Detection of the fungicide transformation product 4-hydroxychlorothalonil in serum of pregnant women from Sweden and Costa Rica.

BACKGROUND: 4-hydroxychlorothalonil (HCT, R182281), a transformation product of the fungicide chlorothalonil, was recently identified in human serum and breast milk. There are indications that HCT may be more toxic and environmentally persistent than chlorothalonil. OBJECTIVE: Our aim was to investigate serum concentrations of HCT in pregnant women in Sweden and Costa Rica. METHODS: We developed a quantitative analytical method for HCT using liquid chromatography tandem mass spectrometry. We measured HCT in 1808 serum samples from pregnant women from the general population in Sweden (1997-2015) and in 632 samples from 393 pregnant women from an agricultural population in Costa Rica (2010-2011). In Swedish samples, we assessed time trends and investigated seasonality. In the Costa Rican samples, we evaluated variability between and within women and explanatory variables of HCT concentrations. RESULTS: HCT was detected in all serum samples, and the limit of detection was 0.1 µg/L. The median HCT concentration in the Swedish samples was 4.1 µg/L (interquartile range [IQR] of 2.9 - 5.8 µg/L), and 3.9 times higher in the Costa Rican samples (median: 16.1 µg/L; IQR: 10.6 - 25.0 µg/L). We found clear seasonal variation with higher concentrations in the first half of each year among Swedish women. In the Costa Rican study, women working in agriculture and living near banana plantations had higher HCT concentrations, whilst higher parity and having a partner working in agriculture were associated with decreased HCT, and no clear seasonal pattern was observed. IMPACT STATEMENT: For the first time, this study quantifies human exposure to the fungicide chlorothalonil and/or its transformation product 4-hydroxychlorothalonil (HCT, R182281) and finds higher serum concentrations in women from a tropical agricultural setting as compared with women from the general population in Sweden.

A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis.

Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.

Mycobacteria Manipulate G-Protein-Coupled Receptors to Increase Mucosal Rac1 Expression in the Lungs.

Mycobacterium bovis bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB). BCG mimics M. tuberculosis (Mtb) in its persistence in the body and is used as a benchmark to compare new vaccine candidates. BCG was originally designed for mucosal vaccination, but comprehensive knowledge about its interaction with epithelium is currently lacking. We used primary airway epithelial cells (AECs) and a murine model to investigate the initial events of mucosal BCG interactions. Furthermore, we analysed the impact of the G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, in this process, as these receptors were previously shown to be important during TB infection. BCG infection of AECs induced GPCR-dependent Rac1 up-regulation, resulting in actin redistribution. The altered distribution of the actin cytoskeleton involved the MAPK signalling pathway. Blocking of the CXCR1 or CXCR2 prior to infection decreased Rac1 expression, and increased epithelial transcriptional activity and epithelial cytokine production. BCG infection did not result in epithelial cell death as measured by p53 phosphorylation and annexin. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs.

Innate Immune Responses after Airway Epithelial Stimulation with Mycobacterium bovis Bacille-Calmette Guérin.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is used as a benchmark to compare the immunogenicity of new vaccines against tuberculosis. This live vaccine is administered intradermal, but several new studies show that changing the route to mucosal immunisation represents an improved strategy. We analysed the immunomodulatory functions of BCG on human neutrophils and primary airway epithelial cells (AECs), as the early events of mucosal immune activation are unclear. Neutrophils and the primary epithelial cells were found to express the IL-17A receptor subunit IL-17RA, while the expression of IL-17RE was only observed on epithelial cells. BCG stimulation specifically reduced neutrophil IL-17RA and epithelial IL-17RE expression. BCG induced neutrophil extracellular traps (NETs), but did not have an effect on apoptosis as measured by transcription factor forkhead box O3 (FOXO3). BCG stimulation of AECs induced CXCL8 secretion and neutrophil endothelial passage towards infected epithelia. Infected epithelial cells and neutrophils were not found to be a source of IL-17 cytokines or the interstitial collagenase MMP-1. However, the addition of IFNγ or IL-17A to BCG stimulated primary epithelial cells increased epithelial IL-6 secretion, while the presence of IFNγ reduced neutrophil recruitment. Using our model of mucosal infection we revealed that BCG induces selective mucosal innate immune responses that could lead to induction of vaccine-mediated protection of the lung.

Impaired CXCR1-dependent oxidative defence in active tuberculosis patients.

Much of the pronounced host inflammatory response that occurs in tuberculosis (TB) is related to failed immunity against the invading pathogen. The G-protein coupled receptors CXCR1 and CXCR2 are implicated in important signal transduction pathways in lung inflammatory responses. We investigated the expression and function of these receptors in a simple whole blood model from 24 patients with pulmonary TB and in subjects with latent TB infection (LTBI). Healthy controls were recruited from close contacts to the pulmonary index patients. We found that pulmonary TB patients had significantly increased CXCR1 expression on blood cells compared to LTBI subjects and controls (p < 0.001). In contrast, LTBI subjects had a significant increase in CXCR2 expression compared to pulmonary TB patients (p < 0.001) and controls (p < 0.01). Leukocyte function, measured as oxidative capacity, was decreased in pulmonary TB patients compared to LTBI and controls (p < 0.001) and correlated with the increased CXCR1 expression. Leukocyte recruitment, measured as the expression of microRNA-223 was increased in pulmonary TB patients compared to LTBI (p < 0.05). We found that variations in receptor expression are linked to disease progression and affect the immune response against Mycobacterium tuberculosis (Mtb).

HTS followed by NMR based counterscreening. Discovery and optimization of pyrimidones as reversible and competitive inhibitors of xanthine oxidase.

The identification of novel, non-purine based inhibitors of xanthine oxidase is described. After a high-throughput screening campaign, an NMR based counterscreen was used to distinguish actives, which interact with XO in a reversible manner, from assay artefacts. This approach identified pyrimidone 1 as a reversible and competitive inhibitor with good lead-like properties. A hit to lead campaign gave compound 41, a nanomolar inhibitor of hXO with efficacy in the hyperuricemic rat model after oral dosing.