Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.
Recent studies suggest that the hydroxyl radical (.OH) plays a pathogenetic role in postischemic ventricular dysfunction (myocardial "stunning"). This concept, however, is predicated exclusively on results obtained in anesthetized open-chest preparations, which are subject to the confounding influence of many unphysiological conditions and in which both myocardial stunning and free radical generation are greatly exaggerated. The lack of supporting evidence in more physiological animal models represents a major limitation of the .OH hypothesis of stunning. Furthermore, concern has been raised that myocardial stunning may be a period of "rest" necessary for full recovery, so that attenuation of the early phase of stunning by antioxidant therapy may have subsequent detrimental effects on the resting function and/or on the return of myocardial contractile reserve. To address these issues, in phase 1 of this study conscious unsedated dogs undergoing a 15-minute coronary artery occlusion received an intravenous infusion of normal saline (n = 22), of the .OH scavenger N-2-mercaptopropionyl glycine (MPG, n = 17), or of the iron chelator desferrioxamine (DF, n = 14). Compared with control dogs, the dogs treated with MPG or DF exhibited significantly greater postischemic wall thickening throughout the first 6 hours of reperfusion; the total deficit of wall thickening during this time interval was reduced 50% by MPG and 50% by DF. The magnitude of this beneficial effect was a function of the severity of ischemia, so that the dogs with the lowest collateral flows had the greatest improvement of wall thickening. The accelerated recovery produced by MPG and DF in the first 6 hours was not followed by any deterioration of resting wall thickening at 24 or 48 hours. Furthermore, in dogs treated with MPG or DF, the increase in wall thickening elicited by maximal inotropic stimulation (isoproterenol or dopamine) was similar before stunning and shortly after resting wall thickening had normalized (24 or 48 hours after reflow); thus, despite the fact that most of the early postischemic dysfunction had been eliminated by antioxidant therapy, there was no subsequent impairment of either resting function or contractile reserve. In phase 2, production of free radicals (measured with the spin trap alpha-phenyl N-tert-butyl nitrone) was markedly (> 80%) inhibited by the same doses of MPG and DF that attenuated stunning in phase 1.(ABSTRACT TRUNCATED AT 400 WORDS)
PEGylation of B-domain deleted factor VIII (PEG-FVIII-BDD) prolongs the half-life of the molecule by approximately twofold in animals (Mei et al., Blood 2010; 116: 270). To investigate the role of von Willebrand factor (vWF) in the catabolism of PEG-FVIII-BDD in vivo, a FVIII-BDD mutant (F8V), which is incapable of binding vWF, was generated by deleting the vWF-binding region in the a3 domain of FVIIII-BDD. F8V was expressed, purified and PEGylated by site-specific conjugation. The biochemical and biological properties of F8V and PEGylated F8V (PEG-F8V) were evaluated in vitro and in vivo. The specific activity of purified F8V by a chromogenic assay was similar to FVIII-BDD and PEGylation had minimal impact on the specific activity of F8V in this assay. Analysis by Biacore indicated that both F8V and PEG-F8V display greatly reduced vWF binding in vitro. Pharmacokinetic studies in FVIII knockout (HaemA) mice showed that the terminal half-life (T1/2 ) of F8V was dramatically reduced relative to FVIII-BDD (0.6 h vs. 6.03 h). PEGylation of F8V promoted a significant increase in T1/2 , although PEGylation did not fully compensate for the loss in vWF binding. PEG-F8V showed a shorter T1/2 than PEG-FVIII-BDD both in HaemA mice (7.7 h vs. 14.3 h) and in Sprague-Dawley male rats (2.0 ± 0.3 h vs. 6.0 ± 0.5 h). These data demonstrated that vWF contributes to the longer T1/2 of PEG-FVIII-BDD. Furthermore, this suggests that the clearance of the FVIII:vWF complex, through vWF receptors, is not the sole factor which places an upper limit on the duration of PEG-FVIII circulation in plasma.
Portal fibroblasts have been considered responsible for biliary fibrosis. Since lipocytes show differentiation toward myofibroblast‐like cells in hepatic fibrogenesis, we studied whether similar differentiation of portal fibroblasts could be observed in biliary fibrosis. We examined rat livers after bile duct ligation by double immunofluorescent staining of α‐smooth‐muscle actin (α‐smA) and desmin and also by electronmicroscopy. In the portal tract of normal livers, α‐smA‐positive cells were noted only in the vessel wall, whereas desmin‐positive cells were occasionally seen in the connective tissue as well. With the development of biliary fibrosis, α‐smA was remarkably expressed in the portal connective tissue, while desmin was seen in a small portion of α‐smA‐positive cells around proliferating bile ducts. In normal livers, portal fibroblasts presented quiescent features, such as a small Golgi complex and a few cisternal profiles of endoplasmic reticulum under electron microscopy. After 7 days of bile duct ligation, portal fibroblasts proliferated, were arrayed in multilayers, and were associated with collagen bundles. Some of these fibroblasts had numerous cytoskeletal components, and developed rough endoplasmic reticulum and a dense body. These data suggest that portal fibroblasts appear to differentiate toward myofibroblasts in biliary fibrosis.
Postoperative delirium is a common complication that often results in poor outcomes in surgical and elderly patients. Accumulating evidences suggest that the pathophysiology of delirium results from multiple neurotransmitter system dysfunctions. To further clarify the effects of the selective serotonin (5-HT) (1A) antagonist WAY-100635 on the behaviors in scopolamine induced-delirium rats and to explore the molecular mechanism, in this study, we investigated the change of monoamine levels in the cerebrospinal fluid (CSF) and different brain regions using high-performance liquid chromatography and assessed the behavioral retrieval of delirium rats treated with WAY-100635. It was found that 5-hydroxy-3-indoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid, and homovanillic acid concentrations in the CSF of scopolamineinduced delirium rats were significantly increased, among which 5-HIAA was also increased in hippocampus and basolateral amygdala (BLA), and 5-HT(1A) receptor was significantly higher in the hippocampuses and BLA than other brain regions. Furthermore, intrahippocampus and intra-BLA stereotactic injection of WAY-100635 improved the delirium-like behavior of rats. Mechanistically, after WAY-100635 treatment, significant reduction of IL-1b release into CSF and NOD-like receptor family, pyrin domain containing 3 (NLRP3) expression, phosphorylated phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), and S6K was observed. Altogether, these results suggest that delirium rats induced by scopolamine may be correlated with an increased cerebral concentration of 5-HT and dopamine neurotransmitters system; the selective 5-HT(1A) antagoniszts can reverse the delirium symptoms at some extent through tendering PI3K/Akt/mammalian target of rapamycin complex 1 (mTOR) activation-induced NLRP3 activity and then reducing IL-1b release. V C 2016 IUBMB Life, 68(4):311-319, 2016Keywords: neurotransmitter; neuroinflammation; 5-HT(1A) receptor antagonist; hippocampus; amygdala Abbreviations: POD, postoperative delirium; HPLC, high-performance liquid chromatography; NE, noradrenaline; DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HIAA, 5-hydroxy-3-indoleacetic acid; 5-HT, serotonin; BLA, basolateral amygdala; PCR, polymerase chain reaction; CSF, cerebrospinal fluid; i.p., intraperitoneally; WAY-100635, N-{2-[4-(2-methoxyphenyl)21-piperazinyl]ethyl}-N-(2-pyridyl)cyclohexanecarboxamide; IL-1b, interleukin-1 beta; NLRP3, NOD-like receptor family, pyrin domain containing 3; PI3K, phosphatidylinositol-3-kinase; Akt, protein kinase B (PKB); mTORC1, mammalian target of rapamycin complex 1; S6K, ribosomal S6 protein kinase.
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