A. Robert Turner

Migration of Bone Marrow and Cord Blood Mesenchymal Stem Cells In Vitro Is Regulated by Stromal‐Derived Factor‐1‐CXCR4 and Hepatocyte Growth Factor‐c‐met Axes and Involves Matrix Metalloproteinases

Human mesenchymal stem cells (MSCs) are increasingly being considered in cell‐based therapeutic strategies for regeneration of various organs/tissues. However, the signals required for their homing and recruitment to injured sites are not yet fully understood. Because stromal‐derived factor (SDF)‐1 and hepatocyte growth factor (HGF) become up‐regulated during tissue/organ damage, in this study we examined whether these factors chemoattract ex vivo‐expanded MSCs derived from bone marrow (BM) and umbilical cord blood (CB). Specifically, we investigated the expression by MSCs of CXCR4 and c‐met, the cognate receptors of SDF‐1 and HGF, and their functionality after early and late passages of MSCs. We also determined whether MSCs express matrix metalloproteinases (MMPs), including membrane type 1 (MT1)‐MMP, matrix‐degrading enzymes that facilitate the trafficking of hematopoietic stem cells. We maintained expanded BM‐ or CB‐derived MSCs for up to 15–18 passages with monitoring of the expression of 1) various tissue markers (cardiac and skeletal muscle, neural, liver, and endothelial cells), 2) functional CXCR4 and c‐met, and 3) MMPs. We found that for up to 15–18 passages, both BM‐ and CB‐derived MSCs 1) express mRNA for cardiac, muscle, neural, and liver markers, as well as the vascular endothelial (VE) marker VE‐cadherin; 2) express CXCR4 and c‐met receptors and are strongly attracted by SDF‐1 and HGF gradients; 3) express MMP‐2 and MT1‐MMP transcripts and proteins; and 4) are chemo‐invasive across the reconstituted basement membrane Matrigel. These in vitro results suggest that the SDF‐1‐CXCR4 and HGF‐c‐met axes, along with MMPs, may be involved in recruitment of expanded MSCs to damaged tissues.

Physical exercise and quality of life in cancer patients following high dose chemotherapy and autologous bone marrow transplantation

Preliminary evidence indicates that physical exercise may be an effective strategy for the rehabilitation of cancer patients following high dose chemotherapy (HDC) and bone marrow transplantation (BMT), but the focus of this research has been on physical fitness and medical outcomes. In the present study, we employed a prospective design to examine the relationship between physical exercise and various quality of life (QOL) indices in 25 BMT patients. Participants completed weekly self‐administered questionnaires upon being admitted to hospital, and monitored the frequency and duration of their exercise during hospitalization. Statistical analyses indicated that exercise during hospitalization was significantly correlated with almost all QOL indices, including physical well‐being, psychological well‐being, depression, anxiety and days hospitalized. Moreover, only some of the correlations were attenuated after controlling for relevant demographic and medical variables. It was concluded that physical exercise may be related to QOL in BMT patients, but that experimental research is needed before any definitive conclusions can be drawn. Copyright

A randomized trial of dasatinib 100 mg versus imatinib 400 mg in newly diagnosed chronic-phase chronic myeloid leukemia

Tyrosine kinase inhibitor therapy with imatinib (IM), dasatinib (DAS), or nilotinib is very effective in chronic-phase chronic myeloid leukemia. Two hundred fifty-three patients with newly diagnosed chronic-phase chronic myeloid leukemia were randomized to IM 400 mg/day or DAS 100 mg/day. The proportion of patients achieving a complete cytogenetic remission rate was superior with DAS (84% vs 69%), as was the 12-month molecular response by the proportions of patients achieving > 3-log, > 4-log, and > 4.5-log reduction in BCR-ABL transcript levels. Overall and progression-free survival was similar in the 2 arms. Among patients who achieved hematologic CR, 3-year relapse-free survival was 91% with DAS and 88% with IM 400 mg. Grade 3 and 4 toxicities were most commonly hematologic, including thrombocytopenia in 18% and 8% of DAS and IM patients, respectively. DAS induced more complete cytogenetic response and deeper molecular responses after 12 months, compared with IM 400 mg, and with a median follow-up of 3.0 years there have been very few deaths, relapses, or progressions in the 2 arms. In summary, DAS compared with IM appeared to have more short-term cytogenetic and molecular response, more hematologic toxicity, and similar overall survival. This trial is registered at www.clinicaltrials.gov as NCT00070499.

Randomized Comparison of Gemcitabine, Dexamethasone, and Cisplatin Versus Dexamethasone, Cytarabine, and Cisplatin Chemotherapy Before Autologous Stem-Cell Transplantation for Relapsed and Refractory Aggressive Lymphomas: NCIC-CTG LY.12

PURPOSE For patients with relapsed or refractory aggressive lymphoma, we hypothesized that gemcitabine-based therapy before autologous stem-cell transplantation (ASCT) is as effective as and less toxic than standard treatment. PATIENTS AND METHODS We randomly assigned 619 patients with relapsed/refractory aggressive lymphoma to treatment with gemcitabine, dexamethasone, and cisplatin (GDP) or to dexamethasone, cytarabine, and cisplatin (DHAP). Patients with B-cell lymphoma also received rituximab. Responding patients proceeded to stem-cell collection and ASCT. Coprimary end points were response rate after two treatment cycles and transplantation rate. The noninferiority margin for the response rate to GDP relative to DHAP was set at 10%. Secondary end points included event-free and overall survival, treatment toxicity, and quality of life. RESULTS For the intention-to-treat population, the response rate with GDP was 45.2%; with DHAP the response rate was 44.0% (95% CI for difference, -9.0% to 6.7%), meeting protocol-defined criteria for noninferiority of GDP (P = .005). Similar results were obtained in a per-protocol analysis. The transplantation rates were 52.1% with GDP and 49.3% with DHAP (P = .44). At a median follow-up of 53 months, no differences were detected in event-free survival (HR, 0.99; stratified log-rank P = .95) or overall survival (HR, 1.03; P = .78) between GDP and DHAP. Treatment with GDP was associated with less toxicity (P < .001) and need for hospitalization (P < .001), and preserved quality of life (P = .04). CONCLUSION For patients with relapsed or refractory aggressive lymphoma, in comparison with DHAP, treatment with GDP is associated with a noninferior response rate, similar transplantation rate, event-free survival, and overall survival, less toxicity and hospitalization, and superior quality of life.

Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal antibodies (MCA) were obtained by immunizing BALB/c mice with 99% pure granulocytes from normal donors or with a whole leukocyte suspension obtained from a chronic myelogenous leukemia (CML) patient, and then fusing the mouse spleen cells with a 315-43 myeloma cell clone. Four MCA were selected and studied using ELISA, immunofluorescence, cytotoxicity assays, and FACS analysis. Antibodies 80H.1, 80H.3, and 80H.5 (from normals) and 81H.1 (from CML) detected antigens expressed on neutrophils. Antibodies 80H.1 and 80H.3 (IgG) also reacted with monocytes but not with other blood cell subsets. Antibodies 80H.5 and 81H.1 (IgM) were cytotoxic and reacted strongly with most of the cells of the neutrophil maturation sequence, i.e., myeloblasts, promyelocytes, myelocytes, and mature granulocytes. Antibodies 80H.5 and 81H.1 also inhibited CFU-GM growth stimulated by leukocyte feeder layers or placental conditioned media, but did not inhibit BFU-E and CFU-E. Antigens recognized by 80H.3, 80H.5, and 81H.1 were expressed both on a proportion of cells from HL.60, KG.1, ML.1, and K562 myeloid cell lines, and on a proportion of blast cells isolated from patients with acute myelogenous leukemia. They were not found on lymphoid cell lines or lymphoid leukemia cells. These MCA recognize either late differentiation antigens expressed on mature neutrophils and monocytes (80H.1 and 80H.3) or early differentiation antigens (80H.5 and 81H.1) specific to the granulocytic lineage. They may be useful for a better definition of those antigens specific to hematopoietic stem cells and their relationship with normal or neoplastic hematopoiesis.

Es nucleoside transporter content of acute leukemia cells: role in cell sensitivity to cytarabine (araC).

Nucleoside analogs are important components of treatment regimens for acute leukemia in adults. Plasma membrane permeation of the nucleoside analog molecules, the initial event in the cellular conversion of nucleosides to active agents, is mediated by nucleoside-specific membrane transporters. The widely-expressed es nucleoside transporter accepts as substrates diverse nucleoside analogs, including cytarabine (araC), 2-chlorodeoxyadenosine, and fludarabine. The cellular content of es transporter sites has been measured in blasts from patients with acute lymphoblastic leukemia and acute myelogenous leukemia, by a sensitive, quantitative flow cytometry assay that employs the tightly-bound es ligand, SAENTA fluorescein. Values for es transporter expression varied ten-fold among samples from patients with acute myelogenous leukemia. In this article, we review current findings that document, in confocal fluorescence microscopy images and in flow cytometry assays of SAENTA fluorescein-stained cells, the patient-to-patient variance of es transporter expression in leukemic blasts from patients. Our data show a correlation between the expression of es transporters and the in vitro sensitivity to nucleoside drugs of blasts from acute leukemia patients. These findings show that the flow cytometry assay of es expression provides a facile means of predicting resistance of leukemia cells to the cytotoxicity of araC and other nucleosides.

Fifth complement cascade protein (C5) cleavage fragments disrupt the SDF-1/CXCR4 axis: Further evidence that innate immunity orchestrates the mobilization of hematopoietic stem/progenitor cells

OBJECTIVE Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/(desArg)C5a) in human HSPC mobilization. MATERIALS AND METHODS C5 cleavage fragments in the plasma were evaluated by enzyme-linked immunosorbent assay using human anti-(desArg)C5a antibody, and expression of the C5a/(desArg)C5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and expression of stromal cell-derived factor-1 (SDF-1)-degrading proteases that perturb retention of HSPC in bone marrow, namely matrix metalloproteinase (MMP)-9, membrane type (MT) 1-MMP, and carboxypeptidase M. RESULTS We found that plasma levels of (desArg)C5a are significantly higher in patients who are good mobilizers and correlate with CD34(+) cell and white blood cell counts in mobilized peripheral blood. C5 cleavage fragments did not chemoattract myeloid progenitors (colony-forming unit granulocyte-macrophage), but (desArg)C5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34(+) cells, but appeared on more mature myeloid precursors, monocytes, and granulocytes. Moreover, granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells and polymorphonuclear cells had a significantly higher percentage of cells expressing CD88 than nonmobilized peripheral blood. Furthermore, C5a stimulation of granulocytes and monocytes decreased CXCR4 expression and chemotaxis toward an SDF-1 gradient and increased secretion of MMP-9 and expression of MT1-MMP and carboxypeptidase M. CONCLUSION C5 cleavage fragments not only induce a highly proteolytic microenvironment in human bone marrow, which perturbs retention through the CXCR4/SDF-1 axis, but also strongly chemoattracts granulocytes, promoting their egress into mobilized peripheral blood, which is crucial for subsequent mobilization of HSPC.

Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

BackgroundCytoglobin (Cygb) and neuroglobin (Ngb) are recently identified globin molecules that are expressed in vertebrate tissues. Upregulation of Cygb and Ngb under hypoxic and/or ischemic conditions in vitro and in vivo increases cell survival, suggesting possible protective roles through prevention of oxidative damage. We have previously shown that Ngb is expressed in human glioblastoma multiforme (GBM) cell lines, and that expression of its transcript and protein can be significantly increased after exposure to physiologically relevant levels of hypoxia. In this study, we extended this work to determine whether Cygb is also expressed in GBM cells, and whether its expression is enhanced under hypoxic conditions. We also compared Cygb and Ngb expression in human primary tumor specimens, including brain tumors, as well as in human normal tissues. Immunoreactivity of carbonic anhydrase IX (CA IX), a hypoxia-inducible metalloenzyme that catalyzes the hydration of CO2 to bicarbonate, was used as an endogenous marker of hypoxia.ResultsCygb transcript and protein were expressed in human GBM cells, and this expression was significantly increased in most cells following 48 h incubation under hypoxia. We also showed that Cygb and Ngb are expressed in both normal tissues and human primary cancers, including GBM. Among normal tissues, Cygb and Ngb expression was restricted to distinct cell types and was especially prominent in ductal cells. Additionally, certain normal organs (e.g. stomach fundus, small bowel) showed distinct regional co-localization of Ngb, Cygb and CA IX. In most tumors, Ngb immunoreactivity was significantly greater than that of Cygb. In keeping with previous in vitro results, tumor regions that were positively stained for CA IX were also positive for Ngb and Cygb, suggesting that hypoxic upregulation of Ngb and Cygb also occurs in vivo.ConclusionsOur finding of hypoxic up-regulation of Cygb/Ngb in GBM cell lines and human tumor tissues suggests that these globin molecules may be part of the repertoire of defense mechanisms that allow cancer cells to survive in hypoxic microenvironments.

Vinblastine, bleomycin, and cisplatin in malignant germ cell tumors of the ovary

Fourteen patients with malignant ovarian germ cell tumors were treated with vinblastine, bleomycin, and cisplatin. A complete clinical response was achieved in all 14 patients; however, 1 patient had small macroscopic disease present at second‐look laparotomy. One patient died of bleomycin pulmonary toxicity. The remaining 13 patients are alive and free of disease from 20 months to 8 years and 8 months after initial diagnosis. Serum alpha‐fetoprotein and beta‐human chorionic gonadotropin levels were monitored in all patients and were found to be reliable indicators of response to treatment and disease status. The uninvolved ovary was preserved in seven patients without compromising the response to treatment, and one patient subsequently became pregnant. Vinblastine, bleomycin, and cisplatin chemotherapy appears to be a safe, effective combination and is recommended as the primary treatment of choice in the management of patients with malignant ovarian germ cell tumors.

The Ins and Outs of Hematopoietic Stem Cells: Studies to Improve Transplantation Outcomes

Deciphering the mechanisms of hematopoietic stem/progenitor cell (HSPC) mobilization and homing is important for the development of strategies to enhance the efficacy of HSPC transplantation and achieve the full potential of HSPC-based cellular therapy. Investigation of these mechanisms has revealed interdependence among the various molecules, pathways and cellular components involved, and underscored the complex nature of these two processes. This review summarizes recent progress in identifying the specific factors implicated in HSPC mobilization and homing, with emphasis on our own work. Particularly, we will discuss our studies on stromal cell-derived factor-1 and its interaction with its receptor CXCR4, proteases (matrix metalloproteinases and carboxypeptidase M), complement proteins (C1q, C3a, C5a, membrane attack complex), sphingosine-1-phosphate, and pharmacologic agents such as the histone deacetylase inhibitor valproic acid and hyaluronic acid.