Guidelines for mouse and human DC functional assays

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists

Application of two different nasal CPAP levels for the treatment of respiratory distress syndrome in preterm infants-"The OPTTIMMAL-Trial"-Optimizing PEEP To The IMMAture Lungs: study protocol of a randomized controlled trial.

BACKGROUND Nasal continuous positive airway pressure (CPAP) applies positive end-expiratory pressure (PEEP) and has been shown to reduce the need for intubation and invasive mechanical ventilation in very low birth weight infants with respiratory distress syndrome. However, CPAP failure rates of 50% are reported in large randomized controlled trials. A possible explanation for these failure rates is the application of insufficient low levels of PEEP during nasal CPAP treatment to maintain adequate functional residual capacity shortly after birth. The optimum PEEP level to treat symptoms of respiratory distress in very low birth weight infants has not been assessed in clinical studies. The aim of the study is to compare two different PEEP levels during nasal CPAP treatment in preterm infants. METHODS In this randomized multicenter trial, 216 preterm infants born at 26 + 0-29 + 6 gestational weeks will be allocated to receive a higher (6-8 cmH2O) or a lower (3-5 cmH2O) PEEP during neonatal resuscitation and the first 120 h of life. The PEEP level within each group will be titrated throughout the intervention based on the FiO2 (fraction of inspired oxygen concentration) requirements to keep oxygenation within the target range. The primary outcome is defined as the need for intubation and mechanical ventilation for > 1 h or being not ventilated but reaching one of the two pre-defined CPAP failure criteria (FiO2 > 0.5 for > 1 h or pCO2 ≥ 70 mmHg in two consecutive blood gas analyses at least 2 h apart). DISCUSSION Based on available data from the literature, the optimum level of PEEP that most effectively treats respiratory distress syndrome in preterm infants is unknown, since the majority of large clinical trials applied a wide range of PEEP levels (4-8 cmH2O). The rationale for our study hypothesis is that the early application of a higher PEEP level will more effectively counteract the collapsing properties of the immature and surfactant-deficient lungs and that the level of inspired oxygen may serve as a surrogate marker to guide PEEP titration. Finding the optimum noninvasive continuous distending pressure during early nasal CPAP is required to improve CPAP efficacy and as a consequence to reduce the exposure to ventilator-induced lung injury and the incidence of chronic lung disease in this vulnerable population of very preterm infants. TRIAL REGISTRATION drks.de DRKS00019940 . Registered on March 13, 2020