Early macrophage response to obesity encompasses Interferon Regulatory Factor 5 regulated mitochondrial architecture remodelling.

Adipose tissue macrophages (ATM) adapt to changes in their energetic microenvironment. Caloric excess, in a range from transient to diet-induced obesity, could result in the transition of ATMs from highly oxidative and protective to highly inflammatory and metabolically deleterious. Here, we demonstrate that Interferon Regulatory Factor 5 (IRF5) is a key regulator of macrophage oxidative capacity in response to caloric excess. ATMs from mice with genetic-deficiency of Irf5 are characterised by increased oxidative respiration and mitochondrial membrane potential. Transient inhibition of IRF5 activity leads to a similar respiratory phenotype as genomic deletion, and is reversible by reconstitution of IRF5 expression. We find that the highly oxidative nature of Irf5-deficient macrophages results from transcriptional de-repression of the mitochondrial matrix component Growth Hormone Inducible Transmembrane Protein (GHITM) gene. The Irf5-deficiency-associated high oxygen consumption could be alleviated by experimental suppression of Ghitm expression. ATMs and monocytes from patients with obesity or with type-2 diabetes retain the reciprocal regulatory relationship between Irf5 and Ghitm. Thus, our study provides insights into the mechanism of how the inflammatory transcription factor IRF5 controls physiological adaptation to diet-induced obesity via regulating mitochondrial architecture in macrophages.

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy.

BACKGROUND: Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. METHODS: Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II-restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)-binding assays. CD4+ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. RESULTS: MS analysis of group 5 pollen allergens reveals considerable intra- and inter-species variability in amino acid sequence, with 30-50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N- and O-glycosylation contribute to the variability of group 1 allergens, yielding 5-10 main isoforms, depending on the species. Out of 14 MHC class II-restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA-DR molecules result in variable CD4+ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species-restricted (or semi-restricted) epitopes associated with group 1 or 5 allergens, respectively. CONCLUSION: Major pollen allergens from distinct grass species bear both shared and species-restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized.

Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine.

6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, (13)C or (15)N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-(13)C(2)-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37 degrees C. CML was extensively studied by (1)H- and (13)C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on (1)H-NMR and tandem MS using lysine as an internal reference.

Comparison of analytical techniques to quantify malondialdehyde in milk powders.

Several analytical methods were compared to quantify malondialdehyde (MDA) in milk powders. Modified thiobarbituric acid (TBA) methods, using either visible spectrophotometry (direct absorbance reading or after third derivative transformation of the spectrum) or HPLC, required derivatisation at elevated temperature, which appeared to catalyse artefactual MDA formation and thus overestimate the MDA content. In contrast to the TBA derivatisation method, the measurement of MDA as the dinitrophenylhydrazone derivative by HPLC or as the phenylhydrazone product by GC-MS with a deuterated internal standard resulted in lower estimates in the ranges of 2-17- and 3-30-fold, respectively; apparently due to the milder derivatisation conditions. The estimates of MDA determined by both HPLC-UV and GC-MS techniques result in lower values which are similar in magnitude even though the GC-MS technique is more sensitive.