ABSTRACT
Background and Aims: Myasthenia gravis (MG) is a T-cell dependent antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen, comprising several T and B cell auto-epitopes. We hypothesized that an efficacious drug candidate for antigen-specific therapy in MG should comprise a broad range of these auto-epitopes and be administered in a noninflammatory and tolerogenic context. Methods: We used a soluble mutated form of the extracellular domain of the α1 chain of the AChR (α1-ECDm), which represents the major portion of auto-epitopes involved in MG, and investigated, in a well-characterized rat model of experimental autoimmune myasthenia gravis (EAMG) whether its intravenous administration could safely and efficiently treat the autoimmune disease. Results: We demonstrated that intravenous administration of α1-ECDm abrogates established EAMG, in a dose and time dependent manner, as assessed by clinical symptoms, body weight, and compound muscle action potential (CMAP) decrement. Importantly, the effect was more pronounced compared to drugs representing current standard of care for MG. The protein had a short plasma half-life, most of what could be recovered was sequestered in the liver, kidneys and spleen. Further, we did not observe any signs of toxicity or intolerability in animals treated with α1-ECDm. Conclusion: We conclude that intravenous treatment with α1-ECDm is safe and effective in suppressing EAMG. α1-ECDm is in preclinical development as a promising new drug candidate for MG.
Subject(s)
Myasthenia Gravis, Autoimmune, Experimental , Receptors, Nicotinic , Animals , Epitopes, B-Lymphocyte , Myasthenia Gravis, Autoimmune, Experimental/drug therapy , Rats , Receptors, Cholinergic , Receptors, Nicotinic/genetics , T-LymphocytesABSTRACT
OBJECTIVE: We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. METHODS AND RESULTS: ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites. CONCLUSIONS: We hypothesize that ADAMTS-1 may promote atherogenesis by cleaving extracellular matrix proteins such as versican and promoting VSMC migration.
Subject(s)
Arteriosclerosis/pathology , Carotid Artery, Common/pathology , Chondroitin Sulfate Proteoglycans/metabolism , Disintegrins/physiology , Immunohistochemistry/methods , Metalloendopeptidases/physiology , Peptide Hydrolases/metabolism , ADAM Proteins , ADAMTS1 Protein , Adolescent , Animals , Arteriosclerosis/metabolism , Carotid Artery, Common/chemistry , Carotid Artery, Common/metabolism , Carotid Artery, Common/surgery , Cell Line , Disease Models, Animal , Disintegrins/biosynthesis , Disintegrins/immunology , Disintegrins/metabolism , Humans , Hydrolysis , Lectins, C-Type , Ligation/methods , Male , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/immunology , Metalloendopeptidases/metabolism , Mice , Mice, Inbred C57BL , Middle Aged , Muscle, Smooth, Vascular/chemistry , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/chemistry , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Neovascularization, Pathologic/pathology , Reverse Transcriptase Polymerase Chain Reaction/methods , VersicansABSTRACT
BACKGROUND: Carboxypeptidase U (EC 3.4.17.20, TAFIa) is a new member of the metallocarboxypeptidase family circulating in human plasma as a zymogen. It is activated during coagulation and is considered as an important player in the regulation of fibrinolysis. METHODS: Heterologous expression of human plasma procarboxypeptidase U (proCPU, TAFI) was obtained in mammalian cells (C127 and DON) and in insect cells (Sf21 and H5 cells). Conditioned media were purified by cation-exchange chromatography and plasminogen affinity chromatography to yield an essentially pure protein. RESULTS: All systems gave high expression levels (6-20 mg/l). Due to differences in glycosylation of the activation peptide, the recombinant variants of proCPU migrated differently on SDS-PAGE (52-65 kDa). However, after activation, all active recombinant enzymes migrated at 35 kDa, similar to native CPU and no evidence for post-translational modification of the catalytic domains could be detected. For the mammalian cell produced variants, activation was more efficient after desialylation. After activation, CPU showed low solubility (0.2 mg/ml) but was inhibited similarly as native CPU. CONCLUSIONS: Mammalian cell systems were the most efficient for the production of human plasma recombinant proCPU. The obtained zymogen differs with respect to the extent and the heterogeneity of glycosylation but, after activation, the experiments did not reveal any alteration between the recombinant and native protein.