Blood current issue

T regulatory cells turn on T regulatory cells

Scott, D. W. — Thu, 05 Nov 2009 09:03:48 PST

Heterogeneity in the AML stem cell pool

Hays, L. E. — Thu, 05 Nov 2009 09:03:48 PST

MAL: not just a leukemia inducer

Ravid, K. — Thu, 05 Nov 2009 09:03:48 PST

Denser matters

Ariens, R. A. S. — Thu, 05 Nov 2009 09:03:48 PST

Immunogenicity reloaded

Flegel, W. A. — Thu, 05 Nov 2009 09:03:48 PST

Are 2 cords better than 1?

Delaney, M. — Thu, 05 Nov 2009 09:03:48 PST

Tophaceous gout with congenital dyserythropoietic anemia

Thu, 05 Nov 2009 09:03:48 PST

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal

Thu, 05 Nov 2009 09:03:48 PST

Although the cancer stem cell (CSC) concept implies that CSCs are rare, recent reports suggest that CSCs may be frequent in some cancers. We hypothesized that the proportion of leukemia stem cells would vary as a function of the number of dysregulated pathways. Constitutive expression of MN1 served as a 1-oncogene model, and coexpression of MN1 and a HOX gene served as a 2-oncogene model. Leukemia-initiating cell (LIC) number and in vitro expansion potential of LICs were functionally assessed by limiting dilution analyses. LIC expansion potential was 132-fold increased in the 2- compared with the 1-oncogene model, although phenotypically, both leukemias were similar. The 2-oncogene model was characterized by granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity and activated STAT/ERK signaling. GM-CSF hypersensitivity of the 2-oncogene model (MN1/HOXA9) was lost in Stat5b^–/– cells, and the LIC expansion potential was reduced by 86- and 28-fold in Stat5b^–/– and Stat1^–/– cells, respectively. Interestingly, in 201 acute myeloid leukemia (AML) patients, coexpression of MN1 and HOXA9 was restricted to patients with the poorest prognosis and was associated with highly active STAT signaling. Our data demonstrate the functional heterogeneity of LICs and show that STAT signaling is critical for leukemia stem cell self-renewal in MN1- and HOXA9-expressing leukemias.

The pleiotropic role of the fibrinogen {gamma}' chain in hemostasis

Uitte de Willige, S., Standeven, K. F., Philippou, H., Ariens, R. A. S. — Thu, 05 Nov 2009 09:03:48 PST

A fraction of fibrinogen contains a differently spliced chain called ', which presents itself mainly as heterodimer with the common A chain as A/' fibrinogen. The ' chain differs from the A chain in its C-terminus and has important functional implications for fibrinogen. The presence of the ' chain modulates thrombin and FXIII activity, influences clot architecture, and eliminates a platelet-binding site. Associations of A/' fibrinogen levels with arterial and venous thrombosis have been reported, indicating that the functional effects of A/' fibrinogen may contribute to the pathology of thrombosis. This review summarizes the key biologic aspects of this interesting variant of fibrinogen and discusses inconsistencies in current reports.

How I treat EBV lymphoproliferation

Heslop, H. E. — Thu, 05 Nov 2009 09:03:48 PST

Epstein-Barr virus (EBV)–associated B-cell lymphoproliferation is a life-threatening complication after hematopoietic stem cell or solid organ transplantation resulting from outgrowth of EBV-infected B cells that would normally be controlled by EBV-cytotoxic T cells. During the past decade, early detection strategies, such as serial measurement of EBV-DNA load in peripheral blood samples, have helped to identify high-risk patients and to diagnose early lymphoproliferation. Treatment options include manipulation of the balance between outgrowing EBV-infected B cells and the EBV cytotoxic T lymphocyte response and targeting the B cells with monoclonal antibodies or chemotherapy. Major challenges remain for defining indications for preemptive therapies and integrating novel and conventional therapies.

Deferasirox pharmacokinetics in patients with adequate versus inadequate response

Thu, 05 Nov 2009 09:03:48 PST

Tens of thousands of transfusion-dependent (eg, thalassemia) patients worldwide suffer from chronic iron overload and its potentially fatal complications. The oral iron chelator deferasirox has become commercially available in many countries since 2006. Although this alternative to parenteral deferoxamine has been a major advance for patients with transfusional hemosiderosis, a proportion of patients have suboptimal response to the maximum approved doses (30 mg/kg per day), and do not achieve negative iron balance. We performed a prospective study of oral deferasirox pharmacokinetics (PK), comparing 10 transfused patients with inadequate deferasirox response (rising ferritin trend or rising liver iron on deferasirox doses > 30 mg/kg per day) with control transfusion-dependent patients (n = 5) with adequate response. Subjects were admitted for 4 assessments: deferoxamine infusion and urinary iron measurement to assess readily chelatable iron; quantitative hepatobiliary scintigraphy to assess hepatic uptake and excretion of chelate; a 24-hour deferasirox PK study following a single 35-mg/kg dose of oral deferasirox; and pharmacogenomic analysis. Patients with inadequate response to deferasirox had significantly lower systemic drug exposure compared with control patients (P < .00001). Cmax, volume of distribution/bioavailability (Vd/F), and elimination half-life (t_1/2) were not different between the groups, suggesting bioavailability as the likely discriminant. Effective dosing regimens for inadequately responding patients to deferasirox must be determined. This trial has been registered at http://www.clinicaltrials.gov under identifier NCT00749515.

A protective role for early oral exposures in the etiology of young adult Hodgkin lymphoma

Thu, 05 Nov 2009 09:03:48 PST

The pattern of adolescent/young adult Hodgkin lymphoma (YAHL) suggests causation by a relatively late infection with a common childhood virus, but no causal virus has been found. Susceptibility is heritable and linked to lower interleukin 12 (IL12) levels, which can also result from fewer fecal-oral microbial exposures early in life. We studied twin pairs discordant for YAHL to examine exposures capable of altering the IL12 response and T-helper type 1 (Th1)–Th2 balance. One hundred eighty-eight YAHL-discordant twin pairs from the International Twin Study returned questionnaires (70% response). Exposure history of YAHL case-twins was compared with that of their unaffected control-twins using conditional logistic regression for matched pairs to calculate odds ratios (ORs). Behaviors likely to produce oral exposure to microbes conveyed decreases in risk (univariable OR range = 0.2-0.5, P = .003-.11). Significant adjusted ORs were seen for appendectomy (OR = 4.3, P = .001), eczema (OR = 4.2, P = .025), smoking (OR = 2.2, P = .054), and relatively more frequent behaviors associated with oral exposures (OR = 0.1; P = .004). Kappa statistics for intrapair agreement were higher than 0.8 for each significant finding. Our observations support a protective role for increased early oral exposure to the microbiome, suggesting that factors associated with increased Th2 and decreased Th1 cytokines are etiologically relevant to YAHL.

Pancreatic iron loading predicts cardiac iron loading in thalassemia major

Noetzli, L. J., Papudesi, J., Coates, T. D., Wood, J. C. — Thu, 05 Nov 2009 09:03:48 PST

Diabetes mellitus and cardiomyopathy are common in chronically transfused thalassemia major patients, occurring in the second and third decades of life. We postulated that pancreatic iron deposition would precede cardiac iron loading, representing an environment favorable for extrahepatic iron deposition. To test this hypothesis, we examined pancreatic and cardiac iron in 131 thalassemia major patients over a 4-year period. Cardiac iron (R2* > 50 Hz) was detected in 37.7% of patients and pancreatic iron (R2* > 28 Hz) in 80.4% of patients. Pancreatic and cardiac R2* were correlated (r² = 0.52), with significant pancreatic iron occurring nearly a decade earlier than cardiac iron. A pancreatic R2* less than 100 Hz was a powerful negative predictor of cardiac iron, and pancreatic R2* more than 100 Hz had a positive predictive value of more than 60%. In serial analysis, changes in cardiac iron were correlated with changes in pancreatic iron (r² = 0.33, P < .001), but not liver iron (r² = 0.025, P = .25). As a result, pancreatic R2* measurements offer important early recognition of physiologic conditions suitable for future cardiac iron deposition and complementary information to liver and cardiac iron during chelation therapy. Staging abdominal and cardiac magnetic resonance imaging examinations could significantly reduce costs, magnet time, and need for sedation in young patients.

Phase 3 randomized, placebo-controlled, double-blind study of high-dose continuous infusion cytarabine alone or with laromustine (VNP40101M) in patients with acute myeloid leukemia in first relapse

Thu, 05 Nov 2009 09:03:48 PST

Laromustine is a sulfonylhdrazine alkylator with significant antileukemia activity. An international, randomized (2:1), double-blind, placebo-controlled study was conducted to compare complete remission (CR) rates and overall survival (OS) in patients with first relapse acute myeloid leukemia (AML) treated with laromustine and high-dose cytarabine (HDAC) versus HDAC/placebo. Patients received 1.5 g/m² per day cytarabine continuous infusion for 3 days and laromustine 600 mg/m² (n = 177) or placebo (n = 86) on day 2. Patients in CR received consolidation with laromustine/HDAC or HDAC/placebo as per initial randomization. After interim analysis at 50% enrollment, the Data Safety Monitoring Board (DSMB) expressed concern that any advantage in CR would be compromised by the observed on-study mortality, and enrollment was held. The CR rate was significantly higher for the laromustine/HDAC group (35% vs 19%, P = .005). However, the 30-day mortality rate and median progression-free survival were significantly worse in this group compared with HDAC/placebo (11% vs 2%; P = .016; 54 days vs 34; P = .002). OS and median response durations were similar in both groups. Laromustine/HDAC induced significantly more CR than HDAC/placebo, but OS was not improved due to mortality associated with myelosuppression and its sequelae. The DSMB subsequently approved a revised protocol with laromustine dose reduction and recombinant growth factor support. The study was registered as NCT00112554 at http://www.clinicaltrials.gov.

CD4+FOXP3+ regulatory T cells confer long-term regulation of factor VIII-specific immune responses in plasmid-mediated gene therapy-treated hemophilia mice

Thu, 05 Nov 2009 09:03:48 PST

Gene transfer of a factor VIII (FVIII) plasmid into hemophilia A (HemA) mice achieved supraphysiologic FVIII expression, but triggered production of high-titer FVIII-specific antibodies and loss of functional FVIII activity. To test whether FVIII-specific regulatory T cells (Tregs) can modulate immune responses against FVIII, we developed a HemA mouse model in which all T cells overexpressed Foxp3 (HemA/Foxp3-Tg). FVIII plasmid therapy did not induce antibody production in HemA/Foxp3-Tg mice. CD4⁺Foxp3⁺ T cells isolated from plasmid-treated HemA/Foxp3-Tg mice significantly suppressed proliferation of FVIII-stimulated CD4⁺ effector T cells. The percentage of CD4⁺ T cells expressing CD25, glucocorticoid-induced tumor necrosis factor receptor, and cytotoxic T lymphocyte antigen 4 increased significantly in spleen and peripheral blood for 9 weeks. Mice receiving adoptively transferred Tregs from FVIII-exposed HemA/Foxp3-Tg mice produced significantly reduced antibody titers compared with controls after initial challenge with FVIII plasmid and second challenge 16 weeks after first plasmid treatment. Adoptively transferred Tregs engrafted and distributed at 2% to 4% in the Treg compartment of blood, lymph nodes, and spleens of the recipient mice and induced activation of endogenous Tregs; the engraftment decreased to negligible levels over 8 to 12 weeks. Antigen-specific Tregs can provide long-lasting protection against immune responses in vivo and limit recall responses induced by a second challenge via infectious tolerance.

Erythroid dysplasia, megaloblastic anemia, and impaired lymphopoiesis arising from mitochondrial dysfunction

Thu, 05 Nov 2009 09:03:48 PST

Recent reports describe hematopoietic abnormalities in mice with targeted instability of the mitochondrial genome. However, these abnormalities have not been fully described. We demonstrate that mutant animals develop an age-dependent, macrocytic anemia with abnormal erythroid maturation and megaloblastic changes, as well as profound defects in lymphopoiesis. Mice die of severe fatal anemia at 15 months of age. Bone-marrow transplantation studies demonstrate that these abnormalities are intrinsic to the hematopoietic compartment and dependent upon the age of donor hematopoietic stem cells. These abnormalities are phenotypically similar to those found in patients with refractory anemia, suggesting that, in some cases, the myelodysplastic syndromes are caused by abnormalities of mitochondrial function.

In vivo prostaglandin E2 treatment alters the bone marrow microenvironment and preferentially expands short-term hematopoietic stem cells

Thu, 05 Nov 2009 09:03:48 PST

Microenvironmental signals can determine hematopoietic stem cell (HSC) fate choices both directly and through stimulation of niche cells. In the bone marrow, prostaglandin E₂ (PGE₂) is known to affect both osteoblasts and osteoclasts, whereas in vitro it expands HSCs and affects differentiation of hematopoietic progenitors. We hypothesized that in vivo PGE₂ treatment could expand HSCs through effects on both HSCs and their microenvironment. PGE₂-treated mice had significantly decreased number of bone trabeculae, suggesting disruption of their microarchitecture. In addition, in vivo PGE₂ increased lineage^– Sca-1⁺ c-kit⁺ bone marrow cells without inhibiting their differentiation. However, detailed immunophenotyping demonstrated a PGE₂-dependent increase in short-term HSCs/multipotent progenitors (ST-HSCs/MPPs) only. Bone marrow cells transplanted from PGE₂ versus vehicle-treated donors had superior lymphomyeloid reconstitution, which ceased by 16 weeks, also suggesting that ST-HSCs were preferentially expanded. This was confirmed by serial transplantation studies. Thus in vivo PGE₂ treatment, probably through a combination of direct and microenvironmental actions, preferentially expands ST-HSCs in the absence of marrow injury, with no negative impact on hematopoietic progenitors or long-term HSCs. These novel effects of PGE₂ could be exploited clinically to increase donor ST-HSCs, which are highly proliferative and could accelerate hematopoietic recovery after stem cell transplantation.

Dysfunctional expansion of hematopoietic stem cells and block of myeloid differentiation in lethal sepsis

Thu, 05 Nov 2009 09:03:48 PST

Severe sepsis is one of the leading causes of death worldwide. High mortality rates in sepsis are frequently associated with neutropenia. Despite the central role of neutrophils in innate immunity, the mechanisms causing neutropenia during sepsis remain elusive. Here, we show that neutropenia is caused in part by apoptosis and is sustained by a block of hematopoietic stem cell (HSC) differentiation. Using a sepsis murine model, we found that the human opportunistic bacterial pathogen Pseudomonas aeruginosa caused neutrophil depletion and expansion of the HSC pool in the bone marrow. "Septic" HSCs were significantly impaired in competitive repopulation assays and defective in generating common myeloid progenitors and granulocyte-monocyte progenitors, resulting in lower rates of myeloid differentiation in vitro and in vivo. Delayed myeloid-neutrophil differentiation was further mapped using a lysozyme–green fluorescent protein (GFP) reporter mouse. Pseudomonas's lipopolysaccharide was necessary and sufficient to induce myelosuppresion and required intact TLR4 signaling. Our results establish a previously unrecognized link between HSC regulation and host response in severe sepsis and demonstrate a novel role for TLR4.

Spatial gradients of blood vessels and hematopoietic stem and progenitor cells within the marrow cavities of the human skeleton

Thu, 05 Nov 2009 09:03:48 PST

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34⁺ and CD117⁺ hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34⁺ or CD117⁺ HSPC to the nearest trabecular surface was measured and binned in 50-µm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34⁺ HSPC population, 60% of the CD117⁺ HSPC population, and 72% of the BVFs were found within 100 µm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.

Fibronectin maintains survival of mouse natural killer (NK) cells via CD11b/Src/{beta}-catenin pathway

Thu, 05 Nov 2009 09:03:49 PST

Tissue microenvironment and stroma-derived extracellular matrix (ECM) molecules play important roles in the survival and differentiation of cells. Mouse natural killer (NK) cells usually die within 24 hours once isolated ex vivo. Exogenous cytokines such as interleukin-12 (IL-12) and IL-15 are required to maintain the survival and activity of mouse NK cells cultured in vitro. Whether and how ECM molecules such as fibronectin can support the survival of NK cells remain unknown. We demonstrate that fibronectin, just like IL-15, can maintain survival of mouse NK cells in vitro. Furthermore, we show that fibronectin binds to the CD11b on NK cells, and then CD11b recruits and activates Src. Src can directly interact with β-catenin and trigger nuclear translocation of β-catenin. The activation of β-catenin promotes extracellular signal-related kinase (ERK) phosphorylation, resulting in the increased expression of antiapoptotic protein B-cell leukemia 2 (Bcl-2), which may contribute to the maintenance of NK-cell survival. Consistently, fibronectin cannot maintain the survival of CD11b^– NK cells and β-catenin–deficient NK cells in vitro, and the number of NK cells is dramatically decreased in the β-catenin–deficient mice. Therefore, fibronectin can maintain survival of mouse NK cells by activating ERK and up-regulating Bcl-2 expression via CD11b/Src/β-catenin pathway.

Interleukin-21 restores immunoglobulin production ex vivo in patients with common variable immunodeficiency and selective IgA deficiency

Thu, 05 Nov 2009 09:03:49 PST

Interleukin-21 (IL-21) is an important promoter for differentiation of human B cells into immunoglobulin (Ig)–secreting cells. The objective of this study was to evaluate an IL-21–based approach to induce immunoglobulin production in B cells from patients with common variable immunodeficiency (CVID) or selective IgA deficiency (IgAD). We show that a combination of IL-21, IL-4, and anti-CD40 stimulation induces class-switch recombination to IgG and IgA and differentiation of Ig-secreting cells, consisting of both surface IgG⁺ (sIgG⁺) and sIgA⁺ B cells and CD138⁺ plasma cells, in patients with CVID or IgAD. Stimulation with IL-21 was far more effective than stimulation with IL-4 or IL-10. Moreover, spontaneous apoptosis of CD19⁺ B cells from patients with CVID or IgAD was prevented by a combination of IL-21, IL-4, and anti-CD40 stimulation. Analysis of IL-21 and IL-21 receptor (IL-21R) mRNA expression upon anti-CD3 stimulation of T cells, however, showed no evidence for defective IL-21 expression in CVID patients and sequencing of the coding regions of the IL21 gene did not reveal any mutations, suggesting a regulatory defect. Thus, our work provides an initial basis for a potential therapeutic role of IL-21 to reconstitute immunoglobulin production in CVID and IgAD.

Comprehensive assessment of T-cell receptor {beta}-chain diversity in {alpha}{beta} T cells

Thu, 05 Nov 2009 09:03:49 PST

The adaptive immune system uses several strategies to generate a repertoire of T- and B-cell antigen receptors with sufficient diversity to recognize the universe of potential pathogens. In β T cells, which primarily recognize peptide antigens presented by major histocompatibility complex molecules, most of this receptor diversity is contained within the third complementarity-determining region (CDR3) of the T-cell receptor (TCR) and β chains. Although it has been estimated that the adaptive immune system can generate up to 10¹⁶ distinct β pairs, direct assessment of TCR CDR3 diversity has not proved amenable to standard capillary electrophoresis-based DNA sequencing. We developed a novel experimental and computational approach to measure TCR CDR3 diversity based on single-molecule DNA sequencing, and used this approach to determine the CDR3 sequence in millions of rearranged TCRβ genes from T cells of 2 adults. We find that total TCRβ receptor diversity is at least 4-fold higher than previous estimates, and the diversity in the subset of CD45RO⁺ antigen-experienced β T cells is at least 10-fold higher than previous estimates. These methods should prove valuable for assessment of β T-cell repertoire diversity after hematopoietic cell transplantation, in states of congenital or acquired immunodeficiency, and during normal aging.

Uptake of CCR7 and acquisition of migratory properties by human KIR+ NK cells interacting with monocyte-derived DC or EBV cell lines: regulation by KIR/HLA-class I interaction

Thu, 05 Nov 2009 09:03:49 PST

C-C chemokine receptor type 7 (CCR7) is a chemokine receptor playing a pivotal role in the induction of human natural killer (NK)–cell migration to lymph nodes. We show that "licensed" peripheral blood killer immunoglobulin-like receptor–positive (KIR⁺) NK-cell populations, as well as KIR⁺ NK-cell clones, de novo express CCR7 upon coculture with mature dendritic cells (mDCs) or Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines. As a consequence, they become capable of migrating in response to the CCR7-specific chemokines C-C chemokine ligand (CCL)–19 and/or CCL21. The acquisition of CCR7 by NK cells requires direct cell-to-cell contact, is detectable within a few minutes, and is due to receptor uptake from CCR7⁺ cells. This mechanism is tightly regulated by KIR-mediated recognition of human leukocyte antigen (HLA) class I as well as by adhesion molecules including leukocyte function-associated antigen 1 (LFA-1) and CD2. Analysis of NK-cell clones revealed that alloreactive (KIR-ligand mismatched) but not autologous NK cells acquire CCR7. These data have important implications in haploidentical hematopoietic stem cell transplantation (HSCT), in which alloreactive NK cells may acquire the ability to migrate to secondary lymphoid compartments (SLCs), where they can kill recipient antigen-presenting cells (APCs) and T cells thus preventing graft-versus-host (and host-versus-graft) reactions.

Different NK cell-activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity

Thu, 05 Nov 2009 09:03:49 PST

The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial hemophagocytic lymphohistiocytosis type 3 (FHL3) are associated with loss-of-function mutations in RAB27A (encoding Rab27a) and UNC13D (encoding Munc13-4). Munc13-4 deficiency abrogates NK-cell release of perforin-containing lytic granules induced by signals for natural and antibody-dependent cellular cytotoxicity. We demonstrate here that these signals fail to induce degranulation in resting NK cells from Rab27a-deficient patients. In resting NK cells from healthy subjects, endogenous Rab27a and Munc13-4 do not colocalize extensively with perforin. However, phorbol 12-myristate 13-acetate and ionomycin stimulation or conjugation to susceptible target cells induced myosin-dependent colocalization of Rab27a and Munc13-4 with perforin. Unexpectedly, individual engagement of receptors leukocyte functional antigen-1, NKG2D, or 2B4 induced colocalization of Rab27a, but not Munc13-4, with perforin. Conversely, engagement of antibody-dependent cellular cytotoxicity receptor CD16 induced colocalization of Munc13-4, but not Rab27a, with perforin. Furthermore, colocalization of Munc13-4 with perforin was Rab27a-dependent. In conclusion, Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals, demonstrating that individual target cell ligands regulate discrete molecular events for lytic granule maturation. The data suggest Rab27a facilitates degranulation at an early step yet highlight a reciprocal relationship between Munc13-4 and Rab27a for degranulation.

The role of the human cytomegalovirus UL111A gene in down-regulating CD4+ T-cell recognition of latently infected cells: implications for virus elimination during latency

Thu, 05 Nov 2009 09:03:49 PST

The capacity of human cytomegalovirus (HCMV) to establish and maintain a latent infection from which it can later reactivate ensures its widespread distribution in the population, but the mechanisms enabling maintenance of latency in the face of a robust immune system are poorly understood. We examined the role of the HCMV UL111A gene, which encodes homologs of the immunosuppressive cytokine interleukin-10 in the context of latent infection of myeloid progenitor cells. A UL111A deletion virus was able to establish, maintain, and reactivate from experimental latency in a manner comparable with parental virus, but major histocompatibility complex class II levels increased significantly on the surfaces of cells infected with the deletion virus. Importantly, there was an increase in both allogeneic and autologous peripheral blood mononuclear cells and CD4⁺ T-cell responses to UL111A deletion virus-infected myeloid progenitors, indicating that loss of the capacity to express viral interleukin-10 during latency results in latently infected cells becoming more readily recognizable by a critical arm of the immune response. The detection of a viral gene that suppresses CD4⁺ T-cell recognition of latently infected cells identifies an immune evasion strategy that probably enhances the capacity of HCMV to persist in a latent state within the human host.

Wild-type FOXP3 is selectively active in CD4+CD25hi regulatory T cells of healthy female carriers of different FOXP3 mutations

Thu, 05 Nov 2009 09:03:49 PST

Forkhead box P3 (FOXP3) is constitutively expressed by CD4⁺CD25^hi regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)–FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4⁺T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

Vaccine site inflammation potentiates idiotype DNA vaccine-induced therapeutic T cell-, and not B cell-, dependent antilymphoma immunity

Qin, H., Cha, S.-c., Neelapu, S. S., Lou, Y., Wei, J., Liu, Y.-J., Kwak, L. W. — Thu, 05 Nov 2009 09:03:49 PST

Lymphoma idiotype protein vaccines have shown therapeutic potential in previous clinical studies, and results from a completed pivotal, phase 3 controlled trial are promising. However, streamlined production of these patient-specific vaccines is required for eventual clinical application. Here, we show that second-generation, chemokine-fused idiotype DNA vaccines, when combined with myotoxins that induced sterile inflammation with recruitment of antigen-presenting cells at vaccination sites, were exceptional in their ability to provoke memory antitumor immunity in mice, compared with several TLR agonists. The combined vaccination strategy elicited both antigen-specific T-cell responses and humoral immunity. Unexpectedly, vaccine-induced tumor protection was intact in B cell–deficient mice but was abrogated completely by T-cell depletion in vivo, suggesting T-cell dependence. Furthermore, the optimal effect of myotoxins was observed with fusion vaccines that specifically targeted antigen delivery to antigen-presenting cells and not with vaccines lacking a targeting moiety, suggesting that the rational vaccine design will require combination strategies with novel, proinflammatory agents and highly optimized molecular vaccine constructs. These studies also challenge the paradigm that antibody responses are the primary of idiotype-specific antitumor effects and support the optimization of idiotype vaccines designed to induce primarily T-cell immunity.

Pim kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells

Chen, L. S., Redkar, S., Bearss, D., Wierda, W. G., Gandhi, V. — Thu, 05 Nov 2009 09:03:49 PST

Pim kinases are involved in B-cell development and are overexpressed in B-cell chronic lymphocytic leukemia (CLL). We hypothesized that Pim kinase inhibition would affect B-cell survival. Identified from a screen of imidazo[1,2-b]pyridazine compounds, SGI-1776 inhibits Pim-1, Pim-2, and Pim-3. Treatment of CLL cells with SGI-1776 results in a concentration-dependent induction of apoptosis. To elucidate its mechanism of action, we evaluated the effect of SGI-1776 on Pim kinase function. Unlike in replicating cells, phosphorylation of traditional Pim-1 kinase targets, phospho-Bad (Ser112) and histone H3 (Ser10), and cell-cycle proteins were unaffected by SGI-1776, suggesting an alternative mechanism in CLL. Protein levels of total c-Myc as well as phospho-c-Myc(Ser62), a Pim-1 target site, were decreased after SGI-1776 treatment. Levels of antiapoptotic proteins Bcl-2, Bcl-X_L, XIAP, and proapoptotic Bak and Bax were unchanged; however, a significant reduction in Mcl-1 was observed that was not caused by caspase-mediated cleavage of Mcl-1 protein. The mechanism of decline in Mcl-1 was at the RNA level and was correlated with inhibition of global RNA synthesis. Consistent with a decline in new RNA synthesis, MCL-1 transcript levels were decreased after treatment with SGI-1776. These data suggest that SGI-1776 induces apoptosis in CLL and that the mechanism involves Mcl-1 reduction.

E{micro}-BCL10 mice exhibit constitutive activation of both canonical and noncanonical NF-{kappa}B pathways generating marginal zone (MZ) B-cell expansion as a precursor to splenic MZ lymphoma

Thu, 05 Nov 2009 09:03:49 PST

BCL10, required for nuclear factor B (NF-B) activation during antigen-driven lymphocyte responses, is aberrantly expressed in mucosa-associated lymphoid tissue-type marginal zone (MZ) lymphomas because of chromosomal translocations. Eµ-driven human BCL10 transgenic (Tg) mice, which we created and characterize here, had expanded populations of MZ B cells and reduced follicular and B1a cells. Splenic B cells from Tg mice exhibited constitutive activation of both canonical and noncanonical NF-B signaling pathways is associated with increased expression of NF-B target genes. These genes included Tnfsf13b, which encodes the B-cell activating factor (BAFF). In addition, levels of BAFF were significantly increased in sera from Tg mice. MZ B cells of Tg mice exhibited reduced turnover in vivo and enhanced survival in vitro, indicative of lymphoaccumulation rather than lymphoproliferation as the cause of MZ expansion. In vivo antibody responses to both T-independent, and especially T-dependent, antigens were significantly reduced in Tg mice. Mortality was accelerated in Tg animals, and some mice older than 8 months had histologic and molecular findings indicative of clonal splenic MZ lymphoma. These results suggest that, in addition to constitutive activation of BCL10 in MZ B cells, other genetic factors or environmental influences are required for short latency oncogenic transformation.

miR-128b is a potent glucocorticoid sensitizer in MLL-AF4 acute lymphocytic leukemia cells and exerts cooperative effects with miR-221

Thu, 05 Nov 2009 09:03:49 PST

MLL-AF4 acute lymphocytic leukemia (ALL) has a poor prognosis. MicroRNAs (miRNA) are small noncoding RNAs that posttranscriptionally regulate expression of target mRNAs. Our analysis of previously published data showed that expression of miR-128b and miR-221 is down-regulated in MLL-rearranged ALL relative to other types of ALL. Reexpression of these miRNAs cooperatively sensitizes 2 cultured lines of MLL-AF4 ALL cells to glucocorticoids. Target genes down-regulated by miR-128b include MLL, AF4, and both MLL-AF4 and AF4-MLL fusion genes; miR-221 down-regulates CDKN1B. These results demonstrate that down-regulation of miR-128b and miR-221 is implicated in glucocorticoid resistance and that restoration of their levels is a potentially promising therapeutic in MLL-AF4 ALL.

Aberrant splicing of the E-cadherin transcript is a novel mechanism of gene silencing in chronic lymphocytic leukemia cells

Sharma, S., Lichtenstein, A. — Thu, 05 Nov 2009 09:03:49 PST

Premature termination codon (PTC) mutations are due to insertion or deletion of nucleotides causing a frameshift and premature termination codon in RNA. These transcripts are degraded by the nonsense-mediated decay pathway and have a very short half-life. We used a microarray technique to screen for genes that up-regulate their RNA signal upon nonsense-mediated decay pathway blockade in chronic lymphocytic leukemia (CLL) specimens and identified an E-cadherin transcript with PTC. Sequencing revealed an aberrant E-cadherin transcript lacking exon 11, resulting in a frameshift and PTC. The aberrant E-cadherin transcript was also identified in normal B cells, but occurred at a much lower level compared with CLL cells. In CLL specimens, E-cadherin expression was depressed more than 50% in 62% cases (relative to normal B cells). By real-time polymerase chain reaction analysis, the relative amounts of wild-type transcript inversely correlated with amounts of aberrant transcript (P = .018). Ectopic expression of E-cadherin in CLL specimens containing high amounts of aberrant transcript resulted in down-regulation of the wnt–β-catenin pathway reporter, a pathway known to be up-regulated in CLL. Our data point to a novel mechanism of E-cadherin gene inactivation, with CLL cells displaying a higher proportion of aberrant nonfunctional transcripts and resulting up-regulation of the wnt–β-catenin pathway.

BMS-214662 induces mitochondrial apoptosis in chronic myeloid leukemia (CML) stem/progenitor cells, including CD34+38- cells, through activation of protein kinase C{beta}

Thu, 05 Nov 2009 09:03:49 PST

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder maintained by cancer stem cells. To target this population, we investigated the mechanism of action of BMS-214662, developed as a farnesyl transferase inhibitor (FTI) and unique in inducing apoptosis in these cells. By contrast, a related congener and equally effective FTI, BMS-225975 does not induce apoptosis, indicating a novel mechanism of action. BMS-214662 significantly and selectively induced apoptosis in primitive CD34⁺38^– CML compared with normal cells. Apoptosis proceeded via the intrinsic pathway: Bax conformational changes, loss of mitochondrial membrane potential, generation of reactive oxygen species, release of cytochrome c, and caspase-9/3 activation were noted. Up-regulation of protein kinase Cβ (PKCβ), down-regulation of E2F1, and phosphorylation of cyclin A–associated cyclin-dependent kinase 2 preceded these changes. Cotreatment of CML CD34⁺ and CD34⁺38^– cells with PKC modulators, bryostatin-1, or hispidin markedly decreased these early events and the subsequent apoptosis. None of these events was elicited by BMS-214662 in normal CD34⁺ cells or by BMS-225975 in CML CD34⁺ cells. These data suggest that BMS-214662 selectively elicits a latent apoptotic pathway in CML stem cells that is initiated by up-regulation of PKCβ and mediated by Bax activation, providing a molecular framework for development of novel therapeutics.

E3 ligase-defective Cbl mutants lead to a generalized mastocytosis and myeloproliferative disease

Thu, 05 Nov 2009 09:03:50 PST

Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold. We recently identified the first c-Cbl mutation in human disease in an acute myeloid leukemia patient, called Cbl-R420Q. Here we analyzed the role of Cbl mutants on Kit-mediated transformation. Coexpression of Cbl-R420Q or Cbl-70Z with Kit induced cytokine-independent proliferation, survival, and clonogenic growth. Primary murine bone marrow retrovirally transduced with c-Cbl mutants and transplanted into mice led to a generalized mastocytosis, a myeloproliferative disease, and myeloid leukemia. Overexpression of these Cbl mutants inhibited stem cell factor (SCF)–induced ubiquitination and internalization of Kit. Both Cbl mutants enhanced the basal activation of Akt and prolonged the ligand-dependent activation. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on receptor tyrosine kinases, but independent of their kinase activity. Instead, transformation depends on the Src family kinase Fyn, as c-Cbl coimmunoprecipitated with Fyn and inhibition abolished transformation. These findings may explain primary resistance to tyrosine kinase inhibitors targeted at receptor tyrosine kinases.

A fundamental role of mAbp1 in neutrophils: impact on {beta}2 integrin-mediated phagocytosis and adhesion in vivo

Thu, 05 Nov 2009 09:03:50 PST

The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several β₂ integrin (CD11/CD18)–mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein–tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during β₂ integrin–mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised β₂ integrin–mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor . Functional analysis showed that the down-regulation of mAbp1 diminished the number of β₂ integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.

MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9

Thu, 05 Nov 2009 09:03:50 PST

Megakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell–derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9.

Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5' exons of protein 4.1R gene

Thu, 05 Nov 2009 09:03:50 PST

The tightly regulated production of distinct erythrocyte protein 4.1R isoforms involves differential splicing of 3 mutually exclusive first exons (1A, 1B, 1C) to the alternative 3' splice sites (ss) of exon 2'/2. Here, we demonstrate that exon 1 and 2'/2 splicing diversity is regulated by a transcription-coupled splicing mechanism. We also implicate distinctive regulatory elements that promote the splicing of exon 1A to the distal 3' ss and exon 1B to the proximal 3' ss in murine erythroleukemia cells. A hybrid minigene driven by cytomegalovirus promoter mimicked 1B-promoter–driven splicing patterns but differed from 1A-promoter–driven splicing patterns, suggesting that promoter identity affects exon 2'/2 splicing. Furthermore, splicing factor SF2/ASF ultraviolet (UV) cross-linked to the exon 2'/2 junction CAGAGAA, a sequence that overlaps the distal U2AF³⁵-binding 3' ss. Consequently, depletion of SF2/ASF allowed exon 1B to splice to the distal 3' ss but had no effect on exon 1A splicing. These findings identify for the first time that an SF2/ASF binding site also can serve as a 3' ss in a transcript-dependent manner. Taken together, our results suggest that 4.1R gene expression involves transcriptional regulation coupled with a complex splicing regulatory network.

Plasmodium falciparum-infected erythrocytes induce NF-{kappa}B regulated inflammatory pathways in human cerebral endothelium

Tripathi, A. K., Sha, W., Shulaev, V., Stins, M. F., Sullivan, D. J. — Thu, 05 Nov 2009 09:03:50 PST

Cerebral malaria is a severe multifactorial condition associated with the interaction of high numbers of infected erythrocytes to human brain endothelium without invasion into the brain. The result is coma and seizures with death in more than 20% of cases. Because the brain endothelium is at the interface of these processes, we investigated the global gene responses of human brain endothelium after the interaction with Plasmodium falciparum–infected erythrocytes with either high- or low-binding phenotypes. The most significantly up-regulated transcripts were found in gene ontology groups comprising the immune response, apoptosis and antiapoptosis, inflammatory response, cell-cell signaling, and signal transduction and nuclear factor B (NF-B) activation cascade. The proinflammatory NF-B pathway was central to the regulation of the P falciparum–modulated endothelium transcriptome. The proinflammatory molecules, for example, CCL20, CXCL1, CXCL2, IL-6, and IL-8, were increased more than 100-fold, suggesting an important role of blood-brain barrier (BBB) endothelium in the innate defense during P falciparum–infected erythrocyte (Pf-IRBC) sequestration. However, some of these diffusible molecules could have reversible effects on brain tissue and thus on neurologic function. The inflammatory pathways were validated by direct measurement of proteins in brain endothelial supernatants. This study delineates the strong inflammatory component of human brain endothelium contributing to cerebral malaria.

Chromosome looping at the human {alpha}-globin locus is mediated via the major upstream regulatory element (HS -40)

Thu, 05 Nov 2009 09:03:50 PST

Previous studies in the mouse have shown that high levels of -globin gene expression in late erythropoiesis depend on long-range, physical interactions between remote upstream regulatory elements and the globin promoters. Using quantitative chromosome conformation capture (q3C), we have now analyzed all interactions between 4 such elements lying 10 to 50 kb upstream of the human cluster and their interactions with the -globin promoter. All of these elements interact with the -globin gene in an erythroid-specific manner. These results were confirmed in a mouse model of human globin expression in which the human cluster replaces the mouse cluster in situ (humanized mouse). We have also shown that expression and all of the long-range interactions depend largely on just one of these elements; removal of the previously characterized major regulatory element (called HS –40) results in loss of all the interactions and -globin expression. Reinsertion of this element at an ectopic location restores both expression and the intralocus interactions. In contrast to other more complex systems involving multiple upstream elements and promoters, analysis of the human -globin cluster during erythropoiesis provides a simple and tractable model to understand the mechanisms underlying long-range gene regulation.

Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome

Thu, 05 Nov 2009 09:03:50 PST

The factor H–related protein family (CFHR) is a group of minor plasma proteins genetically and structurally related to complement factor H (fH). Notably, deficiency of CFHR1/CFHR3 associates with protection against age-related macular degeneration and with the presence of anti-fH autoantibodies in atypical hemolytic uremic syndrome (aHUS). We have developed a proteomics strategy to analyze the CFHR proteins in plasma samples from controls, patients with aHUS, and patients with type II membranoproliferative glomerulonephritis. Here, we report on the identification of persons carrying novel deficiencies of CFHR1, CFHR3, and CFHR1/CFHR4A, resulting from point mutations in CFHR1 and CFHR3 or from a rearrangement involving CFHR1 and CFHR4. Remarkably, patients with aHUS lacking CFHR1, but not those lacking CFHR3, present anti-fH autoantibodies, suggesting that generation of these antibodies is specifically related to CFHR1 deficiency. We also report the characterization of a novel CFHR1 polymorphism, resulting from a gene conversion event between CFH and CFHR1, which strongly associates with aHUS. The risk allotype CFHR1*B, with greater sequence similarity to fH, may compete with fH, decreasing protection of cellular surfaces against complement damage. In summary, our comprehensive analyses of the CFHR proteins have improved our understanding of these proteins and provided further insights into aHUS pathogenesis.

Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives

Thu, 05 Nov 2009 09:03:50 PST

We tested the hypothesis that fibrin structure/function is unfavorably altered in patients after idiopathic venous thromboembolism (VTE) and their relatives. Ex vivo plasma fibrin clot permeability, turbidimetry, and efficiency of fibrinolysis were investigated in 100 patients with first-ever VTE, including 34 with pulmonary embolism (PE), 100 first-degree relatives, and 100 asymptomatic controls with no history of thrombotic events. Known thrombophilia, cancer, trauma, and surgery were exclusion criteria. VTE patients and their relatives were characterized by lower clot permeability (P < .001), lower compaction (P < .001), higher maximum clot absorbancy (P < .001), and prolonged clot lysis time (P < .001) than controls, with more pronounced abnormalities, except maximum clot absorbance, in the patients versus relatives (all P < .01). Fibrin clots obtained for PE patients were more permeable, less compact, and were lysed more efficiently compared with deep-vein thrombosis patients (all P < .05) with no differences in their relatives. Being VTE relative, fibrinogen, and C-reactive protein were independent predictors of clot permeability and fibrinolysis time in combined analysis of controls and relatives. We conclude that altered fibrin clot features are associated with idiopathic VTE with a different profile of fibrin variables in PE. Similar features can be detected in VTE relatives. Fibrin properties might represent novel risk factors for thrombosis.

Immunogenicity of blood group antigens: a mathematical model corrected for antibody evanescence with exclusion of naturally occurring and pregnancy-related antibodies

Tormey, C. A., Stack, G. — Thu, 05 Nov 2009 09:03:50 PST

Blood group antigen immunogenicity is a crucial factor in red blood cell alloimmunization. Previous calculated estimates of immunogenicity suffered from several key shortcomings. To address these issues we have (1) introduced a correction factor for antibody persistence rates into traditional immunogenicity calculations, (2) calculated immunogenicities only in men to eliminate pregnancy-related antibodies, and (3) excluded antibodies reactive only at room temperature to minimize the contribution of naturally occurring antibodies. With these corrections, we have calculated the immunogenicities of common blood group antigens using data collected on clinically significant alloantibodies (n = 452) in a male patient population. We observed a 3- to 5-fold increase in immunogenicity for some antigens (ie, Jk^a, C^w, Lu^a) and smaller changes in others compared with traditionally calculated estimates. In addition, we have calculated the transfusion-related immunogenicities of antigens traditionally associated with naturally occurring antibodies (eg, anti-Le^a, -Le^b, -M, and -P₁).

Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation

Thu, 05 Nov 2009 09:03:50 PST

Viral infection or reactivation remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. We now show that infusions of single cytotoxic T lymphocyte (CTL) lines (5 x 10⁶-1.35 x 10⁸ cells/m²) with specificity for 2 commonly detected viruses, Epstein-Barr virus (EBV) and adenovirus, can be safely administered to pediatric transplantation recipients receiving partially human leukocyte antigen–matched and haploidentical stem cell grafts (n = 13), without inducing graft-versus-host disease. The EBV-specific component of the CTLs expanded in vivo and persisted for more than 12 weeks, but the adenovirus-specific component only expanded in vivo in the presence of concomitant adenoviral infection. Nevertheless, adenovirus-specific T cells could be detected for at least 8 weeks in peripheral blood, even in CTL recipients without viral infection, provided the adenovirus-specific component of their circulating lymphocytes was first expanded by exposure to adenoviral antigens ex vivo. After infusion, none of these 13 high-risk recipients developed EBV-associated lymphoproliferative disease, while 2 of the subjects had resolution of their adenoviral disease. Hence, bispecific CTLs containing both EBV- and adenovirus-specific T cells can safely reconstitute an antigen responsive "memory" population of CTLs after human leukocyte antigen–mismatched stem cell transplantation and may provide antiviral activity. This trial was registered at www.clinicaltrials.gov as #NCT00590083.

Relapse risk after umbilical cord blood transplantation: enhanced graft-versus-leukemia effect in recipients of 2 units

Thu, 05 Nov 2009 09:03:50 PST

Umbilical cord blood (UCB) transplantation is potentially curative for acute leukemia. This analysis was performed to identify risk factors associated with leukemia relapse following myeloablative UCB transplantation. Acute leukemia patients (n = 177; 88 with acute lymphoblastic leukemia and 89 with acute myeloid leukemia) were treated at a single center. Patients received a UCB graft composed of either 1 (47%) or 2 (53%) partially human leukocyte antigen (HLA)–matched unit(s). Conditioning was with cyclophosphamide and total body irradiation with or without fludarabine. The incidence of relapse was 26% (95% confidence interval [CI], 19%-33%). In multivariate analysis, relapse was higher in advanced disease patients (≥ third complete remission [CR3]; relative risk [RR], 3.6; P < .01), with a trend toward less relapse in recipients of 2 UCB units (RR = 0.6; P = .07). However, relapse was lower for CR1-2 patients who received 2 UCB units (RR 0.5; P < .03). Leukemia-free survival was 40% (95% CI, 30%-51%) and 51% (95% CI, 41%-62%) for single- and double-unit recipients, respectively (P = .35). Although it is known that transplantation in CR1 and CR2 is associated with less relapse risk, this analysis reveals an enhanced graft-versus-leukemia effect in acute leukemia patients after transplantation with 2 partially HLA-matched UCB units. This trial was registered at http://clinicaltrials.gov as NCT00309842.

Repulsive axon guidance molecule Slit3 is a novel angiogenic factor

Zhang, B., Dietrich, U. M., Geng, J.-G., Bicknell, R., Esko, J. D., Wang, L. — Thu, 05 Nov 2009 09:03:50 PST

Slits are large, secreted repulsive axon guidance molecules. Recent genetic studies revealed that the Slit3 is dispensable for neural development but required for non-neuron–related developmental processes, such as the genesis of the diaphragm and kidney. Here we report that Slit3 potently promotes angiogenesis, a process essential for proper organogenesis during embryonic development. We observed that Slit3 is expressed and secreted by both endothelial cells and vascular smooth muscle cells in vasculature and that the Slit cognate receptors Robo1 and Robo4 are universally expressed by endothelial cells, suggesting that Slit3 may act in paracrine and autocrine manners to regulate endothelial cells. Cellular function studies revealed that Slit3 stimulates endothelial-cell proliferation, promotes endothelial-cell motility and chemotaxis via interaction with Robo4, and accelerates endothelial-cell vascular network formation in vitro with a specific activity comparable with vascular endothelial growth factor. Furthermore, Slit3 stimulates neovessel sprouting ex vivo and new blood vessel growth in vivo. Consistent with these observations, the Slit3 knockout mice display disrupted angiogenesis during embryogenesis. Taken together, our studies reveal that the repulsive axon guidance molecule Slit3 is a novel and potent angiogenic factor and functions to promote angiogenesis in coordinating organogenesis during embryonic development.

Bone marrow stem and progenitor cell contribution to neovasculogenesis is dependent on model system with SDF-1 as a permissive trigger

Thu, 05 Nov 2009 09:03:50 PST

Adult bone marrow (BM) contributes to neovascularization in some but not all settings, and reasons for these discordant results have remained unexplored. We conducted novel comparative studies in which multiple neovascularization models were established in single mice to reduce variations in experimental methodology. In different combinations, BM contribution was detected in ischemic retinas and, to a lesser extent, Lewis lung carcinoma cells, whereas B16 melanomas showed little to no BM contribution. Using this spectrum of BM contribution, we demonstrate the necessity for site-specific expression of stromal-derived factor-1 (SDF-1) and its mobilizing effects on BM. Blocking SDF-1 activity with neutralizing antibodies abrogated BM-derived neovascularization in lung cancer and retinopathy. Furthermore, secondary transplantation of single hematopoietic stem cells (HSCs) showed that HSCs are a long-term source of neovasculogenesis and that CD133⁺CXCR4⁺ myeloid progenitor cells directly participate in new blood vessel formation in response to SDF-1. The varied BM contribution seen in different model systems is suggestive of redundant mechanisms governing postnatal neovasculogenesis and provides an explanation for contradictory results observed in the field.

Cutaneous tumor lysis syndrome in a patient with HTLV-1 adult T-cell lymphoma/leukemia

Bouaziz, J.-D., Cordel, N., Hickman, G., Fieschi, C., Ortonne, N., Bagot, M. — Thu, 05 Nov 2009 09:03:50 PST

Integrity of the CBL gene in mature B-cell malignancies

McKeller, M. R., Robetorye, R. S., Dahia, P. L. M., Aguiar, R. C. T. — Thu, 05 Nov 2009 09:03:51 PST