Identification of Greb1l as a genetic determinant of crisscross heart in mice showing torsion of the heart tube by shortage of progenitor cells.

Despite their burden, most congenital defects remain poorly understood, due to lack of knowledge of embryological mechanisms. Here, we identify Greb1l mutants as a mouse model of crisscross heart. Based on 3D quantifications of shape changes, we demonstrate that torsion of the atrioventricular canal occurs together with supero-inferior ventricles at E10.5, after heart looping. Mutants phenocopy partial deficiency in retinoic acid signaling, which reflect overlapping pathways in cardiac precursors. Spatiotemporal gene mapping and cross-correlated transcriptomic analyses further reveal the role of Greb1l in maintaining a pool of dorsal pericardial wall precursor cells during heart tube elongation, likely by controlling ribosome biogenesis and cell differentiation. Consequently, we observe growth arrest and malposition of the outflow tract, which are predictive of abnormal tube remodeling in mutants. Our work on a rare cardiac malformation opens novel perspectives on the origin of a broader spectrum of congenital defects associated with GREB1L in humans.

Hemostasis defects underlying the hemorrhagic syndrome caused by mammarenaviruses in a cynomolgus macaque model.

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.

Rapid protection induced by a single-shot Lassa vaccine in male cynomolgus monkeys.

Lassa fever hits West African countries annually in the absence of licensed vaccine to limit the burden of this viral hemorrhagic fever. We previously developed MeV-NP, a single-shot vaccine protecting cynomolgus monkeys against divergent strains one month or more than a year before Lassa virus infection. Given the limited dissemination area during outbreaks and the risk of nosocomial transmission, a vaccine inducing rapid protection could be useful to protect exposed people during outbreaks in the absence of preventive vaccination. Here, we test whether the time to protection can be reduced after immunization by challenging measles virus pre-immune male cynomolgus monkeys sixteen or eight days after a single shot of MeV-NP. None of the immunized monkeys develop disease and they rapidly control viral replication. Animals immunized eight days before the challenge are the best controllers, producing a strong CD8 T-cell response against the viral glycoprotein. A group of animals was also vaccinated one hour after the challenge, but was not protected and succumbed to the disease as the control animals. This study demonstrates that MeV-NP can induce a rapid protective immune response against Lassa fever in the presence of MeV pre-existing immunity but can likely not be used as therapeutic vaccine.

A MOPEVAC multivalent vaccine induces sterile protection against New World arenaviruses in non-human primates.

Pathogenic New World arenaviruses (NWAs) cause haemorrhagic fevers and can have high mortality rates, as shown in outbreaks in South America. Neutralizing antibodies (Abs) are critical for protection from NWAs. Having shown that the MOPEVAC vaccine, based on a hyperattenuated arenavirus, induces neutralizing Abs against Lassa fever, we hypothesized that expression of NWA glycoproteins in this platform might protect against NWAs. Cynomolgus monkeys immunized with MOPEVACMAC, targeting Machupo virus, prevented the lethality of this virus and induced partially NWA cross-reactive neutralizing Abs. We then developed the pentavalent MOPEVACNEW vaccine, expressing glycoproteins from all pathogenic South American NWAs. Immunization of cynomolgus monkeys with MOPEVACNEW induced neutralizing Abs against five NWAs, strong innate followed by adaptive immune responses as detected by transcriptomics and provided sterile protection against Machupo virus and the genetically distant Guanarito virus. MOPEVACNEW may thus be efficient to protect against existing and potentially emerging NWAs.

Case presentation and management of Lyme disease patients: a 9-year retrospective analysis in France.

Introduction: Lyme borreliosis (LB) is the most common vector disease in temperate countries of the northern hemisphere. It is caused by Borrelia burgdorferi sensu lato complex. Methods: To study the case presentation of LB in France, we contacted about 700 physicians every year between 2003 and 2011. An anonymous questionnaire was established allowing the collection of 3,509 cases. The information collected was imported or directly entered into databases and allowed identifying variables that were validated in a multiple correspondence analysis (MCA). Results: Sixty percent of the cases were confirmed, 10% were probable, 13.5% doubtful, 10.2% asymptomatic seropositive and 6.3% were negative. The clinical manifestations reported were cutaneous (63%), neurological (26%), articular (7%), ocular (1.9%) and cardiac (1.3%). Almost all patients were treated. When focusing more particularly on confirmed cases, our studies confirm that children have a distinct clinical presentation from adults. There is a gender effect on clinical presentation, with females presenting more often with erythema migrans or acrodermatitis chronica atrophicans than males, while males present more often with neurological signs or arthritis than females. Discussion: This is the first time that a comprehensive study of suspected Lyme borreliosis cases has been conducted over several years in France. Although we were not able to follow the clinical course of patients after treatment, these results suggest the interest of refining the questionnaire and of following up a cohort of patients over a sufficiently long period to obtain more information on their fate according to different parameters.

The cnf1 gene is associated with an expanding Escherichia coli ST131 H30Rx/C2 subclade and confers a competitive advantage for gut colonization.

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by Escherichia coli, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of E. coli genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, cnf1, is preferentially distributed in four common sequence types (ST) encompassing the pandemic E. coli MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of cnf1-positive multidrug-resistant ST131 strains from subclade H30Rx/C2, accounting for a rising prevalence of cnf1-positive strains in ST131. Despite the absence of phylogeographical signals, cnf1-positive isolates segregated into clusters in the ST131-H30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same cnf1 allele. The suggested dominant expansion of cnf1-positive strains in ST131-H30Rx/C2 led us to uncover the competitive advantage conferred by cnf1 for gut colonization to the clinical strain EC131GY ST131-H30Rx/C2 versus cnf1-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of cnf1 was confirmed for another clinical strain ST131-H30Rx/C2. In addition, functional analysis of the cnf1-positive clinical strain EC131GY ST131-H30Rx/C2 and a cnf1-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-H30Rx/C2 and suggested expansion of cnf1-positive MDR isolates in subclade ST131-H30Rx/C2.

Pathogenesis of recent Lassa virus isolates from lineages II and VII in cynomolgus monkeys.

The area of Lassa virus (LASV) circulation is expanding, with the emergence of highly pathogenic new LASV lineages. Benin recently became an endemic country for LASV and has seen the emergence of a new LASV lineage (VII). The first two outbreaks in 2014 and 2016 showed a relatively high mortality rate compared to other outbreaks. We infected cynomolgus monkeys with two strains belonging to lineage II and lineage VII that were isolated from deceased patients during the 2016 outbreak in Benin. The lineage VII strain (L7) caused uniform mortality. Death was associated with uncontrolled viral replication, unbalanced inflammatory responses characterized by increased concentrations of pro- and anti-inflammatory mediators, and the absence of efficient immune responses, resembling the pathogenesis associated with the prototypic Josiah strain in monkeys. The lineage II strain (L2) showed apparently lower virulence than its counterpart, with a prolonged time to death and a lower mortality rate. Prolonged survival was associated with better control of viral replication, a moderate inflammatory response, and efficient T-cell responses. Transcriptomic analyses also highlighted important differences in the immune responses associated with the outcome. Both strains caused strong inflammation in several organs. Notably, meningitis and encephalitis were observed in the cerebral cortex and cerebellum in all monkeys, independently of the outcome. Due to their apparently high pathogenicity, emerging strains from lineage VII should be considered in preclinical vaccine testing. Lineage II would also be beneficial in pathogenesis studies to study the entire spectrum of Lassa fever severity.

Infection of Human Endothelial Cells with Lassa Virus Induces Early but Transient Activation and Low Type I IFN Response Compared to the Closely-Related Nonpathogenic Mopeia Virus.

Lassa virus (LASV), an Old World arenavirus, is responsible for hemorrhagic fevers in western Africa. The privileged tropism of LASV for endothelial cells combined with a dysregulated inflammatory response are the main cause of the increase in vascular permeability observed during the disease. Mopeia virus (MOPV) is another arenavirus closely related to LASV but nonpathogenic for non-human primates (NHPs) and has never been described in humans. MOPV is more immunogenic than LASV in NHPs and in vitro in human immune cell models, with more intense type I IFN and adaptive cellular responses. Here, we compared the transcriptomic and proteomic responses of human umbilical vein endothelial cells (HUVECs) to infection with the two viruses to further decipher the mechanisms involved in their differences in immunogenicity and pathogenicity. Both viruses replicated durably and efficiently in HUVECs, but the responses they induced were strikingly different. Modest activation was observed at an early stage of LASV infection and then rapidly shut down. By contrast, MOPV induced a late but more intense response, characterized by the expression of genes and proteins mainly associated with the type I IFN response and antigen processing/presentation. Such a response is consistent with the higher immunogenicity of MOPV relative to LASV, whereas the lack of an innate response induced in HUVECs by LASV is consistent with its uncontrolled systemic dissemination through the vascular endothelium.

The Sec61 translocon is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy characterized by the uncontrolled expansion of plasma cells in the bone marrow. While proteasome inhibitors like bortezomib efficiently halt MM progression, drug resistance inevitably develop, and novel therapeutic approaches are needed. Here, we used a recently discovered Sec61 inhibitor, mycolactone, to assess the interest of disrupting MM proteostasis via protein translocation blockade. In human MM cell lines, mycolactone caused rapid defects in secretion of immunoglobulins and expression of pro-survival interleukin (IL)-6 receptor and CD40, whose activation stimulates IL-6 production. Mycolactone also triggered pro-apoptotic endoplasmic reticulum stress responses synergizing with bortezomib for induction of MM cell death and overriding acquired resistance to the proteasome inhibitor. Notably, the mycolactone-bortezomib combination rapidly killed patient-derived MM cells ex vivo, but not normal mononuclear cells. In immunodeficient mice engrafted with MM cells, it demonstrated superior therapeutic efficacy over single drug treatments, without inducing toxic side effects. Collectively, these findings establish Sec61 blockers as novel anti-MM agents and reveal the interest of targeting both the translocon and the proteasome in proteostasis-addicted tumors.

Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry.

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of ß-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control.

Gene expression analysis in EBV-infected ataxia-telangiectasia cell lines by RNA-sequencing reveals protein synthesis defect and immune abnormalities.

BACKGROUND: Epstein-Barr virus (EBV) targets B-cells where it establishes a latent infection. EBV can transform B-cells in vitro and is recognized as an oncogenic virus, especially in the setting of immune compromise. Indeed, immunodeficient patients may fail to control chronic EBV infection, leading to the development EBV-driven lymphoid malignancies. Ataxia telangiectasia (AT) is a primary immune deficiency caused by mutations in the ATM gene, involved in the repair of double-strand breaks. Patients with AT are at high risk of developing cancers, mostly B-cell lymphoid malignancies, most of which being EBV-related. Aside from immune deficiency secondary to AT, loss of ATM function could also hinder the control of the virus within B-cells, favoring lymphomagenesis in AT patients. RESULTS: We used RNA sequencing on lymphoblastoid cell lines derived from patients with AT and healthy donors to analyze and compare both cellular and viral gene expression. We found numerous deregulated signaling pathways involving transcription, translation, oncogenesis and immune regulation. Specifically, the translational defect was confirmed in vitro, suggesting that the pathogenesis of AT may also involve a ribosomal defect. Concomitant analysis of viral gene expression did not reveal significant differential gene expression, however, analysis of EBV interactome suggests that the viral latency genes EBNA-3A, EBNA-3C and LMP1 may be disrupted in LCL from AT patients. CONCLUSION: Our data support the notion that ATM deficiency deregulates cellular gene expression possibly disrupting interactions with EBV latent genes, promoting the oncogenic potential of the virus. These preliminary findings provide a new step towards the understanding of EBV regulation and of AT pathogenesis.

Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques.

Lassa virus (LASV) is endemic in West Africa and induces a viral hemorrhagic fever (VHF) with up to 30% lethality among clinical cases. The mechanisms involved in control of Lassa fever or, in contrast, the ensuing catastrophic illness and death are poorly understood. We used the cynomolgus monkey model to reproduce the human disease with asymptomatic to mild or fatal disease. After initial replication at the inoculation site, LASV reached the secondary lymphoid organs. LASV did not spread further in nonfatal disease and was rapidly controlled by balanced innate and T-cell responses. Systemic viral dissemination occurred during severe disease. Massive replication, a cytokine/chemokine storm, defective T-cell responses, and multiorgan failure were observed. Clinical, biological, immunological, and transcriptomic parameters resembled those observed during septic-shock syndrome, suggesting that similar pathogenesis is induced during Lassa fever. The outcome appears to be determined early, as differentially expressed genes in PBMCs were associated with fatal and non-fatal Lassa fever outcome very early after infection. These results provide a full characterization and important insights into Lassa fever pathogenesis and could help to develop early diagnostic tools.

Patient-derived glioblastoma stem cells transfer mitochondria through tunneling nanotubes in tumor organoids.

Glioblastoma (GBM) is the most aggressive brain cancer and its relapse after surgery, chemo and radiotherapy appears to be led by GBM stem cells (GSCs). Also, tumor networking and intercellular communication play a major role in driving GBM therapy-resistance. Tunneling Nanotubes (TNTs), thin membranous open-ended channels connecting distant cells, have been observed in several types of cancer, where they emerge to drive a more malignant phenotype. Here, we investigated whether GBM cells are capable to intercommunicate by TNTs. Two GBM stem-like cells (GSLCs) were obtained from the external and infiltrative zone of one GBM from one patient. We show, for the first time, that both GSLCs, grown in classical 2D culture and in 3D-tumor organoids, formed functional TNTs which allowed mitochondria transfer. In the organoid model, recapitulative of several tumor's features, we observed the formation of a network between cells constituted of both Tumor Microtubes (TMs), previously observed in vivo, and TNTs. In addition, the two GSLCs exhibited different responses to irradiation in terms of TNT induction and mitochondria transfer, although the correlation with the disease progression and therapy-resistance needs to be further addressed. Thus, TNT-based communication is active in different GSLCs derived from the external tumoral areas associated to GBM relapse, and we propose that they participate together with TMs in tumor networking.

The impact of rainfall on drinking water quality in Antananarivo, Madagascar.

Low-income cities that are subject to high population pressure and vulnerable to climate events often have a low capacity to continuously deliver safe drinking water. Here we reported the results of a 32-year survey on the temporal dynamics of drinking water quality indicators in the city of Antananarivo. We analyzed the long-term evolution of the quality of the water supplied and characterized the interactions between climatic conditions and the full-scale water supply system. A total of 25,467 water samples were collected every week at different points in the supplied drinking water system. Samples were analyzed for total coliforms (TC), Escherichia coli (EC), intestinal Enterococci (IE), and Spores of Sulphite-Reducing Clostridia (SSRC). Nine-hundred-eighty-one samples that were identified as positive for one or more indicators were unevenly distributed over time. The breakpoint method identified four periods when the time series displayed changes in the level and profile of contamination (i) and the monthly pattern of contamination (ii), with more direct effects of rainfall on the quality of supplied drinking water. The modeling showed significantly different lags among indicators of bacteria occurrence after cumulative rainfall, which range from 4 to 8 weeks. Among the effects of low-income urbanization, a rapid demographic transition and the degradation of urban watersheds have gradually affected the quality of the water supplied and resulted in the more direct effects of rainfall events. We focused on the need to adopt an alternative perspective of drinking water and urban watersheds management.

A functional generalized F-test for signal detection with applications to event-related potentials significance analysis.

Motivated by the analysis of complex dependent functional data such as event-related brain potentials (ERP), this paper considers a time-varying coefficient multivariate regression model with fixed-time covariates for testing global hypotheses about population mean curves. Based on a reduced-rank modeling of the time correlation of the stochastic process of pointwise test statistics, a functional generalized F-test is proposed and its asymptotic null distribution is derived. Our analytical results show that the proposed test is more powerful than functional analysis of variance testing methods and competing signal detection procedures for dependent data. Simulation studies confirm such power gain for data with patterns of dependence similar to those observed in ERPs. The new testing procedure is illustrated with an analysis of the ERP data from a study of neural correlates of impulse control.

Vaccines inducing immunity to Lassa virus glycoprotein and nucleoprotein protect macaques after a single shot.

Lassa fever is a major threat in Western Africa. The large number of people living at risk for this disease calls for the development of a vaccine against Lassa virus (LASV). We generated live-attenuated LASV vaccines based on measles virus and Mopeia virus platforms and expressing different LASV antigens, with the aim to develop a vaccine able to protect after a single shot. We compared the efficacy of these vaccines against LASV in cynomolgus monkeys. The vaccines were well tolerated and protected the animals from LASV infection and disease after a single immunization but with varying efficacy. Analysis of the immune responses showed that complete protection was associated with robust secondary T cell and antibody responses against LASV. Transcriptomic and proteomic analyses showed an early activation of innate immunity and T cell priming after immunization with the most effective vaccines, with changes detectable as early as 2 days after immunization. The most efficacious vaccine candidate, a measles vector simultaneously expressing LASV glycoprotein and nucleoprotein, has been selected for further clinical evaluation.

IFITM proteins inhibit placental syncytiotrophoblast formation and promote fetal demise.

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.

Infection of Ixodes ricinus by Borrelia burgdorferi sensu lato in peri-urban forests of France.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. In Europe, it is transmitted by Ixodes ticks that carry bacteria belonging to the Borrelia burgdorferi sensu lato complex. The objective of this work was to explore eco-epidemiological factors of Lyme borreliosis in peri-urban forests of France (Sénart, Notre-Dame and Rambouillet). We investigated whether the introduction of Tamias sibiricus in Sénart could alter the density of infected ticks. Moreover, the density and tick infection were investigated according to the tree species found in various patches of Sénart forest. For this purpose, ticks were sampled during 3 years. In the Sénart forest, the density of nymph and adult ticks showed no significant difference between 2008, 2009 and 2011. The nymph density varied significantly as a function of the month of collection. Regarding the nymphs, a higher rate of infection and infected density were found in 2009. Plots with chipmunks (C) presented a lower density of both nymphs and adult ticks than plots without chipmunks (NC) did. A higher rate of infection of nymphs with Borrelia was seen in C plots. The prevalence of the various species of Borrelia was also found to vary between C and NC plots with the year of the collect. The presence of chestnut trees positively influenced the density of both nymphs and adults. The infected nymph density showed a significant difference depending on the peri-urban forest studied, Sénart being higher than Rambouillet. The prevalence of Borrelia species also differed between the various forests studied. Concerning the putative role that Tamias sibiricus may play in the transmission of Borrelia, our results suggest that its presence is correlated with a higher rate of infection of questing ticks by Borrelia genospecies and if its population increases, it could play a significant role in the risk of transmission of Lyme borreliosis.

Quality of life is predictive of relapse in schizophrenia.

BACKGROUND: The objective of this study was to evaluate whether quality of life (QoL), as measured by the SF36 and the Quality of Life Interview (QoLI), is predictive of relapse for patients with schizophrenia. METHODS: Using data from a multicenter cohort study conducted in France, Germany, and the United-Kingdom (EuroSC), we performed Cox proportional-hazards models to estimate the associations between QoL at baseline and the occurrence of relapse over a 24-month period, with adjustment for age; gender; positive, negative and general psychopathology PANSS factors; functioning (GAF); medication; side-effects; and compliance measures. RESULTS: Our sample consisted of 1,024 patients; 540 (53%) had at least one period of relapse, and 484 (47%) had no relapse. QoL levels were the most important features predicting relapse. We found that a higher level of QoL predicts a lower rate of relapse at 24 months: HR = 0.82 (0.74; 0.91), p < 0.001 for the SF36-Physical Composite Score; and HR = 0.88 (0.81; 0.96), p = 0.002 for the SF36-Mental Composite Score. These results were not confirmed using the QoLI: HR = 0.91 (0.81; 1.01), p = 0.083. To a lesser extent, older age, better functioning, and a higher compliance score also predict a lower rate of relapse at 24 months (HRs from 0.97 to 0.98; p < 0.05). CONCLUSIONS: QoL, as assessed by the SF36, is an independent predictor of relapse at a 24-month follow-up in schizophrenia. This finding may have implications for future use of the QoL in psychiatry. Moreover, our findings may support the development and monitoring of complementary therapeutic approaches, such as 'recovery-oriented' combined with traditional mental health cares to prevent relapse.