Citrullinated and malondialdehyde-acetaldehyde modified fibrinogen activates macrophages and promotes an aggressive synovial fibroblast phenotype in patients with rheumatoid arthritis.

Objective: Post-translational protein modifications with malondialdehyde-acetaldehyde (MAA) and citrulline (CIT) are implicated in the pathogenesis of rheumatoid arthritis (RA). Although precise mechanisms have not been elucidated, macrophage-fibroblast interactions have been proposed to play a central role in the development and progression of RA. The purpose of our study was to evaluate the downstream effects of macrophage released soluble mediators, following stimulation with fibrinogen (FIB) modified antigens, on human fibroblast-like synoviocytes (HFLS). Methods: PMA-treated U-937 monocytes (Mϕ) and macrophage-differentiated peripheral blood mononuclear cells (MP) were stimulated with FIB, FIB-MAA, FIB-CIT, or FIB-MAA-CIT. HFLS-RA cells were stimulated directly with FIB antigens or with supernatants (SN) from macrophages (Mϕ-SN or MP-SN) stimulated with FIB antigens. Genes associated with an aggressive HFLS phenotype, extracellular matrix proteins, and activated signaling pathways were evaluated. Results: HFLS-RA cells treated with Mϕ-SNFIB-CIT and Mϕ-SNFIB-MAA-CIT demonstrated significant increases in mRNA expression of genes associated with an aggressive phenotype at 24-h as compared to direct stimulation with the same antigens. Similar results were obtained using MP-SN. Cellular morphology was altered and protein expression of vimentin (p<0.0001 vs. Mϕ-SNFIB) and type II collagen (p<0.0001) were significantly increased in HFLS-RA cells treated with any of the Mϕ-SN generated following stimulation with modified antigens. Phosphorylation of JNK, Erk1/2, and Akt were increased most substantially in HFLS-RA treated with Mϕ-SNFIB-MAA-CIT (p<0.05 vs Mϕ-SNFIB). These and other data suggested the presence of PDGF-BB in Mϕ-SN. Mϕ-SNFIB-MAA-CIT contained the highest concentration of PDGF-BB (p<0.0001 vs. Mϕ-SNFIB) followed by Mϕ-SNFIB-CIT then Mϕ-SNFIB-MAA. HFLS-RA cells treated with PDGF-BB showed similar cellular morphology to the Mϕ-SN generated following stimulation with modified FIB, as well as the increased expression of vimentin, type II collagen, and the phosphorylation of JNK, Erk1/2 and Akt signaling molecules. Conclusion: Together, these findings support the hypothesis that in response to MAA-modified and/or citrullinated fibrinogen, macrophages release soluble factors including PDGF-BB that induce fibroblast activation and promote an aggressive fibroblast phenotype. These cellular responses were most robust following macrophage activation with dually modified fibrinogen, compared to single modification alone, providing novel insights into the combined role of multiple post-translational protein modifications in the development of RA.

Intravascular ultrasound to aid in the diagnosis and revision of an intra-aortic pedicle screw: illustrative case.

BACKGROUND: Pedicle screw impingement on vessel walls has the potential for complications due to pulsatile effects and wall erosion. Artifacts from spinal instrumentation create difficulty in accurately evaluating this interface. The authors present the first case of intravascular ultrasound (IVUS) used to characterize a pedicle screw breach into the aortic lumen. OBSERVATIONS: A 21-year-old female with surgically corrected scoliosis underwent computed tomography angiography (CTA) 3 years postoperatively, which revealed a pedicle screw within the thoracic aorta lumen. Metal artifact distorted the CTA images, which prompted the decision to use intraoperative IVUS. The IVUS confirmed the noninvasive imaging findings and guided final decisions regarding aortic endograft size and location during spine hardware revision. LESSONS: For asymptomatic patients presenting with pedicle screws malpositioned in or near the aorta, treatment decisions revolve around the extent of vessel wall penetration. Intraluminal depth can be obscured by artifact on computed tomography or magnetic resonance imaging or inadequately evaluated by a transesophageal echocardiogram. In our intraoperative experience, IVUS confirmed the depth of vessel lumen violation by a single pedicle screw and no wall penetration by two additional screws of concern. This was useful in deciding on thoracic endovascular aortic repair graft size and landing zone and facilitated safe spinal instrumentation removal and revision.

Promiscuous dye binding by a light-up aptamer: application for label-free multi-wavelength biosensing.

Light-up DNA aptamers are promising label-free signal-transducers for biosensing applications due to their high chemical stability and low synthetic cost. Herein, we demonstrate that a dapoxyl DNA aptamer DAP-10-42 can be converted into a sensor generating a fluorescence signal at different wavelengths in the range of 500-660 nm depending on the dye that is present. This results from the discovered promiscuity of DAP-10-42 in binding fluorogenic dyes including arylmethane dyes. We have designed a split DAP-10-42 aptasensor for the detection of a katG gene fragment from Mycobacterium tuberculosis with a point mutation causing isoniazid resistance. Efficient interrogation of the gene fragment after nucleic acid sequence-based amplification (NASBA) is achieved directly in a protein-containing NASBA sample. This report lays a foundation for the application of the DAP-10-42 aptamer as a versatile sensing platform.