Makoto Kawakita

Effect of recombinant human interleukin-11 on rat megakaryopoiesis and thrombopoiesis in vivo : comparative study with interleukin-6

Summary. The ability of recombinant human interleukin‐11 (IL‐11) to stimulate rat megakaryopoiesis and thrombopoiesis in vivo was investigated. Once daily subcutaneous injections of IL‐11 at doses of 2, 8 and 20 μg/rat for 5 d caused dose‐dependent increases in platelet counts. The chronic administration of 20 μg/rat/d for 14 d resulted in biphasic increases in platelet counts with peaks at days 8 and 15 of up to 30% over the control, continuing for more than 5 d after cessation of IL‐11 injections. Moreover, a striking increase in megakaryocytic size and ploidy in bone marrow in response to IL‐11 was elicited. IL‐11 induced a dose‐dependent elevation in bone marrow cell numbers but not in splenic weight and cell numbers. Modifications of these parameters were noted as soon as 24 h after the first IL‐11 injections. IL‐11 had a same potency of thrombopoietic effect in rats as compared with IL‐6. However, elevation of acute phase protein such as immunosuppressive acidic protein was 2–2‐fold in rats given 20 μg/d of IL‐6 over those receiving a same dose of IL‐11 (470 v 210 μg/ml). In addition, the rate of body‐weight increase in rats receiving IL‐11 for 5 d as well as 14 d did not differ from that in control animals. In IL‐6 treated rats, the increase in body weight was significantly slower than the controls, which was observed even in the group given 8 μg/d of IL‐6. These results suggest that IL‐11 may be an effective strategy for the treatment of thrombocytopenia.

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack

A 47‐year‐old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement‐sensitivity lysis tests, 80% of her erythrocytes were markedly complement‐sensitive (PNH‐III). Laser cytofluorimetry with a monoclonal antibody against decay‐accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF‐negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patients erythrocytes. When the patients serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

The expression of functional erythropoietin receptors on an interleukin-3 dependent cell line

We report the expression of the erythropoietin receptors on an interleukin-3 dependent cell line. By the transfer into medium supplemented with erythropoietin, DA-1 cells were converted to an erythropoietin dependent growth state. [125I] erythropoietin was used to detect receptors specific for this hormone on the cell surface. Binding studies revealed that erythropoietin bound to 131 +/- 23 receptors/cell with a Kd of 0.54 +/- 0.2 nM. When the cells were incubated at 37 degrees C, trichloroacetic acid soluble radioactivity appeared in the medium after [125I] erythropoietin binding began to decrease, suggesting that the decline represents the degradation of cell associated [125I] erythropoietin- receptor complexes.

Soluble c-kit molecule in serum from healthy individuals and patients with haemopoietic disorders

Summary The proto‐oncogeae, c‐kit, encodes a transmembrane tyrosine kinase receptor (KIT) and plays an important role in haemopoiesis. We have identified a 95kD soluble form of KIT (S‐KIT) in culture supernatant of human megakaryoblastic cell line, CMK. To study the physiological significance of S‐KIT, we have established a sensitive sandwich ELISA system. Serum samples from healthy individuals contained detectable amounts of S‐KIT. Next, we determined a total of 220 samples from 134 patients with haemopoietic disorders. A considerable number of patients with acute myeloid leukaemia (AML), especially those with more immature phenotypes (MO, Ml or M2) had elevated levels of serum S‐KIT. Those levels decreased to the normal range after effective chemotherapy. In chronic myeloid leukaemia, patients with myeloid blastic crisis showed markedly elevated levels of serum S‐KIT. In contrast, S‐KIT levels decreased in cases with either acute or chronic lymphoid leukaemia. There was a tendency for patients with severe aplastic anaemia to show decreased levels, but it was not significant. In myelodysplastic syndrome, S‐KIT levels appeared to vary by subsets, with higher concentration in more advanced forms of the disease. Although the functional role of S‐KIT is not yet elucidated, these results suggest that the serum S‐KIT levels may reflect the pathological states of various haematological disorders.

Thrombopoiesis‐ and Megakaryocyte Colony‐stimulating Factors in the Urine of Patients with Idiopathic Thrombocytopenic Purpura

The urinary extract from patients with severe, chronic idiopathic thrombocytopenic purpura (ITP) was capable of inducing significant thrombocytosis in rats in vivo and enhancing megakaryocyte colony formation in culture of mouse bone marrow cells. The apparent specific activity of megakaryocyte colony stimulating factor (MEG‐CSF) in the ITP extract was approximately one‐half of that of the urinary extract from patients with aplastic anaemia (AA). Daily injections of ITP extract did not cause an increase in Hb concentration, while rats receiving AA urinary extract revealed profound erythropoiesis 3 weeks later. In vitro assay of erythropoietin (EPO) failed to show significant EPO activity in the extract from patients with ITP. These findings excluded the possibility that the observed activities of thrombopoiesis‐stimulating factor (TSF) and MEG‐CSF in the ITP urinary extract are due to contaminating EPO. Urine of patients with severe ITP appears to be a good source of TSF and MEG‐CSF.

Purification of megakaryocyte differentiation activity from a human fibrous histiocytoma cell line: N-terminal sequence homology with activin A

We purified a protein possessing a potent ability to induce the differentiation of a murine megakaryoblastic cell line, L8057, from the supernatant of a human fibrous histiocytoma cell line, KHM-5M. The protein, a homodimer of a molecular weight of 25-kDa, has an N-terminal sequence identical with that of the beta A-chain of inhibin, indicating that it is identical to or highly homologous with activin A. Thus, it is speculated that activin A or its homologue is involved in megakaryocytic differentiation.

Thrombopoiesis and Megakaryocyte Colony Stimulating Factor in the Urine of Patients with Aplastic Anaemia

Summary The urinary extracts from patients with aplastic anaemia and from healthy donors were investigated in vivo and in vitro for their ability to stimulate megakaryopoiesis and platelet production. There was a significantly higher concentration of thrombopoiesis stimulating factor (TSF) and megakaryocyte colony stimulating factor (MEG‐CSF) in the urine from patients with aplastic anaemia than in that from healthy donors. Neuraminidase treatment did not affect the thrombopoietic activity of TSF, whereas coexisting erythropoictin (EPO) in the extract lost its activity in vivo. These findings suggest that TSF and/or MEG‐CSF seems to be different from EPO and that the urine from aplastic anaemia patients would be a good source of TSF and MEG‐CSF for purification and characterization.

Recombinant human interferon α-2b (rh IFNα-2b) therapy for steroid resistant idiopathic thrombocytopenic purpura (ITP)

The efficacy of recombinant human interferon α‐2b (rh IFNα‐2b) in the treatment of steroid resistant idiopathic thrombocytopenic purpura (ITP) was studied in 50 cases.

Apparent heterogeneity of human megakaryocyte colony‐ and thrombopoiesis‐stimulating factors: studies on urinary extracts from patients with aplastic anaemia and idiopathic thrombocytopenic purpura

Summary. Megakaryocyte colony‐stimulating factor (MEG‐CSF) in the urinary extracts from patients with aplastic anaemia (AA) revealed two distinct peaks of activity on Sephadex G‐200 gel filtration with apparent molecular weights of 155 000 and 76 000. Both fractions induced significant thrombocytosis in peripheral blood and megakaryocytosis in the spleen of rats. Heterogeneity of MEG‐CSF was also found in the extracts from the urine of patients with idiopathic thrombocytopenic purpura. The higher molecular weight MEG‐CSF was significantly reduced when the gel filtration was performed under the dissociating conditions. Ion‐exchange chromatography indicated that the higher molecular weight MEG‐CSF had a different charge from the lower molecular weight MEG‐CSF. These results suggest that the apparent heterogeneity of MEG‐CSF is due to interaction of MEG‐CSF with other proteins in the urinary extracts.

Altered Expression of Gangliosides in Erythrocytes of Paroxysmal Nocturnal Hemoglobinuria

In paroxysmal nocturnal hemoglobinuria (PNH), impaired glycosyl-phosphatidylinositol (PI)-anchoring of membrane proteins such as decay-accelerating factor has been known to lead to increased susceptibility to complement. Moreover, abnormal expression of non-PI-anchoring glycoproteins such as C3b/C4b receptor (CR1) or glycophorin-alpha also has been shown in PNH. Therefore, we biochemically analyzed glycosphingolipids (GSL) as one of the membrane glycoconjugates of PNH erythrocytes. Erythrocytes of all seven PNH patients showed altered expression of sialosyl GSL (gangliosides) as compared with the control erythrocytes of healthy donors. Both a sialosylparagloboside (IV6NeuAc-nLc4Cer) among four major gangliosides and some minor gangliosides in normal erythrocytes variably disappeared in erythrocytes from the peripheral blood of PNH patients. As one of the possible mechanisms of altered expression of gangliosides in PNH erythrocytes, structural analysis suggested impaired sialylation of GSL. These results suggest not only the altered metabolism of gangliosides in PNH erythrocytes, but also a metabolic disorder of membrane glycoconjugates as a new feature of PNH.