Fluorescent Labeling of Helminth Extracellular Vesicles Using an In Vivo Whole Organism Approach

In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species

Levels of N-methyl-2-pyrrolidone (NMP) and its metabolites in plasma and urine from volunteers after experimental exposure to NMP in dry and humid air.

Department of Occupational and Environmental Medicine, Institute of Laboratory Medicine, Lund University Hospital, S-221 85 Lund, Sweden. The aim of this study was to investigate if the uptake of N-methyl-2-pyrrolidone (NMP), a widely used industrial chemical, increases after exposure to NMP in humid air compared to dry air. NMP has been described to be an airway irritant and a developmentally toxic compound. Six male volunteers were exposed to NMP, three at the time, for 8h in an exposure chamber. They were each exposed on four different occasions to air levels of 0 and 20mg NMP/m(3) in dry and humid air. Blood and urine were sampled before, during and up to 5 days after the end of the 8-h exposure. Plasma and urine were analysed for NMP and its metabolites, using liquid chromatography-tandem mass spectrometry. There was no statistically significant increase in the total cumulated excretion of NMP and its metabolites in urine after exposure in humid air as compared to dry air. Furthermore, there were no differences in the levels of peak concentrations in either plasma or urine. Also, no differences were found in AUC between the exposures. However, there were large individual differences, especially for the exposure in humid air. A not previously identified metabolite in human, 2-pyrrolidone (2-P), was identified. The results do not support a significantly higher absorption of NMP at exposure in humid air as compared to dry air. However, the large individual differences support the use of biological monitoring for assessment of NMP exposure. In addition, 2-P was confirmed to be an NMP metabolite in humans. This may be of importance for the developmental toxicity of NMP since 2-P have been described to be a reproductively toxic substance

Analysis, metabolism, effects and biological monitoring of N-methyl-2-pyrrolidone

N-methyl-2-pyrrolidone (NMP) is an organic solvent widely used in the industry. Irritation of the eyes and in the respiratory system has been reported after occupational exposure to low air levels of NMP. The EU has also classified it as a developmental toxicant. NMP is easily absorbed in the gastrointestinal tract and airways and through skin. The general aim of this thesis was to develop and evaluate methods for biological monitoring of NMP exposure. For the simultaneous analysis of NMP and its metabolites in plasma and urine, a fast analytical method using liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS) was developed. The method has high accuracy and precision, good recovery and sufficient detection limits for the analysis of NMP and its metabolites in plasma and urine from occupationally exposed workers. In a study where male and female volunteers were dermally exposed to NMP, no differences in toxicokinetics were found between the sexes. Dermal exposure to an aqueous NMP-solution caused a delay of the peaks for NMP and its metabolites. However, the impact of the delay on the biological levels was small for the metabolite 2-hydroxy-N-methylsuccinimide (2-HMSI). It was concluded that density was preferred over creatinine for adjustment of urinary dilution for some of the compounds. No differences in the total accumulated excretion were found in a study of volunteers after air exposure to dry and humid air, but the individual differences were larger after exposure in humid air, indicating a higher uptake for some of the subjects. However, it was suggested that a large dermal uptake was also present at air exposure. A new NMP metabolite, 2-Pyrrolidone (2-P), was identified. This may be of importance since 2-P has been reported to be developmentally toxic. Significant effects related to the NMP exposure were found for ECP in nasal lavages and for tear film BUT of the eyes. In one study, rats were shown to have a similar metabolism as humans. However, the relative biological levels differed. Thus, the plasma concentrations and area under the curve (AUC) following a developmentally toxic dose were much higher for NMP and 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP), while the levels for 2-HMSI were only about four times the levels found in humans experimentally exposed to 20 mg/m3 for 8 h. Based on the results in this thesis, 2-HMSI is suggested as a biomarker of NMP exposure. Post-shift sampling in the end of the working week, will monitor the last three days of exposure

Evaluation of exposure biomarkers from percutaneous absorption of N-methyl-2-pyrrolidone.

OBJECTIVES: The aim of this study was to evaluate different biomarkers of exposure to N-methyl-2-pyrrolidone (NMP), a widely used industrial chemical. For this purpose, differences in toxicokinetics between men and women and between pure and water-mixed NMP were evaluated after dermal absorption. METHODS: Six female and six male volunteers (groups 1 and 2) were topically exposed for 6 hours to 300 mg of NMP. An additional group of six male volunteers (group 3) was exposed to 300 mg of NMP in a 50% water solution. Blood and urine were sampled before, during, and up to 9 days after the exposure. Plasma and urine were analyzed using mass spectrometry. RESULTS: For groups 1 and 2, 16% and 18% of the applied dose were recovered in the urine as the sum of NMP and its metabolites. For group 3, 4% was recovered. The maximal concentration of 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) was 10, 8.1, and 2.1 micromol/l for groups 1, 2 and 3, respectively, in plasma and 420, 360 and 62 micromol/l in urine adjusted for density. For 2-hydroxy-N-methylsuccinimide (2-HMSI), the maximal concentration was 5.4, 4.5, and 1.3 micromol/l for groups 1, 2 and 3, in plasma, respectively, and 110, 82 and 19 micromol/l in urine adjusted for density. For 5-HNMP there was a difference in time to reach the maximal concentration depending on whether pure NMP or 50% NMP in water was used. No such difference was seen for 2-HMSI. The differences in kinetics between male and female volunteers were small. CONCLUSIONS: Preferably 2-HMSI should be used as the biomarker of exposure to NMP