Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation. fluorescent proteins ͉ lentiviral vectors ͉ silencing ͉ stoichiometry

Objectives To increase understanding of the aetiology and pathogenesis of rheumatoid arthritis (RA), genetic and environmental risk factors for RA subsets, defi ned by the presence or absence of different anticitrullinated protein/peptide antibodies (ACPAs) targeting citrullinated peptides from α-enolase, vimentin, fi brinogen and collagen type II, were investigated. Methods 1985 patients with RA and 2252 matched controls from the EIRA case-control cohort were used in the study. Serum samples were assayed by ELISA for the presence of anticyclic citrullinated peptides (anti-CCP) antibodies and four different ACPA fi ne specifi cities. Cross-reactivity between ACPAs was examined by peptide absorption experiments. Genotyping was performed for HLA-DRB1 shared epitope (SE) alleles and the PTPN22 gene, while information regarding smoking was obtained by questionnaire. The association of genetic and environmental risk factors with different subsets of RA was calculated by logistic regression analysis. Results Limited cross-reactivity was observed between different ACPA fi ne specifi cities. In total, 17 RA subsets could be identifi ed based on their different ACPA fi ne specifi city profi les. Large differences in association with genetic and environmental determinants were observed between subsets. The strongest association of HLA-DRB1 SE, PTPN22 and smoking was identifi ed for the RA subset which was defi ned by the presence of antibodies to citrullinated α-enolase and vimentin. Conclusion This study provides the most comprehensive picture to date of how HLA-DRB1 SE, PTPN22 and smoking are associated with the presence of specifi c ACPA reactivities rather than anti-CCP levels. The new data will form a basis for molecular studies aimed at understanding disease development in serologically distinct subsets of RA.

Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.

IntroductionA major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint).MethodsPlasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry.ResultsApproximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA.ConclusionsWe have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.