Inhibitory Effect of Tetramerized Single-Chain Variable Fragment of Anti-Cyclic Citrullinated Peptide Antibodies on the Proliferation, Activation, and Secretion of Cytokines of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis In Vitro Co-Culture System
Tetramerized single chain variable fragment (scFv) anti-cyclic citrullinated peptide (TeAb-CCP) is built tetramerized scFv anti-cyclic peptide citrullinated (CCP) antibody to p53 domain tetrameric, aimed to investigate the effect on fibroblast-like synoviocytes (FLSs) proliferation, migration, invasion, and production of inflammatory mediators in the co-culture system in vitro peripheral mononuclear cells (PBMC) and FLSs. TeAb-CCP constructed by modifying the monovalent scFv antibodies to PKC with tetrameric domain p53 to increase affinity. FLSs isolated and cultured from rheumatoid arthritis (RA) patients and control subjects.
A co-culture system peripheral mononuclear cells (PBMC) and FLSs used. FLSs proliferation, migration and invasion were measured by MTT, the initial tests, and Transwell chamber. The supernatant was measured for cytokines, chemokines, metalloproteinases, and anti-CCP antibodies by Luminex liquid phase protein chip and ELISA. TeAb-CCP FLSs significantly inhibit proliferation in a dose-dependent fashion, with maximal action at a concentration of 100 mg / ml on day 7 in a co-culture with PBMCs and FLSs, but not the same as just FLSs.
TeAb-CCP was significantly suppressed FLSs migration and invasive ability compared with controls. Significantly lower levels of interleukin (IL) -6, IL-8, RANKL, a protein arginine deiminase (PAD) -2, PAD4, metalloproteinase (MMP) -1 and MMP-3 and anti-CCP antibodies found in the supernatant co -budaya TeAb-CCP group. Instead, transforming growth factor-β (TGF-β) and tissue inhibitor of metalloproteinase-2 (TIMP-2) increased significantly in the group TeAb-CCP.
There is no significant difference of IL-1a, IL-10, IL-17, TNF, VEGF and FGF was found between the two groups. As a blocking antibody, TeAb-CCP can significantly inhibit RA PBMC to produce pro-inflammatory mediators, and furthermore, inhibit proliferation, activation, migration, and invasion in vitro FLSs. In turn, it is suggested that a modified self citrullinated epitopes may be a new target for the treatment of RA.
Inhibitory Effect of Tetramerized Single-Chain Variable Fragment of Anti-Cyclic Citrullinated Peptide Antibodies on the Proliferation, Activation, and Secretion of Cytokines of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis In Vitro Co-Culture System
Mst1 promote mitochondrial dysfunction and apoptosis in oxidative stress-induced rheumatoid arthritis synoviocytes
In this study, we investigated the role of macrophage stimulating 1 (mst1) SIRT1 and AMPK-signaling pathway in oxidative stress-induced mitochondrial dysfunction and apoptosis seen in rheumatoid arthritis associated fibroblast-like synoviocytes (RA-FLSs). Mst1 mRNA and protein expression was significantly higher in hydrogen peroxide (H2O2) -treated RA-FLSs of untreated control. H2O2 treatment induced mitochondrial apoptotic pathway by activating caspase3 / 9 and Bax in RA-FLSs.
In addition, H2O2 treatment significantly reduced the mitochondrial membrane potential and mitochondrial state 3 and state 4 respiration, but increased reactive oxygen species (ROS). Mst1 silencing significantly reduce oxidative stress induced mitochondrial dysfunction and apoptosis in RA-FLSs. SIRT1 expression was reduced significantly in H2O2-treated RA-FLSs, but higher in H2O2-treated mst1-RA-FLSs silenced.
Pretreatment with selisistat (SIRT1-specific inhibitor) or compound C (AMPK antagonist) significantly reduces the survival and function of mitochondria in H2O2-treated mst1-RA-FLSs silenced by inhibiting the function or expression of SIRT1 SIRT1, respectively.
Human Fibroblast-Like Synoviocyte Normal Tissue, Adult
Description: Human Fibroblast-Like Synoviocytes (HFLS) are isolated from normal synovial tissues. They are cryopreserved at second passage and can be cultured and propagated at least 5 population doublings.
Human Fibroblast-Like Synoviocyte Osteo Arthritis, Adult
Description: Human Fibroblast-Like Synoviocytes (HFLS) are isolated from synovial tissues obtained from patients with Rheumatoid Arthritis (RA) / Osteoarthritis (OA). They are cryopreserved at second passage and can be cultured and propagated at least 5 population doubling.
Human Fibroblast-Like Synoviocyte Rheumatoid Arthritis, Adult
Description: Human Fibroblast-Like Synoviocytes (HFLS) are isolated from synovial tissues obtained from patients with Rheumatoid Arthritis (RA) / Osteoarthritis (OA). They are cryopreserved at second passage and can be cultured and propagated at least 5 population doubling.
Description: Human synoviocytes (HS), the predominant cell type of healthy synovial tissue, are fibroblast-like cells. The Synoviocytes form a distinct structure called the synovial lining layer. Electron microscopy revealed extensive cell-to-cell contacts within the lining layer. The Synoviocytes produce synovial fluid components and are responsible for absorption from the joint cavity, and for blood/synovial fluid exchanges. The synoviocytes proliferate, show anchorage-independent growth, and also secrete a variety of effector molecules that promote inflammation and joint destruction and, themselves are part of a complex network of autocrine and paracrine acting factors. The synoviocytes express cadherin-11 providing new insight into synovial tissue organization and morphogenesis and of interest as a therapeutic target.HS from Gentaur Research Laboratories are isolated from human synovium. HS are cryopreserved at passage one cultures and delivered frozen. Each vial contains 5×10^5 cells in 1 ml volume. HS are characterized by their fibroblast-like morphology, growth pattern and immunocytochemistry of CD 90 and fibronectin. HS are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi. HS are guaranteed for 15 population doublings at the conditions provided by Gentaur Research Laboratories.
Description: Cell Type iPSCPrimary Tissue Activated fibroblast from primary keratocytesReprogramming Method Episomal PlasmidDisease Keratoconus; Cells are only guaranteed with purchase of Gentaur Media and Gentaur Extra Cellular Matrix for appropriate cell culture, for 30 days from the date of shipment.
Description: Cell Type iPSCPrimary Tissue FibroblastReprogramming Method Episomal PlasmidDisease Huntington's Disease Like; Cells are only guaranteed with purchase of Gentaur Media and Gentaur Extra Cellular Matrix for appropriate cell culture, for 30 days from the date of shipment.
Description: A Rabbit polyclonal antibody against Rat Fibroblast Growth Factor Receptor Like Protein 1 (FGFRL1). This antibody is labeled with APC-Cy7.
Rat Fibroblast Growth Factor Receptor Like Protein 1 (FGFRL1) Antibody Pair Kit (with Standard)
These findings suggest that oxidative stress associated upregulation and activation mst1 promote mitochondrial dysfunction and apoptosis in RA-FLSs by inhibiting AMPK-SIRT1 pathway signaling. This shows mst1 axis-AMPK-SIRT1 is a potential target for the treatment of RA.
Shelby
