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1.  Sustained Induction of Fetal Hemoglobin by Pulse Butyrate Therapy in Sickle Cell Disease 
Blood  1999;93(6):1790-1797.
High levels of fetal hemoglobin (Hb F) protect from many of the complications of sickle cell disease and lead to improved survival. Butyrate and other short chain fatty acids were previously shown to increase Hb F production in erythroid cells in vitro and in animal models in vivo. However, butyrates are also known to inhibit the proliferation of many cell types, including erythroid cells. Experience with the use of butyrate in animal models and in early clinical trials demonstrated that the Hb F response may be lost after prolonged administration of high doses of butyrate. We hypothesized that this loss of response may be a result of the antiproliferative effects of butyrate. We designed a regimen consisting of intermittent or pulse therapy in which butyrate was administered for 4 days followed by 10 to 24 days with no drug exposure. This pulse regimen induced fetal globin gene expression in 9 of 11 patients. The mean Hb F in this group increased from 7.2% to 21.0% (P < .002) after intermittent butyrate therapy for a mean duration of 29.9 weeks. This was associated with a parallel increase in the number of F cells and F reticulocytes. The total hemoglobin levels also increased from a mean of 7.8 g/dL to a mean of 8.8 g/dL (P < .006). The increased levels of Hb F were sustained in all responders, including 1 patient who has been on pulse butyrate therapy for more than 28 months. This regimen, which resulted in a marked and sustained increase in Hb F levels in more than two thirds of the adult sickle cell patients enrolled in this study, was well tolerated without adverse side effects. These encouraging results require confirmation along with an appropriate evaluation of clinical outcomes in a larger number of patients with sickle cell disease.
PMCID: PMC4269326  PMID: 10068649
2.  Short-chain fatty acid derivatives induce fetal globin expression and erythropoiesis in vivo 
Blood  2002;100(13):4640-4648.
Orally bioactive compounds that induce γ globin gene expression at tolerable doses are needed for optimal treatment of the β-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce γ globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce γ globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which γ globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce γ globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human β globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, γ globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate γ globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the β hemoglobinopathies and useful for the oral treatment of other anemias.
doi:10.1182/blood-2002-02-0353
PMCID: PMC4269367  PMID: 12393583
3.  Short-chain fatty acid derivatives stimulate cell proliferation and induce STAT-5 activation 
Blood  2001;97(10):3259-3267.
Current chemotherapeutic and butyrate therapeutics that induce fetal hemoglobin expression generally also suppress erythropoiesis, limiting the production of cells containing fetal hemoglobin (F cells). Recently, selected short-chain fatty acid derivatives (SCFADs) were identified that induce endogenous γ-globin expression in K562 cells and human burst-forming units–erythroid and that increase proliferation of human erythroid progenitors and a multilineage interleukin-3–depen-dent hematopoietic cell line. In this report, γ-globin inducibility by these SCFADs was further demonstrated in mice transgenic for the locus control region and the entire β-globin gene locus in a yeast artificial chromosome and in 2 globin promoter-reporter assays. Conditioned media experiments strongly suggest that their proliferative activity is a direct effect of the test compounds. Investigation of potential mechanisms of action of these SCFADs demonstrates that these compounds induce prolonged expression of the growth-promoting genes c-myb and c-myc. Both butyrate and specific growth-stimulatory SCFADs induced prolonged signal transducer and activator of transcription (STAT)-5 phosphorylation and activation, and c-cis expression, persisting for more than 120 minutes, whereas with IL-3 alone phosphorylation disappeared within minutes. In contrast to butyrate treatment, the growth-stimulating SCFADs did not result in bulk histone H4 hyperacetylation or induction of p21 Waf/Cip, which mediates the suppression of cellular growth by butyrate. These findings suggest that the absence of bulk histone hyperacetylation and p21 induction, but prolonged induction of cis, myb, myc, and STAT-5 activation, contribute to the cellular proliferation induced by selected SCFADs.
PMCID: PMC4263369  PMID: 11342457
4.  Small sizes and indolent evolutionary dynamics challenge the potential role of P2RY8-CRLF2–harboring clones as main relapse-driving force in childhood ALL 
Blood  2012;120(26):5134-5142.
The P2RY8-CRLF2 fusion defines a particular relapse-prone subset of childhood acute lymphoblastic leukemia (ALL) in Italian Association of Pediatric Hematology and Oncology Berlin-Frankfurt-Münster (AIEOP-BFM) 2000 protocols. To investigate whether and to what extent different clone sizes influence disease and relapse development, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high hyperdiploidy, representing approximately 50% of all cases, 67 (25%) were P2RY8-CRLF2 positive. The respective clone sizes were ≥ 20% in 27% and < 20% in 73% of them. The cumulative incidence of relapse of the entire fusion-positive group was clone size independent and significantly higher than that of the fusion-negative group (35% ± 8% vs 13% ± 3%, P = .008) and primarily confined to the non–high-risk group. Of 22 P2RY8-CRLF2–positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2–positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2–positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone.
doi:10.1182/blood-2012-07-443218
PMCID: PMC4194314  PMID: 23091296
5.  RNAi-mediated silencing of TEL/AML1 reveals a heat-shock protein— and survivin–dependent mechanism for survival 
Blood  2006;109(6):2607-2610.
The TEL/AML1 fusion gene results from the most frequent t(12;21)(p13;q22) translocation in childhood acute lymphoblastic leukemia (ALL). Its contribution to transformation is largely unknown, in particular with respect to survival and apoptosis. We therefore silenced TEL/AML1 expression in leukemic REH cells by RNA inhibition, which eventually led to programmed cell death. Microarray and 2D gel electrophoresis data demonstrated a differential regulation of heat-shock proteins (HSPs), among them HSP90, as well as of its client, survivin. Consistent with these findings, ectopic expression of TEL/AML1 in Ba/F3 cells increased protein levels of HSP90 and survivin and conferred resistance to apoptotic stimuli. Our data suggest that TEL/AML1 not only contributes to leukemogenesis by affecting an antiapoptotic network but also seems to be indispensable for maintaining the malignant phenotype. The functional relationship between TEL/AML1, HSP90, and survivin provides the rational for targeted therapy, be it the fusion gene or the latter 2 proteins.
doi:10.1182/blood-2006-04-019612
PMCID: PMC4194423  PMID: 17095626
6.  Immune evasion by oncogenic proteins of acute myeloid leukemia 
Blood  2014;123(10):1535-1543.
PML-RARA and AML1-ETO are important oncogenic fusion proteins that play a central role in transformation to acute myeloid leukemia (AML). Whether these fusion proteins render the tumor cells with immune evasion properties is unknown. Here we show that both oncogenic proteins specifically downregulate the expression of CD48, a ligand of the natural killer (NK) cell activating receptor 2B4, thereby leading to decreased killing by NK cells. We demonstrate that this process is histone deacetylase (HDAC)-dependent, that it is mediated through the downregulation of CD48 messenger RNA, and that treatment with HDAC inhibitors (HDACi) restores the expression of CD48. Furthermore, by using chromatin immuoprecepitation (ChIP) experiments, we show that AML1-ETO directly interacts with CD48. Finally, we show that AML patients who are carrying these specific translocations have low expression of CD48.
doi:10.1182/blood-2013-09-526590
PMCID: PMC3981677  PMID: 24449212
7.  A Randomized Controlled Phase III Trial of Recombinant Human Granulocyte Colony-Stimulating Factor (Filgrastim) for Treatment of Severe Chronic Neutropenia 
Blood  1993;81(10):2496-2502.
Patients with idiopathic, cyclic, and congenital neutropenia have recurrent severe bacterial infections. One hundred twenty-three patients with recurrent infections and severe chronic neutropenia (absolute neutrophil count < 0.5 × 109/L) due to these diseases were enrolled in this multi-center phase III trial. They were randomized to either immediately beginning recombinant human granulocyte colony-stimulating factor (filgrastim) (3.45 to 11.50 μg/kg/d, subcutaneously) or entering a 4-month observation period followed by filgrastim administration. Blood neutrophil counts, bone marrow (BM) cell histology, and incidence and duration of infection-related events were monitored. Of the 123 patients enrolled, 120 received filgrastim. On therapy, 108 patients had a median absolute neutrophil count of ≥ 1.5 × 109/L. Examination of BM aspirates showed increased proportions of maturing neutrophils. Infection-related events were significantly decreased (P < .05) with approximately 50% reduction in the incidence and duration of infection-related events and almost 70% reduction in duration of antibiotic use. Asymptomatic splenic enlargement occurred frequently: adverse events frequently reported were bone pain, headache, and rash, which were generally mild and easily manageable. These data indicate that treatment of patients with severe chronic neutropenia with filgrastim results in a stimulation of BM production and maturation of neutrophils, an increase in circulating neutrophils, and a reduction in infection-related events.
PMCID: PMC4120868  PMID: 8490166
8.  Osteoclast-gene expression profiling reveals osteoclast-derived CCR2 chemokines promoting myeloma cell migration 
Blood  2010;117(4):1280-1290.
Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells (multiple myeloma cells, MMC), primarily in the bone marrow (BM). Osteolytic bone lesions are detected in 80% of patients, due to increased osteoclastic bone resorption and reduced osteoblastic bone formation. MMC are found closely associated to sites of increased bone resorption. Osteoclasts strongly support MMC survival and vice versa in vitro. To further elucidate the mechanisms involved in osteoclast/MMC interaction, we have identified 552 genes overexpressed in osteoclasts compared to other BM cell subpopulations. Osteoclasts express specifically genes coding for four CCR2-targeting chemokines, and genes coding for MMC growth factors (IGF-1, APRIL). An anti-CCR2 MoAb blocked osteoclast chemoattractant activity for MMC and CCR2-chemokines are also MMC growth factors, promoting MAPK activation in MMC. An anti-IGF-1 receptor MoAb completely blocked the osteoclast-induced survival of MMC suppressing both osteoclast and MMC survival. Specific APRIL or IL-6 inhibitors partially blocked osteoclast-induced MMC survival. These in-vitro data may explain why newly-diagnosed patients whose MMC express high levels of CCR2 present numerous bone lesions.
Taken together, this study displays additional mechanisms involved in osteoclast/MMC interaction and suggests using CCR2 and/or IGF-1 targeting strategies to block this interaction and prevent drug resistance.
doi:10.1182/blood-2010-04-279760
PMCID: PMC4009699  PMID: 21097672
Aged; Bone Resorption; metabolism; pathology; Cell Communication; genetics; physiology; Cell Movement; genetics; Cells, Cultured; Chemotactic Factors; genetics; metabolism; Disease Progression; Gene Expression Profiling; Humans; Microarray Analysis; Middle Aged; Multiple Myeloma; genetics; metabolism; pathology; Neoplasm Metastasis; Osteoclasts; metabolism; physiology; Receptors, CCR2; genetics; metabolism; physiology; Multiple Myeloma; osteoclast gene
9.  Does increased red blood cell deformability raise the risk for osteonecrosis in sickle cell anemia? 
Blood  2013;121(15):3054-3056.
doi:10.1182/blood-2013-01-480277
PMCID: PMC3988032  PMID: 23580637
Aged; Anemia, Sickle Cell; blood; drug therapy; Antisickling Agents; therapeutic use; Erythrocyte Deformability; Female; Humans; Hydroxyurea; therapeutic use; Logistic Models; Male; Middle Aged; Multivariate Analysis; Osteonecrosis; blood; Risk Assessment; Risk Factors; Young Adult
10.  CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice 
Blood  2012;119(17):4073-4082.
Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class II-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knock-out of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.
doi:10.1182/blood-2011-08-374645
PMCID: PMC3986681  PMID: 22394599
11.  Interferons limit inflammatory responses by induction of tristetraprolin 
Blood  2006;107(12):4790-4797.
Interferons (IFNs) are cytokines with pronounced proinflammatory properties. Here we provide evidence that IFNs play a key role also in decline of inflammation by inducing expression of tristetraprolin (TTP). TTP is an RNA-binding protein that destabilizes several AU-rich element-containing mRNAs including TNFα. By promoting mRNA decay TTP significantly contributes to cytokine homeostasis. Now we report that IFNs strongly stimulate expression of TTP if a co-stimulatory stress signal is provided. IFN-induced expression of TTP depends on the IFN-activated transcription factor STAT1, and the co-stimulatory stress signal requires p38 MAPK. Within the TTP promoter we have identified a functional gamma interferon-activated sequence that recruits STAT1. Consistently, STAT1 is required for full expression of TTP in response to LPS that stimulates both p38 MAPK and, indirectly, interferon signaling. We demonstrate that in macrophages IFN-induced TTP protein limits LPS-stimulated expression of several proinflammatory genes such as TNFα, IL-6, Ccl2 and Ccl3. Thus, our findings establish a link between interferon responses and TTP-mediated mRNA decay during inflammation, and propose a novel immunomodulatory role of IFNs.
doi:10.1182/blood-2005-07-3058
PMCID: PMC3963709  PMID: 16514065
Immunobiology; innate immunity; monocyte and macrophage biology
12.  HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell–mediated killing 
Blood  2009;115(7):1354-1363.
HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/ stress-sensing ATR kinase and promotes G2 cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4+ T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1–infected cells to NK cell–mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell–mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1–induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1–induced CD4+ T-lymphocyte depletion but may also take part in HIV-1–induced NK-cell dysfunction.
doi:10.1182/blood-2009-08-237370
PMCID: PMC3955183  PMID: 20008788 CAMSID: cams4124
13.  Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells 
Blood  2004;105(1):145-152.
Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD.
doi:10.1182/blood-2004-02-0464
PMCID: PMC3938105  PMID: 15331450
14.  Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of dyskeratosis congenita 
Blood  2012;120(15):2990-3000.
TRF1 is part of the shelterin complex, which binds telomeres and it is essential for their protection. Ablation of TRF1 induces sister telomere fusions and aberrant numbers of telomeric signals associated with telomere fragility. Dyskeratosis congenita is characterized by a mucocutaneous triad, bone marrow failure (BMF), and presence of short telomeres because of mutations in telomerase. A subset of patients, however, show mutations in the shelterin component TIN2, a TRF1-interacting protein, presenting a more severe phenotype and presence of very short telomeres despite normal telomerase activity. Allelic variations in TRF1 have been found associated with BMF. To address a possible role for TRF1 dysfunction in BMF, here we generated a mouse model with conditional TRF1 deletion in the hematopoietic system. Chronic TRF1 deletion results in increased DNA damage and cellular senescence, but not increased apoptosis, in BM progenitor cells, leading to severe aplasia. Importantly, increased compensatory proliferation of BM stem cells is associated with rapid telomere shortening and further increase in senescent cells in vivo, providing a mechanism for the very short telomeres of human patients with mutations in the shelterin TIN2. Together, these results represent proof of principle that mutations in TRF1 lead to the main clinical features of BMF.
doi:10.1182/blood-2012-03-418038
PMCID: PMC3918664  PMID: 22932806
15.  Multiple Myeloma: Increasing Evidence for a Multistep Transformation Process 
Blood  1998;91(1):3-21.
Multiple Myeloma (Mm) is a clonal B-cell neoplasm that affects terminally differentiated B cells (ie, plasma cells) and may proceed through different phases: an inactive phase in which tumor cells are nonproliferating mature plasma cells, an active phase with a small percentage (<1%) of proliferating plasmablastic cells, and a fulminant phase with the frequent occurrence of extramedullary proliferation and an increase in plasmablastic cells. During the past years, considerable progress has been made in identifying some of the critical components of neoplastic transformation in MM. This review intends to propose a model of a stepwise malignant transformation during MM pathogenesis. Both diagnostic and therapeutic implications of this model will be discussed.
PMCID: PMC3901996  PMID: 9414264
16.  NKG2D receptor–mediated NK cell function is regulated by inhibitory Ly49 receptors 
Blood  2004;105(1):233-240.
Interaction of the activating ligand H60 with NKG2D receptor constitutes a major stimulatory pathway for natural killer (NK) cells. The influence of inhibitory Ly49 receptors on NKG2D-mediated activation is not clearly understood. Here we show that the magnitude of NKG2D-mediated cytotoxicity is directly proportional to both the levels of H60 and the nature of major histocompatibility complex (MHC) class I molecules expressed on the target cells. The expression levels of H60 on the target cells determined the extent to which the inhibition by Ly49C/I receptors can be overridden. In contrast, even a higher expression of H60 molecule on the target cells failed to overcome the inhibition mediated by Ly49A/G receptors. Also, the level of interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) generated by NK cells through anti-NKG2D monoclonal antibody (mAb)–mediated activation is significantly reduced by the presence of immobilized anti-Ly49A/G mAbs. Thus, NKG2D-mediated cytotoxicity and cytokine secretion results from the fine balance between activating and inhibitory receptors, thereby defining the NK cell–mediated immune responses.
doi:10.1182/blood-2004-03-1075
PMCID: PMC3889208  PMID: 15328154
17.  Histone deacetylase (HDAC) 1 and 2 are Essential for normal T cell Development and Genomic Stability in Mice 
Blood  2013;121(8):10.1182/blood-2012-07-441949.
Histone deacetylase 1 and 2 (HDAC1/2) regulate chromatin structure as the catalytic core of the Sin3A, NuRD and CoREST co-repressor complexes. To better understand the key pathways regulated by HDAC1/2 in the adaptive immune system and inform their exploitation as drug targets, we have generated mice with a T cell specific deletion. Loss of either HDAC1 or HDAC2 alone has little effect, while dual inactivation results in a 5-fold reduction in thymocyte cellularity, accompanied by developmental arrest at the double-negative to double-positive transition. Transcriptome analysis revealed 892 mis-regulated genes in Hdac1/2 knock-out thymocytes, including down-regulation of LAT, Themis and Itk, key components of the T cell receptor (TCR) signalling pathway. Down-regulation of these genes suggests a model in which HDAC1/2 deficiency results in defective propagation of TCR signalling, thus blocking development. Furthermore, mice with a single Hdac2 allele, develop a lethal pathology by 3-months of age, a result of neoplastic transformation of immature T cells in the thymus. Tumor cells become aneuploid, express increased levels of c-Myc and show elevated levels of the DNA damage marker, γH2AX. These data demonstrate a crucial role for HDAC1/2 in T cell development and the maintenance of genomic stability.
doi:10.1182/blood-2012-07-441949
PMCID: PMC3836254  PMID: 23287868
Deacetylase; chromatin; T cell; development and cancer
18.  Siglec-9 is a novel leukocyte ligand for Vascular Adhesion Protein-1 and can be utilized in PET-imaging of inflammation and cancer 
Blood  2011;118(13):10.1182/blood-2010-09-311076.
Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular Adhesion Protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1 dependent manner, the counter-receptor(s) on this leukocyte population is not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography the 68Gallium- labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging such conditions as inflammation and cancer.
doi:10.1182/blood-2010-09-311076
PMCID: PMC3833035  PMID: 21821708
19.  Hedgehog signalling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signalling in zebrafish 
Blood  2012;120(2):10.1182/blood-2011-10-383729.
Multiple signalling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), Vascular Endothelial Growth Factor (VEGF) and Notch signalling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF or Notch inhibition suggest that not all of the effects of Hh on arterial-venous specification, are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting and haematopoietic stem cell (HSC) gene expression. Importantly, whilst VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signalling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signalling during arteriovenous specification. Finally, our experiments establish a dual function of Hedgehog during induction of runx1+ HSCs.
doi:10.1182/blood-2011-10-383729
PMCID: PMC3833038  PMID: 22668851
20.  MST1 mutations in autosomal recessive primary immunodeficiency characterized by defective naive T-cell survival 
Blood  2011;119(15):10.1182/blood-2011-09-378364.
The molecular mechanisms that underlie T-cell quiescence are poorly understood. In the present study, we report a primary immunodeficiency phenotype associated with MST1 deficiency and primarily characterized by a progressive loss of naive T cells. The in vivo consequences include recurrent bacterial and viral infections and autoimmune manifestations. MST1-deficient T cells poorly expressed the transcription factor FOXO1, the IL-7 receptor, and BCL2. Conversely, FAS expression and the FAS-mediating apoptotic pathway were up-regulated. These abnormalities suggest that increased cell death of naive and proliferating T cells is the main mechanism underlying this novel immunodeficiency. Our results characterize a new mechanism in primary T-cell immunodeficiencies and highlight a role of the MST1/FOXO1 pathway in controlling the death of human naive T cells.
doi:10.1182/blood-2011-09-378364
PMCID: PMC3824282  PMID: 22174160
21.  Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex 
Blood  2012;119(17):10.1182/blood-2011-09-382556.
Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1–dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter’s secretion at the immune synapse.
doi:10.1182/blood-2011-09-382556
PMCID: PMC3824283  PMID: 22308290
22.  Human iNKT and MAIT cells exhibit a PLZF-dependent proapoptotic propensity that is counterbalanced by XIAP 
Blood  2012;121(4):10.1182/blood-2012-09-456095.
Invariant natural killer (iNKT) T cells and mucosal-associated invariant T (MAIT) cells represent peculiar T-lymphocyte subpopulations with innate-like properties that differ from conventional T cells. iNKT are reduced in the primary immunodeficiency caused by mutations in the X-linked inhibitor of apoptosis (XIAP). By studying the mechanism of this depletion, we herein report that iNKT cells exhibit a high susceptibility to apoptosis that is not observed with conventional T cells. Elevated expression of caspases 3 and 7 accounts for the proapoptotic phenotype of iNKT cells, which is inhibited by XIAP although it exerts a moderate effect in conventional T cells. Similarly, MAIT cells exhibit a proapoptotic propensity with elevated expression of activated caspases and are decreased in XIAP-deficient individuals. Knockdown of the transcription factor PLZF/ZBTB-16, which is involved in the effector program of iNKT cells, diminishes their proapoptotic phenotype. Conversely, overexpression of PLZF/ZBTB-16 in conventional T cells leads to a proapoptotic phenotype. Our findings identify a previously unknown pathway of regulation of innate-like T-cell homeostasis depending on XIAP and PLZF. The proapoptotic feature of iNKT cells also gives a reliable explanation of their exhaustion observed in different human conditions including the XIAP immunodeficiency.
doi:10.1182/blood-2012-09-456095
PMCID: PMC3824284  PMID: 23223428
23.  MYC+ diffuse large B-cell lymphoma is not salvaged by classical R-ICE or R-DHAP followed by BEAM plus autologous stem cell transplantation 
Blood  2012;119(20):4619-4624.
Approximately 5–10% of diffuse large B-cell lymphomas (DLBCL) harbor a 8q24/MYC rearrangement (MYC +). We determined the prognostic significance of MYC rearrangement in patients with relapsed/refractory DLBCL prospectively treated by R-ICE or R-DHAP followed by high-dose therapy and autologous stem cell transplantation. Twenty-eight (17%) of the 161 patients analyzed presented a MYC + rearrangement, targeted as either simple hit (25%) or complex hits (n=75%) including MYC/BCL2, MYC/BCL6, and MYC/BCL2/BCL6. Results were statistically highly concordant in matched primary and relapsed biopsies (n=45). Compared to the MYC − DLBCL patients, the MYC + DLBCL patients presented with a more elevated lactico-deshydrogenase level (p=.0006) and a more advanced age-adjusted international pronostic index (p=.0039). The 4-year PFS and OS were significantly lower in the MYC + DLBCL patients than those in the MYC − DLBCL patients, with rates of 18% vs. 42% (p=.0322), and of 29% vs. 62% (p=.0113), respectively. Type of treatment, R-DHAP or R-ICE had no impact on survivals, with 4-year PFS rates of 17% vs. 19% and 4-year OS rates of 26% vs. 31%. In conclusion, MYC rearrangement is an early event in DLBCL. MYC + DLBCL patients have a significant inferior prognosis than MYC − DLBCL patients. Their outcome was not influenced by the proposed salvage therapy.
doi:10.1182/blood-2012-01-406033
PMCID: PMC3815438  PMID: 22408263
Adult; Aged; Antibodies, Monoclonal, Murine-Derived; administration & dosage; adverse effects; Antineoplastic Combined Chemotherapy Protocols; administration & dosage; adverse effects; Carmustine; administration & dosage; adverse effects; Chemotherapy, Adjuvant; Cisplatin; administration & dosage; adverse effects; Combined Modality Therapy; Cytarabine; administration & dosage; adverse effects; Dexamethasone; administration & dosage; adverse effects; Etoposide; administration & dosage; adverse effects; Female; Genes, myc; physiology; Hematopoietic Stem Cell Transplantation; methods; Humans; Ifosfamide; administration & dosage; adverse effects; Immunotherapy; Lymphoma, Large B-Cell, Diffuse; drug therapy; genetics; mortality; therapy; Male; Melphalan; administration & dosage; adverse effects; Middle Aged; Podophyllotoxin; administration & dosage; adverse effects; Salvage Therapy; Transplantation, Autologous; Treatment Failure; Young Adult
24.  A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy 
Blood  2012;119(8):10.1182/blood-2011-09-380048.
Regulatory T cells (CD4+CD25hiCD127lo-FOXP3+ T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4+CD25− [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.
doi:10.1182/blood-2011-09-380048
PMCID: PMC3816350  PMID: 22219224
25.  Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells 
Blood  2003;101(10):4201-4208.
Rodent bone marrow cells can contribute to liver. If these findings are applicable to humans, marrow stem cells could theoretically be harvested from a patient and used to repair his/her damaged liver. To explore this potential, CD34+ or highly purified CD34+CD38−CD7−human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice. One month after transplantation, carbon tetrachloride (CCl4) was administered into the mice to induce liver damage and hepatocyte proliferation. Mice were analyzed in comparison with CCl4-injured mice that did not receive transplants and noninjured controls that received transplants with the same stem cell populations, one month after liver damage. Human-specific albumin mRNA and protein were expressed in the mouse liver and human albumin was detected in the serum of mice that had received CCl4 injury. Human alpha-fetoprotein was never expressed, but in some mice, human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin-secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations in injured mice that did not receive transplants nor in noninjured mice that had received transplants of human stem cells. Human albumin expression was detected only in CCl4-treated mice that received transplants of human stem cells, and recovery was increased by administration of human hepatocyte growth factor 48 hours after the CCl4-mediated liver injury. Our studies provide evidence that human “hematopoietic” stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression.
doi:10.1182/blood-2002-05-1338
PMCID: PMC3781008  PMID: 12560238

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