Confusion in the interpretation of prolactin levels caused by inappropriately low reference intervals.

OBJECTIVE: Serum prolactin measurements are important in the differential diagnosis of pituitary masses and subfertility. We observed discrepancies in serum prolactin levels in several patients measured with different immunoassays. Despite differences in assay results, the reference intervals derived by the manufacturers were similar. In this study we aimed to investigate prolactin assay differences and to re-establish RIs for different prolactin immunoassays. METHODS: For the assay comparison, serum samples were collected from men and women visiting our the Amsterdam UMC hospital. Prolactin levels were measured using the AtellicaTM IM analyzer (Siemens Healthineers) and the Cobas (Roche Diagnostics) immunoassay. Reference intervals for prolactin were re-established for men, premenopausal women and postmenopausal women for both the Atellica and Cobas immunoassay. RESULTS: Prolactin levels measured using the Cobas immunoassay were 1.75 times higher than those measured using the Atellica immunoassay. The re-established reference intervals for Atellica and Cobas confirmed these findings and were <0.32 U/L; <0.55 U/L for men; <0.64 U/L; <0.86 U/L for premenopausal women and <0.31 U/L; <0.59 U/L for postmenopausal women, respectively for Atellica and Cobas assays. The re-established RIs of the Atellica assay matched the current and manufacturer RIs whereas those for Cobas differed substantially. CONCLUSIONS: Prolactin levels are assay dependent and the re-established reference intervals are different for the Atellica and Cobas assays. We recommend that laboratory specialists and manufacturers periodically review prolactin assay RIs as incorrect reference intervals can lead to misinterpretation of prolactin levels and unnecessary referrals and further laboratory testing, as we have experienced.

Imprinting of bronchial epithelial cells upon in vivo rhinovirus infection in people with asthma.

Background: Defective translocation of the translational repressor TIAR (T-cell internal antigen receptor) in bronchial epithelial cells (BECs) from asthma patients underlies epithelial hyperresponsiveness, reflected by an exaggerated production of a select panel of inflammatory cytokines such as CXCL-8, interleukin (IL)-6, granulocyte colony-stimulating factor, CXCL-10, upon exposure to tumour necrosis factor (TNF) and IL-17A. With this study we aimed to clarify whether epithelial hyperresponsiveness is a consistent finding, is changed upon in vivo exposure to rhinovirus (RV)-A16 and applies to the bronchoconstrictor endothelin-1. Methods: BECs were obtained from asthma patients (n=18) and healthy individuals (n=11), 1 day before and 6 days post-RV-A16 exposure. BECs were cultured and stimulated with TNF and IL-17A and inflammatory mediators were analysed. The bronchoalveolar lavage fluid (BALF) was obtained in parallel with BECs to correlate differential cell counts and inflammatory mediators with epithelial hyperresponsiveness. Results: Epithelial hyperresponsiveness was confirmed in sequential samples and even increased in BECs from asthma patients after RV-A16 exposure, but not in BECs from healthy individuals. Endothelin-1 tended to increase in BECs from asthma patients collected after RV-A16 exposure, but not in BECs from healthy individuals. In vitro CXCL-8 and endothelin-1 production correlated. In vivo relevance for in vitro CXCL-8 and endothelin-1 production was shown by correlations with forced expiratory volume in 1 s % predicted and CXCL-8 BALF levels. Conclusion: Epithelial hyperresponsiveness is an intrinsic defect in BECs from asthma patients, which increases upon viral exposure, but not in BECs from healthy individuals. This epithelial hyperresponsiveness also applies to the bronchoconstrictor endothelin-1, which could be involved in airway obstruction.

The Dietary Intake of Carrot-Derived Rhamnogalacturonan-I Accelerates and Augments the Innate Immune and Anti-Viral Interferon Response to Rhinovirus Infection and Reduces Duration and Severity of Symptoms in Humans in a Randomized Trial.

Acute respiratory infections are an important health concern. Traditionally, polysaccharide-enriched extracts from plants, containing immunomodulatory rhamnogalacturonan-I (RG-1), were used prophylactically. We established the effects of dietary supplementation with carrot-derived RG-I (cRG-I, 0-0.3-1.5 g/day) in 177 healthy individuals (18-65 years) on symptoms following infection with rhinovirus strain 16 (RV16). Primary outcomes were changes in severity and duration of symptoms, and viral load in nasal lavage. Secondary outcomes were changes in innate immune and anti-viral responses, reflected by CXCL10 and CXCL8 levels and cell differentials in nasal lavage. In a nested cohort, exploratory transcriptome analysis was conducted on nasal epithelium. Intake of cRG-I was safe, well-tolerated and accelerated local cellular and humoral innate immune responses induced by RV16 infection, with the strongest effects at 1.5 g/d. At 0.3 g/d, a faster interferon-induced response, induction of the key anti-viral gene EIF2AK2, faster viral clearance, and reduced symptom severity (-20%) and duration (-25%) were observed. Anti-viral responses, viral clearance and symptom scores at 1.5 g/d were in between those of 0 and 0.3 g/d, suggesting a negative feedback loop preventing excessive interferon responses. Dietary intake of cRG-I accelerated innate immune and antiviral responses, and reduced symptoms of an acute respiratory viral infection.

Phenotypic comparison of human alveolar macrophages before and after in vivo rhinovirus 16 challenge.

We used mass cytometry to extensively characterize bronchoalveolar lavage macrophages before and two days after in vivo rhinovirus 16 infection in a heterogeneous population of healthy and asthma/COPD subjects. Multivariate partial least squares discriminant analysis revealed distinct clusters of alveolar macrophages before versus after the virus, suggesting changes in overall phenotype.

Metabolic differences between bronchial epithelium from healthy individuals and patients with asthma and the effect of bronchial thermoplasty.

BACKGROUND: Asthma is a heterogeneous disease with differences in onset, severity, and inflammation. Bronchial epithelial cells (BECs) contribute to asthma pathophysiology. OBJECTIVE: We determined whether transcriptomes of BECs reflect heterogeneity in inflammation and severity in asthma, and whether this was affected in BECs from patients with severe asthma after their regeneration by bronchial thermoplasty. METHODS: RNA sequencing was performed on BECs obtained by bronchoscopy from healthy controls (n = 16), patients with mild asthma (n = 17), patients with moderate asthma (n = 5), and patients with severe asthma (n = 17), as well as on BECs from treated and untreated airways of the latter (also 6 months after bronchial thermoplasty) (n = 23). Lipidome and metabolome analyses were performed on cultured BECs from healthy controls (n = 7); patients with severe asthma (n = 9); and, for comparison, patients with chronic obstructive pulmonary disease (n = 7). RESULTS: Transcriptome analysis of BECs from patients showed a reduced expression of oxidative phosphorylation (OXPHOS) genes, most profoundly in patients with severe asthma but less profoundly and more heterogeneously in patients with mild asthma. Genes related to fatty acid metabolism were significantly upregulated in asthma. Lipidomics revealed enhanced levels of lipid species (phosphatidylcholines, lysophosphatidylcholines. and bis(monoacylglycerol)phosphate), whereas levels of OXPHOS metabolites were reduced in BECs from patients with severe asthma. BECs from patients with mild asthma characterized by hyperresponsive production of mediators implicated in neutrophilic inflammation had decreased expression of OXPHOS genes compared with that in BECs from patients with mild asthma with normoresponsive production. BECs obtained after thermoplasty had significantly increased expression of OXPHOS genes and decreased expression of fatty acid metabolism genes compared with BECs obtained from untreated airways. CONCLUSION: BECs in patients with asthma are metabolically different from those in healthy individuals. These differences are linked with inflammation and asthma severity, and they can be reversed by bronchial thermoplasty.

Eosinophils capture viruses, a capacity that is defective in asthma.

BACKGROUND: Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils. METHODS: To follow eosinophil-virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti-IL-5 (mepolizumab), followed by a challenge with rhinovirus-16 (RV16). RESULTS: DiD-RSV and DiD-influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus-induced loss of asthma control. CONCLUSIONS: This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations.

IL-33 drives influenza-induced asthma exacerbations by halting innate and adaptive antiviral immunity.

BACKGROUND: Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive. OBJECTIVE: We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbations. METHODS: Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti-IL-33 receptor ST2, anti-TSLP, or both. RESULTS: We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite-sensitized mice to dampen IFN-ß expression and prevent the TH1-promoting dendritic cell phenotype. IL-33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the TH2 response. CONCLUSION: Interventions targeting the IL-33/ST2 axis could prove an effective acute short-term therapy for virus-induced asthma exacerbations.

Anti-IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses (MATERIAL). A Placebo-controlled, Double-Blind Study.

RATIONALE: Eosinophils drive pathophysiology in stable and exacerbating eosinophilic asthma, and therefore treatment is focused on the reduction of eosinophil numbers. Mepolizumab, a humanized monoclonal antibody that neutralizes IL-5 and efficiently attenuates eosinophils, proved clinically effective in severe eosinophilic asthma but not in mild asthma. OBJECTIVES: To study the effect of mepolizumab on virus-induced immune responses in mild asthma. METHODS: Patients with mild asthma, steroid-naive and randomized for eosinophil numbers, received 750 mg mepolizumab intravenously in a placebo-controlled double-blind trial, 2 weeks after which patients were challenged with rhinovirus (RV) 16. FEV1, FVC, fractional exhaled nitric oxide, symptom scores (asthma control score), viral load (PCR), eosinophil numbers, humoral (luminex, ELISA), and cellular (flow cytometry) immune parameters in blood, BAL fluid, and sputum, before and after mepolizumab and RV16, were assessed. MEASUREMENTS AND MAIN RESULTS: Mepolizumab attenuated baseline blood eosinophils and their activation, attenuated trendwise sputum eosinophils, and enhanced circulating natural killer cells. Mepolizumab did not affect FEV1, FVC, and fractional exhaled nitric oxide, neither at baseline nor after RV16. On RV16 challenge mepolizumab did not prevent eosinophil activation but did enhance local B lymphocytes and macrophages and reduce neutrophils and their activation. Mepolizumab also enhanced secretory IgA and reduced tryptase in BAL fluid. Finally, mepolizumab affected particularly RV16-induced macrophage inflammatory protein-3a, vascular endothelial growth factor-A, and IL-1RA production in BAL fluid. CONCLUSIONS: Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-induced immune responses in mild asthma. Although these latter effects likely are caused by attenuated eosinophil numbers, we cannot exclude a role for basophils. Clinical trial registered with www.clinicaltrials.gov (NCT 01520051).

Neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease.

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA-rich mucus plugs are characteristic features of severe RSV-LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first-line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti-viral effect in vitro. Second, we studied NET formation in vivo during severe RSV-LRTD in infants and bovine RSV-LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV-LRTD. Detailed histopathological examination of calves with RSV-LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV-LRTD contributes to airway obstruction.