The general characteristics of both patients and controls sub jects are summarized in Table 1. All patients were recruited from the baseline and had not yet been treated with disease modifying anti rheumatic drugs and/or steroids before their blood samples were col lected. This study was approved by the Ethical Commit tee of the First Affiliated Hospital of Nanjing Medical University, and all donors provided written informed consent. Blood samples were collected from peripheral veins. PBMCs were isolated by Ficoll Hypaque density centri fugation. Serum and SF samples were stored at 80oC. Synovial biopsies were stored in liquid nitrogen for mRNA analysis or in Carnoys fixative for histological analysis. Real time PCR Total RNA was extracted from PBMC, synovial tissue and fibroblasts using TRIzol.

Reverse transcription reaction was conducted at 37oC for 15 min, 85oC for 5 sec in a 20 uL mixture con taining 1 ug of total RNA, and PrimeScript RT Master Mix. Each real time PCR was prepared in a 20 uL reac tion mixture containing 10 uL SYBR Green PCR Master Mix, 1 uL cDNA, 0. 8 uL primers, and conducted on a ABI Prism 7900 sequence detector. Cycling conditions consisted of initial denaturation 30 sec at 95oC, followed by 40 cycles of 5 sec at 95oC, and 30 sec at 60oC. The primer sequences are summarized in Table 2. All samples of RA and controls were assayed in triplicate. Relative gene expression was determined by the 2 ct method. ELISA Levels of IL 29 in serum and SF were measured by ELISA according to the manufacturers instructions.

The correlation was analyzed between IL 29 and laboratory values, including erythrocyte sedimentation rate, C reactive protein, anti cyclic citrullinated pep tides and rheumatoid factor in serum of RA patients. Immunohistochemical analysis Specimens were fixed in Carnoys fixative and embedded in paraffin wax. Paraffinized synovial tissues were sec tioned to 3um thickness, deparaffinized in xylene and rehydrated through a series of concentrations of ethanol. After inactivation of Anacetrapib endogenous peroxidase, sections were blocked by incubation with 5% bovine serum album for 30 min at room temperature, then incubated with rabbit anti human IL 29 antibody at 4oC overnight in a humidified chamber. After washing, sections were next incubated with peroxidase conjugated goat anti rabbit secondary antibody for 1 h at room temperature.

The reactions were developed using a DAB substrate kit, with hematoxylin as counterstain. Each slide was evaluated by one of the authors under a microscope . Tissue sections were scored for staining of the lining on a 0 to 5 scale as follows 0 no staining, 1 few of the cells positively stained, 2 some of the cells stained, 3 approxi mately half of the cells stained, 4 more than half of the cells stained, and 5 all cells stained.