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. The serum TPO concentrations from 29 normal males and 21 females were 0.79 +/- 0.35 and 0.70 +/- 0.26 fmol/ml, respectively. Serum TPO levels in patients with aplastic anaemia (AA), acute lymphocytic leukemia (ALL) and essential thrombocythaemia (ET) were measured using the ELISA. The serum TPO levels in the patients with ET (n = 6, 2.80 +/- 1.55 fmol/ml) were higher than the normal level. The patients with AA (n = 7, 18.53 +/- 12.37 fmol/ml) and ALL (n = 5, 10.36 +/- 5.57 fmol/ml) had significantly higher serum TPO levels than normal individuals. These results indicate that the ELISA specific to TPO should prove useful in measuring the TPO concentration in serum samples.

Dendritic cells (DC) are a distinct population of leukocytes and specialized antigen-presenting cells for T cell responses. Prior work has shown that GM-CSF can induce the development of large numbers of DC from proliferating progenitors in mouse bone marrow. We have monitored the effects of potentially enhancing and suppressive cytokines in these cultures. In this system, many immature DC develop from proliferating precursors during the first six days of culture, and between days 6-8 maturation of typical nonadherent and nonreplicating DC takes place. The maturation is accompanied by a large increase in the expression of major histocompatibilities complex class II (MHC II) and B7-2/CD86, and in mixed leukocyte reaction stimulating activity. Tumor necrosis factor-α (TNF-α), previously shown to be required for development of human DC, was found to enhance the maturation of mouse DC in the last two days of culture. Transforming growth factor-β1(TGF-β1), on the other hand, almost totally blocked DC maturation, but it had to be given in the first six days of culture when the DC were actively proliferating. TGF-β1 did not block the production of immature, MHC II-positive but B7-2/CD86-negative DC. Maturation would take place between days 6-8 as long as the cultures were depleted of Fc-receptor-bearing cells, or if TNF-α were added. In both instances, maturation was not blocked even when TGF-β1 remained in the culture. We conclude that the development of DC, in response to GM-CSF, can be modified by other cytokines. TGF-β1 is suppressive but only indirectly via Fc-receptorbearing suppressive cells, presumably suppressive macrophages, while TNF-α enhances the final maturation of DC.

The methylotrophic yeast Ogataea minuta IFO 10746 was selected as a suitable strain for producing human-compatible glycoproteins by means of analyses of its cell-wall mannoproteins. First, the OmURA3 gene encoding an orotidine-5'-phosphate decarboxylase was cloned and disrupted to generate a host strain with a uracil auxotrophic marker. Second, both the promoters and the terminators from the OmAOX1 gene encoding an alcohol oxidase for an inducible promoter, or those from the OmTDH1 gene encoding a glyceraldehyde-3-phosphate dehydrogenase for a constitutive promoter, were isolated to construct an expression vector system for heterologous genes. Next, the OmOCH1 gene encoding a starting enzyme with alpha-1,6-mannosyltransferase activity to form a backbone of the N-linked outer sugar chain peculiar to yeast was disrupted, and an alpha-1,2-mannosidase gene from Aspergillus saitoi with an endoplasmic reticulum retention signal (HDEL) under the control of the OmAOX1 promoter was introduced to convert the sugar chain to Man5GlcNAc2 in O. minuta. As a result, we succeeded in breeding a new methylotrophic yeast, O. minuta, producing a Man5GlcNAc2-high-mannose-type sugar chain as a prototype of a human-compatible sugar chain. We also elucidate here the usefulness of the strategy for producing human-compatible sugar chains in yeast.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 A resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded 8-sheets on the inside and six a-helices on the outside. There is a 5-A-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor.Each subunit has a new class of a + 13 sandwich structure consisting of two P-a-P3 motifs. These P-a-38 motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.Glycosylation-inhibiting factor (GIF), is a 13-kDa cytokine involved in the formation of IgE-suppressive factor (1). GIF inhibits N-glycosylation of IgE-binding factors, and the unglycosylated IgE-binding factor then selectively suppresses IgE synthesis. Furthermore, GIF facilitates generation of antigenspecific suppressor T cells (2), and appears to be a subunit of antigen-specific suppressor T-cell factors (1). Indeed, the major cell source of GIF activity is antigen-specific suppressor T cells (3). After molecular cloning of GIF, however, it was found that various cell line cells contained 0.6-kb mRNA, which hybridized with GIF cDNA (4), and secreted the 13-kDa protein that reacted with polyclonal antibodies against recombinant GIF (5). Nevertheless, only the peptide from suppressor T cell had GIF bioactivity. More recent experiments provided evidence that the bioactive GIF and inactive GIF have an identical amino acid sequence and suggested that the bioactivity of GIF is generated by posttranslational modifications of the inactive protein (5, 6). Since neither N-glycosylation nor phosphorylation is involved in the generation of bioactive GIF, we speculated that the heterogeneity of GIF is a conformational transition of the protein (5). The present experiments were undertaken to determine the three-dimensional structure of recombinant human GIF in order to get an insight into the structural basis of its bioactivity. It was found that the crystal structure of GIF is a novel structure with a trimeric barrel with three 6-stranded P-sheets. ¶ MATERIALS AND METHODSCrystallization of GIF. The recombinant human GIF was expressed in Escherichia coli and was purified as described (6 Table 2.

We examined the effects of recombinant human thrombopoietin (rhTPO) on myelosuppressive chemotherapy-induced thrombocytopenia in cynomolgus monkeys. After treatment with nimustine (ACNU) on day 0, the monkeys intravenously received rhTPO at a dose of 0.04, 0.2 or 1 microgram/kg/d or monkey's serum once each day from day 1 to day 28. Administration of rhTPO reduced the severity of thrombocytopenia and accelerated the rate of platelet recovery in a dose-dependent fashion. Treatment with the highest rhTPO dose completely prevented thrombocytopenia and stimulated a marked increase in platelet counts over the normal values. Animals treated with ACNU also became neutropenic and slightly anaemic. Administration of rhTPO following ACNU treatment significantly improved neutropenia with increasing doses of rhTPO, but had no effect on anaemia. Compared to the control animals, rhTPO-treated animals exhibited no significant changes in several serum parameters. C-reactive protein concentration and some blood coagulation profiles within the study period. These results suggest a therapeutic efficacy of rhTPO in improving chemotherapy-induced thrombocytopenia.