Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor γ-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific autoAbs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.
renal urea transporters during LPS-induced acute renal failure is mediated by proinflammatory cytokines and is independent from renal ischemia due to sepsis-induced hypotension.Background Studies reveal that regulatory T (T reg ) cells control immune responses; therefore these responses must be controlled to enable effective protection against infections and cancer. CCR4 knockout (CCR4 -/-) mice are more resistant to lipopolysaccharide shock. So, our aim is to study the mechanisms involved in the resistance of CCR4 -/mice subjected to severe sepsis by cecal ligation and puncture (CLP) and how T reg cells modulate this effect. Methods C57/BL6 mice were subjected to a CLP model, whereby the cecum was partially ligated and punctured nine times with a 21 G needle. Sham-operated mice were used as control. Mice subjected to CLP and sham surgery were treated with antibiotic from 6 hours after surgery until 3 days. Results CCR4 -/mice subjected to CLP presented an increase in the survival rate (78%) compared with wild-type mice (17%), and presented a marked improvement in the innate response concerning neutrophil migration to the peritoneum and lung, bacterial load and cytokine levels compared with wild-type mice. Besides, T reg cells from CCR4 -/-CLP mice did not inhibit proliferation of T effector cells as observed for T reg cells from wild CLP mice, at a proportional ratio of T effector:T reg cells. Interesting, T reg cells from CCR4 -/-CLP mice inhibit 30% of neutrophil migration to bronchoalveolar lavage when co-injected with fungal challenge as secondary infection in sham recipient mice, while the T reg cells from wild CLP mice inhibit 80%, much more than expected. Conclusion These results suggest that T reg cells from CCR4 -/mice did not present a suppressive response and this could be an important factor in their survival. These results are strong evidence for the role of T reg cells in immunosuppression following severe sepsis. P3Abdominal sepsis: efficacy of passive immunotherapy
In order to clarify the effective regions that are substantially involved in the gelling process of plasma glycoprotein fibrinogen, we examined the aggregating properties of plasmin-treated fibrinogen, fragment-X. Two types of fragment-X were prepared by the digestion at 6 o C and 37 o C (fragment-XY and -XN, respectively). αC regions were cleaved thoroughly in both samples, but the amount of cleavage of BβN region differed between them (higher in the fragment-XN). Thrombin-and reptilase-catalyzed fibrin polymerizations were studied for both of the samples. Although the B-knob:b-hole interaction does neither work in the reptilase-catalyzed fibrin polymerization nor the αC-αC interaction is present, network formation proceeded in the fragment-XY with delayed polymerization. In the fragment-XN, protofibril formation occurred, but lateral aggregation did not. Mixing effect of intact fibrinogen showed that BβN region might play an important role in the lateral aggregation process cooperatively with αC-domain. .
The role of N-linked carbohydrate chains, bound to the polypeptide chains Bβ and γ of fibrinogen, in the process of fibrin gel formation has not been understood well yet, although it has been well known that the deglycosylation accelerates the fibrin polymerization. We investigated the time course of the fibrin polymerization employing the turbidity and light scattering measurements focusing on the role of carbohydrate chains. Deglycosylated bovine fibrinogen was prepared by using peptide N-glycosidase F, PNGF, and the effects of its mixing with the intact one were examined. Fibrinopeptide release and protofibril growth were not affected by the deglycosylation at all. However, only a slight mixing of deglycosylated fibrinogen by 5 and 10 % resulted in a markedly accelerated polymerization and the highly promoted switchover from the protofibril growth to the lateral aggregation of protofibrils. It was suggested that N-linked carbohydrate chains could interact with αC and BβN regions so as to suppress their releases from the central region of fibrinogen molecule, and play a regulating role of the switchover from the protofibril growth to the lateral aggregation.
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