A real-time assay for neutrophil chemotaxis.

Neutrophils are the predominant cells during acute phases of inflammation, and it is now recognized that these leukocytes play an important role in modulation of the immune response. Directed migration of these cells to the sites of injury, known as chemotaxis, is driven by chemoattractants present at the endothelial cell surface or in the extracellular matrix (ECM). Since uncontrolled or excessive neutrophil chemotaxis is involved in pathological conditions such as atherosclerosis and severe asthma, studying the chemical cues triggering neutrophil migration is essential for understanding the biology of these cells and developing new anti-inflammatory therapies. Although several methods have been developed to evaluate neutrophil chemotaxis, these techniques are generally labor-intensive or alter the native form of these cells and their physiological response. Here we report a rapid, non-invasive, impedance-based, and label-free assay for real-time assessment of neutrophil chemotaxis.

New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: application on Aspergillus fumigatus grown on wheat.

Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins.

Deoxynivalenol as a new factor in the persistence of intestinal inflammatory diseases: an emerging hypothesis through possible modulation of Th17-mediated response.

BACKGROUND/AIMS: Deoxynivalenol (DON) is a mycotoxin produced by Fusarium species which is commonly found in temperate regions worldwide as a natural contaminant of cereals. It is of great concern not only in terms of economic losses but also in terms of animal and public health. The digestive tract is the first and main target of this food contaminant and it represents a major site of immune tolerance. A finely tuned cross-talk between the innate and the adaptive immune systems ensures the homeostatic equilibrium between the mucosal immune system and commensal microorganisms. The aim of this study was to analyze the impact of DON on the intestinal immune response. METHODOLOGY: Non-transformed intestinal porcine epithelial cells IPEC-1 and porcine jejunal explants were used to investigate the effect of DON on the intestinal immune response and the modulation of naive T cells differentiation. Transcriptomic proteomic and flow cytometry analysis were performed. RESULTS: DON induced a pro-inflammatory response with a significant increase of expression of mRNA encoding for IL-8, IL-1α and IL-1ß, TNF-α in all used models. Additionally, DON significantly induced the expression of genes involved in the differentiation of Th17 cells (STAT3, IL-17A, IL-6, IL-1ß) at the expenses of the pathway of regulatory T cells (Treg) (FoxP3, RALDH1). DON also induced genes related to the pathogenic Th17 cells subset such as IL-23A, IL-22 and IL-21 and not genes related to the regulatory Th17 cells (rTh17) such as TGF-ß and IL-10. CONCLUSION: DON triggered multiple immune modulatory effects which could be associated with an increased susceptibility to intestinal inflammatory diseases.