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.

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.

1 The effect of KRN2391, a novel vasodilator, on the changes of electrocardiogram caused by endothelin-1 (ET-1) was studied in anaesthetized rats and compared with the effects of nicorandil and diltiazem. In addition, the effect of KRN2391 on the action potential of guinea-pig papillary muscle was studied. 2 The intracoronary administration (i.c.) of ET-1 (5 jg) induced not only ST segment elevation of the electrocardiogram due to contraction of the coronary artery, but also arrhythmias involving atrioventricular block (A-V block), ventricular premature contraction (VPC) and ventricular fibrillation (VF), and resulted in death in most animals. However, the administration of methacholine (3 jig, i.c.)produced ST segment elevation alone without developing arrhythmias.3 Pretreatment with intravenous administration of KRN2391 (30 1g kg-') inhibited the ST segment elevation and the development of arrhythmias induced by ET-1, and decreased the incidence of death.4 Nicorandil (1000 tLg kg-') prevented the ST segment elevation without suppression of the occurrence of VF. Diltiazem (100 pg kg-') suppressed both the ST segment elevation and the occurrence of VF but not other arrhythmias. Nicorandil at 3000 jig kg-' and diltiazem at 300 tg kg-' produced not only a suppression of ST segment elevation and VF incidence but also a decrease in the occurrence of arrhythmias. These doses of nicorandil and diltiazem produced a decrease in death in a dose-dependent manner.5 KRN2391 (10 and 30 ig kg-'), nicorandil (1000 and 3000 ig kg-') and diltiazem (100 and 300 fig kg-') significantly decreased mean blood pressure in a dose-dependent manner. Heart rate was decreased by nicorandil (3000 Igkg-') and diltiazem (100 and 300 gkg-') but was not affected by KRN2391 (10 and 30 Ag kg-').6 KRN2391 (30pjM) significantly shortened the action potential duration of guinea-pig ventricle at 50% and 90% repolarization (APD50 and APD%0). The effect of KRN2391 was inhibited by a K+ channel blocker, glibenclamide (301iM). 7 These results suggest that the occurrence of ST segment elevation and arrhythmias induced by ET-1 are due to a dual direct action on both coronary vascular smooth muscle and myocardium. Therefore, the protective effects of KRN2391, nicorandil and diltiazem on ET-l-induced heart disorders appear to be due to their direct actions on coronary vascular smooth muscle and the myocardium.

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Limited endogenous nitric oxide synthesis and elevated endogenous endothelin-1 production during the first few days of life may contribute to pulmonary hypertension in infants with persistent pulmonary hypertension of the newborn.