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In status asthmaticus (SA), severe bronchial inflammation is associated with acute respiratory failure. Neutrophils are the prominent cells found in bronchi from SA patients, but eosinophils are also recruited within the first 48 h after the beginning of mechanical ventilation (MV). Interleukin (IL)-5 and CC chemokines have been directly implicated in the pathophysiology of allergic asthma. However, their involvement in SA had not been determined. The aim of this study was to evaluate the production of CC chemokines and of IL-5 in airways from ventilated patients with SA as compared with mild asthma (A), and to assess the role of these mediators in eosinophil recruitment. We measured levels of the chemokines monocyte chemotactic proteins (MCPs)-1 and -3; regulated on activation, normal T-cell expressed and secreted (RANTES); macrophage inflammatory peptide (MIP)-1alpha; and eotaxin; and of the cytokine IL-5 in bronchial lavage fluid (BLF) from 10 SA patients, four patients without respiratory disease but undergoing ventilation (V) who were receiving MV, 11 patients with A, and eight healthy volunteers (C). We further evaluated in vitro eosinophil chemotactic activity of BLF from the various groups. Levels of MCP-1, MIP-1alpha, RANTES, and IL-5 were significantly higher in the SA than in the V, A, and C groups. MCP-3 and eotaxin values were not significantly different in the SA and other groups; however, their levels, as well as those of MIP-1alpha, RANTES, and IL-5 correlated with eosinophil influx. Eosinophil chemotactic activity in BLF was increased in asthmatic subjects (A and SA groups) as compared with the other groups, and in SA patients as compared with A patients. Addition of neutralizing anti-IL-5, anti-MCP-3, anti-eotaxin, and anti-RANTES antibodies significantly inhibited the eosinophil chemotactic activity as compared with that of native BLF. This study shows that the levels of various CC chemokines and IL-5 are increased in airways of SA patients, and are potentially involved in eosinophil recruitment.

BackgroundThe interpretation of germline TP53 variants is critical to ensure appropriate medical management of patients with cancer and follow-up of variant carriers. This interpretation remains complex and is becoming a growing challenge considering the exponential increase in TP53 tests. We developed a functional assay directly performed on patients’ blood.MethodsPeripheral blood mononuclear cells were cultured, activated, exposed to doxorubicin and the p53-mediated transcriptional response was quantified using reverse transcription–multiplex ligation probe amplification and RT-QMPSF assays, including 10 p53 targets selected from transcriptome analysis, and two amplicons to measure p53 mRNA levels. We applied this blood functional assay to 77 patients addressed for TP53 analysis.ResultsIn 51 wild-type TP53 individuals, the mean p53 functionality score was 12.7 (range 7.5–22.8). Among eight individuals harbouring likely pathogenic or pathogenic variants, the scores were reduced (mean 4.8, range 3.1–7.1), and p53 mRNA levels were reduced in patients harbouring truncating variants. We tested 14 rare unclassified variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro191Arg), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del), c.-117G>T) and 12 yielded functionally abnormal scores. Remarkably, the assay revealed that the c.*1175A>C polymorphic variant within TP53 poly-adenylation site can impact p53 function with the same magnitude as a null variant, when present on both alleles, and may act as a modifying factor in pathogenic variant carriers.ConclusionThis blood p53 assay should therefore be a useful tool for the rapid clinical classification of germline TP53 variants and detection of non-coding functional variants.

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