L. Garratt

Interpretation of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine is adversely affected by methodological inaccuracies when using a commercial ELISA

The DNA lesion 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a urinary marker of oxidative stress, is produced from reactions of reactive oxygen species with host DNA 2-deoxyribonucleotides. The current gold-standard assessment is by complex chromatographic methods using HPLC or LC-MS/MS. Several studies have reported that commercial 8-oxodG ELISA kits correlate sufficiently with chromatographic techniques to be an easier alternative for laboratories without access to gold-standard techniques. However, the assumption that significant correlation translates into a similar ability to differentiate disease categories or treatment groups is yet to be tested. Using LC-MS/MS and two variants of a commercial ELISA, we measured urinary 8-oxodG and creatinine concentrations in young children with cystic fibrosis, a disease associated with oxidative stress, and age-matched controls. We show that, despite significant correlation, both ELISAs overestimate the levels of 8-oxodG, and neither ELISA accurately depicted the difference in group means that was observed by gold-standard LC-MS/MS. The implications of these findings for study outcomes add further support for chromatographic techniques, despite their cost and complexity, to remain the gold standard in urinary 8-oxodG assessment.

Sexual dimorphism in lung function responses to acute influenza A infection

Please cite this paper as: Larcombe et al. (2011) Sexual dimorphism in lung function responses to acute influenza A infection. Influenza and Other Respiratory Viruses 5(5), 334–342.

Stability of interleukin 8 and neutrophil elastase in bronchoalveolar lavage fluid following long-term storage

BACKGROUNDnInterleukin-8 (IL-8) and neutrophil elastase (NE) are commonly measured markers of inflammation in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis. Longitudinal analysis assumes uniform stability during storage, however the effect of extended low-temperature storage on these markers remains unclear.nnnMETHODSnBAL fluid from 104 children with cystic fibrosis was assayed for IL-8 and NE after storage at 4 ° C for 7 days and -80 ° C for up to 6 years and compared with the initial assays performed soon after collection.nnnRESULTSnIL-8 levels were stable after any measured length of time at -80 ° C or 4 ° C. NE levels were stable for 6 months at -80 ° C but decreased beyond that or after 7 days at 4 ° C.nnnCONCLUSIONSnOur data support the stability of IL-8 in BAL stored at -80 ° C for prolonged periods. NE in BAL decreases with storage and should be assayed as soon as practical after collection.

Small macrophages are present in early childhood respiratory disease

BACKGROUNDnRecently, an established small macrophage phenotype has been observed in the sputum of patients with CF and COPD. However, little is known about the prevalence of this phenotype in the airways of young children. Since respiratory inflammation begins early in CF, we hypothesised that these small macrophages would be increased in paediatric CF bronchoalveolar lavage (BAL).nnnMETHODSnMacrophage populations in CF and disease control BAL were assessed by multicolour flow cytometry. BAL inflammatory indices were collected as part of the AREST-CF programme.nnnRESULTSnSmall macrophages were present in CF (n=35, mean 36±12% of BAL macrophages) but not significantly different to the respiratory disease controls (n=7, mean 40±21%). Number of small macrophages correlated significantly with number of BAL neutrophils (r=0.44, p<0.01) but not infection or IL-8.nnnCONCLUSIONSnIn paediatric patients small macrophages are not unique to CF, but their establishment as the dominant phenotype in adults may be due to chronicity of inflammation and infection.

33 CFTR modulators alter innate immune responses by primary airway epithelial cells challenged with rhinovirus

organoid model. Quality and repeatability of the assay are important to ensure the validity of results across laboratories. Therefore, procedures have to be standardized to aim for minimal variability in outcome between labs. The aim of this study is to standardize procedures and to detect, describe and tackle the causes of variability in the assay. Methods: To standardize working procedures, we first exchanged protocols from the currently existing organoid laboratories (UMC Utrecht, KUZ Leuven andUniversity of Lisboa). Next, we detected differences in protocols andmutually decidedwhichworking procedurewas preferred.Wemerged the protocols into one European standard protocol. We exchanged organoids with genotypes ranging from severe to wild-type across the three laboratories. We will perform the Forskolin-induced-swelling (FIS) assay with several different measuring conditions (locally produced growth medium, identical growth medium, and after identical and different amounts of passages. Results of the FIS assay will be compared to detect differences in handling, growth medium and other factors, and their effect on assay variability. Subsequently, important factors that influence variability will be adapted in the protocol. Results: A European standard protocol has been generated. This protocol has been implemented across three laboratories. Measurements are currently taking place. The results of these analyses will be presented at the conference. Conclusion: The organoid model will be validated in three different laboratories, and one final protocol will be established to ensure quality and standardization of measurement outcomes.