Experimental Hematology - Articles in Press

Effects Of High Dose Chemotherapy On Bone Marrow Multipotent Mesenchymal Stromal Cells Isolated From Lymphoma Patients - Accepted Manuscript

2010-02-08

Abstract: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effect of this therapy on multipotent mesenchymal stromal cells (MSCs)Objective: Characterize morphologically and functionally MSCs isolated from bone marrow aspirates of patients after HDCTMethods: We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by ASCT, 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing α-MEM plus 15% fetal bovine serum.Results: The cell population isolated showed a typical MSC morphology, immunophenotype and differentiation capacity into adipogenic, osteogenic and chondrogenic lineages. The MSCs obtained from patients with Hodgkin's disease and non-Hodgkin lymphoma showed decreased CFU-F count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity or hematopoiesis support capability between control and patient group.Conclusions: The results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time and cell expansion rates when compared to controls.

Hypoxia mediates low cell cycle activity and increases the proportion of long term-reconstituting hematopoietic stem cells during in vitro culture - Accepted Manuscript

2010-02-04

Abstract: Objective: Recent evidence suggests that hematopoietic stem cells (HSCs) in the bone marrow (BM) are located in areas where the environment is hypoxic. Although previous studies have demonstrated positive effects by hypoxia, its role in HSC maintenance has not been fully elucidated, neither has the molecular mechanisms been delineated. Here, we have investigated the consequence of in vitro incubation of HSCs in hypoxia prior to transplantation and analyzed the role of hypoxia inducible factor (HIF)-1α.Materials and Methods: HSC and progenitor populations isolated from mouse BM were cultured in 20% or 1% O2, and analyzed for effects on cell cycle, expression of cyclin-dependent kinase inhibitors (CDKI) genes, and reconstituting ability to lethally irradiated mice. The involvement of HIF-1α was studied using methods of protein stabilization and gene silencing.Results: When long-term FLT3-CD34- Lin-Sca-1+c-Kit+ (LSK) cells were cultured in hypoxia, cell numbers were significantly reduced in comparison to normoxia. This was due to a decrease in proliferation and more cells accumulating in G0. Moreover, the proportion of HSCs with long-term engraftment potential was increased. Whereas expression of the CDKI genes p21cip1, p27Kip1, and p57Kip2 increased in LSK cells by hypoxia, only p21cip1 was upregulated in FLT3-CD34-LSK cells. We could demonstrate that expression of p27Kip1 and p57Kip2 was dependent of HIF-1α. Surprisingly, overexpression of constitutively active HIF-1α or treatment with the HIF stabilizer agent FG-4497 led to a reduction in HSC reconstituting ability.Conclusions: Our results imply that hypoxia, in part via HIF-1α, maintains HSCs by decreasing proliferation and favouring quiescence.

Timing of captopril administration determines radiation protection or radiation sensitization in a murine model of total body irradiation - Accepted Manuscript

2010-01-29

Abstract: Objective: Angiotensin II (Ang II), a potent vasoconstrictor, affects the growth and development of hematopoietic cells. Mixed findings have been reported for the effects of ACE inhibitors on radiation-induced injury to the hematopoietic system. We investigated the consequences of different regimens of the ACE inhibitor captopril on radiation-induced hematopoietic injury.Methods: C57BL/6 mice were either sham irradiated or were exposed to 60Co total body irradiation (0.6 Gy/min). Captopril was provided in the water for different time periods relative to irradiation.Results: In untreated mice, the survival rate from 7.5 Gy was 50% at 30 days postirradiation. Captopril treatment for 7 days prior to irradiation resulted in radiosensitization with 100% lethality and a rapid decline of mature blood cells. In contrast, captopril treatment beginning 1 hour postirradiation and continuing for 30 days resulted in 100% survival, with improved recovery of mature blood cells and multilineage hematopoietic progenitors. In nonirradiated control mice captopril biphasically modulated Lin- marrow progenitor cell cycling. After 2 days, captopril suppressed G0-G1 transition and a greater number of cells entered a quiescent state. However, after 7 days of captopril treatment Lin- progenitor cell cycling increased compared to untreated control mice.Conclusion: These findings suggest that ACE inhibition affects hematopoietic recovery following radiation by modulating the hematopoietic progenitor cell cycle. The timing of captopril treatment relative to radiation exposure differentially affects the viability and repopulation capacity of spared hematopoietic stem cells and therefore can result in either radiation protection or radiation sensitization.

Extrinsic signals determine myeloid-erythroid lineage switch in MN1 leukemia - Uncorrected Proof

2010-01-21

Objective: Transcriptional control of hematopoietic lineage fate relies on the integration of many intra- and extracellular signals. To test whether the microenvironment impacts on leukemic phenotype, we exploited the MN1 model of acute myeloid leukemia under defined genetically modified microenvironmental conditions.Materials and Methods: The requirement of both FLT3 and c-Kit signaling for MN1 leukemias was investigated using retroviral infection of bone marrow cells from wild-type, c-Kit–mutated (W41), and Flt3-ligand knockout cells, and bone marrow transplantation into wild-type, c-Kit–mutated, or Flt3-ligand knockout mice.Results: Genetic disruption of both FLT3 and c-Kit signaling in the MN1-leukemia model was dispensable for MN1-induced leukemogenesis. However, it induced a switch from myeloid to erythroid phenotype that was preserved, when FLT3 signaling was restored by secondary transplantation of leukemic cells into wild-type recipients.Conclusions: Our findings underscore the importance of microenvironmental signals for lineage choice in leukemia and identify signals that are important in myeloid-erythroid lineage decisions.

Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription - Uncorrected Proof

2010-01-15

Objective: Microvesicles have been shown to mediate intercellular communication. Previously, we have correlated entry of murine lung-derived microvesicles into murine bone marrow cells with expression of pulmonary epithelial cell-specific messenger RNA (mRNA) in these marrow cells. The present studies establish that entry of lung-derived microvesicles into marrow cells is a prerequisite for marrow expression of pulmonary epithelial cell-derived mRNA.Materials and Methods: Murine bone marrow cells cocultured with rat lung, but separated from them using a cell-impermeable membrane (0.4-μm pore size), were analyzed using species-specific primers (for rat or mouse).Results: These studies revealed that surfactant B and C mRNA produced by murine marrow cells were of both rat and mouse origin. Similar results were obtained using murine lung cocultured with rat bone marrow cells or when bone marrow cells were analyzed for the presence of species-specific albumin mRNA after coculture with rat or murine liver. These studies show that microvesicles both deliver mRNA to marrow cells and mediate marrow cell transcription of tissue-specific mRNA. The latter likely underlies the longer-term stable change in genetic phenotype that has been observed. We have also observed microRNA in lung-derived microvesicles, and studies with RNase-treated microvesicles indicate that microRNA negatively modulates pulmonary epithelial cell-specific mRNA levels in cocultured marrow cells. In addition, we have also observed tissue-specific expression of brain, heart, and liver mRNA in cocultured marrow cells, suggesting that microvesicle-mediated cellular phenotype change is a universal phenomena.Conclusion: These studies suggest that cellular systems are more phenotypically labile then previously considered.

Human embryonic stem cell−derived vascular progenitor cells capable of endothelial and smooth muscle cell function - Uncorrected Proof

2010-01-11

Objective: Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34+ cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization.Materials and Methods: hESCs are cocultured on M2-10B4 stromal cells or Wnt1-expressing M2-10B4 for 13 to 15 days to generate a CD34+ cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin-coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24 to 48hours.Results: CD34+ vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell-specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Human umbilical vein endothelial cells cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1-overexpressing stromal cells produced an increased number of progenitor cells.Conclusions: The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The stepwise differentiation outlined here is an efficient, reproducible method with potential for large-scale cultures suitable for clinical applications.

B-lymphoma cells escape rituximab-triggered elimination by NK cells through increased HLA class I expression - Uncorrected Proof

2010-01-06

Objective: Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is a major effector mechanism of the monoclonal anti-CD20 antibody rituximab in eliminating B-cell lymphomas. Resistance to this treatment occurs, although CD20 antigen is expressed on the tumor cells.Materials and Methods: A model of ADCC was established by stimulating human bulk NK cells and inhibitory killer immunoglobulin receptor (KIR)−defined NK cells from human leukocyte antigen (HLA)−typed donors. NK-cell activation was triggered via stimulation of the Fc receptor with immunoglobulin G aggregates, rituximab-labeled HLA-defined CD20-positive B-lymphoblast cell lines or CD20-positive B-lymphoma cell lines. The effect of KIR ligation by anti-KIR antibodies and HLA, the HLA expression density and rituximab concentrations on the efficacy of ADCC were analyzed in granzyme B ELISPOT measuring NK-cell activation and fluorescein-activated cell sorting cytotoxicity assay.Results: HLA, but not CD20, expression density correlated with NK-cell activity against rituximab-labeled targets. ADCC was increased or decreased following HLA shielding or KIR activation by anti-KIR antibodies, respectively. Herein we show that rituximab-induced ADCC is attenuated upon ligation of KIR by HLA molecules expressed on human B-lymphoma target cells. Moreover, anti-KIR antibodies do not only block KIR/HLA interactions, but display agonistic effects at the KIR, which has to be considered for therapeutical applications.Conclusion: KIR activation and HLA expression density are critical determinants for the efficacy of rituximab treatment. An explanation for the failure of rituximab treatment may be the protection of the tumor cells from ADCC by inhibiting NK-cell function with their surface HLA.

Eriocalyxin B induces apoptosis in lymphoma cells through multiple cellular signaling pathways - Uncorrected Proof

2010-01-04

Objective: Eriocalyxin B (EriB) is a natural diterpenoid purified from Isodon eriocalyx var. laxiflora and possesses strong antileukemic activity. In this study, we further investigated its effect and mechanism of action in human lymphoma.Materials and Methods: In vitro, a series of B- and T-lymphoma cells were treated with EriB. Cell apoptosis was analyzed using flow cytometric assay. Expression of proteins related to apoptosis and cell signal transduction were assessed using Western blot. In vivo antitumor activity of EriB was examined in murine xenograft B- and T-lymphoma models, with in situ cell apoptosis detected by terminal deoxytransferase-catalyzed DNA nick-end labeling assay.Results: EriB significantly inhibited lymphoma cell proliferation and induced apoptosis in association with caspase activation. Antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL were downregulated, with proapoptotic member Bax stable or upregulated, resulting in reduced Bcl-2/Bax and Bcl-xL/Bax ratios. Meanwhile, multiple signal transduction pathways were involved in lymphoma cell apoptosis in response to EriB, including inhibition of nuclear factor (NF)-κB and AKT pathways, and the activation of extracellular signal-related kinase (ERK) pathway. AKT inactivation was related to increased expression of cyclin-dependent kinase inhibitor P21, decreased expression of antiapoptotic phosphorylated form of Bad, and NF-κB activator IκB kinase α/β. ERK activation corresponded to reactive oxygen species production and could be blocked by antioxidant dithiothreitol. In murine xenograft lymphoma models, EriB remarkably inhibited tumor growth and induced in situ tumor cell apoptosis.Conclusion: These findings broaden the value of EriB as a promising candidate targeting apoptosis cascade in treatment of hematological malignancies.

Auto-reconstitution of the T-cell compartment by radioresistant hematopoietic cells following lethal irradiation and bone marrow transplantation - Uncorrected Proof

Nabil Bosco, Lee Kim Swee, Angèle Bénard, Rhodri Ceredig, Antonius Rolink — 2010-01-04

Objective: In lethally irradiated bone marrow chimeras, part of the reconstituted T-cell compartment is derived from the irradiated host, but the detailed origin and functional activity of host-derived T cells has not been thoroughly analyzed. Herein, we determine the origin and function of radioresistant host-derived T cells.Materials and Methods: Lethally irradiated thymectomized or nonthymectomized C57BL/6 host mice were reconstituted with syngeneic bone marrow, itself incapable of generating T cells. Using fetal thymic organ cultures, bulk and limiting dilution assays on OP9-DL1 stromal cells, unambiguous cohorts of thymus-derived and peripheral T-cell−derived T cells were phenotypically characterized by flow cytometry and functionally characterized by their ability to participate in a T-cell−dependent antibody response.Results: Both thymus-derived and peripheral T-cell−derived host T cells are functional and can reconstitute 35% of the normal T-cell pool. By comparing thymectomized vs nonthymectomized hosts, host-derived T cells were shown to comprise a major (70%) subpopulation of de novo generated, thymus-derived, polyclonal, naïve cells, and a minor subpopulation of surviving, peripheral, oligoclonal, memory-like cells. Unlike euthymic recipients, mice whose T cells were derived from surviving peripheral T cells were frequently incapable of mounting a T-cell−dependent antibody response. Host-derived thymocytes regenerated in an interleukin-7−dependent fashion from conventional DN2 thymocytes and their differentiation recapitulated normal thymic ontogeny.Conclusion: We characterized, for the first time, functional radioresistant DN2-phenotype thymic T-cell precursors, the T-cell progeny of which might provide a first line of defense against infections during the lymphopenic phase post−bone marrow transplantation.

Impact of CXCR4 inhibition on FLT3-ITD−positive human AML blasts - Uncorrected Proof

2009-12-25

Objective: Internal tandem duplication (ITD) mutations of the FLT3 receptor are associated with a high incidence of relapse in acute myeloid leukemia (AML). Expression of the CXCR4 receptor in FLT3-ITD−positive AML is correlated with poor outcome, and inhibition of CXCR4 was shown to sensitize AML blasts toward chemotherapy. The aim of this study was to evaluate the impact of FLT3-ITD on cell proliferation and CXCR4-dependent migration in human hematopoietic progenitor cells and to investigate their response to CXCR4 inhibition.Materials and Methods: We used primary blasts from patients with FLT3-ITD or FLT3 wild-type AML. In addition, human CD34+ hematopoietic progenitor cells were transduced to >70% with retroviral vectors containing human FLT3-ITD.Results: We found that FLT3-ITD transgene overexpressing human hematopoietic progenitor cells show strongly reduced migration toward stromal-derived factor−1 in vitro and display significantly reduced bone marrow homing in nonobese diabetic severe combined immunodeficient mice. Cocultivation of FLT3-ITD−positive AML blasts or hematopoietic progenitor cells on bone marrow stromal cells resulted in a strong proliferation advantage and increased early cobblestone area−forming cells compared to FLT3−wild-type AML blasts. Addition of the CXCR4 inhibitor AMD3100 to the coculture significantly reduced both cobblestone area−forming cells and proliferation of FLT3-ITD−positive cells, but did not affect FLT3−wild-type cells—highlighting the critical interaction between CXCR4 and FLT3-ITD.Conclusion: CXCR4 inhibition to decrease cell proliferation and to control the leukemic burden may provide a novel therapeutic strategy in patients with advanced FLT3-ITD−positive AML.

Abnormalities of the αβ T-cell receptor repertoire in advanced myelodysplastic syndrome - Uncorrected Proof

2009-12-25

Objective: Analysis of the αβ T-cell receptor (TCR) repertoire in patients with myelodysplastic syndrome (MDS) using the technique of TCR β-chain spectratyping has provided valuable insight into the pathophysiology of cytopenias in a subset of patients with this heterogeneous disorder. TCR β-chain spectratypes are complex data sets, however, and statistical tools for their comprehensive analysis are limited. The objective of the present work was to develop a method to enable quantitative evaluation and global comparison of spectratype data from different individuals and to study the prevalence of TCR β repertoire abnormalities in MDS patients.Materials and Methods: We developed a robust statistical method based on k-means clustering analysis, and applied this method to analysis of the αβ TCR repertoires in 50 MDS patients and 23 age-matched healthy controls.Results: Cluster analysis identified a subset of 11 MDS patients with profoundly abnormal αβ TCR repertoires. This group of patients was characterized by advanced disease by International Prognostic Scoring System and World Health Organization criteria, increased expression of the Wilms' tumor-1 oncogene, increased bone marrow myeloblast count, and older age.Conclusions: We have developed a robust analytic algorithm that enables the comparison of αβ TCR repertoires between individuals and have shown that abnormal αβ TCR repertoire is a feature of a subset of patients with advanced MDS.

CD154 expression triggered by purine analogues in vitro: Correlation with treatment response and autoimmune events in chronic lymphocytic leukemia - Corrected Proof

2009-12-21

Objective: Despite a fludarabine-based treatment is the first choice of therapy in chronic lymphocytic leukemia (CLL), not all patients achieve a partial or complete response and some of them develop autoimmune manifestations. The aim of this study was to evaluate the influence of CD154 on these adverse effects because CD154 is involved in both B-cell survival and autoimmunity.Materials and Methods: Peripheral blood mononuclear cells (PBMC) from 36 patients with CLL were cultured in vitro with fludarabine or 2-chlorodeoxyadenosine for 24, 48, and 72hours.Results: Seven patients (19.4%) presented CD154 expression in PBMC cultured with purine analogues in vitro for 24 and/or 48hours, while no expression was found when cultured in media alone. These seven patients showed a decreased apoptotic rate in vitro after purine analogues compared with those patients who did not express CD154 (p=0.01 for fludarabine; p<0.001 for 2-chlorodeoxyadenosine). CD154 expression was found to have prognostic value for response to fludarabine in vivo and was associated with the development of autoimmune manifestations (odds ratio=25; 95% confidence interval=3.5−166.7; p<0.001).Conclusion: Our preliminary results suggest that CD154 expression in CLL patients, which may be induced by purine analogues, is associated with resistance to fludarabine and with development of autoimmune manifestations.

Antiplatelet antibodies in WASP(−) mice correlate with evidence of increased in vivo platelet consumption - Uncorrected Proof

2009-09-04

Objective: To study the role of antiplatelet antibodies in the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS).Materials and Methods: A flow cytometric method was developed for detection of serum antiplatelet antibodies via their binding to intact target platelets lacking surface antibodies. Platelets were labeled with 5-chloromethylfluorescein diacetate in order to track their clearance from the circulation. WASP(−)μMT(−/−) mice were generated by standard breeding methods.Results: Serum antiplatelet antibodies were detected in approximately 40% of WASP(−) males. Mean level of reticulated platelets is significantly increased in these antibody(+) males. While WASP(−) males show an approximately 50% reduction in platelet counts, 5% to 10% show a more severe thrombocytopenia associated with increased reticulated platelets, suggesting the presence of clearance-inducing antiplatelet antibodies. In support of that inference, 90% of the latter mice show detectable serum antiplatelet antibodies. The antibodies are primarily immunoglobulin G, and are also detected in >30% of CD47(−/−) males. WASP(−)μMT(−/−) males, which demonstrate no serum- or platelet-associated antibodies, show a degree of thrombocytopenia similar to that of WASP(−) males. Their platelet clearance rates remain accelerated—more so in WASP(−)μMT(−/−) than WASP(+)μMT(−/−) recipients.Conclusions: These findings suggest that platelet WASP deficiency results in an increase in platelet clearance rates by two mechanisms, i.e., an antibody-independent mechanism that largely requires WASP deficiency in trans, and an antibody-dependent mechanism that does not. Both an increased incidence of antiplatelet antibodies and an increased susceptibility to their effects contribute to antibody-dependent clearance of WASP(−) platelets.