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1.  Avian erythrocytes have functional mitochondria, opening novel perspectives for birds as animal models in the study of ageing 
Frontiers in Zoology  2013;10:33.
Background
In contrast to mammalian erythrocytes, which have lost their nucleus and mitochondria during maturation, the erythrocytes of almost all other vertebrate species are nucleated throughout their lifespan. Little research has been done however to test for the presence and functionality of mitochondria in these cells, especially for birds. Here, we investigated those two points in erythrocytes of one common avian model: the zebra finch (Taeniopygia guttata).
Results
Transmission electron microscopy showed the presence of mitochondria in erythrocytes of this small passerine bird, especially after removal of haemoglobin interferences. High-resolution respirometry revealed increased or decreased rates of oxygen consumption by erythrocytes in response to the addition of respiratory chain substrates or inhibitors, respectively. Fluorometric assays confirmed the production of mitochondrial superoxide by avian erythrocytes. Interestingly, measurements of plasmatic oxidative markers indicated lower oxidative stress in blood of the zebra finch compared to a size-matched mammalian model, the mouse.
Conclusions
Altogether, those findings demonstrate that avian erythrocytes possess functional mitochondria in terms of respiratory activities and reactive oxygen species (ROS) production. Interestingly, since blood oxidative stress was lower for our avian model compared to a size-matched mammalian, our results also challenge the idea that mitochondrial ROS production could have been one actor leading to this loss during the course of evolution. Opportunities to assess mitochondrial functioning in avian erythrocytes open new perspectives in the use of birds as models for longitudinal studies of ageing via lifelong blood sampling of the same subjects.
doi:10.1186/1742-9994-10-33
PMCID: PMC3686644  PMID: 23758841
Red blood cell; Ageing; Mitochondria; ROS; Oxidative stress; Electron transport chain
2.  Macroautophagy is deregulated in murine and human lupus T lymphocytes 
Autophagy  2012;8(7):1113-1123.
Macroautophagy was recently shown to regulate both lymphocyte biology and innate immunity. In this study we sought to determine whether a deregulation of autophagy was linked to the development of autoimmunity. Genome-wide association studies have pointed out nucleotide polymorphisms that can be associated with systemic lupus erythematosus, but the potential role of autophagy in the initiation and/or development of this syndrome is still unknown. Here, we provide first clues of macroautophagy deregulation in lupus. By the use of LC3 conversion assays and electron microscopy experiments, we observed that T cells from two distinct lupus-prone mouse models, i.e., MRLlpr/lpr and (NZB/NZW)F1, exhibit high loads of autophagic compartments compared with nonpathologic control CBA/J and BALB/c mice. Unlike normal mice, autophagy increases with age in murine lupus. In vivo lipopolysaccharide stimulation in CBA/J control mice efficiently activates T lymphocytes but fails to upregulate formation of autophagic compartments in these cells. This argues against a deregulation of autophagy in lupus T cells solely resulting from an acute inflammation injury. Autophagic vacuoles quantified by electron microscopy are also found to be significantly more frequent in T cells from lupus patients compared with healthy controls and patients with non-lupus autoimmune diseases. This elevated number of autophagic structures is not distributed homogeneously and appears to be more pronounced in certain T cells. These results suggest that autophagy could regulate the survival of autoreactive T cell during lupus, and could thus lead to design new therapeutic options for lupus.
doi:10.4161/auto.20275
PMCID: PMC3429547  PMID: 22522825
systemic lupus erythematosus; lupus-prone mice; macroautophagy; T lymphocytes
3.  Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis 
PLoS Pathogens  2011;7(12):e1002405.
Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.
Author Summary
MiRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally via binding to complementary sites in target mRNAs. This evolutionary conserved regulatory system is present in most eukaryotes, and it has recently been shown that certain viruses have evolved to express their own miRNAs. Due to their non-immunogenic nature, viral miRNAs represent an efficient tool for the virus to control its environment. Here we show that KSHV miRNAs are involved in the control of apoptosis both when expressed in stable cell lines and in the context of viral infection. Using a microarray based approach we identified putative cellular targets, among which the effector caspase 3 is targeted by three of the viral miRNAs. Finally, we showed that blocking these miRNAs in infected cells resulted both in increased Casp3 levels and a higher apoptosis rate. These findings indicate that miRNAs of viral origin are key players in cell death inhibition by KSHV.
doi:10.1371/journal.ppat.1002405
PMCID: PMC3234232  PMID: 22174674
4.  HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus 
Annals of the Rheumatic Diseases  2010;70(5):837-843.
Background
The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells.
Objective
To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells.
Methods
P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers.
Results
P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux.
Conclusion
The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.
doi:10.1136/ard.2010.139832
PMCID: PMC3070272  PMID: 21173017
5.  Soluble HLA-G Molecules Are Increased during Acute Leukemia, Especially in Subtypes Affecting Monocytic and Lymphoid Lineages1 
Neoplasia (New York, N.Y.)  2006;8(3):223-230.
Abstract
Human leukocyte antigen G (HLA-G) molecules corresponding to nonclassic class I genes of the major histocompatibility complex exhibit immunomodulatory properties. They are either membrane-bound or solubly expressed during certain tumoral malignancies. Soluble human leukocyte antigen G (sHLA-G) molecules seem more frequently expressed than membrane-bound isoforms during hematologic malignancies, such as lymphoproliferative disorders. Assay of these molecules by enzyme-linked immunosorbent assay in patients suffering from another hematologic disorder (acute leukemia) highlights increased sHLA-G secretion. This increased secretion seems more marked in acute leukemia subtypes affecting monocytic and lymphoid lineages such as FABM4 and FABM5, as well as both B and T acute lymphoblastic leukemia (ALL). Moreover, this study uses in vitro cytokine stimulations and reveals the respective potential roles of granulocyte-macrophage colony-stimulating factor and interferon-γ in increasing this secretion in FABM4 and ALL. Correlations between sHLA-G plasma level and clinical biologic features suggest a link between elevated sHLA-G level and 1) the absence of anterior myelodysplasia and 2) high-level leukocytosis. All these findings suggest that sHLA-G molecules could be a factor in tumoral escape from immune survey during acute leukemia.
PMCID: PMC1578523  PMID: 16611416
Soluble human leukocyte antigen G (sHLA-G); acute myeloblastic leukemia (AML); acute lymphoblastic leukemia (ALL); myelodysplasia; immunomodulation

Results 1-5 (5)