Experimental Hematology - Articles in Press

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγnull (NSG) mice - Accepted Manuscript

2012-02-03

Abstract: Delayed engraftment remains a major hurdle following cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the BM microenvironment. Since fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the BM microvasculature can occur, a simple 30 minute ex vivo incubation of CB HPC with fucosyltransferase (FT) - VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB HPC hypo-fucosylation was investigated in vivo in NOD-SCID IL-2Rγnull (NSG) mice. By isolating fucosylated and non-fucosylated CD34+ cells from CB we show that only fucosylated CD34+ cells are responsible for engraftment in NSG mice. Further, since the proportion of CD34+ cells that are fucosylated in CB is significantly less than in BM and PB, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed following CB transplantation. Since engraftment appears to be correlated with the fucosylation of CD34+ cells, we hypothesized that increasing the proportion of CD34+ cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase (FT)-VI significantly increases the levels of CD34+ fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells, or pattern of long-term, multi-lineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

A Functional Role For The Histone Demethylase Utx In Normal And Malignant Hematopoietic Cells - Accepted Manuscript

2012-02-03

Abstract: UTX, an H3K27Me2/3 demethylase, has been implicated in development, self-renewal and differentiation of various organs and embryonic stem (ES) cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral shRNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells (BM), and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine BM cells impairs their colony forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1 and Scl in primary murine BM cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1 and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Maternal Administration of Busulfan Before in Utero Transplantation of Human Hematopoietic Stem Cells Enhances Engraftments in Sheep - Accepted Manuscript

Yutaka Hanazono — 2012-02-03

Inside This Issue - Uncorrected Proof

2012-01-25

See Khan et al., pages XXX–XXX.

Lymphoid progenitors in normal mouse lymph nodes develop into NK cells and T cells in vitro and in vivo - Accepted Manuscript

Kathrin Warner, Claudia Luther, Fumio Takei — 2012-01-24

Abstract: We have identified a population of normal mouse LN cells, termed LN lymphoid progenitor (LNLP), resembling common lymphoid progenitor (CLP) in the BM. LNLPs lack lineage markers, express CD127, low levels of CD117 (c-Kit) and Sca-1, but lack fms-related tyrosine kinase 3 (Flt3). They efficiently differentiate in vitro into NK cells and T cells, but not mature B cells. LNLPs injected into non-irradiated lymphopenic mice that have no LN develop into mostly splenic T cells with low numbers of NK cells and B cells. When injected into irradiated mice, they generate NK cells and T cells, but not B cells, in the LN. By contrast, BM CLPs develop into mostly B cells with very small numbers of T and NK cells in recipients' spleen and LN. Thus, LNLPs have NK and T cell potentials but little B cell potential, and they may develop into NK cells within the LN of normal mice, whereas their contribution to the T cell lineage is unknown.

A novel and simple hollow-fiber assay for in vivo evaluation of nonpeptidyl thrombopoietin receptor agonists - Accepted Manuscript

Cheng-Ying Xie, Yong-Ping Xu, Hong-Bing Zhao, Li-Guang Lou — 2012-01-24

Abstract: Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult owing to the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to pre-clinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for non-peptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. The cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a non-peptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signalling transducers and activators of transcription (STAT) in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.

The adaptor protein NTAL enhances proximal signaling and potentiates corticosteroid-induced apoptosis in T-ALL - Accepted Manuscript

2012-01-24

Abstract: The biology of T-ALL is characterized by functional pre-T-cell receptor (TCR) signaling. NTAL is a non-enzymatic transmembrane adaptor molecule that is involved in the proximal signaling of lymphocytes. In our previous work, we found an association between high NTAL expression in T-cell ALL blasts and a favorable response to initial glucocorticoid treatment. In the present study, we confirm our previous observation in an experimental model. Moreover, the molecular mechanism of the contribution of NTAL to malignant T-ALL blast signaling and to methylprednisolone-induced cell death is analyzed. In the in vitro experiments, we used the T-ALL Jurkat cell line (Jurkat/wt), and derived Jurkat cell line with stable NTAL expression (Jurkat/NTAL+). Cell signaling and cell death after methylprednisolone treatment and after TCR stimulation was analyzed using flow cytometry, western blot and quantitative PCR. Jurkat/NTAL+ cells are significantly more sensitive to both methylprednisolone treatment and TCR-induced stimulation. Moreover, after TCR stimulation, Jurkat/NTAL+ cells show a higher level of intracellular ERK phosphorylation and increased expression of the CD69 activation marker on the cell surface than the Jurkat/wt cells. The ERK inhibitor U0126 almost completely abrogates TCR-induced cell death and, importantly, reverses the sensitizing effect of the NTAL protein on methylprednisolone-induced cell death. In conclusion, NTAL acts as a tumor suppressor that enhances the proximal signaling of leukemic blasts. The key downstream molecule responsible for the biological effect of TCR signaling is ERK. Higher ERK phosphorylation leads to enhanced cell death after TCR stimulation and increases cell sensitivity to methylprednisolone-induced cell death.

Consolidative therapy with Stem Cell Transplantation Improves Survival of patients with Mantle Cell Lymphoma following any Induction Regimen - Accepted Manuscript

2012-01-24

Abstract: Intensive induction regimen followed by high dose chemotherapy and autologous stem cell transplantation (auto-SCT) is frequently used to improve outcome in patients with mantle-cell lymphoma (MCL). The comparative impact of conventional vs. intensive induction regimen before transplantation is unknown. Forty eight patients with MCL receiving SCT at our institution between January 2000 and December 2010 were included in this study. At the time of initial presentation, 43 (89.5%) had stage IV disease and 18(37.5%) received more than one chemotherapy regimens prior to transplantation. Forty patients underwent auto-SCT, 7 allo-SCT; one patient had an allo-SCT for relapsed disease after auto-SCT. At the time of this analysis (median follow-up 6 years from diagnosis and 4 years from transplantation), 40 patients (88%) were alive with a 5 year disease free survival of 74.8%. Age, disease stage, number of regimens pre-SCT, pre-SCT disease status, and type of SCT had no impact on long-term outcome. Importantly, there was no difference among the type of induction regimen on outcome in this cohort receiving SCT. Based on our data, we believe that future studies should focus on strategies to prevent disease relapse rather than comparing induction regimens prior to stem cell transplantation.

The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability - Accepted Manuscript

2012-01-24

Abstract: Erythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia (XLSA). Human ALAS2 protein consists of 587 amino acids, and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in pathogenesis of XLSA.In vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with XLSA. Val562Ala showed the higher catalytic activity in vitro, but a shorter half-life in vivo, compared to those of wild-type ALAS2 (WT). In contrast, the in vitro activity of Met567Ile mutant was about 25% of WT, while its half-life was longer than that of WT. However, in vivo catalytic activity of each mutant was lower than that of WT. Furthermore, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in vitro and in vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses the catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.

Immunomodulatory nonablative conditioning regimen for B-cell lymphoid malignancies - Accepted Manuscript

2012-01-24

Abstract: Twenty six patients with recurrent CD20+ B-cell lymphoid malignancies received fludarabine, cyclophosphamide, and rituximab (FCR) based non-ablative conditioning (NST) followed by either matched related (n=18) or unrelated (n=8) donor allogeneic stem cell transplantation (allo-SCT) between March 2008 and May 2011. The median age of patients at transplantation was 59 years (range 41-64). At diagnosis, 20 (77%) had stage IV disease; 23 (88%) received ≥3, 14 (54%) ≥4 regimens and 4 (15%) had prior autologous-SCT. All patients had either chemosensitive or stable disease and 9 (35%) were in complete remission before transplantation. At the time of analysis, 17 patients were alive with an estimated 2-year overall survival and progression-free-survival of 63%, and non-relapse mortality 25%. Grade II-IV acute graft-verus-host-disease (GVHD) occurred in 8 (31%) and chronic GVHD in 6 (23%) patients (extensive=3). Causes of death include progressive disease 4, acute GVHD 2 (both after receiving donor lymphocyte infusion for mixed chimerism with residual disease), infection 1 and others 2 (substance abuse, leucoencephalopathy). Six patients required re-hospitalization within 100 days of SCT, (average 10 days; range, 3-18). Our data support FCR NST allo-SCT in CD20+ B-cell lymphoid malignancies and it is time to compare this regimen with alternative reduced-intensity conditioning regimen in B-cell malignancies.

Vitamin-D deficiency, autoimmunity and graft-versus-host-disease risk:Implication for preventive therapy - Accepted Manuscript

Mona Benrashid, Kim Moyers, Mohamad Mohty, Bipin N. Savani — 2012-01-19

Abstract: The majority of patients after allogeneic stem cell transplantation (HSCT) are expected to have vitamin-D deficiency early post-HSCT due to poor nutritional status and limited sun exposure. The importance of vitamin D in the immune system has been well defined over the past several years, as vitamin-D has demonstrated modulatory effects on the immune system through B- and T-lymphocyte, macrophage, monocyte and dendritic cell regulations, which are the effector cells involved in graft-versus-host-disease (GVHD) pathophysiology after HSCT. High dose early replacement of vitamin D might attenuate autoimmune reactions and may decrease the severity of GVHD. In this article, we discuss the hypothetical link between early vitamin-D deficiency and GVHD and its potential therapeutic role in GVHD and long term bone loss after HSCT.

Abnormal Differentiation of Erythroid Precursors in p45 NF-E2 -/- Mice - Accepted Manuscript

Jadwiga J. Gasiorek, Zaynab Nouhi, Volker Blank — 2012-01-19

Abstract: The transcription factor p45 NF-E2 plays major roles in erythroid and megakaryocytic lineages. Here, we investigated the role of p45 NF-E2 in erythoid differentiationin vivo. Absence of p45 NF-E2 in mice leads to a two-fold increase of serum erythropoietin levels. In the bone marrow of these animals, we found a different distribution of precursor populations compared to wild-type mice, suggesting abnormal differentiation. Loss of p45 NF-E2 was also associated with an increase in splenic erythropoiesis, as evidenced by an accumulation of early precursors, namely late basophilic and polychromatic erythroblasts. These observations are consistent with a stress erythropoiesis phenotype and indicate that the spleen is likely compensating for ineffective erythropoiesis in the bone marrow. Analysis of bone marrow samples revealed increased GATA1 levels as well as an increased proportion of erythroid cells arrested at the G1 stage of cell cycle in p45 NF-E2 deficient mice. These results suggest that p45 NF-E2 is required for the differentiation of erythroid precursors.

Production of large numbers of plasmacytoid dendritic cells with functional activities from CD34+ hematopoietic progenitor cells: Use of interleukin-3 - Uncorrected Proof

Stéphanie Demoulin, Patrick Roncarati, Philippe Delvenne, Pascale Hubert — 2012-01-12

Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type-I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases, such as chronic viral infections (e.g., hepatitis C virus, human immunodeficiency virus), autoimmunity (e.g., psoriasis, systemic lupus erythematosus), and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34+ cord blood progenitors to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity, and cancer. Human CD34+ hematopoietic progenitor cells isolated from cord blood were cultured with a combination of Flt3-ligand (Flt3L), thrombopoietin (TPO), and one of the following cytokine: interleukin (IL)-3, interferon-β, or prostaglandin E2. Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34+ hematopoietic progenitor cells. Indeed, Flt3L/TPO alone or supplemented with prostaglandin E2 or interferon-β produced smaller amounts of pDC from hematopoietic progenitor cells. In addition, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical, and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for large-scale generation of pDC. This culture condition generated, starting from 2 × 105 CD34+ cells, up to 2.6 × 106 pDC presenting features of blood pDC.

Critical role of CD4+CD25+ regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia - Uncorrected Proof

Tetsuya Nishimoto, Takashi Satoh, Tsutomu Takeuchi, Yasuo Ikeda, Masataka Kuwana — 2012-01-11

Autoimmune response suppression by regulatory T cells (Tregs) helps to maintain peripheral immune tolerance, and defects in this mechanism are thought to play a role in the pathogenesis of various autoimmune diseases. In patients with immune thrombocytopenia, naturally occurring CD4+CD25+ Tregs are both functionally impaired and reduced in number. This study was undertaken to investigate Tregs’ role in preventing immune thrombocytopenia in mice. Treg-deficient mice were prepared by inoculation of Treg-depleted CD4+CD25− T cells isolated from BALB/c mice into syngeneic nude mice intravenously. Platelet count, proportion of reticulated platelets, platelet-associated IgG, platelet-associated anti-platelet antibodies, and IgG anti-platelet antibody production in splenocyte cultures were examined by flow cytometry. Of 69 Treg-deficient mice, 25 (36%) spontaneously developed thrombocytopenia that lasted at least 5 weeks. The platelet-associated IgG level and proportion of reticulated platelets were elevated in the thrombocytopenic mice. Platelet eluates and splenocyte culture supernatants prepared from thrombocytopenic mice, but not from nonthrombocytopenic mice, contained IgG antibodies capable of binding to intact platelets. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. Treatment with IgG anti-cytotoxic T lymphocyte−associated antigen 4 antibody canceled this Treg-governed suppressive effect. In summary, these results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging cytotoxic T lymphocyte−associated antigen 4.

Sonic hedgehog maintains survival and growth of chronic myeloid leukemia progenitor cells through β-catenin signaling - Uncorrected Proof

Wenxia Su, Fankai Meng, Lifang Huang, Miao Zheng, Wenli Liu, Hanying Sun — 2012-01-11

Sonic hedgehog (Shh) signaling plays an important role in many human cancers and cancer stem cells. Here we investigate the activity and functional role of Shh signaling in chronic myeloid leukemia (CML) and leukemia progenitor cells. Differential activation of Shh signaling was found in about 50% CML-chronic phase samples, 70% of CML-accelerated phase samples, and >80% CML-BP samples. Deregulated activation of Shh signaling was observed in CD34+ and c-kit+ leukemia progenitor cells. Stimulation of Shh signaling with exogenous Shh peptide induced expansion of CD34+ and c-kit+ progenitor cells (p < 0.05) inversely, blocking the pathway with signal inhibitor−induced cell apoptosis (p < 0.05). Low level of Shh protein was observed in CML bone marrow stromal cells, and CD34+ progenitor cells are less sensitive to exogenous Shh peptide and more sensitive to cyclopamine than CD34− cells (p < 0.05), implying cell-autonomous activation of Shh signaling play a predominant role in progenitor cells. Coactivation of Shh and β-catenin signaling was found in CD34+ and c-kit+ progenitor cells. Administration of Shh-neutralizing antibody or Wnt3a-neutralizing antibody in c-kit+ progenitor cells induced cell apoptosis; however, Wnt3a peptide could salvage anti−Shh-induced cell apoptosis, while Shh peptide failed to revert anti−Wnt3a-induced cell apoptosis. C-MYC, GLI1, BCL-2, AND P21 were also found to be downstream targets of Shh signaling, mediating apoptosis or G2/M cell cycle arrest of progenitor cells. Our results demonstrate that autoactivated Shh signaling provides survival and proliferative cues in CML progenitor cells through downstream β-catenin signaling, suggesting a novel therapeutic approach in CML.

ABT-737 increases tyrosine kinase inhibitor-induced apoptosis in chronic myeloid leukemia cells through XIAP down-regulation and sensitizes CD34+ CD38- population to imatinib - Accepted Manuscript

2012-01-11

Abstract: Chronic myeloid leukemia (CML) tumorigenicity is driven by the oncogenic BCR-ABL tyrosine kinase. Specific tyrosine kinase inhibitors (TKI) have been designed and are now used for the treatment of CML. These TKI induce apoptosis in leukemic cells in a BIM-dependent mechanism. We hypothesized that an increase in BIM activity could sensitize CML cells to TKI. Thus, we blocked the anti-apoptotic proteins of the Bcl-2 family, by using ABT-737, a Bcl-2 and Bcl-XL inhibitor. ABT-737 modified Bcl-2 protein interactions towards a pro-apoptotic phenotype. Its combination with TKI resulted in a strong synergism in CML cell lines. The association also induced a large decrease in XIAP, followed by caspase 3 activation. This XIAP decrease was due to post-translational events. The mitochondrial serine protease HtrA2/Omi was identified as being responsible for this off-target effect. Then, ABT-737 and TKI cooperate at several levels to induce apoptosis of CML cells lines and the benefit of this association was also observed in CML hematopoietic progenitors. Interestingly, a lethal effect was also observed in the more immature CD34+CD38- TKI insensitive population. Thus, the combination therapy could by an interesting strategy for the treatment of CML patients.

Tocopherol succinate– and AMD3100-mobilized progenitors mitigate radiation-induced gastrointestinal injury in mice - Uncorrected Proof

2012-01-11

The goal of this study was to elucidate the role of α-tocopherol succinate (TS)– and AMD3100-mobilized progenitors in mitigating the ionizing-radiation–induced gastrointestinal syndrome in mice. We demonstrate the efficacy of a bridging therapy that will allow the lymphohematopoietic system of severely immunocompromised victims exposed to ionizing radiation to recover from high doses of radiation. CD2F1 mice were irradiated with a high dose of radiation causing gastrointestinal syndrome (11 Gy, cobalt-60 γ-radiation) and then transfused intravenously (retro-orbital sinus) with whole blood or peripheral blood mononuclear cells (PBMC) from TS- and AMD3100-injected mice 2, 24, or 48 hours post irradiation and monitored for 30-day survival. Jejunum sections were analyzed for tissue area, surviving crypts, villi, mitotic figures, and basal lamina enterocytes. Our results demonstrate that infusion of whole blood or PBMC from TS- and AMD3100-injected mice significantly improved survival of mice receiving a high dose of radiation. Histopathology and immunostaining of jejunum from irradiated and TS- and AMD3100-mobilized PBMC-transfused mice reveal significant protection of gastrointestinal tissue from radiation injury. We demonstrate that TS and AMD3100 mobilize progenitors into peripheral circulation and that the infusion of mobilized progenitor-containing blood or PBMC acts as a bridging therapy for immune-system recovery in mice exposed to high, potentially fatal, doses of ionizing radiation.

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34+ cells - Corrected Proof

2011-12-22

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type–dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)–mediated very rapid apoptosis occurs within 1 hour in CD34+ cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34+ cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34+ cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34+ cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34+ cells and neutralization or blocking of type-I IFN receptor does not rescue CD34+ cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TRL3, signaling triggers poly I:C–induced rapid apoptosis of human CD34+ cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis - Corrected Proof

2011-12-22

Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology—haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia.

Mice heterozygous for CREB binding protein are hypersensitive to γ-radiation and invariably develop myelodysplastic/myeloproliferative neoplasm - Corrected Proof

2011-12-22

Myelodysplastic syndrome is a complex family of preleukemic diseases in which hematopoietic stem cell defects lead to abnormal differentiation in one or more blood lineages. Disease progression is associated with increasing genomic instability and a large proportion of patients go on to develop acute myeloid leukemia. Primarily a disease of the elderly, it can also develop after chemotherapy. We have previously reported that CREB binding protein (Crebbp) heterozygous mice have an increased incidence of hematological malignancies, and others have shown that CREBBP is one of the genes altered by chromosomal translocations found in patients suffering from therapy-related myelodysplastic syndrome. This led us to investigate whether hematopoietic tumor development in Crebbp+/− mice is preceded by a myelodysplastic phase and whether we could uncover molecular mechanisms that might contribute to its development. We report here that Crebbp+/− mice invariably develop myelodysplastic/myeloproliferative neoplasm within 9 to 12 months of age. They are also hypersensitive to ionizing radiation and show a marked decrease in poly(ADP-ribose) polymerase-1 activity after irradiation. In addition, protein levels of XRCC1 and APEX1, key components of base excision repair machinery, are reduced in unirradiated Crebbp+/− cells or upon targeted knockdown of CREBBP levels. Our results provide validation of a novel myelodysplastic/myeloproliferative neoplasm mouse model and, more importantly, point to defective repair of DNA damage as a contributing factor to the pathogenesis of this currently incurable disease.

Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment - Corrected Proof

2011-12-22

Strategies that increase homing to the bone marrow and engraftment efficacy of ex vivo expended CD34+ cells are expected to enhance their clinical utility. Here we report that nicotinamide (NAM), a form of vitamin B-3, delayed differentiation and increased engraftment efficacy of cord blood–derived human CD34+ cells cultured with cytokines. In the presence of NAM, the fraction of CD34+CD38− cells increased and the fraction of differentiated cells (CD14+, CD11b+, and CD11c+) decreased. CD34+ cells cultured with NAM displayed increased migration toward stromal cell derived factor–1 and homed to the bone marrow with higher efficacy, thus contributing to their increased engraftment efficacy, which was maintained in competitive transplants with noncultured competitor cells. NAM is a known potent inhibitor of several classes of ribosylase enzymes that require NAD for their activity, as well as sirtuin (SIRT1), class III NAD+-dependent-histone-deacetylase. We demonstrated that EX-527, a specific inhibitor of SIRT1 catalytic activity, inhibited differentiation of CD34+ cells similar to NAM, while specific inhibitors of NAD-ribosylase enzymes did not inhibit differentiation, suggesting that the NAM effect is SIRT1-specific. Our findings suggest a critical function of SIRT1 in the regulation of hematopoietic stem cell activity and imply the clinical utility of NAM for ex vivo expansion of functional CD34+ cells.

Aldehyde dehydrogenases are regulators of hematopoietic stem cell numbers and B-cell development - Corrected Proof

2011-12-22

High levels of the aldehyde dehydrogenase isoform ALDH1A1 are expressed in hematopoietic stem cells (HSCs); however, its importance in these cells remains unclear. Consistent with an earlier report, we find that loss of ALDH1A1 does not affect HSCs. Intriguingly, however, we find that ALDH1A1 deficiency is associated with increased expression of the ALDH3A1 isoform, suggesting its potential to compensate for ALDH1A1. Mice deficient in ALDH3A1 have a block in B-cell development as well as abnormalities in cell cycling, intracellular signaling, and gene expression. Early B cells from these mice exhibit excess reactive oxygen species and reduced metabolism of reactive aldehydes. Mice deficient in both ALDH3A1 and ALDH1A1 have reduced numbers of HSCs as well as aberrant cell cycle distribution, increased reactive oxygen species levels, p38 mitogen-activated protein kinase activity and sensitivity to DNA damage. These findings demonstrate that ALDH3A1 can compensate for ALDH1A1 in bone marrow and is important in B-cell development, both ALDH1A1 and 3A1 are important in HSC biology; and these effects may be due, in part, to changes in metabolism of reactive oxygen species and reactive aldehydes.

Infection of bone marrow cells by dengue virus in vivo - Corrected Proof

2011-12-21

Abnormal bone marrow (BM) suppression is one of the hallmarks of dengue virus (DENV) infection in patients. Although the etiology remains unclear, direct viral targeting of the BM has been reasoned to be a contributing factor. The present studies were carried out in an effort to determine the potential effect of DENV infection on the cellularity of BM using a previously established nonhuman primate model of DENV-induced coagulopathy. BM aspirates were collected at various times from the infected nonhuman primate and cells were phenotypically defined and isolated using standard flow cytometry (fluorescence-activated cell sorting). These isolated cells were subjected to detection of DENV utilizing quantitative real-time reverse transcription polymerase chain reaction, electron microscopy, and immunostaining techniques. DENV RNA was detectable by quantitative real-time reverse transcription polymerase chain reaction in BM specimens and the presence of DENV-like particles within platelet was confirmed by electron microscopy. Enumeration of BM cells revealed a transient surge in cellularity at day 1, followed by a gradual decline from days 2 to 10 post infection. Detailed phenotypic studies showed similar kinetics in the frequencies of CD41+CD61+ cells, regardless of CD34 and CD45 expression. The CD61+ cells were not only the predominant cells that stained for DENV antigen but fluorescence-activated cell sorting–assisted isolation of CD61+ cells from the BM were shown to contain infectious DENV by coculture with Vero cells. These data support the view that intravenous infection of nonhuman primate with DENV leads to direct infection of the BM, which is likely to be a contributing factor for transient cell suppression in the peripheral blood characteristic of acute DENV infection.

Bone marrow−derived cells do not engraft into skeletal muscle microvasculature but promote angiogenesis after acute injury - Corrected Proof

Nicholas Ieronimakis, Aislinn Hays, Morayma Reyes — 2011-12-12

The skeletal muscle is supported by a vast network of microvessels with the capacity to regenerate in response to injury. However, the dynamics of microvascular repair and the origin of reconstituted endothelial cells in the skeletal muscle are poorly understood. A growing body of literature exists to indicate bone marrow (BM)−derived cells engraft into regenerating vascular endothelium and muscle macrovasculature. Therefore, we investigated the extent of BM contribution to skeletal muscle microvasculature after acute injury. Because reporters and markers commonly used to trace donor BM cells are not endothelial specific but are also expressed by leukocytes, we generated novel BM chimeras utilizing Tie2-green fluorescent protein BM cells transplanted into CD31 and Caveolin-1 knockout recipients. In turn, we surveyed BM vascular contribution, not just by the presence of green fluorescent protein, but also CD31 and Caveolin-1, respectively. After stable BM reconstitution, chimera limb muscles were cardiotoxin (CTX) injured and examined 21 days post-injury for the presence of green fluorescent protein, CD31, and Caveolin-1. Acute muscle injury by CTX is characterized by initial microvasculature death followed by rapid endothelial regeneration within 14 days post-damage. Histological analysis of injured and uninjured contralateral limb muscles revealed a complete absence of BM engraftment in the muscle vasculature of wild-type and CD31/Caveolin-1 knockout chimeras. In contrast, F4/80+ cells isolated from CTX-injured muscle, expressed endothelial-related markers and promoted angiogenesis in vitro. Therefore, despite the absence of BM engraftment to regenerated skeletal muscle microvasculature, macrophages recruited after injury promote angiogenesis and, in turn, vascular regeneration.

Hematopoietic stem cell expansion facilitates multilineage engraftment in a nonhuman primate cord blood transplantation model - Corrected Proof

2011-12-09

The use of umbilical cord blood for allogeneic transplantation has increased dramatically over the past years. However, the limited number of cells available in a single cord blood unit remains a serious obstacle. Here, we wished to establish a nonhuman primate cord blood transplantation model that would allow us to test various hematopoietic stem cell expansion and gene therapy strategies. We implemented HOXB4-mediated expansion based on our previous experience with HOXB4 in autologous cells. Cord blood units were divided into two equal parts; half of the cells were transduced with a yellow fluorescent protein control vector and cryopreserved, and half were transduced with a HOXB4GFP vector, expanded, and cryopreserved. Both fractions of cells were transplanted into Macaca nemestrina subjects. We found that neutrophil recovery occurred within 19 days in all animals, and both neutrophil and platelet recovery were substantially accelerated compared to human single unit cord blood transplants. In addition, HOXB4-transduced and expanded cells resulted in superior engraftment of all hematopoietic lineages in all animals over nonexpanded controls. In conclusion, we have successfully established a nonhuman primate cord blood transplantation model and demonstrated that HOXB4 stimulates expansion and engraftment of repopulating cells. The availability of such a model has significant implications for developing and testing strategies to improve clinical cord blood transplantation, as it will allow comparison of different stem cell expansion methodologies within a single animal. Furthermore, it can be used in long-term follow-up studies to determine how specific expansion techniques affect engraftment of various hematopoietic lineages.

Fak depletion in both hematopoietic and nonhematopoietic niche cells leads to hematopoietic stem cell expansion - Corrected Proof

2011-12-09

Hematopoietic stem cells (HSCs) reside in complex bone marrow microenvironments, where niche-induced signals regulate hematopoiesis. Focal adhesion kinase (Fak) is a nonreceptor protein tyrosine kinase that plays an essential role in many cell types, where its activation controls adhesion, motility, and survival. Fak expression is relatively increased in HSCs compared to progenitors and mature blood cells. Therefore, we explored its role in HSC homeostasis. We have used the Mx1-Cre−inducible conditional knockout mouse model to investigate the effects of Fak deletion in bone marrow compartments. The total number as well as the fraction of cycling Lin−Sca-1+c-kit+ (LSK) cells is increased in Fak−/− mice compared to controls, while hematopoietic progenitors and mature blood cells are unaffected. Bone marrow cells from Fak−/− mice exhibit enhanced, long-term (i.e., 20-week duration) engraftment in competitive transplantation assays. Intrinsic Fak function was assessed in serial transplantation assays, which showed that HSCs (Lin−Sca-1+c-kit+CD34−Flk-2− cells) sorted from Fak−/− mice have similar self-renewal and engraftment ability on a per-cell basis as wild-type HSCs. When Fak deletion is induced after engraftment of Fakfl/flMx1-Cre+ bone marrow cells into wild-type recipient mice, the number of LSKs is unchanged. In conclusion, Fak inactivation does not intrinsically regulate HSC behavior and is not essential for steady-state hematopoiesis. However, widespread Fak inactivation in the hematopoietic system induces an increased and activated HSC pool size, potentially as a result of altered reciprocal interactions between HSCs and their microenvironment.

Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation - Corrected Proof

2011-11-28

Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia, a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29−/− embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes upregulated in the rps29 mutant are enriched for genes upregulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in hematopoietic stem cells and red blood cell development.

Treatment with high-dose simvastatin inhibits geranylgeranylation in AML blast cells in a subset of AML patients - Uncorrected Proof

2011-11-28

It is currently unknown whether the in vitro effects observed with statins in acute myeloid leukemia (AML) cells, including lowering of cholesterol, inhibition of isoprenylation, and sensitization to chemotherapy, also occur in vivo. Therefore, AML mononuclear cells (MNCs) were isolated from 12 patients before and after 7 days of high-dose (7.5–15 mg/kg/day) simvastatin treatment. Parallel mouse studies were performed to have, in addition to AML cells, access to liver tissue, a major target of statins. Serum cholesterol levels were lowered by simvastatin in all patients, however, only limited changes in the messenger RNA expression of cholesterol metabolism genes were seen in patient and mouse MNCs compared to murine liver cells. Still, two out of seven patients displayed an increased in vitro chemosensitivity of their AML cells upon simvastatin treatment. Gene set enrichment analysis on microarray data of AML patient cells and Western blot analysis for the isoprenylated proteins DnaJ and Rap1 on murine and AML patient MNCs demonstrated that in vivo simvastatin treatment resulted in inhibition of geranylgeranylation in murine MNCs and in a subset of patient AML MNCs. In summary, our data demonstrate that simvastatin treatment results in chemosensitization and inhibition of geranylgeranylation in AML cells of a subset of patients.

A subset of chronic lymphocytic leukemia patients display reduced levels of PARP1 expression coupled with a defective irradiation-induced apoptosis - Corrected Proof

2011-11-24

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by defects in the DNA damage response and apoptosis. Among the factors involved in these pathways, we focused on the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and on its substrate Che-1 by evaluating their basal expression and functional changes upon irradiation (IR). Microarray experiments were performed on 98 untreated CLL cases. Next, freshly isolated primary cells from 21 untreated patients were analyzed for in vitro response to irradiation through Western blot, PARP activity assay, Annexin-V analysis, and PARP1 basal expression by quantitative polymerase chain reaction. Microarray analysis showed that PARP1 and CHE1 were constitutively expressed in CLL and had a high degree of correlation with each other and with TP53. PARP1 and TP53 downmodulation was associated with worse clinical outcomes, especially in TP53-mutated cases. Next, CLL samples from 21 untreated patients were classified as responders and nonresponders based on IR-induced PARP1 cleavage. Notably, while responder samples were characterized by Che-1 and p53 induction at 8 hours and reduction at 24 hours post-IR, nonresponders included both samples with p53 dysfunctions and cases with a normal IR-induced Che-1 and/or p53 response. Finally, we observed that PARP1 was downregulated in nonresponder vs responder samples and that its basal expression was positively correlated with PARP1 cleavage after IR. In conclusion, we showed that reduced expression of PARP1 is associated with an impairment of CLL responsiveness to cell death.

Targeted overexpression of an activated N-ras gene results in B-cell and plasma cell lymphoproliferation and cooperates with c-myc to induce fatal B-cell neoplasia - Corrected Proof

Michael A. Linden, Nicole Kirchhof, Cathy S. Carlson, Brian G. Van Ness — 2011-11-24

Multiple myeloma is an incurable malignant expansion of plasma cells in the bone marrow. Although there is no pathognomonic genetic lesion among multiple myeloma patients, activation of the ras gene has been identified as a common mutation. We have previously described the use of the 3′ κ immunoglobulin light chain enhancer (3′KE) to target transgenic expression in murine B and plasma cells, resulting in bcl-XL and c-myc–driven murine models of multiple myeloma. In this report, we characterize the role of activated mutant N-ras in B and plasma cells in transgenic mice. We constructed transgenic mice that use 3′KE to direct expression of a mutant activated N-ras. We also crossed the N-ras mice with mice bearing a c-myc transgene to study the cooperative effects of the transgenic constructs. Mice were sacrificed when moribund or at specific time intervals and characterized by serology, light microscopy, and flow cytometry. The transgenic N-ras animals develop B- and plasma cell lymphoproliferation, and aged mice develop immunoglobulinemia, renal hyaline tubular casts, and microscopic foci of abnormal plasma cells in extramedullary sites, including the liver and kidney. Bitransgenic 3′KE/N-Ras V12 × Eμ-c-Myc mice develop fatal B-cell neoplasia, with a median survival of 10 weeks. These data indicate that activated N-ras can play a role in B- and plasma cell homeostasis and that activated N-Ras and c-Myc can cooperate to induce B-cell neoplasia.

Para-NO-aspirin inhibits NF-κB and induces apoptosis in B-cell progenitor acute lymphoblastic leukemia - Corrected Proof

2011-11-18

Although patients with acute lymphoblastic leukemia (ALL) usually achieve complete remission, disease relapse is common and difficult to treat. Para-NO-aspirin (para-NO-ASA) is a novel drug with demonstrated efficacy against a number of solid tumors and most recently chronic lymphocytic leukemia. In this study, we used ALL cell lines to assess the effects on cell viability by flow cytometry and investigated the mechanism of cell death using chemical inhibitors of key molecules and assessed the effects by flow cytometry, electrophoretic mobility shift assay, Western blotting, and quantitative reverse transcription polymerase chain reaction. Para-NO-ASA induced cell death in the pre-B ALL cell lines in association with increased reactive oxygen species, and suppression of nuclear factor−κB (NF-κB) activity. Chemical inhibitors of NF-κB similarly induced apoptosis in ALL cells, suggesting a role for suppression of NF-κB in para-NO-ASA−induced cell death. Modulation of NF-κB was not via regulation of IκB but potentially through suppression of ROCK1 and loss of reduced glutathione. Our results demonstrate that para-NO-ASA potently induces apoptosis in B-lineage ALL cells via a reactive oxygen species−dependent mechanism that is associated with suppression of NF-κB activity.

Erratum to Experimental Hematology 2010;38:61-70 - Uncorrected Proof

2011-07-15

Title of the publication: Preclinical development of a bridging therapy for radiation casualties In the original publication an incorrect figure was printed as figure 4. Figure 3 was printed twice: once as figure 3 and again as figure 4. As a correction the above figure should be viewed as the proper figure 4. The published caption for figure 4 is correct.

Detection of bone marrow−derived lung epithelial cells - Uncorrected Proof

Susannah H. Kassmer, Diane S. Krause — 2010-05-05

Studies on the ability of bone marrow−derived cells to adopt the morphology and protein expression pattern of epithelial cells in vivo have expanded rapidly during the last decade, and hundreds of publications report that bone marrow−derived cells can become epithelial cells of multiple organs, including lung, liver, gastrointestinal tract, skin, pancreas, and others. In this review, we critically evaluate the literature related to engraftment of bone marrow–derived cells as epithelial cells in the lung. More than 40 articles focused on whether bone marrow cells can differentiate into lung epithelial cells have been published, nearly all of which claim to identify marrow-derived epithelial cells. A few investigations have concluded that no such cells are present and that the phenomenon of marrow-derived epithelial cells is based on detection artifacts. Here we discuss the problems that exist in published articles identifying marrow-derived epithelial cells, and propose standards for detection methods that provide the most definitive data. Identification of bone marrow−derived epithelial cells requires reliable and sensitive techniques for their detection, which must include cell identification based on the presence of an epithelial marker and the absence of blood cell markers as well as a marker for donor bone marrow origin. In order for these studies to be rigorous, they must also use approaches to rule out cell overlap by microscopy or single-cell isolation. Once these stringent criteria for identification of marrow-derived epithelial cells are used universally, then the field can move forward to address the critical questions about which bone marrow−derived cells are responsible for engraftment as epithelial cells, the mechanisms by which this occurs, whether these cells play a role in normal tissue repair, and whether specific cell subsets can be used for therapeutic benefit.

Stem cell plasticity: Recapping the decade, mapping the future - Uncorrected Proof

Neil D. Theise — 2010-05-03

In slightly more than a decade of stem cell plasticity research, 24 peer-reviewed articles have demonstrated plasticity across organ and/or embryonic lineage boundaries at the single-cell level, with only 1 article showing negative results. These data, taken together with data about reversibility of gene restrictions that have also accumulated during the same period, indicate that postnatal cells, even “terminally differentiated” ones, have a degree of plasticity not appreciated previously. This review looks back at the four known pathways of cell plasticity and at previously described “plasticity principles” of Genomic Completeness, Cellular Uncertainty, Stochasticity of Cell Origin and Fate, relating these to issues of experimental design and discourse that are key to understanding and evaluating plasticity data. Although the physiologic roles played by such plasticity may still be debated, the manipulations of these phenomena for therapeutic or industrial purposes should finally be considered ripe for exploration. For the future, plasticity, indeed all stem cell biology, must be considered as part of a larger web of cell-to-cell and cell-to-matrix interactions that function fully only at the tissue level; thus, the success of stem cell biology necessarily must involve assembling data from cell and molecular biology research into systems of interactions that might be reasonably called “tissue biology.” Interdisciplinary collaborations with complexity and chaos theorists, using mathematical/computer modeling of cell behaviors, will be vital to fully exploring stem cell behaviors in the coming decades.