The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion.

Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency.

T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7-stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34+ cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC-/- engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC-/- recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.

[Oxygen toxicity in acute care settings]. / Toxicité de l'oxygène dans les soins aigus.

Oxygen therapy is widespread in acute care settings as adequate oxygen supplementation is essential in case of hypoxemia. Excessive oxygen supplementation has several unrecognized deleterious effects. This article reviews the deleterious effects of hyperoxemia and sums up the actual recommendations for safe oxygen supplementation.

L'administration libérale d'oxygène dans les soins aigus est fréquente, mais n'est pas anodine. En cas d'hypoxémie, elle est bien sûr indispensable, mais son utilisation abusive peut résulter en une hyperoxie, avec de nombreux effets secondaires. Cet article résume les principes de l'oxygénothérapie ainsi que les effets néfastes provoqués par l'hyperoxie qui sont souvent méconnus.

Gene Therapy in Fanconi Anemia: A Matter of Time, Safety and Gene Transfer Tool Efficiency.

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive marrow failure. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential cure since it is a monogenetic disease with mutations in the FANC genes, coding for DNA repair enzymes [1]. However, the collection of hCD34+-cells in FA patients implies particular challenges because of the reduced numbers of progenitor cells present in their bone marrow (BM) [2] or mobilized peripheral blood [3-5]. In addition, the FA genetic defect fragilizes the HSCs [6]. These particular features might explain why the first clinical trials using murine leukemia virus derived retroviral vectors conducted for FA failed to show engraftment of corrected cells. The gene therapy field is now moving towards the use of lentiviral vectors (LVs) evidenced by recent succesful clinical trials for the treatment of patients suffering from adrenoleukodystrophy (ALD) [7], ß-thalassemia [8], metachromatic leukodystrophy [9] and Wiskott-Aldrich syndrome [10]. LV trials for X-linked severe combined immunodificiency and Fanconi anemia (FA) defects were recently initiated [11, 12]. Fifteen years of preclinical studies using different FA mouse models and in vitro research allowed us to find the weak points in the in vitro culture and transduction conditions, which most probably led to the initial failure of FA HSC gene therapy. In this review, we will focus on the different obstacles, unique to FA gene therapy, and how they have been overcome through the development of optimized protocols for FA HSC culture and transduction and the engineering of new gene transfer tools for FA HSCs. These combined advances in the field hopefully will allow the correction of the FA hematological defect in the near future.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent.

Hematopoietic stem cell (HSC)-based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein-displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)-LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc-/- (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering.

Surface engineering of lentiviral vectors for gene transfer into gene therapy target cells.

Since they allow gene integration into their host genome, lentiviral vectors (LVs) have strong therapeutic potentials, as emphasized by recent clinical trials. The surface-display of the pantropic vesicular stomatitis virus G glycoprotein (VSV-G) on LVs resulted in powerful tools for fundamental and clinical research. However, improved LVs are required either to genetically modify cell types not permissive to classical VSV-G-LVs or to restrict entry to specific cell types. Incorporation of heterologous viral glycoproteins (gps) on LVs often require modification of their cytoplasmic tails and ligands can be inserted into their ectodomain to target LVs to specific receptors. Recently, measles virus (MV) gps have been identified as strong candidates for LV-retargeting to multiple cell types, with the potential to evolve toward clinical applications.

Exclusive Transduction of Human CD4+ T Cells upon Systemic Delivery of CD4-Targeted Lentiviral Vectors.

Playing a central role in both innate and adaptive immunity, CD4(+) T cells are a key target for genetic modifications in basic research and immunotherapy. In this article, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4(+) cells by surface engineering. When applied to PBMCs, CD4-LV transduced CD4(+) but not CD4(-) cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMCs or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4(+) human lymphocytes. In bone marrow and spleen, memory T cells were preferentially hit. Toward therapeutic applications, we also show that CD4-LV can be used for HIV gene therapy, as well as for tumor therapy, by delivering chimeric Ag receptors. The potential for in vivo delivery of the FOXP3 gene was also demonstrated, making CD4-LV a powerful tool for inducible regulatory T cell generation. In summary, our work demonstrates the exclusive gene transfer into a T cell subset upon systemic vector administration opening an avenue toward novel strategies in immunotherapy.

Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs.

Hematopoietic stem cell (HSC)-based gene therapy holds promise for the cure of many diseases. The field is now moving toward the use of lentiviral vectors (LVs) as evidenced by 4 successful clinical trials. These trials used vesicular-stomatitis-virus-G protein (VSV-G)-LVs at high doses combined with strong cytokine-cocktail stimulation to obtain therapeutically relevant transduction levels; however, they might compromise the HSC character. Summarizing all these disadvantages, alternatives to VSV-G-LVs are urgently needed. We generated here high-titer LVs pseudotyped with a baboon retroviral envelope glycoprotein (BaEV-LVs), resistant to human complement. Under mild cytokine prestimulation to preserve the HSC characteristics, a single BaEV-LV application at a low dose, resulted in up to 90% of hCD34(+) cell transduction. Even more striking was that these new BaEV-LVs allowed, at low doses, efficient transduction of up to 30% of quiescent hCD34(+) cells, whereas high-dose VSV-G-LVs were insufficient. Importantly, reconstitution of NOD/Lt-SCID/γc(-/-) (NSG) mice with BaEV-LV-transduced hCD34(+) cells maintained these high transduction levels in all myeloid and lymphoid lineages, including early progenitors. This transduction pattern was confirmed or even increased in secondary NSG recipient mice. This suggests that BaEV-LVs efficiently transduce true HSCs and could improve HSC-based gene therapy, for which high-level HSC correction is needed for life-long cure.

Mutations in the H, F, or M Proteins Can Facilitate Resistance of Measles Virus to Neutralizing Human Anti-MV Sera.

Although there is currently no evidence of emerging strains of measles virus (MV) that can resist neutralization by the anti-MV antibodies present in vaccinees, certain mutations in circulating wt MV strains appear to reduce the efficacy of these antibodies. Moreover, it has been hypothesized that resistance to neutralization by such antibodies could allow MV to persist. In this study, we use a novel in vitro system to determine the molecular basis of MV's resistance to neutralization. We find that both wild-type and laboratory strain MV variants that escape neutralization by anti-MV polyclonal sera possess multiple mutations in their H, F, and M proteins. Cytometric analysis of cells expressing viral escape mutants possessing minimal mutations and their plasmid-expressed H, F, and M proteins indicates that immune resistance is due to particular mutations that can occur in any of these three proteins that affect at distance, rather than directly, the native conformation of the MV-H globular head and hence its epitopes. A high percentage of the escape mutants contain mutations found in cases of Subacute Sclerosing Panencephalitis (SSPE) and our results could potentially shed light on the pathogenesis of this rare fatal disease.

Virus-like particle vaccine induces cross-protection against human metapneumovirus infections in mice.

Human metapneumovirus (HMPV) is a paramyxovirus that causes acute respiratory-tract infections in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We engineered HMPV viral-like particles (HMPV-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two HMPV viruses of either lineage A or B. These VLPs functionally display F and G HMPV surface glycoproteins. When injected in mice, HMPV-VLPs induce strong humoral immune response against both homologous and heterologous strains. Moreover, the induced neutralizing antibodies prevented mortality upon subsequent infection of the lungs with both homologous and heterologous viruses. Upon challenge, viral titers in the lungs of immunized animals were significantly reduced as compared to those of control animals. In conclusion, a HMPV-VLP vaccine that induces cross-protective immunity in mice is a promising approach to prevent HMPV infections.

Lentiviral vectors displaying modified measles virus gp overcome pre-existing immunity in in vivo-like transduction of human T and B cells.

Gene transfer into quiescent T and B cells is important for gene therapy and immunotherapy approaches. Previously, we generated lentiviral vectors (LVs) pseudotyped with Edmonston (Ed) measles virus (MV) hemagglutinin (H) and fusion (F) glycoproteins (H/F-LVs), which allowed efficient transduction of quiescent human T and B cells. However, a major obstacle in the use of H/F-LVs in vivo is that most of the human population is vaccinated against measles. As the MV humoral immune response is exclusively directed against the H protein of MV, we mutated the two dominant epitopes in H, Noose, and NE. LVs pseudotyped with these mutant H-glycoproteins escaped inactivation by monoclonal antibodies (mAbs) but were still neutralized by human serum. Consequently, we took advantage of newly emerged MV-D genotypes that were less sensitive to MV vaccination due to a different glycosylation pattern. The mutation responsible was introduced into the H/F-LVs, already mutated for Noose and NE epitopes. We found that these mutant H/F-LVs could efficiently transduce quiescent lymphocytes in the presence of high concentrations of MV antibody-positive human serum. Finally, upon incubation with total blood, mimicking the in vivo situation, the mutant H/F-LVs escaped MV antibody neutralization, where the original H/F-LVs failed. Thus, these novel H/F-LVs offer perspectives for in vivo lymphocyte-based gene therapy and immunotherapy.

Measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both SLAM and CD46 entry receptors.

Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with the Edmonston measles virus (MV) hemagglutinin and fusion glycoproteins (Hgps and Fgps) (H/F-LVs), which, for the first time, allowed efficient transduction of quiescent human B and T cells. These target cells express both MV entry receptors used by the vaccinal Edmonston strain, CD46 and signaling lymphocyte activation molecule (SLAM). Interestingly, LVs pseudotyped with an MV Hgp, blind for the CD46 binding site, were completely inefficient for resting-lymphocyte transduction. Similarly, SLAM-blind H mutants that recognize only CD46 as the entry receptor did not allow stable LV transduction of resting T cells. The CD46-tropic LVs accomplished vector-cell binding, fusion, entry, and reverse transcription at levels similar to those achieved by the H/F-LVs, but efficient proviral integration did not occur. Our results indicate that both CD46 and SLAM binding sites need to be present in cis in the Hgp to allow successful stable transduction of quiescent lymphocytes. Moreover, the entry mechanism utilized appears to be crucial: efficient transduction was observed only when CD46 and SLAM were correctly engaged and an entry mechanism that strongly resembles macropinocytosis was triggered. Taken together, our results suggest that although vector entry can occur through the CD46 receptor, SLAM binding and subsequent signaling are also required for efficient LV transduction of quiescent lymphocytes to occur.

Advances in the field of lentivector-based transduction of T and B lymphocytes for gene therapy.

Efficient gene transfer into quiescent T and B lymphocytes for gene therapy or immunotherapy purposes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as immunodeficiencies, and the development of novel therapeutic strategies for cancers and acquired diseases. Lentiviral vectors (LVs) can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T and B cells. In T cells, completion of reverse transcription (RT), nuclear import, and subsequent integration of the vesicular stomatitis virus G protein pseudotyped LV (VSVG-LV) genome does not occur efficiently unless they are activated via the T-cell receptor (TCR) or by survival-cytokines inducing them to enter into the G(1b) phase of the cell cycle. Lentiviral transduction of B cells is another matter because even B-cell receptor-stimulation inducing proliferation is not sufficient to allow efficient VSVG-LV transduction. Recently, a new LV carrying the glycoproteins of measles virus (MV) at its surface was able to overcome vector restrictions in both quiescent T and B cells. Importantly, naive as well as memory T and B cells were efficiently transduced while no apparent activation, cell-cycle entry, or phenotypic switch were detected, which opens the door to a multitude of gene therapy and immunotherapy applications as reported here.

Improving anticoagulation control in hospitalized elderly patients on warfarin.

OBJECTIVES: To determine the effect of patient characteristics and of specific guidelines that were developed for managing warfarin therapy in older adults and included in an in-house computer program on anticoagulation quality. DESIGN: Thirteen-month observational study. SETTING: Acute care, extended care, and rehabilitation geriatric wards of a teaching hospital in Paris, France. PARTICIPANTS: Hospitalized patients (N=307, mean age 86.1 +/- 6.1) treated with warfarin with a therapeutic international normalized ratio range of 2.0 to 3.0. INTERVENTION: Patients were assigned according to care unit to the computer-generated dosing group (CGD) or the standard management group (SM; usual physician-based care). MEASUREMENTS: Relationships between anticoagulation quality criteria and covariates (age, sex, warfarin indication, treatment phase, follow-up duration, model of care). RESULTS: According to multivariate analysis, only model of care and follow-up duration independently influenced anticoagulation control; the proportion of time within therapeutic INR range 2.0 to 3.0 was significantly greater in the CGD group than in the SM group (59% vs 48%, P=.004). When a wider INR range was analyzed (1.8-3.2), the proportion of time within range was 73% versus 64% (P=.006). Use of the computer was associated with fewer days with INRs greater than 3, a smaller percentage of INRs of 4 or greater, a longer time to the first INR of 4.0 or greater, and a smaller mean number of INRs per month than SM (all P<.01). CONCLUSION: Initiation regimen and long-term rules that have specifically been developed and included in a computerized dosage program improve quality of anticoagulation in elderly inpatients, allowing them to benefit from a quality of care as high as that of younger ambulatory patients.

Efficient and stable transduction of resting B lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors.

Up to now, no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes, which hampers its application in gene therapy and immunotherapy areas. Here, we report that LVs incorporating measles virus (MV) glycoproteins, H and F, on their surface allowed transduction of 50% of quiescent B cells, which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover, the naive and memory phenotypes of transduced resting B cells were maintained. Importantly, H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells, B-cell chronic lymphocytic leukemia cells, blocked in G(0)/G(1) early phase of the cell cycle. Thus, H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells.

Use of the bioMérieux VIDAS troponin I ultra assay for the diagnosis of myocardial infarction and detection of adverse events in patients presenting with symptoms suggestive of acute coronary syndrome.

BACKGROUND: We demonstrate the performance of the bioMérieux VIDAS Troponin I Ultra assay for diagnostic accuracy for detection of myocardial infarction (MI) and risk stratification. METHOD: cTnI was measured in 545 patients from 2 clinical centers with symptoms suggestive of ACS at admission, with an additional specimen at 4-12 h (453 patients). The 99th percentile value (0.01 microg/l) was used to assess clinical accuracy for diagnosis of acute MI. Primary endpoint for risk stratification was first of cardiac event or death in 302 patients (one center) followed for 60 days. RESULTS: 157 (28.8%) patients ruled in for an MI during index hospitalization. Sensitivities and specificities were 88.1% (95% CI 81.9 to 92.4%) and 79.9% (CI 75.5 to 83.6%) for baseline and 100% (CI 96.5 to 100%) and 79.4% (CI 74.4 to 83.4%) for follow-up specimens. ROC curve areas increased from 0.912 (CI 0.879 to 0.944) at baseline to 0.994 (CI 0.988 to 0.999) at second sampling (n=453, p<0.01); with no differences between sites. Primary endpoint rate for the 223 patients (74%) with normal cTnI on presentation was lower than the 79 patients (26%) with cTnI>0.01 ug/l (5.9% vs. 42.3%, p<0.0001). The relative risk for the >0.01 ug/l group was 8.9 (CI 4.6 to 17). CONCLUSION: The VIDAS cTnI assay is a sensitive diagnostic method for the early detection of MI and predicts increased risk for adverse events in patients with symptoms suggestive of ACS.