Search

Hematopoietic stem cell transplantation (HSCT) is an important treatment option for patients with malignant and nonmalignant hematologic diseases. Methods to improve transplant efficiency are being explored with the intent to improve engraftment and immune reconstitution post-HSCT. A current approach under investigation involves treatment of donor cells with inhibitors that target the protease CD26, a negative regulator of the chemokine CXCL12/stromal cell-derived factor-1. CD26 inhibitor treatment has been shown to improve the functional response of CD34(+) cord blood (CB) cells, but not CD34(+) granulocyte colony-stimulating factor-mobilized peripheral blood stem cells, to CXCL12/stromal cell-derived factor-1. The effect of CD26 inhibitors on unfractionated CB, bone marrow, or granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells has not been evaluated previously. We observed that although CB had greater CD26 expression than bone marrow or mobilized peripheral blood, treatment with a CD26 inhibitor (Diprotin A) resulted in increased responsiveness to stromal cell-derived factor-1 for all three mononuclear cell sources tested. This suggests that clinical therapeutic benefit might be gained by using CD26 inhibitors as a strategy to improve engraftment of unfractionated mobilized peripheral blood cells as well as CB cells.

BACKGROUND: A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/HPC) trafficking is critical to improve transplant efficiency and immune reconstitution during hematopoietic stem cell transplantation (HSCT). Our earlier findings suggested that suppression of CD26 (dipeptidyl peptidase IV) proteolytic activity in the donor cell population can be utilized as a method for increasing transplant efficiency. However, factors in the recipient should not be overlooked, given the potential for the bone marrow (BM) microenvironment to regulate HSCT. STUDY DESIGN AND METHODS: We first evaluated CD26 expression and then investigated the effects of the CD26 inhibitor diprotin A and the absence of CD26 (CD26−/−) in recipient mice on HSC/HPC homing and engraftment using an in vivo congenic mouse model of HSCT. RESULTS: A significant increase in donor cell engraftment into the peripheral blood (PB), and to a lesser extent homing into the BM, was observed in CD26−/− mice or CD26 inhibitor–treated mice. Increased PB engraftment of CD26−/− mice was significant at 3 and 6 months, but not 1 month, after transplant. It was noted that the increased homing was statistically greater with donor cell manipulation (CD26−/− donor cells) than with recipient manipulation (CD26−/− recipient mice). Conversely, donor and recipient manipulation both worked well to increase PB engraftment at 6 months. CONCLUSION: These results provide preclinical evidence of CD26, in the HSCT recipient, as a major regulator of HSC/HPC engraftment with minor effects on HSC/HPC homing and suggest the potential use of CD26 inhibitors in HSCT patients to improve transplant efficiency.

The field of hematopoietic stem cell transplantation (HSCT) has overcome many obstacles that have led to our current clinical ability to utilize cells collected from marrow, mobilized peripheral blood, or umbilical cord blood for the treatment of malignant and nonmalignant hematologic diseases. It is in this context that it becomes evident that future progress will lie in our development of an understanding of the biology by which the process of HSCT is regulated. By understanding the cellular components and the mechanisms by which HSCT is either enhanced or suppressed it will then be possible to design therapeutic strategies to improve rates of engraftment that will have a positive impact on immune reconstitution post-HSCT. In this review we focus primarily on allogeneic hematopoietic stem cell transplantation (allo-HSCT), the current challenges associated with allo-HSCT, and some developing strategies to improve engraftment in this setting.

Summary Objective To review the literature on modulation of chondrocyte activities in the osteoarthritic joint, and to discuss these changes in relation to established hard and soft tissue repair paradigms, with an emphasis on transforming growth factor beta (TGFβ1)-mediated signaling which can promote either a chondrogenic or fibrogenic phenotype. Methods Papers addressing the close relationship between repair in general, and the specific post-injury response of joint tissues are summarized. Different interpretations of the role of TGFβ1 in the emergence of an "osteoarthritic" chondrocyte are compared and the phenotypic plasticity of "reparative" progenitor cells is examined. Lastly, emerging data on a central role for A-Disintegrin-And-Metalloproteinase-with-Thrombospondin-like-Sequences-5 (ADAMTS5) activity in modulating TGFβ1 signaling through activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5) pathways is discussed. Results The review illustrates how a transition from ALK5-mediated fibrogenic signaling to ALK1-mediated chondrogenic signaling in joint cells represents the critical transition from a non-reparative to a reparative cell phenotype. Data from cell and in vivo studies illustrates the mechanism by which ablation of ADAMTS5 activity allows the transition to reparative chondrogenesis. Multiple large gene expression studies of normal and osteoarthritis (OA) human cartilages (CAs) also support an important role for TGFβ1-mediated pro-fibrogenic activities during disease progression. Conclusions We conclude that progressive articular CA damage in post-injury OA results primarily from biomechanical, cell biologic and mediator changes that promote a fibroblastic phenotype in joint cells. Since ADAMTS5 and TGFβ1 appear to control this process, agents which interfere with their activities may not only enhance endogenous CA repair in vivo , but also improve the properties of tissue-engineered CA for implantation.

y Department of Internal Medicine (Rheumatology), Rush University Medical Center, Chicago, IL, USAz Department of Biochemistry, Rush University Medical Center, Chicago, IL, USAx Department of Internal Medicine (Hematology/Cell Therapy), Rush University Medical Center, Chicago, IL, USAk Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL, USA

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a−/− embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a −/− mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a−/− mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.

Our objective was to explore associations of circulating factors implicated in insulin-like growth factor- 1 receptor (IGF-1R) signaling with clinical outcomes of patients with acute myeloid leukemia (AML). Pretreatment blood samples from patients with non-M3 AML (n = 30) were collected prospectively and levels of IGF binding proteins (IGFBPs) 1-7 and IGF-1 (free and total) were established at diagnosis and statistically evaluated. Baseline levels of IGFBP-1 and -6 below respective thresholds of 8.8 ng/mL and 237 ng/mL were associated (p = 0.0347 and 0.0099, respectively) with superior progression-free survival, whereas baseline levels of IGFBP -1, -2, -6 and -7 below the respective thresholds of 8.8, 28.8, 237 and 119 ng/mL were strongly associated (p = 0.0004, 0.0085, 0.0031, 2.46 × 10(- 7), respectively) with improved overall survival. These findings provide promising evidence that IGFBP signatures could be used as predictive tools in AML, with applications in remission surveillance and the development of IGFBP-directed biologic therapy.

The chemokine CXCL12 (stromal cell derived factor-1/SDF-1) stimulates hematopoietic stem and progenitor cells (HSCs/HPCs) through the corresponding chemokine receptor CXCR4. CXCL12 is thought to be important for both proper HSC homing, retention, and engraftment into the bone marrow (BM) and mobilization out of the BM. Previous studies suggest that breaking the CXCL12-CXCR4 interaction mobilizes HPCs, blocking CXCR4 inhibits HSC homing, and overexpression increases HSC/HPC repopulation. The efficiency of mobilization and engraftment therefore appears to be dependent on the response of HSCs/HPCs to CXCL12, which is in turn dependent upon levels of CXCR4 expressed on HSCs/HPCs. However, expression of CXCR4 on the surface of HSCs/HPCs appears to be variable. To study the function of CXCR4 on HSCs/HPCs, we used the MSCV-based bicistronic (EGFP) retroviral vector MIEG3 to overexpress CXCR4 on M07e cells, an established model of human HPC. CXCR4 overexpression resulted in significant increases in CXCL12-induced chemotaxis and cell survival. Most importantly, cells overexpressing CXCR4 responded to CXCL12 at levels typically too low induce a response. These data suggest that an increased transplant efficiency resulting from CXCR4 overexpression is likely a function of increased HSC/HPC homing and increased HSC/HPC survival in the recipient's BM. These experiments also validate the ability of the MIEG3-CXCR4 retroviral construct to overexpress CXCR4 efficiently and the use of MIEG3-CXCR4 M07e cells for further study. Finally, this information may have future potential therapeutic implications for improvements in transplant efficiency.

We isolated and characterized a novel AML1 (also termed Runx1) fusion transcript from a radiation-associated acute myeloid leukemia with a t(19;21). This fusion transcript, termed AML1–AMPl9, was joined out of frame, resulting in a truncated AML1 protein that inhibits activation of AML1 target promoters. It is now becoming clear that truncations of AMLl are more common in leukemia than previously thought. To analyze the effect of truncated AML1 species on myeloid differentiation and proliferation, AML1–AMPl9 was retrovirally transduced into the IL-3-dependent 32D cells. 32D cells over-expressing AML1–AMPl9 failed to differentiate normally when stimulated with G-CSF, but continued to proliferate and maintained a primitive phenotype. However, AML1–AMPl9 did not transform the cells to cytokine independence, implying that for full transformation of a myeloid progenitor by truncated AML1 another genetic lesion is required.

Compelling evidence now exists supporting the involvement of chemokines in the pathogenesis of autoimmune diseases. Examples of chemokines and chemokine receptors being involved in mediating autoimmune disease exist for rheumatoid arthritis, multiple sclerosis, allograft rejection, systemic lupus erythematosus, psoriasis, atopic dermatitis, lichen planus, and graft-versus-host-disease. Expression of chemokines by endothelial cells appears to be an important step in the development of these diseases. Since chemokines are small molecular weight molecules that act through G-protein coupled receptors, they make attractive drug targets. Several antagonists of chemokine - chemokine receptor interactions have been used to successfully alleviate some or all of the symptoms associated with many of these diseases in animal models. Further investigation of the involvement of chemokines in the pathogenesis or progression of autoimmune diseases may lead to practical clinical advances in diagnosis, prognosis, and therapy of such diseases.

CXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1α (SDF-1α) at its position two proline. CXCL12/SDF-1α induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34+ hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34+ hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1α lacks the ability to induce the migration of CD34+ cord blood cells and acts to inhibit normal CXCL12/SDF-1α-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34+ cells enhances the migratory response of these cells to CXCL12/SDF-1α. This process of CXCL12/SDF-1α cleavage by CD26/DPPIV on a subpopulation of CD34+ cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.

Chemokines are a large family of cytokines that direct normal leukocyte migration. They also have been implicated in leukocyte development and in the pathogenesis of many diseases. The CC chemokine CCL21, also known as Exodus-2, SLC, 6Ckine, and TCA4 induces both the adhesion and migration of human T cells. CCL21 is hypothesized to regulate the trafficking of T cells through secondary lymphoid tissues. To test this hypothesis, a transgenic mouse model was generated that placed the expression of mouse CCL21 (mCCL21) under the control of the T cell-specific lck promoter to abrogate the concentration gradient to which T cells normally respond. Overexpression of mCCL21 in T cells resulted in defects in CCL21- and CCL19-induced T-cell chemotaxis, node T-cell subpopulations, and lymph node architecture. The regulation of T-cell trafficking in secondary lymphoid tissues by CCL21 is therefore a tightly regulated system that can be altered by changes in the level of environmental CCL21 protein.

Chemokines are small basic proteins that are the major mediators of all leukocyte migration. There are at least 46 distinct chemokines, and 19 chemokine receptors, making it easily the largest cytokine family. Chemokines can be both beneficial and harmful, by either stimulating an appropriate immune response to microbial invasion, or by mediating pathologic tissue destruction in many types of human disease. Chemokines have been implicated in the tissue destruction seen in autoimmune diseases, atherosclerosis, allograft rejection, and neoplasia. Chemokines also play essential roles in normal lymphocyte trafficking to primary and secondary lymphoid organs for antigen presentation and lymphocyte maturation. Chemokines also regulate hematopoietic stem and progenitor cell homing and proliferation. Therefore, it is likely that chemokines will become important targets for pharmacologic intervention in a wide variety of human diseases in the future.

Natural killer (NK) cells play an important role in innate and adaptive immune responses to obligate intracellular pathogens. Nevertheless, the regulation of NK cell trafficking and migration to inflammatory sites is poorly understood. Exodus-1/MIP-3alpha/LARC, Exodus-2/6Ckine/SLC, and Exodus-3/MIP-3beta/ELC/CKbeta-11 are CC chemokines that share a unique aspartate-cysteine-cysteine-leucine motif near their amino terminus and preferentially stimulate the migration of T lymphocytes. The effects of Exodus chemokines on human NK cells were examined. Exodus-1, -2, and -3 did not induce detectable chemotaxis of resting peripheral blood NK cells. In contrast, Exodus-2 and -3 stimulated migration of polyclonal activated peripheral blood NK cells in a dose-dependent fashion. Exodus-2 and -3 also induced dose-dependent chemotaxis of NKL, an IL-2-dependent human NK cell line. Results of modified checkerboard assays indicate that migration of NKL cells in response to Exodus-2 and -3 represents true chemotaxis and not simply chemokinesis. Exodus-1, -2, and -3 did not induce NK cell proliferation in the absence of other stimuli. Nevertheless, Exodus-2 and -3 significantly augmented IL-2-induced proliferation of normal human CD56(dim) NK cells. In contrast, Exodus-1, -2, and -3 did not affect the cytolytic activity of resting or activated peripheral blood NK cells. Expression of message for CCR7, a shared receptor for Exodus-2 and -3, was detected in activated polyclonal NK cells and NKL cells but not resting NK cells. Taken together, these results indicate that Exodus-2 and -3 can participate in the recruitment and proliferation of activated NK cells. Exodus-2 and -3 may regulate interactions between T cells and NK cells that are crucial for the generation of optimal immune responses.

Specific cytokine signatures correlate with genetic aberrations in CLL/SLL, reflecting a pattern of Th1/Th2/Treg dysregulation that may predict aggressive disease, and a need for therapy. Early treatment of CLL/SLL does not impact survival-reflecting limitations in detecting progression early and identifying asymptomatic patients likely to benefit from early treatment. Improved understanding of CLL/SLL biology would identify better prognostic/predictive markers. This study attempts to address these issues by determining the relationship between cytokine aberrations and poor clinical outcomes in CLL/SLL in the context of a genetic–based prognostic model. Fifty-nine serum cytokines/chemokines were measured in 28 untreated CLL/SLL patients. Patients were stratified as GR or int/PR using cytogenetics. Comparison of CLL/SLL with 28 HCs revealed increased expression of Th2 cytokines (IL-10, IL-5, sIL-2Rα; P≤0.01) and decreased levels of Th1 cytokines (IL-17, IL-23, IFN-γ; P≤0.003). In a multivariate analysis of GR versus int/PR groups, differential expression of sIL-2Rα maintained significance with increased expression in int/PR CLL/SLL. With median follow-up of 54.3 months after diagnosis, four patients incurred disease progression, with an IL-17/sIL-2Rα model predicting need for treatment in all cases. In summary, specific cytokine signatures are associated with genetically defined aggressive disease and predict need for therapy. This suggests utility in detecting disease progression early, identifying those likely to incur a survival advantage with early treatment, and directing future therapy.

The role of mesenchymal stem cells (MSCs) on breast cancer progression, growth and tumorigenesis remains controversial or unknown. In the present study, we investigated the role of MSCs on breast tumor induction and growth in a clinically relevant somatic breast cancer model. We first conducted in vitro studies and found that conditioned media (CM) of RCAS-Neu and RCAS-PyMT breast cancer cell lines and tumor cells themselves dramatically increased the proliferation and motility of MSCs and induced morphological changes of MSCs and differentiation into fibroblast-like cells. In contrast, the CM of MSCs inhibited the proliferation of two breast cancer cell lines by arresting the cell cycle at the G0/G1 phase. In vivo studies revealed that fluorescence dye-labeled MSCs migrated into tumor tissues. Unexpectedly, single or multiple intravenous injections of MSCs did not affect the latency of breast cancer in TVA- transgenic mice induced by intraductal injection of the RCAS vector encoding polyoma middle-T antigen (PyMT) or Neu oncogenes. Moreover, MSCs had no effect on RCAS-Neu tumor growth in a syngeneic ectopic breast cancer model. While our studies consistently demonstrated the ability of breast cancer cells to profoundly induce MSCs migration, differentiation, and proliferation, the anti-proliferative effect of MSCs on breast tumor cells observed in vitro could not be translated into an antitumor activity in vivo, probably reflecting the antagonizing or complex effects of MSCs on tumor environment and tumor cells themselves.

In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d−/− mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d−/−, BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.

Stem cell mobilization, which is defined as the forced egress of stem cells from the bone marrow to the peripheral blood (PB) using chemokine receptor agonists, is an emerging concept for enhancing tissue regeneration. However, the effect of stem cell mobilization by a single injection of the C-X-C chemokine receptor type 4 (CXCR4) antagonist AMD3100 on intramembranous bone regeneration is unclear.We therefore asked: Does AMD3100 mobilize adult stem cells in C57BL/6 mice? Are stem cells mobilized to the PB after marrow ablation? And does AMD3100 enhance bone regeneration?Female C57BL/6 mice underwent femoral marrow ablation surgery alone (n = 25), systemic injection of AMD3100 alone (n = 15), or surgery plus AMD3100 (n = 57). We used colony-forming unit assays, flow cytometry, and micro-CT to investigate mobilization of mesenchymal stem cells, endothelial progenitor cells, and hematopoietic stem cells to the PB and bone regeneration.AMD3100 induced mobilization of stem cells to the PB, resulting in a 40-fold increase in mesenchymal stem cells. The marrow ablation injury mobilized all three cell types to the PB over time. Administration of AMD3100 led to a 60% increase in bone regeneration at Day 21.A single injection of a CXCR4 antagonist lead to stem cell mobilization and enhanced bone volume in the mouse marrow ablation model of intramembranous bone regeneration.

In this issue of Blood , Juarez and colleagues reveal a need for the Sphingosine-1-Phosphate/S1P receptor 1 (S1P/S1P1) axis in the egress of hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) from the bone marrow after CXCR4 antagonist mediated mobilization in mice.[1][1] The

Achieving improvements in survival and reducing relapse remains a challenge in acute myelogenous leukemia (AML) patients. This study evaluated thein vitroefficacy of the active form of novel agent sapacitabine, CNDAC, compared to current chemotherapeutic drugs Ara-C and mitoxantrone using two AML cell lines, HL-60 (promyelocytic) and THP-1 (monocytic), as well as bone marrow (BM) and peripheral blood (PB) cells collected from AML patients. Cell lines were exposed to compound for 3–6 days and primary cells for 4 days. The viability of primary cells was additionally evaluated 3, 7, and 31 days after removal of tested compound to determine the durability of the response. Our studies indicate that CNDAC and mitoxantrone have a greater impact on viability than ara-C in primary AML cells and AML cell lines. CNDAC is more effective at reducing viability and inducing apoptosis than ara-C at equivalent concentrations in the THP-1 cell line, which is defined as displaying resistance to ara-C. As sapacitabine has shownin vivoactivity at clinically achievable doses, future studies are warranted to assess the potential for combining it with ara-C and/or mitoxantrone, with an emphasis on cells and patients insensitive to ara-C treatment.

Objective Megakaryopoiesis involves commitment of hematopoietic stem cells (HSC) toward the myeloid lineage in combination with the proliferation, maturation, and terminal differentiation of progenitors into megakaryocytes. The exact mechanism of megakaryocyte development from HSC is unknown, but growth factors such as thrombopoietin have been identified as critical. Additionally, it has been suggested that the chemokine CXCL12/stromal-cell derived factor-1α has a role in regulating megakaryopoiesis and thrombopoiesis. We recently reported the importance of the extracellular protease CD26 (dipeptidylpeptidase IV) in regulating HSC responses to CXCL12, as well as modulating HSC trafficking into and out of the bone marrow. However, the importance of CD26 for megakaryopoiesis has not been reported. We therefore compared megakaryocyte development between CD26-deficient (CD26 −/− ) mice and C57BL/6 control mice. Materials and Methods Adult CD26 −/− mice and C57BL/6 control mice were evaluated using blood differentials, histological analysis, flow cytometric analysis, and progenitor colony assays. Results Bone marrow from CD26 −/− mice has a significantly expanded megakaryocyte and megakaryocyte progenitor population compared to control C57BL/6 mice bone marrow. Conclusions Our results indicate that endogenous CD26 normally suppresses megakaryopoiesis and that loss of CD26 activity results in expansion of the megakaryocyte progenitor population in vivo. This suggests the potential use of CD26 inhibitors to improve megakaryocyte progenitor function and/or reconstitution of the megakaryocyte cell population.

Objective We previously reported that inhibition or loss of CD26 (DPPIV/dipeptidylpeptidase IV) results in a defect in normal mobilization of hematopoietic stem and progenitor cells induced by granulocyte-colony stimulating factor (G-CSF). This suggests that CD26 is a necessary component of the mobilization pathway. Our goal in this study was to determine whether mobilization can be induced by the CXCR4 antagonist AMD3100 in mice lacking CD26 (CD26 −/− ). Materials and Methods Ten week old CD26 −/− and C57BL/6 mice received a subcutaneous injection of AMD3100. One hour post-injection the mice were euthanized and peripheral blood and bone marrow were collected and evaluated. Results AMD3100 mobilizes hematopoietic progenitors into the peripheral blood of CD26 −/− and mice. Conclusions Our finding that AMD3100 rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery in CD26-deficient transgenic mice that otherwise exhibit a mobilization defect in response to G-CSF suggests that: (1) CD26 is downstream of G-CSF but upstream of the CXCL12-CXCR4 axis and (2) AMD3100 can be used as a single agent to mobilize hematopoietic stem and progenitor cells in normal donors or patients that have an intrinsic defect in their response to G-CSF treatment. Stem cell transplants are often the only curative treatment in some cancer patients. The ability to perform the transplantation and its success is dependent on the ability to mobilize adequate numbers of hematopoietic progenitor cells. The use of AMD3100 as a single agent would give patients or donors an additional option for a successful stem cell transplant.

LXRs are known to play an important role in the control of lipid homeostasis. Specifically, LXRs have been implicated in the regulation of cholesterol and fatty acid metabolism. They have also been suggested to be immunomodulatory and potentially important in cancer biology. As such, LXRs have promise as a target for future therapeutic strategies for solid tumors and hematologic malignancies. It is therefore important to understand the role that LXRs play in a given tumor type to develop new, clinical, therapeutic strategies effectively. LXRα and LXRβ are nuclear receptors that have a primary purpose in regulating lipid and cholesterol metabolism. LXRα (also known as NR1H3) is highly expressed in liver, adipose tissue, kidney, and macrophages [1]. LXRβ (also known as NR1H2) is expressed ubiquitously [1]. LXRs are activated by oxysterols and may also be stimulated using synthetic ligands such as T0901317 or GW3965. In this issue of the Journal of Leukocyte Biology, a paper by Geyeregger et al. [2] examines the influence of LXRα on cell proliferation and survival in the Kit225 T-CLL cell line, in T cell blasts from healthy donors, and in primary T and B cells from CLL patients. This paper focuses primarily on the effect of LXR activation via LXRα agonist treatment on the function of cytokine stimulation using in vitro cell culture-based models.

Hematopoietic stem cells (HSCs) are routinely obtained from marrow, mobilized peripheral blood, and umbilical cord blood. Traditionally, adult marrow has been utilized as a source of mesenchymal stem cells (MSCs). Successful expansion of transplantable HSCs is thought to be possible by coculture of HSCs with cells believed to be representative of the stem cell “niche.” Contact with a stromal component or with MSCs1,2 may fulfill the requirement of the niche by preserving the necessary hematopoietic microenvironment to maintain stem cell function. Bone marrow–derived MSCs (BM-MSCs) have previously been shown to maintain the growth of HSCs obtained from cord blood and have been utilized for cord blood expansion purposes.3 Coculture with BM-MSCs has also been reported to promote engraftment of CD34+ defined cord blood hematopoietic stem and progenitor cells (HSCs/HPCs) into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.4 However, it is possible that the use of BM-MSCs as a feeder layer to support the long-term culture of cord blood HSCs may not be ideal for the clinical transplant setting. For clinical transplantation, it may be preferred that HSCs and MSCs be obtained from the same donor, thereby eliminating the potential for complications resulting from a HSC and MSC genetic mismatch. There may alternatively be an advantage of obtaining HLA-matched or unmatched donor MSC from a nonmarrow or nonadult tissue source. However, it has been previously reported that the numbers of MSCs obtainable from cord blood are small in comparison to marrow.5 As an alternative, the possibility of obtaining MSCs from placenta or from the umbilical cord is attractive. Others have shown that adherent cells from the placenta can be cultured in such a way that they proliferate and also show osteogenic and adipogenic differentiation potential.6 Isolation of fibroblastlike cells from the Wharton’s jelly of the umbilical cord was originally described in 1991.7 At the time these cells were not evaluated in the context of stem cell biology. More recently, putative MSCs were obtained from the umbilical cord itself using two different dissection methods, either from the subendothelial layer of the cord vein8 or from the Wharton’s jelly, the connective tissue of the umbilical cord.9–11 Importantly, MSCs isolated from the umbilical cord were shown to have the ability to differentiate down multiple lineages, including adipose,8 bone,8,10 and neuronal lineages,9,11 thereby suggesting that these cells are likely to have mesenchymal stem and/or progenitor cell potential. Most recently, umbilical cord–derived MSCs (UC-MSCs) were shown to secrete several important cytokines and growth factors, including granulocyte–colony-stimulating factor, granulocyte-macrophage–colony-stimulating factor (GM-CSF), interleukin (IL)-6, and IL-8 and that UC-MSCs–produced cytokines were capable of enhancing CFU-GM colony formation during a standard methylcellulose-based myeloid colony assay supplemented with 30 percent fetal bovine serum (FBS), stem cell factor, GM-CSF, IL-3, and erythropoietin (EPO).12 Cotransplantation of UC-MSCs along side CD34+ cord blood cells were also shown to increase the numbers of human CD45+ cells detectable in the marrow of a lethally irradiated NOD/SCID recipient 6 to 8 weeks after transplant.12 Having accepted the previous reports suggesting that cells isolated from the Wharton’s jelly showed characteristics of MSCs we sought to evaluate another important functional characteristic of MSCs, the ability to support the maintenance of blood. We therefore hypothesized that UC-MSCs would have the ability to act as stromal cells and support ex vivo the long-term growth and maintenance of cord blood–derived HSCs, similar to that previously reported for BM-MSCs. To test this hypothesis, MSCs were isolated from the Wharton’s jelly of umbilical cord segments and defined morphologically utilizing established cell surface markers.13 UC-MSCs were then tested for their ability to support the growth of CD34+ cord blood cells in bulk long-term culture-initiating cell (LTC-IC) assays.

Given the tremendous need for and potential of umbilical cord blood (CB) to be utilized as a donor source for hematopoietic stem cell (HSC) transplantation in adults, there is a strong push to overcome the constraints created by the limited volumes and subsequent limited HSC and hematopoietic progenitor cell (HPC) numbers available for HSC transplantation from a single collection. We have previously described the use of CD26 inhibitor treatment of donor cells as a method to increase the transplant efficiency of mouse HSCs and HPCs into a mouse recipient. To study the use of CD26 inhibitors as a method of improving the transplantation of human CB HSCs and HPCs, we utilized the nonobese diabetic/severe combined immunodeficient/beta 2 microglobulin null (NOD/SCID/B2m(null)) immunodeficient mouse model of HSC transplantation. We report here significant improvements in the engraftment of long-term repopulating cells following the treatment of either CD34(+) or lineage negative (lin()) donor CB with the CD26 inhibitor, Diprotin A, prior to transplant. These results establish a basis on which to propose the use of CD26 inhibitor treatment of donor CB units prior to transplantation for the purpose of improving transplant efficiency and subsequently patient outcome.

Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis.

Objective The primary function of chemokines is the regulation of leukocyte trafficking by stimulating directional chemotaxis. The chemokine CXCL14 (BRAK) is highly expressed in all normal tissues, but is not expressed in most malignant tissues. The chemotactic activity of CXCL14 has been difficult to characterize. Recently it was reported that CXCL14 is a chemoattractant for activated monocytes and immature dendritic cells. Given that CXCL14 is downregulated upon transition to malignancy, we sought to characterize whether CXCL14 might play a role in NK cell chemotaxis. Methods Human natural killer (NK) cells were isolated from buffy coats obtained from normal volunteers and were activated with lymphocyte conditioned media, IL-2, and ionomycin. Standard transwell chemotaxis assays, proliferation assays, and chromium release cell cytotoxicity assays were performed. Results CXCL14 was found to stimulate migration of activated human NK cells in transwell chemotaxis assays by 1.4-fold. Similarly, it increased migration of an IL-2-dependent natural killer leukemia (NKL) cell line by 1.9-fold. Antisera against CXCL14 or pertussis toxin blocked this chemotactic effect. However, CXCL14 did not affect the proliferation or cytotoxic activity of normal human NK cells. CXCL14 also stimulated the chemotaxis of immature monocyte-derived dendritic cells. Conclusions CXCL14 may play a role in the trafficking of NK cells to sites of inflammation or malignancy. In addition, the downregulation of the expression of CXCL14 might be an important step in successful oncogenesis to prevent NK immune surveillance of the malignancy.

Objective Cytokine treatment with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) is a mainstay of current and future clinical and research protocols for peripheral blood stem cell mobilization, therapeutic care after hematopoietic stem cell transplantation (HSCT), and ex vivo hematopoietic stem and progenitor cell (HSC/HPC) expansion. We have previously shown that the peptidase CD26 (DPPIV/dipeptidylpeptidase IV) negatively regulates HSC/HPC and that inhibition of CD26 improves the chemotactic ability and trafficking of HSC/HPC. We set out to establish whether short-term in vitro G-CSF, GM-CSF, or SCF treatment upregulates CD26 and thereby has a detrimental effect on the chemotactic potential of HSC/HPC that could be reversed by CD26 inhibitor treatment. Materials and Methods CD34 + or CD34 + CD38 − cells, a population enriched in HSC, were isolated from human umbilical cord blood and subjected to G-CSF, GM-CSF, or SCF treatment. We then evaluated CD26 expression, CD26 activity, and CXCL12 (SDF-1)-induced migration in the presence or absence of a CD26 inhibitor, Diprotin A. Results Treatment with G-CSF and GM-CSF but not SCF upregulates CD26 expression and activity resulting in a CD26 inhibitor-reversible downregulation of CXCL12-induced chemotactic response. Conclusions Short-term in vitro G-CSF and GM-CSF treatment upregulates the peptidase CD26, resulting in downregulation of the functional ability of CD34 + CD38 − cells to respond to the chemokine CXCL12. This suggests that current and future clinical protocols utilizing G-CSF and GM-CSF may have unforeseen detrimental effects on the trafficking of HSC/HPC during HSCT that can be overcome through the use of CD26 inhibitors.

Hematopoietic stem cell homing and engraftment are crucial to transplantation efficiency, and clinical engraftment is severely compromised when donor-cell numbers are limiting. The peptidase CD26 (DPPIV/dipeptidylpeptidase IV) removes dipeptides from the amino terminus of proteins. We present evidence that endogenous CD26 expression on donor cells negatively regulates homing and engraftment. By inhibition or deletion of CD26, it was possible to increase greatly the efficiency of transplantation. These results suggest that hematopoietic stem cell engraftment is not absolute, as previously suggested, and indicate that improvement of bone marrow transplant efficiency may be possible in the clinic.

In spite of the wide usage of mobilized peripheral blood hematopoietic stem and progenitor cells (HSC/HPC) for transplantation, the mechanism of granulocyte colony-stimulating factor (G-CSF)-induced HSC/HPC mobilization has yet to be fully determined. Our previous studies suggested that that G-CSF-induced mobilization may involve the extracellular peptidase CD26 (DPPIV/dipeptidylpeptidase IV). We set out to establish whether CD26 was an essential component of normal G-CSF-induced mobilization of HSC/HPC.Normal wild-type (WT) control C57BL/6 mice and CD26 knockout (CD26(-/-)) mice, also on a C57BL/6 background, were treated with or without G-CSF and peripheral blood cells, bone marrow cells, and spleen cells were assessed for CFU-GM, BFU-E, and CFU-GEMM content.No statistical difference in the number of CFU-GM, BFU-E, or CFU-GEMM in the peripheral blood, bone marrow, or spleen of untreated WT vs untreated CD26(-/-) mice was observed. G-CSF treatment of WT mice resulted in the mobilization of HPC into the peripheral blood. The number of HPC detected in G-CSF-treated CD26(-/-) mouse peripheral blood was significantly less than the corresponding number of HPC detected in G-CSF-treated WT mouse peripheral blood after either a 2- or 4-day G-CSF regimen.CD26 plays a critical role in G-CSF-induced mobilization of HPC.

Metastasis of cancer cells is a complex process involving multiple steps including invasion, angiogenesis, and trafficking of cancer cells through blood vessels, extravasations, organ-specific homing, and growth. While matrix metalloproteinases, urokinase-type plasminogen activator, and cytokines play a major role in invasion and angiogenesis, chemokines such as stromal derived factor-1alpha (SDF-1alpha) and their receptors such as CXCR4 are thought to play a critical role in motility, homing, and proliferation of cancer cells at specific metastatic sites. We and others have previously reported that the extracellular signal-activated transcription factor NF-kappaB up-regulates the expression of matrix metalloproteinases, urokinase-type plasminogen activator, and cytokines in highly metastatic breast cancer cell lines. In this report, we demonstrate that NF-kappaB regulates the motility of breast cancer cells by directly up-regulating the expression of CXCR4. Overexpression of the inhibitor of kappaB (IkappaB) in breast cancer cells with constitutive NF-kappaB activity resulted in reduced expression of CXCR4 and a corresponding loss of SDF-1alpha-mediated migration in vitro. Introduction of CXCR4 cDNA into IkappaB-expressing cells restored SDF-1alpha-mediated migration. Electrophoretic mobility shift assays and transient transfection assays revealed that the NF-kappaB subunits p65 and p50 bind directly to sequences within the -66 to +7 region of the CXCR4 promoter and activate transcription. We also show that the cell surface expression of CXCR4 and the SDF-1alpha-mediated migration are enhanced in breast cancer cells isolated from mammary fat pad xenografts compared with parental cells grown in culture. A further increase in CXCR4 cell surface expression and SDF-1alpha-mediated migration was observed with cancer cells that metastasized to the lungs. Taken together, these results implicate NF-kappaB in the migration and the organ-specific homing of metastatic breast cancer cells.

The signals that mediate T-cell infiltration during T-cell autoimmune diseases are poorly understood. The chemokine CCL21 (originally isolated by us and others as Exodus-2/6Ckine/SLC/TCA4) is highly potent and highly specific for stimulating T-cell migration. However, it is thought to be expressed only in secondary lymphoid organs, directing naive T cells to areas of antigen presentation. It is not thought to play a role in T-cell effector function during a normal immune response. In this study we tested the expression of T-cell chemokines and their receptors during T-cell autoimmune infiltrative skin diseases. By using immunohistology it was found that the expression of CCL21 but not CCL19 or 20 was highly induced in endothelial cells of T-cell autoimmune diseases. The receptor for CCL21, CCR7, was also found to be highly expressed on the infiltrating T cells, most of which expressed the memory CD45Ro phenotype. These data imply that the usual loss of CCL21 responsiveness in the normal development of memory T-cell effector function does not hold for autoimmune skin diseases.

The chemokine CCL21, also known as Exodus-2/6-Ckine/secondary lymphoid-tissue chemokine/T cell activator protein-4, is the most potent stimulator of T cell migration and adhesion yet described. Endothelial heparin-like glycosaminoglycans (GAGs) are thought to present chemokines at sites of inflammation, maintaining a local concentration gradient to which leukocytes can respond. In contrast, this study found that GAGs markedly inhibit the ability of CCL21 to stimulate T cell adhesion and chemotaxis. Enzymes, such as heparinase, that split GAGs into component-sulfated saccharides abrogate this inhibition, suggesting a mechanism for local tissue regulation of CCL21 function. Low-molecular-weight heparins also strongly inhibit CCL21 adhesion and chemotaxis. Therefore, low-molecular-weight heparins may be effective therapeutic agents in decreasing the pathology of T cell-infiltrative autoimmune diseases by targeting the CCL21 regulation of T cell infiltration.

Background: The impact of mesenchymal stem cells (MSCs) on breast cancer (BC) progression and growth remains controversial. Some studies using xenograft BC models in nude mice suggest that MSCs stimulate BC metastases, whereas other studies in syngeneic rat BC models suggest that MSCs suppress BC development and growth. In addition, whether MSCs have a role in BC initiation has not been tested. The objective of this study was to determine if bone marrow-derived MSCs affect BC initiation and progression in vitro and in clinically relevant somatic and synegeneic BC mouse models. Materials and Methods: MSCs were isolated from bone marrows of FVB wild-type mice, cultured, and characterized for their potential to differentiate into adipocytes and express flow-cytometric cell surface markers. MSC-conditioned medium (CM) was used to culture RCAS-Neu and RCAS-PyMT BC cell lines, derived from TVA-transgenic mice infected with an avian retroviral vector encoding Neu or polyoma middle T antigen (PyMT). Cell proliferation was analyzed with CellTiter 96 proliferation assay and 5-bromo-2-deoxyuridine labeling. The effect of RCAS-Neu and RCAS-PyMT cells and their CM on MSC migration was determined with Boyden chamber migration assay. In a somatic BC model, TVA-transgenic mice expressing the receptor for an avian retrovirus vector, RCAS, were infected with RCAS-PyMT vector by intraductal injection and treated with MSC (2x106cells/mouse) by i.v. injection. Mice were observed for BC development by palpation. In a syngeneic BC model, RCAS-Neu BC cells were co-implanted with MSCs (5x105/gland) at the ratios 1:0, 1:0.2, or 1:1 into the fat pad of FVB female mice. BC growth was monitored for 9 weeks. Results: CM from MSC did not significantly affect the proliferation of RCAS-Neu and RCAS-PyMT cells. However, RCAS-Neu and RCAS-PyMT cells and their CM induced morphologic changes in MSC and dramatically increased their motility. In somatic model, MSCs had no effect on BC initiation or growth with the mean tumor latency 27.5±7.5 days in MSC-treated mice and 29±5.5 days in control mice treated with PBS. In syngeneic BC model, there was no significant difference in the growth of RCAS-Neu alone or RCAS-Neu cells co-implanted with MSCs. Since BC in these models does not metastasize to distant organs, it could not be determined whether MSCs affect BC metastases. Discussion: Our results demonstrated that BC cell lines and their CM are able to induce MSCs migration and possibly differentiation. However, MSCs had no effect on BC initiation in a somatic BC model and on tumor growth in a syngeneic BC model. It is possible that the number of MSCs and schedule of MSC injection were not optimized or strong PyMT oncogene rapidly induced BC and overcame the effect of MSCs on BC formation. Experiments are ongoing to determine if multiple administrations of MSCs affect BC development induced by PyMT and Neu oncogenes. Lack of effect of MSCs in BC initiation and progression, if confirmed, will suggest that MSCs are safe for delivering novel antitumor agents for BC treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-04.

We and other workers have recently isolated three novel CC chemokines termed Exodus-1/LARC/Mip-3alpha, Exodus-2/6Ckine/SLC/TCA4, and Exodus-3/Mip-3beta/CKbeta11/ELC. These chemokines share an amino terminal Asp-Cys-Cys-Leu sequence, unique among all chemokines. They also selectively regulate migration of adult T cells. Indeed, there is evidence that Exodus-2 and -3 are critical for adult T-cell adhesion to high endothelial venules in lymph nodes, a rate-limiting step for T-cell trafficking through nodal tissue. Less is known of the factors controlling migration of naive human fetal T cells. We tested whether these chemokines could regulate chemotaxis in cord blood T-cell populations, and compared that efficacy with normal peripheral blood adult T cells. The findings indicated that naive CD45RA+ cord blood T-cell migration is stimulated by Exodus-2 and -3, and CD4+ cord blood T cells are attracted preferentially by Exodus-2 or -3 as compared with CD8+. Exodus-2 and -3 are likely to be critical in regulating the flux of naive CD4 + fetal T-cell population of secondary lymphoid tissue.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report here, the isolation of a novel murine CC chemokine termed ALP for its amino terminal peptide sequence. This novel chemokine is distantly related to other CC chemokines (37% identity with murine Exodus-1/LARC/Mip-3alpha), but has a unique carboxy terminal extension. It is expressed preferentially in testis, heart, and liver, which is atypical for CC chemokines.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report the PCR cloning of a novel human chemokine termed BRAK for its initial isolation from breast and kidney cells. This novel chemokine is distantly related to other CXC chemokines (30% identity with MIP-2alpha and beta) and shares several biological activities. BRAK is expressed ubiquitously and highly in normal tissue. However, it was expressed in only 2 of 18 cancer cell lines. BRAK is located on human chromosome 5q31.

Introduction Low risk MDS is a heterogeneous disease, with limited and short lived responses to available therapeutic options. We hypothesized that the combination of lenalidomide and azacitidine may result in higher response rates and longer duration of such responses than those achieved with either agent. Methods This was an investigator- initiated phase 2 trial, seeking to evaluate the safety , tolerability and response rate of the combination of lenalidomide and azacitidine in patients with low- int-1 risk MDS (excluding those with del 5q). Patients had to have up to three months of lenalidomide monotherapy, followed by response assessment per IWG 2006 criteria. Responders were to continue on lenalidomide until progression or failure. Non responders would receive 2 cycles of combination therapy with azacitidine at 25 mg/m2 for 5 days every 28 days, together with lenalidomide at the same dose they received in cycle 3. If no grade 4 toxicity developed, or if the initiation of the next cycle of therapy was not delayed by more than 2 weeks, patients would continue combination therapy, with dose escalation of azacitidine to 50 mg/m² on days 1 through 5 every 28 days for 4 more cycles along with same dose of lenalidomide received in prior cycles. Response assessment was to be performed after 6 cycles of combination therapy, with responders continuing to receive treatment until disease progression, intolerable toxicities or return to baseline transfusion dependence. Results Between January of 2011 and November of 2012, we enrolled 2 patients, a 69 year old male (1), and a 65 year old female (2), both patients had RARS, normal karyotype, IPSS was 0, and 1. Patient (1) was previously treated with ESA/G-CSF, the other was treatment naïve. Both patients started Lenalidomide at 5 mg daily, due to reduced GFR (1). Treatment of patient (1) was complicated by severe anemia after 2 cycles of therapy, necessitating blood transfusion, dose reduction, and eventually holding therapy. The patient declined participation and was taken off study in September of 2011. Patient (2) was enrolled in August of 2012, received 3 cycles of lenalidomide at 5 mg daily, resulting in stable disease. She went on to receive two cycles of combination therapy. Treatment was complicated by a rash, attributed to revlimid, and hematological toxicity resulting in treatment delay by more than 2 weeks. Per protocol, she was taken off study. Conclusion Our study was closed prematurely, and our sample size is very small, but this experience suggests that Combination therapy in patients with low risk MDS is challenging on multiple levels: Accrual, tolerability of two myelosuppressive agents, and potentially, an unacceptable risk/benefit ratio. Disclosures: Shammo: celgene: Consultancy, Honoraria, Research Funding. Off Label Use: The use of lenalidomide and azacitidine in combination for the treatment of low risk MDS.

Abstract 4162 Background: Graft versus host disease (GVHD) remains the leading cause of the treatment failures following an otherwise successful allogeneic Stem Cell Transplant. Alemtuzumab – based prophylactic regimens is evolving as an important option for prevention of this devastating complication. Multiple phase II studies suggested that patients who received Alemtuzumab –containing GHVD prophylactic regimens tends to have less acute GVHD though the utility is limited by increased incidence of mixed chimerism often requiring donor lymphocytes infusion and cytomegalovirus reactivation. Hypothesis: We hypothesize that if we could identify an optimal dose of Alemtuzumab; we could maintain the benefit of lowering the incidence of GVHD and also minimize mixed chimerism and CMV reactivation following the transplant. Method: To test our hypothesis, we conducted a pilot study utilizing the combination of Alemtuzumab (20 Related Donor: 30 mg on Day -1; 10 Unrelated donor: 30 mg daily on Day -2 & Day -1) and cyclosporine as GVHD prophylaxis. Between 5/11 and 5/12, 28 pts were included in the study. There were 15 females and 13 males. The diagnosis included 16 AML, 3 NHL, 2 MDS, 1 CML, 1 CLL, 1 ALL, 1 HD, 1 PLL, 1 MPD and 1 MM. Conditioning regimens consisted of Fludarabine/Melphalan in 20, Fludarabine/Busulfan in 5, TBI/Cy in 2 and Fludarabine/Clofarabine in 1. Upon achievement of engraftment, we monitor CMV by PCR weekly and engraftment by Short tandem repeat (STR) monthly with T-cell and Myeloid subsets analysis. Results: 26/28 pts achieved neutrophils engraftment except 1 died shortly after SCT from heart failure and 1 experienced primary graft failure with day 30 STR showing only 3% donor T-cell chimerism. On Day 30 post-SCT, all evaluable pts had predominately donor T-cell (median 99%; 19/27 (70%) ≥ 99%; range 82 to > 99%) and Myeloid Chimerism (24/26 (92%) ≥ 99% except one pt who developed rapidly progressive leukemia phase PTLC after transplant. 25 pts (89%) survived beyond Day 90 post-SCT and have engraftment data available for analysis. On Day 90 analysis, while most pts maintain predominant donor myeloid chimerism (> 95%; median 99%); only 10/25 (40%) had maintained 99% donor T-cell chimerism. In addition, 76% pts had their T-cell donor chimerism worsened by Day 90 when compared with Day 30 analysis (9 pts dropped below 80%; range 8–75%). On the other hand, no pt developed secondary graft failure. 5 pts (17%) developed acute GVHD with only 1 (4%) Grade IV aGVHD. 8 pts (29%) developed CMV reactivation within day 100 post-SCT. Full donor T-cell chimerism did not predict for GVHD or CMV reactivation. With longer follow up, some pts' have donor T-Cell chimerism increased with tapering of immunosuppressive therapy with or without GVHD or after receiving DLI. Conclusion: 1. Our preliminary data suggested that using a lower dose of Alemtuzumab (30 mg for RD and 60 mg for MUD) as GVHD prophylaxis is effective to prevent GVHD though still associated with high incidence of CMV reactivation. 2. Most pts achieve predominant Donor T cell chimerism on day 30 though many worsen on subsequent follow-up. 3. Although it appears that there's no impact on durability of engraftment, longer follow-up is required to determine the impact on relapse, chronic GVHD and survival. Disclosures: Fung: Genzyme: Honoraria. Off Label Use: Alemtuzumab for GVHD prophylaxis.

6552 Background: Plerixafor for mobilization of autologous stem cells (ASC) has increased the yield of transplanted ASC and is evolving as an important option for mobilization. A prior study reported a 10% incidence of SN after AHSCT (BMT 2009 Aug; 44(3): 175-83). Incidence and outcome of SN in the setting of Plerixafor/G-CSF (P/G), is unknown. We report the incidence and possible etiology for the development of SN in pts undergoing AHSCT for lymphoma at our institution. Methods: Data from 80 consecutive AHSCT pts over a 2 year period were reviewed. All pts were mobilized using P/G. Demographics, AHSCT indication, prior therapies (Rx), conditioning regimen (CR), engraftment and post-transplant complications were identified and collected. SN was defined as ANC 2 Rx, 18 (25.7%) with loco-regional XRT and 3 (4.3%) with RIT. CR included BEAM, BEC and Benda-EAM +/- Rituxan. # of CD34+ cells given ranged; 1.77-19.99 x 10^6/kg. Neutrophil engraftment occurred at a median of 11 days. 26 (37.14%) pts developed SN possibly from PCP prophylactic antibiotics and infections. Prior BM involvement, Rx or XRT had no role. 5 (45.4%) pts with Benda-EAM CR had SN. Associated morbidity/mortality were not noted. Conclusions: We conclude that secondary neutropenia is common after autologous stem cell transplant using the Plerixafor/GCSF combination for mobilization and is higher than reported in the literature (37% vs 10%). Patients who received this combination for mobilization should be followed up closely in the post-transplant period for secondary neutropenia. About half the patients who received the Benda-EAM conditioning regimen developed secondary neutropenia warranting its use with caution outside of a clinical trial.

Introduction: Achieving improvements in survival and reducing relapse remains a challenge in acute myelogenous leukemia (AML) patients. This study evaluated the in vitro efficacy of the active form of novel agent sapacitabine, 2′-C-Cyano-2′-deoxy-1-α-d-arabino-pentofuranosylcytosine (CNDAC, Cyclacel Ltd, Dundee, UK), as compared to current chemotherapeutic drugs Ara-C (Cytarabine) and Mitoxantrone (Mit, synthetic anthracenedione) using two AML cell lines, HL-60 (promyelocytic) and THP-1 (monocytic), as well as bone marrow (BM) and peripheral blood (PB) cells collected from 5 AML patients. Methods: Cells lines and AML patient cells were treated in vitro with Ara-C (1-100 µM), CNDAC (1-100 µM) or Mit (0.005-0.5 µM) for 4 days. During treatment, HL-60, THP-1, and PB AML cells were cultured in suspension. BM AML cells were co-cultured with M2-10B4 mouse stromal cells. Cell lines were assessed immediately. BM and PB AML cells were assessed after an additional 3, 7, or 31 days of co-culture on M2-10B4 cells. Treated cells were assessed for sensitivity (cell death by trypan blue and apoptosis by 7AAD/Annexin V) as compared to untreated cells and IC50 values (50% of maximum possible effective response) were calculated. Results: In HL-60 cells, the amount of cell death was greater with CNDAC compared to Ara-C at all doses tested (p≤0.05, n=3). In THP-1 cells, CNDAC and Mit, but not Ara-C, induced a significant apoptotic response. At a 10-fold lower seeding density, which correlates to higher proliferation rates, the response of THP-1 cells to Ara-C, CNDAC and Mit reached significance compared to untreated cells (p≤0.05, n=3). However, the IC50 values for the 3 drugs in THP-1 cells reflect the observation that this cell line is in essence resistant to AraC (IC50 = 7.77 µM) but sensitive to CNDAC (IC50 = 0.929 µM) and Mit (IC50 = 0.003 µM). Using PB AML cells, a significant response to 1 µM CNDAC and 0.005 µM Mit but not 1 µM Ara-C was observed, as compared to untreated cells at all days post drug removal (p≤0.05, n=5). A significantly greater apoptotic response to CNDAC was observed compared to Ara-C at low doses (p≤0.05, n=5). At higher doses, all 3 drugs induced significant cell death (p≤0.05, n=5). In BM AML cells, overall survival with 1 µM CNDAC or 0.005 µM Mit, but not 1 µM Ara-C, was significantly less than untreated cells 31 days post-treatment (p≤0.05, n=5). Higher doses induced cell death (p≤0.05, n=5) with all 3 drugs. Conclusion: Low dose CNDAC and Mitoxantrone induce a greater response than low dose Ara-C in patient AML cells and AML cell lines. CNDAC also exhibits a greater activity in cell lines (THP-1) that are less sensitive to Ara-C. The in vitro sensitivity of AML cells to CNDAC supports the ongoing clinical evaluation of sapacitabine in AML patients. Future studies are warranted to assess the potential for combining sapacitabine with Ara-C and/or Mitoxantrone, with an emphasis on cells and patients insensitive to Ara-C treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4668. doi:1538-7445.AM2012-4668

Abstract 2646 Introduction: Chronic lymphocytic leukemia (CLL)/Small lymphocytic leukemia (SLL) is one of the most common forms of indolent lymphomas in elderly adults. Currently, CLL is not treated until it develops into later stage disease. An increase in the knowledge of the biology of CLL could aid in the development of new treatment strategies for early stage CLL, thus positively impacting disease progression. We therefore investigated the cytokine levels present in peripheral blood (PB) serum of CLL patients and compared them to levels in healthy donors. Methods: PB was obtained from 25 untreated CLL and 3 untreated SLL patients and 29 normal healthy donors under an IRB approved protocol. Serum was stored at −80°C until analyzed. 86% of the patients were Rai 0/1, 3.5% Rai stage 2, and 10.5% unknown at diagnosis. Risk stratification based on cytogenetics and FISH found of the enrolled patients, 47% good, 25% intermediate, and 14% poor risk. The risk factor was unknown in 14%. β-2 microglobulin and 54 cytokine protein levels in 2 different serum samples per patient (collected at least one year post diagnosis) were measured in duplicate using MILLIPLEX™, a multiplex Luminex based technology. Genespring 11.5.1 software was used to convert the data into base-2 logarithmic values and then apply a median baseline transformation across all samples. Data is grouped according to the source of the sample (CLL or normal PB serum). A Filter on Volcano Plot analysis is then done. This filter analysis performs an unpaired t-test, which yields a p-value as well as computes the fold change of each cytokine. Results: As expected, β-2 microglobulin was significantly higher in CLL patients compared to normal samples (p=5.17×10−7, 1.79 fold). Using a minimum of a 1.5 fold change and p-value≤0.05, 16 cytokines had significantly higher expression and 9 cytokines had significantly lower expression in CLL samples compared to normal samples (see Table 1). Conclusion: These changes in cytokine levels help provide insight to the peripheral blood microenvironment, in which circulating CLL cells reside. Some cytokines were substantially higher in CLL patients; soluble IL-2 receptor alpha (sIL-2Rα) and stem cell factor (SCF). The substantially higher levels of sIL-2Rα have also been observed in follicular lymphoma (FL) and appear to predict which FL patients will have a reduced survival (Yang et al., Blood 2011). Levels of sIL-2Rα correlating to survival may be a phenomenon seen in all B-cell lymphomas. SCF is thought to be important in early B-cell development but its receptor c-kit (CD117) has not been reported to be expressed on B-CLL cells. The chemokine receptors CXCR4, CXCR5 and CCR7 are expressed on B-CLL cells. Interestingly, their corresponding ligands, CXCL12, CXCL13, and CCL21 are significantly higher (Table 1) in the PB serum of CLL patients. It is likely that existing immunomodulatory therapies, such as IMiDs, lenalidomide or thalidomide alter the PB levels of the cytokine milieau. Other successful hematological malignancy therapies involve targeted monoclonal antibodies. Therefore, a deeper understanding of the cytokine microenvironment will provide guidance in the development of future targeted therapies or highlight current therapies for other malignancies that could be promising for CLL. Disclosures: No relevant conflicts of interest to declare.

Abstract 4061 Background: High-dose chemotherapy and/or radiotherapy are effective treatment strategies for patients with life-threatening hematologic malignancies. A critical step to the success of transplantation is achieving adequate mobilization of CD34+ stem cells from the bone marrow into the peripheral blood to provide sufficient cell yield after apheresis for the transplant. Plerixafor combined with granulocyte colony-stimulating factor (G-CSF) has proven efficacious in mobilizing CD34+ stem cells in patients with lymphoma and myeloma prior to autologous stem cell transplantation. In the phase 3 clinical trials of plerixafor plus G-CSF for SCM, plerixafor was administered at 10:00 pm on days prior to apheresis. This dosing schedule is based on the peak level of CD34+ cells in the peripheral blood (PB) at 11–14 hours after administration; however PB CD34+ cell levels were elevated from 4–18 hours after plerixafor administration. Due to inconvenience in dosing plerixafor at 10:00pm, we took advantage of its pharmacodynamic profile and explored an alternative dosing schedule, giving plerixafor at 5:00 pm. Here, we report our updated experience with the efficacy of this schedule. Method: We performed a retrospective study using our Stem Cell Harvest database. A total of 58 patients (31 lymphoma, 27 myeloma) underwent mobilization with G-CSF + plerixafor, either as front-line (n=51) or as salvage (n=7) mobilization strategies between February 2009 through May 2010. Mobilization consisted of G-CSF 10 μg/kg SC administered daily at 6:00 am day 1 through 4 plus plerixafor 0.24 mg/kg SC given once daily at 5:00 pm in an outpatient clinic beginning on day 4. For patients with renal impairment (ie, creatinine clearance ≤50 mL/min), the dose of plerixafor was lowered by a third to 0.16 mg/kg. Daily apheresis began at 8:30 am on the morning of day 5 and continued for up to 4 days, with a minimum collection goal defined as ≥ 2 × 106 CD34+ cells/kg for lymphoma patients and ≥ 4 × 106 CD34+ cells/kg for myeloma patients. Mobilization with G-CSF plus plerixafor and apheresis were halted once the minimum goal was reached between day 2 and 4 of apheresis, or after a single collection achieved the optimal goal which was defined as ≥ 4 × 106 (lymphoma) or ≥ 8 × 106 (myeloma) CD34+ cells/kg. The mobilization strategy was considered a failure if patients did not reach the minimum CD34+ cell collection goal within the 4 days of apheresis. Results: G-CSF + plerixafor mobilization yielded a median 5.13 × 106 CD34+ cells/kg (range, 0.06–25.8) in a median of 2 apheresis days. Forty-five of 58 (78%) patients achieved the minimum CD34+ cells/kg required for transplantation, including 30 (52%) patients who achieved this goal on the first day of apheresis. Overall, 52 (90%) patients proceeded to transplantation, with median neutrophil and platelet engraftment times of 11 and 18 days, respectively. Conclusion: In summary, the alternative 5:00 pm dosing of plerixafor for stem cell mobilization provided a more convenient dosing schedule while ensuring that a majority of lymphoma and myeloma patients achieved the minimum CD34+ cell yield required to proceed to transplantation. Disclosures: Tornatta: Genzyme: Consultancy, Honoraria, Speakers Bureau. Fung:Genzyme: Consultancy, Honoraria, Speakers Bureau.

Abstract 3685 Introduction: Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia (CLL/SLL) is a lymphoproliferative disorder that is characterized by the slow accumulation of malignant B cells. Patients follow heterogeneous clinical courses. The effectiveness of Rituximab, an anti-CD20 monoclonal antibody (mAb), is limited because target B cells from CLL patients express low levels of CD20. We previously reported that human serum suppresses CD20 expression ex vivo. Therefore, understanding mechanisms of low expression of CD20 as well as other target surface receptors would benefit CLL patients. New therapies such as epratuzumab (anti-CD22 mAb), lumiliximab (anti-CD23 mAb), and TRU-016 (anti-CD37 SMIP protein) are being investigated as alternative means to treat CLL. We therefore investigated CD20, CD22, CD23, and CD37 expression during ex vivo culture of CLL B cells from thirteen patients with and without human serum albumin (HSA), normal donor serum (NS), autologous CLL serum (CS), fetal bovine serum (FBS), IL2, IL4, IL13, IL15, IL21, TNFα, IFNα, IFNγ, G-CSF, or GM-CSF. Methods: Peripheral blood (PB) from thirteen CLL patients and five healthy normal donors was obtained with IRB approval. Nine CLL patients were Rai 0/1, one Rai 4, and three unknown. Prognosis, as determined by cytogenetics and/or FISH, was: four good, three intermediate, one poor, and five unknown. Beta-2 Microglobulin (B2M) ranged from 1.4–7.1 mg/L. CD19+ cells, isolated by positive magnetic selection, were cultured in AIM-V serum-free media at 37°C, 5% CO2, 100% humidity for 0, 24, 48, and 96 hours. Cells were either untreated, treated with 5% serum, or 5ng/mL cytokine. Receptor expression was measured by multivariate flow cytometry and calculated as percent loss or increase in response to treatment. Data was presented as mean ± SEM and analyzed using the Mann-Whitney U test as compared to HSA treatment. Results: Prior to culture, CD19+ CLL cells had 11.7±1.7% CD20, 40.3±7.9% CD22, 60.6±6.8% CD23, and 81.6±2.7% CD37 positive expression (N=13). Analysis of untreated CLL cells cultured for 96 hours showed that the CLL patients subdivided into two groups based on changes in CD20 expression: variable (72.5±7.4% CD20+, N=9) and stable (27.2±4.2% CD20+, N=4). Within the variable group, losses in CD20 expression resulted from treatment as compared to HSA were: NS (40.2±3.6%, p Disclosures: No relevant conflicts of interest to declare.

6588 Background: A major challenge in developing treatment regimens for AML is identifying strategies to prevent disease relapse. Advances in understanding the biology of AML leukemogenesis have re...