Novel loci associated with usual sleep duration: the CHARGE Consortium Genome-Wide Association Study.

Usual sleep duration is a heritable trait correlated with psychiatric morbidity, cardiometabolic disease and mortality, although little is known about the genetic variants influencing this trait. A genome-wide association study (GWAS) of usual sleep duration was conducted using 18 population-based cohorts totaling 47 180 individuals of European ancestry. Genome-wide significant association was identified at two loci. The strongest is located on chromosome 2, in an intergenic region 35- to 80-kb upstream from the thyroid-specific transcription factor PAX8 (lowest P=1.1 × 10(-9)). This finding was replicated in an African-American sample of 4771 individuals (lowest P=9.3 × 10(-4)). The strongest combined association was at rs1823125 (P=1.5 × 10(-10), minor allele frequency 0.26 in the discovery sample, 0.12 in the replication sample), with each copy of the minor allele associated with a sleep duration 3.1 min longer per night. The alleles associated with longer sleep duration were associated in previous GWAS with a more favorable metabolic profile and a lower risk of attention deficit hyperactivity disorder. Understanding the mechanisms underlying these associations may help elucidate biological mechanisms influencing sleep duration and its association with psychiatric, metabolic and cardiovascular disease.

Adoptive T cell immunotherapy for treatment of ganciclovir-resistant cytomegalovirus disease in a renal transplant recipient.

Cytomegalovirus (CMV) is a significant cause of morbidity, mortality and graft loss in solid organ transplantation (SOT). Treatment options for ganciclovir-resistant CMV are limited. We describe a case of ganciclovir-resistant CMV disease in a renal transplant recipient manifested by thrombotic microangiopathy-associated glomerulopathy. Adoptive T cell immunotherapy using CMV-specific T cells from a donor bank was used as salvage therapy. This report is a proof-of-concept of the clinical and logistical feasibility of this therapy in SOT recipients.

Haploidentical peripheral blood stem cell infusion in combination with chemotherapy for acute myeloid leukaemia in elderly patients.

Elderly patients with acute myeloid leukaemia (AML) have a poor prognosis with standard chemotherapy. Two elderly AML patients treated with infusion of family-derived partially human leukocyte antigen (HLA)-matched peripheral blood stem cells following each cycle of chemotherapy entered morphological complete remission without graft versus host disease or major toxicity. Our results support this as a non-toxic approach for inducing a graft versus leukaemia effect in patients not suitable for allogeneic transplantation. Additional resources required for donor assessment and harvest may be reduced by using banked partially HLA-matched mononuclear cells from unrelated donors.

A risk score for early cytomegalovirus reactivation after allogeneic stem cell transplantation identifies low-, intermediate-, and high-risk groups: reactivation risk is increased by graft-versus-host disease only in the intermediate-risk group.

BACKGROUND: This retrospective study was aimed at establishing a clinical score to stratify the risk of cytomegalovirus (CMV) reactivation in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) in order to direct strategies for post-transplant CMV monitoring and therapy. PATIENTS AND METHODS: In total, 335 adult patients undergoing HSCT were analyzed and divided into a training set (n = 235) and a validation set (n = 100). Logistic regression analysis on the training set identified recipient and donor CMV seropositivity, acute graft-versus-host disease (GVHD), and use of anti-thymocyte globulin or alemtuzumab as significant risk factors for CMV reactivation. Weighted scores were assigned to each factor. A weighted score (CMV scoring index [CSI]) was calculated for each patient using the scores of all risk factors except for GVHD. The index was collapsed into 3 risk groups - low risk (score of 0-2), intermediate risk (score of 3-5), and high risk (score of 6-7) - and reactivation rates were calculated. In the training set, CMV reactivation occurred in 5.8% in the low-risk group, 44.8% in the intermediate-risk group, and 67.7% in the high-risk group. RESULTS: In patients with an intermediate CSI only, significantly higher reactivation rates were seen in the presence of corticosteroid treatment for GVHD (57.8% vs. 24.5%, P < 0.01). These findings were similar in the validation set with reactivation rates of 0% in the low-risk, 46% in the intermediate-risk, and 68.4% in the high-risk groups. As seen in the training set, the presence of GVHD was associated with higher CMV reactivation rates only in the intermediate-risk group (64% vs. 28% in the absence of GVHD, P = 0.02). CONCLUSIONS: Identification of these 3 risk groups in association with the presence or absence of GVHD will help transplant units to make pre-transplant policy decisions about prophylactic, pre-emptive, or experimental CMV prevention strategies in groups of patients undergoing HSCT, as well as in those developing GVHD post transplant.

How we mobilize haemopoietic stem cells.

Mobilization and collection of haemopoietic stem and progenitor cells (HSPC) is the cornerstone of autologous and allogeneic stem cell transplantation for a wide variety of haematological and some non-haematological malignancies. Centres providing this service face the challenge of optimizing the likelihood of successful collection of transplantable doses of cells, while maximizing the efficiency of the apheresis unit and minimizing the risk of toxicity as well as mobilization failure. Recent developments in the understanding of the molecular mechanisms of mobilization have led to the emergence of novel strategies for HSPC mobilization, which may assist in meeting these imperatives. The task for clinicians is how to incorporate the use of these strategies into practice, in the light of emerging evidence for efficacy and safety of these agents. Herein, the literature is reviewed, and a proposed algorithm for HSPC mobilization is presented.

Failure to achieve a threshold dose of CD34+ CD110+ progenitor cells in the graft predicts delayed platelet engraftment after autologous stem cell transplantation.

In this study, we retrospectively analysed the utility of CD110 expression on CD34(+) cells as a predictor of delayed platelet transfusion independence in 39 patients who underwent autologous peripheral blood stem cell transplantation. Absolute CD34(+) cells and CD34(+) subsets expressing CD110 were enumerated using flow cytometry. Of the 39 patients, 7 required 21 days or more to achieve platelet transfusion independence. Six of the seven patients received a dose of CD34(+)CD110(+) cells below 6.0 x 10(4)/kg while 30 of 32 patients who achieved platelet transfusion independence in <21 days received a dose of CD34(+)CD110(+) cells >6.0 x 10(4)/kg (P<0.001). Patients with delayed platelet engraftment received a median dose of 5.2 x 10(4) CD34(+)CD110(+) cells/kg compared with a median dose of 16.4 x 10(4) cells/kg for those engrafting within 21 days (P=0.003). Further analysis showed that >6.0 x 10(4) CD34(+)CD110(+) cells/kg was highly sensitive (93.8%) and highly specific (85.7%) for achieving platelet transfusion independence within 21 days. Delay in platelet transfusion independence translated into an increased requirement for platelet transfusion (median 6 vs 2 transfusions, P<0.0001). The dose of CD34(+)/CD110(+) cells/kg infused at time of transplantation appears to be an important factor identifying patients at risk of delayed platelet engraftment.

Unrelated umbilical cord blood transplantation for adults with haematological malignancies: results from a single Australian centre.

BACKGROUND: A number of haematological malignancies can be cured by allogeneic stem cell transplantation but only approximately 30% of Australians have a suitable histocompatible related donor. Matched donors can be found on international registries of unrelated volunteers for a proportion of the remaining patients. For those patients in need of an allogeneic transplant, but for whom a suitable matched related or unrelated adult donor cannot be found, the use of banked unrelated umbilical cord blood has emerged as a potential option. However, there is uncertainty about the applicability of this technique for the majority of adult patients as a result of limitations in the number of cells in banked cord blood units and the degree of mismatching for histocompatibility antigens. AIMS: The aim of this study was to define the feasibility of allogeneic stem cell transplantation using single unrelated cord blood units in a cohort of adults with poor prognosis leukaemia or lymphoma. METHODS: Nine patients with haematological malignancies (five with acute myeloid leukaemia, one with acute lymphoblastic leukaemia, one with Hodgkin lymphoma and two with non-Hodgkin lymphomas) received transplants of cryopreserved cord blood after conditioning therapy with high-dose cyclophosphamide, total body irradiation and antithymocyte globulin. Cord units contained a median 2.6 x 10(7) nucleated cells/kg recipient bodyweight and were matched for four (seven cases) or five (two cases) major histocompatibility complex class 1 and 2 antigens. Patients were given post-transplant immunosuppression with cycosporin and methylprednisolone. RESULTS: Neutrophil recovery to 0.5 x 10(9)/L was seen by median day 30 after transplant in all seven patients who survived more than 1 month post-transplant. Platelet recovery to 50 x 10(9)/L occurred by median day 81 in five evaluable patients. Acute graft versus host disease (GVHD) grades II-IV was seen in four of seven evaluable patients and limited chronic GVHD was seen in four of five. Infection was the most common complication. Four patients died before day 100 of infection (methicillin-resistant Staphylococcus aureus septicaemia, respiratory syncitial virus pneumonia), GVHD and multi-organ failure, and intracranial bleeding. Five patients survived 7-69 months post-transplant, without evidence of relapse of the underlying malignancy. CONCLUSION: Unrelated cord blood transplantation is feasible in adults with high-risk malignancy, with infection relating to immunocompromise being the major limitation.

Mutators in Saccharomyces cerevisiae: MUT1-1, MUT1-2 and MUT2-1.

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.

Human acute myeloid leukaemia cells express adhesion proteins and bind to bone marrow fibroblast monolayers and extracellular matrix proteins.

Adhesion protein expression by acute myeloid leukaemia (AML) cells may affect bone marrow stromal localization and determine exposure of leukaemic cells to stromal derived myeloid growth factors. We have analysed the surface expression by myeloid leukaemic cells of proteins with known adhesive function and the ability of AML cells to adhere to bone marrow fibroblasts and the extracellular matrix proteins fibronectin and laminin. Cells from all six patients tested adhered to bone marrow fibroblast monolayers (mean binding 28.8 +/- 12.8%) and to purified fibronectin in five cases studied (mean binding 33.8 +/- 15.3%). Cells from four patients with AML also adhered to laminin (mean binding 20.9 +/- 4.0%). AML cells from the majority of patients with leukaemia at diagnosis or relapse expressed the ligand pair LFA-1 and ICAM-1, the CD2 ligand LFA-3, alpha and beta chains of the integrins VLA-4, VLA-5 and VLA-6, and the hyaluronate receptor CD44. Antibodies to CD11a, CD18, VLA-4 alpha, and VLA-5 alpha failed to inhibit binding of AML cells to bone marrow fibroblasts but anti-VLA-5 alpha antibodies inhibited AML cell binding to fibronectin by approximately 50%. The ability of AML cells to adhere to bone marrow fibroblasts and extracellular matrix proteins such as fibronectin and laminin may to help explain the capacity of AML cells to persist in the marrow during periods of apparent complete remission and to subsequently proliferate under the influence of locally secreted myeloid growth factors.

GM-CSF enhances IL-2-activated natural killer cell lysis of clonogenic AML cells by upregulating target cell expression of ICAM-1.

Acute myeloid leukemia (AML) cells express the surface adhesion proteins intercellular adhesion molecule-1 (ICAM-1, CD54) and lymphocyte function associated molecule-3 (LFA-3, CD58). Exposure to the myeloid growth-promoting cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulates expression of ICAM-1 and LFA-3 on AML cells but does not increase their sensitivity to lysis by interleukin-2-activated natural killer cells (LAK) in 51Cr assays. However when AML cells are exposed to GM-CSF prior to incubation with LAK, their subsequent clonogenic activity is significantly reduced. If a blocking antibody to ICAM-1 is added during the incubation period of AML with LAK, the inhibitory effect is completely ablated. A less pronounced effect is observed with an antibody to LFA-3. ICAM-1 is expressed on a greater proportion of CD34+ than CD34- AML cells and exposure to GM-CSF induces a significantly greater upregulation of ICAM-1 on leukemic CD34+ cells than their CD34- counterparts. These data suggest that the inhibitory effect of IL-2-activated natural killer cells on clonogenic AML cells is mediated principally via the lymphocyte function associated molecule-1 (LFA-1)/ICAM-1 interaction. Interleukin-2 upregulates LFA-1 expression on natural killer cells. Simultaneous administration of effector cell activators such as IL-2 and target cell modulators such as GM-CSF may have a therapeutic benefit in patients with minimal residual myeloid leukemia.

Natural killer cells adhere to bone marrow fibroblasts and inhibit adhesion of acute myeloid leukemia cells.

In this study we have demonstrated that natural killer (NK) cells adhere to elements of the bone marrow stroma (BMS) including fibroblasts, fibronectin and laminin but not to collagen type I, vitronectin and hyaluronic acid. NK cells bind to fibronectin and laminin using the beta 1 integrins VLA-4 and VLA-5, and VLA-6 respectively. The mechanism of adhesion to bone marrow fibroblasts is more complicated with beta 1 and beta 2 integrins being partially responsible but the majority of adhesion remaining unexplained. IL-2 stimulation of NK cells resulted in an increase in the expression of adhesion molecules involved in binding of NK cells to bone marrow fibroblasts (BMF) and extracellular matrix (ECM) proteins including the beta 1 chain CD29, alpha chains of VLA-4 and 5, beta 2 chain CD18 and alpha L chain CD11a. A marked increase in expression of beta 7 was also observed. There was a significant increase in the adhesion of NK cells to fibronectin in response to IL-2 treatment. NK cells also bound more strongly to BMF following IL-2 treatment although the development of cytotoxicity appeared to interfere with the adhesion assay. NK cells competitively inhibit the binding of AML blasts but not ALL blasts to BMF. Mechanisms underlying the inhibition of leukemic growth by NK and LAK cells may include direct and cytokine mediated cytotoxicity and perturbation of the interaction of leukemic blasts with the bone marrow stroma which is essential for blast cell survival.

Expression of CD44 variant exons in acute myeloid leukemia is more common and more complex than that observed in normal blood, bone marrow or CD34+ cells.

CD44 is an adhesion molecule that is expressed on hematopoietic cells and has been implicated in the interactions between bone marrow stromal layers and hematopoietic progenitors. The expression of variant forms of CD44, particularly forms containing exon v6, have been associated with poor prognosis in a number of hematological malignancies. The expression of CD44 variants on normal bone marrow (BM), peripheral blood (PBMC) and CD34+ hematopoietic progenitors was compared with those expressed on blasts from 30 patients with acute myeloid leukemia (AML). Normal BM, PBMC and CD34+ progenitor cells were negative for all variants tested by flow cytometry. In contrast exon v3 was expressed on 13%, v4 on 67%, v5 on 19%, v6 on 7% and v7 on 65% of AML cases. RT-PCR and Southern blotting revealed the expression of exons v3, v6, v8, v9 and v10 in normal bone marrow and peripheral blood mononuclear cells and the expression of exons v3, v6, v8 and v10 in CD34+ progenitors. A more complex pattern of variant exon expression was observed in leukemic samples in comparison to normal hematopoietic cells. Sixty-two percent of AML cases expressed exon v3 and 70% exon v6. Exons v4 and v5 were not detected while exons v7, v8, v9 and v10 were detected in 21, 83, 71 and 92% of cases, respectively. In summary, our data demonstrate a striking increase in the complexity of CD44 variant expression in cells from patients with AML, along with surface expression of some variant CD44 proteins. Further analysis will be directed at how these alter the interaction of leukemic blasts with the bone marrow microenvironment and their diagnostic, prognostic and therapeutic potential.

Effects of inhibitors of the chemokine receptor CXCR4 on acute lymphoblastic leukemia cells in vitro.

Stromal cell-derived factor-1 (SDF-1) is a key regulator of the behavior of normal and leukemic precursor-B (pre-B) cells. It is possible that inhibiting SDF-1-driven processes in pre-B acute lymphoblastic leukemia (ALL) may have therapeutic implications. In this study, we examined the ability of SDF-1 inhibitors to modulate pre-B ALL cell responses to SDF-1, including chemotaxis, migration into bone marrow stroma, and stroma-supported survival and proliferation on human bone marrow stromal layers. The polyphemusin II-derived inhibitors, T140, TC140012, and T134, and the bicyclam AMD3100, effectively inhibited binding of the anti-CXCR4 monoclonal antibody 12G5 on the pre-B ALL cell line NALM6, with IC(50) values of 0.9, 0.9, 0.9, and 1.9 nM, respectively. Similar results were obtained with ALL samples. T140 (0.1 micro M) and AMD3100 (1 micro M) completely blocked SDF-1-induced chemotaxis and attenuated the migration of pre-B ALL cells into bone marrow stromal layers. AMD3100 and TC140012 at a concentration of 50 micro M significantly inhibited stroma-dependent proliferation of six and four of the eight cases tested, respectively, without reducing the cell viability. In addition, AMD3100 and TC140012 enhanced the cytotoxic and antiproliferative effects of the cytotoxic agents vincristine and dexamethasone. The ability of SDF-1 inhibitors to modulate these biologically important functions of leukemic cells warrants further investigation.

Stem cell factor enhances the adhesion of AML cells to fibronectin and augments fibronectin-mediated anti-apoptotic and proliferative signals.

Acute myeloid leukaemia (AML) cells express the SCF receptor c-kit (CD117) on their cell surface and demonstrate enhanced adhesion to fibronectin (FN) following exposure to stem cell factor (SCF). Increased adhesion occurs within 5 min, is dose dependent, and persists beyond 2 h. Baseline and enhanced adhesion occur through the surface FN receptor very late antigen-5 (VLA-5, CD49e/CD29) which is expressed by AML cells. Unstimulated AML cells exposed to FN undergo less apoptosis than controls (inhibition 22.5 +/- 7.0%, P = 0.02, n = 8). Exposure to SCF alone without FN also inhibits AML cell apoptosis (by 19.0 +/- 7.7% compared to controls, P = 0.06, n = 8). Simultaneous exposure to SCF and FN increases the inhibition of AML cell apoptosis to 37.8 +/- 7.9% (P = 0.005 compared to control, P = 0.04 compared to FN alone, P = 0.06 compared to SCF alone) demonstrating that SCF not only enhances the propensity of AML cells to adhere to FN, but also results in an additive survival benefit following FN contact. Some but not all the reduction in apoptosis is mediated through VLA-5. The combination of SCF and FN also affects proliferation, resulting in a synergistic enhancement of AML cell proliferation in half the cases studied. When normal CD34+ human haemopoietic progenitors were studied, FN had little effect on their apoptosis and failed to enhance the anti-apoptotic effect of SCF. It did, however, synergise with SCF in promoting CD34+ cell proliferation. Exposure of AML cells to SCF and FN, both of which can be found in high concentration in the bone marrow stroma, inhibits apoptosis. Cytokines and extracellular matrix proteins augment each others' effects since SCF enhances adhesion to fibronectin, which in turn augments the survival signal delivered by the cytokine alone. Cytokine and adhesion receptors can combine to affect cell characteristics including proliferation and survival.

Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers.

Acute lymphoblastic leukemia (ALL) blasts undergo migration into layers of bone marrow fibroblasts (BMF) in vitro, utilizing the beta1 integrins VLA-4 and VL-5 as adhesion molecules. However, it has been unclear as to whether this is a selective process mediated by specific chemoattractant molecules, or simply a reflection of the highly motile nature of early B cell precursors. We further characterized this process using a transwell culture system, in which the two chambers were separated by an 8 microm diameter microporous membrane, through which leukemic cells could move. When a BMF layer was grown on the upper surface of the membrane there was an 84.1% reduction in transmigration of the human pre-B ALL cell line NALM-6 into the lower chamber, compared to control membrane with no BMF layer. Localization of leukemic cells under the BMF layer was confirmed ultrastructurally, suggesting the possibility that the migration of leukemic cells was directed by a chemotactic agent secreted by BMF. The involvement of the chemokine stromal cell-derived factor-1 (SDF-1) in this process was next investigated. BMF were shown to express m-RNA for SDF-1. Addition of SDF-1 at 100 ng/ml into the lower chamber increased transmigration of NALM-6 across the membrane by 2.2-fold, and also induced a 1.4- to 6.1-fold increase in movement of NALM-6 through a BMF layer into the lower chamber. The receptor for SDF-1, CXCR4, was demonstrated by flow cytometry on all 10 cases of precursor-B ALL analyzed, as well as on NALM-6, KM-3 and REH lines. An inhibitory antibody to CXCR4 was able to block the migration of NALM-6 cells into BMF monolayers grown on plastic by 51%, and in nine cases of ALL by 8-40%, as well as partially inhibit transmigration of leukemic cells through BMF layers along an SDF-1 concentration gradient. These results confirm that precursor-B ALL cells selectively localize within bone marrow stroma in vitro, and that this process is partially due to the stromal chemokine SDF-1 binding to its receptor CXCR4 on leukemic cells. SDF-1 may be important in influencing the localization of precursor-B ALL cells in marrow microenvironmental inches which regulate their survival and proliferation.

MUC18, a member of the immunoglobulin superfamily, is expressed on bone marrow fibroblasts and a subset of hematological malignancies.

Despite the importance of bone marrow stromal cells in hemopoiesis, the profile of surface molecule expression is relatively poorly understood. Mice were immunized with cultured human bone marrow stromal cells in order to raise monoclonal antibodies to novel cell surface molecules, which might be involved in interactions with hemopoietic cells. Three antibodies, WM85, CC9 and EB4 were produced, and were found to identify a 100-110 kDa antigen on bone marrow fibroblasts. Molecular cloning revealed the molecule to be MUC18 (CD146), a member of the immunoglobulin superfamily, previously described as a marker of metastatic melanoma. In addition to the expected expression on melanoma cell lines and endothelial cells, a number of human leukemic cell lines were found to express MUC18, including all six T leukemia lines tested, one of five B lineage lines and one of four myeloid lines. Analysis of bone marrow samples from patients revealed positivity in 20% of B lineage ALL (n = 20), one of three T-ALL, 15% of AML (n = 13) and 43% of various B lymphoproliferative disorders (n = 7). No apparent reactivity was observed with mononuclear cells from normal peripheral blood or bone marrow, including candidate hemopoietic stem cells characterized by their expression of the CD34 antigen. However, positive selection of bone marrow mononuclear cells labeled with MUC18 antibody revealed a rare subpopulation (<1%) containing more than 90% of the stromal precursors identified in fibroblast colony-forming assays. The structure and tissue distribution of MUC18 suggest a functional role in regulation of hemopoiesis.

Effect of a change in house staff work schedule on resource utilization and patient care.

Concern is frequently expressed by health care providers and consumers that the work environment of physicians-in-training may adversely affect their performance. This article documents the effects of changing from a traditional rotational overnight call schedule for house staff to a schedule designed to reduce sleep deprivation, distribute admissions more evenly throughout the week, and improve continuity of inpatient care on the internal medicine service of a large, university-affiliated Veterans Affairs Medical Center. In a prospective, time-series study, the hypothesis that this change would improve the efficiency and quality of medical care was evaluated by comparing the hospital course of the patients admitted during 4-week periods prior to and following the change in work schedule. The patients in the preintervention group do not differ significantly from those in the postintervention group in any identifiable clinical characteristics. The length of stay was shorter (10.9 vs 9.3 days) and the number of laboratory tests ordered per patient was smaller (24.0 vs 19.0) for patients cared for under the new work schedule compared with those cared for under the traditional work schedule. Resident physicians also committed fewer medication errors under the new work schedule (16.9 vs 12.0 per 100 patients discharged). We conclude that altering the house staff work schedule affects patient care and can lead to a decrease in utilization of health care resources.

Bone marrow fibroblast exposure to the inflammatory cytokines tumor necrosis factor-alpha and interferon-gamma increases adhesion of acute myeloid leukemia cells and alters the adhesive mechanism.

Human acute myeloid leukemia (AML) cells adhere to bone marrow fibroblasts (BMF) and extracellular matrix proteins including fibronectin. Adhesion is increased when fibroblast monolayers are exposed to tumor necrosis factor-alpha (TNF) alone and in combination with interferon-gamma (IFN) or interleukin-4 (IL-4). The combination of TNF and IFN caused enhanced AML cell adhesion to treated BMFs, from a mean of 25.0 +/- 4.1% to 36.3 +/- 5.4% (p = 0.0007). Enhanced binding was partially a result of upregulated vascular cell adhesion molecule-1 expression on BMFs. Intercellular adhesion molecule-1 was also upregulated, but did not appear to play a role in the increased binding to cytokine-stimulated BMFs. In contrast to observed adhesion to resting BMFs, AML cells binding to TNF/IFN-stimulated BMFs rely more heavily on the VLA-4 alpha chain (CD49d). In some cases, alpha4 integrin chain antibody was more effective than beta1 antibody in blocking binding, suggesting that a non-beta1 alpha4 integrin, possibly alpha4 beta7, on AML cells may act as a stromal ligand. The addition of alpha4 antibody to beta1 and beta2 antibodies significantly increased the inhibition of AML cells to stimulated BMFs. The myeloid cytokines granulocyte colony stimulating factor, granulocyte-monocyte colony stimulating factor, interleukin-3 and stem cell factor enhanced the adhesion of AML blast cells to BMFs in some cases. The phorbol ester PMA, however, consistently upregulated AML cell-binding to BMFs, the increase being mediated entirely via beta1 and beta2 integrins without altering AML cell integrin expression. Binding of AML cells to marrow stroma can be enhanced by influences on leukemic cell or stroma. Enhanced binding under these conditions occurs via different pathways, illustrating the heterogeneity of mechanisms underlying leukemic cell retention within the bone marrow stroma.

The chemokine receptor CXCR4 enhances integrin-mediated in vitro adhesion and facilitates engraftment of leukemic precursor-B cells in the bone marrow.

OBJECTIVE: It has been demonstrated that acute lymphoblastic leukemia (ALL) blasts migrate into layers of bone marrow fibroblasts (BMF) in vitro using the beta1 integrins VLA-4 and VLA-5, and that the chemokine SDF-1 and its receptor CXCR4 influences ALL migration. We investigated whether this effect was due to SDF-1-mediated induction of adhesion through beta1 integrins. METHODS: Adhesion of pre-B ALL cells or the cell line NALM6 to extracellular matrix proteins was examined using short-term in vitro binding assays. The effects of exposure of cells to SDF-1, antibodies to CXCR4, and the G protein inhibitor pertussis toxin (PTX) were assessed. The consequences of down regulation of CXCR4 on the in vivo behavior of pre-B ALL cells after injection into sublethally irradiated NOD/SCID mice was studied. RESULTS: Treatment with SDF-1 of NALM6 cells or cells from cases of precursor-B ALL resulted in a doubling of adhesion to fibronectin, laminin, and VCAM-1, but had no effect on binding to collagens I or IV. Antibodies to CXCR4 and PTX inhibited SDF-1-induced adhesion on these substrates. NALM6 cells with CXCR4 expression downregulated by SDF-1 exposure demonstrated a reduced capacity to engraft into the bone marrow of NOD/SCID mice, with only 22 +/- 11% of marrow cells being of human origin in mice receiving SDF-1-treated cells compared to 48 +/- 5% in mice receiving untreated cells (p < 0.001). The homing of SDF-1-treated cells to the bone marrow after 24 hours was also reduced by 72 +/- 16% compared to control cells. CONCLUSIONS: These data show that SDF-1 and CXCR4 are involved in regulation of beta1 integrin function, and are important for the localization of pre-B cells to the bone marrow in vivo.

Bone marrow adherent layers inhibit apoptosis of acute myeloid leukemia cells.

Human acute myeloid leukemia (AML) cells, like normal hematopoietic progenitors, die rapidly by apoptosis when cultured under serum-free conditions. Apoptosis was demonstrated by electron microscopy and agarose gel electrophoresis and quantified by flow cytometry. Culturing AML blasts in the presence of a bone marrow fibroblast (BMF) monolayer reduced the percentage of AML blasts undergoing apoptosis in the majority of cases studied. The effect was more pronounced when AML cells were cultured in the presence of an adherent long-term bone marrow (LTBM) stroma rather than BMF. Overall, the mean percentage of AML cells with fragmented DNA fell from 85 +/- 8% in control cultures to 20 +/- 9% in cultures with adherent stroma (p = 0.0004, n = 7). Supplementation of serum-free medium with recombinant cytokines, including stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha or with human placenta-conditioned medium (HPCM) matched the degree of inhibition of apoptosis induced by BMF in only 50% of cases. Granulocyte colony-stimulating factor (G-CSF), interleukin-1 beta (IL-1 beta), and IL-6 were completely ineffective. Consistent with this observation, direct contact between leukemic cells and adherent layers was essential for maximum inhibition of leukemic-cell DNA fragmentation. Separation by a porous membrane allowing passage of soluble growth factors, but interrupting direct cell contact, was associated with significantly greater DNA fragmentation and cell death. Inhibition of leukemic-cell apoptosis correlated with improved survival and growth of malignant clonogenic cells. Colonies grown in cultures were identified as leukemic by morphology and by fluorescence in in situ hybridization to demonstrate numerical chromosomal abnormalities identified at diagnosis. Close contact between leukemic cells and bone marrow inhibits blast cell apoptosis and directly promotes survival of clonogenic AML cells.