Anti-TNF monoclonal antibody, clone UO4-20 Functional Grade

Background Because of the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. Objectives The primary objective of this study is to develop and functionally characterize a novel megakaryocyte nuclear factor kappa B (NF-kappa B) reporter cell line to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. Methods A Meg-01-NF-kappa B-GFP-Luc (Meg-01R) cell line was developed by inserting a reporter NF-kappa B-GFP-Luc cassette into normal Meg-01 cells to produce luciferase following activation of NF-kappa B to enable easy detection of pro-inflammatory and reparative signalling. Results and conclusions Meg-01 and Meg-01R cells have comparable characteristics, including the expression of both GPIb alpha and integrin beta(3). Meg-01R cells responded to various inflammatory molecules as measured by NF-kappa B-dependent bioluminescence. For example, inflammatory molecules such as tumor necrosis factor-alpha and Pam3CSK4 increased NF-kappa B activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg-01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg-01R cells were able to respond to lipopolysaccharide (LPS; non-ultrapure), although it was not able to react to ultrapure LPS because of the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterization of a novel megakaryocyte NF-kappa B reporter cell line (Meg-01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF-kappa B activity.

Objectives The aim of this study was to evaluate the effect of specific inhibition of MMP-13 on inflammation and inflammatory bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontitis. Materials and methods Periodontitis was induced in mice by micro-injections of LPS into the gingival tissues adjacent to the palatal surfaces of maxillary molars twice a week for 15 days. Matrix metalloproteinase-13 (Mmp-13) shRNA or a specific biochemical inhibitor were also injected into the same sites in alternating days with the LPS injections. Efficacy of shRNA-mediated silencing of Mmp-13 was verified by quantitative real-time polymerase chain reaction (qPCR) and immunoblot. Bone resorption was assessed by microcomputed tomography (uCT). Histological sections stained with hematoxylin/eosin (H/E) were used in the stereometric analysis of the inflammatory infiltrate. Gingival tissues were used to evaluate expression of Mmp-13, Il-6, Tnf-alpha, Ptgs2, and Rankl (qPCR). Protein levels of TGF-beta and IL-10 in the tissues were determined by enzyme-linked immunosorbent assays (ELISA) or by MMP-13 and p38 immunoblot. Results Silencing Mmp-13 expression reduced bone resorption significantly. Expression of Mmp-13, Il-6, and Tnf-alpha, as well as the protein levels of IL-6 and TNF-alpha, was reduced in the animals treated with adenovirus-delivered shRNA; however, these effects were not associated with modulation of p38 MAPK signaling. Interestingly, inhibition Mmp-13 did not affect the severity of inflammatory infiltrate. Conclusions Site-specific inhibition of MMP-13 reduced bone resorption and production of inflammatory mediators associated with periodontal disease.