Hnp1 3

The nasal secretions were analyzed for the presence of antimicrobial peptides and proteins, IL-8 (as a measurement for inflammation) and albumin (as a measurement for vascular leakage). In addition, hCAP18/LL-37 levels were analyzed in plasma. Commercially available ELISA kits were used to detect LCN2 (Bioporto), hCAP18/LL-37 and HNP1-3 (Hycult Biotech), and IL-8 (Sanquin). The SLPI ELISA was developed in our laboratory at the Leiden University Medical Center[21 ]. The absorbance was measured at 450 nm using a Microplate reader (model 680; Bio-Rad, Hercules, CA) and Microplate Manager software (version 5.2.1, Bio-Rad). The lower limits of detection were: LCN2 10 pg/ml; SLPI 10 ng/ml; HNP1-3 150 ng/ml; LL-37 20 ng/ml and IL-8 200 pg/ml. Albumin levels were determined using nephelometry (Siemens BN Prospec) with a lower limit of detection of 17 μg/ml. Inter and intra-assay variability for all assays was < 10%.
Serum 1,25(OH)₂D3 and 25(OH)D3 were assessed at the central clinical chemistry laboratory of the LUMC. Quantification of the 25(OH)D3 concentration in the serum was done using a DiaSorin ¹²⁵I RIA Kit (DIASORIN, INC.). Quantification of the 1,25-(OH)D3 concentration in the serum was done using a DiaSorin ¹²⁵I RIA Kit (DIASORIN, INC.) preceded by extraction and column separation.

Salivary immunity was evaluated using commercially available enzyme-linked immunosorbent assay (ELISA) kits. First, mucosal immune competency was characterized by measuring salivary sIgA and alpha-amylase (Salimetrics, State College, PA, USA). Next, salivary antimicrobial proteins (AMP) HNP1-3, LL-37 (Hycult Biotech, Uden, The Netherlands), and lactoferrin (Biomatik, Kitchener, Ontario, Canada) concentrations were determined. Finally, lung inflammation, a marker of overall lung health, was assessed by salivary surfactant protein A (SP-A) (Biomatik, Kitchener, Ontario, Canada), secretory leucocyte protease inhibitor (SLPI) (R&D Systems, Minneapolis, MN, USA) and salivary neutrophil Elastase-alpha-1-antitrypsin complex (Hycult Biotech, Uden, The Netherlands) concentrations^{47 (link),48 (link)}. According to manufacturers’ instructions, all samples were tested in duplicate, read on a plate reader (SprectraMax i3x, Molecular Devices; San Jose, CA) and concentrations were calculated on absorbance reading based on standard curves. Salivary biomarker concentrations were then converted to secretion rates by multiplying the concentrations by salivary flow rate. Unfortunately, technical limitations associated with saliva collection led to low volume recovery in some participants, and some analyses were conducted on a reduced sample size.

A range of antimicrobial peptides which are commonly associated with the oral cavity were selected for assessment in our patient’s saliva [32 (link)–34 (link)]. The following AMPs were assessed by ELISA, LL-37, Calprotectin, Lactoferrin, HNP1-3 (Hycult biotech, The Netherlands), Histatin 5 (Stratech scientific, Suffolk, UK) and Beta defensin 1 (BD1) (Peprotech, London, UK) as per manufacturer’s instructions. Clarified saliva samples were diluted 1:5 in assay buffer (PBS, 0.5% BSA, 0.1% Tween20). Results were calculated using a 4-parameter curve fit, quantifying colorimetric changes at 630 nm (BMG-Labtech, Ortenberg, Germany).