BackgroundNeutrophil extracellular traps (NETs), extracellular structures composed of decondensed chromatin and antimicrobial molecules, are released in a process called NETosis. NETs, which are part of normal host defense, have also been implicated in multiple human diseases. Unfortunately, methods for quantifying NETs have limitations which constrain the study of NETs in disease. Establishing optimal methods for NET quantification holds the potential to further elucidate the role of NETs in normal and pathologic processes.ResultsTo better quantify NETs and NET-like structures, we created DNA Area and NETosis Analysis (DANA), a novel ImageJ/Java based program which provides a simple, semi-automated approach to quantify NET-like structures and DNA area. DANA can analyze many fluorescent microscope images at once and provides data on a per cell, per image, and per sample basis. Using fluorescent microscope images of Sytox-stained human neutrophils, DANA quantified a similar frequency of NET-like structures to the frequency determined by two different individuals counting by eye, and in a fraction of the time. As expected, DANA also detected increased DNA area and frequency of NET-like structures in neutrophils from subjects with rheumatoid arthritis as compared to control subjects. Using images of DAPI-stained murine neutrophils, DANA (installed by an individual with no programming background) gave similar frequencies of NET-like structures as the frequency of NETs determined by two individuals counting by eye. Further, DANA quantified more NETs in stimulated murine neutrophils compared to unstimulated, as expected.ConclusionsDANA provides a means to quantify DNA decondensation and the frequency of NET-like structures using a variety of different fluorescent markers in a rapid, reliable, simple, high-throughput, and cost-effective manner making it optimal to assess NETosis in a variety of conditions.Electronic supplementary materialThe online version of this article (10.1186/s12575-018-0072-y) contains supplementary material, which is available to authorized users.

Objective. Anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly present in rheumatoid arthritis (RA) without a clear rationale for their coexistence. Moreover, autoantibodies develop against proteins with different posttranslational modifications and native proteins without obvious unifying characteristics of the antigens. We undertook this study to broadly evaluate autoantibody binding in seronegative and seropositive RA to identify novel features of reactivity.Methods. An array was created using a total of 172,828 native peptides, citrulline-containing peptides, and homocitrulline-containing peptides derived primarily from proteins citrullinated in the rheumatoid joint. IgG and IgM binding to peptides were compared between cyclic citrullinated peptide (CCP)-positive RF+, CCP+RF−, CCP−RF+, and CCP−RF− serum from RA patients (n = 48) and controls (n = 12). IgG-bound and endogenously citrullinated peptides were analyzed for amino acid patterns and predictors of intrinsic disorder, i.e., unstable 3-dimensional structure. Binding to IgG-derived peptides was specifically evaluated. Enzyme-linked immunosorbent assay confirmed key results.Results. Broadly, CCP+RF+ patients had high citrulline-specific IgG binding to array peptides and CCP+RF− and CCP−RF+ patients had modest citrulline-specific IgG binding (median Z scores 3.02, 1.42, and 0.75, respectively; P < 0.0001). All RA groups had low homocitrulline-specific binding. CCP+RF+ patients had moderate IgG binding to native peptides (median Z score 2.38; P < 0.0001). The highest IgG binding was to citrulline-containing peptides, irrespective of protein identity, especially if citrulline was adjacent to glycine or serine, motifs also seen in endogenous citrullination in the rheumatoid joint. Highly bound peptides had multiple features predictive of disorder. IgG from CCP+RF+ patients targeted citrulline-containing IgG-derived peptides.Conclusion. Disordered antigens, which are frequently citrullinated, and common epitopes for ACPAs and RF are potentially unifying features for RA autoantibodies.

Decision paralysis (DP) can be defined as a patient’s inability to commit to a physician and/or initiate appropriate treatment for their condition. An incessant search for greater physician opinions often leads to treatment delay, disease progression, and initiation of care at more advanced stages. Despite the harms associated with DP, a dearth of research on the issue remains. There are no guidelines that assist in both recognition and rectification of DP, leaving patients with chronic illnesses and diagnoses without well-characterized treatment algorithms especially vulnerable. This paper analyzes why patients are inclined toward DP and the clinical implications. Review of the literature affirms that the patient–physician relationship holds considerable influence; physicians identifying DP can improve therapeutic outcomes for their patients. Using these findings, we then propose a framework for broaching this topic with a method that supports patients while respecting their autonomy. A practical approach to both recognition and patient-centered discourse is introduced, providing a foundation for physicians to host these conversations and understand their patients’ perspectives. This approach toward recognition and discourse on DP holds clinical importance, given that there is a paucity of established guidance. A future uniform approach may generate optimal patient care recommendations, which will hold far-reaching impact on both the patient–physician relationship and overall patient outcomes.