Vitrification of Human Oocytes Before or After Rescue-IVM Does not Impair Maturation Kinetics but Induces Meiotic Spindle Alterations.

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16% and IVM post-VIT: 59.38%, p = 0.72) and timings (17.73 h in fresh IVM, 17.33 h in IVM post-VIT, p = 0.72) were observed whether IVM is performed freshly or after vitrification. Meiotic spindles were shorter in VIT + IVM (10.47 µm vs 11.23 µm in IVM and 11.40 µm in IVM + VIT, p = 0.012 and p = 0.043) and wider in IVM + VIT (9.37 µm vs 8.12 µm in IVM and 8.16 µm VIT + IVM, p = 0.027 and p = 0.026). The length-to-width ratio was lower in vitrified groups (IVM + VIT: 1.19 and VIT + IVM: 1.26) compared to IVM (1.38), p = 0.013 and p = 0.014. No differences in multipolar spindle and chromosome misalignment occurrence and cytoplasmic F-actin filament length and density were observed between groups. Our results suggest vitrification before or after rescue-IVM does not seem to impair maturation rates and kinetics parameters but induces meiotic spindle alterations.

Potential implication of new torque teno mini viruses in parapneumonic empyema in children.

An unexplained increase in the incidence of parapneumonic empyema (PPE) in pneumonia cases has been reported in recent years. The present study investigated the genetic and biological specifications of new isolates of torque teno mini virus (TTMV) detected in pleural effusion samples from children hospitalised for severe pneumonia with PPE. A pathogen discovery protocol was applied in undiagnosed pleural effusion samples and led to the identification of three new isolates of TTMV (TTMV-LY). Isolated TTMV-LY genomes were transfected into A549 and human embryonic kidney 293T cells and viral replication was assessed by quantitative real-time PCR and full-length genome amplification. A549 cells were further infected with released TTMV-LY virions and the induced-innate immune response was measured by multiplex immunoassays. Genetic analyses of the three TTMV-LY genomes revealed a classic genomic organisation but a weak identity (<64%) with known sequences. We demonstrated the in vitro replication of TTMV-LY in alveolar epithelial cells and the effective release of infectious viral particles. We also showed a selective production of inflammatory mediators in response to TTMV infection. This study reports the description of replicative TTMV-LY isolated from parapneumonic effusions of children hospitalised with PPE, suggesting a potential role of the virus in the pathogenesis of pneumonia.

Freezing Does Not Alter Sperm Telomere Length despite Increasing DNA Oxidation and Fragmentation.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

Protein transfer into human cells by VSV-G-induced nanovesicles.

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic, nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors, allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this, gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents.

Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality.

BACKGROUND: Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with ("H+" arm) or without ("H-" arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. RESULTS: Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and +  21.6% respectively, p < 0.005) than spermatozoa from the control "H-" arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (- 16.1%, p < 0.05), DNA fragmentation (- 18.7%, p < 0.05) and nuclear vacuolization (- 20.8%, p < 0.05). CONCLUSION: Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. TRIAL REGISTRATION: Clinical Trial, NCT04011813 . Registered 19 May 2019 - Retrospectively registered.

RéSUMé: CONTEXTE: Bien que largement utilisée, la congélation lente modifie considérablement les fonctions des spermatozoïdes humains. La cryoconservation induit des altérations nucléaires du sperme et une cryocapacitation, réduisant les chances de grossesse. L'hypotaurine est. naturellement présente dans les voies génitales masculines et féminines et possède des propriétés capacitantes, osmotiques et anti-oxydantes. Les mesures ont été réalisées sur le reliquat de sperme d'hommes avec des paramètres spermatiques normaux (n = 19) ou anormaux (n = 14). Les spermatozoïdes ont été sélectionnés par centrifugation sur gradient de densité (test de migration survie) avant congélation lente. Pour chaque prélèvement, ces étapes ont été réalisées en parallèle avec des milieux supplémentés en hypotaurine (bras « H+ ¼) ou sans hypotaurine (bras « H- ¼). Après décongélation, nous avons mesuré la mobilité totale et progressive, la vitalité, l'intégrité de l'acrosome, des marqueurs de la voie de signalisation de la capacitation et la qualité nucléaire. Pour cette dernière, nous nous sommes concentrés sur la condensation de la chromatine, la fragmentation de l'ADN et la présence de vacuoles dans le noyau du sperme. RéSULTATS: Post-décongélation, les spermatozoïdes sélectionnés et congelés en présence d'hypotaurine avaient une vitalité plus élevée (+ 16,7%, p < 0,001), une motilité progressive et totale (+ 39,9% et + 21,6% respectivement, p < 0,005) que les spermatozoïdes du bras « H- ¼ sans suplémentation. L'hypotaurine a également réduit la phosphorylation non spécifique des marqueurs protéiques de capacitation P110 et P80 (p < 0,01), indiquant une diminution de la cryocapacitation. La supplémentation en hypotaurine a réduit la décondensation de la chromatine, mesurée par la chromomycine A3 (− 16,1%, p < 0,05), la fragmentation de l'ADN (− 18,7%, p < 0,05) et la vacuolisation nucléaire (− 20,8%, p < 0,05). CONCLUSION: Notre étude est. la première à démontrer les effets bénéfiques de la supplémentation en hypotaurine dans les milieux de préparation et de congélation sur la capacité de fécondation des spermatozoïdes humains et leur qualité nucléaire. La supplémentation en hypotaurine a limité la cryocapacitation, augmenté la proportion de spermatozoïdes vivants et progressivement mobiles et réduit le pourcentage de spermatozoïdes présentant une décondensation de la chromatine, une fragmentation de l'ADN et une vacuolisation nucléaire. ENREGISTREMENT DE L'ESSAI: essai clinique, NCT04011813 . Enregistré le 19 mai 2019 - Enregistré rétrospectivement.

High-density lipoprotein phospholipids interfere with dendritic cell Th1 functional maturation.

Lipoproteins are both lipid carriers in the blood and regulators of essential biological processes. Several studies demonstrated that lipoproteins modified during pathological conditions could alter dendritic cell (DC) maturation. Here the immune function of non-pathological lipoproteins is addressed by analysing their impact on human DC maturation triggered by TLR ligands. Upon TLR4 stimulation, low- and high-density lipoproteins (LDL and HDL) strongly inhibited the ability of DC to induce a Th1 response of T cells, characterized by high levels of IFNγ secretion, whereas the effect of very low-density lipoprotein was subject to variations. HDL also inhibited the Th1 function of DC stimulated by TLR1/2 and TLR2/6 ligands. The phospholipid fraction from HDL retained the inhibitory activity of the lipoprotein. We identified the 1-palmitoyl-2-linoleyl-phosphatidylcholine (PLPC) as one active phospholipid that inhibited the Th1 function of mature DCs whereas the dipalmitoyl-phosphatidylcholine had no significant effect. The treatment of DC by PLPC, 24h before TLR4 stimulation, resulted in reduced activation of NF-κB. This study shows that some HDL phospholipids have a direct immunoregulatory function, by modulating DC ability to activate a Th1 response of T cells.

Human group X secreted phospholipase A2 induces dendritic cell maturation through lipoprotein-dependent and -independent mechanisms.

OBJECTIVE: Increased secreted phospholipase A(2) (sPLA(2)) activity has been documented in several inflammatory disorders. Among sPLA(2)s, the human group X (hGX)-sPLA(2) has the highest catalytic activity towards phosphatidylcholine (PC), the major phospholipid of cell membranes and blood lipoproteins. hGX-sPLA(2) has been detected in human atherosclerotic lesions, indicating that sPLA(2)s are an important link between lipids and inflammation, both involved in atherosclerosis. The presence of dendritic cells (DC), the most potent antigen presenting cells, in atherosclerotic lesions has raised the question about their role in disease progression. METHODS AND RESULTS: In this study, we show that hGX-sPLA(2)-treated LDL induces human monocyte-derived DC maturation, resulting in a characteristic mature DC phenotype and enhanced DC ability to activate IFNγ secretion from T cells. hGX-sPLA(2) phospholipolysis of LDL produces high levels of lipid mediators, such as lysophosphatidylcholine (LPC) and free fatty acids (FFAs), which also modulate DC maturation. The major molecular species of LPC containing a palmitic or stearic acid esterified in the sn-1 position induce DC maturation, whereas the FFAs can positively or negatively modulate DC maturation depending on their nature. hGX-sPLA(2) added alone can also activate DC in vitro through the hydrolysis of the DC membrane phospholipids leading, however, to a different cytokine profile secretion pattern than the one observed with hGX-sPLA(2)-phospholipolysed LDL. CONCLUSION: hGX-sPLA(2) secreted in inflamed tissues can contribute to local DC maturation, resulting in pro-Th1 cells, through the production of various lipid mediators from hydrolysis of either LDL and/or cell plasma membrane.

Viral and bacterial pathogens identification in children hospitalised for severe pneumonia and parapneumonic empyema.

Pneumonia is caused by respiratory bacteria and/or viruses. Little is known if co-infections are an aggravating factor in hospitalised children with severe pneumonia. We studied the impact of respiratory pathogens on the severity of pneumonia. Between 2007 and 2009, 52 children hospitalised with a well-documented diagnosis of community-acquired pneumonia (CAP), with or without parapneumonic empyema (PPE), were enrolled in the study. The patients were classified into 2 groups: CAP + PPE (n = 28) and CAP (n = 24). The identification of respiratory viruses and bacteria in nasopharyngeal aspirates and pleural effusion samples were performed using conventional bacterial techniques and molecular assays. Using real-time multiplex PCR and antigen detection, Streptococcus pneumoniae was the main agent identified in 76% of the cases by molecular tests and BinaxNOW® in pleural fluid. A total of 8% of pleural fluid samples remained undiagnosed. In nasopharyngeal aspirates, rhinovirus, parainfluenza viruses, human metapneumovirus, and respiratory syncytial virus were detected in both CAP and CAP + PPE populations; however, the percentage of viral co-detection was significantly higher in nasopharyngeal aspirates from CAP + PPE patients (35%) compared with CAP patients (5%). In conclusion, viral co-detection was observed mainly in patients with more severe pneumonia. Molecular biology assays improved the pathogens detection in pneumonia and confirmed the S. pneumoniae detection by BinaxNOW® in pleural effusion samples. Interestingly, the main S. pneumoniae serotypes found in PPE are not the ones targeted by the heptavalent pneumococcal conjugate vaccine.

Th1 disabled function in response to TLR4 stimulation of monocyte-derived DC from patients chronically-infected by hepatitis C virus.

BACKGROUND: Lack of protective antibodies and inefficient cytotoxic responses are characteristics of chronic hepatitis C infection. A defect in dendritic cell (DC) function has thus been suspected, but this remains a controversial issue. METHODS AND FINDINGS: Here we show that monocyte-derived DC (MoDC) from chronically-infected patients can mature in response to TLR1/2, TLR2/6 or TLR3 ligands. In contrast, when stimulated with the TLR4 ligand LPS, MoDC from patients show a profound defect in inducing IFNgamma secretion by allogeneic T cells. This defect is not due to defective phenotypic maturation or to the presence of HCV-RNA in DC or monocytes but is correlated to reduced IL-12 secretion by DC. Restoration of DC ability to stimulate IFNgamma secretion can be obtained by blocking MEK activation in DC, indicating that MEK/ERK pathway is involved in the Th1 defect of MoDC. Monocytes from HCV patients present increased spontaneous secretion of cytokines and chemokines, especially MIP-1beta. Addition of MIP-1beta on healthy monocytes during differentiation results in DC that have Th1 defect characteristic of MoDC from HCV patients, suggesting that MIP-1beta secretion by HCV monocytes participates in the Th1 defect of DC. CONCLUSIONS: Our data indicate that monocytes from HCV patients are activated in vivo. This interferes with their differentiation into DC, leading to deficient TLR4 signaling in these cells that are enable to induce a Th1 response. This specific defect is linked to the activation of the MEK/ERK pathway.

Differential induction of gene promoter constructs by constitutively active human TLRs.

Antigen presenting cells can sense microorganisms through activation of members of the Toll like receptor family (TLRs), which initiate signals leading to transcription of many inflammation-associated genes. TLRs and IL-1R, through their TIR domains, activate NFkappaB and mitogen-activated protein kinase pathways and upregulate a set of specific target genes. Recent evidence points to several differences in signaling pathways activated by individual TLRs. To evaluate the basic signaling potential of individual TIR signaling domains, we generated constitutively active versions of all known human TLRs by fusing mouse CD4 extracellular portion with the TLR transmembrane and TIR domains. A panel of promoters from genes known to be activated by TLRs as well as artificial promoter constructs with transcription factor binding sites were selected to measure their response in the presence of constitutively active CD4TLR fusion molecules. These studies show for the first time that a unique panel of promoters appears to be highly induced by CD4TLR1, 6 (TLRs that usually function through heterodimerisation with TLR2), and CD4TLR10. We also observed that CD4TLR4 is the most potent gene activator compared to all other ten human TLRs. Preliminary analyses of several promoter deletions showed that TLRs use different sequence elements to activate these reporters. In addition, since different ligands for a single TLR (e.g., TLR9) can induce different pathways, the CD4TLR fusions seem to activate all the pathways and therefore can be used to assess the overall signaling capacity of a given TLR. Finally, analysis of promoter constructs induced by the only orphan TLR, TLR10, allowed the identification of the ENA78 promoter as a tool for screening its ligands.

Infantile-onset ascending hereditary spastic paralysis is associated with mutations in the alsin gene.

We studied 15 patients, from 10 families, who presented with severe spastic paralysis with an infantile onset and an ascending progression. Spastic paraplegia began during the first 2 years of life and extended to upper limbs within the next few years. During the first decade of life, the disease progressed to tetraplegia, anarthria, dysphagia, and slow eye movements. Overall, the disease was compatible with long survival. Signs of lower motor-neuron involvement were never observed, whereas motor-evoked potentials and magnetic resonance imaging demonstrated a primitive, pure degeneration of the upper motor neurons. Genotyping and linkage analyses demonstrated that this infantile-onset ascending hereditary spastic paralysis (IAHSP) is allelic to the condition previously reported as juvenile amyotrophic lateral sclerosis at the ALS2 locus on chromosome 2q33-35 (LOD score 6.66 at recombination fraction 0). We analyzed ALS2, recently found mutated in consanguineous Arabic families presenting either an ALS2 phenotype or juvenile-onset primary lateral sclerosis (JPLS), as a candidate gene. In 4 of the 10 families, we found abnormalities: three deletions and one splice-site mutation. All the mutations lead to a truncated alsin protein. In one case, the mutation affected both the short and the long alsin transcript. In the six remaining families, absence of cDNA ALS2 mutations suggests either mutations in regulatory ALS2 regions or genetic heterogeneity, as already reported in JPLS. Alsin mutations are responsible for a primitive, retrograde degeneration of the upper motor neurons of the pyramidal tracts, leading to a clinical continuum from infantile (IAHSP) to juvenile forms with (ALS2) or without (JPLS) lower motor-neuron involvement. Further analyses will determine whether other hereditary disorders with primitive involvement of the central motor pathways, as pure forms of spastic paraplegia, could be due to alsin dysfunction.