consensus-brain-derived-protein-extraction-protocol-for-study-human

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

Faculté de Médecine - Pôle Recherche

The FIP1L1-PDGFRA (F/P) fusion gene, which was identified as a recurrent molecular finding in hypereosinophilic syndrome (HES), lead to a constitutively increased tyrosine kinase activity of the fusion protein. Despite data obtained in animals or cell lines models, the mechanisms underlying the predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. To define more precisely intrinsic molecular events associated with F/P gene, we performed a proteomic analysis comparing F/P+ eosinophils (F/P-Eos) and eosinophils from healthy donors (C-Eos). Using 2D-DIGE and mass spectrometry techniques, we identified 41 proteins significantly overexpressed between F/P-Eos and C-Eos. Among them, 17.8% belonged to the oxidoreductase family. We further observed a down-expression of peroxiredoxin-2 (PRX-2) and an overexpression of src-homology-2 domain containing tyrosine phosphatase (SHP-1), enzymes regulating PDGFR downstream pathways, and especially intracellular reactive oxygen species (ROS) production. This profile, confirmed in immunoblot analysis, appears specific to F/P-Eos compared to controls and patients with idiopathic HES. In this clonal disorder possibly involving a pluripotent hematopoietic stem cell, we postulate that the well documented relationships between PDGFRA downstream signals and intracellular ROS levels might influence the phenotype of this leukemia.

Comparative proteomic analysis of blood eosinophils reveals redox signaling modifications in patients with FIP1L1-PDGFRA-associated chronic eosinophilic leukemia.

Background. Absence of acquired protective immunity in endemic areas children leads to higher susceptibility to severe malaria. To investigate the involvement of regulatory process related to self-reactivity, we evaluated potent changes in auto-antibody reactivity profiles in children and older subjects living in malaria-endemic zones comparatively to none-exposed healthy controls. Methods. Analysis of IgG self-reactive footprints was performed using Western blotting against healthy brain antigens. Plasmas of 102 malaria exposed individuals (MEIs) from endemic zone, with or without cerebral malaria (CM) were compared to plasmas from non-endemic controls (NECs). Using linear discriminant and principal component analysis, immune footprints were compared by counting the number, the presence or absence of reactive bands. We identified the most discriminant bands with respect to age and clinical status. Results. A higher number of bands were recognized by IgG auto-antibodies in MEI than in NEC. Characteristic changes in systemic self-IgG-reactive repertoire were found with antigenic bands that discriminate Plasmodium falciparum infections with or without CM according to age. 8 antigenic bands distributed in MEI compared with NEC were identified while 6 other antigenic bands were distributed within MEI according to the age and clinical status. Such distortion might be due to evolutionary processes leading to pathogenic/protective events.

Changes Related to Age in Natural and Acquired Systemic Self-IgG Responses in Malaria.

IL-1 receptor antagonist anakinra is usually highly efficient in Schnitzler syndrome (SS), a rare inflammatory condition associating urticaria, fever, and IgM monoclonal gammopathy. In this study, we aimed to assess lipopolysaccharide (LPS)-induced production of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs) before and after 1 month of anakinra in patients with SS. LPS-induced production of IL-1β, IL-6 and TNFα was assessed by enzyme-linked immunosorbent assay with and without anakinra in vitro, and before and after 1 month (in vivo condition) of treatment in 2 patients with SS. Spontaneous production of IL-1β, IL-6 and TNF-α by PBMCs was similar in the patients and the healthy controls and was almost undetectable. Stimulation with LPS caused a higher release of cytokines from the patients than from the healthy controls. Before in vivo anakinra start, in vitro adjunction of anakinra reduced the high LPS-induced production of IL-1β and TNFα in both patients and of IL-6 in one patient. After 1 month of treatment with anakinra, while the patients had dramatically improved, there was also a marked reduction in LPS-induced cytokines production, which was almost normalized in one patient. This study shows an abnormal LPS-induced inflammatory cytokines production in SS, which can be decreased or even normalized by in vitro and in vivo anakinra.

Virginie Dutoit-Lefevre

EA2686-Laboratorie d'Immunologie

Faculté de Médecine - Pôle Recherche

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting