BLOOD First Edition Papers

Cytokine storm in a mouse model of IgG-mediated hemolytic transfusion reactions

2008-05-15

Cytokines are hypothesized to play a central role in the pathophysiology of IgG-mediated hemolytic transfusion reactions (HTRs), and deeper understanding is required for improving therapy for these events. After establishing well-defined mouse models of HTRs, we tested whether cytokines were involved. Red blood cells (RBCs) from human glycophorin A transgenic (hGPA-Tg) or wild-type (WT) mice were transfused into non-Tg recipients passively immunized with monoclonal antibodies (Mabs). Only transfusions of incompatible RBCs induced IgG-mediated HTRs, exemplified by rapid clearance and hemoglobinuria. Very high plasma levels of monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-6, and lower levels of tumor necrosis factor- (TNF-), were induced after incompatible transfusion. No significant changes in IL-10, IL-12, or interferon- (IFN-) levels were observed. The proinflammatory cytokines elaborated in this in vivo mouse model are also implicated in the systemic inflammatory response syndrome (SIRS) and confirm the hypothesis that cytokine storm occurs as a result of HTRs.

Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma

2008-05-15

Cyclin D1 (CCND1) is a well known regulator of cell cycle progression. It is overexpressed in several types of cancer including breast, lung, squamous, neuroblastoma, and lymphomas. The most well known mechanism of overexpression is the t(11;14)(q13;q32) translocation found in mantle cell lymphoma (MCL). Weistner et al¹ have shown that truncated CCND1 mRNA in MCL correlates with poor prognosis. We hypothesized that truncations of the CCND1 mRNA alters its ability to be down regulated by microRNAs in MCL. MicroRNAs are a new class of abundant small RNAs that play important regulatory roles at the post transcriptional level by binding to the 3' untranslated region (UTR) of mRNAs blocking either their translation or initiating their degradation. In this study, we have identified the truncation in CCND1 mRNA in MCL cell lines. We also found that truncated CCND1 mRNA leads to increased CCND1 protein expression and increased S-phase cell fraction. Furthermore, we demonstrated that this truncation alters miR-16-1 binding sites, and through the use of reporter constructs, we were able to show that miR-16-1 regulates CCND1 mRNA expression. This study introduces the role of miR-16-1 in the regulation of CCND1 in MCL.

Primary cystic lung light chain deposition disease : a clinicopathologic entity derived from unmutated B cells with a stereotyped IGHV4-34/IGKV1 receptor

2008-05-15

We have recently described a new form of light chain deposition disease (LCDD) presenting as a severe cystic lung disorder requiring lung transplantation. There was no bone marrow plasma cell proliferation. Because of the absence of disease recurrence after bilateral lung transplantation and of serum free light chain ratio normalization after the procedure, we hypothesized that monoclonal light chain synthesis occurred within the lung. The aim of this study was to look for the monoclonal B-cell component in three patients with cystic lung LCDD. Histological examination of the explanted lungs showed diffuse non-amyloid light chain deposits associated with a mild lymphoid infiltrate mainly composed of small CD20+, CD5-, CD10- B lymphocytes arranged in nodules reminiscent of bronchus-associated lymphoid tissue. Using PCR, we identified a dominant B-cell clone in the lung in the three studied patients. The clonal expansion of each patient shared a unmutated antigen receptor variable region sequence characterized by the use of VH4-34 and VK1 gene family with heavy and light chain CDR3 sequences of more than 80% amino acid identity, a feature evocative of an antigen-driven process. Combined with clinical and biological data, our results strongly argue for a new antigen-driven primary pulmonary lymphoproliferative disorder.

Angiopoietin-2 predicts disease-free survival after allogeneic stem-cell transplantation in patients with high-risk myeloid malignancies

2008-05-15

Emerging data suggest a critical role for bone marrow (BM) angiogenesis in hematologic malignancies. The Angiopoietin/Tie ligand-receptor system is an essential regulator of this process. We evaluated whether circulating Angiopoietin-2 (Ang-2) is a predictor for the probability of disease-free survival (DFS) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for high-risk AML or MDS. Ang-2 was measured by ELISA in serum from 20 healthy controls and 90 patients with AML or MDS before initiation of combined cytoreduction and reduced-intensity conditioning for HSCT. Circulating Ang-2 was elevated in patients (median (range): 2.21 (0.18-48.84) ng/ml) compared to controls (0.87 (0.27-4.51) ng/ml; p<0.0001). Multivariate analyses confirmed the independent prognostic impact of Ang-2 (hazard ratio 2.46, 95% CI 1.27-4.76, p = 0.005), percentage of BM infiltration (HR 1.14, 95% CI 1.01-1.29, p = 0.033), and chemotherapy cycles prior to HSCT (HR 1.38, 95% CI 1.01-1.08, p = 0.048). Regression tree analysis detected optimal cut-off values for Ang-2, and recursively identified BM blasts and Ang-2 as the best predictors for DFS. As few predictors for DFS exist in the setting of allo-HSCT, Ang-2 may be used as a readily available biomarker to pre-estimate DFS and may open new perspectives for risk-adapted treatment of high-risk myeloid malignancies.

Identification of a novel type of ITD mutations located in non-juxtamembrane domains of the FLT3 tyrosine kinase receptor

2008-05-15

In AML, internal tandem duplications (ITDs) of the juxtamembrane (JM) of FLT3 have been demonstrated to play a crucial role in driving proliferation and survival of the leukemic clone. Here, we report the identification of FLT3_ITD mutations located in non-JM domains of the FLT3-receptor. This novel type of FLT3_ITD mutations was found in 216 of 753 (28.7%) of unselected FLT3_ITD-positive AML cases. An FLT3 receptor harbouring a prototypic non-JM ITD (FLT3_ITD627E) mediated constitutive phosphorylation of FLT3 and of STAT5 suggesting that non-JM ITDs confer constitutive activation of the receptor. FLT3_ITD627E induced transformation of hematopoietic 32D cells and led to a lethal myeloproliferative disease in a syngeneic mouse model. Our results indicate that a significant proportion of activating FLT3_ITD mutations is not confined to the JM-domain of FLT3. Further studies are warranted to define the biological and clinical characteristics of non-JM ITDs.

Regulation of D6 chemokine scavenging activity by ligand and Rab11-dependent surface upregulation

2008-05-14

The decoy receptor D6 plays a non-redundant role in the control of inflammatory processes through scavenging of inflammatory chemokines. However it remains unclear how it is regulated. Here we show that D6 scavenging activity relies on unique trafficking properties. Under resting conditions, D6 constitutively recycled through both a rapid wortmannin (WM)-sensitive and a slower brefeldin A (BFA)-sensitive pathway maintaining low levels of surface expression that required both Rab4 and Rab11 activities. In contrast to "conventional" chemokine receptors that are downregulated by cognate ligands, chemokine engagement induced a dose-dependent BFA-sensitive Rab11-dependent D6 redistribution to the cell membrane and a corresponding increase in chemokine degradation rate. Thus, the energy-expensive constitutive D6 cycling through Rab11 vesicles allows a rapid, ligand concentration-dependent, increase of chemokine scavenging activity by means of receptor redistribution to the plasma membrane. D6 is not regulated at a transcriptional level in a variety of cellular contexts, thus ligand-dependent optimization of its scavenger performance represents a rapid and unique mechanism allowing D6 to control inflammation.

Resolvin E1, an EPA-derived mediator in whole blood, selectively counterregulates leukocytes and platelets

2008-05-14

Resolvin E1 (RvE1) is an omega-3 eicosapentaenoic acid (EPA)-derived lipid mediator generated during resolution of inflammation and in human vasculature via leukocyte-endothelial cell interactions. RvE1 possesses anti-inflammatory and pro-resolving actions. Here, we report that RvE1 in human whole blood rapidly regulates leukocyte expression of adhesion molecules. RvE1 in the 10-100 nM range stimulated L-selectin shedding, while reducing CD18 expression in both neutrophils and monocytes. When added to whole blood, RvE1 did not stimulate reactive oxygen species by either neutrophils or monocytes, nor did it directly stimulate cytokine/chemokine production in heparinized blood. Intravital microscopy (IVM) demonstrated that RvE1 rapidly reduced leukocyte rolling (~40%) in venules of mice. In human platelet-rich plasma (PRP), RvE1 selectively blocked both ADP-stimulated and thromboxane receptor agonist U46619-stimulated platelet aggregation in a concentration-dependent manner. In contrast, 6,14-trans-RvE1 isomer was inactive. RvE1 did not block collagen-stimulated aggregation, and regulation of ADP-induced platelet aggregation was not further enhanced with aspirin treatment. These results indicate RvE1 is a potent modulator of leukocytes as well as selective platelet responses in blood and PRP, respectively. Moreover, they demonstrate novel agonist-specific anti-platelet actions of RvE1 that are potent and may underlie some of the beneficial actions of EPA in humans.

The incidence of and risk factors for venous thromboembolism (VTE) and bleeding among 1,514 patients undergoing hematopoietic stem cell transplantation: implications for VTE prevention

Gerber, D. E, Segal, J. B, Levy, M. Y., Kane, J., Jones, R. J., Streiff, M. B — 2008-05-14

Venous thromboembolism (VTE) is increasingly diagnosed among individuals with hematologic malignancies. However, the risk of VTE among patients undergoing hematopoietic stem cell transplantation (HSCT) is unclear. We examined the incidence and risk factors for VTE and bleeding among 1,514 patients undergoing inpatient HSCT. No protocolized VTE prophylaxis was used. By HSCT Day 180, 75 symptomatic VTE events occurred in 70 patients (4.6%; 95% CI, 3.6-5.8%). Fifty-five (3.6%) were catheter-associated, 11 (0.7%) were non-catheter-associated deep venous thromboses, and 9 (0.6%) were pulmonary emboli. Thirty-four percent of VTE events occurred at a platelet count less than 50 K/mm³; 13% occurred at a platelet count less than 20 K/mm³. In multivariate analysis, VTE was associated with prior VTE (OR 2.9; 95% CI, 1.3-6.6) and with graft-versus-host-disease (GVHD) (OR 2.4; 95% CI, 1.4-4.0). Clinically significant bleeding occurred in 230 patients (15.2%; 95% CI, 13.4%-17.1%); 55 patients (3.6%; 95% CI, 2.7%-4.7%) had fatal bleeding. Bleeding was primarily associated with anticoagulation (OR 3.1; 95% CI, 1.8-5.5), GVHD (OR 2.4; 95% CI, 1.8-3.3), and veno-occlusive disease (OR 2.2; 95% CI, 1.4-3.6). In HSCT patients, VTE is primarily catheter-related and three-fold less common than clinically significant bleeding. These findings warrant consideration when selecting VTE prophylaxis in HSCT patients.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in {beta}-thalassemia

2008-05-14

In β-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by β-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared to normal mice. Therefore, we sought to determine if IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, Ki-67 and the anti-apoptotic protein Bcl-X_L. They also differentiated less that normal erythroid ones in vitro. In order to investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to normal and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. While our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Dasatinib crosses the blood-brain barrier and is an efficient therapy for central nervous system Philadelphia chromosome-positive leukemia

2008-05-13

Although imatinib, a BCR-ABL tyrosine kinase inhibitor, is used to treat acute Philadelphia chromosome-positive (Ph+) leukemia, it does not prevent central nervous system (CNS) relapses due to poor drug penetration through the blood-brain barrier. Imatinib and dasatinib (a dual-specific SRC/BCR-ABL kinase inhibitor) were compared in a preclinical mouse model of intracranial Ph+ leukemia. Clinical dasatinib treatment in patients with CNS Ph+ leukemia was assessed. In preclinical studies, dasatinib increased survival, while imatinib failed to inhibit intracranial tumor growth. Stabilization and regression of CNS disease was achieved with continued dasatinib administration. The drug also demonstrated substantial activity in 11 adult and pediatric patients with CNS Ph+ leukemia. Eleven evaluable patients had clinically significant, long-lasting responses, which were complete in seven patients. In three additional patients, isolated CNS relapse occurred during dasatinib therapy and in two of them it was caused by expansion of a BCR-ABL-mutated dasatinib-resistant clone, implying selection pressure exerted by the compound in the CNS. Dasatinib has promising therapeutic potential in managing intracranial leukemic disease, and substantial clinical activity in patients who experience CNS relapse while on imatinib therapy. This study is registered at ClinicalTrials.gov as CA180006 (NCT00108719) and CA180015 (NCT00110097).

Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes

2008-05-13

T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3+ CD8+ cells. Leukemic LGL correspond to terminally differentiated effector-memory cytotoxic T-lymphocytes (CTL) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from thirty LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGL. Pathway based microarray analysis indicated that balance of pro-apoptotic and anti-apoptotic sphingolipid mediated signaling was deregulated in leukemic LGL. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGL and that its inhibition induced apoptosis of leukemic LGL. We also showed that S1P₅ is the predominant S1P-receptor in leukemic LGL, while S1P₁ is downregulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGL and also sensitized leukemic LGL to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTL. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Vascular dysfunction in a murine model of severe hemolysis

2008-05-13

Spectrin is the backbone of the erythroid cytoskeleton; sph/sph mice have severe hereditary spherocytosis (HS) due to a mutation in the murine erythroid -spectrin gene. sph/sph mice have a high incidence of thrombosis and infarction in multiple tissues, suggesting significant vascular dysfunction. In the current study, we provide evidence for both pulmonary and systemic vascular dysfunction in sph/sph mice. We found increased levels of soluble cell adhesion molecules in sph/sph mice, suggesting activation of the vascular endothelium. We hypothesized that plasma hemoglobin released by intravascular hemolysis initiates endothelial injury through nitric oxide (NO) scavenging and oxidative damage. In agreement, electron paramagnetic resonance spectroscopy showed that plasma hemoglobin is much greater in sph/sph mice. Moreover, plasma from sph/sph mice had significantly higher oxidative potential. Finally, xanthine oxidase, a potent superoxide generator, is decreased in subpopulations of liver hepatocytes and increased on liver endothelium in sph/sph mice. These results indicate that vasoregulation is abnormal, and NO-based vasoregulatory mechanisms particularly impaired, in sph/sph mice. Together, these data indicate that sph/sph mice with severe HS have increased plasma hemoglobin and NO scavenging capacity, likely contributing to aberrant vasoregulation and initiating oxidative damage.

Inhibition of the TGF-{beta} receptor I kinase promotes hematopoiesis in MDS

2008-05-12

MDS is characterized by ineffective hematopoiesis that leads to peripheral cytopenias. Development of effective treatments has been impeded by limited insight into pathogenic pathways governing dysplastic growth of hematopoietic progenitors. We demonstrate that smad2, a downstream mediator of TGF-β receptor I kinase (TBRI) activation, is constitutively activated in MDS bone marrow (BM) precursors and is overexpressed in gene expression profiles of MDS CD34+ cells, providing direct evidence of overactivation of TGF-β pathway in this disease. Suppression of the TGF-β signaling by lentiviral shRNA mediated downregulation of TBRI leads to in vitro enhancement of hematopoiesis in MDS progenitors. Pharmacological inhibition of TBRI (alk5) kinase by a small molecule inhibitor, SD-208, inhibits smad2 activation in hematopoietic progenitors, suppresses TGF-β mediated gene activation in BM stromal cells and reverses TGF-β mediated cell cycle arrest in BM CD34+ cells. Furthermore, SD-208 treatment alleviates anemia and stimulates hematopoiesis in vivo in a novel murine model of bone marrow failure generated by constitutive hepatic expression of TGF-β1. Moreover, in vitro pharmacologic inhibition of TBRI kinase leads to enhancement of hematopoiesis in varied morphologic MDS subtypes. These data directly implicate TGF-β signaling in the pathobiology of ineffective hematopoiesis and identify TBRI as a potential therapeutic target in low risk MDS.

Constitutive expression of IL-12RB2 on human multiple myeloma cells delineates a novel therapeutic target

2008-05-12

The IL-12 receptor(R)B2 gene acts as tumor suppressor in human acute and chronic B cell leukemias/lymphomas and IL-12rb2 deficient mice develop spontaneously localized plasmacytomas. With this background, we have investigated the role of IL-12RB2 in multiple myeloma (MM) pathogenesis. Here we show that i) IL12Rβ2 was expressed in primary MM cells, but downregulated in comparison with normal polyclonal plasmablastic cells (PPC) and plasma cells (PC). IL-6 dampened IL-12Rβ2 expression on PPC and MM cells, and ii) IL-12 reduced the pro-angiogenic activity of primary MM cells in vitro and decreased significantly (P=0.0001) the tumorigenicity of the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The latter phenomenon was found to depend on abolished expression of a wide panel of pro-angiogenic genes and up-regulated expression of the antiangiogenic genes IFN-, IFN-, platelet factor-4 and TIMP-2. Inhibition of the angiogenic potential of primary MM cells was related to down-regulated expression of the pro-angiogenic genes CCL11, vascular endothelial-cadherin, CD13 and AKT and to up-regulation of an IFN- related anti-angiogenic pathway. Thus, IL-12Rβ2 restrains directly MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy.

TGF-{beta} signaling in thymic epithelial cells regulates thymic involution and post-irradiation reconstitution

2008-05-12

The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiological process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-β signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiological process of thymic senescence is mitigated in mice deficient for the expression of TGF-βRII on thymic epithelial cells. Moreover, TGF-β signaling in these stromal cells transiently hinders the early phase of thymic reconstitution following myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-β signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis following hematopoietic stem cell transplantation.

Role of VEGF-D and VEGFR-3 in developmental lymphangiogenesis, a chemicogenetic study in Xenopus tadpoles

2008-05-12

The importance of the lymphangiogenic factor VEGF-D and its receptor VEGFR-3 in early lymphatic development remains largely unresolved. We therefore investigated their role in Xenopus laevis tadpoles, a small animal model allowing chemicogenetic dissection of developmental lymphangiogenesis. Single morpholino antisense oligo knockdown of VEGF-D did not affect lymphatic commitment, but transiently impaired lymphatic endothelial cell (LEC) migration. Notably, combined knockdown of VEGF-D with VEGF-C at suboptimal morpholino concentrations, resulted in more severe migration defects and lymphedema formation than the corresponding single knockdowns. Knockdown of VEGFR-3 or treatment with the VEGFR-3 inhibitor MAZ51 similarly impaired lymph vessel formation and function and caused pronounced edema. VEGFR-3 silencing by morpholino knockdown, MAZ51 treatment, or VEGF-C/D double knockdown also resulted in dilation and dysfunction of the lymph heart. These findings document a critical role of VEGFR-3 in embryonic lymphatic development and function, and reveal a previously unrecognized modifier role of VEGF-D in the regulation of embryonic lymphangiogenesis in frog embryos.

Dynamics of factor VIII interactions determine its immunological fate in hemophilia A

2008-05-09

Pro-coagulant factor VIII (FVIII) is either produced endogenously under physiological conditions, or administered exogenously as a therapeutic hemostatic drug in patients with hemophilia A. In the circulation, FVIII interacts with a multitude of glycoproteins, and may be used for coagulation at the sites of bleeding, eliminated by scavenger cells or be processed by the immune system, either as a self-constituent or as a foreign antigen. The fate of FVIII is dictated by the immune status of the individual, the location of FVIII in the body at a given time-point, and the inflammatory microenvironment. It also depends on the local concentration of FVIII and of each interacting partner, and on the affinity of the respective interactions. FVIII, by virtue of its promiscuity, thus constitutes the core of a dynamic network that links the coagulation cascade, cells of the immune system and, presumably, the inflammatory compartment. We describe the different interactions that FVIII is prone to establish during its life cycle, with a special focus on players of the innate and adaptive immune response. Lessons can be learned from understanding the dynamics of FVIII interactions; lessons that should pave the way to the conception of long-lasting hemostatic drugs devoid of iatrogenic immunogenicity.

Is allergic disease curable or transferable with allogeneic hematopoietic cell transplantation?

Khan, F., Hallstrand, T. S, Geddes, M. N, Henderson, W. R, Storek, J. — 2008-05-09

In the pathogenesis of allergic disease, two main types of cells play a role: hematolymphatic cells (eosinophils, T cells, B cells) and nonhematolymphatic cells (airway smooth muscle cells, epithelial cells). It is not known which one of the two cell types plays the primary role. Here we review the literature on allergy transfer and cure with allogeneic hematopoietic cell transplantation (HCT), as transferability and curability would support the primary role of hematolymphatic cells, which would have implications for donor selection for HCT and possible future treatment of severe allergic disease with HCT. A total of 16 nonallergic recipients with allergic donors were reported to develop allergic disease posttransplant, however, conclusive information was available for only 5 cases. Allergic disease was reported to abate in 3 allergic recipients with nonallergic donors, however, conclusive information was available for only 2 cases. Problems in interpreting the reports include incomplete data on allergic disease in the donor or recipient pretransplant, not knowing the denominator, and the lack of controls. In summary, review of the literature generates the hypothesis that allergic disease is transferable and curable with HCT. A prospective study, including appropriate controls, is needed to evaluate this hypothesis.

Co-activator function of RIP140 for NF{kappa}B/RelA-dependent cytokine gene expression

2008-05-09

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected co-activator function for the nuclear receptor interacting protein (RIP) 140 for nuclear factor (NF) B, a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that RIP140 suppresses the expression of metabolic gene networks but we have found that genetic as well as acute deficiency of RIP140, leads to the inhibition of the pro-inflammatory program in macrophages. The ability of RIP140 to function as a coactivator for cytokine gene promoter activity relies on direct protein-protein interactions with the NFB subunit RelA and histone acetylase CREB-binding protein (CBP). RIP140-dependent control of pro-inflammatory gene expression via RelA/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.

Down-regulation of TCF8 is involved in the leukemogenesis of adult-T cell leukemia/lymphoma

2008-05-08

Adult T-cell leukemia/lymphoma (ATLL) is caused by latent human T-lymphotropic virus-1 (HTLV-1) infection. To clarify the molecular mechanism underlying leukemogenesis after viral infection, we precisely mapped 605 chromosomal breakpoints in 61 ATLL cases by spectral karyotyping and identified frequent chromosomal breakpoints in 10p11, 14q11, and 14q32. Single nucleotide polymorphism (SNP) array-comparative genomic hybridization (CGH), genetic, and expression analyses of the genes mapped within a common breakpoint cluster region in 10p11.2 revealed that in ATLL cells, transcription factor 8 (TCF8) was frequently disrupted by several mechanisms, mainly including epigenetic dysregulation. TCF8-mutant mice frequently developed invasive CD4⁺ T-cell lymphomas in the thymus or in ascitic fluid in vivo. Down-regulation of TCF8 expression in ATLL cells in vitro was associated with resistance to transforming growth factor (TGF)-β1, a well-known characteristic of ATLL cells, suggesting that escape from TGF-β1-mediated growth inhibition is important in the pathogenesis of ATLL. These findings indicate that TCF8 has a tumor suppressor role in ATLL.

How I treat acquired factor VIII inhibitors

Franchini, M., Lippi, G. — 2008-05-07

Acquired hemophilia A is a rare bleeding diathesis caused by autoantibodies directed against clotting factor VIII and associated with an increased morbidity and mortality. This autoimmune disorder most commonly occurs in the elderly. Although it may be associated with several underlying pathologies, up to 50% of reported cases remain idiopathic. In contrast with congenital hemophilia, which is commonly characterized by hemarthroses, hemorrhages in patients with acquired hemophilia involve most frequently soft tissues. The two treatment priorities are to arrest the acute bleeding and to eradicate the factor VIII autoantibody. Acute bleeding episodes in patients with low-titer inhibitors can be treated using human factor VIII concentrates, whereas factor VIII bypassing agents, such as activated prothrombin complex concentrates or recombinant activated factor VII, are effective for the treatment of those with high-titer inhibitors. An analysis of the literature shows that the most effective first-line treatment for the eradication of factor VIII autoantibodies is the combination of steroids and cyclophosphamide. However, there is increasing evidence on the effectiveness of other treatment approaches, such as immune tolerance regimens and rituximab. If confirmed by large controlled studies, these innovative therapies might become a valid option for long-term eradication of factor VIII inhibitors.

Rituximab efficacy and safety in adult splenectomy candidates with chronic immune thrombocytopenic purpura - results of a prospective multicenter phase 2 study

2008-05-07

Whether rituximab could effectively and safely avoid splenectomy for adults with chronic immune thrombopenic purpura (ITP) remains unresolved. A multicenter, prospective, open-label, single-arm, phase 2 trial was conducted to assess rituximab safety and efficacy in adult splenectomy candidates with chronic ITP. Sixty patients with chronic (≥6 months) ITP and platelet counts <30x10⁹/L received a weekly intravenous infusion of rituximab (375 mg/m²) for 4 weeks. All other ITP treatments were stopped. A good response was defined as a platelet count ≥50x10⁹/L, with at least a doubling of the initial value at 1 and 2 years after the first rituximab infusion. Patients who required another treatment during follow-up were considered nonresponders. Sixteen patients experienced transient side effects that necessitated treatment discontinuation for only 1. Good 1-year responses were obtained in 40% of the patients (24/60 [95% confidence interval: 28-52%]). At 2 years, 33.3% (20/60 patients) had good responses and 6.7% (4/60) has sustained platelet counts ≥30x10⁹/L without treatment. Thirty-six (60%) patients failed to respond; 25 of them underwent splenectomy. Based on these results, rituximab was an apparently safe and effective splenectomy-avoiding option in some adults with chronic ITP. This trial is registered at http://clinicaltrials.gov as NCT00225875.

Interleukin-3 (IL-3) promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation

2008-05-06

Interleukin-3 (IL-3) released by infiltrating inflammatory cells in different pathological settings contributes to organ and tumor angiogenesis. Herein we demonstrate that IL-3 expands a subset of CD45+ circulating angiogenic cells clonally derived from the hemopoietic progenitors. Moreover, CD45+ cells exposed to IL-3 acquire arterial specification and directly contribute to the formation of vessels in vivo. Depletion of STAT5 provides evidences that IL-3-mediated cell expansion and arterial morphogenesis rely on STAT5 activation. In addition, by means of Tie2-transgenic mice we demonstrate that STAT5 also regulates IL-3-induced expansion and arterial specification of bone-marrow-derived CD45+ cells. Thus, our data provide the first evidence that, in inflammatory microenvironment containing IL-3, angiogenic cells derived from hemopoietic precursors can act as adult vasculogenic cells. Moreover, the characterization of the signalling pathway regulating these events provides the rational for therapeutically targeting STAT5 in these pathological settings.

Osteolineage niche cells initiate hematopoietic stem cell mobilization

Mayack, S. R, Wagers, A. J — 2008-05-02

Recent studies using gene-targeted and hormone-treated mice have implicated bone-lining osteoblasts as important regulators of hematopoietic stem cell (HSC) self-renewal and differentiation; however, as much of the evidence supporting this notion derives from indirect in vivo experiments, which are unavoidably complicated by the presence of other cell types within the complex bone marrow milieu, the sufficiency of osteoblasts in modulating HSC activity has remained controversial ¹. To address this issue, we prospectively isolated mouse osteoblasts, using a novel, flow cytometry-based approach, and directly tested their activity as HSC niche cells and their role in cyclophosphamide/G-CSF (Cy/G) induced HSC proliferation and mobilization. We found that osteoblasts expand rapidly following Cy/G treatment and exhibit significant phenotypic and functional changes that directly influence their ability to support HSC proliferation and maintenance of reconstituting potential. Furthermore, these effects of mobilization on osteoblast number and function depend on the function of ATM, the product of the ataxia telangiectasia mutated (Atm) gene, demonstrating a new role for this kinase in supporting stem cell niche activity. Taken together, these studies demonstrate that signals from osteoblasts can directly initiate and modulate HSC proliferation in the context of mobilization. More broadly, this work establishes that direct interaction with the osteolineage niche cell itself - in the absence of additional environmental inputs - is sufficient to modulate stem cell activity.

Idiopathic CD4+ lymphocytopenia: natural history and prognostic factors

2008-05-02

Idiopathic CD4+ lymphocytopenia (ICL) is a rare non-HIV related syndrome with unclear natural history and prognosis. This prospective natural history cohort study describes the clinical course, CD4 T lymphocyte kinetics, outcome and prognostic factors of ICL. Thirty nine patients (17 men, 22 women), 25 to 85 years old with ICL were evaluated between 1992 and 2006 and thirty six were followed for a median of 49.5 months. Cryptococcal and non-tuberculous mycobacterial infections were the major presenting opportunistic infections. Seven patients presented with no infection. In thirty two, CD4 T cell counts remained below 300/mm³ throughout the study period and in seven normalized after an average of 31 months. Overall, fifteen (41.6%) developed an opportunistic infection in follow up, five (13.8%) of which were "AIDS defining clinical conditions", and four (11.1%) developed autoimmune diseases. Seven patients died, four from ICL related opportunistic infections within 42 months after diagnosis. Immunologic analyses revealed increased activation and turnover in CD4 but not CD8 T lymphocytes. CD8 T lymphocytopenia (<180/mm³) and the degree of CD4 T cell activation (measured by HLA-DR expression) at presentation were associated with adverse outcome (Opportunistic infection related death) (p=0.003 and 0.02 respectively). This trial is registered at http://clinicaltrials.gov as NCT00001319.

MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort

2008-05-01

Activating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET) but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA and identify four different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific PCR was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F-negative patients and a single V617F-positive patient. Patients carrying the W515K allele had a significantly higher allele burden than those with the W515L allele, suggesting a functional difference between the two variants. Compared to V617F-positive ET patients, those with MPL mutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth and reduced bone marrow erythroid and overall cellularity. Compared to V617F-negative patients, those with MPL mutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinico-pathological subgroup. MPL mutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

Increased risks of polycythemia vera, essential thrombocythemia, and myelofibrosis among 24577 first-degree relatives of 11039 patients with myeloproliferative neoplasms in Sweden

2008-05-01

Previous small studies have reported familial clustering of myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). We identified 6,217 PV, 2,838 ET, 1,172 MF, and 812 MPN unclassifiable (NOS) patients diagnosed in Sweden, 43,550 controls, and first-degree relatives of cases (n=24,577) and controls (n=99,542). Using a marginal survival model we calculated relative risks (RR) and 95% confidence intervals as measures of familial aggregation. Relatives of MPN patients had significantly increased risks of PV (RR=5.7; 3.5-9.1), ET (RR=7.4; 3.7-14.8), and MPN NOS (RR=7.5; 2.7-20.8). Analyses stratified by type of first-degree relative revealed consistently higher risks for siblings compatible with a model of recessive genetic inheritance, although this can only be confirmed by identifying the actual susceptibility gene(s). Mean age at MPN diagnosis was not different (p=0.20, t-test) for affected relatives of cases (57.5 years) versus controls (60.6 years) and risk of MPN by age was not different for parents versus offspring of MPN cases (p=0.10, log-rank test) providing no support for anticipation. Relatives of MPN patients had a borderline increased risk of chronic myeloid leukemia (CML) (RR=1.9; 0.9-3.8; p=0.09). Our findings of 5- to 7-fold elevated risk of MPNs among first-degree relatives of MPN patients support the hypothesis that there are common, strong, shared susceptibility genes predisposing to PV, ET, MF and possibly CML.

ATF4-dependent transcription is a key mechanism in VEGF upregulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response

2008-05-01

We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators such as VEGF. We now address the pathways mediating upregulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR upregulated VEGF, while inhibition of UPR by chemical chaperones or knock-down of co-chaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of transcription factor ATF4, an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGF gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating upregulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathological angiogenesis in atherosclerotic plaques.

The microenvironment for erythropoiesis is regulated by HIF-2{alpha} through VCAM-1 in endothelial cells

2008-05-01

Erythropoiesis is a dynamic process regulated by oxygen in vertebrates. Recent evidence has indicated that erythropoietin (Epo) expression is regulated by hypoxia-inducible transcription factors (HIFs), HIF-2 in particular. In this study, we report that knockdown mutation of HIF-2 in mice (kd/kd) results in normocytic anemia, despite Epo induction in response to hypoxia not being severely affected. Transplantation analyses clearly demonstrated that the hematopoietic microenvironment, but not the hematopoietic cells, was altered in kd/kd. Furthermore, cell-type specific recovery of HIF-2 expression in endothelial cells (EC) abrogated the anemic condition of the kd/kd mice, indicating that HIF-2 in EC plays an essential role in supporting erythropoiesis. In the absence of HIF-2, the expression of vascular adhesion molecule-1 (VCAM-1) was reduced significantly and restoration of VCAM-1 expression in kd/kd EC enhanced the development of erythroid progenitors. Finally, a chromatin immunoprecipitation assay and a reporter assay indicated that VCAM-1 gene transcription is directly regulated by HIF-2. These data suggest that the hematopoietic microenvironment required for erythropoiesis is dynamically regulated by oxygen through the functions of HIF-2 in EC.

Reduced-toxicity conditioning with fludarabine, BCNU and melphalan in allogeneic hematopoietic cell transplantation: particular activity against advanced hematological malignancies

2008-05-01

Toxicity reduced conditioning is being used for allogeneic stem cell transplantation in older and/or comorbid patients. Here we report on the treatment of 133 patients (median age: 55.6 [23-73 years]) with AML/MDS (n=81), ALL (n=3), CML/MPS (n=20), NHL/CLL/MM (n=29) using the FBM preparative regimen: fludarabine (5x30 mg/m²), BCNU (2x200 mg/m²), melphalan (140 mg/m²). Patients ≥ 55 years received fludarabine with reduced BCNU (2x150 mg/m²) and melphalan (110 mg/m²). After engraftment, chimerism analyses revealed complete donor hematopoiesis in 95.7% of patients. With a median follow-up of 58.5 months, 3- and 5- year overall survival (OS) was 53.0% and 46.1%, event free survival (EFS) was 46.4% and 41.9%. No significant differences in OS and EFS were evident considering disease status (early v. advanced), patient age (<55 v. ≥ 55 years), or donor (related v. unrelated) in uni- and multivariate analyses. The cumulative incidence of death due to relapse was 20.1% at 5 years. Non-relapse mortality (NRM) after 100 days and 1 year was 15.8% and 26.3%. Among patients with AML/MDS, advanced cases (n=64, including 61 patients with active disease) showed an OS of 44.6% and 42.4% after 3 and 5 years. Therefore, FBM conditioning combines effective disease control with low NRM.

SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom Macroglobulinemia

2008-04-30

Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells as well as endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, We further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R-CHOP

2008-04-29

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease characterized by variable clinical outcomes. Outcome prediction at the time of diagnosis is of paramount importance. Previously, we constructed a 6-gene model for outcome prediction of DLBCL patients treated with anthracycline-based chemotherapies (Lossos et al NEJM 2004, 350:1829). However, the standard therapy has evolved into rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Herein, we evaluated the predictive power of a paraffin-based 6-gene model in R-CHOP treated DLBCL patients. RNA was successfully extracted from 132 formalin-fixed paraffin-embedded (FFPE) specimens. Expression of the 6 genes comprising the model was measured and the mortality predictor score was calculated for each patient. The mortality predictor score divided patients into low-risk (below median) and high-risk (above median) subgroups with significantly different overall survival (OS) (p=0.0021) and progression-free survival (PFS) (p=0.038). The model also predicted OS and PFS when the mortality predictor score was considered as a continuous variable (p=0.0022 and 0.0098, respectively) and was independent of the IPI for prediction of OS (P=0.0083). These findings demonstrate that the prognostic value of the 6-gene model remains significant in the era of R-CHOP treatment and that the model can be applied to routine FFPE tissue from initial diagnostic biopsies.

Role of ephrinB2 expression in endothelial cells during arteriogenesis: Impact on smooth muscle cell migration and monocyte recruitment

Korff, T., Braun, J., Pfaff, D., Augustin, H. G, Hecker, M. — 2008-04-29

Expression of the arterial marker gene ephrinB2 in endothelial cells is a prerequisite for adequate remodeling processes of the developing or angiogenic vasculature. Although its role in these processes has been extensively studied, the impact of ephrinB2 on the remodeling of adult arteries is largely unknown. To this end, we analyzed its expression during a biomechanically induced arteriolar remodeling process known as arteriogenesis and noted a significant increase in ephrinB2 expression under these conditions. By examining those biomechanical forces presumed to drive arteriogenesis, we identified cyclic stretch as a critical inducer of ephrinB2 expression in endothelial cells. Subsequent functional analyses in vitro revealed that endothelial cells expressing ephrinB2 limit the migration of smooth muscle cells, thereby enhancing segregation of both cell types. Moreover, MCP-1 induced transmigration of monocytes through a monolayer of endothelial cells over-expressing a truncated variant of ephrinB2 was clearly impeded. Taken together, these data suggest that expression of ephrinB2 in adult endothelial cells is up-regulated during arterial remodeling and controlled by cyclic stretch - a well known inducer of such processes. This stretch-induced ephrinB2 expression may be pivotal for arteriogenesis as it limits smooth muscle cell migration within defined borders and controls monocyte extravasation.

Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T cell responses

2008-04-29

Mesenchymal stem cells (MSC) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSC appears a promising therapy for graft-versus-host-disease (GvHD). Little is known about the specificity of immunosuppression by MSC, in particular the effect on immunity to pathogens. We have studied how MSC affect Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific T-cell responses. We found that EBV and CMV-induced proliferation and interferon- (IFN-) production from peripheral blood mononuclear cells (PBMC) was less affected by third-party MSC than the response to alloantigen and that MSC had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T-cells (CTL) or CMV-CTL cultured with MSC retained the ability to proliferate and produce IFN- in response to their cognate antigen and to kill virally infected targets. Finally, PBMC from 2 patients who received MSC for acute GvHD showed persistence of CMV-specific T-cells and retained IFN- response to CMV post MSC infusion. In summary, MSC have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSC and suggest that the effector functions of virus-specific T-cells may be retained after MSC infusion.

Epidemiology of Myelodysplastic Syndromes and Chronic Myeloproliferative Disorders in the United States, 2001-2004: Utilizing Data from the NAACCR and SEER Programs

2008-04-28

Reporting of myelodysplastic syndromes (MDS) and chronic myeloproliferative disorders (CMD) to population-based cancer registries in the U.S. was initiated in 2001. In this first analysis of data from the North American Association of Central Cancer Registries (NAACCR), encompassing 82% of the U.S. population, we evaluated trends in MDS and CMD incidence, estimated case numbers for the entire U.S., and assessed trends in diagnostic recognition and reporting. Based on >40,000 observations, average annual age-adjusted incidence rates of MDS and CMD for 2001-2003 were 3.3 and 2.1 per 100,000, respectively. Incidence rates increased with age for both MDS and CMD (p<0.05) and were highest among Whites and non-Hispanics. Based on follow-up data through 2004 from the Surveillance Epidemiology and End Results Program, overall relative 3-year survival rates for MDS and CMD were 45% and 80%, respectively, with males experiencing poorer survival than females. Applying the observed age-specific incidence rates to U.S. Census population estimates, approximately 9,700 MDS cases and 6,300 CMD cases were estimated for the entire U.S. in 2004. MDS incidence rates significantly increased with calendar year in 2001-2004, and only 4% of cases were reported to registries by physicians offices. Thus, MDS disease burden in the U.S. may be underestimated.

Priming of T cells to Fas-mediated proliferative signals by Interleukin-7

2008-04-25

T cell depletion associated with HIV infection or cytoreductive therapies triggers potential T cell regenerative mechanisms such as peripheral T lymphocyte expansion to weak antigenic stimuli and the increased availability of IL-7, a cytokine with potent anti-apoptotic and proliferative activities. Deleterious mechanisms also associated with lymphopenia, such as increased Fas expression and apoptosis of T cell, may however result in opposing effects. In this study we show that Fas molecules, primarily associated with T cell depletion in lymphopenic settings, may also contribute to compensatory T cell expansion through transmitting costimulatory signals to suboptimally activated T cells. Proliferation of T lymphocytes in response to concomitant Fas and TCR triggering was shown to be increased in HIV-infected individuals as compared to non-infected controls. As IL-7 levels are often elevated in lymphopenic individuals in association with increased Fas expression we analyzed whether IL-7 would influence Fas-mediated proliferative signals in T cells. We show that IL-7 is able to increase the efficacy of Fas to induce proliferation of suboptimally activated T cells. Thus high IL-7 levels associated with lymphopenic conditions may simultaneously induce sensitivity to Fas-mediated apoptosis in non-activated T cells and increase Fas-induced costimulatory signals in T cells recognizing low affinity antigens.

H3K79 di-methylation marks developmental activation of the {beta}-globin gene but is reduced upon LCR-mediated high-level transcription

2008-04-25

Genome-wide analyses of the relationship between H3 K79 di-methylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels at wild type and transcriptionally impaired β-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/LCR heterozygous mice reveals no significant H3 K79 di-methylation of the β-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and LCR alleles, and both alleles are located in proximity to H3 K79 di-methylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the LCR allele compared to the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 di-methylation is inversely correlated with β-globin gene expression levels. Thus, while our results support a link between H3 K79 di-methylation and gene expression, high levels of this mark are not essential for high level β-globin gene transcription. We propose that H3 K79 di-methylation is destabilized on a highly transcribed template.

Genomic complexity identifies patients with aggressive chronic lymphocytic leukemia

2008-04-24

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes. We studied 178 CLL patients that are enrolled in a prospective study at the University of Michigan, of which 139 and 39 were previously untreated and treated, respectively. We obtained unbiased, high-density, genome-wide measurements of sub-chromosomal copy number changes in highly purified DNA from sorted CD19+ cells and buccal cells using the Affymetrix XbaI 50K SNP-array platform. Genomic complexity scores were derived and correlated with the surrogate clinical endpoints time to first therapy (TTFT) and time to subsequent therapy (TTST): measures of disease aggressiveness and/or therapy efficaciousness. In univariate analysis, progressively increasing complexity scores in previously untreated CLL patients identified patients with short TTFT at high significance levels. Similarly, TTST was significantly shorter in pre-treated patients with high as opposed to low genomic complexity. In multivariate analysis, genomic complexity emerged as an independent risk factor for short TTFT and TTST. Finally, algorithmic sub-chromosomal complexity determination was developed, facilitating automation and future routine clinical application of CLL whole-genome analysis.

Role of decorin in the antimyeloma effects of osteoblasts

Li, X., Pennisi, A., Yaccoby, S. — 2008-04-24

Building on our previous report that osteoblasts and increased bone formation have a negative impact on myeloma cell growth in a subset of patients, we investigated the role of decorin, the main small leucine-rich proteoglycan (SLRP) expressed and produced by osteoblasts, in the antimyeloma effects of osteoblasts. In coculture experiments with osteoblasts, primary myeloma cell survival was significantly higher when decorin expression in osteoblasts was knocked down by short-hairpin RNA. Coculture experiments of myeloma cells and supporting osteoclasts in the presence of osteoblast-conditioned medium showed reduced myeloma cell survival, an effect that was attenuated by decorin-neutralizing antibody. Decorin overexpression in mesenchymal stem cells or use of recombinant decorin in coculture with osteoclasts reduced the ability of osteoclasts to support primary myeloma cell survival. The antimyeloma effect of decorin involved direct induction of apoptosis and activation of p21WAF. Decorin also inhibited myeloma cell-induced tube formation and osteoclast differentiation. Decorin expression was insignificantly lower in patients' than donors' osteoblasts and slightly increased by bortezomib. We conclude that certain SLRPs are involved in the antimyeloma effect of osteoblasts directly and indirectly through inhibition of angiogenesis and osteoclastogenesis; therefore, increasing endogenous or exogenous SLRPs in myelomatous bone may help control myeloma.

CXCR4 dimerization and {beta}-arrestin-mediated signaling account for the enhanced chemotaxis to CXCL12 in WHIM syndrome

2008-04-24

WHIM syndrome is an immune deficiency linked in many cases to heterozygous mutations causing truncations in the cytoplasmic tail of the chemokine receptor CXCR4. Leukocytes expressing truncated CXCR4 display enhanced responses to the receptor ligand CXCL12, including chemotaxis, which likely impair their trafficking and contribute to the immuno-hematological clinical manifestations of the syndrome. CXCR4 desensitization and endocytosis are dependent on β-arrestin recruitment to the cytoplasmic tail, so that the truncated CXCR4 are refractory to these processes and so have enhanced G-protein-dependent signaling. Here, we show that the augmented responsiveness of WHIM leukocytes is also accounted for by enhanced β-arrestin2-dependent signaling downstream of the truncated CXCR4 receptor. Indeed, the WHIM-associated receptor CXCR4¹⁰¹³ maintains association with β-arrestin2 and triggers augmented and prolonged β-arrestin2-dependent signaling, as revealed by ERK1/2 phosphorylation kinetics. Evidence is also provided that CXCR4¹⁰¹³-mediated chemotaxis critically requires β-arrestin2, and disrupting the SHSK motif in the third intracellular loop of CXCR4¹⁰¹³ abrogates β-arrestin2-mediated signaling, but not coupling to G-proteins, and normalizes chemotaxis. We also demonstrate that CXCR4¹⁰¹³ spontaneously forms heterodimers with wild-type CXCR4. Accordingly, we propose a model where enhanced functional interactions between β-arrestin2 and receptor dimers account for the altered responsiveness of WHIM leukocytes to CXCL12.

Macrophage mannose receptor on lymphatics controls cell trafficking

2008-04-23

Macrophage mannose receptor (MR) participates in pathogen recognition, clearance of endogenous serum glycoproteins and antigen presentation. MR is also present on lymphatic vessels, where its function is unknown. Here we show that migration of lymphocytes from the skin into the draining lymph nodes through the afferent lymphatics is reduced in MR-deficient mice, whilst the structure of lymphatic vasculature remains normal in these animals. Moreover, in a tumor model the primary tumors grow significantly bigger in MR-/- mice than in the wild-type (WT) controls, whereas the regional lymph node metastases are markedly smaller. Adhesion of both normal lymphocytes and tumor cells to lymphatic vessels is significantly decreased in MR deficient mice. Ability of macrophages to present tumor antigens is indistinguishable between the two genotypes. Thus, MR on lymphatic endothelial cells is involved in leukocyte trafficking and contributes to the metastatic behavior of cancer cells. Blocking of MR may provide a new approach to controlling inflammation and cancer metastasis by targeting the lymphatic vasculature.

SAGE analysis demonstrates increased expression of TOSO contributing to Fas mediated resistance in CLL

2008-04-23

To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19+). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19+ libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time RT-PCR in 78 CLL and 12 nCD19+ cases (p<0.0001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6 fold increase of TOSO protein in circulating CLL cells (p=0.013) and lymph nodes (p=0.0057). Our SAGE results have demonstrated that TOSO is a novel over-expressed anti-apoptotic gene in CLL.

Rapid mobilization of functional donor hematopoietic cells without G-CSF using plerixafor, an antagonist of the CXCR4/SDF-1 interaction

2008-04-21

Allografts from HLA-matched sibling donors were mobilized and collected without G-CSF using Plerixafor (AMD3100), a direct antagonist of CXCR4/SDF-1. Donors (N=25) were treated with AMD3100 at a dose of 240 µg/kg by subcutaneous injection and leukapheresis was then initiated just 4 hours later. Two-thirds of the donors collected an allograft with a CD34+ cell dose sufficient for transplantation after just one dose of AMD3100. No donor experienced more than grade one toxicity. Following a myeloablative regimen, twenty patients with hematological malignancies received allografts collected after AMD3100 alone. All patients engrafted neutrophils (median day +10) and platelets (median day +12) promptly. Acute GVHD grades 2-4 occurred in 35% of patients. One patient died due to complications related to acute GVHD. No unexpected adverse events were observed in any of the recipients. All 14 patients surviving in remission have robust trilineage hematopoiesis and are transfusion-free with a median follow-up of 277 days (range 139-964 days). Direct antagonism of CXCR4 by AMD3100 may provide a more rapid and possibly less toxic and cumbersome alternative to traditional G-CSF-based mobilization in normal donors. This trial was registered as #NCT00241358 at www.ClinicalTrials.gov

MDM2 SNP309 and TP53 Arg72Pro interact to alter therapy-related acute myeloid leukemia susceptibility

2008-04-21

The p53 tumor suppressor directs the cellular response to many mechanistically distinct DNA damaging agents and is selected against during the pathogenesis of therapy-related acute myeloid leukemia (t-AML). We hypothesized that constitutional genetic variation in the p53 pathway would affect t-AML risk. Therefore, we tested associations between patients with t-AML (n=171) and two common functional p53-pathway variants, the MDM2 SNP309 and the TP53 codon 72 polymorphism. Although neither polymorphism alone influenced the risk of t-AML, an interactive effect was detected such that MDM2 TT TP53 Arg/Arg double homozygotes, and individuals carrying both a MDM2 G allele and a TP53 Pro allele were at increased risk of t-AML (p for interaction=0.009). This interactive effect was observed in patients previously treated with chemotherapy but not in patients treated with radiotherapy, and in patients with loss of chromosomes 5 and/or 7, acquired abnormalities associated with prior exposure to alkylator chemotherapy. Additionally, there was a trend towards shorter latency to t-AML in MDM2 GG versus TT homozygotes in females but not in males, and in younger but not older patients. These data indicate that the MDM2 and TP53 variants interact to modulate responses to genotoxic therapy and are determinants of risk for t-AML.

Insight into the pathogenesis of chronic lymphocytic leukemia (CLL) through analysis of IgVH gene usage and mutation status in familial CLL

2008-04-18

To address the proposition that familial B-cell chronic lymphocytic leukemia (CLL) may exhibit a more restricted phenotype than sporadic CLL with respect to immunoglobulin gene usage or ontogenic development we compared immunoglobulin (Ig) heavy chain variable region (VH) gene usage and IgVH mutation status in 327 cases of CLL from 214 families, with 724 sporadic cases. The frequency of mutated CLL was higher in familial CLL (P<0.001) and there was evidence of intra-familial concordance in mutation status (P<0.001). The repertoire and frequency of IgVH usage was, however, not significantly different between familial and sporadic CLL. Furthermore, IgVH usage was not correlated between affected members of the same family. These observations provide evidence that familial CLL is essentially indistinguishable from sporadic CLL favoring a genetic basis to disease development in general rather than a simple environmental etiology.

CD38 gene polymorphism and chronic lymphocytic leukemia: a role in transformation to Richter's syndrome?

2008-04-18

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n=232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter's syndrome (RS). 5-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

Human mast cell activation with virus associated stimuli leads to the selective chemotaxis of natural killer cells by a CXCL8 dependent mechanism

2008-04-18

Human mast cells are found in skin and mucosal surfaces and next to blood vessels. They play a sentinel cell role in immunity, recognizing invading pathogens and producing pro-inflammatory mediators. Mast cells can recruit granulocytes, and monocytes in allergic disease and bacterial infection, but their ability to recruit anti-viral effector cells such as natural killer (NK) cells and T cells has not been fully elucidated. To investigate the role of human mast cells in response to virus associated stimuli, human cord blood-derived mast cells (CBMC) were stimulated with polyinosinic·polycytidylic acid, a double-stranded RNA analogue, or infected with the doublestranded RNA virus, reovirus serotype 3 Dearing for 24 hours. CBMC responded to stimulation with polyinosinic·polycytidylic acid by producing a distinct chemokine profile, including CCL4, CXCL8, and CXCL10. CBMC produced significant amounts of CXCL8 in response to low levels of reovirus infection, while both skin and lung derived fibroblasts were unresponsive unless higher doses of reovirus were used. Supernatants from CBMC infected with reovirus induced substantial NK cell chemotaxis that was highly dependent on CXCL8 and CXCR1. These results suggest a novel role for mast cells in the recruitment of human NK cells to sites of early viral infection via CXCL8.

Flk2+ common lymphoid progenitors possess equivalent differentiation potential for the B and T lineages

Karsunky, H., Inlay, M. A, Serwold, T., Bhattacharya, D., Weissman, I. L — 2008-04-18

Mature blood cells develop from multipotent hematopoietic stem cells through a series of sequential intermediates in which the developmental potential for particular blood lineages is progressively extinguished. We previously reported the identification of one of these developmental intermediates, the common lymphoid progenitor (CLP), which can give rise to T, B, dendritic, and NK cells but lacks myeloid and erythroid potential. Recently, several studies, have suggested that the T and dendritic cell potential of CLP is limited or absent, and/or that CLP contain significant myeloid potential. Here, we show that the originally identified CLP population can be divided into functionally distinct subsets based on the expression of the tyrosine kinase receptor, Flk2. The Flk2⁺ subset, contains robust in vivo and in vitro T, B, DC, and NK potential, but lacks myeloid potential and therefore, represents an oligopotent, lymphoid-restricted progenitor. This population of cells does not appear to be B cell-biased and robustly reconstitutes both B and T lineages in vivo, consistent with it being a physiological progenitor of both of these subsets. Thus, Flk2 expression defines a homogeneous, readily obtainable subset of bone marrow CLP that is completely lymphoid committed and can differentiate equivalently well into both B and T lineages.