C-mpl Ligand Research Articles

The Impact of Eltrombopag Administration on the Clinical Course of Severe Refractory Fatal Acquired Aplastic Anemia

The Impact of Eltrombopag Administration on the Clinical Course of Severe Refractory Fatal Acquired Aplastic Anemia

Erythropoiesis‐stimulating agents are effective in myelodysplastic syndromes, but are they safe?

69. 11. Gotlib J, Lavori P, Quesada S, et al. A phase II intra-patient dose-escalation trial of weight-based darbepoetin alfa with or without granulocyte-colony-stimulating factor (G-CSF) in myelodysplastic syndromes. Am J Hematol 10.1002/

Humanized Sc(Fv)2 Minibody against C-Mpl Successfully Increased Platelet Number in Monkey and Increased Engraftment of Human Umbilical Cord Blood Cells in NOD/SCID Mouse.

Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody, confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.

CML Developed in a Japanese Family Transmitting a Novel Point Mutation in the Thrombopoietin Gene(TPO).

Introduction: Four families are reported to have hereditary thrombocythemia (HT) with a mutation in TPO. Their clinical manifestation is essentially thrombocytosis without leukemia. CML is one of myeloproliferative disorders, and shows leukocytosis and thrombocytosis associated with a proliferation of malignant clone originated from a hematopoietic stem cell (HSC). The incidence of CML is about 5 per 100,000 in Japan. Mutations of cytokine receptor including c-kit, flt-3 and G-CSF receptor are reported as a cause of AML. Especially flt-3 abnormalities are found in about 20% of AML. However, abnormality of c-mpl or TPO is not reported as a cause of leukemia. In this paper, we analyzed a CML case with novel point mutation in the TPO who still had thrombocytosis after cytogenetic complete response.Case: Japanese, 35 y.o., male, complained leukocytosis. He had a family history of thrombocytosis in 4 individuals over 3 generations. A physical examination revealed a moderate splenomegaly. Laboratory tests at the time of diagnosis were as follows; WBC 141,000/μl (blast 1.8%, promyelo 2.4%, myelo 20.0%, meta 8.2%, stab 24.2%, seg 22.2%, immature eosinophils 1.8%, eosinophil 3.6%, immature basophils 0.4%, basophils 10.4%, mono 1.0%, lymphocytes 4.0%, erythroblast 3%), PLT 641,000/μl and NAP score 53 (nl; 156–271). Bone marrow showed hypercellularity with the increased megakaryocytes(Meg), bcr-abl fusion mRNA positive, Ph1 chromosome positive. After 5 months treatment with STI571, most of clinical findings including karyotype and fusion mRNA turned to be normal, but thrombocyte(PLT) still showed more than 1,000,000/μl. At this time, serum TPO concentration was 8.14 f mole/ml (nl; 0.40 +/− 0.28 f mole/ml, mean +/− SD). Genetic analysis of TPO revealed novel point mutation at splicing donor site of 3′-end of the exon3. A point mutation at splicing donor site is reported to cause an exon-skipping and intron-retention, which induce a malfunction of a suppressive post-transcriptional and translational regulation, and consequent high-level expression of functional TPO protein.Discussion: TPO was cloned as a c-mpl ligand, which leads to the production of PLTs. Its receptor is a c-mpl proto-oncogene product, which is expressed not only in Meg, but also in HSC. Thus, TPO can stimulate HSC. The c-mpl transgenic mice are reported to have the increased Meg, its committed progenitor and PLT. Knockout mice of TPO presented not only the decreased Meg, but also multi-lineage committed progenitors. Thus, a modulation of c-mpl or its ligand function affects on both Meg and HSC. The c-mpl was cloned as a cellular homolog of a viral oncogene, v-mpl of myeloproliferative leukemia virus (MPL). The MPL causes myeloproliferative leukemia syndrome through v-mpl function in mice. Since v-mpl and c-mpl indicate high homology, it is possible that abnormal c-mpl function causes v-mpl like response. Through continuous stimulation of c-mpl signal, high TPO concentration may have induced a malignant transformation of HSC or supported a survival of an immature malignant clone in the present case. Improvement of thrombocytosis in CML is one of hematological responses to an anti-CML treatment such as STI571. In such a case who had good response other than thrombocytosis, an existence of HT might be considered. On the other hand, during following up HT family, occurrence of CML should be noted.

Targeting c-Mpl for revival of human immunodeficiency virus type 1-induced hematopoietic inhibition when CD34+ progenitor cells are re-engrafted into a fresh stromal microenvironment in vivo.

The inhibition of multilineage hematopoiesis which occurs in the severe combined immunodeficiency mouse with transplanted human fetal thymus and liver tissues (SCID-hu Thy/Liv) due to human immunodeficiency virus type 1 (HIV-1) infection is also accompanied by a severe loss of c-Mpl expression on these progenitor cells. Inhibition of colony-forming activity (CFA) of the CD34(+) progenitor cells is partially revived to about 40% of mock-infected Thy/Liv implants, following reconstitution of the CD34(+) cells that were exposed to HIV-1 infection, in a new Thy/Liv stromal microenvironment of irradiated secondary SCID-hu recipients at 3 weeks post-re-engraftment. In addition, in these reconstituted animals, the proportion of c-Mpl(+) CD34(+) cells relative to c-Mpl(-) CD34(+) cells increased by about 25%, to 35% of mock-infected implants, suggesting a reacquirement of c-Mpl phenotype by the c-Mpl(-) CD34(+) cells. These results suggest a correlation between c-Mpl expression and multilineage CFA of the human CD34(+) progenitor cells that have experienced the effects of HIV-1 infection. Treatment of the secondary-recipient animals with the c-Mpl ligand, thrombopoietin (Tpo), further increased c-Mpl expression and CFA of re-engrafted CD34(+) cells previously exposed to virus in the primary implants to about 50 to 70% over that of those re-engrafted CD34(+) cells derived from implants of untreated animals. Blocking of c-Mpl with anti-c-Mpl monoclonal antibody in vivo by injecting the SCID-hu animals resulted in the reduction or loss of CFA. Thus, inhibition, absence, or loss of c-Mpl expression as in the c-Mpl(-) CD34(+) subset of cells is the likely cause of CFA inhibition. Further, CFA of the CD34(+) cells segregates with their c-Mpl expression. Therefore, c-Mpl may play a role in hematopoietic inhibition during HIV-1 infection, and control of its expression levels may aid in hematopoietic recovery and thereby reduce the incidence of cytopenias occurring in infected individuals.

919. Gene Therapy for Platelet Disorders: Targeting Transgene Expression in Canine Platelets

Platelets mediate the primary response to vascular injury; as such molecular defects in some platelet proteins can disrupt normal hemostasis and lead to uncontrolled hemorrhage. To develop a large animal model to examine lineage-specific gene therapy for disorders affecting platelets, canine CD34+ peripheral blood cells (PBC) were transduced with a self-inactivating, human immunodeficiency type-1 lentivirus-derived vector controlled by the megakaryocyte-specific human integrin αIIb gene promoter. This construct, αIIb-GFP-WPT, utilized the woodchuck hepatitis virus postregulatory element (W), and the central polypurine tract (PT) to enhance the efficiency of transgene expression. cDNA encoding the green fluorescent protein (GFP) was subcloned into the construct to analyze transgene expression in the progeny of transduced cells. A second vector (EF1-GFP-WPT) under control of the promoter from the housekeeping gene, Elongation Factor-1, which drives constitutively active transgene expression was transduced into CD34+ PBC as a control for lineage non-specific transgene expression. Cells were immuno-magnetically selected for the CD34+ antigen from granulocyte-colony stimulating factor mobilized PBC, which were collected by apheresis of a normal dog. CD34+ PBC were prestimulated for 2 days in media containing cytokines (flk2/flt3-ligand, SCF, IL-3, IL-6 and c-mpl ligand), transduced for 2 days and induced for 5 days with culture media containing the cocktail of cytokines used for prestimulation and IL-11 to induce the cells to differentiate into a population comprised of several lineages including megakaryocytes. Flow cytometric analysis using an antibody specific for the canine β3-subunit revealed that the vectors (EF1- and αIIb-) transduced megakaryocytes with equal efficiency with 60% of megakaryocytes expressing GFP. Only 14% of the cell population expressing GFP was megakaryocytes when using the EF1-promoter, while 60% of the cells expressing GFP under control of the αIIb-promoter were megakaryocytes. When αIIb-GFP transduced PBC were xenotransplanted into immune-compromised (NOD/SCID) mice, flow cytometric analysis demonstrated that GFP-positive platelets were circulating within the vasculature and could be isolated at the site of a tail vein injury at two weeks post-transplant of canine PBC. These studies demonstrate that a lentivirus construct under control of the human αIIb promoter can target expression of GFP in canine megakaryocytes with high specificity compared to the EF1-promoter's ability to drive lineage-specific transgene expression. Furthermore, transduced PBC are capable of forming megakaryocytes and platelet progeny expressing transgene products in vivo. This work suggests that there is potential feasibility for using a lineage-specific αIIb gene promoter to develop gene therapy for disorders affecting platelets.

Structure of the receptor-binding domain of human thrombopoietin determined by complexation with a neutralizing antibody fragment.

The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.

Differential role of Stat5 isoforms in effecting hematopoietic recovery induced by Mpl-ligand in lethally myelosuppressed mice

Objective To determine the role of the c-terminal half of c-Mpl in Mpl-L-induced myeloprotection and the importance of Stat5 isoforms in the survival signaling pathways induced by Mpl ligand. Materials and methods Δ60-Mpl knockin mice, Stat5a −/−/b −/−, Stat5a −/−, and Stat5b −/− mice and wild-type (WT) controls were given a lethal myelosuppressive regimen: 80 mg/kg carboplatin intravenously followed by 7.5 or 6.5 Gy 137Cs total-body irradiation. A single dose of PEG-rmMGDF (65 μg/kg) was intravenously injected immediately after myelosuppression. Mice survival and blood counts were monitored for 22 days posttreatment. Results Knockin Δ60-Mpl mice lacking the c-terminal half of the intracellular domain of c-Mpl show reduced ability of Mpl-L to prevent lethal myelosuppression and an impaired thrombopoietic response to exogenous c-Mpl ligand. The survival of Mpl-L-treated Stat5a −/−/b −/− mice exposed to the lethal myelosuppressive regimen was substantially compromised compared to that of WT mice. Reduced survival of Stat5a −/−/b −/− mice was due to more severe hematopoietic suppression. Deletion of Stat5a did not result in a defect in hematopoietic recovery. In contrast, Mpl-L-treated Stat5b-deficient mice demonstrated significantly delayed hematopoietic recovery compared to WT controls. Conclusions Myeloprotective signaling transduced by the terminal 60 amino acids of the intracellular domain of c-Mpl is essential for complete protection from lethal myelosuppression provided by Mpl-L. Our studies differentiate the functions of Stat5 isoforms in hematopoietic stress and reveal a pivotal role of Stat5b in Mpl-L-induced hematopoietic recovery in this lethal myelosuppression model.

Thrombopoietin as a drug: biologic expectations, clinical realities, and future directions.

After the cloning of thrombopoietin (c-mpl ligand, Tpo) in 1994, 2 recombinant thrombopoietic growth factors, full-length glycosylated recombinant human Tpo (reHuTPO) and polyethylene glycol conjugated megakaryocyte growth and development factor (PEG-reHuMGDF), have been studied in humans in a variety of clinical settings. Both thrombopoietins are generally well tolerated if administered intravenously (IV). The c-mpl ligands produce a dose-related enhancement of platelet levels, reduce nonmyeloablative chemotherapy-induced mild thrombocytopenia, and mobilize hematopoietic progenitors. On September 11, 1998, the development of PEG-reHuMGDF was suspended in the U.S., due to formation of the neutralizing anti-Tpo antibody. Those neutralizing antibodies lead to thrombocytopenia and pancytopenia in some patients receiving subcutaneous (SC) PEG-reHuMGDF. Japanese investigators indicate that the probability of antibody formation against PEG-reHuMGDF is low when the drug is administered IV instead of SC. reHuTPO has a more favorable safety profile from the point of antibody production. The c-mpl ligands can improve apheresis yields when administered to normal platelet donors. Preliminary data about the use of PEG-reHuMGDF in myelodysplasia, aplastic anemia, and immune thrombocytopenic purpura are promising. Tpo is usually not effective in myeloablative thrombocytopenia when bone marrow hematopoietic progenitors are not present. The major obstacle for the thrombopoietins is their delayed action for managing clinical thrombocytopenia. This review will focus on the biologic basis, current clinical experience, and future directions for the use of thrombopoietic molecules as drugs. The identification of a safe, effective, and potent pharmacologic platelet growth factor could significantly improve the management of thrombocytopenia-induced bleeding.

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.

Promegapoietin, a family of chimeric growth factors, supports megakaryocyte development through activation of IL-3 and c-Mpl ligand signaling pathways.

The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.

Efficient ex vivo generation of human dendritic cells from mobilized CD34+ peripheral blood progenitors.

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.

Thrombopoietin in the fetus and neonate.

C-mpl ligand or thrombopoietin (Tpo) is increasingly recognised as the major regulator of platelet homeostasis in humans. Relatively little is known about Tpo in the fetus and neonate but no evidence has yet been found to suggest any fundamental difference in Tpo structure, function and regulation in the fetus and neonate compared to older age groups. Tpo mRNA transcripts have been detected in the fetus as early as 6 weeks post conception and the liver appears to be the main site of Tpo production in both the fetus and neonate. The vast majority of healthy newborns have detectable levels of circulating Tpo and raised Tpo levels are commonly, but not consistently, found in thrombocytopenic neonates. In adults receptor binding and subsequent metabolism of Tpo is proposed as the main method of regulation of the circulating Tpo level. Preliminary studies in neonates showing increased Tpo levels most often during thrombocytopenia accompanied by reduced megakaryocytopoiesis supports this concept. In addition to this demonstrable fetal and neonatal endogenous Tpo production megakaryocyte progenitor and precursor cells from the fetus and from preterm and term newborns proliferate and differentiate extensively in-vitro in response to exogenous Tpo. Furthermore a recent study has shown a marked rise in platelet count in newborn rhesus monkeys administered one form of recombinant Tpo. Although these studies remain at an early stage together these findings strongly suggest that, as in adults, Tpo is the major regulator of platelet homeostasis in the fetus and neonate. Thrombocytopenia is common in sick neonates and progress in understanding this important clinical problem is likely to be greatly enhanced by the current and future research into Tpo production, function and regulation in the healthy and thrombocytopenic fetus and neonate.

Differential CD34+ engraftment of nod-scid mice with ex vivo expanded human CD34+ cord blood stem cells correlates with CD38 phenotype

Ex vivo expansion of cord blood [CB] hematopoietic stem cells [HSC] may increase the number of patients eligible for allogeneic transplants, by making CB a reliable source of HSC for transplantation. We previously showed that CB-HSC can be significantly expanded with flt-3 ligand [FL] and c-mpl ligand [ML]; 106 input CD34+ cells increase to 108 CD34+ cells by 8-9 weeks of culture. However, it is essential to know the function of expanded CB HSC cells prior to clinical usage. To assess the in vivo function of expanded CB HSC, cells from expansion cultures were injected into irradiated [375 cGy] non-obese diabetic/severe combined immune deficient [NOD-scid] mice. Uncultured CD34+ UCB served as engraftment controls. Engraftment was analyzed 5-8 weeks post-injection by two-color staining of bone marrow cells with antibodies to human CD45 [FITC] and selected CD antigens [PE]. Cells grown from 2 to 16 weeks engraft NOD-scid mice, as evidenced by human CD45+ cells in the marrow, giving rise to mature human leukocytes. Surprisingly, cells from 2-week cultures generate few CD34+ cells in the marrow of NOD-scid mice, while mice engrafted with cells from cultures ≥3 weeks appear similar to uncultured UCB controls. Kinetic analysis of CD38 expression on ex vivo expanded CB-HSC using 3-color flow cytometry shows that the CD34+ cells from uncultured UCB are predominantly CD38lo, but there is a shift to CD34+/CD38+ cells during the first 2 weeks of culture. At later time points, the CD38+ population diminishes, so that virtually all CD34+ cells are CD38lo. Since CD34+/CD38+ stem cells are considered more mature than CD34+/CD38lo HSC, our results correlated CD38 expression to CD34+ engraftment potential. These findings indicate that short-term cultures (10-14 days) lead to a decline in CB HSC activity and impaired engraftment, but extended culture conditions lead to a return of CD34+/CD38lo stem cells, and restoration of normal engraftment potential by these cultured CB HSC.

Environmental Research, Section B

C-mpl ligand or thrombopoietin (Tpo) is increasingly recognised as the major regulator of platelet homeostasis in humans. Relatively little is known about Tpo in the fetus and neonate but no evidence has yet been found to suggest any fundamental difference in Tpo structure, function and regulation in the fetus and neonate compared to older age groups. Tpo mRNA transcripts have been detected in the fetus as early as 6 weeks post conception and the liver appears to be the main site of Tpo production in both the fetus and neonate. The vast majority of healthy newborns have detectable levels of circulating Tpo and raised Tpo levels are commonly, but not consistently, found in thrombocytopenic neonates. In adults receptor binding and subsequent metabolism of Tpo is proposed as the main method of regulation of the circulating Tpo level. Preliminary studies in neonates showing increased Tpo levels most often during thrombocytopenia accompanied by reduced megakaryocytopoiesis supports this concept. In addition to this demonstrable fetal and neonatal endogenous Tpo production megakaryocyte progenitor and precursor cells from the fetus and from preterm and term newborns proliferate and differentiate extensively in-vitro in response to exogenous Tpo. Furthermore a recent study has shown a marked rise in platelet count in newborn rhesus monkeys administered one form of recombinant Tpo. Although these studies remain at an early stage together these findings strongly suggest that, as in adults, Tpo is the major regulator of platelet homeostasis in the fetus and neonate. Thrombocytopenia is common in sick neonates and progress in understanding this important clinical problem is likely to be greatly enhanced by the current and future research into Tpo production, function and regulation in the healthy and thrombocytopenic fetus and neonate.

Biologic and structural differences of thrombopoietic growth factors

The search for a thrombopoietic agent has resulted in the identification of numerous cytokines and growth factors with thrombopoietic activity. However, with the exception of interleukin (IL)-11 and thrombopoietin (TPO), the megakaryopoietic activity of most of these molecules has not produced clearly identifiable clinical benefits. Despite the relatively modest effect of IL-11 on megakaryocyte and platelet production in vitro and in vivo, it does reduce the need for platelet transfusions in specialized clinical settings. In contrast, the c-Mpl ligand TPO has been shown to be a potent stimulator of megakaryocyte and platelet production both in vitro and in vivo. Clinical studies are being conducted with two different preparations of the c-Mpl ligand: recombinant human thrombopoietin (rhTPO) and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF). A recombinant form of the complete human molecule, rhTPO is glycosylated and produced in mammalian cells. PEG-rHuMGDF consists of only the receptor-binding domain linked to a polyethylene glycol (PEG) moiety and is generated in Escherichia coli. Although c-Mpl ligands are still being evaluated, preliminary evidence indicates that these molecules can elevate platelet counts and may be useful in a range of clinical contexts. This report discusses aspects of the biology behind the clinical actions of IL-11 and the c-Mpl ligands.

Pharmacologic treatment options in patients with thrombocytopenia

Thrombocytopenia that results from chemotherapy has become an increasingly important issue in the treatment of cancer and remains a difficult clinical problem. The identification of a safe and effective platelet growth factor could significantly improve the management of severe chemotherapy-induced thrombocytopenia. Over the past decade, a number of hematopoietic growth factors with thrombopoietic activity have been identified, including stem-cell factor (c- kit ligand), interleukin (IL)-1, IL-3, IL-6, and IL-11, as well as thrombopoietin (TPO) and its derivatives. Only a few of these agents have shown acceptable tolerability and sufficient ability to stimulate thrombopoiesis to justify testing in randomized clinical trials. Currently, IL-11 is the only cytokine licensed in the United States for treatment of chemotherapy-induced thrombocytopenia. However, its thrombopoietic activity is modest and its use is often associated with unfavorable side effects. Identification of TPO, the c-Mpl ligand, as the primary physiologic regulator of megakaryocyte and platelet development offers important promise for treatment of thrombocytopenia. Preliminary clinical studies of recombinant human TPO (rhTPO), a full-length glycosylated molecule, indicate that it is safe and biologically active in reducing severe chemotherapy-induced thrombocytopenia. In addition to rhTPO, the future may see the development of novel genetically engineered, high-affinity cytokine receptor agonists and c-Mpl ligand mimetic peptides.

Clinical experience with recombinant human thrombopoietin in chemotherapy-induced thrombocytopenia

Since the identification and cloning of c-Mpl ligand, two forms of recombinant human thrombopoietin have undergone clinical development. Both the full-length molecule, known as rhTPO, and the truncated version of the molecule, known as pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), have been evaluated in phase I/II clinical trials in cancer patients receiving myelosuppressive chemotherapy. Early clinical trials with PEG-rHuMGDF in cancer patients demonstrated its clinical safety and platelet-stimulating activity. However, the development of neutralizing antibodies and clinically significant thrombocytopenia in some patients and normal donors who received PEG-rHuMGDF have led to discontinuation of clinical trials with this molecule in the United States. Clinical experience with rhTPO so far indicates that this full-length glycosylated molecule is remarkably well tolerated and has a favorable safety profile. In these studies, rhTPO exhibited dose-dependent increases in circulating platelet counts and bone marrow megakaryocytes before chemotherapy. In addition, there was an increase in the frequency and proliferation of bone marrow progenitor cells and mobilization of progenitors into the peripheral blood. Early results also showed that rhTPO can attenuate chemotherapy-induced severe thrombocytopenia and reduce the need for platelet transfusions. However, in this setting, the optimal schedule of rhTPO administration may depend on the length of the regimen and anticipated timing of the platelet nadir. These initial results indicate that rhTPO is a safe and potentially useful agent in the prevention and management of chemotherapy-induced thrombocytopenia. Results of larger randomized clinical trials will determine the therapeutic potential of this novel growth factor in various clinical settings.

Clinical experience with recombinant human thrombopoietin in chemotherapy-induced thrombocytopenia.

Since the identification and cloning of c-Mpl ligand, two forms of recombinant human thrombopoietin have undergone clinical development. Both the full-length molecule, known as rhTPO, and the truncated version of the molecule, known as pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), have been evaluated in phase I/II clinical trials in cancer patients receiving myelosuppressive chemotherapy. Early clinical trials with PEG-rHuMGDF in cancer patients demonstrated its clinical safety and platelet-stimulating activity. However, the development of neutralizing antibodies and clinically significant thrombocytopenia in some patients and normal donors who received PEG-rHuMGDF have led to discontinuation of clinical trials with this molecule in the United States. Clinical experience with rhTPO so far indicates that this full-length glycosylated molecule is remarkably well tolerated and has a favorable safety profile. In these studies, rhTPO exhibited dose-dependent increases in circulating platelet counts and bone marrow megakaryocytes before chemotherapy. In addition, there was an increase in the frequency and proliferation of bone marrow progenitor cells and mobilization of progenitors into the peripheral blood. Early results also showed that rhTPO can attenuate chemotherapy-induced severe thrombocytopenia and reduce the need for platelet transfusions. However, in this setting, the optimal schedule of rhTPO administration may depend on the length of the regimen and anticipated timing of the platelet nadir. These initial results indicate that rhTPO is a safe and potentially useful agent in the prevention and management of chemotherapy-induced thrombocytopenia. Results of larger randomized clinical trials will determine the therapeutic potential of this novel growth factor in various clinical settings.

Thrombopoietin, Interleukin-11, and Early-Acting Megakaryocyte Growth Factors in Human Myeloid Leukemia Cells

In this study we report our data on effects of early-acting megakaryocyte growth factors, particularly the c-mpl ligand also known as thrombopoietin (TPO) and interleukin-11 (IL-11), on cell proliferation and apoptosis (Apo) of primary acute myeloid leukemia (AML) cells. A proliferative response to TPO was noticed in the majority of AML samples (17/19) with an average increase of S-phase cells from 7.8% ± 1.5 to 14.5% ± 2.1 (p=0.0006). Resulting cell cycle activation did not always correlate with expression of the c-mpl receptor, although it was coupled, in the majority of samples, by an average decrease of apoptotic cells from 13% ± 0.7 to 8.8% ± 1.8 (p=0.05). Clonogenic cell growth (CFU-L) was confirmed in 5/17 of the samples with a mean colony number of 21.4 ± 9.6 × 105 cells plated. Conversely, effects of IL-11 on AML cells demonstrated that cell cycle changes (recruitment from G0 to S phase) were promoted only in a minority of samples (2/14) and there was little, if any, effect on CFU-L growth (mean colony number=17.5 ± 9.5) or Apo (from 13% ± 0.7 to 13.3±1.9). Combination of TPO with IL-11 induced a slight increase of clonogenic cell growth, while the addition of IL-3 or SCF to the c-mpl ligand significantly raised the mean colony numbers up to 119.2 ± 68.3 and 52.9±22.1 × 105 cells plated, respectively. In summary, TPO shows activity on AML cells by stimulating their proliferation in a significant proportion of cases and generally protecting the majority of AML blast cells from induction of Apo. Conversely, IL-11 exerts little effect on the cell cycle activation and Apo. These data help to understand regulation of myeloid leukemia cell growth and should be considered in the clinical use of early-acting megakaryocyte growth factors in acute leukemia.