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Biomedical subjects

K Kaushansky

Publications and source records attributed to K Kaushansky.

At least 91 records · Page 5Linked to original sources
PubMed1995

Accelerated reconstitution of platelets and erythrocytes after syngeneic transplantation of bone marrow cells derived from thrombopoietin pretreated donor mice.

The recent cloning of the ligand of the c-Mpl hematopoietin receptor has indicated a major role for this cytokine in the development of megakaryocytes. In this study we have applied c-Mpl ligand (thrombopoietin [TPO]) in the setting of syngeneic transplantation in an attempt to accelerate the reconstitution of platelets. Donor mice were treated with 20 kilounits (kU)/d TPO intraperitoneally (ip) for 5 days. This resulted in a 2.5-fold increment in platelet counts from 1,119 x 10(9)/L to 2,582 x 10(9)/L (mean, n = 7). Total numbers of hematopoietic progenitor cells in bone marrow (BM) and spleen, as assessed in a colony-forming unit-granulocyte erythroid monocyte macrophage (CFU-GEMM) colony assay (55.3 v 38.6 x 10(3) CFU/femur; 27.3 v 16.3 x 10(3) CFU/spleen, mean, n = 7) as well as total numbers of burst-forming unit-erythroid (BFU-E) (24.0 v 16.4 x 10(3)/femur; 10.2 v 1.9 x 10(3)/spleen, mean, n = 7), were significantly higher in TPO-treated donors than in saline-treated controls. Female Balb-C mice were lethally (8.5 Gy) irradiated and transplanted with 10(5) BM cells. After transplantation, groups of mice were treated with recombinant murine TPO at a dose of 20 to 30 kU/d ip or subcutaneously (SC) for 5 to 14 days. Using this dose and schedule, TPO did not stimulate the recovery of platelets in comparison with control animals transplanted with equal cell numbers but given vehicle alone. In other experiments, 10(5) BM cells were procured from TPO-treated donor mice and transplanted into lethally irradiated recipient mice. In comparison with animals transplanted with an equal number of BM cells derived from saline-treated controls, recipients of TPO-treated BM cells had significantly faster platelet recovery and higher platelet nadir counts (88 v 30 x 10(9)/L, mean, n = 20). Transplantation of TPO-treated BM cells also resulted in an accelerated recovery of erythrocytes and increased erythrocyte nadir counts (7.2 v 5.0 x 10(12)/L, mean, n = 20). At the day of platelet nadir (day 12 after transplantation) these animals had higher numbers of BFU-Es (770 v 422, mean, n = 5) in the marrow and also had higher reticulocyte counts (44 / 1000 v 8 / 1000 mean, n = 5) in the blood. Therefore, the accelerated recovery of erythrocytes may be a direct effect of TPO on erythropoiesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Interleukin-4 induces endothelial vascular cell adhesion molecule-1 (VCAM-1) by an NF-kappa b-independent mechanism.

While all features of the inflammatory response induced by IL-1 are not observed following IL-4 stimulation, suboptimal concentrations both cytokines result in synergistic VCAM-1 expression in HUVEC. We have shown that, while IL-1 stimulated HUVEC express GM-CSF, tissue factor and VCAM-1, only VCAM-1 is detectable after exposure to IL-4. While kB was found essential for both basal and IL-1-mediated activity of VCAM-1, IL-4 induction was kB-independent. Inducible kB-binding proteins were identified in IL-1-, but not IL-4-stimulated nuclear extracts. Our results indicate that IL-4 exerts its transcriptional effects on the VCAM-1 gene through element(s) which do not require kB.

Cells, Cultured↗
PubMed1995

Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development.

The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that early-acting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to late-acting MK potentiators, including IL-6 and IL-11. Recently, the Mp1 ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.

Animals↗
PubMed1995

Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro.

Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU-Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with > or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg.

Animals↗
PubMed1995

Recombinant antibodies in bioactive peptide design.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is important in many immune and inflammatory processes. GM-CSF binds to specific cellular receptors which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design, and such design depends on a molecular understanding of ligand-receptor interactions. One approach to dissecting out critical intermolecular interactions is to develop analogs of specific interaction sites of potential importance. Monoclonal antibodies have been employed for these purposes in prior studies. Here we present application of recombinant antibody technology to the development of analogs of a site on GM-CSF bound by a neutralizing anti-GM-CSF monoclonal antibody. Polyclonal antisera with high titer neutralizing activity against human GM-CSF were developed in BALB/c mice. Purified immunoglobulins were prepared and used to immunize syngeneic mice. Anti-anti-GM-CSF was developed which demonstrated biological antagonist activity against GM-CSF-dependent cellular proliferation. RNA was extracted from spleen cells of mice with biologically active anti-anti-GM-CSF, cDNA synthesized, and polymerase chain reaction performed with primers specific for murine kappa light chain V regions. Polymerase chain reaction products were cloned into the pDABL vector and an expression library developed. This was screened with anti-GM-CSF neutralizing mAb 126.213, and several binding clones isolated. One clone (23.2) which inhibited 126.213 binding to GM-CSF was sequenced revealing a murine kappa light chain of subgroup III. Comparison of the 23.2 sequence with the human GM-CSF sequence revealed only weak sequence similarity of specific complementarity determining regions (CDRs) with human GM-CSF. Structural analysis revealed potential mimicry of specific amino acids in the CDR I, CDR II and FR3 regions of 23.2 with residues on the B and C helices of GM-CSF. A synthetic peptide analog of the CDR I was bound by 126.213, specifically antagonized GM-CSF binding to cells and blocked GM-CSF bioactivity. These studies indicate the feasibility of using recombinant antibody libraries as sources of interaction site analogs.

Amino Acid Sequence↗
PubMed1995

In vitro characterization of the human recombinant soluble granulocyte-macrophage colony-stimulating factor receptor.

We have cloned, expressed, and partially purified a naturally occurring, truncated, soluble form of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha subunit to investigate its biochemical and biologic properties. The soluble receptor species lacks the transmembrane and cytoplasmic domains that are presumably removed from the intact receptor cDNA by a mechanism of alternative splicing. The resulting soluble 55- to 60-kD glycosylated receptor species binds GM-CSF with a dissociation constant (kd) of 3.8 nmol/L. The soluble GM-CSF receptor successfully competes for GM-CSF binding not only with the transmembrane-anchored GM-CSF receptor alpha subunit but also with the native oligomeric high-affinity receptor complex. In addition, in human bone marrow colony-forming assays, the soluble GM-CSF receptor species can antagonize the activity of GM-CSF. Our data suggest that the soluble GM-CSF receptor may be capable of acting in vivo as a modulator of the biologic activity of GM-CSF.

Binding, Competitive↗
PubMed1995

The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl.

c-Mpl is a member of the cytokine receptor superfamily, expressed primarily on hematopoietic cells. Recently, the c-Mpl ligand was cloned and found to have thrombopoietic activity. In this paper we report that ligand binding induced tyrosine phosphorylation in BaF3 cells engineered to express the murine Mpl receptor (BaF3/mMpl). Phosphorylation occurred within 1 min at cytokine concentrations sufficient for proliferation of receptor-bearing cells. Using specific antibodies for immunoprecipitation and Western blotting, several of these phosphorylated proteins were identified. Shc and Jak2, known cytokine signaling molecules, and the c-Mpl receptor were shown to be major substrates for tyrosine phosphorylation. In contrast, phospholipase C-gamma and phosphatidylinositol 3-kinase displayed little and no tyrosine phosphorylation, respectively, after thrombopoietin stimulation. Co-immunoprecipitation studies demonstrated that Jak2 became physically associated with c-Mpl relatively late in the observed time course (20-60 min), significantly later than tyrosine phosphorylation of Jak2 (1-5 min). These results suggest that c-Mpl induces signal transduction pathways similar to those of other known cytokines. Additionally, in light of its late physical association with c-Mpl following ligand binding, Jak2 may not be the initiating tyrosine kinase in the thrombopoietin-induced signaling cascade.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Thrombopoietin, the c-mpl ligand, is a major regulator of platelet production.

Hematopoiesis is regulated by a family of glycoproteins, the hematopoietic growth factors. Although the cytokines that influence the late stages of granulopoiesis (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor) or erythropoiesis (erythropoietin) have been identified, characterized, and cloned, and have been in clinical use since that late 1980's, the cytokine that stimulates thrombopoiesis had remained elusive. By using strategies based on the c-mpl receptor, several groups have recently succeeded in purifying and cloning thrombopoietin [1-6]. This study outlines the cloning and biological characterization in vitro and in vivo of thrombopoietin, a major regulator of platelet production.

Amino Acid Sequence↗
PubMed1995

Thrombopoietin: basic biology, clinical promise.

Cytokines which regulate thrombopoiesis have been the subject of intense study and debate over the past 35 years. Recently, based on recognition of the orphan cytokine receptor c-mpl, thrombopoietin (Tpo), the primary regulator of platelet production has been cloned. Its biological properties form the basis of this review. Tpo promotes both the proliferation of megakaryocytic progenitor cells and their maturation into platelet-producing megakaryocytes. It acts to expand megakaryocyte size and ploidy, and increases the expression of platelet-specific membrane glycoproteins. In vivo, Tpo greatly augments marrow and splenic megakaryocytic progenitor cells and large megakaryocytes, and leads to profound increases in platelet production. In addition, the hormone hastens the recovery or platelet counts following cytoreductive therapies, and has been associated with improved survival in certain murine models. Clearly, our growing understanding of the biology or Tpo will lead to important new insights into megakaryopoiesis, and to clinical improvements in the therapy of both natural and acquired states of marrow failure.

Animals↗
PubMed1995

Structure and function of the cytokine receptor superfamily.

The cytokine receptor superfamily is a group of transmembrane proteins, characterized by a common extracellular structure--two barrels composed of seven beta strands each. Over the past year, several new members of the cytokine receptor family have been described, and new insights have been made into how related receptors share common subunits. Also this year, the ligand for the orphan cytokine receptor c-Mpl was identified and shown to stimulate megakaryocyte development and thrombopoiesis. Progress has been made in unraveling the precise atomic basis for ligand-receptor interactions and the role that subunit association plays in receptor activation. Finally, advances have been made in understanding the organization of the intracellular domain and how signaling to the nucleus is achieved. Together, these new results have led to a greater appreciation of the role that cytokine receptors play in the regulation of proliferation and lineage-specific differentiation during hematopoiesis.

Animals↗
PubMed1995

Physiology and preclinical studies of thrombopoietin.

Until recently, the molecular basis for the control of platelet production was largely unknown. In the past year, several groups have obtained complementary DNA for thrombopoietin, the substance first theorized nearly 40 years ago to regulate this process. Cellular and molecular studies have confirmed many of the properties previously attributed to this molecule, and have revealed some surprises. It is now clear that thrombopoietin is the critical regulator of platelet production. Detailed study of the molecule will likely yield important physiologic insights into megakaryocyte biology, and its application to states of iatrogenic and natural marrow failure will almost certainly provide therapeutic advances.

Blood Platelets↗
PubMed1995

Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy.

Thrombopoietin (TPO), the ligand for the receptor protooncogene c-mpl, has been cloned and shown to be the critical regulator of platelet production. Several features of c-Mpl expression, including its presence on erythroid cell lines, and the panmyeloid transformation characteristic of myeloproliferative leukemia (MPL) viral disease led us to investigate whether this receptor-ligand system may play a role in erythropoiesis. We report that although TPO alone did not support the growth of either early or late erythroid progenitors, it acted in synergy with erythropoietin to expand these populations. Moreover, while the effects on erythropoiesis in normal animals were modest, TPO greatly expanded the number of erythroid progenitors and blood reticulocytes and was associated with accelerated red cell recovery in myelosuppressed mice. Together, these data strongly suggest that erythroid progenitors respond to TOP and that this newly cloned cytokine, critical for platelet production, can augment erythropoiesis in states of marrow failure.

Animals↗
PubMed1995

The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases.

Thrombopoietin (TPO) is a growth and differentiation factor for megakaryocyte-lineage cells. The receptor for TPO, c-MPL, is a member of the hematopoietic cytokine receptor family and has previously been shown to rapidly activate one or more cytoplasmic tyrosine kinases after ligand binding. In this study, we found that activation of the TPO receptor rapidly induced tyrosine phosphorylation of two members of the Jak tyrosine kinase family, JAK2 and TYK2, but not JAK1 or JAK3, in two different factor-dependent hematopoietic cell lines. The activation of both JAK2 and TYK2 was dose- and time-dependent and was associated with rapid tyrosine phosphorylation of a series of STAT proteins including STAT1, STAT3, and STAT5. Gel-shift assays indicated that one or more of these STATs is likely to participate in the formation of specific DNA-binding complexes. The activation of tyrosine kinases and signal propagation through tyrosine phosphorylation are likely to represent important initial steps in mediating the activities of TPO in myeloid cells.

Animals↗
PubMed1995

Thrombopoietin: the primary regulator of megakaryocyte and platelet production.

The development of blood cells requires the interplay of hematopoietic stem and progenitor cells, marrow stroma and polypeptide growth factors. Although many proteins support the expansion of megakaryocytic precursor cells, identification of the late acting, lineage specific growth factor for platelet production, termed thrombopoietin, has remained elusive. Recently, characterization of the proto-oncogene c-mpl revealed structural homology with the hematopoietic cytokine receptor family. Based on the cell of origin of its cDNA, we hypothesized that the ligand for c-Mpl might be identical with Tpo, and together with scientists at ZymoGenetics, Inc. recently cloned its cDNA. Using recombinant protein we have shown that the mpl-ligand displays all of the expected biological properties of the major regulator of megakaryocyte development, and proposed that it be termed thrombopoietin.

Amino Acid Sequence↗
PubMed1994

Mapping of human granulocyte-macrophage-colony-stimulating-factor domains interacting with the human granulocyte-macrophage-colony-stimulating-factor-receptor alpha-subunit.

The high-affinity granulocyte-macrophage-colony-stimulating-factor (GM-CSF) receptor (R) is composed of at least two subunits, termed alpha and beta. The alpha subunit is crucial for initiating ligand/receptor interaction and for ligand specificity. The experiments reported in this study sought to identify the domains of human (h)GM-CSF which are responsible for interaction with hGM-CSFR alpha. Anti-(human-GM-CSF) mAb were used as competitors in a 125I-GM-CSF receptor-binding assay on cells expressing the recombinant hGM-CSFR alpha chain. Inhibition of 125I-GM-CSF binding to the GM-CSFR alpha chain was demonstrated by mAb which mapped to the middle third of the third alpha helix (amino acids 78-83), the distal two-thirds of the third alpha helix and the initial 7 residues of the loop between the third and fourth helix (amino acids 78-94), and the extreme carboxy terminus. No inhibition of binding occured with an antibody whose domain begins in the first beta-pleated sheet (amino acids, 39-43), continues through the second helix (amino acids 55-64) and into the proximal third of the third helix (to amino acid 77). mAb which mapped to the distal half of the fourth helix and the carboxy-terminal tail (amino acids 110-127) increased the binding of 125I-GM-CSF to GM-CSFR alpha. Due to the known discontinous epitopes the possibility that the distal portion of the fourth helix contributes to binding cannot be eliminated. However, the domains of hGM-CSF most clearly involved in binding to the hGM-CSFR alpha are the distal two-thirds of the third alpha helix, the immediate downstream residues and the extreme carboxy terminus of hGM-CSF.

Animals↗