Reasons for multiple biologic and targeted synthetic DMARD switching and characteristics of treatment refractory rheumatoid arthritis.

OBJECTIVE: Switching biologic and targeted synthetic DMARD (b/tsDMARD) medications occurs commonly in RA patients, however data are limited on the reasons for these changes. The objective of the study was to identify and categorize reasons for b/tsDMARD switching and investigate characteristics associated with treatment refractory RA. METHODS: In a multi-hospital RA electronic health record (EHR) cohort, we identified RA patients prescribed ≥1 b/tsDMARD between 2001 and 2017. Consistent with the EULAR "difficult to treat" (D2T) RA definition, we further identified patients who discontinued ≥2 b/tsDMARDs with different mechanisms of action. We performed manual chart review to determine reasons for medication discontinuation. We defined "treatment refractory" RA as not achieving low disease activity (<3 tender or swollen joints on <7.5 mg of daily prednisone equivalent) despite treatment with two different b/tsDMARD mechanisms of action. We compared demographic, lifestyle, and clinical factors between treatment refractory RA and b/tsDMARD initiators not meeting D2T criteria. RESULTS: We identified 6040 RA patients prescribed ≥1 b/tsDMARD including 404 meeting D2T criteria. The most common reasons for medication discontinuation were inadequate response (43.3 %), loss of efficacy (25.8 %), and non-allergic adverse events (13.7 %). Of patients with D2T RA, 15 % had treatment refractory RA. Treatment refractory RA patients were younger at b/tsDMARD initiation (mean 47.2 vs. 55.2 years, p < 0.001), more commonly female (91.8% vs. 76.1 %, p = 0.006), and ever smokers (68.9% vs. 49.9 %, p = 0.005). No RA clinical factors differentiated treatment refractory RA patients from b/tsDMARD initiators. CONCLUSIONS: In a large EHR-based RA cohort, the most common reasons for b/tsDMARD switching were inadequate response, loss of efficacy, and nonallergic adverse events (e.g. infections, leukopenia, psoriasis). Clinical RA factors were insufficient for differentiating b/tsDMARD responders from nonresponders.

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer.

As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. Therefore, a better understanding of macrophage phenotype conversion in relevant models is needed to elucidate its potential roles in disease. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each cancer cell line with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that iNos production is enhanced in these cancer models using PBI1, tracking increased fluorescence. Collectively, this work demonstrates that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.