Experimental Hematology - Articles in Press

Erratum - Uncorrected Proof

2010-07-30

Dr. Kazuhiro Nishii was inadvertently left out of the author line-up in the recently published article (Exp Hematol. 2010;38:685-696) entitled “A potential activity of valproic acid in the stimulation of interleukin-3−mediated megakaryopoiesis and erythropoiesis” by Bing Liu, et al. The correct author listing should be as follows:

Resistance of T-cell acute lymphoblastic leukemia (T-ALL) to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis - Accepted Manuscript

2010-07-28

Abstract: Objective: Cytotoxic ligands are involved in tumor immunity and graft-versus-leukemia effect after allogeneic stem cell transplantation for leukemia. To clarify the susceptibility of T-cell acute lymphoblastic leukemia (T-ALL) to tumor immunity, sensitivity to recombinant human soluble Fas ligand (rhsFasL) and tumor necrosis factor-related apoptosis-inducing ligand (rhsTRAIL) was determined.Materials and Methods: Sensitivity to rhsFasL and rhsTRAIL and cell surface expression of their receptors were tested in T-ALL cell lines (n=7) and patients' samples (n=17), and compared with those in B-precursor ALL cell lines (n=30). Expression of components of the death-inducing signaling complex (DISC) and the TRAIL receptor genes (DR4/DR5), and the methylation status and promoter activity of the DR4/DR5 gene were tested in T-ALL cell lines.Results: T-ALL cell lines showed higher level of Fas expression and higher sensitivity to rhsFasL than did B-precursor ALL cell lines. Despite comparable expression of components of DISC, cell lines and patients' samples of T-ALL showed TRAIL-resistance associated with low cell surface expression of DR4/DR5. Gene expression of DR4/DR5 in T-ALL cell lines was significantly lower than that in B-precursor ALL cell lines, and the methylation status of the gene promoter in T-ALL cell lines was associated with the gene expression level at least for DR4. The demethylating agent, 5-aza 2'deoxycytidine, upregulated the gene expression of DR4/DR5, but was insufficient for their surface expression due to low basal promoter activity.Conclusions: In contrast to higher sensitivity to FasL, T-ALL showed resistance to TRAIL, which might be responsible for resistance to TRAIL-mediated cellular immunity.

The HoxA cluster is haploinsufficient for activity of hematopoietic stem and progenitor cells - Accepted Manuscript

2010-07-26

Abstract: Objective: Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knock-outs. Since HoxB genes are dispensable for hematopoiesis, and most HoxA genes are expressed an order of magnitude higher than other cluster genes in hematopoietic stem cell (HSC) enriched populations, we hypothesize that maintenance of HoxA cluster expression is important for adult hematopoiesis and that global decrease of HoxA gene expression levels affects steady-state hematopoiesis.Methods: Expression levels of HoxA cluster genes have been determined in primitive hematopoietic populations derived from adult mice using quantitative reverse transcriptase (RT)-polymerase chain reaction (PCR). Furthermore, the functional effect of single allelic deletion of the entire HoxA cluster on hematopoietic cells was analysed by competitive repopulation assays using HoxA+/- mice.Results: We show that the HoxA cluster is predominantly expressed in long-term (LT)-HSCs and that expression declines with progression to short-term (ST)-HSCs and early progenitors in a quantifiable manner. Monoallelic deletion of the HoxA cluster caused a general increase in primitive hematopoietic cell populations, but a decrease in side populations. In addition exhaustion of B-cell progenitors with age was observed, resulting in less mature B-cells. Moreover, bone marrow of HoxA+/- mice had a significant larger population of Mac1/Gr1 neutrophils, which might be caused by accelerated maturation of myeloid progenitors. Transplantation assays demonstrated that HoxA+/- HSCs were less competitive in long-term repopulation of myeloablated recipients, which appeared intrinsic to HSCs.Conclusions: These results show for the first time that maintenance of adult HSCs and progenitors is particularly sensitive to HoxA gene levels, suggesting a specific role for the HoxA cluster in primary regulation of definitive hematopoiesis.

Activation of ephrin A proteins influences Hematopoietic Stem Cell Adhesion and Trafficking patterns - Accepted Manuscript

Michael J. Ting, Bryan W. Day, Mark D. Spanevello, Andrew W. Boyd — 2010-07-26

Abstract: Objective: To determine if Eph receptors and the ephrins can modulate the homing of hematopoietic cells in a murine bone marrow transplantation model.Materials and Methods: EphA and ephrin A gene expression by mouse HSC and the progenitor cell line FDCP-1 was determined by real time RT-PCR and flow cytometry. The effect of ephrin A activation on adhesion of hematopoietic progenitors was determined by in vitro adhesion assays in which cells were exposed to fibronectin or VCAM-1 and an increasing gradient of immobilized EphA3-Fc. Adhesion to fibronectin and VCAM-1 was further investigated using soluble preclustered EphA3-Fc. We used soluble unclustered EphA3-Fc as an antagonist to block endogenous EphA-ephrin A interactions in vivo. The effect of injecting soluble EphA3-Fc on the mobilization of hematopoietic progenitor cells was examined. We determined the effect on short term homing by pre-treating bone marrow cells with EphA3-Fc or the control IgG prior to infusion into lethally irradiated mice.Results: Preclustered and immobilized EphA3-Fc increased adhesion of progenitor cells and FDCP-1 to fibronectin and VCAM-1 (1.6 to 2 fold higher adhesion p<0.05) relative to control (0 mg/cm2 EphA3-Fc ECM alone). Injection of the antagonist soluble EphA3-Fc increased progenitor cell and CFU-S cells in the peripheral blood (42% greater CFU-C p<0.05, 3.8 fold higher CFU-S) relative to control. Treating bone marrow cells with EphA3-Fc resulted in reduction by 31% in donor stem cells homing to the BM and accumulation of donor cells in recipient spleens (50% greater than control) and greater recovery of donor stem cells from the peripheral blood.

Oxidative stress is increased in primary and post-polycythemia vera myelofibrosis - Accepted Manuscript

2010-07-23

Abstract: Background: Increased cell turnover in chronic myeloproliferative disorders (CMPD) may lead to hyperhomocysteinemia as a result of folate and/or cobalamin depletion, and may contribute to oxidative stress.Design and Methods: The clinical role of oxidative stress was investigated by measuring reactive oxygen species (ROS), total antioxidant capacity (TAC), and total homocysteine (tHcy), folate, cobalamin, holotranscobalamin (HoloTC) levels in 51 CMPD patients [M/F: 1.1; median age 64 years, range: 40-84], 42 with primary myelofibrosis (PMF), and nine with post-polycythemia vera myelofibrosis (post-PV MF).Results: Myelofibrotic patients had higher tHcy (p=0.0201) and an unbalanced oxidative status (higher ROS and lower TAC levels, p<0.0001) than controls. The presence of diabetes or another neoplasia was associated with higher ROS levels (p<0.05), splenomegaly, hepatomegaly and peripheral blasts with lower HoloTC levels (p<0.005). The most severe forms of myelofibrosis (MF 2-3) were associated with lower TAC (p=0.045) and HoloTC levels (p=0.017). The patients with JAK-2 mutations had lower HoloTC levels (p=0.0059). HoloTC deficiency was more frequently associated with JAK-2 homozygosity (p<0.0003).Conclusions: Our findings suggest that the determination of HoloTC, tHcy, ROS concentrations and TAC, can identify latent cobalamin deficiency and provide a rational basis for correcting the increased oxidation associated with disease progression.

Irf8-driven myeloid differentiation is regulated by 12/15-lipoxygenase-mediated redox signaling - Accepted Manuscript

2010-07-21

Abstract: Objectives: Several transcription factors determine the cell fate decision between granulocytes and monocytes, but the upstream signal transduction pathways that govern myelopoiesis are largely unknown. Based on our observation of aberrant myeloid cell representation in hematopoietic tissues of 12/15-lipoxygenase (12/15-LOX)-deficient (Alox15) mice, we tested the hypothesis that polyunsaturated fatty acid metabolism regulates myelopoiesis.Methods: Multi-color flow cytometric analysis and methylcellulose assays were used to compare myelopoiesis and the differentiative capacity of progenitors from Alox15 and wild-type mice. Furthermore, we elucidated the mechanism by which 12/15-LOX is involved in regulation of myelopoiesis.Results: Granulopoiesis in Alox15 mice is increased while monopoiesis is reduced. Moreover, there is an accumulation of granulocyte-macrophage progenitors that exhibit defective differentiation. Mechanistically, we demonstrate that transcriptional activity of Irf8, which regulates myelopoiesis, is impaired in Alox15 progenitors and bone marrow-derived macrophages due to loss of 12/15-LOX-mediated redox regulation of Irf8 nuclear accumulation. Restoration of redox signaling in Alox15 bone marrow cells and GMP reversed the defect in myeloid differentiation.Conclusions: These data establish 12/15-LOX-mediated redox signaling as a novel regulator of myelopoiesis and Irf8.

Engraftment of syngeneic bone marrow is not more efficient after intrafemoral transplantation than following traditional intravenous administration - Accepted Manuscript

2010-07-20

Abstract: Objectives: Hematopoietic stem cells (HSCs) are key elements for life-long production of mature blood cells. The success of clinical stem cell transplantation may be improved when the number of stem cells that engraft after transplantation can be increased. Here, we investigated in a syngeneic mouse model whether engraftment and reconstitution can be improved by transplantation directly into the bone marrow.Methods: In this study we directly compared syngeneic transplantation of hematopoietic stem cells into the bone marrow with intravenous administration and assessed reconstitution kinetics and engraftment by bioluminescent imaging (BLI) and chimerism determination.Results: Surprisingly, only about 10% of cells injected directly into the femur (intrafemoral, IF) could be retrieved within 5 minutes after injection. Only in the first 48 hours after transplantation engraftment in IF transplanted animals was higher compared to intravenous (IV) injection. However, at all later time points no differences could be detected using whole body bioluminescence or measuring blood cell reconstitution. Most importantly, we found that IF transplanted cells did not outcompete cells transplanted intravenously when co-transplanted in the same recipient.Conclusion: In conclusion, IF transplantation in a murine syngeneic setting revealed no enhanced engraftment. Previous reports on IF transplantation may have relied on escape from immune rejection in xenogeneic or allogeneic models. Therefore, we conclude that stem cells can find the proper microenvironment irrespective of the route of administration.

Streptamer-based selection of WT1-specific CD8+ T cells for specific donor lymphocyte infusions - Accepted Manuscript

2010-07-16

Abstract: Objective: Donor lymphocyte infusions may generate a desirable graft-versus-leukemia effect, but also elicit a noxious graft-versus-host disease. A positive selection of leukemia (antigen)-specific T cells would be highly desirable. In this study, we focused on the immunogenic leukemia-antigen Wilms' Tumor gene 1 (WT1).Methods: We employed the technology of streptamers available at good manufacturing practice level to first determine the frequency of HLA-A2 restricted WT1-specific CD8+ T cells. Then, specific cells were labeled with streptamers and selected by magnetic cell separation. Purities and the immunophenotype of selected cells were analyzed.Results: 21/40 healthy donors had naive WT1-specific CD8+ T cell frequencies of more than 0.5%, and 8/40 even more than 1.0% of all CD8+ T cells. In seven of ten acute myeloid leukemia patients, the frequencies were 0.5 to 3.65%. After positive selection by magnetic cell separation, a 60-fold increase with a purity of up to 17.79% in the lymphocyte gate and 86.18% in the CD8+ T cell gate could be achieved for CD8+WT1streptamer+CD28+/-CD45RA+CCR7- effector T cells.Conclusions: Streptamer technology allows selection of pure WT1-specific effector T cells. This is a prerequisite for clinical applications targeting tumor-specific antigens such as adoptive T cell transfer.

Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules - Accepted Manuscript

2010-07-15

Abstract: Objectives: Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi).Materials and Methods: SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR.Results: Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ˜40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells.Conclusion: Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma.

Assessment of human MAPCs for stem cell transplantation and cardiac regeneration after myocardial infarction in SCID mice - Uncorrected Proof

2010-07-12

Objective: Clinical studies suggest that transplantation of total bone marrow (BM) after myocardial infarction (MI) is feasible and potentially effective. However, focusing on a defined BM-derived stem cell type may enable a more specific and optimized treatment. Multilineage differentiation potential makes BM-derived multipotent adult progenitor cells (MAPCs) a promising stem cell pool for regenerative purposes. We analyzed the cardioregenerative potential of human MAPCs in a murine model of myocardial infarction.Materials and Methods: Human MAPCs were selected by negative depletion of CD45+/glycophorin+ BM cells and plated on fibronectin-coated dishes. In vitro, stem cells were analyzed by reverse transcription polymerase chain reaction. In vivo, we transplanted human MAPCs (5 × 105) by intramyocardial injection after MI in immunodeficient severe combined immunodeficient (SCID) beige mice. Six and 30 days after the surgical procedure, pressure-volume relationships were investigated in vivo. Heart tissues were analyzed immunohistochemically.Results: Reverse transcription polymerase chain reaction experiments on early human MAPC passages evidenced an expression of Oct-4, a stem cell marker indicating pluripotency. In later passages, cardiac markers (Nkx2.5, GATA4, MLC-2v, MLC-2a, ANP, cTnT, cTnI,) and smooth muscle cell markers (SMA, SM22α) were expressed. Transplantation of human MAPCs into the ischemic border zone after MI resulted in an improved cardiac function at day 6 (ejection fraction, 26% vs 20%) and day 30 (ejection fraction, 30% vs 23%). Confirmation of human MAPC marker vimentin in immunohistochemistry demonstrated that human MAPC integrated in the peri-infarct region. The proliferation marker Ki67 was absent in immunohistochemistry and teratoma formation was not found, indicating no tumorous potential of transplanted human MAPCs in the tumor-sensitive immunodeficient SCID model.Conclusion: Transplantation of human MAPCs after MI ameliorates myocardial function, which may be explained by trophic effects of human MAPCs. Lack of evidence of tumorous potential in the tumor-sensitive SCID model indicates that human MAPCs may deliver an effective and safe stem cell pool for potential treatment of ischemic heart disease.

The cytokine/chemokine pattern in the bone marrow environment of multiple myeloma patients - Uncorrected Proof

2010-07-08

Objective: The interaction of multiple myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing pattern, survival, and proliferation of malignant plasma cells.Materials and Methods: We determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines, as well as in the plasma derived from BM and peripheral blood samples of 10 newly diagnosed MM patients, 20 MM patients who had received allogeneic stem cell transplantation (alloSCT), and 20 healthy donors.Results: Besides cytokines/chemokines known to be secreted by myeloma cell lines, such as interleukin-1 receptor antagonist (IL-1RA), IL-8, monocyte chemotactic protein−1 (MCP-1), macrophage inflammatory protein (MIP)−1α, MIP-1β, and MIP-3α, we also detected significant levels of epidermal growth factor, hepatocyte growth factor (HGF), IL2R, IL-12p40/p70, IL-22, interferon-γ (IFN-γ)−inducible protein 10 (IP-10), monokine induced by IFN-γ, and regulated on activation normally T-cell expressed and secreted in culture supernatants. The BM environment in MM patients evidenced elevated concentrations of HGF, IL-2R, IL-16, epidermal growth factor, IL-1RA, IP-10, MCP-1, and monokine induced by IFN-γ. Additionally, in the BM of MM patients post alloSCT, we found selectively elevated concentration of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin. Eotaxin levels were particularly high in patients with chronic graft-vs-host disease.Conclusion: Our study demonstrates characteristic cytokine/chemokine patterns in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin, might not only be developed into diagnostic instruments and/or predictive biomarkers, but are also potential targets for future myeloma- or graft-vs-host disease−specific therapies.

Cell-dose−dependent increases in circulating levels of immune effector cells in rhesus macaques following intracranial injection of allogeneic MSCs - Uncorrected Proof

Iryna A. Isakova, Jason Dufour, Calvin Lanclos, Julie Bruhn, Donald G. Phinney — 2010-07-02

Objective: Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity, but whether they evade immune surveillance in an allogeneic transplant setting remains controversial. Herein we evaluated whether administration of major histocompatibility (MHC) class I−mismatched MSCs induce an immune response in rhesus macaques.Materials and Methods: MSCs from a male donor were injected intracranially at two different doses into eight immunocompetent female infant rhesus macaques. Blood cell counts and circulating levels of lymphocyte subpopulations were quantified prior to surgery and at 10, 30, and 90 to 180 days postsurgery by flow cytometry. Immunoreactivity of recipient peripheral blood mononuclear cells to donor MSCs was evaluated in vitro and alloantibody production in vivo was determined by enzyme-linked immunosorbent assay and flow cytometry.Results: MSC transplantation induced transient but significant increases in circulating white blood cells, lymphocytes, and neutrophils in most transplant recipients, but not sham-operated control animals. Flow cytometric analysis revealed a strong correlation between expansion of CD8+ve, CD16+ve, and CD8+ve/CD16+ve lymphocyte subpopulations in peripheral blood, the dose of administered MSCs, and degree of antigenic mismatch between donor and recipient. MSC-specific alloantibodies were also detected in several transplant recipients. However, peripheral blood mononuclear cells harvested from transplant recipients postsurgery exhibited no lytic activity against donor MSCs in vitro upon rechallenge.Conclusions: MSCs induced an allograft response in rhesus macaques that involved principally CD8+ve, CD16+ve, and CD8+ve/CD16+ve lymphocyte subpopulations and was cell-dose− and haplotype-dependent. This study demonstrates that MSCs are weakly immunogenic in vivo when transplanted across MHC class I barriers.

Multipotent mesenchymal stem cell grafting to treat cutaneous radiation syndrome: development of a new minipig model - Uncorrected Proof

2010-06-28

Objective: Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Recent grafting of three ionizing radiation−burned patients has suggested the benefit of local bone marrow mesenchymal stem cell (MSC) injection in favor of wound healing and pain control. Here, we have developed a new minipig model of severe CRS to study underlying mechanisms of this cell therapy approach.Materials and Methods: Göttingen minipigs were locally irradiated using a 60Co gamma source as follows: ungrafted 50 and 60 Gy (n = 4) and grafted 50 and 60 Gy (n = 3). Bone marrow MSCs were cultured in minimum essential medium with 10% fetal calf serum and basic fibroblast growth factor (2 ng·mL−1). Autologous MSCs were intradermally injected twice or three times from days 27 to 96 (range, 99−128.5 × 106 MSCs per injection).Results: All animals exhibited a clinical evolution similar to humans after a latency phase of several weeks, including early erythema, hair loss, and dry/moist desquamation followed by necrosis during 81 to 222 days post−ionizing radiation. Skin damage in higher exposed animals appeared slightly earlier. Immunohistology revealed severe skin damage in all animals and rhabdomyolysis in the muscle tissue below the entry area, with the latter being more severe in controls. In grafted animals, MSCs led to local accumulation of lymphocytes at the dermis/subcutis border and improved vascularization.Conclusion: This study establishes a new minipig model that is close to human and allows development of stem cell therapy strategies that can be applied in treatment of human radiation burns.

Limited functional capacity of microchimeric fetal hematopoietic progenitors acquired by mothers during pregnancy - Uncorrected Proof

2010-06-28

Studies have now definitely shown that fetal cells are transferred into the maternal circulation during human, murine, and other mammalian species pregnancy and could persist long-term in maternal niches, mainly the marrow . Indeed, decades after delivery, it is possible to detect fetal-derived cells among the T, B, natural killer, or monocyte cell populations of women with a history of pregnancy . It has therefore been hypothesized that fetal hematopoietic progenitors/stem cells transferred during gestation engraft in maternal bone marrow and give rise to cells detected in the maternal circulation. Numerous studies have indeed described the transfer and persistence of CD34+ cells from the fetus to the mother and claimed their stemness based on the expression of this marker . Of note, CD34+CD38+ lymphoid progenitors also seem to be detected more often after delivery . Finally, after mobilization of hemopoietic progenitors, fetal cells can be found among the CD34+ apheresis product . Others have also shown the capacity of these fetal cells to form colonies if cultured from maternal blood, mostly during or immediately after gestation .

Synchrony of telomere length among hematopoietic cells - Uncorrected Proof

2010-06-28

Objective: Little is known about the relationship of telomere length among leukocyte subsets and leukocytes and cells up the hematopoietic hierarchy. This information is relevant because telomere dynamics in granulocytes were postulated to mirror those of hematopoietic stem cells (HSCs).Materials and Methods: In newborn umbilical cord blood (UCB), we examined the relationships of telomere length in hematopoietic progenitor cells (HPCs) (CD34+CD45−) with those in T lymphocytes and granulocytes. In addition, we correlated telomere length in granulocytes with those in whole leukocyte samples of individuals ranging in age from birth to 100 years.Results: In the UCB, we found strong correlations of telomere length in HPCs with telomere length in T lymphocytes (r ranging from 0.882 to 0.935; p ranging from 0.0038 to 0.0007) and in granulocytes (r = 0.930; p = 0.0072). At birth, strong correlations were also observed between telomere length in granulocytes and those in all leukocytes (r = 0.979; p = 0.0003). Throughout the human lifespan, the relationship between telomere length in granulocytes and that in all leukocytes was r > 0.980 and p < 0.0001.Conclusions: Robust synchrony exists among leukocyte subsets throughout the human lifespan; individuals with relatively long (or short) telomeres in one leukocyte subset have long (or short) telomeres in other leukocyte subsets. Moreover, telomere length in leukocytes reflects its length in cells up the hematopoietic hierarchy, i.e., HPCs and, by inference, HSCs. Strong links have been found by many studies between leukocyte telomere length and a host of aging-related diseases. Our findings suggest, therefore, that that these links might be traced to telomere dynamics in HSCs.

Identification of E74-like factor 1 (ELF1) as a transcriptional regulator of the Hox cofactor MEIS1 - Uncorrected Proof

2010-06-24

Objective: Myeloid ectropic viral integration site 1 (MEIS1) is a Hox cofactor known for its role in development and is strongly linked to normal and leukemic hematopoiesis. Although previous studies have focused on identifying protein partners of MEIS1 and its transcriptionally regulated targets, little is known about the upstream transcriptional regulators of this tightly regulated gene. Understanding the regulation of MEIS1 is important to understanding normal hematopoiesis and leukemogenesis.Materials and Methods: Here we describe our studies focusing on the evolutionary conserved putative MEIS1 promoter region. Phylogenetic sequence analysis followed and reporter assays in MEIS1-expressing (K562) and nonexpressing (HL60) leukemic cell line models were used to identify key regulatory regions and potential transcription factor binding sites within the candidate promoter region followed by functional and expression studies of one identified regulator in both cell lines and primary human cord blood and leukemia samples.Results: Chromatin status of MEIS1 promoter region is associated with MEIS1 expression. Truncation and mutation studies coupled with reporter assays revealed that a conserved ETS family member binding site located 289 bp upstream of the annotated human MEIS1 transcription start site is required for promoter activity. Of the three ETS family members tested, only ELF1 was enriched on the MEIS1 promoter as assessed by both electrophoretic mobility shift assay and chromatin immunoprecipitation experiments in K562. This finding was confirmed in MEIS1-expressing primary human samples. Moreover, small interfering RNA−mediated knockdown of ELF1 in K562 cells was associated with a decreased MEIS1 expression.Conclusions: We conclude that the ETS transcription factor ELF1 is an important positive regulator of MEIS1 expression.

Abnormal regulation of soluble and anchored IL-6 receptor in monocytes from patients with essential thrombocythemia - Uncorrected Proof

2010-06-24

Objective: In a previous study, we found increased plasma soluble receptor for interleukin-6 (sIL-6R) levels in patients with essential thrombocythemia (ET) that could promote megakaryocytopoiesis through IL-6 binding and further interaction with the signal transducer gp130. Here we have searched for the cell source of sIL-6R within mononuclear cells in these patients and the underlying abnormalities involved in its overproduction.Materials and Methods: Thirty patients with the diagnosis of ET were studied. sIL-6R levels were measured by enzyme-linked immunosorbent assay technique in the supernatants of peripheral monocyte and lymphocyte cultures. Expression of membrane-anchored IL-6R was determined by flow cytometry. In order to study the mechanism of sIL-6R production, tumor necrosis factor−α protease inhibitor was added to specifically block IL-6R shedding. Gene expression of sIL-6R levels were evaluated by reverse transcription polymerase chain reaction.Results: Monocytes were the main source of sIL-6R. Besides, in ET patients, monocyte sIL-6R release was higher than that of controls (p = 0.0014). Lymphocytes enhanced monocyte sIL-6R production by cell-mediated contact in normal controls, but this cooperation could not be seen in patients. Membrane expression of IL-6R was increased after monocyte adhesion in ET. sIL-6R synthesis was upregulated in most patients, while messenger RNA was normal.Conclusion: Our results indicate that ET monocytes are responsible for sIL-6R overproduction within mononuclear cells through synthesis upregulation. In addition, the lack of cooperation of lymphocytes in monocyte sIL-6R production in ET could be due to a monocyte abnormality. The agonistic effect of sIL-6R on IL-6 action could contribute to the exacerbated megakaryocytic growth in ET.

Production of proinflammatory cytokines without invocation of cytotoxic effects by an Epstein-Barr virus−infected natural killer cell line established from a patient with hypersensitivity to mosquito bites - Uncorrected Proof

2010-06-23

Objective: Cumulative evidence supports that Epstein-Barr virus (EBV)−infected natural killer (NK) cells induce severe systemic and cutaneous inflammation in patients with hypersensitivity to mosquito bites (HMB). In order to understand the pathogenesis of HMB, we established an EBV-infected cell line and characterized the cytological profiles.Materials and Methods: A novel EBV-infected NK-cell line, designated NKED, was established from a patient with HMB and used for the present study along with two other NK-cell lines, KAI3 and KHYG-1.Results: NKED expressed the latency II−related transcripts. NKED cells were positive for CD2 and CD161 antigens, and negative for CD3, CD16, CD34, CD56, and T-cell receptor α/β and γ/δ antigens. Although NKED cells contained several cytotoxic molecules, the cells had an extremely poor cytotoxic activity. The majority of NKED cells were negative for perforin, major histocompatibility complex class I−restricted NK-cell receptors, CD94 and KIR2D, and an activating receptor, NKG2D. NKED cells, however, secreted higher levels of tumor necrosis factor−α. Stimulation with phorbol 12-myristate 13-acetate or tumor necrosis factor−α induced expression of BZLF1 messenger RNA in the NKED and KAI3 cells, indicating the transition from the latent- to the lytic-cycle infection.Conclusion: These data suggested that NKED cells revealed a very low cytotoxic effect probably because of the low expression levels of perforin, but had the ability to release proinflammatory cytokines. NKED cells did not reflect the characteristics of HMB, as they were different from pathogenic NK cells proliferating in the HMB patient, but the difference indicated that pathogenic NK cells could change their character in the presence of interleukin-2.

IL-17 is a potent synergistic factor with GM-CSF in mice in stimulating myelopoiesis, dendritic cell expansion, proliferation, and functional enhancement - Uncorrected Proof

2010-06-21

Objective: Interleukin (IL)-17, which now defines the Th17 immune response, is a critical cytokine expressed and required for stress granulopoiesis during microbial invasion. Dendritic cells (DC) can instigate this response by inducing IL-17 expression in CD4+ T cells. Besides IL-17, microbial invasion also stimulates production of the DC growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). The objective was the in vitro and in vivo investigation of IL-17 on DC proliferation and function in mice.Materials and Methods: Murine IL-17 (mIL-7) or murine GM-CSF (mGM-CSF), or both, was expressed in C57BL6 mice using adenoviral technology to assess hematopoietic and DC changes. The E-22 tymoma tumor cell line using a previously described vaccinia virus ovalbumin/LacZ murine tumor model was employed to study effects on tumor rejection.Results: The combination of mIL-17 and mGM-CSF increased peripheral neutrophila by 28-fold and splenic colonies by 11- and 14-fold over each individual factor in mice, respectively. The effect of mIL-17 by itself on murine DCs in vitro and in vivo was minimal; however, the combination greatly enhanced the stimulating effects of mGM-CSF, increasing the total numbers of CD14b/c+ spleen DC by fourfold, as well as their function measured by enhanced endocytosis. Mixed lymphocyte reactions using mIL-17/mGM-CSF cultured DCs stimulator cells enhanced lymphocyte responses by twofold over mGM-CSF alone. Vaccination against LacZ in the C57BL6 E22 syngenic thymoma tumor model effectively delayed tumor growth in animals pretreated with the mIL-17/mGM-CSF combination prior to vaccination.Conclusions: mIL-17 effectively synergizes with mGM-CSF in stimulating granulopoiesis and DC expansion, as well as in functional enhancement of DCs.

Slow-cycling/quiescence balance of hematopoietic stem cells is related to physiological gradient of oxygen - Uncorrected Proof

2010-06-14

Objective: Regulation of hematopoiesis depends on cytokines, cellular interactions, transcription, and metabolic factors. Among the latter, O2 has been neglected for a long time. Recently, an increasing number of publications evidenced the regulatory role of physiological low O2 concentrations (0.1−5%; similar to those in bone marrow) on the in vitro behavior of hematopoietic stem cells. This brief review utilizes the article of Eliasson and colleagues in this Journal to summarize the major results and questions about the relationships between O2 and hematopoiesis.Materials and Methods: In order to be synthetic and interesting for readers unfamiliar with this field, we selected only the most significant data that either reinforce or contradict the conclusions of Eliasson et al., but we also provide references of reviews with a more detailed bibliography.Results: A critical analysis of some key publications provides partial answers to three important questions: is the term hypoxia appropriate to describe physiological low O2 concentrations? Is a very low O2 level sufficient to control the quiescence/slow cycling balance of hematopoietic stem cells? Is the O2 concentration able to modify the effect of cytokines on hematopoietic stem cells?Conclusion: We propose to use in situ normoxia instead of the confusing term hypoxia when working with normal cells at physiological low O2 concentrations. We suggest that a very low O2 concentration is necessary but not sufficient to induce hematopoietic stem cell quiescence. We review some articles showing that O2 variations modify the effect of cytokines.

Contact with the bone marrow microenvironment readdresses the fate of transplanted hematopoietic stem cells - Uncorrected Proof

2010-06-14

Objective: Despite enormous advancements in our comprehension of molecular mechanisms governing hematopoietic stem cells (HSCs) engraftment in the bone marrow, current clinical protocols of intravenous (IV) transplantation suffer from a relatively low seeding efficiency. To solve this problem, intrabone (IB) injection of HSCs has been proposed. However, the mechanisms underlying the benefit provided by this procedure remain unknown. This study aims to evaluate the effect of IB on HSCs trafficking and homing features in the living rat.Materials and Methods: A total of 35 Lewis rats underwent IB or IV administration of HSCs harvested from syngeneic animals and purified according to CD90 expression. These cells were labeled with 37 MBq 99mTc-exametazime and injected either IV or IB. Cell trafficking and distribution in heart, lung, spleen, liver, and forelimb was evaluated by dynamic radionuclide imaging. Logan graphical approach was used to estimate tissue recruitment of HSCs.Results: More than 90% of cells escaped from the injected bone to the bloodstream in <15 seconds. However, this short contact profoundly modified HSCs kinetics, reducing their lung sequestration and shortening their blood persistence with respect to IV. More importantly, IB passage resulted in reduced lung uptake and in a fourfold increase in homing of remote bone marrow sites. CD90+ cells transplantation restored hematopoiesis in eight further rats previously exposed to lethal irradiation.Conclusion: The first-entry contact with the hematopoietic microenvironment immediately readdresses the fate of transplanted HSCs, providing them with “the final destination stamp” to define their bone marrow homing.

Induction of pluripotency in human cord blood−unrestricted somatic stem cells - Uncorrected Proof

2010-06-11

Objective: Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)−derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells.Materials and Methods: Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation.Results: CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential.Conclusions: Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen−matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.

Glycogen synthase kinase−3β inhibitors suppress leukemia cell growth - Uncorrected Proof

2010-06-10

Objective: The objective of this study was to investigate the effect of small molecule inhibitors of glycogen synthase kinase−3β (GSK-3β) on leukemia cell growth and survival.Materials and Methods: Analysis of cytotoxicity and cell proliferation was conducted using the MTS assay, cell-cycle analysis, and division tracking. Apoptosis was investigated by Annexin-V/7-aminoactinomycin D and caspase-3 expression. The effect of GSK-3β inhibitors was also tested in vivo in an animal model of leukemia. Gene expression analysis was performed to identify the genes modulated by GSK-3β inhibition in leukemia cells.Results: GSK-3β inhibitors suppress cell growth and induce apoptosis in seven leukemia cell lines of diverse origin, four acute myeloid leukemia, one myelodysplastic syndrome, and one acute lymphoblastic leukemia samples. GSK-3β inhibitors are cytotoxic for rapidly dividing clonogenic leukemia blasts, and higher doses of the inhibitors are needed to eliminate primitive leukemia progenitor/stem cells. Slow cell-division rate, low drug uptake, and interaction with bone marrow stroma make leukemia cells more resistant to apoptosis induced by GSK-3β inhibitors. Global gene expression analysis combined with functional approaches identified multiple genes and specific signaling pathways modulated by GSK-3β inhibition. An important role for Bcl2 in the regulation of apoptosis induced by GSK-3β inhibitors was defined by expression analysis and confirmed by using pharmacological inhibitors of the protein. In vivo administration of GSK-3β inhibitors delayed tumor formation in a mouse leukemia model. GSK-3β inhibitors did not affect hematopoietic recovery following irradiation.Conclusion: Our data support further evaluation of GSK-3β inhibitors as promising novel agents for therapeutic intervention in leukemia and warrant clinical investigation in leukemia patients.

KIT polymorphisms and mutations determine responses of neoplastic mast cells to bafetinib (INNO-406) - Corrected Proof

2010-05-31

Objective: Advanced systemic mastocytosis (SM) is characterized by uncontrolled growth of neoplastic mast cells (MC) and drug resistance. The tyrosine kinase receptor KIT is often mutated and activated and thus contributes to malignant growth of MC. Therefore, KIT-targeting drugs are currently tested for their ability to block growth of malignant MC.Materials and Methods: We determined the effects of the multikinase inhibitor INNO-406 (bafetinib) on primary neoplastic MC, the canine mastocytoma cell line C2, the human MC leukemia cell line HMC-1.1 bearing the KIT mutant V560G, and HMC-1.2 cells harboring KIT V560G and KIT D816V.Results: INNO-406 was found to inhibit proliferation in HMC-1.1 cells (IC50: 30−40 nM), but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM. In canines, growth-inhibitory effects of INNO-406 were seen in C2 cells (IC50: 50−100 nM) exhibiting a KIT exon 11 internal tandem-duplication and in primary neoplastic MC harboring wild-type exon 11, whereas no effects were seen in MC exhibiting a polymorphism at amino acid 581 in exon 11. INNO-406 was found to block KIT phosphorylation and expression in HMC-1.1 cells and C2 cells, but not in HMC-1.2 cells, whereas Lyn-phosphorylation was blocked by INNO-406 in all types of MC.Conclusions: In neoplastic MC, the major target of INNO-406 appears to be KIT. Drug responses may depend on the presence and type of KIT mutation. In human MC, the KIT D816V mutant introduces resistance, and in canine mastocytomas, an exon 11 polymorphism may be indicative of resistance against INNO-406.

Of mice and men: Human RNA polymerase III promoter U6 is more efficient than its murine homologue for shRNA expression from a lentiviral vector in both human and murine progenitor cells - Corrected Proof

Roland Roelz, Ingo H. Pilz, Manuel Mutschler, Heike L. Pahl — 2010-05-31

Objective: RNA interference mediated by transcription of short hairpin RNAs (shRNAs) from lentiviral expression vectors has emerged as an efficient method to effectively and specifically silence gene expression in a vast variety of mammalian cells. shRNA expression is routinely driven by a RNA polymerase III promoter, most often by the U6 promoter. Here we demonstrate that U6 promoter activity—and consequently gene silencing success—differs significantly among species.Materials and Methods: We have modified pLeGO-G, an HIV-based third-generation lentivector, to express a 19nt shRNA sequence against the human transcription factor nuclear factor erythroid 2 or against its murine homologue, as well as an shRNA against murine JAK2, from either the human or the murine U6 promoter. Gene silencing efficiency was analyzed in a human erythroleukemic cell line, in primary human CD34+ cells, as well as in a murine erythroleukemic cell line and in primary murine bone marrow.Results: ShRNA expression from the human U6 promoter resulted in a fourfold increase in knockdown efficiency compared to expression from the murine U6 promoter in both human and murine cells.Conclusions: The U6 promoter constitutes an important determinant for efficient gene silencing by shRNAs.

In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V - Uncorrected Proof

2010-05-31

Objective: In most patients with systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell (MC) leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V, which confers resistance to imatinib. Cladribine (2-chlorodeoxyadenosine [2CdA]) is a nucleoside analog that has been introduced as a promising agent for treatment of advanced SM.Materials and Methods: We examined the in vitro effects of cladribine (2CdA) on growth of neoplastic MC, and the in vivo effects of 2CdA (0.13 mg/kg/day intravenously, days 1−5; three to eight cycles) in seven patients with advanced SM.Results: Cladribine was found to inhibit growth of primary MC and the MC line HMC-1 in a dose-dependent manner, with lower IC50 values recorded in HMC-1.2 cells harboring KIT D816V (IC50: 10 ng/mL) compared to HMC-1.1 cells lacking KIT D816V (IC50: 300 ng/mL). In two patients with progressive smoldering SM, cladribine produced a long-lasting response with a sustained decrease in serum tryptase levels, whereas in patients with progressive ASM or MCL, cladribine showed little if any effects. The drug was well-tolerated in most cases. However, one patient developed a massive generalized purulent long-lasting skin rash. The antiproliferative effects of cladribine on MC were found to be associated with morphologic signs of apoptosis and caspase cleavage. Cladribine did not counteract the kinase activity of KIT D816V or KIT-downstream signaling molecules.Conclusions: Cladribine may be a promising agent for treatment of progressive smoldering KIT D816V+ SM. In rapidly progressing ASM or MCL, additional or alternative drugs are required to induce antineoplastic effects.

Modulated expression of adhesion molecules and galectin-1: Role during mesenchymal stromal cell immunoregulatory functions - Corrected Proof

2010-05-31

Objective: As mesenchymal stromal cells (MSCs) have been proposed as a tool for management or prevention of graft-vs-host disease, we investigated their immunoregulatory properties, their expression of adhesion molecules and galectin-1, and the impact of environment context on these functions.Materials and Methods: The effects of MSCs on T-cell proliferation were analyzed using carboxyfluorescein diacetate N-succinimidyl ester labeling. We evaluated the expression of adhesion molecules and galectin-1 by MSCs and the impact of an inflammatory or infectious environment on these expressions. Using neutralizing antibodies against adhesion molecules and a galectin-1 inhibitor, we assessed the role of these molecules in MSC functions.Results: MSCs inhibition of T-cell proliferation depended on MSC concentrations, cell contact, and culture environment. Expression of adhesion molecules and secretion of galectin-1 by MSCs are tightly regulated. Coculture with activated T cells upregulated expression of CD54 (intercellular adhesion molecule 1) and CD58 (lymphocyte function−associated antigen 3) and secretion of galectin-1 by MSCs. Interestingly, in an inflammatory or infectious environment, expression of adhesion molecules and galectin-1 by MSCs was differentially modulated. Furthermore, blocking galectin-1 activity prevented the suppressive potential of MSCs. Neutralization of adhesion molecule activity had no effect on MSC inhibition.Conclusion: Galectin-1 plays an important role in MSC immunoregulatory functions, which are depending on cell environment. The present study provides new insights concerning MSC physiology and will increase the safety and efficiency of MSCs in clinical settings.

H1-receptor antagonists terfenadine and loratadine inhibit spontaneous growth of neoplastic mast cells - Uncorrected Proof

2010-05-31

Objective: In mast cell (MC) neoplasms, clinical problems requiring therapy include local aggressive and sometimes devastating growth of MCs and mediator-related symptoms. A key mediator of MCs responsible for clinical symptoms is histamine. Therefore, use of histamine receptor (HR) antagonists is an established approach to block histamine effects in these patients.Materials and Methods: We screened for additional beneficial effects of HR antagonists and asked whether any of these agents would also exert growth-inhibitory effects on primary neoplastic MCs, the human MC line HMC-1, and on two canine MC lines, C2 and NI-1.Results: We found that the HR1 antagonists terfenadine and loratadine suppress spontaneous growth of HMC-1, C2, and NI-1 cells, as well as growth of primary neoplastic MCs in all donors tested (human patients, n = 5; canine patients, n = 8). The effects of both drugs were found to be dose-dependent (IC50: terfenadine, 1–20 μM; loratadine, 10−50 μM). Both agents also produced apoptosis in neoplastic MCs and augmented apoptosis-inducing effects of two KIT-targeting drugs, PKC412 and dasatinib. The other HR1 antagonists (fexofenadine, diphenhydramine) and HR2 antagonists (famotidine, cimetidine, ranitidine) tested did not exert substantial growth-inhibitory effects on neoplastic MCs. None of the histamine receptor blockers were found to modulate cell-cycle progression in neoplastic MCs.Conclusions: The HR1 antagonists terfenadine and loratadine, in addition to their antimediator activity, exert in vitro growth-inhibitory effects on neoplastic MCs. Whether these drugs (terfenadine) alone, or in combination with KIT inhibitors, can also affect in vivo neoplastic MC growth remains to be determined.

Identification of defects in the transcriptional program during lineage-specific in vitro differentiation of CD34+ cells selected from patients with both low- and high-risk myelodysplastic syndrome - Uncorrected Proof

2010-05-31

Objective: Development of myelodysplastic syndrome (MDS) is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells, resulting in aberrant differentiation and proliferation.Materials and Methods: To detect alterations within the transcriptional program in MDS-derived CD34+ cells during lineage-specific differentiation, we performed serial gene expression analysis of in vitro differentiated erythro-, granulo-, and megakaryopoietic cells using oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA, USA). For selected genes, expression data were confirmed using real-time polymerase chain reaction.Results: We identified genes with altered expression during lineage-specific differentiation in either low- or high-risk MDS cells compared to the expression patterns of continuously up- or downregulated genes from the normal transcriptional program of hematopoiesis. In cluster analyses, we could show that MDS samples have a distinct expression pattern of a set of selected genes compared to normal cells, which allows prediction of the affiliation of a sample to one group. Furthermore, this study gives an overview of genes that are differentially expressed in MDS cells compared to normal hematopoiesis.Conclusion: Our data provide the first comprehensive transcriptional analysis of differentiating human CD34+ cells derived from MDS patients compared to normal individuals. It gives new insights into the alteration of differentiation and proliferation of MDS stem cells.

Analysis of migratory and prosurvival pathways induced by the homeostatic chemokines CCL19 and CCL21 in B-cell chronic lymphocytic leukemia - Uncorrected Proof

2010-05-20

Objective: The CCR7 chemokine receptor has been reported to promote homing of B-cell chronic lymphocytic leukemia (CLL) cells into lymph nodes and support their survival, but the mechanisms mediating these effects are largely unknown. We investigated the role of different signaling pathways triggered by CCR7 engagement by its ligands, the chemokines CCL19 and CCL21, in the control of CLL migration and survival.Materials and Methods: Chemotaxis and apoptosis assays were performed in the presence of pharmacologic inhibitors and genetic mutants of the phosphatidylinositol-3-OH kinase (PI3K), Rho guanosine triphosphatase, and mitogen-activated protein kinase (MAPK) signaling cascades to assess the role of these pathways on primary CLL migration and survival in response to CCR7 activation. Kinase activation was determined by immunoblotting and pull-down experiments.Results: CLL chemotactic activity induced by CCL19 or CCL21 was markedly reduced by inhibitors of PI3K and the Rho effector molecule Rho-associated coiled-coil forming protein kinases (ROCK), and also by the expression of dominant negative forms of PI3K and RhoA, whereas constitutively activated PI3K and RhoA mutants strongly promoted CLL migration. In contrast, MAPKs were not significantly involved in CLL migration to CCL19/CCL21. Conversely, extracellular signal-regulated kinase and c-Jun-N-terminal kinase, along with PI3K, had a role in CCR7-mediated CLL cell survival. Biochemical experiments confirmed that CCL19/21 induced PI3K-dependent phosphorylation of Akt/protein kinase B, activation of the Rho/Rho-associated coiled-coil forming protein kinases/myosin light chain pathway and MAPKs phosphorylation.Conclusions: The role of PI3K, Rho guanosine triphosphatases, and MAPKs in CCR7-mediated CLL cells migration and survival suggests that these signal transduction pathways could represent promising targets for CLL therapy.

MT1-MMP association with membrane lipid rafts facilitates G-CSF−induced hematopoietic stem/progenitor cell mobilization - Uncorrected Proof

2010-05-14

Objective: Soluble matrix metalloproteinases (MMPs) facilitate the egress of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF)−induced mobilization. Because membrane-type (MT)1-MMP, which is localized on the leading edge of migrating cells, activates the latent forms of soluble MMPs, we investigated its role in HSPC mobilization.Materials and Methods: We examined the effect of G-CSF on the expression of MT1-MMP and its activities (proMMP-2 activation and migration) in hematopoietic cells. We also investigated the subcellular localization of MT1-MMP and the signaling pathways that regulate its expression and function in hematopoietic cells after exposure to G-CSF.Results: We found that G-CSF increases MT1-MMP transcription and protein synthesis in hematopoietic cells; proMMP-2 activation in cocultures of HSPC with BM fibroblasts; chemoinvasion across reconstituted basement membrane Matrigel toward a stromal cell−derived factor-1 gradient, which is reduced by small interfering RNA silencing of MT1-MMP; and localization of MT1-MMP to membrane lipid rafts through a mechanism that is regulated by the phosphatidylinositol 3-kinase signaling pathway. Disruption of raft formation (by the cholesterol-sequestering agent methyl-β-cyclodextrin) abrogated phosphatidylinositol 3-kinase phosphorylation and MT1-MMP incorporation into lipid rafts resulting in reduced proMMP-2 activation and HSPC migration.Conclusion: G-CSF−induced upregulation of MT1-MMP in hematopoietic cells and its enhanced incorporation into membrane lipid rafts contributes to proMMP-2 activation, which facilitates mobilization of HSPC.

Establishment of a new Philadelphia chromosome−positive acute lymphoblastic leukemia cell line (SK-9) with T315I mutation - Uncorrected Proof

Seiichi Okabe, Tetsuzo Tauchi, Kazuma Ohyashiki — 2010-05-14

Objective: The BCR-ABL mutation, T315I, is a common mutation and is resistant to both imatinib and second-generation Abl kinase inhibitors. Although strategies to overcome resistance-mediated T315I mutation may improve the survival of BCR-ABL−positive leukemia patients, there is little information on cell-based studies.Materials and Methods: We established a new human BCR-ABL−positive acute lymphoblastic leukemia (ALL) cell line, SK-9 with the T315I mutation, from the peripheral blood of a 36-year-old female patient.Results: Growth kinetic studies revealed an approximate population doubling time of 48 hours. The common B-cell phenotype is a feature of the SK-9 cell line. Cells have the Philadelphia chromosome (Ph) with many structural abnormalities, as well as the T315I mutation in the BCR-ABL gene. Insertion of SK-9 cells into athymic nude mice induced the formation of tumors in the lymph node that infiltrated into the spleen and bone marrow. We examined the drug sensitivity of imatinib, dasatinib, and nilotinib using a cell proliferation assay and an immunoblot assay. Cell proliferation did not decrease after imatinib, dasatinib, or nilotinib treatment as compared to the BCR-ABL−positive chronic myeloid leukemia cell line K562. Because phosphorylation of BCR-ABL and Crk-L did not decrease after imatinib and dasatinib treatment, it is suggested that SK-9 is resistant to imatinib, dasatinib, and nilotinib.Conclusion: This cell line may provide a useful model for in vitro and in vivo cellular and molecular studies of BCR-ABL−positive ALL with T315I mutation.

VE-cadherin and PECAM-1 enhance ALL migration across brain microvascular endothelial cell monolayers - Corrected Proof

2010-05-13

Objective: Infiltration of the central nervous system (CNS) by leukemia is a problematic disease manifestation of acute lymphoblastic leukemia (ALL). The mechanisms by which leukocytes interact with human brain−derived microvasculature endothelial cells (HBMEC) and enter the CNS are largely derived from models of inflammation. However, our data indicate that ALL cells do not elicit an inflammatory phenotype by HBMEC. Our current investigation focuses on the contribution of the unique coexpression of vascular endothelial (VE)−cadherin and platelet endothelial cell adhesion molecule−1 (PECAM-1) by ALL in mediating leukemic cell interactions with HBMEC as an in vitro model of the blood-brain barrier.Materials and Methods: Primary ALL and ALL cell lines were evaluated for VE-cadherin and PECAM-1 expression. Lentiviral-mediated transduction of VE-cadherin and PECAM-1 into REH cells and antibody neutralization of VE-cadherin and PECAM-1 in SUP-B15 cells was used to delineate the role of these two proteins in mediating ALL adhesion to, and migration through, HBMEC monolayers.Results: Although cell line models indicate that VE-cadherin and PECAM-1 expression is found on the surface Philadelphia chromosome−positive ALL, evaluation of primary ALL demonstrates that VE-cadherin and PECAM-1 are expressed independent of Philadelphia status. Expression of VE-cadherin and PECAM-1 by ALL enhanced the adhesion of ALL to HBMEC, while expression of PECAM-1 enhanced ALL adhesion to, and migration through, HBMEC.Conclusions: Expression of VE-cadherin and PECAM-1 by ALL cells positions them to interact with HBMEC. By increasing our understanding of molecular mechanisms through which ALL cells gain entry into the CNS, new strategies may be designed to prevent leukemia cell entry into the CNS.

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.

Osteopoietic engraftment after bone marrow transplantation: Effect of inbred strain of mice - Corrected Proof

2010-05-05

Objective: Transplantable osteoprogenitors, as well as hematopoietic progenitors, reside in bone marrow. We previously reported the first clinical trial of bone marrow transplantation (BMT) for a genetic disorder of bone, osteogenesis imperfecta. Although the patients demonstrated striking clinical benefits after transplantation, measured osteopoietic engraftment was low and did not seem to be durable. Therefore, we sought an animal model, which closely reflects the clinical experience, to facilitate development of strategies to improve the efficiency of osteoprogenitor engraftment after BMT.Materials and Methods: We transplanted unfractionated bone marrow cells from green fluorescent protein−transgenic mice into lethally irradiated recipients in four combinations of inbred mouse strains: from C57BL/6 into C57BL/6 (C-C), from C57BL/6 into FVB/N (C-F), from FVB/N into C57BL/6 (F-C), and from FVB/N into FVB/N (F-F). At 2 weeks after transplantation, we assessed donor hematopoietic and osteopoietic engraftment by flow cytometry, using a novel mean fluorescence assay, and by immunohistochemical staining for green fluorescent protein.Results: Hematopoietic reconstitution by donor cells was complete in all four combinations. Although osteopoietic engraftment of the transplanted cells was also documented in all the four groups, the magnitude of osteopoietic engraftment differed markedly among the strains where F-F > C-F > F-C > C-C.Conclusion: Our findings indicate that the genetic background of inbred mouse strains affects efficiency of osteopoietic engraftment after BMT. Thus, the murine strain must be considered when comparing experimental outcomes. Moreover, comparing the genetic variation among murine strains may lend insight into the factors governing osteopoietic differentiation of transplanted marrow cells.

Gene transfer into human cord blood−derived CD34+ cells by adeno-associated viral vectors - Uncorrected Proof

2010-05-05

Objective: Bone marrow−derived CD34+ cells are currently used in clinical trials in patients with ischemic heart disease. An option to enhance activity of injected progenitors may be offered by genetic engineering of progenitor cells with angiogenic growth factors. Recombinant adeno-associated viral vectors (rAAV) have emerged as a leading gene transfer systems. In contrast to other vector systems in use for genetic engineering of CD34+ cells, rAAV-mediated gene expression does not depend on vector integration. This is relevant for application in regenerative medicine of ischemic tissues, where transient transgene expression is likely sufficient to achieve therapeutic benefits.Materials and Methods: We compared three different human AAV serotypes, packaged as pseudotypes by a helper virus-free production method, for their transduction efficiency in human cord blood−derived CD34+ cells. We further assessed the impact of vector genome conformation, of αvβ5 and α5β1 integrin availability and of the transcription-modulating drugs retinoic acid and Trichostatin A on rAAV-mediated human CD34+ cell transduction.Results: We provide, for the first time, evidence that hCD34+ cells can be reproducibly transduced with high efficiency by self-complementary rAAV2 without inducing cytotoxicity or interfering with their differentiation potential. We further show the involvement of α5β1 integrin as a crucial AAV2 internalization receptor and a function for transcription-modulating drugs in enhancing rAAV-mediated transgene expression.Conclusion: This study represents a first step toward translation of a combined cellular/rAAV-based therapy of ischemic disease.

Establishment of a new Glivec-resistant chronic myeloid leukemia cell line, SNUCML-02, using an in vivo model - Corrected Proof

2010-05-03

Objective: In this study, we report a newly established chronic myeloid leukemia (CML) cell line, SNUCML-02, which is resistant to imatinib and describe its biological characteristics.Materials and Methods: Mononuclear cells were obtained from the bone marrow of a CML patient in blast crisis and were cultured in Dulbecco's modified Eagle's medium/F12 containing 20% fetal bovine serum. After 2 months of primary culture, these cells were injected into nonobese diabetic/severe combined immune-deficient mice via tail vein. Eight weeks after injection, mice were sacrificed and ex vivo culture was performed from the bone marrow cells isolated from the mice. The established cell line was named as SNUCML-02 and the biological features were characterized by cytogenetic analysis, fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, sequencing analysis, cell proliferation assay, and Western blot analysis.Results: Cytogenetic studies using conventional G-banding and fluorescent in situ hybridization of SNUCML-02 demonstrated classical Philadelphia chromosome, (9;22)(q34;q11.2), and other abnormalities, such as add(11)(q23), +19 and +der(9;22). SNUCML-02 has the same BCR-ABL fusion transcript as was seen in K562 cells, but has no mutations in the ABL kinase domain. SNUCML-02 was more resistant to imatinib (STI571, Gleevec, Glivec) than other CML cell lines (K562, Kcl22, and BV173). SNUCML-02 has constitutive activation of extracellular signal-regulated kinase phosphorylation. In addition, interleukin-3 induced c-ABL phosphorylation and constitutively enhanced extracellular signal-regulated kinase phosphorylation was not inhibited by imatinib in SNUCML-02.Conclusion: SNUCML-02 is a new established cell line with a relatively high level of resistance to imatinib, which is useful for investigating the pathogenesis of CML progression, and will be useful in developing optimal therapeutic strategies for this ailment.

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.

A retroviral vector common integration site between leupaxin and zinc finger protein 91 (ZFP91) observed in baboon hematopoietic repopulating cells - Corrected Proof

Hans-Peter Kiem, Christina Ironside, Brian C. Beard, Grant D. Trobridge — 2010-04-30

Objective: Retroviral vector proviruses can lead to aberrant expression of nearby genes in hematopoietic repopulating cells, leading to an over-representation of clones with dysregulated genes that affect hematopoiesis. Common integration sites (CISs) identified using the vector provirus as a molecular tag can be used to identify these genes. Here we characterized a retroviral CIS observed at high frequency in baboon hematopoietic repopulating cells that has not been described previously.Materials and Methods: Gammaretroviral vector integration sites in baboon repopulating cells identified by polymerase chain reaction amplification were localized to the human genome to identify a CIS. The presence of each clone was tracked over time using allele-specific polymerase chain reaction.Results: In three different animals that received gammaretrovirally transduced CD34-enriched bone marrow cells, vector proviruses were identified at three distinct sites within a window of 664 base pairs between leupaxin and zinc finger protein 91 (ZFP91). All three integrants of the CIS occurred within a CpG island between leupaxin and zinc finger protein 91 (ZFP91).Conclusions: We describe a novel CIS between leupaxin and ZFP91 in hematopoietic repopulating cells. Our data suggest that leupaxin and/or ZFP91 may play a role in hematopoietic repopulating cells.