BRAF Drives Synovial Fibroblast Transformation in Rheumatoid Arthritis

Synovial fibroblasts destroy articular cartilage and bone in rheumatoid arthritis, but the mechanism of fibroblast transformation remains elusive. Because gain-of-function mutations of BRAF can transform fibroblasts, we examined BRAF in rheumatoid synovial fibroblasts. The strong gain-of-function mutation, V600R, of BRAF found in melanomas and other cancers was identified in first passage synovial fibroblasts from two of nine rheumatoid arthritis patients and confirmed by restriction site mapping. BRAF-specific siRNA inhibited proliferation of synovial fibroblasts with V600R mutations. A BRAF aberrant splice variant with an intact kinase domain and partial loss of the N-terminal autoinhibitory domain was identified in fibroblasts from an additional patient, and fibroblast proliferation was inhibited by BRAF-specific siRNA. Our finding is the first to establish mechanisms for fibroblast transformation responsible for destruction of articular cartilage and bone in rheumatoid arthritis and establishes a new target for therapeutic intervention.     

Isolation and partial characterization of a 39 kDa major outer membrane protein of Actinobacillus actinomycetemcomitans Y4

The outer membrane fractions of Actinobacillus actinomycetemcomitans, which were extracted from whole cells with cetyl trimethyl ammonium bromide and CaCl2, contained four major outer membrane proteins (MOMP) of 39, 37, 36 and 30 kDa. The 39 kDa MOMP of A. actinomycetemcomitans was sequentially purified by extraction with Zwittergent 3-14 detergent, anion-exchange chromatography and gel filtration chromatography. Analysis of amino acid composition and N-terminal amino acid sequence of 20 residues of purified 39 kDa MOMP was performed. Although some of the periodontitis patient sera reacted strongly with 39 kDa and 30 kDa MOMP in crude outer membrane fractions, purified 39 kDa MOMP showed decreased immunoreactivity with the human sera.     

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.     

Site-specific mutagenesis of human interleukin-6 and its biological activity

Amino acid substitutions of human interleukin-6 (IL-6) were performed. Single substitution Met162----Ala and double substitutions Leu159.166----Val resulted in a significant decrease of IL-6 activity in the production of immunoglobulin (Ig) from B-cells. Single substitution Leu166----Val or Leu159----Val gave a slight or no significant decrease in the Ig-induction activity, respectively. The receptor-binding activity of each IL-6 mutant was also examined. It was observed that the decrease of the receptor-binding activity was generally in parallel with that of the Ig-induction activity. We therefore suggest that hydrophobic side-chains existing in Met162, Leu159, and Leu166 are significantly involved in the receptor-binding of IL-6.