ALBERT

Description: Patients with myeloproliferative disorders (MPD) are at a high risk of developing thrombotic events. We hypothesize that one of the contributory factors to this thrombotic tendency is the involvement of vascular endothelial cells (EC) by the malignant process. In vitro and in vivo assays were used to determine the involvement of EC in patients with MPD. Endothelial progenitor cells (EPC) were assayed from the peripheral blood (PB) mononuclear cells (MNCs) of 3 normal controls (NC) and 16 patients with MPD (12 polycythemia vera (PV), 4 primary myelofibrosis (PMF). MNC were cultured for 2 days in EC growth media on fibronectin(FN)-coated plates. The non-adherent cells were then harvested and transferred to a secondary FN-coated plate for additional 5–14 days. EC colonies were identified by their morphological appearance. The colonies were plucked and analyzed for PECAM-1(CD31), VE-Cadherin(CD144), VEGFR-2, vWF, Endoglin(CD105), ULEX-1, CD45, CD14 by flow cytometry and acetylated LDL(Ac-ADL) uptake. EC colonies were CD31+CD144+VEGFR2+ULEX-1+vWF+CD105+CD45+CD14+ and capable of taking up Ac-LDL and when exposed to TNF-α and IL-1β, expressing ICAM(CD54) and E-selectin(CD62e). MPD MNC formed fewer numbers of EC colonies than normal MNC (31.1±34.2 vs 78.8±28.9; p

Description: Primary myelofibrosis (PMF) is a myeloproliferative disorder characterized by abnormal trafficking of CD34+ cells, resulting in their constitutive mobilization. This abnormal trafficking of PMF CD34+ cells has been related to the reduced expression of the chemokine receptor CXCR4 (Shi et al, Cancer Research67: 6417, 2007). We further tested this hypothesis by studying the homing of PMF CD34+ cells to the marrow and the spleens of sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and determining if this defect could be corrected by treatment with chromatin modifying agents. Following the infusion of PMF (n=10) or G-CSF mobilized peripheral blood (mPB) (n=4) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and assayable human CFU-GM were detected in the marrow of these mice (139±47 PMF CD34+ cells/106 BMCs vs. 1493±946 mPB CD34+ cells/106 BMCs, 2.0±0.8% of input PMF CFU-GM vs. 12.6±6.6% of input mPB CFU-GM, P

Description: Polycythemia vera (PV) is a Philadelphia chromosome negative myeloproliferative disorder characterized by the JAK2V617F mutation as well as constitutive mobilization of hematopoietic progenitor cells. In order to identify the cell of origin of the neoplastic event which leads to PV, CD34+ or CD34− cells from the peripheral blood (PB) of 10 PV patients and 6 G-CSF mobilized normal donors were transplanted into NOD/SCID mice. PV PB CD34+ cells produced after 8 weeks donor cells belonging to multiple hematopoietic lineages, but to a lesser degree than that obtained with normal mobilized CD34+ cells (0.6% versus 12.7%). The human CD45+ cells in the BM of the recipient mice transplanted with normal and PV CD34+ cells contained donor derived CD33+ and CD19+ cells, but not CD3+ cells. By contrast, the transplantation of CD34− cells into NOD/SCID mice resulted in a far more robust degree of human cell chimerism in both the bone marrow (2%–68%) and spleen (3%–97%) of the recipient mice. Engraftment with CD34− cells was first observed in recipient NOD/SCID mice 4 weeks post-transplantation and increased progressively. Normal CD34− cells were incapable of engrafting in the NOD/SCID mice. Further analysis demonstrated that the human cells within the mouse marrow and spleen were CD3+ (100%), CXCR3 (100%), but did not express either CXCR4 or CD56. The engrafted T cells contained CD4, CD8, TCRαβ and TCRγδ single positive cells, CD4 and CD8 as well as TCRαβ and TCRγδ double positive cells. When subpopulations of CD3+ and CD3− cells within the PV CD34− cell population were isolated and transplanted into the NOD/SCID mouse, CD3+ cells were shown to be exclusively responsible for the human cell chimerism. The engraftment of CD3+ cells in the NOD/SCID mice was unique to PV, since CD3+ cells from normal volunteers and patients with idiopathic myelofibrosis did not behave in a similar fashion. An increased proliferative response of PV CD3+ cells was demonstrated in response to the lymphocyte mitogen, PHA as compared to normal PB CD3+ cells. In order to determine if this defect in T cell behavior was a consequence of the JAK2V617F mutation, the presence of this mutation was sought using quantitative PCR. The T cells of 9 of the 10 PV patients did not contain this mutation while the ratio of JAK2V617F to total JAK2 allele was only 7.6% in the genomic DNA of the T cells of the remaining PV patient. Primary T cells from patients with PV were phenotypically similar to that of normal controls. In order to determine if the functional abnormal PV T cells affected blood cell production, media conditioned by PV and normal PB CD3+ cells was prepared and incubated with cord blood (CB) and PV PB CD34+ cells in the absence of cytokines and the number of hematopoietic colonies enumerated. Both PV and normal CD3+ cell conditioned media promoted CFU-GM colony formation to a similar extent, while only PV CD3+ cell conditioned media promoted BFU-E (0 versus13±3 per 400 CD34+ cells plated), CFU-MK (0 versus14±4), and CFU-Mix (0 versus 6±3) colony formation by CB CD34+ cells in the absence of the addition of exogenous cytokines. PV CD3+ cell conditioned media had a similar effect on the cloning efficiency of PV PB CD34+ cells. These data indicate that small numbers of CD34+ SCID repopulating cells are present in the PB of PV patients but that a previously unrecognized functional abnormality of T cells exist in PV which might contribute to the excessive production of erythroid and megakaryocytic cells belonging to the malignant PV clone.

Description: In this study, cord blood CD34+ cells expressed CD81, a member of the transmembrane 4 superfamily, and were classified into 3 subpopulations on the basis of their expression levels: CD34+CD81+, CD34lowCD81+, and CD34+CD81high. The lymphohematopoietic activity of each subpopulation was then examined by using suspension and clonogenic cultures for hematopoietic potential, coculture with MS-5 cells for B-cell potential, organ cultures of thymus lobes from nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) fetal mice, coculture with stromal cells derived from NOD/SCID fetal-mouse liver tissue for natural killer (NK) cell and mast cell potentials, and xenotransplantation into NOD/SCID mice for long-term repopulating (LTR) ability. CD34+CD81+ cells represented a heterogeneous population that had all the lymphohematopoietic activities, including NOD/SCID mouse-repopulating ability. CD34lowCD81+ cells were enriched in erythroid, megakaryocytic, and NK lineage potentials but had lost T-cell and B-cell potentials and LTR ability. The CD34+CD81high fraction was depleted of most lymphohematopoietic potentials except NK cell and mast cell potentials. Thus, along the differentiation cascade from CD34+CD81+ lymphohematopoietic stem cells, an up-regulation of CD81 or a down-regulation of CD34 results in a change in lymphohematopoietic properties. CD81 may serve as a marker for defining developmental stages of lymphohematopoietic stem cells.

Description: Pruritus is a common symptom in patients with Philadelphia chromosome–negative myeloproliferative disorders (MPDs). The pathophysiology of MPD-associated pruritus is unclear. We have demonstrated that MPD mast cells (MCs) are involved by the malignant process. In the present study, we explored the hypothesis that MCs play an important role in the development of pruritogenesis in MPDs. We found that MPD MCs released significantly greater amounts of pruritogenic factors, including histamine, leukotrienes, and interleukin-31 (IL-31) than normal MCs. Elevated levels of IL-31 were also observed in MPD CD3+ cell-conditioned media. MPD MCs exhibited increased migratory behavior in response to stem cell factor or interleukin-8, which was associated with increased filamentous-actin content. Furthermore, the presence of pruritus in MPDs was statistically correlated with a greater number of MCs being generated by CD34+ cells, a greater number of MC colonies being formed by CD34+ cells, decreased apoptosis and prostaglandin D2 release by cultured MCs, and higher plasma levels of IL-31. These data demonstrate that functional abnormalities of MPD MCs probably lead to pruritogenesis in patients with MPDs. These studies provide cellular and molecular targets for the development of antipruritus drugs for patients with MPDs.

Description: The Philadelphia chromosome negative (Ph−) myeloproliferative disorders (MPD), which include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are thought to result from the transformation of a multipotent hematopoietic stem cell (HSC). These Ph− MPDs are characterized by trilineage bone marrow hyperplasia with increased production of red cells, granulocytes and platelets which largely determines their clinical manifestations. Pruritus is common in Ph− MPDs occurring in approximately 50% of cases. The itching occurs spontaneously or appears when taking a hot shower or following other sudden environmental changes. In the present study, we explored the hypothesis that mast cells (MCs) play an important role in pruritogenesis in Ph− MPDs. PB CD34+ cells from 18 PV, 11 PMF, and 7 G-CSF mobilized (Gmob) volunteers were cultured in the presence of SCF (100ng/ml) and IL-6 (50ng/ml) for 7–8 weeks. After 49 days of culture, Gmob PB CD34+ cells generated significantly greater numbers of MCs (range: 5.6–20.1×106; mean±SD: 11.3±5.2×106) than PV (0.5–3.8×106; 1.2±0.9×106; p

Description: The JAK2V617F mutation has been shown to occur in the overwhelming majority of patients with polycythemia vera (PV). To study the role of the mutation in the excessive production of differentiated hematopoietic cells in PV, CD19+, CD3+, CD34+, CD33+, and glycophorin A+ cells and granulocytes were isolated from the peripheral blood (PB) of 8 patients with PV and 3 healthy donors mobilized with G-CSF, and the percentage of JAK2V617F mutant allele was determined by quantitative real-time polymerase chain reaction (PCR). The JAK2V617F mutation was present in cells belonging to each of the myeloid lineages and was also present in B and T lymphocytes in a subpopulation of patients with PV. The proportion of hematopoietic cells expressing the JAK2V617F mutation decreased after differentiation of CD34+ cells in vitro in the presence of optimal concentrations of SCF, IL-3, IL-6, and Epo. These data suggest that the JAK2V617F mutation may not provide a proliferative and/or survival advantage for the abnormal PV clone. Although the JAK2V617F mutation plays an important role in the biologic origins of PV, it is likely not the sole event leading to PV.

Description: In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF), peripheral blood CD34+ cells isolated from patients with IMF, polycythemia vera (PV), and G-CSF–mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is, therefore, likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-β (TGF-β) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9, therefore, likely contribute to the development of many pathological epiphenomena associated with IMF.

Description: The CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin−c-Kit+ long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin−c-Kit+CD34− marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin−c-Kit+ CD34high HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin−c-Kit+CD34− cells after 16 weeks, indicating that adult Lin−c-Kit+CD34− HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.

Description: Megakaryocytic (MK) hyperplasia is a pathological hallmark of the Philadelphia chromosome-negative myeloproliferative disorders. We studied the mechanisms of MK hyperplasia in idiopathic myelofibrosis (IMF) and evaluated the role of MKs in the generation of marrow fibrosis and increased microvessel density in IMF. Isolated CD34+ cells from normal mobilized peripheral blood (NMPB) and peripheral blood from IMF patients (IMFPB) were cultured for 15 days using serum-free media in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO). 5 out of 11 IMF patients were JAK2V617F positive. In the absence of TPO, after 11 days of culture, the percentage of CD41+ cells generated in IMFPB CD34+ cell cultures was 17.3% while only 1.1% of the cells were CD41+ in cultures of NMPB CD34+ cells. The addition of TPO (5ng/ml) led to a 19-fold increase in the percentage of NMPB CD41+ cells as compared to a 1.5 fold increase of IMF CD41+ cells. The absolute numbers of CD41+ cells generated from JAK2V617F positive and negative IMF CD34+ cells in the presence of TPO by day 11 were 5.4 fold and 4 fold greater respectively than normal control CD34+ cells. IMF MKs were smaller and had a lower degree of ploidy as determined flow cytometrically. The percentage of MK undergoing apoptosis was assessed by CD41/propidium iodide (PI) staining and TUNEL assay and revealed that far fewer IMF MKs underwent apoptosis by day 15 (7.7% +/− 1.6(IMF) vs 16.8 +/− 3.2 normal controls, p