No abstract

Mucosal-associated invariant T (MAIT) cells are a subset of innate-like lymphocytes that are restricted by major histocompatibility complex-related molecule 1 (MR1). In this study, we investigated the role of MAIT cells in the pathogenesis of lupus in FcγRIIb−/−Yaa mice, a spontaneous animal model of lupus. Using two approaches of MAIT cell deficiency, MR1 knockout animals and a newly synthesized inhibitory MR1 ligand, we demonstrate that MAIT cells augment the disease course of lupus by enhancing autoantibody production and tissue inflammation. MR1 deficiency reduced germinal center responses and T cell responses in these mice. Suppression of MAIT cell activation by the inhibitory MR1 ligand reduced autoantibody production and lupus nephritis in FcγRIIb−/−Yaa mice. MAIT cells directly enhanced autoantibody production by B cells in vitro. Our results indicate the contribution of MAIT cells to lupus pathology and the potential of these cells as novel therapeutic targets for autoimmune diseases such as lupus.

Recent studies have revealed that the platelet adhesive process under flow is tightly regulated by multiple ligand-receptor interactions. However, platelet morphological changes during this process, particularly its physiological relevance, remain unknown under blood flow conditions. Using epifluorescence and scanning electron microscopy, we evaluated the real-time changes in platelet morphology during a platelet adhesive process on a von Willebrand factor-coated surface under physiological high shear flow in a perfusion chamber. Here, we show that dynamic platelet shape changes occurring during distinct phases of the adhesive process are precisely regulated by "inside-out" and "outside-in" integrin signals and are also a key regulatory element in successful platelet thrombogenesis opposing rapid blood flow in vivo.

The metalloprotease ADAMTS13 is assumed to regulate the functional levels of von Willebrand factor (VWF) appropriate for normal hemostasis in vivo by reducing VWF multimer size, which directly represents the thrombogenic activity of this factor. Using an in vitro perfusion chamber system, we studied the mechanisms of ADAMTS13 action during platelet thrombus formation on a collagen surface under whole blood flow conditions. Inhibition studies with a functionblocking anti-ADAMTS13 antibody, combined with immunostaining of thrombi with an anti-VWF monoclonal antibody that specifically reflects the VWFcleaving activity of ADAMTS13, provided visual evidence for a shear ratedependent action of ADAMTS13 that limits thrombus growth directly at the site of the ongoing thrombus generation process. Our results identify an exquisitely specific regulatory mechanism that prevents arterial occlusion under high shear rate conditions during mural thrombogenesis. IntroductionThe adhesive protein von Willebrand factor (VWF) plays a major role in platelet thrombogenesis, a process crucial for hemostasis. However, the excessive function of VWF is thought to increase the risk of fatal arterial thrombosis. 1,2 The thrombogenic activity of VWF is strictly dependent upon its multimeric structure, which is thought to be regulated in vivo by the metalloprotease ADAMTS13 through its cleavage of the A2 domain of the VWF subunit. 3,4 Indeed, patients with congenital deficiency of ADAMTS13 suffer repeated thrombotic complications attributed to excessive function of the ultra-large VWF (ULVWF) multimer, which is not found in normal blood circulation. [3][4][5][6] This concept was recently confirmed by knock-out mouse studies, in which ADAMTS13 Ϫ/Ϫ mice exhibited enhanced thrombogenicity in the ex vivo or in vitro experimental blood flow conditions tested. 7,8 The mechanisms by which ADAMTS13 regulates VWF remain poorly understood. However, recent studies showing that ADAMTS13 under flow conditions can rapidly cleave ULVWF secreted from and anchored to cultured endothelial cell layers 9,10 have raised the possibility that blood flow is critical in activating ADAMTS13. 11 Indeed, the VWF-cleaving activity of ADAMTS13 cannot be reproduced in vitro under static conditions unless the substrate VWF molecule is somewhat modified (eg, denatured by guanidine-HCl or urea). 3,4 Further, the question arises of whether ADAMTS13, in addition to its known action on ULVWF freshly released from endothelial cells, might also act directly at the local sites of thrombus generation to regulate thrombus growth.To address these issues, we analyzed the role and mechanisms of ADAMTS13 action in mural platelet thrombogenesis on a collagen-coated glass surface in an in vitro perfusion chamber system. Our visual evidence demonstrates that ADAMTS13 cleaves VWF and down-regulates mural thrombus growth at the site of ongoing thrombus generation in a shear rate-dependent manner under whole blood flow conditions. Methods Blood collectionThe present work was approved by the i...

Implantation of a stented elephant trunk into the descending aorta combined with replacement of the ascending aorta and total arch for acute type A aortic dissection is effective in closing the residual false lumen of the descending aorta and in preventing expansion of the descending aorta. However, further technical modifications, such as using a short stented elephant trunk, eliminating aortic clamping, shortening CPB and spinal cord ischemic time, and reconstruction of left subclavian artery, are needed to prevent neurologic complications.

Using a perfusion chamber and confocal laser scanning microscopy, we analyzed the interplay of von Willebrand factor (VWF) and fibrinogen during thrombus growth on a collagen surface under physiologic high shear rate conditions. During initial thrombogenesis, platelet thrombi were constructed totally by VWF, not by fibrinogen. Fibrinogen accumulated predominantly inside the growing thrombi as a function of time, whereas the thrombus surfaces directly exposed to flow were occupied constantly by VWF throughout the observation period. In perfusion of afibrinogenemia (AF) blood lacking both plasma and platelet fibrinogen, the final height and volume of thrombi were significantly reduced compared with controls, albeit the area of surface coverage was normal. The impaired thrombus growth in AF was only partially corrected by the addition of purified fibrinogen to AF blood, whereas the addition of purified VWF to blood of severe von Willebrand disease (VWD) completely normalized the defective thrombus growth in this disease.Thus, the initial 2-dimensional thrombus expansion involves only VWF, whereas the time-dependent accumulation of fibrinogen, released from activated platelets, acts as a core adhesive ligand, increasing thrombus strength and height and resulting in 3-dimensional thrombus development against rapid blood flow. (Blood. 2002;100:3604-3610)

To explore the mechanisms that underlie the bleeding tendency in type 2A and 2B von Willebrand disease (VWD), we analyzed the mural thrombus generation process on a collagen surface under physiologic blood flow in a perfusion chamber using whole blood from these VWD patients. At a low shear rate (50 s ؊1 ), thrombus generation in all type 2A and 2B VWD patients was comparable to that of healthy controls. At a high shear rate (1500 s ؊1 ), thrombus generation was impaired in all type 2A patients, whereas that in type 2B VWD patients varied from normal to significantly defective, as judged by epifluorescence microscopy of thrombus surface coverage. However, in type 2B patients who showed normal thrombus generation at 1500 s ؊1 , the height and volume of thrombi was significantly reduced, albeit with the normal surface coverage, compared with control thrombi, and von Willebrand factor (VWF) was poorly distributed within the type 2B thrombus mass when analyzed in detail by confocal laser scanning microscopy. Addition of purified VWF to patient blood completely reversed the defective spatial thrombus growth in type 2B VWD. Thus, our results confirm the impaired thrombus generation in type 2B VWD, which has never been demonstrable in previous in vitro soluble-phase platelet aggregation assays, and point to the critical function of larger VWF multimers in the proper spatial growth of mural thrombi under high shear rate conditions. (Blood. 2003; 101:915-920)

Ventricular dilation after myocardial infarction can cause heart failure. Increasing strength and elasticity in the infarct region might prevent ventricular dilation. Because elastin provides strength, extensibility, and resilience to tissues and maintains tissue architecture, we studied the effect of elastin expression in the infarct on scar expansion and heart function. COS-7 cells transfected with a plasmid with an elastin gene fragment or a vector were seeded into a Gelfoam mesh and cultured. Mechanical stretch test (n = 5/group) showed that the elastin mesh was more elastic (P < 0.05) and tensile (P < 0.05) than the vector mesh. In an in vivo study in rats, 6 days after left anterior descending coronary artery ligation, COS-7 cells (Cell group, n = 7) or COS-7 cells with elastin gene (Elastin group, n = 9) or vector (Vector group, n = 9) were transplanted into the infarct; infarcted rats served as controls (n = 7). Over 8 wk the Cell group did not demonstrate effects on scar expansion and deterioration of heart function vs. controls. In contrast, infarct expansion was smaller and heart function was better maintained in the Elastin group vs. the Vector group (P < 0.05). At 8 wk after cell transplantation Langendorff data showed that the Elastin group had greater (P < 0.01) developed pressure and a smaller left ventricular volume than the Vector group. Western blot and histology showed accumulated elastin in the Elastin group infarct. Changing the extracellular matrix composition of a myocardial infarct by increasing elastin fragment content attenuated scar expansion, ventricular dilation, and onset of heart dysfunction.