Extracellular NK histones promote immune cell anti-tumor activity by inducing cell clusters through binding to CD138 receptor.

BACKGROUND: Natural killer (NK) cells are important anti-tumor cells of our innate immune system. Their anti-cancer activity is mediated through interaction of a wide array of activating and inhibitory receptors with their ligands on tumor cells. After activation, NK cells also secrete a variety of pro-inflammatory molecules that contribute to the final immune response by modulating other innate and adaptive immune cells. In this regard, external proteins from NK cell secretome and the mechanisms by which they mediate these responses are poorly defined. METHODS: TRANS-stable-isotope labeling of amino acids in cell culture (TRANS-SILAC) combined with proteomic was undertaken to identify early materials transferred between cord blood-derived NK cells (CB-NK) and multiple myeloma (MM) cells. Further in vitro and in vivo studies with knock-down of histones and CD138, overexpression of histones and addition of exogenous histones were undertaken to confirm TRANS-SILAC results and to determine functional roles of this material transferred. RESULTS: We describe a novel mechanism by which histones are actively released by NK cells early after contact with MM cells. We show that extracellular histones bind to the heparan sulfate proteoglycan CD138 on the surface of MM cells to promote the creation of immune-tumor cell clusters bringing immune and MM cells into close proximity, and thus facilitating not only NK but also T lymphocyte anti-MM activity. CONCLUSION: This study demonstrates a novel immunoregulatory role of NK cells against MM cells mediated by histones, and an additional role of NK cells modulating T lymphocytes activity that will open up new avenues to design future immunotherapy clinical strategies.

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells.

One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion techniques were compared: (1) CD133+ selection followed by ex vivo liquid culture and (2) co-culture of unmanipulated CB with bone-marrow-derived mesenchymal stem cells (MSCs). Ex vivo culture was performed in medium supplemented with granulocyte colony-stimulating factor, stem cell factor and either thrombopoietin or megakaryocyte growth and differentiation factor. Expansion was followed by measuring total nucleated cell (TNC), CD133+ and CD34+ cell, colony-forming unit and cobblestone area-forming cell output. When compared to liquid culture, CB-MSC co-culture (i) required less cell manipulation resulting in less initial HPC loss and (ii) markedly improved TNC and HPC output. CB-MSC co-culture therefore holds promise for improving engraftment kinetics in CB transplant recipients.

A novel triple purge strategy for eliminating chronic myelogenous leukemia (CML) cells from autografts.

Imatinib-refractory chronic myelogenous leukemia (CML) patients can experience long-term disease-free survival with myeloablative therapy and allogeneic hematopoietic cell transplantation; however, associated complications carry a significant risk of mortality. Transplantation of autologous hematopoietic cells has a reduced risk of complications, but residual tumor cells in the autograft may contribute to relapse. Development of methods for purging tumor cells that do not compromise the engraftment potential of the normal hematopoietic cells in the autograft has been a long-standing goal. Since primitive CML cells differentiate more rapidly in vitro than their normal counterparts and are also preferentially killed by mafosfamide and imatinib, we examined the purging effectiveness on CD34(+) CML cells using a strategy that combines a brief exposure to imatinib (0.5-1.0 microM for 72 h) and then mafosfamide (30-90 microg/ml for 30 min) followed by 2 weeks in culture with cytokines (100 ng/ml each of stem cell factor, granulocyte colony-stimulating factor and thrombopoietin). Treatment with 1.0 microM imatinib, 60 microg/ml mafosfamide and 14 days of culture with cytokines eliminated BCR-ABL(+) cells from chronic phase CML patient aphereses, while preserving normal progenitors. This novel purging strategy may offer a new approach to improving the effectiveness of autologous transplantation in imatinib-refractory CML patients.

Hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor and erythropoietin in the absence of matrix metalloproteinase-9.

The use of mobilized hematopoietic progenitor cells (HPC) has largely replaced the use of bone marrow HPC for autologous and allogeneic transplantation; however, the mechanisms of HPC mobilization remain unclear. A better understanding of these mechanisms, may allow the development of improved (potentially more rapid and/or higher yield) HPC mobilization strategies, especially for patients who mobilize poorly using current mobilization protocols. Clinically, granulocyte colony-stimulating factor (G-CSF) is widely used to induce HPC mobilization, and evidence suggests that metalloproteinase enzymes released by activated granulocytes play an important role in the G-CSF-induced HPC mobilization. These enzymes may act to disrupt putative cell-cell and/or cell-extracellular matrix interactions within the hematopoietic microenvironment thereby releasing HPC into the blood. Matrix metalloproteinase-9 (MMP-9) appears to be important for G-CSF-induced mobilization. Using an MMP-9 knock-out (KO) mouse model, we investigated the role of MMP-9 in G-CSF and erythropoietin (EPO)-based HPC mobilization at clinically relevant cytokine doses. There were few hematologic or hematopoietic differences between the wild-type and MMP-9KO mice during steady-state hematopoiesis. When treated subcutaneously with EPO (500 U/kg per day) and G-CSF (15 microg/kg per day) for 5 days and assayed on day 6, similarly increased extramedullary hematopoiesis and numbers of HPC in the spleen and blood were observed for both the wild-type and MMP-9KO mice. These data demonstrate that MMP-9 is not required for EPO + G-CSF mobilization and that alternative mobilization mechanisms must be active at clinically relevant cytokine concentrations.

Evidence for a qualitative hierarchy within the Hoechst-33342 'side population' (SP) of murine bone marrow cells.

In vitro cobblestone area (CA)-forming cell (CAFC) and in vivo (short-term and competitive repopulation) assays demonstrate that a qualitative hierarchy exists within the Hoechst-33342-defined side population (SP) in murine bone marrow (BM). Consistent with and extending previous studies, we demonstrate that (i) hematopoietic activity found in whole BM (WBM) is concentrated within the SP, rather than the non-SP (NSP); and (ii) within the SP, those cells that more strongly efflux the dye (lower SP, LSP) are qualitatively different from those that less strongly efflux the dye (upper SP, USP). Qualitative differences are highlighted by evidence that (i) CA derived from LSP CAFC persist in culture significantly longer than CA derived from USP CAFC; (ii) short-term, multilineage repopulation of lethally irradiated mice by LSP cells is more rapid than that in mice receiving USP, NSP, whole SP (WSP), or WBM cells and (iii) LSP cells out-compete USP cells in the multilineage hematopoietic repopulation of lethally irradiated recipients. These data suggest that LSP cells are of higher quality than USP cells and potentially provide a means by which qualitative changes in primitive hematopoietic progenitors occurring naturally with aging, or clinically as a consequence of therapeutic manipulation, can be assessed.

Loss of marrow reserve from dose-intensified chemotherapy results in impaired hematopoietic reconstitution after autologous transplantation: CD34(+), CD34(+)38(-), and week-6 CAFC assays predict poor engraftment.

Autologous hematopoietic stem cell transplantation (HSCT) is an increasingly successful modality for treating a variety of malignant disorders in the clinic. Experimental and clinical data suggest that prior exposure to cytotoxic agents that damage primitive stem cells results in impaired hematopoiesis after autologous HSCT. To further investigate the ability to predict for impaired hematopoiesis, we measured different stem/progenitor cell populations transplanted and time to engraftment. Patients with previously untreated, advanced-stage follicular lymphoma were treated in sequential prospective protocols with 6-8 cycles of standard-dose (SD) cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or four cycles of a higher-dose (HD) CHOP and granulocyte colony-stimulating factor, to induce remission prior to high-dose cyclophosphamide, total body irradiation, and autologous bone marrow transplantation (ABMT). Cryopreserved marrow samples obtained prior to ABMT were assayed for CD34(+), CD34(+)38(-), and cobblestone area-forming cell (CAFC) frequencies. Despite receiving similar numbers of nucleated cells at ABMT, HD-CHOP patients took significantly longer to attain platelet engraftment than the SD-CHOP patients. Marrow from the HD-CHOP patients contained significantly lower CD34(+), CD34(+)38(-), and week 6-8 CAFC frequencies than marrow from SD-CHOP-treated patients. Time to platelet engraftment was plotted against progenitor/stem cell numbers transplanted for each patient and threshold values were developed for all three stem/progenitor cell populations. These values were 0.5 x 10(6) CD34(+) cells/kg, 0.14 x 10(6) CD34(+)38(-) cells/kg, and 9500 week-6 CAFC/kg transplanted. Approximately 50% of patients received marrow progenitor/stem cell numbers above the threshold values and all engrafted without delay. However, transplantation of stem/progenitor cell numbers below threshold values did not uniformly predict for delayed platelet engraftment. These data provide further evidence for the association of low marrow reserve at ABMT, low numbers of stem/progenitor cells transplanted, and delayed hematopoietic recovery. However, there remains a group of patients who have rapid platelet engraftment after ABMT despite low numbers of progenitor/stem cells transplanted. These data suggest the presence of a crucial stem cell population not represented by the stem/progenitor cell populations studied in these experiments.

Transmissible cytotoxicity of multiple myeloma cells by cord blood-derived NK cells is mediated by vesicle trafficking.

Natural killer cells (NK) are important effectors of anti-tumor immunity, activated either by the downregulation of HLA-I molecules on tumor cells and/or the interaction of NK-activating receptors with ligands that are overexpressed on target cells upon tumor transformation (including NKG2D and NKP30). NK kill target cells by the vesicular delivery of cytolytic molecules such as Granzyme-B and Granulysin activating different cell death pathways, which can be Caspase-3 dependent or Caspase-3 independent. Multiple myeloma (MM) remains an incurable neoplastic plasma-cell disorder. However, we previously reported the encouraging observation that cord blood-derived NK (CB-NK), a new source of NK, showed anti-tumor activity in an in vivo murine model of MM and confirmed a correlation between high levels of NKG2D expression by MM cells and increased efficacy of CB-NK in reducing tumor burden. We aimed to characterize the mechanism of CB-NK-mediated cytotoxicity against MM cells. We show a Caspase-3- and Granzyme-B-independent cell death, and we reveal a mechanism of transmissible cell death between cells, which involves lipid-protein vesicle transfer from CB-NK to MM cells. These vesicles are secondarily transferred from recipient MM cells to neighboring MM cells amplifying the initial CB-NK cytotoxicity achieved. This indirect cytotoxicity involves the transfer of NKG2D and NKP30 and leads to lysosomal cell death and decreased levels of reactive oxygen species in MM cells. These findings suggest a novel and unique mechanism of CB-NK cytotoxicity against MM cells and highlight the importance of lipids and lipid transfer in this process. Further, these data provide a rationale for the development of CB-NK-based cellular therapies in the treatment of MM.

Effects of low concentrations of benzene on mouse hematopoietic cells in vivo: a preliminary report.

Evaluation of benzene-induced hematotoxicity following exposure to low concentration is important for understanding mechanisms of toxicity and determining the dose response at benzene levels close to the current occupational exposure limit (1 ppm). Male B6C3F1 mice were exposed to 0, 1, 10, 100, or 200 ppm benzene by inhalation for 6 hr/day, 5 days/week, for 1, 2, 4, or 8 weeks. At each sampling time, we evaluated primitive and committed progenitor cells, differentiating and maturing lineage-specific cells, and stromal cells in the bone marrow; T and B lymphocytes of the spleen and thymus; micronucleated reticulocytes and erythrocytes; and standard blood parameters. At 100 and 200 ppm benzene, there were rapid and significant reductions in number of reticulocytes in the blood, B lymphocytes in the bone marrow and spleen, and an increased frequency of micronucleated reticulocytes in the bone marrow. At 10 ppm, the only parameter affected was a transient reduction in the number of splenic B lymphocytes. There were no significant effects induced by 1 ppm benzene in this study. The present study suggests numbers of B lymphocytes and maturing erythrocytes, and frequency of micronucleated reticulocytes are sensitive indicators of benzene-induced hematotoxicity and will be useful in further investigation of the hematotoxicity induced by 10 to 100 ppm benzene.

Delivery of Flt3 ligand (Flt3L) using a poloxamer-based formulation increases biological activity in mice.

Fms-like tyrosine kinase (Flt3L) is a potent stimulator of hematopoietic progenitor cell (HPC) expansion and mobilization; however, this requires 7-10 days of administration. We investigated whether sustained delivery of Flt3L using a poloxamer-based matrix (PG) could accelerate and/or improve the hematopoietic activity of Flt3L in mice. A single injection of PG-Flt3L stimulated significantly more rapid and greater HPC mobilization to the spleen and peripheral blood than the daily injection of Flt3L formulated in saline. Pharmacokinetic analysis demonstrated that the formulation of Flt3L in PG prolonged its elimination (Tbeta) half-life (2.3-fold) and increased its bioavailability (>two fold) and the time to maximum serum concentration (T(max)) (2.7-fold). Further, coadministration of G-CSF and PG-Flt3L allowed lower doses of Flt3L to be active, with significantly greater hematopoietic and mobilization activity, compared to the same total dose of G-CSF, Flt3L or G-CSF and Flt3L formulated in saline. These data demonstrate that formulation of Flt3L in PG significantly accelerates and increases HPC expansion and mobilization. The observation of increased bioactivity by PG-Flt3L in rodents suggests the potential for improved clinical efficacy of Flt3L by reducing the time required for HPC mobilization.

Flt3 ligand bioactivity and pharmacology in neoplasia.

Fms-like tyrosine kinase 3 ligand (Flt3L) has multiple effects on the hematopoietic and immune systems. Further, preclinical studies have suggested potential therapeutic activity against cancer. Flt3L is a potent hematopoietic cytokine, capable of stimulating the expansion and differentiation of hematopoietic progenitor and stem cells. Administration of Flt3L mobilizes hematopoietic cells from the bone marrow (BM) into the blood, lymphoid organs, and parenchymal tissues. This mobilization activity, especially effective in combination with granulocyte colony stimulating factor (G-CSF), has stimulated studies of Flt3L in hematopoietic stem cell (HSC) transplantation. In addition to its effects on hematopoietic stem and progenitor cells, Flt3L has been shown to increase the frequency and number of dendritic cells (DCs) within the circulatory system and solid organs. DC expansion by Flt3L has been the focus of preclinical and clinical studies on antigen (Ag) specific T-cell mediated immunity. The mechanism for the augmentation of T-cell mediated immunity has yet to be completely identified, although Flt3L's ability to expand DCs in lymphoid and non-lymphoid tissues is involved. This expansion occurs primarily with DCs, which secrete interleukin (IL) 12. Consistent with the expansion of this DC population, treatment with Flt3L enhances T-cell mitogenesis and preferentially induces type 1 T-cell responses. However, the DCs resulting from Flt3L administration are immature, leading in some studies to the induction of tolerance. This review focuses on the effects of Flt3L on DCs and other effector populations, and on its potential activity as a therapeutic agent for cancer, alone and in combination with vaccines.

Immunotoxicological effects of benzene inhalation in male Sprague-Dawley rats.

The inhalation of benzene is toxic to various components of the immunologic system in rodents. Spleen and thymus weights, total spleen and femur marrow cell counts, enumeration of spleen B- and T-lymphocytes, and an assessment of humoral immunocompetence, were used to evaluate the immunotoxicity of benzene in male Sprague-Dawley rats. Rats were exposed to 0, 30, 200 or 400 ppm benzene for 6 h/day, 5 days/week for 2 or 4 weeks. An early indicator of immunotoxicity was a reduction in the number of B-lymphocytes after 2 weeks of 400 ppm. After 4 weeks of 400 ppm, there was a reduction in thymus weight and spleen B-, CD4+/CD5+ and CD5+ T-lymphocytes. Rats exposed to 30, 200 or 400 ppm benzene for 2 or 4 weeks and challenged with sheep red blood cells developed a humoral response comparable to that of the control (0 ppm) animals. Enumeration of spleen T- and B-lymphocytes in rats exposed to benzene and challenged with SRBC showed only a transient reduction in spleen B-lymphocytes after 2 weeks of exposure to 400 ppm. These data suggest that there are no immunotoxicological effects of exposure to 200 ppm benzene or less, in rats exposed for 6 h/day, 5 days/week for 2 or 4 weeks.

Effects of benzene on splenic, thymic, and femoral lymphocytes in mice.

Chronic exposure to high concentrations of benzene, primarily by inhalation, can affect the function of the human immune system. Limited data are available on the immunotoxic effects of low concentrations of benzene. This study evaluated the effects of 1, 5, 10, 100, and 200 ppm benzene on lymphocytes in mice exposed by inhalation for up to 8 weeks. Exposure to 100 or 200 ppm benzene induced rapid and persistent reductions in femoral B-, splenic T- and B-, and thymic T-lymphocytes. The percentage of femoral B-lymphocytes and thymic T-lymphocytes in apoptosis was increased 6- to 15-fold by 200 ppm benzene compared to controls. Replication of femoral B-lymphocytes was increased during the exposure period in the bone marrow as a compensation for the lymphocyte loss induced by 100 and 200 ppm benzene. Exposure of mice to 10 ppm benzene or less did not have a statistically significant effect on numbers or replication of the lymphocyte populations evaluated. A reduced number of splenic B-lymphocytes after 2 weeks of exposure to benzene appeared to be the most sensitive end point and time point for evaluating benzene cytotoxicity in this study.

Domestic water treatment appliances and the fluoride ion.

Specific ion metering of fluoride ion levels of water samples obtained after passage through water softeners and a conditioner showed no alteration in concentration when compared with controls. Similar comparison of water samples passed through water filters demonstrated that highly significant amounts of fluoride ion were removed. In one filter tested, 90% of the fluoride content was lost in the filtration process. The findings of this study suggest that, in a household using a filter, it may be necessary to increase the fluoride supplement or in some cases to initiate use of fluoride supplements as children are not receiving as much fluoride as was thought. New guidelines are required to take account of this surprising effect.

Occlusal outcome in patients undergoing orthognathic surgery with internal fixation.

Fifty consecutive patients undergoing orthognathic surgery with internal fixation (IF) were studied retrospectively with a weighted Peer Assessment Rating (PAR) Index to assess occlusal outcome at the end of all active treatment, and compared with 50 patients who had undergone treatment for malocclusion by orthodontic means alone. In the surgically treated patients, the mean percentage reduction in the weighted PAR Index was 83% and 31 out of 38 patients (82%) were 'greatly improved'. This implies a high standard of treatment in terms of the occlusal outcome. There was no difference in the proportion of patients having a final weighted PAR Index of less than 10 and no significant difference in the final weighted PAR Index between the two groups. This suggests that the occlusal outcome is no different whether patients undergo orthognathic surgery or orthodontic treatment alone, and that excellent occlusal results can be achieved in patients undergoing orthognathic surgery with internal fixation.

Extracorporeal shock wave lithotripsy. Use of antibiotics to avoid postprocedural infection.

Extracorporeal shock wave lithotripsy (ESWL) has the potential to revolutionize the treatment of urolithiasis, but its success depends in part upon minimizing potential sequelae. Although ESWL is safe, effective, and relatively economical, one complication can be urosepsis, resulting from liberation of bacteria when the stones disintegrate. Patients who are at increased risk of infection are those who have existing urinary tract infection; perioperative urologic manipulation; infected stones; predisposition for infectious endocarditis; or multiple, large, or complex stones. If urosepsis occurs, it usually requires prolonged hospitalization, which obviates any cost-benefits that can be accrued from ESWL. Prophylactic use of an antibiotic before ESWL is rational and cost-effective. Ideally, the antibiotic should possess a spectrum of activity against the most likely bacteria to be encountered, require a limited number of doses, and offer the flexibility of sustained coverage in the event that the procedure is delayed. Our experience at Presbyterian Hospital of Dallas supports the use of a long-acting cephalosporin for the prevention of infections following ESWL.

Orthodontic treatment and the wind instrument.

Many children are learning to play musical instruments at the same time as they are undergoing orthodontic treatment. If a patient or parent enquires about how orthodontic treatment might affect their child's playing, what advice could you give them?

FOXP3 is a direct target of miR15a/16 in umbilical cord blood regulatory T cells.

Exact mechanism of action of umbilical cord blood (CB)-derived regulatory T cells (Tregs) in the prevention of GVHD remains unclear. On the basis of selective overexpression of peptidase inhibitor 16 in CB Tregs, we explored the related p53 pathway, which has been shown to negatively regulate miR15a/16 expression. Significantly lower levels of miR15a/16 were observed in CB Tregs when compared with conventional CB T cells (Tcons). In a xenogeneic GVHD mouse model, lower levels of miR15a/16 were also found in Treg recipients, which correlated with a better GVHD score. Forced overexpression of miR15a/16 in CB Tregs led to inhibition of FOXP3 and CTLA4 expression and partial reversal of Treg-mediated suppression in an allogeneic mixed lymphocyte reaction that correlated with the reversal of FOXP3 demethylation in CB Tregs. On the other hand, miR15a/16 knockdown in CB Tcons led to expression of FOXP3 and CTLA4 and suppression of allogeneic lymphocyte proliferation. Using a luciferase-based mutagenesis assay, FOXP3 was determined to be a direct target of miR15a and miR16. We propose that miR15a/16 has an important role in mediating the suppressive function of CB Tregs and these microRNAs may have a 'toggle-switch' function in Treg/Tcon plasticity.

Ex vivo expansion of umbilical cord blood for transplantation.

Umbilical cord blood (UCB) has become an important source of hematopoietic stem cell transplant (HSCT) for hematologic malignancies in adults. Its ready availability, allowance of higher HLA disparity and lower incidence of graft-versus-host disease (GVHD) makes it a very attractive source especially for minority populations. The major limitation to a wider use of this source of HSCT is the relative low number of progenitor cells in the graft. For this reason, adult UCB transplants are usually associated with delayed engraftment and increased rates of infectious complications. CB ex vivo expansion holds the promise of delivering higher cell doses and improved outcomes. Here we discuss different methods of expansion, their shortcomings and future directions.

CD3(+) and/or CD14(+) depletion from cord blood mononuclear cells before ex vivo expansion culture improves total nucleated cell and CD34(+) cell yields.

Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle in the use of CB is its low cell dose, which is largely responsible for an elevated risk of graft failure and a significantly delayed neutrophil and platelet engraftment. As a positive correlation has been shown between the total nucleated cell (TNC) and CD34(+) cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with MSC in a cytokine cocktail. We show that this strategy can be further improved if CD3(+) and/or CD14(+) cells are first depleted from the CB MNC before ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible as clinical-grade devices and reagents are available. Ultimately, the aim of improving TNC and CD34(+) transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.