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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  The Effect of Rituximab on Vaccine Responses in Patients with Immune Thrombocytopenia 
Blood  2013;122(11):1946-1953.
B-cell depletion therapy may impair vaccine responses and increase infection risk in patients with immune thrombocytopenia (ITP). Capitalizing on a multicenter randomized placebo-controlled trial, we investigated the effects of rituximab on the antibody and cellular responses to Streptococcus pneumoniae polysaccharide vaccine and Haemophilus influenzae type b (Hib) conjugate vaccine in ITP patients. Of 60 patients in the main trial, 24 patients received both vaccines 6 months after rituximab (n=17) or placebo (n=7). Among 20 evaluable patients, 3/14 (21%) in the rituximab group and 4/6 (67%) in the placebo group achieved a 4-fold increase in anti-pneumococcal antibodies (p=0.12). For anti-Hib antibodies, 4/14 (29%) and 5/6 (83%), respectively, achieved a 4-fold increase (p<0.05). Fewer patients in the rituximab group demonstrated functional Hib killing (2/14 [14%] versus 5/6 [83%], p<0.05). Three of 14 rituximab-treated patients failed to respond to vaccines by any criteria. After vaccinations, pre-plasma cell blasts and interferon-γ secreting T-cells were reduced in rituximab-treated patients. We found that antibody responses were impaired for at least 6 months after rituximab. Cellular immunity was reduced in parallel with the depleted B-cell pool. These findings have implications for the timing of vaccinations and the mechanism of infection after rituximab in patients with ITP.
doi:10.1182/blood-2013-04-494096
PMCID: PMC3773242  PMID: 23851398 CAMSID: cams3208
20.  C/EBPα initiates primitive myelopoiesis in pluripotent embryonic cells 
Blood  2009;114(1):40-48.
The molecular mechanisms that underlie the development of primitive myeloid cells in vertebrate embryos are not well understood. Here we characterize the role of cebpa during primitive myeloid cell development in Xenopus. We show that cebpa is one of the first known hematopoietic genes expressed in the embryo. Loss and gain-of-function studies show that it is both necessary and sufficient for the development of functional myeloid cells. In addition, we show that cebpa misexpression leads to the precocious induction of myeloid cell markers in pluripotent prospective ectodermal cells, without the cells transitioning through a general mesodermal state. Finally we use live imaging to show that cebpa expressing cells exhibit many attributes of terminally differentiated myeloid cells, such as highly active migratory behavior, the ability to quickly and efficiently migrate toward wounds and phagocytose bacteria, and the ability to enter the circulation. Thus C/EPBα is the first known single factor capable of initiating an entire myelopoeisis pathway in pluripotent cells in the embryo.
doi:10.1182/blood-2008-11-189159
PMCID: PMC3747498  PMID: 19420355
blood; myeloid; primitive macrophage; primitive myelopoiesis; Xenopus
21.  Adeno-Associated Virus Vectors and Hematology 
Blood  1999;94(3):864-874.
PMCID: PMC3739711  PMID: 10419876
22.  KAP1 regulates gene networks controlling mouse B lymphoid cell differentiation and function 
Blood  2012;119(20):4675-4685.
Chromatin remodeling is fundamental for B cell differentiation. Here, we explored the role in this process of KAP1, the cofactor of KRAB-ZFP transcriptional repressors. B lymphoid-specific Kap1 knockout mice displayed reduced numbers of mature B cells, lower steady-state levels of antibodies and accelerated rates of decay of neutralizing antibodies following viral immunization. Transcriptome analyses of Kap1-deleted B splenocytes revealed an upregulation of PTEN, the enzymatic counter-actor of PIK3 signaling, and of genes encoding DNA damage response factors, cell-cycle regulators and chemokine receptors. ChIP/seq studies established that KAP1 bound at or close to a number of these genes, and controlled chromatin status at their promoters. Genome-wide, KAP1-binding sites avoided active B cell-specific enhancers and were enriched in repressive histone marks, further supporting a role for this molecule in gene silencing in vivo. Likely responsible for tethering KAP1 to at least some of these targets, a discrete subset of KRAB-ZFPs is enriched in B lymphocytes. This work thus reveals the role of KRAB/KAP1-mediated epigenetic regulation in B cell development and homeostasis.
doi:10.1182/blood-2011-12-401117
PMCID: PMC3683646  PMID: 22452978
B cells; epigenetics; gene expression; KAP1; KRAB-ZFP
23.  p16INK4a deficiency promotes IL-4-induced polarization and inhibits proinflammatory signaling in macrophages 
Blood  2011;118(9):2556-2566.
The CDKN2A locus, which contains the tumor suppressor gene p16INK4a, is associated with an increased risk of age-related inflammatory diseases, such as cardiovascular disease and type 2 diabetes, in which macrophages play a crucial role. Monocytes can polarize towards classically (CAMφ) or alternatively (AAMφ) activated macrophages. However, the molecular mechanisms underlying the acquisition of these phenotypes are not well defined.
Here, we show that p16INK4a-deficiency (p16−/−) modulates the macrophage phenotype. Transcriptome analysis revealed that p16−/− bone marrow-derived macrophages (BMDM) exhibit a phenotype resembling interleukin (IL)-4-induced macrophage polarization. In line with this observation, p16−/− BMDM displayed a decreased response to classically polarizing IFNγ and LPS and an increased sensitivity to alternative polarization by IL-4. Furthermore, mice transplanted with p16−/− bone marrow displayed higher hepatic AAMφ marker expression levels upon Schistosoma mansoni infection, an in vivo model of AAMφ phenotype-skewing. Surprisingly, p16−/− BMDM did not display increased IL-4-induced STAT6 signaling, but decreased IFNγ-induced STAT1 and LPS-induced IKKα,β phosphorylation. This decrease correlated with decreased JAK2 phosphorylation and with higher levels of inhibitory acetylation of STAT1 and IKKα,β. These findings identify p16INK4a as a modulator of macrophage activation and polarization via the JAK2-STAT1 pathway with possible roles in inflammatory diseases.
doi:10.1182/blood-2010-10-313106
PMCID: PMC3677739  PMID: 21636855
Animals; Bone Marrow Transplantation; Cyclin-Dependent Kinase Inhibitor p16; deficiency; physiology; Cytokines; biosynthesis; Genes, p16; I-kappa B Kinase; physiology; Inflammation; genetics; Interferon-gamma; pharmacology; Interleukin-4; pharmacology; Janus Kinase 2; physiology; Lipopolysaccharides; pharmacology; Liver; metabolism; pathology; Macrophage Activation; drug effects; Macrophages; drug effects; physiology; Mice; Mice, Inbred C57BL; Phosphorylation; Protein Processing, Post-Translational; Radiation Chimera; STAT1 Transcription Factor; physiology; STAT6 Transcription Factor; physiology; Schistosomiasis; immunology; Signal Transduction
24.  JAK2V617F homozygosity arises commonly and recurrently in PV and ET, but PV is characterized by expansion of a dominant homozygous subclone 
Blood  2012;120(13):2704-2707.
Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.
doi:10.1182/blood-2012-05-431791
PMCID: PMC3672970  PMID: 22898600
25.  Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets 
Blood  2012;119(24):5795-5806.
Hepatosplenic T-cell lymphoma (HSTL) is a rare entity mostly derived from γδ T cells that shows a fatal outcome. Its pathogenesis remains largely unknown. HSTL samples (7γδ, 2αβ) and the DERL2 HSTL-cell line were subject to combined gene expression profiling and array-based comparative genomic hybridization. Compared to other T-cell lymphomas, HSTL disclosed a distinct molecular signature irrespective of TCR cell lineage. Compared to PTCL,NOS and normal γδ cells, HSTL overexpressed genes encoding NK-cell associated molecules, oncogenes (FOS, VAV3), the Sphingosine-1-phosphatase receptor 5 involved in cell trafficking and the tyrosine kinase SYK, whereas the tumor suppressor gene AIM1 was among the most downexpressed. Methylation analysis of DERL2 cells demonstrated highly methylated CpG islands of AIM1 and decitabine treatment induced significant increase in AIM1 transcripts. Notably, Syk was demonstrated in HSTL cells with its phosphorylated form present in DERL2 cells by Western blot, and in vitro DERL2 cells were sensitive to a Syk inhibitor. Genomic profiles confirmed recurrent isochromosome 7q (n=6/9) without alterations at 9q22 and 6q21 containing SYK and AIM1 genes, respectively. The current study identifies a distinct molecular signature for HSTL and highlights oncogenic pathways which offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.
doi:10.1182/blood-2011-12-396150
PMCID: PMC3779008  PMID: 22510872
Adult; Aged; Base Sequence; Cell Lineage; genetics; Chromosome Aberrations; Cluster Analysis; Crystallins; metabolism; Drug Resistance, Neoplasm; genetics; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; genetics; Humans; Intracellular Signaling Peptides and Proteins; antagonists & inhibitors; metabolism; Isochromosomes; genetics; Liver Neoplasms; drug therapy; genetics; pathology; Lymphoma, T-Cell; drug therapy; genetics; pathology; Male; Membrane Proteins; metabolism; Middle Aged; Molecular Sequence Data; Molecular Targeted Therapy; Protein-Tyrosine Kinases; antagonists & inhibitors; metabolism; Receptors, Antigen, T-Cell, alpha-beta; genetics; Receptors, Antigen, T-Cell, gamma-delta; genetics; Splenic Neoplasms; drug therapy; genetics; pathology; Tumor Markers, Biological; genetics; metabolism; Young Adult

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