Edward Bruno

Assay of an Activity in the Serum of Patients with Disorders of Thrombopoiesis That Stimulates Formation of Megakaryocytic Colonies

We have recently described an in vitro clonal assay system for human megakaryocyte-progenitor cells or megakaryocytic colony-forming units (CFU-M). Serum specimens from patients with quantitative platelet disorders were screened for the capacity to alter in vitro megakaryocyte-colony formation. Serum from 11 patients with hypomegakaryocytic thrombocytopenia significantly enhanced the formation of CFU-M-derived colonies (200 to 1840 per cent). Neither serum from eight patients with thrombocytopenia and normal or increased numbers of marrow megakaryocytes nor serum from 11 patients with thrombocytosis altered colony formation. This stimulatory activity has been termed megakaryocytic-colony-stimulating activity (Meg-CSA). The number of megakaryocytic colonies formed was directly proportional to the quantity of stimulatory serum added. Meg-CSA levels appeared to be inversely related to marrow megakaryocyte numbers. The variations in Meg-CSA levels that were detected in different disease states suggest that alterations in the production of this stem-cell regulator have physiologic importance.

An Antibody Cytotoxic to Megakaryocyte Progenitor Cells in a Patient with Immune Thrombocytopenic Purpura

Chronic idiopathic thrombocytopenic purpura is a syndrome characterized by bleeding due to a reduced number of platelets and normal or increased numbers of marrow megakaryocytes. The thrombocytopen...

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.

CrkL and CrkII participate in the generation of the growth inhibitory effects of interferons on primary hematopoietic progenitors

Interferons are potent regulators of normal and malignant hematopoietic cell proliferation in vitro and in vivo, but the signaling mechanisms by which they exhibit their growth inhibitory effects are unknown. We have recently shown that CrkL is engaged in Type I IFN signaling, as shown by its rapid tyrosine phosphorylation during engagement of the Type I IFN receptor. In the present study, we provide evidence that the related CrkII protein is also rapidly phosphorylated on tyrosine during treatment of U-266 and Daudi cells with IFNalpha or IFNbeta. We also show that both members of the Crk-family, CrkL and CrkII, are phosphorylated in an interferon-dependent manner in primary hematopoietic progenitors. Furthermore, inhibition of CrkL or CrkII protein expression by antisense oligonucleotides, reverses the inhibitory effects of IFNalpha or IFNgamma on the proliferation of normal bone marrow progenitor cells (colony forming units-granulocytic/monocytic [CFU-GM] and burst-forming units-erythroid [BFU-E]). Thus, both CrkL and CrkII are engaged in a signaling pathway (s) that mediates interferon-regulated inhibition of hematopoietic cell proliferation.

Recurrence of clonal hematopoiesis after discontinuing pegylated recombinant interferon-α 2a in a patient with polycythemia vera [11]

Recurrence of clonal hematopoiesis after discontinuing pegylated recombinant interferon- α 2a in a patient with polycythemia vera

Haematopoietic cell lineage distribution of MPLW515L/K mutations in patients with idiopathic myelofibrosis

Idiopathic myelofibrosis (IM) is a clonal haematological malignancy that is thought to originate at the level of the pluripotent haematopoietic stem cell (Baxter et al, 2005). The JAK2V617F mutation has been documented in 50% of patients with IM (Baxter et al, 2005). Pikman et al (2006) and Pardanani et al (2006) have recently defined two gain of function mutations, MPLW515L and MPLW515K (MPLW515L/K), in patients with IM and shown that these mutations play a role in the development of IM. Like JAK2V617F, the MPLW515L/K mutations also lead to activation of JAK-STAT signalling in these IM patients (Pikman et al, 2006). MPLW515L/K mutations are much less common than JAK2V617F but are disease-specific since they solely occur in 5 c. 10% of patients with IM (Lasho et al, 2006; Moliterno et al, 2006; Pardanani et al, 2006; Pikman et al, 2006). MPLW515L/K mutations have been previously detected in DNA from granulocytes of patients with IM (Lasho et al, 2006; Moliterno et al, 2006; Pardanani et al, 2006; Pikman et al, 2006). It is, however, unclear what other haematopoietic cell lineages are involved by these mutations. In the current study, we screened the granulocytes of 38 patients with IM for the MPLW515L/K mutations by DNA sequencing and allele-specific polymerase chain reaction (PCR). All patients met the World Health Organization diagnostic criteria for IM and all human tissue samples were obtained after informed consent was provided. Of the 38 patients, three patients had MPL515 mutations, two

Drosophila forkhead homologues are expressed in CD34+HLA-DR- primitive human hematopoietic progenitors

The Forkhead gene (FKH) regulates morphogenesis in Drosophila. It is the prototype of a new family of transcriptional activators. We used the polymerase chain reaction (PCR) to analyze the expression pattern of this new transcriptional regulatory gene family in primitive hematopoeitic progenitors. Partially degenerate oligonucleotides to two conserved amino acid sequences of this family were used to prime a PCR amplification of cDNA synthesized from CD34+/HLA-DR- hematopoietic cells. Known and novel FKH genes were found to be expressed in these cells.

Interleukin 10-induced thrombocytopenia in normal healthy adult volunteers: evidence for decreased platelet production: IL-10-induced Thrombocytopenia: Decreased Platelet Production

Recombinant human interleukin 10 (rhuIL‐10) inhibits the production of proinflammatory cytokines and has shown promise in the treatment of inflammatory bowel disease. Clinical trials have been accompanied by a reversible decline in platelet counts. We conducted a randomized, double‐blinded, placebo‐controlled, parallel group trial in 12 healthy volunteers to investigate the aetiology of rhuIL‐10‐induced thrombocytopenia. Eight volunteers received 8 μg/kg/d of rhuIL‐10 subcutaneously, while four subjects received a placebo alone for 10 d. A reversible decline in the platelet counts from a mean of 275 × 109/l to 164 × 109/l was observed in the IL‐10‐treated cohort (P = 0·012). A fall in the haemoglobin mean levels was also observed in the IL‐10‐treated cohort from 13·7 to 11·7 g/dl (P = 0·011). No significant change was observed in the bone marrow cellularity or myeloid/erythroid ratio or in the number of megakaryocytes per high‐powered field (HPF). A fall was observed in the number of megakaryocyte colony‐forming units (CFU‐MKs) after the administration of IL‐10 compared with those receiving the placebo (P = 0·068). No difference in the change in granulocyte–macrophage CFUs (CFU‐GMs), mixed lineage CFUs (CFU‐GEMMs) or erythroid burst‐forming units (BFU‐Es) was observed when comparing the IL‐10‐ vs. placebo‐treated groups (P > 0·465). Serum cytokine levels of thrombopoietin (TPO), IL‐6 and granulocyte–macrophage colony stimulating factor (GM‐CSF) were not decreased following IL‐10 administration. In fact, both TPO and GM‐CSF appeared to be slightly increased in the serum. All subjects underwent In111‐labelled platelet survival studies with liver/spleen scans to assess splenic sequestration prior to and then on day 7 of treatment. A significant reduction in splenic sequestration of platelets (P = 0·012) was observed in the IL‐10‐treated group, but not in the placebo‐treated subjects.