The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

Assessment of a defined approach based on a stacking prediction model to identify skin sensitization hazard.

Skin sensitization is an important toxicological endpoint in the safety assessment of chemicals and cosmetic ingredients. Driven by ethical considerations and European Union (EU) legislation, its assessment has progressed from the reliance on traditional animal models to the use of non-animal test methods. It is generally accepted that the assessment of skin sensitization requires the integration of various non-animal test methods in defined approaches (DAs), to cover the mechanistic key events of the adverse outcomes pathway (AOP) (OECD, 2014). Several case studies for DAs predicting skin sensitization hazard or potency have been submitted to the OECD, including a stacking meta-model developed by L'Oréal Research & Innovation (OECD, 2017b; Del Bufalo et al., 2018; Noçairi et al., 2016). The present study evaluated the predictive performance of the defined approach integrating a stacking meta-model incorporating in silico, in chemico and in vitro assays, using the Cosmetics Europe (CE) skin sensitization database. Based on the optimized prediction cut-offs, the defined approach provided a hazard prediction for 97 chemicals with a sensitivity of 91%, a specificity of 76% and accuracy of 86% (kappa of 0.67) against human skin sensitization hazard data and a sensitivity of 85%, specificity of 91% and accuracy of 87% (kappa of 0.67) against Local Lymph Node Assay (LLNA) hazard data. A comparison of the in vivo LLNA with human hazard data for the same 97 chemicals showed a sensitivity of 92%, specificity of 51% and accuracy of 78% (kappa of 0.48). Thus, the defined approach showed a higher degree of concordance, as compared to the LLNA for predicting human skin sensitization hazard. Moreover, a comparison with the six DAs selected for evaluation of their predictivity in the study by Kleinstreuer et al. (2018) showed a similar high accuracy of 86% for 97 overlapping chemicals. The next step will be an independent evaluation of the DA for its integration in the performances based test guidelines (PBTG) for skin sensitization.

Alveolar fluid in acute respiratory distress syndrome promotes fibroblast migration: role of platelet-derived growth factor pathway*.

OBJECTIVES: Fibroblast migration is an initiating step in fibroproliferation; its involvement during acute lung injury and acute respiratory distress syndrome remains poorly understood. The aims of this study were: 1) to determine whether bronchoalveolar lavage fluids from patients with acute lung injury/acute respiratory distress syndrome modulate lung fibroblast migration; 2) to assess lung fibroblast migration's clinical relevance; and 3) to evaluate the role of the platelet-derived growth factor pathway in this effect. DESIGN: Prospective cohort study. SETTING: Three intensive care units of a large tertiary referral center. PATIENTS: Ninety-three ventilated patients requiring bronchoalveolar lavage fluids were enrolled (48 with acute respiratory distress syndrome, 33 with acute lung injury, and 12 ventilated patients without acute lung injury/acute respiratory distress syndrome). INTERVENTIONS: After bronchoalveolar lavage fluids collection during standard care, the patients were followed up for 28 days and clinical outcomes were recorded. Migration assays were performed by using a Transwell model; bronchoalveolar lavage fluids platelet-derived growth factor and soluble platelet-derived growth factor receptor-α were characterized by Western blot and measured by ELISA. MEASUREMENTS AND MAIN RESULTS: Most of the bronchoalveolar lavage fluids inhibited basal fibroblast migration. Bronchoalveolar lavage fluids chemotactic index increased with severity of lung injury (28% in patients without acute lung injury/acute respiratory distress syndrome and with acute lung injury vs. 91% in acute respiratory distress syndrome patients; p = .016). In acute lung injury/acute respiratory distress syndrome patients, inhibition of basal fibroblast migration by bronchoalveolar lavage fluids below 52% was independently associated with a lower 28-day mortality (odds ratio [95% confidence interval] 0.313 [0.10-0.98], p = .046). Platelet-derived growth factor-related peptides and soluble platelet-derived growth factor-Rα were detected in all bronchoalveolar lavage fluids from acute lung injury/acute respiratory distress syndrome patients. The effect of bronchoalveolar lavage fluids stimulating migration was inhibited by a specific platelet-derived growth factor receptor inhibitor (AG1296). Bronchoalveolar lavage fluids inhibiting migration reversed the effect of rh-platelet-derived growth factor-BB and reduced by 40% the binding of 125I-platelet-derived growth factor-BB to fibroblast cell surface in favor of a role for platelet-derived growth factor-sRα. CONCLUSIONS: : Together, our results suggest that during acute lung injury, fibroblast migration is modulated by bronchoalveolar lavage fluids through a platelet-derived growth factor/platelet-derived growth factor-sRα balance. Migration is associated with clinical severity and patient 28-day mortality.

Alveolar fibrocyte percentage is an independent predictor of poor outcome in patients with acute lung injury.

OBJECTIVE: Fibrocytes are mesenchymal progenitors involved in normal and pathologic repair. The aims of this study were: 1) to quantify fibrocytes in bronchoalveolar lavage fluid from patients with or without acute lung injury and acute respiratory distress syndrome; and 2) to evaluate the prognostic value of bronchoalveolar lavage fibrocyte percentage in patients with acute lung injury and acute respiratory distress syndrome. DESIGN: Prospective cohort study. SETTING: Three intensive care units of a large tertiary referral center. PATIENTS: One hundred twenty-two ventilated patients requiring bronchoalveolar lavage were enrolled (62 acute respiratory distress syndrome, 30 acute lung injury, 30-ventilated patients without acute lung injury and acute respiratory distress syndrome). INTERVENTIONS: After bronchoalveolar lavage collection during standard care, the patients were followed up for 28 days and clinical outcome was recorded. Fibrocytes (CD45+/collagen 1+) were quantified in bronchoalveolar lavage by flow cytometry. Comparison of bronchoalveolar lavage fibrocyte percentage from patients with or without acute lung injury and acute respiratory distress syndrome was performed using a Wilcoxon test. A multivariate analysis using a Cox model was performed to study the independent predictors of survival. MEASUREMENTS AND MAIN RESULTS: Fibrocytes were detected in 90 of 92 (98%) bronchoalveolar lavages from patients with acute lung injury and acute respiratory distress syndrome. The median percentage of bronchoalveolar lavage fibrocytes was significantly higher in patients with acute lung injury and acute respiratory distress syndrome (5.0%) in comparison with ventilated control subjects (0.9%, p < .0001). After adjustment for age, comorbidity of malignancy, and severity of illness, a bronchoalveolar lavage fibrocyte percentage >6% was independently associated with a higher 28-day mortality in patients with acute lung injury and acute respiratory distress syndrome (hazard ratio [95% confidence interval] 6.15 [2.78-13.64], p ≤ .0001). Addition of bronchoalveolar lavage fibrocyte percentage in a clinical model predicting mortality in patients with acute lung injury and acute respiratory distress syndrome improved global fit and discriminatory capacity (c-statistic, 0.78-0.85; p = .007). CONCLUSIONS: Fibrocytes are detectable in bronchoalveolar lavage during acute lung injury and acute respiratory distress syndrome. A bronchoalveolar lavage fibrocyte percentage >6% provides an additive prognostic value to clinical predictors and may be useful to identify patients with acute lung injury and acute respiratory distress syndrome at highest risk of an adverse outcome.