Immunofluorescence analysis of human eosinophils.

A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 µg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.

Adherence to Anticoagulation Interruption Guidelines in Patients with Atrial Fibrillation.

INTRODUCTION: Annually, 15% of patients who receive oral anticoagulation require interruption for surgery or an invasive procedure. This study evaluates the adherence of patients with atrial fibrillation with a history of stroke or transient ischemic attack to the Thrombosis Canada Perioperative guidelines for the discontinuation and reinitiation of anticoagulation treatment. METHODS: We collected data from a prospective patient survey at the Stroke Prevention Clinic in the University of Alberta hospital. Patients' charts were reviewed from the electronic medical records, and adherence was looked at according to the Thrombosis Canada Perioperative guidelines for the interruption of anticoagulants. RESULTS: During the study period (2016-2019), there were 509 patients surveyed. Anticoagulation treatment was interrupted in 150 patients with 98 interrupted for surgical or invasive procedures. The interruption was adherent to guidelines in only 29 (29.6%) of patients and inappropriate or nonadherent in 69 (70.4%) patients. There were seven ischemic strokes recorded during the period of interruption. The proportion of strokes was higher in patients whose anticoagulation interruption was longer than what the guidelines recommended (6/61 or 9.8%) when compared to those who adhered to recommended perioperative anticoagulation guidelines (1/29 or 3.4%). CONCLUSION: Our results indicate that significant discrepancy with following the recommended perioperative anticoagulation guidelines is common in real-life practice. Delay in re-anticoagulation may increase the risk of complications.

Cytokine trafficking of IL-9 and IL-13 through TfnRc+ vesicles in activated human eosinophils.

Eosinophils are granulocytes that are elevated in lung mucosa in approximately half of patients with allergic asthma. These highly granulated cells can synthesize and secrete many cytokines, including IL-9 and IL-13. We hypothesized that IL-9 and IL-13 are found as preformed mediators in crystalloid granules and secreted using distinct trafficking pathways. Human eosinophils were purified from peripheral venous blood, adhered to coverslips, and stimulated with platelet activating factor (PAF). Cells were immunolabeled with antibodies to IL-9 or IL-13 and colocalized with markers for secretory organelles, using CD63 for crystalloid granules and transferrin receptor (TfnRc) for vesicles. Fixed cells were imaged using super-resolution microscopy and quantified by colocalization using Pearson's correlation coefficient. IL-9 immunofluorescence increased in a time-dependent manner to PAF, whereas colocalization of IL-9 and CD63 significantly increased from 0.52 to 0.67 after 5 min PAF. Colocalization of IL-9 with TfnRc significantly increased at 60 min of stimulation with PAF (0.54 at 0 min to 0.60 at 60 min). IL-13 showed lower colocalization with CD63 (0.55) than TfnRc (0.63) in unstimulated cells. Upon PAF stimulation, IL-13 intensity transiently decreased at 5 and 60 min, whereas colocalization of IL-13 with CD63 decreased throughout stimulation to 0.43. While colocalization of IL-13 with TfnRc transiently increased to 0.66 at 5 min PAF, it returned to near baseline levels (0.64) after 15 min PAF. Our results suggest that IL-9 and IL-13 are stored in crystalloid granules as well as endosomal structures, and that IL-9 is primarily trafficked to the cell surface via TfnRc+ endosome-like vesicles.