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.

Peptidyl arginine deiminase expression and macrophage polarization following stimulation with citrullinated and malondialdehyde-acetaldehyde modified fibrinogen.

OBJECTIVE: Post-translational modifications of extracellular matrix proteins such as fibrinogen may lead to tolerance loss and have been implicated in rheumatoid arthritis (RA) pathogenesis. The purpose of this study was to determine whether fibrinogen (FIB) modified with citrulline (CIT), malondialdehyde-acetaldehyde (MAA) or both leads to altered macrophage polarization, peptidyl arginine deiminase (PAD) expression, or production of citrullinated proteins. METHODS: PMA-treated U-937 cells (M0 cells) were stimulated with MAA, CIT or MAA-CIT modified FIB. Macrophage (M1/M2) phenotypes were evaluated by flow cytometry, RT-PCR, and ELISA. PAD enzyme expression and protein citrullination was evaluated using RT-PCR and Western Blot. RESULTS: Flow cytometry revealed that M0 macrophages stimulated with FIB-MAA-CIT resulted in mixed M1/M2 phenotypes as demonstrated by cell surface expression and mRNA levels of CD14, CD192, CD163, and CD206 (p < 0.001 vs. others), and the release of IL-18, IP-10, CCL22, and IL-13 (p < 0.001 vs. others). While FIB-MAA treated M0 cells demonstrated a mixed M1/M2 phenotype, cytokine and cell surface markers differed from FIB-MAA-CIT. Finally, M0 cells treated with FIB-CIT demonstrated markers and cytokines consistent with only the M1-like phenotype. Exposure of M0 cells to FIB-MAA-CIT (at 48 h) and FIB-MAA (at 24 h) led to increased mRNA expression and protein expression of PAD2 (p < 0.001) with increased protein citrullination. CONCLUSION: These findings suggest that MAA-modification and citrullination of FIB, in isolation or combination, yield specific effects on macrophage polarization, PAD expression and citrullination that ultimately may induce inflammatory and fibrotic responses associated with RA.

Examining the Otolaryngology Match and Relationships Between Publications and Institutional Rankings.

This study examines associations among publication number, National Institutes of Health (NIH) funding rank, medical school research rank, and otolaryngology department ranks of otolaryngology applicants during the 2018-2019 match cycle. Information regarding 2018-2019 otolaryngology applicants was collected from Otomatch.com and verified via department websites. Information was also collected regarding 2018 NIH funding rank and 2020 US News & World Report research rank of medical schools and otolaryngology departments. T tests and chi-square analyses were performed. Top 40 NIH funding rank, top 40 medical school research rank, and home institution department rank were separately associated with more publications and higher rates of matching into highly reputed otolaryngology departments (all P < .01). Furthermore, applicants who matched into ranked otolaryngology departments averaged significantly more publications (P < .01). Prospective otolaryngology applicants should take into account NIH funding rank, medical school research rank, and otolaryngology department rank, as they are associated with matching into high-ranking institutions.

Antioxidant properties of citric acid interfere with the uricase-based measurement of circulating uric acid.

BACKGROUND: Circulating uric acid (UA) is an important biomarker, not only in the detection and management of gout, but also in assessing the risk of related comorbidity. The impact of collection methods on clinical UA measurements has been the subject of limited study. After observing significant differences between UA concentrations of blood samples obtained by different collection tubes, we began examining the effects of exogenous tube components on measured UA concentrations. We aimed to: (1) demonstrate the variability in uricase-based UA measurements attributable to different collection methods and (2) identify factors influencing this variability. METHODS: Blood samples from human subjects were collected using Serum Separator Tubes (SST tubes), Acid Citrate Dextrose (ACD) tubes, and Sodium Citrate (SC) tubes. Circulating UA concentrations were measured by chemistry analyzers utilizing the uricase method. Absorbance assays were run in order to determine the effects of citric acid, sodium citrate, and dextrose on measured absorbance in the presence of leuco crystal violet dye, hydrogen peroxide, and peroxidase. Statistical analyses-including Student's T tests and ANOVA-were used to compare results. RESULTS: UA concentrations of blood samples collected in ACD tubes were significantly lower than those collected in SST tubes (P < 0.01). Samples collected in SC tubes trended towards lower UA measurements than samples collected in SST tubes, although this difference did not reach statistical significance (P = 0.06). Blood samples spiked with separate concentrations of sodium citrate (3.2 and 22.0 g/L), citric acid (8.0 g/L), and dextrose (24.5 g/L) demonstrated significantly lower UA measurements compared to controls (P < 0.01). Absorbance assays demonstrated that increasing concentrations of citric acid and sodium citrate-in the presence of leuco crystal violet, hydrogen peroxide, and peroxidase-decreased the amount of oxidized dye in the uricase method of UA measurement in a dose-dependent manner (P < 0.01). In contrast, dextrose did not significantly alter the amount of oxidized dye available. DISCUSSION: Our results indicate that citric acid obstructs accurate uricase-based UA measurement, providing falsely low values. Citric acid, a known antioxidant, scavenges hydrogen peroxide, a key intermediate using the uricase method. By scavenging hydrogen peroxide, citric acid decreases the amount of oxidized leuco dye leading to falsely low UA measurements. Therefore, collection tubes, like ACD and SC tubes, which contain concentrations of citric acid or its conjugate base sodium citrate should not be used to measure circulating UA levels when utilizing uricase-based measurement methods.