Search

Epidemiological studies have revealed some alterations in systemic immunity that associate with farm exposure and the risk of respiratory diseases, but in vitro studies focusing on immunological responses in the airways are scarce. Our aim was to assess how cowshed dust affects the integrity and inflammation of human airway tissue in vitro. Cowshed dust samples were collected from four different dairy farms. Lung tissue constructs were exposed to dust samples in air-liquid interface. Transepithelial resistance of the tissue, secreted proteins, and a panel of pro-inflammatory cytokines, growth factors, and chemokines were analysed. Cowshed dust stimulation was associated mainly with increased production of IL-13, IL-15, IP-10 and IFN-γ. Some differences between farms were seen. Only one farm dust sample induced a significant change in transepithelial resistance, whereas dust from two of the farms induced the secretion of proteins. The exposure to cowshed dust affected protein and cytokine secretion, but the response profiles were not uniform between farms. The effect on tight junction dynamics was less pronounced, suggesting the relevance of soluble factors in induced responses in the airways. Our results indicate that in addition to farm type, the contribution of cowshed characteristics to dust composition and its immunomodulatory properties should be taken into account., Competing Interests: Declaration of Competing Interest Authors do not have any actual or potential conflicts of interest including any financial, personal, or other relationships that could inappropriately influence, or be perceived to influence, their work., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Multiple health effects are associated with moisture damage in buildings. Studies explaining these associations and cell-level mechanisms behind the observed health effects are urgently called for., Objectives: We focused on characterizing gene expression in human airway epithelium after exposure to indoor air particulate matter (PM) sampled from houses with and without moisture damage, alongside determination of general toxicological markers., Methods: We performed detailed technical building inspections in 25 residential houses and categorized them based on the detection of moisture damages and the probability of occupant exposure. PM sampling was complemented by microbiological and volatile organic compound assessment. We exposed human airway constructs to three dilutions (1:16, 1:8, 1:4) of collected PM from moisture-damaged (index) and non-moisture-damaged (reference) houses and imaged selected constructs with electron microscopy. We analyzed general toxicological markers and the RNA of exposed constructs was sequenced targeting genes associated with toxicological pathways. We did groupwise comparisons between index and reference houses and pairwise comparisons in matched index/reference houses., Results: In groupwise comparison, gene Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1) was statistically significantly over-expressed in index houses at all dilutions of collected PM and Nuclear Factor Kappa B Subunit 1 (NFKB1) at dilution 1:4 of collected PM. In pairwise index/reference house comparison, several genes related to multiple toxicological pathways were activated, largest expression differences seen for CYP1A1. However, none of the genes was consistently expressed in all the matched pairs, and general toxicological markers did not differentiate index and reference houses., Discussion: The exposure to PM from index houses activated toxicology -related genes in airway constructs. Differential expression was not consistent among all the index/reference pairs, possibly due to compositional differences of bioactive particles. Our study highlights CYP1A1 and NFKB1 as potential targets in moisture damage -associated cellular responses., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In vitro models mimicking the human respiratory system are essential when investigating the toxicological effects of inhaled indoor air particulate matter (PM). We present a pulmonary cell culture model for studying indoor air PM toxicity. We exposed normal human bronchial epithelial cells, grown on semi-permeable cell culture membranes, to four doses of indoor air PM in the air-liquid interface. We analyzed the chemokine interleukin-8 concentration from the cell culture medium, protein concentration from the apical wash, measured tissue electrical resistance, and imaged airway constructs using light and transmission electron microscopy. We sequenced RNA using a targeted RNA toxicology panel for 386 genes associated with toxicological responses. PM was collected from a non-complaint residential environment over 1 week. Sample collection was concomitant with monitoring size-segregated PM counts and determination of microbial levels and diversity. PM exposure was not acutely toxic for the cells, and we observed up-regulation of 34 genes and down-regulation of 17 genes when compared to blank sampler control exposure. The five most up-regulated genes were related to immunotoxicity. Despite indications of incomplete cell differentiation, this model enabled the comparison of a toxicological transcriptome associated with indoor air PM exposure., (© 2019 The Authors. Indoor Air published by John Wiley & Sons Ltd.)

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls., Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV- and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data., Results: Approximately 1 x 10 10 EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p < 0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples., Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Thiol-ene polymer formulations are raising growing interest as new low-cost fabrication materials for microfluidic devices. This study addresses their feasibility for microchip electrophoresis (MCE) via characterization of the effects of UV curing conditions and aging on the surface charge and wetting properties. A detailed comparison is made between stoichiometric thiol-ene (1:1) and thiol-ene formulations bearing 50% molar excess of allyls ("enes"), both prepared without photoinitiator or other polymer modifiers. Our results show that the surface charge of thiol-ene 1:1 increases along with increasing UV exposure dose until a threshold (here, about 200J/cm(2)), whereas the surface charge of thiol-ene 2:3 decreases as a function of increasing UV dose. However, no significant change in the surface charge upon storage in ambient air was observed over a period of 14 days (independent of the curing conditions). The water contact angles of thiol-ene 2:3 (typically 70-75°) were found to be less dependent on the UV dose and storing time. Instead, water contact angles of thiol-ene 1:1 slightly decrease (from initial 90 to 95° to about 70°) as a function of UV increasing exposure dose and storing time. Most importantly, both thiol-ene formulations remain relatively hydrophilic over extended periods of time, which favors their use in MCE applications. Here, MCE separation of biologically active peptides and selected fluorescent dyes is demonstrated in combination with laser-induced fluorescence detection showing high separation efficiency (theoretical plates 8200 per 4cm for peptides and 1500-2700 per 4cm for fluorescent dyes) and lower limits of detection in the sub-μM (visible range) or low-μM (near-UV range) level., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A new modified fused-silica glass whose coefficient of thermal expansion at 20 degrees C is less than 1/25 that of fused silica is described. It should be particularly useful for large, first-surface mirrors and in other applications where extremely low expansion is desirable. Both solid and lightweight telescope mirror blanks can be constructed using fusion techniques. A discussion of a number of physical properties is included.