Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. Here we show that erlotinib, an inhibitor of the tyrosine kinase EGFR, reduces the severity of established collagen-induced arthritis (CIA), a mouse model of RA—and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with CIA and patients with RA. Together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefit in the treatment of RA.
BACKGROUND
Lateral flow assays (LFAs) are popular point-of-care diagnostic tools because they are rapid and easy to use. Nevertheless, they often lack analytical sensitivity and quantitative output and may be difficult to multiplex, limiting their usefulness in biomarker measurement. As a proof-of-concept study, we detail the design of a quantitative, multiplex LFA with readily available near-infrared (NIR) detection to improve analytical sensitivity.
METHODS
NIR dye was conjugated to selected antibodies and incorporated into LFAs. We used singleplex, optimized NIR-LFAs to measure interleukin (IL)-6 from 0 to 200 pg/mL and developed duplex assays to simultaneously measure IL-6 from 0 to 100 pg/mL (0 to 4.5 pmol/L) and C-reactive protein (CRP) from 50 to 2500 ng/mL (0.4 to 20 nmol/L) on a single test strip. Assays were tested on 60 different spiked samples and compared to ELISA results.
RESULTS
NIR-LFAs detected IL-6 in a 10% plasma matrix with a limit of detection of 4 pg/mL (182 fmol/L) and a CV <7%. Duplex NIR-LFAs quantitatively measured IL-6 and CRP concentrations simultaneously. Values strongly correlated to ELISA measurements, with R2 values of 0.9825 and 0.9711 for IL-6 and CRP, respectively.
CONCLUSIONS
NIR-LFAs exhibit quantitative measurement at pg/mL concentrations owing to a high signal-to-BACKGROUND ratio and robust detection antibody clearance through the test strip. Moreover, NIR-LFAs are able to detect molecules present at vastly different concentrations in multiplex format and compare favorably to ELISAs. LFAs with direct NIR detection may be a valuable tool for biomarker evaluation in the point-of-care setting.
As critical regulators of numerous cell signaling pathways, tyrosine kinases are implicated in the pathogenesis of several diseases, including rheumatoid arthritis (RA). In the absence of disease, synoviocytes produce factors that provide nutrition and lubrication for the surrounding cartilage tissue; few cellular infiltrates are seen in the synovium. In RA, however, macrophages, neutrophils, T cells and B cells infiltrate the synovium and produce cytokines, chemokines and degradative enzymes that promote inflammation and joint destruction. In addition, the synovial lining expands owing to the proliferation of synoviocytes and infiltration of inflammatory cells to form a pannus, which invades the surrounding bone and cartilage. Many of these cell responses are regulated by tyrosine kinases that operate in specific signaling pathways, and inhibition of a number of these kinases might be expected to provide benefit in RA.
Paper based diagnostic devices have great potential in the low-cost sensing of blood diseases. However, patient blood testing using these devices is limited by tedious manual intervention and qualitative colorimetric readouts. We present the novel design of an RFID-based anemia detection sensor that integrates a paper-based diagnostic device with a passive Ultra High Frequency (UHF) RFID tag. Differences in red blood cell (RBC) count in a patient's blood manifests itself as a controlled time-dependent change in the tag's signal response. We demonstrate that our sensor is capable of reliably differentiating between blood having 20, 30, 40 and 50% RBC concentration by volume -indicative of anemic vs. healthy blood. Furthermore the sensor can be read using off the shelf RFID equipment allowing for automated screening of blood specimens at large scale. Challenges in sensor design and future research directions are also discussed.
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