: particularly, t(4;14) patients showed specific over-expression of let-7e, miR-125a-5p and miR-99b belonging to a cluster at 19q13.33. The occurrence of other lesions, i.e. 1q gain, 13q and 17p deletions, and hyperdiploidy, was slightly characterized by specific miRNA signatures. Furthermore, the occurrence of several allelic imbalances or loss-of-heterozygosity was found significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 or miR-140-3p at 16q22. Finally, the integrative analysis based on computational target prediction and miRNA/mRNA profiling defined a network of putative functional miRNA-target regulatory relations supported by expression data.

International consensus report on the investigation and management of primary immune thrombocytopenia

Wed, 21 Oct 2009 14:03:29 PDT

Previously published guidelines for the diagnosis and management of primary immune thrombocytopenia (ITP) require updating largely due to the introduction of new classes of therapeutic agents, and a greater understanding of the disease pathophysiology. However, treatment-related decisions still remain principally dependent on clinical expertise or patient preference rather than high quality clinical trial evidence. This consensus document aims to report on new data, and provide consensus-based recommendations relating to diagnosis and treatment of ITP in adults, children and in pregnancy. The inclusion of summary tables within this document, supported by information tables in the online appendices, is intended to aid clinical decision making.

How I treat hairy cell leukemia

Grever, M. R. — Tue, 20 Oct 2009 12:52:47 PDT

The description of hairy cell leukemia as a specific clinical entity was published fifty years ago. The clinical outcome for patients was hampered by ineffective chemotherapy, and splenectomy was the major therapeutic approach to improve peripheral blood counts. The median survival following diagnosis was four years. With the introduction of alpha-interferon in 1984, marked improvements in patient responses were observed. Shortly thereafter, the introduction of the purine nucleoside analogs transformed this disease into a highly treatable form of leukemia, and patients with the classic form of this rare leukemia now have a near normal life expectancy. Other clinical entities mimicking this disease do not respond as well, however, thus accurate diagnosis is important. Immunophenotypic features in classic hairy cell leukemia show that the leukemic cells express CD11c, CD25, CD103, CD123, and display bright CD20. Despite the high percentage of durable complete remissions with modern therapy, the long-term disease-free survival curves have not reached a plateau. Many patients who achieve a complete remission by morphologic criteria have minimal residual disease demonstrable by either flow cytometry or immunohistochemical staining, and this population may be at higher risk for earlier relapse. Continued clinical research is essential to optimize therapy for this disease.

Differential requirement for Gata1 DNA binding and transactivation between primitive and definitive stages of hematopoiesis in zebrafish

Tue, 20 Oct 2009 12:52:37 PDT

The transcription factor Gata1 is required for the development of erythrocytes and megakaryocytes. Previous studies with a complementation rescue approach showed that the zinc finger domains are required for both primitive and definitive hematopoiesis. Here we report a novel zebrafish gata1 mutant with an ENU-induced point mutation in the C-finger (gata1^T301K). The Gata1 protein with this mutation bound to its DNA target sequence with reduced affinity and transactivated inefficiently in a reporter assay. gata1^T301K/T301K fish had decreased number of erythrocytes during primitive hematopoiesis but normal adult hematopoiesis. We crossed the gata1^T301K/T301K fish with those carrying the R339X mutation, also known as vlad tepes (vlt), which abolishes DNA binding and transactivation activities. As we reported before, gata1^vlt/vlt embryos were "bloodless" and died around 11-15 days post fertilization (dpf). Interestingly, the gata1^T301K/vlt fish had nearly complete block of primitive hematopoiesis, but they resumed hematopoiesis between 7-14 dpf and grew to phenotypically normal fish with normal adult hematopoiesis. Our findings suggest that the impact of Gata1 on hematopoiesis correlates with its DNA-binding ability and that primitive hematopoiesis is more sensitive to reduction in Gata1 function than definitive hematopoiesis.

Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites

Ben-Ami, R., Lewis, R. E., Leventakos, K., Kontoyiannis, D. P. — Tue, 20 Oct 2009 12:52:26 PDT

In susceptible hosts, angioinvasion by Aspergillus fumigatus triggers thrombosis, hypoxia and proinflammatory cytokine release, all of which are stimuli for angiogenesis. We sought to determine whether A. fumigatus directly modulates angiogenesis. A. fumigatus culture filtrates profoundly inhibited the differentiation, migration and capillary tube formation of human umbilical vein endothelial cells in vitro. To measure angiogenesis at the site of infection, we devised an in vivo matrigel assay in cyclophosphamide-treated Balb/c mice with cutaneous invasive aspergillosis. Angiogenesis was significantly suppressed in matrigel plugs implanted in A. fumigatus-infected mice compared with plugs from uninfected control mice. The antiangiogenic effect of A. fumigatus was completely abolished by deletion of the global regulator of secondary metabolism, laeA, and to a lesser extent by deletion of gliP, which controls gliotoxin production. Moreover, pure gliotoxin potently inhibited angiogenesis in vitro in a dose-dependent manner. Finally, overexpression of multiple angiogenesis-mediator encoding genes was observed in the lungs of cortisone-treated mice during early invasive aspergillosis, whereas gene-expression returned rapidly to baseline levels in cyclophosphamide/cortisone-treated mice. Taken together, these results indicate that suppression of angiogenesis by A. fumigatus both in vitro and in a neutropenic mouse model is mediated through secondary metabolite production.

IL-21 blockade reduces graft-versus-host-disease mortality by supporting inducible T regulatory cell generation

Tue, 20 Oct 2009 12:52:15 PDT

IL-21 is a pleiotropic cytokine produced by CD4⁺ T-cells and NKT-cells that enhances Th1 and Th17 differentiation while inhibiting the conversion of inducible Tregs from naive T-cells. To determine the role of IL-21 in GVHD, anti-IL-21 Ab was given to recipients of CD25^-CD4⁺ or CD4⁺ and CD8⁺ T-effectors. IL-21 neutralization attenuated GVHD-related weight loss and prolonged survival. Likewise, a majority of mice receiving IL-21^-/- CD25^- T-effectors survived long-term, while those receiving wild-type T cells died. The latter recipients had higher grades of GVHD-related tissue damage in the ileum and colon. Surprisingly, disruption of IL-21 signaling did not affect IL-17 production, although colon-infiltrating T-effector cells had decreased IFN and increased IL-4 production. FoxP3⁺ Tregs were increased in colons of anti-IL-21 Ab-treated recipients of FoxP3^- IL-21^-/- T-cells, indicating Treg conversion. Recipients of congenic FoxP3-deficient T-effectors isolated from chimeras and used as donor T-cells were resistant to the GVHD protective effects of IL-21 blockade. Whereas graft-versus-leukemia (GVL) can occur in the absence of IL-21, loss of both IL-21 and perforin expression abrogated GVL. Together, these data indicate that IL-21 suppresses iTreg conversion and further suggest that IL-21 blockade is an attractive strategy to reduce GVHD-induced injury.

Epidemiological study on survival of chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) patients with BCR-ABL T315I mutation

Tue, 20 Oct 2009 12:51:55 PDT

The BCR-ABL T315I mutation represents a major mechanism of resistance to tyrosine kinase inhibitors (TKIs). The objectives of this retrospective observational study were to estimate overall (OS) and progression-free survival (PFS) for CML in chronic (CP), accelerated (AP), or blastic (BP) phase, and Ph+ ALL patients with T315I mutation. Medical records of 222 patients from nine countries were reviewed; data were analyzed using log-rank tests and Cox proportional hazard models. Median age at T315I mutation detection was 54 years; 57% cases were male. Median time between TKI treatment initiation and T315I mutation detection was 29.2, 15.4, 5.8, and 9.1 months, respectively, for CP, AP, BP, and Ph+ ALL patients. After T315I mutation detection, 2^nd generation TKIs were utilized in 56% of cases, hydroxyurea in 39%, imatinib in 35%, cytarabine in 26%, MK-0457 in 11%, stem cell transplantation in 17%, and interferon alpha in 6% of cases. Median OS from T315I mutation detection was 22.4, 28.4, 4.0, and 4.9 months, and median PFS was 11.5, 22.2, 1.8, and 2.5 months, respectively, for CP, AP, BP, and Ph+ ALL patients. These results confirm that survival of patients harboring a T315I mutation is dependent on disease phase at the time of mutation detection.

Phase 1/2 study of lumiliximab combined with fludarabine, cyclophosphamide, and rituximab in patients with relapsed or refractory chronic lymphocytic leukemia

Tue, 20 Oct 2009 12:51:46 PDT

Preclinical data demonstrate enhanced antitumor effect when lumiliximab, an anti-CD23 monoclonal antibody, is combined with fludarabine or rituximab. Clinical data from a phase 1 trial with lumiliximab demonstrated an acceptable toxicity profile in patients with relapsed or refractory chronic lymphocytic leukemia (CLL). We therefore pursued a phase 1/2 dose escalation study of lumiliximab added to fludarabine, cyclophosphamide, and rituximab (FCR) in previously treated CLL patients. Thirty-one patients received either 375 mg/m² (n = 3) or 500 mg/m² (n = 28) of lumiliximab in combination with FCR for 6 cycles. The toxicity profile was similar to that previously reported for FCR in treatment of relapsed CLL. The overall response rate was 65% with 52% of patients achieving a complete response (CR), which compares favorably with the CR rate previously reported for the FCR regimen alone in relapsed CLL. The estimated median progression-free survival for all responders was 28.7 months. The addition of lumiliximab to FCR therapy is feasible, achieves a high CR rate, and does not appear to enhance toxicity in previously treated patients with CLL. A randomized trial comparing lumiliximab plus FCR with FCR alone is underway to define the benefit of this combination in relapsed CLL. This trial were registered at clinicaltrials.gov as NCT00103558.

How I treat age-related morbidities in elderly persons with hemophilia

Mannucci, P. M., Schutgens, R. E.G., Santagostino, E., Mauser-Bunschoten, E. P. — Tue, 20 Oct 2009 12:51:37 PDT

In persons with hemophilia life expectancy is now approaching that of the general male population, at least in countries that can afford regular replacement therapy with coagulation factor concentrates. The new challenges for comprehensive treatment centers are thus to provide optimal health care for this aging population of patients, who often present not only with the comorbidities typically associated to hemophilia (arthropathy, chronic pain, bloodborne infections), but also with common age-related illnesses such as cardiovascular disease and cancer. There are no evidence-based guidelines on the management of these conditions, that often require drugs that interfere with hemostasis, enhance the bleeding tendency and warrant more intensive replacement therapy. At the moment, elderly patients with hemophilia affected by other diseases should be managed like their age peers without hemophilia, provided replacement therapy is tailored to the heightened risk of bleeding associated with the need for invasive procedures and drugs that further compromise the deranged hemostasis. More detailed advice is provided on the schedules of replacement therapy that are needed to tackle cardiovascular diseases such as acute coronary syndromes and nonvalvular atrial fibrillation, because these conditions will become more and more frequent challenges for the comprehensive treatment centers.

A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic phase chronic myeloid leukemia patients treated with imatinib

Fri, 16 Oct 2009 16:57:42 PDT

In chronic phase chronic myeloid leukemia (CML) patients the lack of a major cytogenetic response (MCyR, <36% Ph+ metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure we performed gene expression array profiling of CD34+ cells from two independent cohorts of imatinib-naive chronic phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response (CCyR) or >65% Ph-positive metaphases within 12 months of imatinib therapy. Based on ANOVA p<0.1 and fold difference >|1.5| we identified 885 probe sets with differential expression between responders and non-responders, from which we extracted a 75-probe set minimal signature (classifier) that separated the two groups. Upon application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of non-responders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated β-catenin in their regulation, suggesting that chronic phase CML patients destined to fail imatinib have more advanced disease than evident by morphological criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit, while sparing good-risk patients from unnecessary toxicity.

EMMPRIN promotes angiogenesis through HIF-2{alpha} mediated regulation of soluble VEGF isoforms and their receptor VEGFR-2

Fri, 16 Oct 2009 16:57:35 PDT

EMMPRIN/CD147 is thought to promote tumor angiogenesis mostly through its protease inducing function and more recently by its ability to increase tumor cell expression of vascular endothelial growth factor (VEGF). In this study we present evidence that EMMPRIN can promote angiogenesis by a direct effect on endothelial cells through a paracrine regulation of the VEGF/VEGF-receptor system. Using HMEC-1 endothelial cells we show that EMMPRIN selectively increased the soluble VEGF isoforms (121 and 165) but not the matrix bound VEGF 189 form. In addition, EMMPRIN upregulated the expression of VEGF receptor type 2 (VEGFR-2) without an effect on VEGFR-1. This increase in VEGFR-2 was responsible for the observed EMMPRIN stimulation of the migratory and tube formation capacity of endothelial cells. EMMPRIN's effects, which were MMPs and uPA independent, were mediated primarily through HIF-2 expression, also upregulated by EMMPRIN. VEGFR-2 increase was also observed in vivo in a mouse model of xenograph tumors overexpressing EMMPRIN. These results suggest that in addition to increasing protease production, EMMPRIN may contribute to the formation of a reactive stroma also through the upregulation of HIF-2, VEGFR-2 and the soluble forms of VEGF in endothelial cells, thus directly regulating the angiogenic process.

Migrating monocytes recruited to the spleen play an important role in control of blood stage malaria

Fri, 16 Oct 2009 16:57:27 PDT

Host responses controlling blood stage malaria include both innate and acquired immune effector mechanisms. During Plasmodium chabaudi infection in mice a population of CD11b^highLy6C⁺ monocytes are generated in bone marrow, the majority of which depend on the chemokine receptor CCR2 for migration from bone marrow to the spleen. In the absence of this receptor mice harbor higher parasitemias. Most importantly, splenic CD11b^highLy6C⁺ cells from P. chabaudi infected wild-type mice significantly reduce acute stage parasitemia in CCR2^-/- deficient mice. The CD11b^highLy6C⁺ cells in this malaria infection display effector functions such as production of iNOS and reactive oxygen intermediates, and phagocytose P. chabaudi parasites in vitro, and in a proportion of the cells, in vivo in the spleen, suggesting possible mechanisms of parasite killing. In contrast to monocyte-derived DC, CD11b^highLy6C⁺ cells isolated from malaria-infected mice express low levels of MHC II and have limited ability to present the P. chabaudi antigen, Merozoite Surface Protein-1, to specific TCR transgenic CD4 T cells, and fail to activate these T cells. We propose that these monocytes, which are rapidly produced in the bone marrow as part of the early defense mechanism against invading pathogens, are important for controlling blood-stage malaria parasites.

Identification of early growth response protein 1 (EGR-1) as a novel target for JUN-induced apoptosis in multiple myeloma

Fri, 16 Oct 2009 16:57:12 PDT

Tumor-bone marrow microenvironment interactions in multiple myeloma (MM) are documented to play crucial roles in plasma cell growth/survival. In-vitro co-culture of MM cells with osteoclasts supported cell survival and significantly downregulated JUN expression. JUN expression in myeloma cells from late-stage and high-risk MM was significantly lower than in plasma cells from healthy donors, monoclonal gammopathy of undetermined significance, smoldering MM and low-risk MM; patients with low-JUN-expressing MM cells had earlier disease-related deaths. JUN overexpression in MM cells induced cell death and growth inhibition, and upregulated expression of early growth response protein 1 (EGR-1), whose low expression also carried unfavorable clinical implications. EGR-1 knockdown in MM cells abrogated JUN-overexpression-induced MM cell death and growth inhibition, indicating that EGR-1 acts directly downstream of JUN. JUN modulates myeloma cell apoptosis through interacting with EGR-1, which downregulates Survivin and triggers caspase signaling. Importantly, high JUN or EGR-1 expression was associated with improved outcome in Total Therapy 3 in which bortezomib is given throughout therapy versus Total Therapy 2 in which bortezomib is given only at relapse. Consistently, JUN or EGR-1 knockdown in cultured MM cells enhanced their resistance to bortezomib, demonstrating the crucial role of low JUN/EGR-1 expression in MM resistance to bortezomib.

Increased tissue factor expression on circulating monocytes in chronic HIV infection: relationship to in vivo coagulation and immune activation

Wed, 14 Oct 2009 14:46:42 PDT

Human immunodeficiency virus (HIV) infection is associated with an increased risk of thrombosis, and as antiretroviral therapy has increased the lifespan of HIV infected patients, their risk for cardiovascular events is expected to increase. A large clinical study found recently that all cause mortality for HIV+ patients was related to plasma levels of interleukin-6 and to D-dimer products of fibrinolysis. We provide evidence that this elevated risk for coagulation may be related to increased proportions of monocytes expressing cell surface tissue factor (TF, thromboplastin) in persons with HIV infection. Monocyte TF expression could be induced in vitro by lipopolysaccharide (LPS) and flagellin, but not by interleukin-6. Monocyte expression of TF was correlated with HIV levels in plasma, with indices of immune activation, and with plasma levels of soluble CD14, a marker of in vivo LPS exposure. TF levels also correlated with plasma levels of D-dimers, reflective of in vivo clot formation and fibrinolysis. Thus, drivers of immune activation in HIV disease, such as HIV replication, and potentially, microbial translocation, may activate clotting cascades and contribute to thrombus formation and cardiovascular morbidities in HIV infection.

Evidence for a novel human-specific xeno-auto-antibody response against vascular endothelium

Wed, 14 Oct 2009 14:46:08 PDT

Humans are genetically unable to synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc). However, Neu5Gc can be metabolically incorporated and covalently expressed on cultured human cell surfaces. Meanwhile, humans express varying and sometimes high titers of polyclonal anti-Neu5Gc antibodies. Here, a survey of human tissues by immunohistochemistry with both a mono-specific chicken anti-Neu5Gc antibody and with affinity-purified human anti-Neu5Gc antibodies demonstrates endothelial expression of Neu5Gc, likely originating from Neu5Gc-rich foods like red meats. We hypothesized that the combination of Neu5Gc incorporation and anti-Neu5Gc antibodies can induce endothelial activation. Indeed, incubation of high-titer human sera with Neu5Gc-fed endothelial cells led to Neu5Gc-dependent antibody binding, complement deposition, endothelial activation, selectin expression, increased cytokine secretion and monocyte binding. The pro-inflammatory cytokine TNF-alpha also selectively enhanced human anti-Neu5Gc antibody reactivity. Anti-Neu5Gc antibodies affinity-purified from human serum also directed Neu5Gc-dependent complement deposition onto cultured endothelial cells. These data indicate a novel human-specific mechanism in which Neu5Gc-rich foods deliver immunogenic Neu5Gc to the endothelium, giving anti-Neu5Gc antibody- and complement-dependent activation, and potentially contributing to human vascular pathologies. In the case of atherosclerosis, Neu5Gc is present both in endothelium overlying plaques and in sub-endothelial regions, providing multiple pathways for accelerating inflammation in this disease.

A novel thromboxane A2 receptor D304N variant which abrogates ligand binding in a patient with a bleeding diathesis

Wed, 14 Oct 2009 14:45:53 PDT

We investigated the cause of mild mucocutaneous bleeding in a 14 year old male (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A₂ receptor (TxA₂R) agonist U46619 were reduced in P1 compared to controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA₂R. In CHO-K1 cells expressing the variant D304N TxA₂R, U46619 did not increase cytosolic free Ca²⁺ concentration indicating loss of receptor function. The TxA₂R antagonist [³H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to CHO-K1 cells expressing variant D304N TxA₂R. This is the second naturally occurring TxA₂R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA₂R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA₂R function in vivo.

T-cell acute lymphoblastic leukemia in adults: clinical features, immunophenotype, cytogenetics and outcome from the large randomised prospective trial (UKALL XII/ECOG 2993)

Wed, 14 Oct 2009 14:45:40 PDT

The biology and outcome of adult T-cell acute lymphoblastic leukemia are poorly understood. We present here the clinical and biological features of 356 patients treated uniformly on the prospective trial (UKALL XII/ECOG 2993) with the aim of describing the outcome and identifying prognostic factors. Complete remission was obtained in 94% of patients and 48% survived 5 years. Positivity of blasts for CD1a and lack of expression of CD13 were associated with better survival (p=0.01 and 0.0005 respectively). NOTCH1 and CDKN2A mutations were seen in 61% and 42% of those tested. Complex cytogenetic abnormalities were associated with poorer survival (19% vs 51% at 5 years, p=0.006). Central nervous system involvement at diagnosis did not affect survival (47% vs 48%, p=NS). For 99 patients randomised between autograft and chemotherapy 5 year survival was 51% in each arm. Patients with a matched sibling donor had superior 5 year survival to those without donors (61% vs 46%, chi-square p=0.02); this was due to less relapse (25% vs 51% at 5 years, p<0.001). Only 8 of 123 relapsed patients survive. This study provides a baseline for trials of new drugs such as nelarabine and may allow risk-adapted therapy in patients with poor-prognosis T-cell ALL.

Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of Arginine245

Wed, 14 Oct 2009 07:49:52 PDT

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among individuals. Here, we demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, IFN- production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in their transmembrane domain, which mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245 negative alleles, KIR2DL1 that included R245 recruited more SHP-2 and β-arrestin 2, showed higher inhibition of lipid rafts polarization at immune synapse, and had less downregulation of cell surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably fundamental understanding of KIR2DL1 alleleic polymorphism in human disease susceptibility, transplant outcome, and donor selection.

Chemokine induction by all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia - triggering the differentiation syndrome

Wed, 14 Oct 2009 07:49:33 PDT

In acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide (ATO) can induce a differentiation syndrome (DS) with massive pulmonary infiltration of differentiating leukemic cells. Since chemokines are implicated in migration and extravasation of leukemic cells, chemokines might play a role in DS. ATRA-stimulation of the APL cell line NB4 induced expression of multiple CC-chemokines (CCLs) and their receptors (> 19-fold), resulting in increased chemokine levels and chemotaxis. Induction of CCL2 and CCL24 was directly mediated by ligand-activated retinoic acid receptors. In primary leukemia cells derived from APL patients at diagnosis, ATRA induced chemokine production as well. Furthermore, in plasma of an APL patient with DS we observed chemokine induction, suggesting that chemokines might be important in DS. Dexamethasone, which efficiently reduces pulmonary chemokine production, did not inhibit chemokine induction in APL cells. Finally, chemokine production was also induced by ATO as single agent or in combination with ATRA. We propose that differentiation therapy may induce chemokine production in the lung and in APL cells, which both trigger migration of leukemic cells. Since dexamethasone does not efficiently reduce leukemic chemokine production, pulmonary infiltration of leukemic cells may induce an uncontrollable hyper-inflammatory reaction in the lung.

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain

Mon, 12 Oct 2009 17:12:24 PDT

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In Heavy-Chain Deposition Disease (HCDD) truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC) deficient (L^-/-) mice. In L^-/- animals the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here we show a novel function of LC in preventing antibody aggregation. L^-/- mice produce truncated HC naturally, C(constant region) and C lack C_H1 and Cµ is without C_H1 or C_H1 and C_H2. Most plasma cells found in these mice are CD138⁺ Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice (µNR), expressing Cµ without C_H1 and C_H2, which only develop an abundance of RBs when LC is absent (µNR L^-/-). These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of HCDD, and in general recognizing the mechanisms, which initiate protein conformational diseases.

The impact of prophylactic fresh frozen plasma and cryoprecipitate on the incidence of CNS thrombosis and hemorrhage in children with acute lymphoblastic leukemia receiving asparaginase

Mon, 12 Oct 2009 17:11:50 PDT

Asparaginase (ASP) therapy is associated with depletion of antithrombin (AT) and fibrinogen (FG). Potential toxicities include CNS thrombosis (CNST) and hemorrhage. Historical practice at the IWK Health Centre (IWK) involves measuring AT and FG levels following ASP administration and transfusing fresh frozen plasma (FFP) or cryoprecipitate (CRY) to prevent thrombotic and hemorrhagic complications. To determine if this practice reduced these complications in children with acute lymphoblastic leukemia (ALL) receiving ASP, incidence, outcome and clinical characteristics of ASP-related CNST in ALL patients at IWK were compared to a similarly treated cohort from B.C. Children's Hospital (BCCH), where prophylaxis was not performed. Costs associated with preventative versus expectant management strategies were estimated. From 1990-2005, 240 patients were treated at IWK and 479 at BCCH. Seven BCCH patients developed venous CNST (1.5%), compared with none at IWK. CNST occurred exclusively during induction. Six patients received anti-coagulation and continued ASP. The remaining patient discontinued ASP. All remain in remission. NCI high risk (HR) ALL predicted CNST risk (p=0.02), while gender, age, race, and BMI did not. Neither FFP nor CRY protected against CNST, suggesting prophylaxis is unwarranted for unselected ALL patients. However, prophylactic replacement for HR patients in induction may be appropriate and cost-effective.

Single unit dominance following double unit umbilical cord blood transplantation coincides with a specific CD8+ T cell response against the non-engrafted unit

Mon, 12 Oct 2009 17:11:38 PDT

We investigated the potential role of an immune reaction in mediating the dominant engraftment of one cord blood unit in fourteen patients that received a double unit cord blood transplant (CBT). In 10 patients, dominant engraftment of a single donor unit emerged by day 28 after CBT. In nine of these 10 patients, a significant subset of CD8⁺ CD45RO^+/-CCR7^- T cells, present in peripheral blood mononuclear cells (PBMC) and derived from the engrafting cord blood unit, produced IFN- in response to the non-engrafting unit. No significant population of IFN- secreting cells were detectable when post transplant PBMC were stimulated against cells from the engrafted unit (p<0.001) or from a random HLA disparate third party (p=0.003). Three patients maintained persistent mixed chimerism following CBT, and no significant IFN- secreting cells were detected following similar stimulations in these patients (p=.0047). Our data provide the first direct evidence in human double unit CBT recipients that immune rejection mediated by effector CD8⁺ T cells developing after CBT from naive precursors is responsible for the failure of one unit to engraft. Future investigations based on these findings may result in strategies to predict a dominant unit and enhance graft-versus-leukemia (GVL) effect.

Differential roles for ETS, CREB and EGR binding sites in mediating VEGF receptor 1 expression in vivo

Mon, 12 Oct 2009 17:11:23 PDT

Vascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between -748 and +284) contains information for expression in the intact endothelium of Hprt-targeted mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for ELF-1 (between -49 and -52), CREB (between -74 and -81) and EGR-1/3 (between -16 to -25) were shown to play a positive role in gene transcription, while a putative ETS motif between -36 and -39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1-binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in virtual loss of expression in the adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the -39 ETS site resulted in increased LacZ staining in multiple vascular beds. All promoters were expressed in the endothelium of tumor xenografts. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.

Allogeneic stem cell transplantation after reduced-intensity conditioning in patients with myelofibrosis: a prospective, multicenter study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT)

Wed, 07 Oct 2009 12:55:45 PDT

From 2002 to 2007, 103 patients with primary myelofibrosis or post ET/PV myelofibrosis and a median age of 55 years (range, 32--68) were included in a prospective multicenter phase II trial to determine efficacy of a busulfan (10 mg/kg) /fludarabine (180mg/m²) -based reduced-intensity conditioning regimen (RIC) followed by allogeneic stem cell transplantation from related (n=33) or unrelated donors (n=70). All but two patients (2%) showed leukocyte and platelet engraftment after a median of 18 and 22 days, respectively. Acute graft-versus-host disease (GvHD) grade II to IV occurred in 27% and chronic GvHD in 43% of the patients. Cumulative incidence of non-relapse mortality at one year was 16% (95% CI: 9--23%) and significantly lower for patients with a completely matched donor (12% vs. 38%) (p=0.003). The cumulative incidence of relapse at three years was 22% (95% CI: 13--31%), and was influenced by Lille-risk profile (low: 14%, intermediate: 22%, and high: 34%) (p=0.02). The estimated five-year event-free and overall survival was 51% and 67%, respectively. In a multivariate analysis, age > 55 years (HR: 2.70; p=0.02) and HLA-mismatched donor (HR; 3.04; p=0.006) remained significant factors for survival. The study was registered at www.clinicaltrials.gov under: NCT 00599547.

Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13

Wed, 07 Oct 2009 12:55:12 PDT

Elevated plasma von Willebrand factor (VWF) and low ADAMTS13 activity have been reported in several inflammatory states, including sepsis and acute respiratory distress syndrome. One hallmark of inflammation is neutrophil activation and production of reactive oxygen species (ROS), including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). HOCl is produced from hydrogen peroxide and chloride ions through the action of myeloperoxidase. HOCl can oxidize methionine to methionine sulfoxide and tyrosine to chlorotyrosine. This is of interest because the ADAMTS13 cleavage site in VWF, the Tyr1605-Met1606 peptide bond, contains both oxidation-prone residues. We hypothesized that HOCl would oxidize either or both of these residues and possibly inhibit ADAMTS13-mediated cleavage. We therefore treated ADAMTS13 substrates with HOCl and examined their oxidative modification by mass spectrometry. Met1606 was oxidized to the sulfoxide in a dose-dependent manner, with complete oxidation at 75 µM HOCl, whereas only a miniscule percentage of Tyr1605 was converted to chlorotyrosine. The oxidized substrates were cleaved much more slowly by ADAMTS13 than the non-oxidized substrates. A similar result was obtained with multimeric VWF. Taken together, these findings indicate that ROS released by activated neutrophils have a prothrombotic effect, mediated in part by inhibition of VWF cleavage by ADAMTS13.

Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation

Tue, 06 Oct 2009 13:52:12 PDT

Platelet-type von Willebrand disease (PT-VWD) is a bleeding disorder of the platelet glycoprotein (GP) Ib-IX/von Willebrand factor (VWF) axis caused by mutations in the GP Ib-IX receptor that lead to an increased affinity with VWF. In this report, platelets from a mouse expressing a mutation associated with PT-VWD have been visualized using state-of-the art image collection and processing. Confocal analysis revealed VWF bound to the surface of single platelets and bridging micro aggregates of platelets. Surface-bound VWF appears as a large, linear structure on the surface of 50% of the PT-VWD platelets. In vivo thrombus formation following chemical injury to the carotid artery revealed a severe impairment to occlusion as a consequence of the PT-VWD mutation. In vitro stimulation of PT-VWD platelets with ADP or thrombin demonstrates a significant block in their ability to bind fibrinogen. The impairment of in vivo thrombus formation and in vitro fibrinogen binding are more significant than might be expected from the observed platelet binding to VWF polymers over a small portion of the plasma membrane. Visualization of the receptor/ligand interaction and characterization of a severe anti-thrombotic phenotype provides a new understanding on the molecular basis of bleeding associated with the PT-VWD phenotype.

AML1/RUNX1 mutations in 470 adult patients with de novo acute myeloid leukemia: prognostic implication and interaction with other gene alterations

Tue, 06 Oct 2009 12:56:03 PDT

Somatic mutation of AML1/RUNX1(RUNX1) gene is seen in acute myeloid leukemia (AML) M0 subtype and AML transformed from myelodysplastic syndrome, but the impact of this gene mutation on survival in AML patients remains unclear. In this study, we sought to determine the clinical implications of RUNX1 mutations in 470 adult patients with de novo non-M3 AML. Sixty-three distinct RUNX1 mutations were identified in 62 individuals (13.2%); 32 were in N-terminal and 31, C-terminal. The RUNX1 mutation was closely associated with male gender, older age, lower LDH value, FAB M0/M1 subtypes and expression of HLA-DR and CD34, but inversely correlated with CD33, CD15, CD19 and CD56 expression. Furthermore, the mutation was positively associated with MLL/PTD, but negatively associated with CEBPA and NPM1 mutations. AML patients with RUNX1 mutations had a significantly lower complete remission rate and shorter disease-free and overall survival than those without the mutation. Multivariate analysis demonstrated that RUNX1 mutation was an independent poor prognostic factor for overall survival. Sequential analysis in 133 patients revealed that none acquired novel RUNX1 mutations during clinical courses. Our findings provide evidences that RUNX1 mutations are associated with distinct biological and clinical characteristics and poor prognosis in patients with de novo AML.

{beta}2 glycoprotein I ({beta}2GPI) binds platelet factor 4 (PF4): implications for the pathogenesis of antiphospholipid syndrome

Mon, 05 Oct 2009 11:39:16 PDT

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by arterial/venous thrombosis and/or pregnancy morbidity in the presence of aPL antibodies which mainly recognize β2glycoprotein I (β2GPI). In order to investigate potential platelet ligands of β2GPI, platelet membrane proteins from healthy individuals and APS patients were passed through a β2GPI-affinity column. Using mass spectrometry, platelet Factor 4 (PF4) appeared as the dominant β2GPI binding protein. PF4 could bind in vitro, with high affinity, recombinant β2GPI and the binding was abrogated by soluble β2GPI. Co-precipitation experiments further confirmed this interaction. In silico molecular docking revealed that PF4 tetramers can bind two β2GPI molecules simultaneously. Size exclusion chromatography confirmed that anti-β2GPI antibodies selectively interact with complexes comprised of (β2GPI)₂-(PF4)₄. In addition, as demonstrated by the β2GPI antigenicity evaluation, the reactivity of APS sera was higher against PF4-β2GPI complex than against β2GPI alone. Upon complex formation, anti-β2GPI-β2GPI-PF4 significantly induced platelet p38MAPK phosphorylation and TXB2 production, mainly through F(ab')₂ fragments of antibodies. In summary, this study makes evident that β2GPI forms stable complexes with PF4, leading to the stabilization of β2GPI dimeric structure which facilitates the antibody recognition. This interaction can probably be involved in the precoagulant tendency of APS.

Depletion of the C3 component of complement enhances the ability of rituximab-coated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model

Mon, 05 Oct 2009 11:38:51 PDT

Growing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced NK cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK cell activation when it was pre-incubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or non-malignant ascites were used as surrogates for extravascular fluid suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted prior to mAb in a syngenetic murine model of lymphoma. Survival of lymphoma-bearing mice following treatment with CVF plus mAb, and a human C3 derivative with CVF-like functions (HC3-1496) plus mAb, were both superior to that of mAb alone. These studies demonstrate that complement depletion enhances NK cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.

The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation

Fri, 02 Oct 2009 10:35:30 PDT

Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused disturbance of polymerized filaments. Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts and fetal liver-derived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, was also reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function.

How I treat acute promyelocytic leukemia

Tallman, M. S., Altman, J. K. — Thu, 01 Oct 2009 13:29:54 PDT

Acute promyelocytic leukemia is the first malignant disease highly curable with targeted therapy directed at a unique molecular abnormality. The characteristic bleeding diathesis is the most notorious manifestation of the disease which historically, has accounted for a high mortality rate during induction. Acute promyelocytic leukemia is one of the few hematological diseases which must be recognized under the microscope by the practicing hematologist because early institution of all-trans retinoic acid (ATRA) at the first suspicion of the disease before confirmation of the diagnosis and aggressive blood product support are critical to reduce early mortality. ATRA plus anthracycline-based chemotherapy for induction and consolidation followed by maintenance ATRA with low-dose chemotherapy is currently the standard of care. However, the combination of ATRA and arsenic trioxide (ATO), with minimal chemotherapy to control leukocytosis, is very effective therapy for newly diagnosed patients. This combination is likely to replace conventional approaches for most, if not all, patients in the very near future. Acute promyelocytic leukemia should be considered in any patient with newly diagnosed acute myeloid leukemia because the treatment is urgent and different from all other subtypes.

In vivo cellular imaging pinpoints the role of reactive oxygen species in the early steps of adult hematopoietic reconstitution

Thu, 01 Oct 2009 13:29:09 PDT

Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) following lethal irradiation. Using a fiberoptic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin^-Sca-1⁺c-Kit⁺CD34^- (LSKCD34^-) hematopoietic stem cells highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34^- hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34^- hematopoietic cells in the femoral head was potent three days after transplantation. Using a development of this fiberoptic imaging system, we show that the transplanted LSKCD34^- hematopoietic cells are associated with vascularized structures as early as five hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of VCAM-1 expression on endothelial cells and is followed by a ROS dependent proliferation of LSKCD34^- hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.

Sickle cell disease and stroke

Verduzco, L. A., Nathan, D. G. — Thu, 01 Oct 2009 13:28:56 PDT

Twenty-four percent of sickle cell disease (SCD) patients suffer a stroke by the age of 45. Blood transfusions decrease stroke risk in patients deemed high risk by transcranial Doppler (TCD). However, TCD has poor specificity, and transfusions are limited by alloimmunization and iron overload. Furthermore, transfusion withdrawal may be associated with an increased rebound stroke risk. Although extended blood typing decreases alloimmunization and hemolytic reactions in SCD, this practice is not universally adopted. Transfusions for thalassemia begun in early childhood are associated with lower rates of alloimmunization than are seen in SCD, suggesting a time window of immune tolerance. Optimal oxygen transport efficiency (OTE) occurs at a relatively low hematocrit (Hct) for SCD patients because of hyperviscosity. Consequently, exchange rather than simple transfusions are more effective in improving OTE, but the former are technically more demanding and require more blood units. Furthermore, though viscosity is of importance in the non-cerebral manifestations of SCD, inflammation may play a larger role than viscosity in the development of large vessel stroke. The future of SCD stroke management lies in the avoidance of transfusion. Hydroxyurea treatment and novel targeted anti-inflammatory measures may reduce the need for transfusion. The results of recent genome-wide association studies may provide an alternative avenue for modulating fetal hemoglobin production enough to attenuate stroke risk and other complications of SCD.

Human induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Thu, 01 Oct 2009 13:28:31 PDT

Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells such as postnatal fibroblasts to iPS cells that resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be re-directed to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of two patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Identification of a myeloid committed progenitor as the cancer initiating cell in acute promyelocytic leukemia

Thu, 01 Oct 2009 13:28:18 PDT

Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15:17) translocation leading to expression of the fusion protein PML-RAR. Treatment with retinoic acid leads to degradation of PML-RAR protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer "stem" cells in hematopoietic organs after treatment. Using a novel sorting strategy, we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34⁺, c-kit⁺, FcRIII/II⁺, Gr1^int) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer initiating cell. These cells downregulate the transcription factor C/EBP possibly through a methylation dependent mechanism, indicating that C/EBP deregulation contributes to transformation of APL cancer initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease.

The effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia

Wed, 30 Sep 2009 13:38:08 PDT

Elevated level of B-cell activating factor (BAFF) has been implicated in the pathogenesis of some autoimmune diseases. Blockade of receptor and ligand binding by decoy receptor has demonstrated clinical benefit in both oncological and immunological diseases. In this report, we have detected plasma BAFF and BAFF mRNA expression in immune thrombocytopenia (ITP) patients by ELISA and real time quantitative PCR (RT-PCR). The effects of recombinant human BAFF (rhBAFF) and BAFF-R-Fc fusion protein (BR3-Fc) on B cells, T cells, platelets, secretion of IFN-gamma (IFN-) and IL-4 were measured by flow cytometry and ELISA. Patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls. In in vitro assays, rhBAFF promoted the survival of CD19⁺ and CD8⁺ cells, increased the apoptosis of platelets and the secretion of IFN-. BR3-Fc successfully corrected the above effects of rhBAFF. These findings suggest that BAFF may play a pathogenic role in ITP by promoting the survival of CD19⁺ and CD8⁺ cells, increasing the apoptosis of platelets and the secretion of IFN-. Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.

In vivo delivery of a microRNA regulated transgene induces antigen-specific regulatory T cells and promotes immunological tolerance

Wed, 30 Sep 2009 13:37:57 PDT

We previously showed that incorporating target sequences for the hematopoietic-specific microRNA miR-142 into an antigen-encoding transgene prevents antigen expression in antigen presenting cells (APCs). To determine whether this approach induces immunological tolerance we treated mice with a miR-142-regulated lentiviral vector encoding GFP, and subsequently vaccinated the mice against GFP. In contrast to control mice, no anti-GFP response was observed indicating that robust tolerance to the transgene-encoded antigen was achieved. Furthermore, injection of the miR-142-regulated vector induced a population of GFP-specific regulatory T cells. Interestingly, an anti-GFP response was observed when microRNA miR-122a was inserted into the vector and antigen expression was de-targeted from hepatocytes as well as APCs. This demonstrates that, in the context of Lentiviral Vector (LV) mediated gene transfer, de-targeting antigen expression from professional APCs, coupled with expression in hepatocytes, can induce antigen-specific immunological tolerance.

Modifiers of von Willebrand factor identified by natural variation in inbred strains of mice

Shavit, J. A., Manichaikul, A., Lemmerhirt, H. L., Broman, K. W., Ginsburg, D. — Tue, 29 Sep 2009 13:32:46 PDT

Type 1 von Willebrand disease (VWD) is the most common inherited human bleeding disorder. However, diagnosis is complicated by incomplete penetrance and variable expressivity, as well as wide variation in VWF levels among the normal population. Previous work has exploited the highly variable plasma VWF levels among inbred strains of mice to identify two major regulators, Mvwf1 and 2 (modifier of VWF). Mvwf1 is a glycosyltransferase and Mvwf2 is a natural variant in Vwf which alters biosynthesis. We report the identification of an additional alteration at the Vwf locus (Mvwf5), as well as two loci unlinked to Vwf (Mvwf6-7) using a backcross approach with the inbred mouse strains WSB/EiJ and C57BL/6J. Through positional cloning, we show that Mvwf5 is a cis-regulatory variant which alters Vwf mRNA expression. A similar mechanism could potentially explain a significant percentage of human VWD cases, especially those with no detectable mutation in the VWF coding sequence. Mvwf6 displays conservation of synteny with potential VWF modifier loci identified in human pedigrees, suggesting that its ortholog may modify VWF in human populations.

Soluble lymphotoxin is an important effector molecule in GVHD and GVL

Tue, 29 Sep 2009 13:32:14 PDT

TNF is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LT3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have employed preclinical models to investigate the role of lymphotoxin in GVHD. We confirm that grafts deficient in LT have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T cell function suggesting that LT3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LT is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LT and TNF deficient grafts. Further impairment in tumor clearance was seen using TNF/LT^-/- donors, suggesting that these molecules play non-redundant roles in GVL. Importantly, donor TNF/LT were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LT3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.

MICA-129 genotype, soluble MICA and anti-MICA antibodies as biomarkers of chronic graft versus host disease

Mon, 28 Sep 2009 11:37:05 PDT

The MHC class I-related chain A (MICA) molecules exist as membrane-bound and soluble isoforms and are encoded by a polymorphic gene. Their genetic and phenotype characteristics have been studied in various pathological settings but not in the context of hematopoietic stem cell transplantation (HSCT). Here we evaluated whether MICA-related features namely MICA-129 gene polymorphism, pre- and post-HSCT serum levels of soluble MICA (sMICA) and anti-MICA antibodies (MICA Abs) could influence the incidence of chronic graft-versus-host disease (cGvHD) and relapse of their disease in 211 HLA-identical sibling pairs and in a subset of 116 recipients respectively. While the MICA-129 val/val genotype and elevated post-HSCT sMICA serum levels are independently associated with the incidence of cGvHD (P = .002 and .001) regardless of history of acute GvHD, the presence of pre-transplant MICA Abs confers protection against cGvHD (P = .04). There is an inverse relationship between MICA Abs and sMICA suggesting an antibody-based neutralization of deleterious effects of sMICA. Similarly, these genetic and phenotype characteristics of MICA influence the incidence of relapse. Altogether, these data suggest that the studied MICA genotype and phenotype specificities could be used as relevant biomarkers for cGvHD monitoring.

Allogeneic hematopoietic cell transplantation after conditioning with 131I-anti-CD45 antibody plus fludarabine and low-dose total body irradiation for elderly patients with advanced acute myeloid leukemia or high-risk myelodysplastic syndrome

Mon, 28 Sep 2009 11:36:52 PDT

We conducted a study to estimate the maximum tolerated dose (MTD) of ¹³¹I-anti-CD45 antibody (Ab; BC8) that can be combined with a standard reduced-intensity conditioning regimen before allogeneic hematopoietic cell transplantation. Fifty-eight patients over 50 years of age with advanced acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) were treated with ¹³¹I-BC8 Ab and fludarabine (FLU) plus 2 Gy total body irradiation (TBI). Eighty-six percent of patients had AML or MDS with >5% marrow blasts at the time of transplant. Treatment produced a complete remission in all patients, and all had 100% donor-derived CD3⁺ and CD33⁺ cells in the blood by day 28 after the transplant. The MTD of ¹³¹I-BC8 Ab delivered to liver was estimated to be 24 Gy. Seven (12%) patients died from non-relapse causes by day 100. The estimated probability of recurrent malignancy at 1 year is 40%, and the 1-year survival estimate is 41%. These results demonstrate that CD45-targeted radiotherapy can be safely combined with a reduced-intensity conditioning regimen to yield encouraging overall survival for older, high-risk, patients with AML or MDS. This study is registered at http://clinicaltrials.gov as NCT00008177.

Class prediction models of thrombocytosis using genetic biomarkers

Tue, 22 Sep 2009 11:56:16 PDT

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia; ET) from non-clonal (reactive thrombocytosis; RT) etiologies. We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V⁶¹⁷F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time PCR (qRT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Gender differences were rare in normal and ET cohorts (<1% of genes), but were male-skewed for ~3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the three cohorts with 86.3% accuracy, with 93.6% accuracy in two-way class prediction (ET vs. RT). Subsequent qRT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2-wild type ET in >85% patient samples using either microarray or qRT-PCR profiling, with lower predictive capacity in JAK2V⁶¹⁷F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy.

A CD44v6 peptide reveals a role of CD44 in VEGFR-2 signaling and angiogenesis

Tue, 22 Sep 2009 11:56:04 PDT

A specific splice variant of the CD44 cell surface protein family, CD44v6, has been shown to act as a co-receptor for the receptor tyrosine kinase (RTK) c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another RTK, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting and tubule formation induced by HGF or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents suggesting that the co-receptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathological conditions.

Interim positron emission tomography (PET) scans in diffuse large B-cell lymphoma: an independent expert nuclear medicine evaluation of the Eastern Cooperative Oncology Group E3404 study

Fri, 18 Sep 2009 12:02:43 PDT

Positive interim-PET scans are thought to be associated with inferior outcomes in diffuse large B-cell lymphoma (DLBCL). In the E3404 DLBCL study, PET scans at baseline and after 3 R-CHOP were centrally reviewed by a single reader. To determine reproducibility of interim PET interpretation, an expert panel of 3 external nuclear medicine physicians visually scored baseline and interim-PET scans independently and blinded to clinical information. The binary ECOG study criteria were based on modifications of the Harmonization Criteria; the London criteria were also applied. Of 38 interim scans, agreement was complete in 68% and 71% by ECOG and London criteria, respectively. The range of PET+ interim scans was 16% to 34% (p=NS) by reviewer. Moderate consistency of reviews was observed: kappa statistic 0.445 using ECOG and 0.502 using London criteria. These data, showing only moderate reproducibility among nuclear medicine experts, indicate the need to standardize PET interpretation in research and practice. This trial has been registered on http://www.clinicialtrials.gov as NCT00274924.

TGFBIp/{beta}ig-h3 activates platelets and promotes thrombogenesis

Tue, 08 Sep 2009 11:54:27 PDT

TGFBIp/βig-h3 is a 68 kDa extracellular matrix protein that is functionally associated with the adhesion, migration, proliferation, and differentiation of various cells. The presence of TGFBIp in platelets led us to study the role of this protein in the regulation of platelet functions. Upon activation, platelet TGFBIp was released and associated with the platelets. TGFBIp mediates not only the adhesion and spread of platelets, but also activates them, resulting in phosphatidylserine exposure, -granule secretion, and increased integrin affinity. The FAS1 domains of TGFBIp are mainly responsible for the activation of platelets. TGFBIp promotes thrombus formation on type I fibrillar collagen under flow conditions in vitro, and induces pulmonary embolism in mice. Moreover, transgenic mice, which have about 1.7-fold higher blood TGFBIp concentration, are significantly more susceptible to collagen- and epinephrine-induced pulmonary embolism than wild-type mice. These results suggest that TGFBIp, a human platelet protein, plays important roles in platelet activation and thrombus formation. Our findings will increase our understanding of the novel mechanism of platelet activation, contributing to a better understanding of thrombotic pathways and the development of new antithrombotic therapies.

Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the eye?

Pullarkat, V. — Wed, 26 Aug 2009 14:42:10 PDT

The role of iron chelation therapy in MDS remains controversial. Averting cardiac dysfunction in low-grade MDS patients who have sufficient longevity to experience deleterious cardiac effects of iron overload has been the major argument in favor of iron chelation. Although there is significant evidence showing the adverse impact of transfusion dependency on survival in MDS, direct evidence linking tissue iron overload to poor survival or in particular to cardiac dysfunction is lacking. Given the heterogeneity of MDS, it is likely that the pathophysiology of iron overload is equally heterogeneous and complex in these patients. In this article, I argue that prevention of cardiac dysfunction in patients with lower grades of MDS may not be the major benefit of iron chelation therapy, and present evidence suggesting a potential benefit of iron chelation on three other outcomes namely (1) lowering infection risk, (2) improving the outcome of allogeneic hematopoietic stem cell transplantation and (3) delaying leukemic transformation. These outcomes have particular relevance for patients with higher grades of MDS and should be evaluated in future prospective clinical trials that include patients with all grades of MDS in order to fully evaluate the benefit of iron chelation therapy.

The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model

Fri, 07 Aug 2009 14:34:54 PDT

Despite the fact that NPM1 gene mutations leading to aberrant cytoplasmic expression of nucleophosmin (NPMc+) are the most frequent genetic lesions in AML, there is yet no experimental model demonstrating their oncogenicity in vivo. We report the generation and characterization of a transgenic mouse model expressing the most frequent human NPMc+ mutation driven by the myeloid-specific human MRP8 promoter (hMRP8-NPMc+). In parallel, we generated a similar wild-type NPM transgenic model (hMRP8-NPM). Interestingly, hMRP8-NPMc+ transgenic mice developed myeloproliferation in bone marrow and spleen, while non-transgenic littermates and hMRP8-NPM transgenic mice remained disease free. These findings provide the first in vivo evidence indicating that NPMc+ confers a proliferative advantage in the myeloid lineage. No spontaneous AML was found in hMPR8-NPMc+ or hMRP8-NPM mice. This model will also aid in the development of therapeutic regimens that specifically target NPMc+.