Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence

BACKGROUND: Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera. METHODS: We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde. RESULTS: We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells. CONCLUSION: Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells

Uncoupling of IL-6 signaling and LC3-associated phagocytosis drives immunoparalysis during sepsis

Contains fulltext : 238107.pdf (Publisher’s version ) (Closed access)Immune deactivation of phagocytes is a central event in the pathogenesis of sepsis. Herein, we identify a master regulatory role of IL-6 signaling on LC3-associated phagocytosis (LAP) and reveal that uncoupling of these two processes during sepsis induces immunoparalysis in monocytes/macrophages. In particular, we demonstrate that activation of LAP by the human fungal pathogen Aspergillus fumigatus depends on ERK1/2-mediated phosphorylation of p47phox subunit of NADPH oxidase. Physiologically, autocrine IL-6/JAK2/Ninein axis orchestrates microtubule organization and dynamics regulating ERK recruitment to the phagosome and LC3(+) phagosome (LAPosome) formation. In sepsis, loss of IL-6 signaling specifically abrogates microtubule-mediated trafficking of ERK, leading to defective activation of LAP and impaired killing of bacterial and fungal pathogens by monocytes/macrophages, which can be selectively restored by IL-6 supplementation. Our work uncovers a molecular pathway linking IL-6 signaling with LAP and provides insight into the mechanisms underlying immunoparalysis in sepsis

Calcium sequestration by fungal melanin inhibits calcium-calmodulin signalling to prevent LC3-associated phagocytosis

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca2+ signalling pathway that depends on intracellular Ca2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca2+-CaM signalling in aspergillosis. Finally, we demonstrate that Ca2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca2+-CaM signalling to inhibit LAP. These findings reveal the important role of Ca2+-CaM signalling in antifungal immunity and identify an immunological function of Ca2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.Onassis Foundation under the ‘Special Grant and Support Program for Scholars’ Association Members’ (Grant no. R ZM 003-1/2016-2017); G.C. was supported by grants from the Greek State Scholarship Foundation (I.K.Y.), the Hellenic General Secretariat for Research and Technology-Excellence program (ARISTEIA) and a Research Grant from Institut Mérieux; J.P.L. was supported by European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement 260338 ALLFUN and ANR-10-BLAN-1309 HYDROPHOBIN, and the Association Vaincre La Mucoviscidose (RF20140501052/1/1/141); H.F. and N.M.N. were supported by the project FROnTHERA (NORTE-01-0145-FEDER-000023), supported by Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e Tecnologia (FCT) project SPARTAN (PTDC/CTM-BIO/4388/2014), funded through the PIDDAC Program. A.C. and C.C. were supported by NORTE 2020, under the Portugal 2020 Partnership Agreement, through the ERDF (NORTE-01-0145-FEDER-000013), and by FCT (IF/00735/2014 and SFRH/BPD/96176/2013). G.S.D. and J.L.F. were supported by NIH grant AI-106269. K.J.K-C is supported by the Division of Intramural Research (DIR), NIAID, NIHinfo:eu-repo/semantics/publishedVersio

Phosphorylation at serine 331 is required for Aurora B activation

Chk1 phosphorylates Aurora B to promote its full activation during mitotic prometaphase

Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence

Abstract Background Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera. Methods We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde. Results We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells. Conclusion Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells.</p