Extracellular traps are evident in Romanowsky-stained smears of bronchoalveolar lavage from children with non-cystic fibrosis bronchiectasis.

BACKGROUND AND OBJECTIVE: The importance of extracellular traps (ETs) in chronic respiratory conditions is increasingly recognized but their role in paediatric bronchiectasis is poorly understood. The specialized techniques currently required to study ETs preclude routine clinical use. A simple and cost-effective ETs detection method is needed to support diagnostic applications. We aimed to determine whether ETs could be detected using light microscopy-based assessment of Romanowsky-stained bronchoalveolar lavage (BAL) slides from children with bronchiectasis, and whether the ETs cellular origin could be determined. METHODS: Archived Romanowsky-stained BAL slides from a cross-sectional study of children with bronchiectasis were examined for ETs using light microscopy. The cellular origin of individual ETs was determined based on morphology and physical contact with surrounding cell(s). RESULTS: ETs were observed in 78.7% (70/89) of BAL slides with neutrophil (NETs), macrophage (METs), eosinophil (EETs) and lymphocyte (LETs) ETs observed in 32.6%, 51.7%, 4.5% and 9%, respectively. ETs of indeterminate cellular origin were present in 59.6% of slides. Identifiable and indeterminate ETs were co-detected in 43.8% of slides. CONCLUSION: BAL from children with bronchiectasis commonly contains multiple ET types that are detectable using Romanowsky-stained slides. While specialist techniques remain necessary to determining the cellular origin of all ETs, screening of Romanowsky-stained slides presents a cost-effective method that is well-suited to diagnostic settings. Our findings support further research to determine whether ETs can be used to define respiratory endotypes and to understand whether ETs-specific therapies may be required to resolve airway inflammation among children with bronchiectasis.

Randomised controlled trial of perinatal vitamin D supplementation to prevent early-onset acute respiratory infections among Australian First Nations children: the 'D-Kids' study protocol.

INTRODUCTION: Globally, acute respiratory infections (ARIs) are a leading cause of childhood morbidity and mortality. While ARI-related mortality is low in Australia, First Nations infants are hospitalised with ARIs up to nine times more often than their non-First Nations counterparts. The gap is widest in the Northern Territory (NT) where rates of both acute and chronic respiratory infection are among the highest reported in the world. Vitamin D deficiency is common among NT First Nations neonates and associated with an increased risk of ARI hospitalisation. We hypothesise that perinatal vitamin D supplementation will reduce the risk of ARI in the first year of life. METHODS AND ANALYSIS: 'D-Kids' is a parallel (1:1), double-blind (allocation concealed), randomised placebo-controlled trial conducted among NT First Nations mother-infant pairs. Pregnant women and their babies (n=314) receive either vitamin D or placebo. Women receive 14 000 IU/week or placebo from 28 to 34 weeks gestation until birth and babies receive 4200 IU/week or placebo from birth until age 4 months. The primary outcome is the incidence of ARI episodes receiving medical attention in the first year of life. Secondary outcomes include circulating vitamin D level and nasal pathogen prevalence. Tertiary outcomes include infant immune cell phenotypes and challenge responses. Blood, nasal swabs, breast milk and saliva are collected longitudinally across four study visits: enrolment, birth, infant age 4 and 12 months. The sample size provides 90% power to detect a 27.5% relative reduction in new ARI episodes between groups. ETHICS AND DISSEMINATION: This trial is approved by the NT Human Research Ethics Committee (2018-3160). Study outcomes will be disseminated to participant families, communities, local policy-makers, the broader research and clinical community via written and oral reports, education workshops, peer-reviewed journals, national and international conferences. TRIAL REGISTRATION NUMBER: ACTRN12618001174279.

Evidence of maternal transfer of antigen-specific antibodies in serum and breast milk to infants at high-risk of S. pneumoniae and H. influenzae disease.

Introduction: Children in low-mid income countries, and First Nations children in high-income countries, experience disproportionately high rates of Streptococcus pneumoniae and Haemophilus influenzae infections and diseases including pneumonia and otitis media. We previously observed that infants from Papua New Guinea had no evidence of waning maternal immunity for H. influenzae-specific antibodies. In this study, we assessed S. pneumoniae and H. influenzae antibody titres in Australian First Nation mothers and infants to determine antigen-specific antibody ontogenies and whether H. influenzae antibody titres in infants were due to low maternal antibody titres or lack of placental transfer. Methods: Breast milk, infant nasopharyngeal swabs and ear assessment data were collected 1-, 2-, 7-months post-birth as well as maternal, cord and 7-month-old infant sera, from 85 Australian Aboriginal and Torres Strait Islander mother-infant pairs. Serum IgG and breast milk IgG and IgA antibody titres to S. pneumoniae antigens (PspA1, PspA2, CbpA, Ply) and H. influenzae antigens (PD, ChimV4, OMP26, rsPilA) were measured. Results: IgG titres in maternal and cord sera were similar for all antigens, except Ply (higher in cord; p=0.004). Sera IgG titres at 7-months of age were lower than cord sera IgG titres for all S. pneumoniae antigens (p<0.001). Infant sera IgG titres were higher than cord sera for H. influenzae PD (p=0.029), similar for OMP26 (p=0.817) and rsPilA (p=0.290), and lower for ChimV4 (p=0.004). Breast milk titres were similar for all antigens at 1, 2 and 7-months except OMP26 IgA (lower at 7-months than 1-month; p=0.035), PspA2 IgG (p=0.012) and Ply IgG that increased by 7-months (p=0.032). One third of infants carried nontypeable Haemophilus influenzae (NTHi), 45% carried S. pneumoniae and 52% had otitis media (OM) observed at least once over the 7-months. 73% of infants who carried either S. pneumoniae or NTHi, also had otitis media observed. Conclusions: Similarities between maternal and cord IgG titres, and absence of waning, support a lack of maternal H. influenzae IgG antibodies available for cross-placental transfer. Increased maternal anti-PD IgG could offer some protection from early carriage with NTHi, and maternal immunisation strategies should be considered for passive-active immunisation of infants to protect against S. pneumoniae and H. influenzae diseases. Trial registration: ClinicalTrials.gov NCT00714064 and NCT00310349.

Can dried blood spots be used to accurately measure vitamin D metabolites?

BACKGROUND: Where conventional blood sampling is challenging, dried blood spots (DBS) provide a practical sample alternative for measuring vitamin D levels. Our study aimed to develop and evaluate a clinical pathology service-based assay suitable for measuring vitamin D in batches of DBS samples collected remote to the testing site. METHODS: A high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with derivatisation was developed to measure 25-hydroxyvitamin D metabolites (25OHD3, 25OHD2 and 3-epi-25OHD3) in DBS samples. The assay was validated using paired DBS and plasma samples from 37 healthy adults. RESULTS: The assay reproducibly (<11.5% coefficient of variation) quantified 25OHD3 (range 1-300 nmol/L), 25OHD2 (range 2-300 nmol/L) and 3-epi-25OHD3 (range 1-200 nmol/L) in DBS samples. The 25OHD3 metabolite was detected in all DBS samples, 3-epi-25OHD3 in six plasma (range 2.1-6.3 nmol/L) and paired DBS samples, and 25OHD2 was not detected. Concentrations of 25OHD3 were highly correlated between paired samples: capillary DBS and venous plasma (r = 0.92), venous DBS and venous plasma (r = 0.93), and capillary DBS and venous DBS (r = 0.97). Ordinary least squares regression was used to characterise (ß = 0.81) and correct the systematic bias in DBS data (compared to paired plasma). Thereafter, Bland-Altman analysis demonstrated robust agreement between sample-methods. CONCLUSION: This simple and rapid DBS-based LC-MS/MS assay accurately quantified serum vitamin D metabolites using a paired-sample 'bridging strategy' to correct for the inherent sample-method bias.

Vitamin D Modulation of the Innate Immune Response to Paediatric Respiratory Pathogens Associated with Acute Lower Respiratory Infections.

Vitamin D is an essential component of immune function and childhood deficiency is associated with an increased risk of acute lower respiratory infections (ALRIs). Globally, the leading childhood respiratory pathogens are Streptococcus pneumoniae, respiratory syncytial virus and the influenza virus. There is a growing body of evidence describing the innate immunomodulatory properties of vitamin D during challenge with respiratory pathogens, but recent systematic and unbiased synthesis of data is lacking, and future research directions are unclear. We therefore conducted a systematic PubMed literature search using the terms "vitamin D" and "Streptococcus pneumoniae" or "Respiratory Syncytial Virus" or "Influenza". A priori inclusion criteria restricted the review to in vitro studies investigating the effect of vitamin D metabolites on human innate immune cells (primary, differentiated or immortalised) in response to stimulation with the specified respiratory pathogens. Eleven studies met our criteria. Despite some heterogeneity across pathogens and innate cell types, vitamin D modulated pathogen recognition receptor (PRRs: Toll-like receptor 2 (TLR2), TLR4, TLR7 and nucleotide-binding oligomerisation domain-containing protein 2 (NOD2)) expression; increased antimicrobial peptide expression (LL-37, human neutrophil peptide (HNP) 1-3 and ß-defensin); modulated autophagosome production reducing apoptosis; and modulated production of inflammatory cytokines (Interleukin (IL) -1ß, tumour necrosis factor-α (TNF-α), interferon-É£ (IFN-É£), IL-12p70, IFN-ß, Regulated on Activation, Normal T cell Expressed (RANTES), IL-10) and chemokines (IL-8 and C-X-C motif chemokine ligand 10 (CXCL10)). Differential modulation of PRRs and IL-1ß was reported across immune cell types; however, this may be due to the experimental design. None of the studies specifically focused on immune responses in cells derived from children. In summary, vitamin D promotes a balanced immune response, potentially enhancing pathogen sensing and clearance and restricting pathogen induced inflammatory dysregulation. This is likely to be important in controlling both ALRIs and the immunopathology associated with poorer outcomes and progression to chronic lung diseases. Many unknowns remain and further investigation is required to clarify the nuances in vitamin D mediated immune responses by pathogen and immune cell type and to determine whether these in vitro findings translate into enhanced immunity and reduced ALRI in the paediatric clinical setting.