Search

BackgroundZoonotic simian foamy viruses (SFVs) establish persistent infections in humans, for whom the long-term consequences for health are poorly described. In this study, we aimed to characterize blood-cell phenotypes and plasma biomarkers associated with gorilla SFV infection in humans.MethodsWe used a case-control design to compare 15 Cameroonian hunters infected with gorilla SFV (cases) to 15 controls matched for age and ethnicity. A flow cytometry-based phenotypic study and quantification of plasma immune biomarkers were carried out on blood samples from all participants. Wilcoxon signed-rank tests were used to compare cases and controls.ResultsCases had a significantly higher percentage of CD8 T lymphocytes than controls (median, 17.6% vs 13.7%; P = .03) but similar levels of B, natural killer, and CD4 T lymphocytes. Cases also had a lower proportion of recent CD4 thymic emigrants (10.9% vs 18.6%, P = .05), a higher proportion of programmed death receptor 1 (PD-1) expressing memory CD4 T lymphocytes (31.7% vs 24.7%, P = .01), and higher plasma levels of the soluble CD163 scavenger receptor (0.84 vs .59 µg/mL, P = .003) than controls.ConclusionsWe show, for the first time, that chronic infection with SFV is associated with T lymphocyte differentiation and monocyte activation.

In perinatally HIV-1-infected youths living in France, we previously reported that Gag-specific CD4 and CD8 T cell proliferation is more frequently detected in patients of black ethnicity than in those of other ethnicities. We observed that black patients had higher levels of dendritic cells (DCs) than other patients. We aimed at studying the association of DC levels with Gag-specific T cell proliferation. The ANRS-EP38-IMMIP study is an observational study of youths aged between 15 and 24 years who were perinatally infected with HIV. A single blood sample was drawn for virological and immunological assays. Data from cART-treated 53 youths with undetectable plasma HIV RNA were analyzed. Gag-specific T cell proliferation was assessed by using a CFSE-based test. Peripheral blood myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) were phenotyped by flow cytometry. Plasma markers were quantified by ELISA or multiplex assays. Logistic regression was used for univariate and multivariate analyses. Patients with Gag-specific CD4 T cell proliferative responses had significantly higher percentages and absolute counts of mDCs and pDCs in the peripheral blood than nonresponding patients. Gag-specific CD4 and CD8 T cell proliferation was associated with lower plasma sCD14 levels. Plasma levels of IFN-α, TRAIL, and chemokines involved in T cell migration to secondary lymphoid organs were not associated with T cell proliferation. Multivariate analysis confirmed the association between Gag-specific CD4 T cell proliferation and pDC levels. In conclusion, DC levels are a robust correlate of the presence of Gag-specific T cell proliferation in successfully treated youths.

International audience; Zika virus (ZIKV) belongs to the large category of arboviruses. Surprisingly, several human-to-human transmissions of ZIKV have been notified, either following sexual intercourse or from the mother to fetus during pregnancy. Importantly, high viral loads have been detected in the human breast milk of infected mothers, and the existence of breastfeeding as a new mode of mother-to-child transmission of ZIKV was recently hypothesized. However, the maternal origin of infectious particles in breast milk is currently unknown. Here, we show that ZIKV disseminates to the mammary glands of infected mice after both systemic and local exposure with differential kinetics. Ex vivo, we demonstrate that primary human mammary epithelial cells were sensitive and permissive to ZIKV infection in this study. Moreover, by using in vitro models, we prove that mammary luminal-and myoepithelial-phenotype cell lines are both able to produce important virus progeny after ZIKV exposure. Our data suggest that the dissemination of ZIKV to the mammary glands and subsequent infection of the mammary epithelium could be one mechanism of viral excretion in human breast milk.

International audience; Cross-species transmission of simian foamy viruses (SFVs) from nonhu-man primates (NHPs) to humans is currently ongoing. These zoonotic retroviruses establish lifelong persistent infection in their human hosts. SFV are apparently non-pathogenic in vivo, with ubiquitous in vitro tropism. Here, we aimed to identify envelope B-cell epitopes that are recognized following a zoonotic SFV infection. We screened a library of 169 peptides covering the external portion of the envelope from the prototype foamy virus (SFVpsc_huHSRV.13) for recognition by samples from 52 Central African hunters (16 uninfected and 36 infected with chimpanzee, gorilla, or Cercopithecus SFV). We demonstrate the specific recognition of peptide N 96-V 110 located in the leader peptide, gp18 LP. Forty-three variant peptides with truncations, alanine substitutions, or amino acid changes found in other SFV species were tested. We mapped the epitope between positions 98 and 108 and defined six amino acids essential for recognition. Most plasma samples from SFV-infected humans cross-reacted with sequences from apes and Old World monkey SFV species. The magnitude of binding to peptide N 96-V 110 was significantly higher for samples of individuals infected with a chimpanzee or gorilla SFV than those infected with a Cercopithecus SFV. In conclusion, we have been the first to define an immunodomi-nant B-cell epitope recognized by humans following zoonotic SFV infection. IMPORTANCE Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. SFVs can be transmitted to humans, in whom they establish persistent infection, like the simian lenti-and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 and human T-lymphotropic virus type 1. This is the first identification of an SFV-specific B-cell epitope recognized by human plasma samples. The immunodominant epitope lies in gp18 LP , probably at the base of the envelope trimers. The NHP species the most genetically related to humans transmitted SFV strains that induced the strongest antibody responses. Importantly, this epitope is well conserved across SFV species that infect African and Asian NHPs.

International audience; Background. A spillover of simian foamy virus (SFV) to humans, following bites from infected nonhuman primates (NHPs), is ongoing in exposed populations. These retroviruses establish persistent infections of unknown physiological consequences to the human host. Methods. We performed a case-control study to compare 24 Cameroonian hunters infected with gorilla SFV and 24 controls matched for age and ethnicity. A complete physical examination and blood test were performed for all participants. Logistic regression and Wilcoxon signed rank tests were used to compare cases and controls. Results. The cases had significantly lower levels of hemoglobin than the controls (median, 12.7 vs 14.4 g/dL; P = .01). Basophil levels were also significantly lower in cases than controls, with no differences for other leukocyte subsets. Cases had significantly higher urea, creatinine, protein, creatinine phosphokinase, and lactate dehydrogenase levels and lower bilirubin levels than controls. Cases and controls had similar frequencies of general, cutaneous, gastrointestinal, neurological, and cardiorespiratory signs. Conclusions. The first case-control study of apparently healthy SFV-infected Cameroonian hunters showed the presence of hematological abnormalities. A thorough clinical and laboratory workup is now needed to establish the medical relevance of these observations because more than half of cases had mild or moderate anemia. Clinical Trials Registration. NCT03225794.

Simian foamy viruses (SFV) are retroviruses that are widespread among nonhuman primates. SFV can be transmitted to humans, giving rise to a persistent infection. Only a few data are available concerning the distribution of SFV in human blood cells. Here we purified blood mononuclear cell subsets from 11 individuals infected with a Gorilla gorilla SFV strain and quantified SFV DNA levels by quantitative PCR. SFV DNA was detected in the majority of the CD8 + , CD4 + , and CD19 + lymphocyte samples and rarely in CD14 + monocyte and CD56 + NK lymphocyte samples. The median (interquartile range [IQR]) SFV DNA counts were 16.0 (11.0 to 49.8), 11.3 (5.9 to 28.3), and 17.2 (2.0 to 25.2) copies/10 5 cells in CD8 + T lymphocytes, CD4 + T lymphocytes, and CD19 + B lymphocytes, respectively. In the CD4 compartment, SFV DNA was detected in both memory and naive CD4 + T lymphocytes. SFV DNA levels in CD4 + T cells were positively correlated with the duration of the infection. Our study shows with a quantitative method that CD8 + , CD4 + , and B lymphocytes are major cellular targets of SFV in the blood of infected humans. IMPORTANCE Investigation of SFV infections in humans is important due to the origin of human immunodeficiency viruses (HIV) and human T cell lymphotropic viruses (HTLV) from cross-species transmission of their simian counterparts to humans. Surprisingly little is known about many aspects of the biology of SFV in infected humans, including quantitative data concerning the cellular targets of SFV in vivo . Here we show that the distribution of SFV DNA among the different leukocyte populations is not homogeneous and that viral load in CD4 + T lymphocytes is correlated with the duration of infection. These new data will help in understanding the biology of retroviral infections in humans and can be useful in the growing field of SFV-based gene therapy.

BackgroundGag-specific T lymphocytes play a key role in the control of human immunodeficiency virus (HIV) replication. Their restoration will be important for future reservoir targeting strategies. In this study, we aimed to identify immune correlates of Gag-specific CD8 T-cell proliferation in youths with perinatally acquired HIV-1 infection.MethodsThe ANRS-EP38-IMMIP study included youths of 15 to 24 years of age. Fifty-three were taking combination anti-retroviral therapy and aviremic at the time of the study and had undergone valid 5-6-carboxyfluorescein diacetate succimidyl ester-based flow cytometry T-cell proliferation assays. Plasma analytes were quantified by enzyme-linked immunosorbent assay or multiplex assays. Peripheral blood cells were phenotyped by flow cytometry. Logistic regression was used to study the association between Gag-specific T-cell proliferation and immune markers.ResultsPatients with Gag-specific CD8 T-cell proliferation had higher levels of plasma transforming growth factor (TGF)-β1, a lower proportion of naive cells among regulatory T cells (Tregs), and higher percentages of CD4 and CD8 T cells expressing the α4β7 integrin or CD161 molecule than those without a Gag-specific response. These associations were significant based on analyses including potential confounders.ConclusionsPreserved Gag-specific CD8 T-cell proliferation was associated with higher TGF-β1 levels and increased percentages of T cells with a gut-homing phenotype at least 15 years after HIV infection during the perinatal period.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4 + T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. IMPORTANCE Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases activated leukocyte cell adhesion molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.

Human diseases of zoonotic origin are a major public health problem. Simian foamy viruses (SFVs) are complex retroviruses which are currently spilling over to humans. Replication-competent SFVs persist over the lifetime of their human hosts, without spreading to secondary hosts, suggesting the presence of efficient immune control. Accordingly, we aimed to perform an in-depth characterization of neutralizing antibodies raised by humans infected with a zoonotic SFV. We quantified the neutralizing capacity of plasma samples from 58 SFV-infected hunters against primary zoonotic gorilla and chimpanzee SFV strains, and laboratory-adapted chimpanzee SFV. The genotype of the strain infecting each hunter was identified by direct sequencing of the env gene amplified from the buffy coat with genotype-specific primers. Foamy virus vector particles (FVV) enveloped by wild-type and chimeric gorilla SFV were used to map the envelope region targeted by antibodies. Here, we showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain. Epitopes recognized by neutralizing antibodies have been conserved during the cospeciation of SFV with their nonhuman primate host. Greater neutralization breadth in plasma samples of SFV-infected humans was statistically associated with smaller SFV-related hematological changes. The neutralization patterns provide evidence for persistent expression of viral proteins and a high prevalence of coinfection. In conclusion, neutralizing antibodies raised against zoonotic SFV target immunodominant and conserved epitopes located in the receptor binding domain. These properties support their potential role in restricting the spread of SFV in the human population., Author summary Foamy viruses are the oldest known retroviruses and have been mostly described to be nonpathogenic in their natural animal hosts. Simian foamy viruses (SFVs) can be transmitted to humans, in whom they establish persistent infection, as have the simian lenti- and deltaviruses that led to the emergence of two major human pathogens, human immunodeficiency virus type 1 (HIV-1) and human T lymphotropic virus type 1 (HTLV-1). Such cross-species transmission of SFV is ongoing in many parts of the world where humans have contact with nonhuman primates. We present the first comprehensive study of neutralizing antibodies in SFV-infected humans. We showed high titers of neutralizing antibodies in the plasma of most SFV-infected individuals. Neutralizing antibodies target the dimorphic portion of the envelope protein surface domain that overlap with the receptor binding domain. SFV-specific antibodies target epitopes conserved over 8 million years of co-speciation with their nonhuman primate host. Greater neutralization potency in infected individuals was statistically associated with smaller SFV-related hematological changes. In conclusion, our results suggest the protective action of neutralizing antibodies against SFV infection and spread in the human population.

International audience; Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (α-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.

Les mecanismes physiopathologiques de la degenerescence maculaire liee a l’âge (DMLA) sont encore peu connus. Comme d’autres pathologies du vieillissement telles que la maladie d’Alzheimer ou l’atherosclerose, la DMLA est associee a l’apparition de depots macromoleculaires anormaux appeles drusen. Ces derniers sont localises au niveau de la membrane de Bruch. De recentes investigations sur la nature de ces depots, leurs relations avec les reactions inflammatoires et immunitaires ainsi que sur le role important du stress oxydatif ont permis d’elargir les hypotheses sur les mecanismes physiopathologiques conduisant aux lesions de DMLA. De nombreuses analogies avec les mecanismes impliques dans la genese des lesions d’atherosclerose suggerent des similitudes physiopathologiques entre ces deux maladies. Le role pro-atherogene du cholesterol et de ses derives oxydes, les oxysterols, a ete largement demontre dans les processus atheromateux. Il semblerait que ces composes interviennent egalement dans la genese de la DMLA entrainant des activites cytotoxiques, pro-oxydantes et pro-inflammatoires.

Resume En cytometrie en flux, les microbilles constituent des outils importants. Initialement developpees pour optimiser les signaux de diffraction et de fluorescence detectes par les cytometres, les microbilles sont maintenant largement utilisees pour la quantification d'antigenes. Actuellement, leur utilisation comme support reactionnel permet aussi de detecter et de quantifier simultanement par cytometrie en flux plusieurs analytes tels que des proteines, des nucleotides, et des ions, mais aussi des agents infectieux dans quelques microlitres d'echantillon. Pour cela, des microbilles de fluorescences et/ou de tailles differentes couplees a des proteines ou a des oligonucleotides sont le plus souvent utilisees et analysees sur des cytometres en flux conventionnels ou specifiquement dedies. Les applications de ces methodes multiplexes d'analyses biomoleculaires par cytometrie en flux ouvrent de nombreuses perspectives en biologie, medecine, agroalimentaire et environnement (biosurveillance).

The detection of hepatitis C virus (HCV)-specific T cell responses in HCV-uninfected, presumably unexposed, subjects could be due to an underestimation of the frequency of spontaneously resolving infections, as most acute HCV infections are clinically silent. To address this hypothesis, HCV-specific cellular immune responses were characterized, in individuals negative for an HCV PCR assay and humoral response, with (n = 32) or without (n = 33) risk of exposure to HCV. Uninfected volunteers (n = 20) with a chronically HCV-infected partner were included as positive controls for potential exposure to HCV and HCV infection, respectively. HCV-specific T cell responses in freshly isolated peripheral blood mononuclear cells were studied ex vivo by ELISPOT and CFSE-based proliferation assays using panels of HCV Core and NS3-derived peptides. A pool of unrelated peptides was used as a negative control, and a peptide mix of human cytomegalovirus, Epstein-Bar virus and Influenza virus as a positive control. Overall, 20% of presumably HCV-uninfected subject tested had detectable T-cell responses to the virus, a rate much higher than previous estimates of HCV prevalence in developed countries. This result would be consistent with unapparent primary HCV infections that either cleared spontaneously or remained undetected by conventional serological assays.

Oxysterols, found in some commonly consumed foods, can induce a wide range of cytotoxic effects, which have been extensively studied. On the other hand, the side effects of phytosterols and oxyphytosterols are less well-known. Over the past few years, different types of foods have been enriched with phytosterols on the basis of the properties of these compounds that reduce circulating cholesterol levels in certain experimental conditions. It is therefore important to gain better knowledge of the risks and benefits of this type of diet. In this study, conducted in human monocytic U937 cells, the ability of phytosterols (sitosterol, campesterol) and oxyphytosterols (7 beta-hydroxysitosterol, 7-ketositosterol) to induce cell death, polar lipid accumulation, and pro-inflammatory cytokine (MCP-1; IL-8) secretion was determined and compared to that of oxysterols (7-ketocholesterol, 7 beta-hydroxycholesterol). Phytosterols and oxyphytosterols had no significant effects on the parameters studied; only 7 beta-hydroxysitosterol slightly increased cell death, whereas at the concentration used (20 mu g/mL), strong cytotoxic effects were observed with the oxysterols. With sitosterol, campesterol, and 7-ketositosterol, IL-8 secretion was decreased, and with campesterol the intracellular polar lipid level was reduced. The data show that phytosterols and oxyphytosterols have no oxysterol-like side effects, and they rather argue in favor of phytosterols' beneficial effects.

Aim: The composition of the meibum of blepharitis patients is characterised by increased levels of branched-chain fatty acids (BCFAs) that return to normal values in patients treated with cyclins and lid hygiene. The aim of this study was to determine if BCFAs had toxic effects on conjunctival cells related to the disease. Methods: Chang and IOBA-NHC conjunctival human cells were treated with BCFAs (isoC16 and isoC20) or palmitic acid as a control for 4 h or 24 h at 50 μM or 100 μM. Morphological and functional changes were investigated by measuring mitochondrial dehydrogenase activity, cell permeability, mitochondrial depolarisation, chromatin condensation, IL-1β and reactive oxygen species production.Results: None of the fatty acids modified the parameters of cytotoxicity in conjunctival cells in Chang or IOBA-NHC cell lines. Only the mitochondrial dehydrogenase activity was significantly decreased in relation to the isoC20 concentration increase.Conclusions: The increase in BCFAs in the tears of blepharitis patients does not consistently participate in the conjunctival cell changes throughout the course of the disease. Instead, it is likely an adaptive response of the ocular surface to the lack of tears, possibly increasing meibum fluidity, thus enhancing lacrimal film stability. Footnotes

Lipopolysaccharides (LPS) are components of Gram-negative bacteria. The cellular response from the host to LPS is mediated through stepwise interactions involving the lipopolysaccharide binding protein (LBP), CD14, and MD-2 which produces the rearrangement of TLR4. In addition to LBP, the lipid transfer/lipopolysaccharide binding protein (LT/LBP) gene family includes the phospholipid transfer protein (PLTP). Here we show that the intravascular redistribution of LPS from the plasma lipoprotein-free fraction towards circulating lipoproteins is delayed in PLTP-deficient mice. In agreement with earlier in vitro studies which predicted the neutralization of the endotoxic properties of LPS when associated with lipoproteins, significant increases in the plasma concentration of pro-inflammatory cytokines were found in PLTP-deficient as compared to wild-type mice. Similar inflammatory damage occurred in tissues from wild-type and PLTP-deficient mice 24 hours after one single intraperitoneal injection of LPS, however with a more severe accumulation of red blood cells in glomeruli of LPS-injected PLTP-deficient mice. Complementary ex vivo experiments on isolated splenocytes from wild-type and PLTP-deficient mice came in further support of the ability of cell-derived PLTP to prevent LPS-mediated inflammation and cytotoxicity when combined with lipoprotein acceptors. Finally, PLTP deficiency in mice led to a significant increase in LPS-induced mortality. It is concluded that increasing circulating levels of PLTP may constitute a new and promising strategy in preventing endotoxic shock.

International audience; Purpose: To characterize the possible cytotoxic effects of oxysterols (7-hydroxycholesterol (7-OH), 25-hydroxycholesterol (25-OH)) in human retinal pigment epithelial cells (ARPE-19) and to detail the relationships between some of these effects. Methods: ARPE-19 cells were treated with 7-OH and 25-OH. Cell viability was measured with the MTT assay. Membrane permeability, mitochondrial potential, and lysosomal integrity were measured by flow cytometry with propidium iodide, DiOC6(3), and acridine orange, respectively. Cell death was characterized by staining with Hoechst 33342, transmission electron microscopy, and analysis of the DNA fragmentation pattern. Caspase activity was examined with fluorochrome-labeled inhibitors of caspases (FLICA) and Western blotting. Immunofluorescence staining was used to visualize the cellular distribution of cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF). The effect of the cathepsin inhibitor (z-FA-fmk) on oxysterol-induced cell death was evaluated. Results: Cell viability of ARPE-19 cells was decreased with 7-OH, whereas 25-OH had no cytotoxic effects. Loss of mitochondrial potential and lysosomal destabilization was associated with 7-OH-induced cell death, few morphologically apoptotic cells were identified, and no internucleosomal DNA fragmentation was found. Slight caspase activation was detected with FLICA, and no caspase-3 activation was revealed. A pronounced relocalization of Cyt-c and AIF was observed. Noteworthy, z-FA-fmk was able to prevent cell death. Conclusion: 7-OH induced a caspase-3-independent mode of cell death associated with lysosomal destabilization, which could play a key role in the signaling pathways leading to cell death, as shown by the ability of z-FA-fmk to counteract the cytotoxic effects of 7-OH.

Oxysterols, mainly those oxidized at the C7 position, induce a complex mode of cell death exhibiting some characteristics of apoptosis associated with a rapid induction of lipid rich multilamellar cytoplasmic structures (myelin figures) observed in various pathologies including atherosclerosis. The aim of this study was to determine the relationships between myelin figure formation, cell death, and lipid accumulation in various cell lines (U937, THP-1, MCF-7 [caspase-3 deficient], A7R5) treated either with oxysterols (7-ketocholesterol [7KC], 7beta-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 25-hydroxycholesterol) or cytotoxic drugs (etoposide, daunorubicin, tunicamycin, rapamycin). Cell death was assessed by the measurement of cellular permeability with propidium iodide, characterization of the morphological aspect of the nuclei with Hoechst 33342, and identification of myelin figures by transmission electron microscopy. Nile Red staining (distinguishing neutral and polar lipids) was used to identify lipid content by flow cytometry and spectral imaging microscopy. Whatever the cells considered, myelin figures were only observed with cytotoxic oxysterols (7KC, 7beta-hydroxycholesterol, cholesterol-5beta, 6beta-epoxide), and their formation was not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. When U937 cells were treated with oxysterols or cytotoxic drugs, polar lipid accumulation was mainly observed with 7KC and 7beta-hydroxycholesterol. The highest polar lipid accumulation, which was triggered by 7KC, was counteracted by z-VAD-fmk. These findings demonstrate that myelin figure formation is a caspase-independent event closely linked with the cytotoxicity of oxysterols, and they highlight a relationship between caspase activity and polar lipid accumulation.

International audience; Aim To investigate the ocular surface inflammatory response to chronic topical treatments in patients with glaucoma by measuring the cytokine level in tears using multiplex bead analysis. Methods Tear samples were collected from 21 patients with glaucoma and 12 healthy volunteers. Tears were analysed for the presence of 17 cytokines: interleukin (IL)1β, IL2, IL4, IL5, IL6, IL7, IL8, IL10, IL12, IL13, IL17, granulocyte‐colony stimulating factor, granulocyte‐macrophage stimulating factor, interferon (INF)γ, monocyte chemotactic protein (MCP)1, macrophage inflammatory protein 1β and tumour necrosis factor (TNF)α. The cytokines in each sample of tears were measured using multiplex bead analysis with microspheres as solid support for immunoassays. Results In the tears of treated patients, proinflammatory cytokines (IL1β, IL6, IL12, TNFα) were significantly increased compared with controls. T helper (Th)1 (INFγ, IL2) and Th2 (IL5, IL10, IL4) type cytokines were also significantly higher (p

Background: Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C-reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7β-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol), and various cytokines. Methods: Different flow cytometric bead-based assays were used to quantify some cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1β, or TNF-α) in the culture media of oxysterol-treated U937 and THP-1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods were used to measure IL-8 mRNA levels, intracellular IL-8 content, and protein phosphorylation in the mitogenic extracellular kinase/extracellular signal-regulated kinase1/2 (MEK/ERK1/2) signaling pathway. Results: All oxysterols investigated are potent in vitro inducers of MCP-1, MIP-1β, TNF-α, and/or IL-8 secretion, the latter involving the MEK/ERK1/2 cell signaling pathway. In healthy and atherosclerotic subjects, no relationships were found between cytokines (IL-8, IL-1β, IL-6, IL-10, TNF-α, IL-12, and MCP-1), CRP, conventional markers of hyperlipidemia, and oxysterols. However, in patients with arterial disorders of the lower limbs, small but statistically significant differences in the circulating levels of CRP, TNF-α, and IL-10 were observed comparatively to healthy subjects and according to the atherosclerotic stage considered. Conclusions: Flow cytometric bead-based assays are well adapted to measure variations of cytokine secretion in the culture media of oxysterol-treated cells and in the sera of healthy and atherosclerotic subjects. They underline the in vitro proinflammatory properties of oxysterols and may permit to distinguish healthy and atherosclerotic subjects, as well as various atherosclerotic stages. © 2006 International Society for Analytical Cytology

Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca 2 + , which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the 'BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK → ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway.

Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, referred to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7alpha-, 7beta-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O2*-) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O2*- overproduction quantified with hydroethidine was identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, with cholesterol-5alpha, 6alpha-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7beta-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.

International audience; Ligation of CD47 by its natural ligand thrombospondin (TSP), or cross-linking by CD47 antibodies, triggers caspase-independent cell death in normal and leukemic cells. This kind of cell death is characterised by the cytoplasmic events of apoptosis including externalisation of phosphatidylserines and mitochondria swelling. We report herein selective mitochondrial changes in CD47-dependent cell death of T cells. After T cell stimulation via CD47, a rapid mitochondrial transmembrane potential (deltapsi(m)) disruption is accompanied by the production of reactive oxygen species (ROS) and phosphatidylserine exposure. Surprisingly, mitochondrial dysfunction does not induce cytochrome c or AIF release. Moreover, the dying cells do not exhibit caspase-3 activation and display intact nuclei without any large-scale, or oligonucleosomal DNA fragmentation. We conclude that DeltaPsi(m) loss and ROS production are an early step in CD47-dependent killing and neither cytochrome c, nor AIF are implicated in this new cell death pathway.

International audience; Simian foamy viruses (SFV) are widespread retroviruses among non-human primates (NHP). SFV actively replicate in the oral cavity of NHP and can be transmitted to humans through NHP bites, in whom they establish a persistent infection. We aimed to study three major properties of these zoonotic retroviruses: replicative status, tropism and variability. In 14 hunters from Cameroon previously shown to be infected with a gorilla SFV strain, viral DNA could be detected by quantitative polymerase chain reaction in most samples of peripheral blood mononuclear cells (PBMCs) and saliva. The SFV DNA levels were 7.1±6.0 SFV DNA copies/105 cells in PBMCs and 2.4±4.3 SFV DNA copies/105 cells in saliva. In contrast, no SFV RNA was detected by qRT-PCR in either PBMCs or saliva. PBMCs populations (T4, T8, B, NK lymphocytes and monocytes) were sorted with magnetic beads before quantification of SFV DNA. Our preliminary results showed the presence of SFV DNA in all PBMCs populations at different levels. We finally assessed the viral diversity in vivo. Although intra-individual SFV genetic variation was low (

But Des mecanismes inflammatoires locaux sont mis en cause dans la genese des lesions de degenerescence maculaire liee a l’âge (DMLA). Les capacites pro inflammatoires des oxysterols ont ainsi ete testees sur des cellules epitheliales pigmentaires en culture. La secretion d’IL-8, chemokine connue pour ses pouvoirs chimioattractif surles cellules de l’inflammation et pour son role pro-angiogenique a ete particulierement etudiee ainsi que les proteines impliquees dans le controle de sa secretion. Materiels et Methodes Des cellules epitheliales pigmentaires en lignee (ARPE-19) ont ete cultivees et traitees par differents oxysterols : le 7-cetocholesterol, le 7- betahydroxycholesterol et le 25-hydroxycholesterol (25-OH). Apres 24 h et 48 h de traitement a differentes concentrations les surnageants etaient recuperes et un dosage de plusieurs cytokines a ete realise d’une part avec une technique d’analyse multiplexee et d’autre part par methode ELISA. L’implication de la voie des MAP-kinases dans la secretion d’IL-8 a ete evaluee en utilisant des inhibiteurs specifiques de cette voie U0126 et PD98059. Resultats Le dosage a ete realise par cytometrie en flux avec analyse simultanee de la secretion de 6 cytokines (IL-8, IL-1s, IL-6, IL-10, TNF- αet IL-12). Seule la secretion d’IL-8 a ete identifiee sous l’influence du 25-OH. Le 7-ceto et le 7- βn’induisent pas la secretion d’IL-8. Ceci a ete confirme par les dosages avec la methode ELISA qui montrent que seul le 25-OH stimule la secretion d’IL-8. L’utilisation des inhibiteurs specifiques de la voie des MAP-kinases diminue la secretion d’IL-8 induite par le 25-OH. Les valeurs mesurees sont alors comparables a celles obtenues sur le surnageant de cellules non traitees. Discussion L’IL-8 est la seule cytokine dont la secretion par les cellules ARPE-19 est stimulee et ce uniquement par le 25-OH. Cet oxysterol qui n’a par ailleurs aucun effet cytotoxique peut donc etre considere comme un promoteur de l’inflammation. La voie des MAP-kinases semble etre une des voies de signalisation majeure impliquee dans le controle de la secretion d’IL-8 par les cellules ARPE-19. Conclusion Le 25-OH semble etre un agent pro-inflammatoire qui favorise au travers de la secretion d’IL-8 le recrutement des cellules inflammatoires contribuant auxlesions observees dans la DMLA. Par ailleurs l’IL-8 possede aussi des proprietes pro-angiogeniques qui pourraient favoriser le developpement des lesions neo vasculaires observees au cours de la DMLA.

Introduction Des travaux preliminaires ont montre une augmentation de la proportion en acides gras branches (acides gras dont la chaine est branchee par un ou plusieurs groupes methyl) dans le film lipidique lacrymal de patients souffrant de blepharite chronique. Or les acides gras branches ont prouve leur toxicite dans certains modeles cellulaires. Le but de notre travail a ete d’etudier les effets de certains acides gras branches sur des cellules conjonctivales en culture. Materiels et Methodes Des cellules conjonctivales humaines immortalisees ont ete incubees avec des solutions contenant les acides gras branches isoC16, isoC20, l’acide phytanique ou un acide gras lineaire : le C16 a des concentrations variant de 50 a 200 μm pendant 4 h ou 24 h. La toxicite des molecules a ete evaluee par la methode MTT (methylthiazol tetrazolium bromide), la croissance cellulaire, la production d’especes reactives de l’oxygene (dichlorodihydrofluoresceine), la permeabilite membranaire (iodure de propidium), la depolarisation mitochondriale (Mititracker) et l’apoptose (Hoechst). L’incorporation des acides gras dans les cellules a elle aussi ete etudiee. Resultats La respiration mitochondiale est diminuee significativement par l’IsoC20 et le C16 apres 4 h d’incubation et par l’isoC20 et l’acide phytanique apres 24h d’incubation (p Discussion L’isoC20, un des acides gras branches presents dans les lipides des larmes a raison de 3,5 % des acides gras totaux affecte la respiration mitochondriale. Cet effet ne semble pas lie au stress oxydatif, a la croissance cellulaire ou a la mort cellulaire. Il pourrait etre du a l’incorporation de cet acide gras au sein des membranes mitochondriales. Conclusion L’isoC16 et l’isoC20 n’ont pas le meme effet sur les cellules de la conjonctive. Cette difference pourrait etre liee a une incorporation preferentielle de l’isoC20 dans les membranes mitochondriales par rapport a l’iso C16.

International audience; BackgroundEarly combined antiretroviral therapy (cART) limits the total HIV-DNA load in children. However, data on its impact in older children and adolescents remain scarce. This study aims to compare HIV reservoirs in children (5-12 years) and adolescents (13-17 years) who started cART before 6 months (early (E-)group) or after 2 years old (late (L-)group). MethodsThe ANRS-EP59-CLEAC study prospectively enrolled 76 HIV-1 perinatally-infected patients who reached HIV-RNA