Tomoyuki Tahara

A sensitive sandwich ELISA for measuring thrombopoietin in human serum: serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders

A sensitive sandwich enzyme‐linked immunosorbent assay (ELISA) has been established to estimate serum thrombopoietin (TPO) concentrations in healthy volunteers and patients with haemopoietic disorders. The ELISA uses a mouse monoclonal antibody (Ab) as the capture Ab and a biotinylated rabbit polyclonal Ab as the detector. The ELISA was reproducible, highly sensitive and specific for human TPO. The coefficients of intra‐ and inter‐assay variation were from 3.0% to 4.9% and from 5.9% to 6.1%, respectively. The quantitative limit of the ELISA was 0.09 fmol/ml in serum. The quantitative limit was lower than the normal level. The dose–response curves of serum samples from healthy volunteers and patients with haemopoietic disorders were parallel to the standard curves. The ELISA did not cross‐react with a variety of blood components and cytokines to produce false‐positive results.

Circulating thrombopoietin level in chronic immune thrombocytopenic purpura

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system. The TPO level (mean±SD) in ITP patients was mildly elevated (1.86±1.17 fmol/ml) compared to that in normal subjects (0.76±0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35±6.42 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.

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 (hTPO163) to a 2.5-Å resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO163 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 hTPO163 interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 × 109 M–1 and 1.1 × 106 M–1. The presence of the neutralizing Fab did not inhibit binding of hTPO163 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.

Functional analysis of the cytoplastic domain of the human Mpl receptor for tyrosine-phosphorylation of the signaling molecules, proliferation and differentiation

To investigate the functional domains for signal transduction of human Mpl, we constructed a series of human c‐mpl cDNAs with various deletions in the cytoplasmic domain, and then introduced each cDNA into murine IL‐3‐dependent myeloid leukemia FDC/P2 cells to establish stable transformants. We examined the growth and differentiation responses and tyrosine phosphorylation of the intracellular signaling proteins including Jak2, Tyk2, Stat3, Stat5, Vav, SHPTP2, Cbl, Shc and Shc‐associated p145 when receptor stimulation occurred after thrombopoietin (TPO) binding. TPO stimulated cell proliferation and induced the expression of megakaryocyte lineage‐specific AP‐51 and CD61 cell surface antigens and tyrosine phosphorylation of the signaling proteins in transformants expressing full length human Mpl. These results suggested that Mpl not only induced proliferation but also transduced megakaryocyte‐specific differentiation signals into FDC/P2 cells. Mutational analysis of human Mpl indicated that the N‐terminal region of its cytoplasmic domain is necessary and sufficient to transduce proliferation and differentiation signals into cells, while the C‐terminal region may also play important roles in transducing the differentiation signals.

Serum Thrombopoietin Level in Various Hematological Diseases

To investigate the pathophysiological role of thrombopoietin (TPO) in thrombopoiesis, we measured its serum levels in 15 healthy individuals, 84 patients with various hematological diseases and 2 patients with liver cirrhosis using an enzyme immunoassay procedure. The TPO level was 0.84 ± 0.40 f mol/ml in normal individuals. TPO levels were considerably elevated in patients with myelosuppression after intensification chemotherapy of acute leukemia in complete remission (postchemotherapy group; n = 18; 18.46 ± 9.70 f mol/ml). When the data of normal individuals and the postchemotherapy group were combined, TPO levels were inversely correlated with the platelet count in this combined group. We compared these data of normal individuals and the postchemotherapy group with various hematological disease states. In aplastic anemia (n = 13; 16.03 ± 9.44 f mol/ml), acute lymphoblastic leukemia (n = 5; 10.36 ± 5.57 f mol/ml), malignant lymphoma (n = 6; 2.79 ± 2.27 f mol/ml), multiple myeloma (n = 3; 3.34 ± 0.20 f mol/ml) and chronic lymphocytic leukemia (n = 2; 1.71 ± 3.91 f mol/ml), the relationship of serum TPO levels and platelet counts was almost the same as in the combined group with normal individuals and the postchemotherapy group. However, the TPO levels were slightly higher in myeloproliferative disorders (n = 12; 1.99 ± 1.47 f mol/ml) and lower in acute myelogenous leukemia (n = 8; 2.27 ± 1.25 f mol/ml), hypoplastic leukemia (n = 3; 2.76 ± 2.23 f mol/ml), myelodysplastic syndrome (n = 2; 0.42 ± 0.60 f mol/ml), liver cirrhosis (n = 2; 1.50 ± 0.92 f mol/ml) and idiopathic thrombocytopenic purpura (n = 12; 2.08 ± 1.41 f mol/ml), when compared to the regression line for the combined group with normal individuals and postchemotherapy group. These findings suggest that TPO might play an important role in regulation of the platelet count in normal and pathological conditions.

Native thrombopoietin: Structure and function

Thrombopoietin (TPO), the c‐Mpl ligand, is produced constitutively in liver and other organs, circulates in the bloodstream, and is delivered to bone marrow, where it stimulates the early development of multiple hematopoietic lineages and megakaryocytopoiesis. The concentration of TPO in blood is regulated by c‐Mpl mass on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including the possible modulation of TPO mRNA abundance in bone marrow, megakaryocytopoiesis and platelet production may be regulated as a result of modulation of TPO activity by proteolytic processing that generates truncated forms of the molecule. Characterization of TPO partially purified from human plasma, however, revealed that the full‐length molecule was the predominant form in the blood of both normal individuals and thrombocytopenic patients, although small amounts of truncated species were detected. Thus, truncation of TPO, at least that in the circulation examined, does not appear to contribute to the direct regulation of platelet production in response to increased demand. Given that native TPO isolated from the plasma of thrombocytopenic animals comprises truncated forms, the truncation of TPO is likely of physiological importance in the life history of this molecule.

Thrombopoietin in Patients with Hepatoblastoma

Marked thrombocytosis (over 50 × 104/μl) is frequently seen in patients with hepatoblastoma. Thrombopoietin (TPO), c‐mpl ligand, has recently been purified as the major physiological regulator of the thrombopoiesis and is mainly produced in the liver. Since it is possible that TPO participates in thrombocytosis and the tumor growth of this particular hepatic tumor, serum TPO levels in addition to interleukin 1β (IL‐1β) and IL‐6 levels were assessed in seven untreated patients by using a sandwich enzyme‐linked immunosorbent assay. High serum TPO levels were observed in all of the examined patients. The level ranged from 3.15 to 11.02 (mean ± standard deviation; 6.08 ± 1.25) fmol/ml. IL‐6 levels were also somewhat higher than normal. Platelet counts, however, appeared to correlate more with serum TPO levels (p = 0.1) than with IL‐1β (p = 0.5) and IL‐6 (p = 0.2) levels. Furthermore, using the reverse transcriptase polymerase chain reaction method, the expression of c‐mpl mRNA was found in five of eight hepatoblastoma tissues as well as TPO mRNA in all eight tissues. These observations suggest that thrombocytosis in hepatoblastoma patients results from the production of cytokine members, including TPO, within tumor tissues. Additionally, it is possible that TPO might act as a type of autocrine and/or paracrine system for cellular growth in this tumor.

Serum thrombopoietin level is mainly regulated by megakaryocyte mass rather than platelet mass in human subjects.

A patient with idiopathic thrombocytopenic purpura (ITP) developed T‐cell lymphoma while undergoing steroid therapy. We examined the relationship between the patients serum thrombopoietin (Tpo) level, platelet count, megakaryocyte number and CFU‐Meg number during the second 5 d course of chemotherapy for lymphoma in which megakaryopoiesis switched from ITP phase to amegakaryocytic phase. The patients platelet count was temporarily elevated but CFU‐Meg numbers were markedly suppressed, and megakaryocyte numbers were decreased in this period, whereas serum Tpo level was not suppressed despite an increased platelet count, indicating that serum Tpo level is mainly regulated by megakaryocyte mass.

Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

ABSTRACT Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.

Complement Activation Plays a Key Role in Antibody-Induced Infusion Toxicity in Monkeys and Rats

Infusion reactions are a major side effect of the administration of therapeutic Abs and are the result of a complex immune reaction. In this study, we report that substitutions of Fc amino acids in the anti-HLA-DR Ab HD8 reduce its ability to induce infusion reactions in rats and monkeys. We first showed that i.v. administration of IgG1- and IgG2-subclass HD8 Abs induces severe infusion reactions in monkeys. These Abs express strong complement-dependent cytotoxicity (CDC), and in vivo depletion of complement in rats by pretreatment with cobra venom factor abrogated the lethal infusion reactions generated by HD8-IgG1. Thus, the infusion reactions appear to be largely driven by the complement system. To reduce the CDC function of HD8-IgG1, its Fc region was modified by two amino acid substitutions at Pro331Ser and Lys322Ala. The modified Ab was incapable of expressing CDC in vitro and did not induce severe infusion reactions in rats and monkeys, even at extremely high doses. The modified Ab retained its Ab-dependent cellular cytotoxicity function as well as its antitumor activity in a tumor-bearing mouse model. In summary, complement appears to drive infusion reactions, and modifications that eliminate the CDC activity of an Ab also reduce its ability to induce infusion reactions.