Summary. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 mg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-b1 (TGF-b1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7-and 2-fold, respectively. The elevated levels of TGF-b1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 mg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-b1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcβ1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

Background: Several studies have shown that cultured eosinophils can be generated from human umbilical cord blood mononuclear cells (UCMC) in the presence of interleukin (IL)–3 and IL–5 in vitro. Other reports have indicated that cellular adhesion to hyaluronic acid (HA) enhances the proliferation of cultured eosinophils derived from CD34+ cells purified from UCMC. The aim of this study was to obtain large numbers of mature eosinophils from UCMC using IL–3, IL–5 and HA, and to investigate their functions. Methods: We examined several combinations of IL–3 and IL–5 and their effect on eosinophil development from UCMC in HA–coated on non–coated flasks. We also examined whether cultured eosinophils degranulated eosinophil–derived neurotoxin (EDN) induced by secretory immunoglobulin A conjugated to sepharose beads (sIgA–beads) and responded to eotaxin. Results: Culture with HA–coated flasks for 35 days (in the presence of IL–3 and IL–5, with IL–3 omitted after day 14 of culture) caused a 11.2–fold augmentation in the proliferation of UCMC. On day 35 of the culture, 98% of cultured cells were eosinophils judging from May–Grünwald and Giemsa staining and transmission electron micrographs. The EDN content of the cultured eosinophils on day 35 was 156 ng/105 cells. Cultured eosinophils degranulated EDN induced by sIgA–beads and responded to eotaxin by chemotaxis and intracellular Ca2+ mobilization. Conclusion: We found a useful culture system to obtain large numbers of eosinophils derived from UCMC, which may facilitate the investigation of eosinophil function, since there was no significant difference in response to sIgA–beads and eotaxin between cultured and peripheral eosinophils.

We examined whether pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) affected 5-fluorouracil-induced thrombocytopenia without inducing more severe intimal thickening after injury to rat carotid arteries. Rat carotid arteries were injured using a balloon catheter on day 0. 5-Fluorouracil (100 mg kg(-1)) or vehicle was intravenously administered on day 1 in balloon-injured rats. PEG-rHuMGDF (100 microg kg(-1)) or vehicle was intravenously administered once a day on days 1-5 to balloon-injured rats given 5-fluorouracil or vehicle. 5-Fluorouracil (100 mg kg(-1), i.v.) caused a significant decrease in the platelet count from day 3 and peaked on days 7-9 in balloon-injured rats. PEG-rHuMGDF (100 microg kg(-1), i.v.) reduced this decrease on days 9 and 11. The administration of PEG-rHuMGDF did not accelerate the intimal thickening of balloon-injured arteries in rats treated with 5-fluorouracil compared with control balloon-injured rats. PEG-rHuMGDF did not increase plasma tumour growth factor-beta1 (TGF-beta1) from days 0-9 in balloon-injured rats compared with control balloon-injured rats. These results suggest that PEG-rHuMGDF ameliorated 5-fluorouracil-induced thrombocytopenia without accelerating the intimal thickening of balloon-injured arteries.

Abstract:We studied the time courses of the number of osteoclasts, the expression of osteoblast differentiation markers and TGF-β1 levels in the platelet-poor plasma (PPP) in order to clarify the mechanism of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)-induced bone formation in the mouse. Mice received PEG-rHuMGDF subcutaneously at a suprapharmacological dose (1.0 mg/kg) daily for 5 days (Day of the first injection: Day 1). PEG-rHuMGDF caused a gradual increase of platelet count, with a maximum increase on day 9. Histological analysis showed an increase of reticulin fibers on the inner side of endosteum on day 9, partial osteoid formation on day 11, new and excessive bone formation on day 13. On day 9, at the early stage of reticulin fiber increase, the number of osteoclasts was decreased. On day 11, osteopontin (OPN) mRNA-expression, a marker of osteoblast production, was observed in the reticulin fiber and osteoid near the substantia of bone marrow, whereas OPN mRNA-expressing cells did not express bone Gla protein (BGP) mRNA, a marker of osteoblasts, indicating that the expression of OPN mRNA was induced earlier than that of BGP mRNA during the course of osteogenesis. Additionally, the level of TGF-β1 in the PPP was increased, with about 2.5-fold elevation compared with the vehicle-treated mice at day 9. These findings suggest that PEG-rHuMGDF causes increase of reticulin fibers and osteogenesis by stimulating osteoblast differentiation and inhibiting osteoclast differentiation via an increase of the TGF-β1 level in the bone marrow. (J T oxicol Pathol 2001; 14: 113-120)