Highly sensitive point-of-care diagnostic tools for detection of all human malaria species are needed to make MSAT an effective strategy in settings where malaria elimination programs are in the pre-elimination phase.

Background The majority of CD4+ T helper (Th) cells found in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) express CXCR3, a receptor associated with Th1 cells. In blood, subsets of Th2 and Th17 cells also express CXCR3, but it is unknown if these cells are present in RA SF or how cytokines from these subsets affect cytokine/chemokine secretion by fibroblast-like synoviocytes (FLS) from patients with RA. Methods We examined the proportions of Th1, Th2, CXCR3+Th2, Th17, CXCR3+Th17, Th1Th17, peripheral T helper (TPh) and T follicular helper (TFh) cells in paired SF and blood, as well as the phenotype of TPh and TFh cells in RA SF (n = 8), by the use of flow cytometry. We also examined the cytokine/chemokine profile in paired SF and plasma (n = 8) and in culture supernatants of FLS from patients with chronic RA (n = 7) stimulated with Th-associated cytokines, by the use of cytometric bead arrays and ELISA. Cytokine receptor expression in FLS (n = 3) were assessed by the use of RNA sequencing and qPCR. Results The proportions of Th1 and CXCR3+Th2 cells were higher in SF than in blood (P < 0.05). TPh and PD-1highTFh in RA SF were primarily of a Th1 and a CXCR3+Th2 phenotype. Moreover, the levels of CXCL9, CXCL10, CCL20, CCL2, CXCL8, IL-6 and IL-10 were higher in SF than in plasma (P < 0.05). Lastly, IL-4, IL-13 and IL-17A induced RA FLS to secrete proinflammatory IL-6, CCL2, CXCL1 and CXCL8, while IFNγ mainly induced CXCL10. Conclusion These findings indicate that not only Th1 but also CXCR3+Th2 cells may have a pathogenic role in RA synovial inflammation.

The autoimmune regulator AIRE controls the negative selection of self-reactive T-cells as well as the induction of regulatory T-cells in the thymus by mastering the transcription and presentation of tissue restricted antigens (TRAs) in thymic cells. However, extrathymic AIRE expression of hitherto unknown clinical significance has also been reported. Genetic polymorphisms of AIRE have been associated with rheumatoid arthritis (RA), but no specific disease-mediating mechanism has been identified. Rheumatoid arthritis is characterized by a systemic immune activation and arthritis. Activated fibroblast-like synoviocytes (FLS) are key effector cells, mediating persistent inflammation, and destruction of joints. In this study, we identified AIRE as a cytokine-induced RA risk gene in RA FLS and explored its role in these pathogenic stroma cells. Using RNA interference and RNA sequencing we show that AIRE does not induce TRAs in FLS, but augments the pro-inflammatory response induced by tumor necrosis factor and interleukin-1β by promoting the transcription of a set of genes associated with systemic autoimmune disease and annotated as interferon-γ regulated genes. In particular, AIRE promoted the production and secretion of a set of chemokines, amongst them CXCL10, which have been associated with disease activity in RA. Finally, we demonstrate that AIRE is expressed in podoplanin positive FLS in the lining layer of synovial tissue from RA patients. These findings support a novel pro-inflammatory role of AIRE at peripheral inflammatory sites and provide a potential pathological mechanism for its association with RA.

BackgroundA key feature of joints in rheumatoid arthritis (RA) is the formation of hyperplastic destructive pannus tissue, which is orchestrated by activated fibroblast-like synoviocytes (FLS). We have demonstrated that the RA risk gene and tumor suppressor Limb bud and heart development (LBH) regulates cell cycle progression in FLS. Methotrexate (MTX) is the first-line treatment for RA, but its mechanisms of action remain incompletely understood. Here, we studied the effects of MTX on mitogen-induced FLS proliferation and expression of cell cycle regulators in vitro.MethodsPrimary FLS from patients with RA or osteoarthritis were stimulated with the mitogen platelet-derived growth factor (PDGF) and the cytokine interleukin-1β (IL-1β) in the presence or absence of MTX. Cells were then subjected to qPCR for gene expression and cell cycle analysis by flow cytometry.ResultsStimulation with PDGF and IL-1β increased the percentage of FLS in the G2/M phase and shifted the cell morphology to a dendritic shape. These effects were inhibited by MTX. Furthermore, PDGF + IL-1β reduced LBH mRNA expression. However, MTX treatment yielded significantly higher transcript levels of LBH, and of CDKN1A (p21) and TP53 (p53), compared to untreated samples upon mitogen stimulation. The expression of DNA methyltransferase-1 (DNMT1) was also higher in the presence of MTX and there was strong correlation between DNMT1 and LBH expression.ConclusionsTherapeutic concentrations of MTX abolish the effects of PDGF and IL-1β on tumor suppressor expression and inhibit mitogen-promoted FLS proliferation. These data demonstrate novel and important effects of MTX on pathogenic effector cells in the joint, which might involve epigenetic mechanisms.

BackgroundActivated fibroblast-like synoviocytes (FLS) are key effector cells in the joint in rheumatoid arthritis (RA). Local FLS proliferation is responsible for synovial hyperplasia, a key feature of the RA synovium correlating with disease activity. PDGF and IL-1b are known FLS mitogens. LBH is a transcription regulator and tumor suppressor, recently identified as a RA risk gene. We have demonstrated that LBH regulates FLS proliferation and that LBH expression is regulated by growth factors and by epigenetic mechanisms[1]. Methotrexate (MTX) is still the first-line treatment of RA but the target cells and mechanism of action of the low dose used in rheumatic diseases is largely unknown. Increased expression of cell cycle checkpoint genes[2] and modified DNA methylation[3] in immune cells have recently been described.ObjectivesThe aim of this study was to investigate the effects of MTX on PDGF and IL1b-induced FLS proliferation in vitro and in particular on the expression of LBH, cell cycle genes (CDKN1A/p21 and CCND1/cyclinD1) and on genes regulating DNA methylation (DNMTs) in order to further understand the pharmacodynamics of this drug in RA and to identify novel markers for drug response.MethodsPrimary FLS from RA patients and from patients with osteoarthritis (OA) were plated on day 0 in DMEM complete, pre-treated 24 hours with MTX or control medium day 1, and stimulated with 20ng/ml PDGF+2 ng/ml IL-1b with or without 1 uM MTX in DMEM with 1% FBS for 24–48 hours starting day 2. Cells were then harvested for qPCR for gene expression and flowcytometry for cell cycle analysis.ResultsStimulating RA-FLS cultures (n=3) with PDGF+IL-1b for 24 hours, pushed 24,5±3,5% cells into G2/M phase compared to 3,4±0,8% in unstimulated controls. Interestingly, treating PDGF+IL1b stimulated FLS with MTX, significantly inhibited cell cycle progression (4,6±1,9% in G2/M phase, p=0,02). PDGF+IL-1b stimulation of FLS for 24 hours reduced LBH mRNA expression. However, in the presence of 1uM MTX the LBH mRNA expression was significantly higher in RA-FLS (3,2±0,5 fold, p=0.002, n=5) and in OA-FLS (2,2±0,5 fold, p=0,02, n=5) after PDGF+IL-1b stimulation compared to untreated controls. In addition, MTX treatment strikingly increased the CDKN1A expression 14,3±4,4 fold (p=0,006) of treated vs untreated stimulated FLS. Furthermore, we found that 1 uM MTX restored and increased a lowered DNMT1 mRNA expression to 144±12% after PDGF+IL1b stimulation. There were no significant effects of MTX on CCND1 or DNMT3a expression at investigated time points.ConclusionsTherapeutic doses of MTX reduce mitogen induced FLS proliferation and significantly revert mitogen-induced reduction of LBH and p21 expression in RA FLS. MTX restores expression of DNMT1 suggesting that MTX might regulate gene expression and proliferation by affecting the epigenome.References Ekwall, A.K., et al., The Rheumatoid Arthritis Risk Gene LBH Regulates Growth in Fibroblast-like Synoviocytes. Arthritis Rheumatol, 2015. 67(5): p. 1193–202.Spurlock, C.F., 3rd, et al., Methotrexa...