Bioactive lipids as biomarkers of adverse reactions associated with apheresis platelet concentrate transfusion

Platelet concentrate (PC) transfusion seeks to provide haemostasis in patients presenting severe central thrombocytopenia or severe bleeding. PCs may induce adverse reactions (AR) that can occasionally be severe (SAR). PCs contain active biomolecules such as cytokines and lipid mediators. The processing and storage of PCs creates so-called structural and biochemical storage lesions that accumulate when blood products reach their shelf life. We sought to investigate lipid mediators as bioactive molecules of interest during storage and review associations with adverse reactions post-transfusion. To facilitate understanding, we focused on single donor apheresis (SDA) PCs with approximately 31.8% of PCs being delivered in our setting. Indeed, pooled PCs are the most widely transfused products, but the study of a single donor lipid mediator is easier to interpret. We are investigating key lipid mediators involved in AR. Adverse reactions were closely monitored in accordance with current national and regional haemovigilance protocols. Residual PCs were analysed post-transfusion in a series of observations, both with and without severe reactions in recipients. A decrease in the lysophosphatidylcholine species to produce the lysophosphatidic acid species has been observed during storage and in the case of AR. Lysophosphatidic acid increased with primarily platelet-inhibitor lipids. Anti-inflammatory platelet-induced inhibition lipids were weakly expressed in cases of severe adverse reactions. We therefore propose that a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid can prospectively predict serious adverse transfusion reactions

Etude in vitro des déterminants moléculaires impliquées dans la réponse autophagique au cours de l'infection à staphylococcus aureus

Staphylococcus aureus is a human pathogenic and commensal bacterium able to trigger its internalization in non-professional phagocytic cells (NPPCs). The presence of S. aureus induced a storng autophagic response in order to eliminate intracellular bacteria. The objective was to identify bacterial virulence factors and host cell response mechanisms that may be associated with autophagy and intracellular survival of S. aureus. Our results showed that the autophagic response against S. aureus promotes the elimination of intracellular bacteria in NPPCs but S. aureus is able to escape and block autophagy in order to replicate. The intracellular replication of S. aureus is associated with the induction of calcium ions (Ca2+) influx from intracellular stores. This Ca2+ influx is associated with an accumulation of autophagosomes, an inhibition of the autophagosomes maturation towards lysosomes and an increase of cytotoxicity induced by S. aureus. Thanks to the development of CRISPR/Cas9 techniques and the generation of S. aureus mutant strains for the esxA, esxB and essH genes from the type VII secretion system (T7SS), we have demonstrated that these toxins promote intracellular S. aureus survival by blocking the maturation of autophagosomes. Finally, we have shown that the inhibition of the transcription factor YAP by the staphylococcal toxin EDIN-B or by genome editing promotes the intracellular replication of S. aureus associated with an accumulation of autophagosomes and a lack of lysosomal acidificationStaphylococcus aureus est une bactérie pathogène et commensale de l’Homme capable de déclencher son internalisation dans des cellules phagocytaires non professionnelles (NPPCs). La présence de S. aureus provoque une réponse autophagique rapide visant à éliminer les bactéries intracellulaires. L’objectif général était d’identifier des facteurs de virulence bactériens et des mécanismes de réponse de la cellule hôte pouvant être associés à l’autophagie et à la survie intracellulaire de S. aureus. Nos résultats ont montré que la réponse autophagique dirigée contre S. aureus favorise l’élimination des bactéries intracellulaires dans les NPPCs mais S. aureus est capable d’échapper et de bloquer l’autophagie afin de se répliquer. La réplication intracellulaire de S. aureus est associée à l’induction d’un influx d’ions calciques (Ca2+) provenant de réserves intracellulaires. Cet influx de Ca2+ est associé à une accumulation des autophagosomes, une inhibition de la maturation des autophagosomes vers les lysosomes et une augmentation de la cytotoxicité induite par S. aureus. Grâce au développement des techniques CRISPR/Cas9 et à la génération de souches de S. aureus mutantes pour les gènes esxA, esxB et essH appartenant au système de sécrétion de type VII (T7SS), nous avons mis en évidence que ces toxines favorisent la survie intracellulaire de S. aureus en bloquant la maturation des autophagosomes. Enfin, nous avons montré que l’inhibition du facteur de transcription YAP par la toxine staphylococcique EDIN-B ou par « genome editing » favorise la réplication intracellulaire de S. aureus associée à une accumulation des autophagosomes et un défaut d’acidification des lysosomes

In vitro study of the molecular determinants involved in autophagic response during Staphylococcus aureus infection

Staphylococcus aureus est une bactérie pathogène et commensale de l’Homme capable de déclencher son internalisation dans des cellules phagocytaires non professionnelles (NPPCs). La présence de S. aureus provoque une réponse autophagique rapide visant à éliminer les bactéries intracellulaires. L’objectif général était d’identifier des facteurs de virulence bactériens et des mécanismes de réponse de la cellule hôte pouvant être associés à l’autophagie et à la survie intracellulaire de S. aureus. Nos résultats ont montré que la réponse autophagique dirigée contre S. aureus favorise l’élimination des bactéries intracellulaires dans les NPPCs mais S. aureus est capable d’échapper et de bloquer l’autophagie afin de se répliquer. La réplication intracellulaire de S. aureus est associée à l’induction d’un influx d’ions calciques (Ca2+) provenant de réserves intracellulaires. Cet influx de Ca2+ est associé à une accumulation des autophagosomes, une inhibition de la maturation des autophagosomes vers les lysosomes et une augmentation de la cytotoxicité induite par S. aureus. Grâce au développement des techniques CRISPR/Cas9 et à la génération de souches de S. aureus mutantes pour les gènes esxA, esxB et essH appartenant au système de sécrétion de type VII (T7SS), nous avons mis en évidence que ces toxines favorisent la survie intracellulaire de S. aureus en bloquant la maturation des autophagosomes. Enfin, nous avons montré que l’inhibition du facteur de transcription YAP par la toxine staphylococcique EDIN-B ou par « genome editing » favorise la réplication intracellulaire de S. aureus associée à une accumulation des autophagosomes et un défaut d’acidification des lysosomes.Staphylococcus aureus is a human pathogenic and commensal bacterium able to trigger its internalization in non-professional phagocytic cells (NPPCs). The presence of S. aureus induced a storng autophagic response in order to eliminate intracellular bacteria. The objective was to identify bacterial virulence factors and host cell response mechanisms that may be associated with autophagy and intracellular survival of S. aureus. Our results showed that the autophagic response against S. aureus promotes the elimination of intracellular bacteria in NPPCs but S. aureus is able to escape and block autophagy in order to replicate. The intracellular replication of S. aureus is associated with the induction of calcium ions (Ca2+) influx from intracellular stores. This Ca2+ influx is associated with an accumulation of autophagosomes, an inhibition of the autophagosomes maturation towards lysosomes and an increase of cytotoxicity induced by S. aureus. Thanks to the development of CRISPR/Cas9 techniques and the generation of S. aureus mutant strains for the esxA, esxB and essH genes from the type VII secretion system (T7SS), we have demonstrated that these toxins promote intracellular S. aureus survival by blocking the maturation of autophagosomes. Finally, we have shown that the inhibition of the transcription factor YAP by the staphylococcal toxin EDIN-B or by genome editing promotes the intracellular replication of S. aureus associated with an accumulation of autophagosomes and a lack of lysosomal acidificatio

Etude in vitro des déterminants moléculaires impliquées dans la réponse autophagique au cours de l'infection à staphylococcus aureus

Staphylococcus aureus is a human pathogenic and commensal bacterium able to trigger its internalization in non-professional phagocytic cells (NPPCs). The presence of S. aureus induced a storng autophagic response in order to eliminate intracellular bacteria. The objective was to identify bacterial virulence factors and host cell response mechanisms that may be associated with autophagy and intracellular survival of S. aureus. Our results showed that the autophagic response against S. aureus promotes the elimination of intracellular bacteria in NPPCs but S. aureus is able to escape and block autophagy in order to replicate. The intracellular replication of S. aureus is associated with the induction of calcium ions (Ca2+) influx from intracellular stores. This Ca2+ influx is associated with an accumulation of autophagosomes, an inhibition of the autophagosomes maturation towards lysosomes and an increase of cytotoxicity induced by S. aureus. Thanks to the development of CRISPR/Cas9 techniques and the generation of S. aureus mutant strains for the esxA, esxB and essH genes from the type VII secretion system (T7SS), we have demonstrated that these toxins promote intracellular S. aureus survival by blocking the maturation of autophagosomes. Finally, we have shown that the inhibition of the transcription factor YAP by the staphylococcal toxin EDIN-B or by genome editing promotes the intracellular replication of S. aureus associated with an accumulation of autophagosomes and a lack of lysosomal acidificationStaphylococcus aureus est une bactérie pathogène et commensale de l’Homme capable de déclencher son internalisation dans des cellules phagocytaires non professionnelles (NPPCs). La présence de S. aureus provoque une réponse autophagique rapide visant à éliminer les bactéries intracellulaires. L’objectif général était d’identifier des facteurs de virulence bactériens et des mécanismes de réponse de la cellule hôte pouvant être associés à l’autophagie et à la survie intracellulaire de S. aureus. Nos résultats ont montré que la réponse autophagique dirigée contre S. aureus favorise l’élimination des bactéries intracellulaires dans les NPPCs mais S. aureus est capable d’échapper et de bloquer l’autophagie afin de se répliquer. La réplication intracellulaire de S. aureus est associée à l’induction d’un influx d’ions calciques (Ca2+) provenant de réserves intracellulaires. Cet influx de Ca2+ est associé à une accumulation des autophagosomes, une inhibition de la maturation des autophagosomes vers les lysosomes et une augmentation de la cytotoxicité induite par S. aureus. Grâce au développement des techniques CRISPR/Cas9 et à la génération de souches de S. aureus mutantes pour les gènes esxA, esxB et essH appartenant au système de sécrétion de type VII (T7SS), nous avons mis en évidence que ces toxines favorisent la survie intracellulaire de S. aureus en bloquant la maturation des autophagosomes. Enfin, nous avons montré que l’inhibition du facteur de transcription YAP par la toxine staphylococcique EDIN-B ou par « genome editing » favorise la réplication intracellulaire de S. aureus associée à une accumulation des autophagosomes et un défaut d’acidification des lysosomes

Etude in vitro des déterminants moléculaires impliquées dans la réponse autophagique au cours de l'infection à staphylococcus aureus

Staphylococcus aureus is a human pathogenic and commensal bacterium able to trigger its internalization in non-professional phagocytic cells (NPPCs). The presence of S. aureus induced a storng autophagic response in order to eliminate intracellular bacteria. The objective was to identify bacterial virulence factors and host cell response mechanisms that may be associated with autophagy and intracellular survival of S. aureus. Our results showed that the autophagic response against S. aureus promotes the elimination of intracellular bacteria in NPPCs but S. aureus is able to escape and block autophagy in order to replicate. The intracellular replication of S. aureus is associated with the induction of calcium ions (Ca2+) influx from intracellular stores. This Ca2+ influx is associated with an accumulation of autophagosomes, an inhibition of the autophagosomes maturation towards lysosomes and an increase of cytotoxicity induced by S. aureus. Thanks to the development of CRISPR/Cas9 techniques and the generation of S. aureus mutant strains for the esxA, esxB and essH genes from the type VII secretion system (T7SS), we have demonstrated that these toxins promote intracellular S. aureus survival by blocking the maturation of autophagosomes. Finally, we have shown that the inhibition of the transcription factor YAP by the staphylococcal toxin EDIN-B or by genome editing promotes the intracellular replication of S. aureus associated with an accumulation of autophagosomes and a lack of lysosomal acidificationStaphylococcus aureus est une bactérie pathogène et commensale de l’Homme capable de déclencher son internalisation dans des cellules phagocytaires non professionnelles (NPPCs). La présence de S. aureus provoque une réponse autophagique rapide visant à éliminer les bactéries intracellulaires. L’objectif général était d’identifier des facteurs de virulence bactériens et des mécanismes de réponse de la cellule hôte pouvant être associés à l’autophagie et à la survie intracellulaire de S. aureus. Nos résultats ont montré que la réponse autophagique dirigée contre S. aureus favorise l’élimination des bactéries intracellulaires dans les NPPCs mais S. aureus est capable d’échapper et de bloquer l’autophagie afin de se répliquer. La réplication intracellulaire de S. aureus est associée à l’induction d’un influx d’ions calciques (Ca2+) provenant de réserves intracellulaires. Cet influx de Ca2+ est associé à une accumulation des autophagosomes, une inhibition de la maturation des autophagosomes vers les lysosomes et une augmentation de la cytotoxicité induite par S. aureus. Grâce au développement des techniques CRISPR/Cas9 et à la génération de souches de S. aureus mutantes pour les gènes esxA, esxB et essH appartenant au système de sécrétion de type VII (T7SS), nous avons mis en évidence que ces toxines favorisent la survie intracellulaire de S. aureus en bloquant la maturation des autophagosomes. Enfin, nous avons montré que l’inhibition du facteur de transcription YAP par la toxine staphylococcique EDIN-B ou par « genome editing » favorise la réplication intracellulaire de S. aureus associée à une accumulation des autophagosomes et un défaut d’acidification des lysosomes

Comparison of bacterial filtration efficiency vs. particle filtration efficiency to assess the performance of non-medical face masks

International audienceAbstract As a result of the current COVID-19 pandemic, the use of facemasks has become commonplace. The performance of medical facemasks is assessed using Bacterial Filtration Efficiency (BFE) tests. However, as BFE tests, require specific expertise and equipment and are time-consuming, the performance of non-medical facemasks is assessed with non-biological Particle Filtration Efficiency (PFE) tests which are comparatively easier to implement. It is necessary to better understand the possible correlations between BFE and PFE to be able to compare the performances of the different types of masks (medical vs. non-medical). In this study BFE results obtained in accordance with the standard EN 14683 are compared to the results of PFE from a reference test protocol defined by AFNOR SPEC S76-001 with the aim to determine if BFE could be predicted from PFE. Our results showed a correlation between PFE and BFE. It was also observed that PFE values were higher than BFE and this was attributed to the difference in particle size distribution considered for efficiency calculation. In order to properly compare these test protocols for a better deduction, it would be interesting to compare the filtration efficiency for a similar granulometric range

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting

OBJECTIVES: Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate the antimicrobial eradication of an intra-osteoblastic Staphylococcus aureus reservoir in the setting of bone and joint infection (BJI).METHODS: MICs of 16 anti-staphylococcal molecules active against methicillin-sensitive S.aureus (MSSA) were evaluated at pH 5 and pH 7. Additionally, the lysosomal alkalizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and ammonium chloride were assessed. The results led to further investigation of clindamycin, cotrimoxazole, daptomycin and levofloxacin-alone or in combination with hydroxychloroquine-in an invitro model of osteoblast infection. The impact of hydroxychloroquine on autophagy was finally investigated using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein.RESULTS: Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone significantly decreased the intracellular staphylococcal reservoir (5.12 log10CFU/100000cells) by 0.14 (95%CI 0.01-0.34), 0.25 (95%CI 0.12-0.43), 0.16 (95%CI 0.004-0.39) and 1.18 (95%CI 1.04-1.38) log10CFU/100000cells, respectively (

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting

OBJECTIVES: Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate the antimicrobial eradication of an intra-osteoblastic Staphylococcus aureus reservoir in the setting of bone and joint infection (BJI).METHODS: MICs of 16 anti-staphylococcal molecules active against methicillin-sensitive S.aureus (MSSA) were evaluated at pH 5 and pH 7. Additionally, the lysosomal alkalizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and ammonium chloride were assessed. The results led to further investigation of clindamycin, cotrimoxazole, daptomycin and levofloxacin-alone or in combination with hydroxychloroquine-in an invitro model of osteoblast infection. The impact of hydroxychloroquine on autophagy was finally investigated using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein.RESULTS: Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone significantly decreased the intracellular staphylococcal reservoir (5.12 log10CFU/100000cells) by 0.14 (95%CI 0.01-0.34), 0.25 (95%CI 0.12-0.43), 0.16 (95%CI 0.004-0.39) and 1.18 (95%CI 1.04-1.38) log10CFU/100000cells, respectively (

Lysosomal alkalization to potentiate eradication of intra-osteoblastic Staphylococcus aureus in the bone and joint infection setting

OBJECTIVES: Beyond intracellular penetration, acidic lysosomal pH might affect the intracellular activity of some antimicrobials. This study evaluated the ability of lysosomotropic alkalizing agents to potentiate the antimicrobial eradication of an intra-osteoblastic Staphylococcus aureus reservoir in the setting of bone and joint infection (BJI).METHODS: MICs of 16 anti-staphylococcal molecules active against methicillin-sensitive S.aureus (MSSA) were evaluated at pH 5 and pH 7. Additionally, the lysosomal alkalizing potential (spectrofluorometry) and cytotoxicity (MTT assay) of hydroxychloroquine, amantadine and ammonium chloride were assessed. The results led to further investigation of clindamycin, cotrimoxazole, daptomycin and levofloxacin-alone or in combination with hydroxychloroquine-in an invitro model of osteoblast infection. The impact of hydroxychloroquine on autophagy was finally investigated using Western blot detection of two autophagic flux indicators, the LC3 membrane protein and the SQSTM1 cargo protein.RESULTS: Daptomycin, cotrimoxazole, clindamycin and levofloxacin alone significantly decreased the intracellular staphylococcal reservoir (5.12 log10CFU/100000cells) by 0.14 (95%CI 0.01-0.34), 0.25 (95%CI 0.12-0.43), 0.16 (95%CI 0.004-0.39) and 1.18 (95%CI 1.04-1.38) log10CFU/100000cells, respectively (

Lipidomic analysis of differently prepared platelet concentrates in additive solution during storage

International audienceBackground: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. Lesions may appear during platelet concentrate storage, some of which may be involved in adverse transfusion reactions. The preparation and storage of platelet concentrates (PC) may modify and even damage the lipid mediator content. The aim of this study was to investigate the lipidomic profile identified in the supernatants of PCs according to processing and storage conditions, both after leukocyte filtration and contained in platelet additive solution (PAS), comparing single donor apheresis (SDA) products with pooled buffy coat (BC) products.Materials and methods: We investigated the accumulation of various lipid mediators including lysophospholipids (LP) and eicosanoids in SDA and BC products stored for 0-5 days. All products were processed following French Blood Establishment (EFS) procedures in accordance with EDQM/GTS European Standards. Both SDA and BC were leukocyte reduced and conserved in 35% autologous donor plasma and 65% platelet additive solution. Lipidomic analysis was performed on PC supernatants using LS/MS spectrometry.Results: Our data demonstrate that lysophosphatidylcholine (LPC) levels were higher in BCs compared to SDAs, with no difference in lysophosphatidic acid (LPA) expression between the two preparation methods. Results for other eicosanoids showed greater similarity; indeed, no clear pattern emerged from analysis of eicosanoids in terms of storage time and process. In general, we observed longitudinal lipid mediator modulation for both SDAs and BCs, particularly at later time points.Discussion: The expression of LPC and some eicosanoids in BCs could be used as novel biomarkers of PC quality. Future studies are needed to explore their impact on adverse transfusion reactions