Integrated Microfluidic Device for Functional Secretory Immunophenotyping of Immune Cells.

Integrated platforms for automatic assessment of cellular functional secretory immunophenotyping could have a widespread use in the diagnosis, real-time monitoring, and therapy evaluation of several pathologies. We present a microfluidic platform with integrated biosensors and culture chambers to measure cytokine secretion from a consistent and uniform number of immune cells. The biosensor relies on a fluorescence sandwich immunoassay enabled by the mechanically induced trapping of molecular interactions method. The platform contains 32 cell culture chambers, each patterned with an array of 492 microwells, to capture and analyze both adherent and nonadherent immune cells. Multiple stimuli can be delivered to a set of culture chambers. Per chamber, we were able to capture consistently 1113 ± 191 of blood-derived monocytes and neutrophils and 348 ± 37 THP-1 monocytes. Good occupancy efficiencies of ∼70% with a uniformity of ∼90% across all of the culture chambers of the device were achieved. Furthermore, we demonstrate that up to 96% of cells remain viable for the first 48 h. The employment of epoxy-modified glass substrates and active mixing enhanced the biosensing performance compared to the use of bare glass and simple diffusion. Finally, we performed functional secretory analysis of interleukin-8 and tumor necrosis factor alpha from human neutrophils and monocytes, stimulated with various doses of lipopolysaccharide and phorbol 12-myristate 13-acetate-ionomycin, respectively. We foresee the employment of our microfluidic platform in the diagnosis of different pathologies where alterations in cytokine secretion patterns can be used as biomarkers.

Candida albicans and non-albicans Isolates from Bloodstream Have Different Capacities to Induce Neutrophil Extracellular Traps.

Neutrophils activated with pathogens or their products induce formation of extracellular traps (NETs), but if this constitutes a general response against all pathogenic species in a single genus or intrageneric differences exist remains unknown, yet this is of great importance for the establishment of effective treatments. To determine this, we analyzed neutrophil extracellular traps formation after the stimulation with bloodstream isolates from different Candida species (Candida albicans, C. tropicalis, C. parapsilosis, and C. glabrata), and found that each species has a different capacity to induce DNA extrusion, which is independent of their morphology (yeast or hyphae). We observed that phospholipase producer's strains and their secretion products were able to induce NETs, a property not observed with phospholipase deficient strains, with exception of some Candida glabrata sensu stricto isolates, which showed no NETs induction although they did show phospholipase production. To further analyze this, we extended our study to include Candida glabrata cryptic species (C. bracarensis and C. nivariensis) and no extracellular traps formation was observed. Here, we contribute to the understanding of how neutrophils initiate NETs, and we found that certain strains may have a differential capacity to trigger these structures, which may explain the high mortality of some isolates.