A novel 4-dimensional live-cell imaging system to study leukocyte-endothelial dynamics in ANCA-associated vasculitis.

Neutrophils, monocytes and the endothelium are critical to ANCA-associated vasculitis (AAV) pathogenesis. This study aimed to develop a 4-dimensional (4D) live-cell imaging system that would enable investigation of spatial and temporal dynamics of these cells in health and disease. We further aimed to validate this system using autologous donor serum from AAV patients and polyclonal ANCA IgG, as well as exploring its potential in the pre-clinical testing of putative therapeutic compounds. Neutrophils and monocytes were isolated from peripheral venous blood of AAV patients or healthy controls and co-incubated on an endothelial monolayer in the presence of autologous serum. Alternatively, polyclonal ANCA IgG was used, following TNF-α priming, and imaged in 4-dimensions for 3 h using a spinning disc confocal microscope. Volocity 6.3® analysis software was used for quantification of leukocyte dynamics. The use of autologous serum resulted in increased neutrophil degranulation (p = .002), transmigration (p = .0096) and monocyte transcellular transmigration (p = .0013) in AAV patients. Polyclonal MPO-ANCA IgG induced neutrophil degranulation (p < .001) in this system. C5aR1 antagonism reduced neutrophil degranulation (p < .0002). We have developed a novel 4D in vitro system that allows accurate quantification of multiple neutrophil- and monocyte-endothelial interactions in AAV in a single assay. This system has the potential to highlight dynamics key to pathophysiology of disease, as well investigating the impact of potential therapeutics on these functions.

Metabolic and Structural Skeletal Muscle Health in Systemic Lupus Erythematosus-Related Fatigue: A Multimodal Magnetic Resonance Imaging Study.

OBJECTIVE: To investigate the potential structural and metabolic role of skeletal muscle in systemic lupus erythematosus (SLE)-related fatigue. METHODS: A case-control, multimodal magnetic resonance imaging (MRI) study was conducted. Cases were patients with inactive SLE who reported chronic fatigue. Controls were age- and sex-matched healthy members of the general population. Patients were clinically characterized and then underwent a 3T whole-body MRI scan. Resting and dynamic 31 P MRI spectroscopy of the calf muscles was applied, from which phosphocreatine (PCr) recovery halftime, a marker of mitochondrial dysfunction, was computed. In addition, microstructural sequences (T1-weighted anatomic images, T2 mapping, and diffusion tensor imaging) were acquired. Descriptive statistics evaluated group differences and within-case physical fatigue correlations were explored. RESULTS: Of the 37 recruits (mean age 43.8 years, 89.2% female), cases (n = 19) reported higher levels of physical fatigue, pain, depression, and sleep disturbance compared to the control group (P < 0.0001). PCr was greater (P = 0.045) among cases (mean ± SD 33.0 ± 9.0 seconds) compared to controls (mean ± SD 27.1 ± 6.6 seconds). No microstructural group differences were observed. Within cases, physical fatigue did not correlate with PCr (r = -0.28, P = 0.25). CONCLUSION: We report preliminary data demonstrating greater skeletal muscle mitochondrial dysfunction among fatigued patients with SLE compared to healthy controls.