Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

The intravenous vancomycin prescription practices of French infectious disease specialists: A cross-sectional observational study.

INTRODUCTION: Vancomycin prescription modalities remain non-consensual. We examined and evaluated the vancomycin prescription habits of infectious disease specialists in France. METHODS: Through an anonymized online questionnaire sent to members of the French Infectious Diseases Society, detailed information on vancomycin prescription modalities was collected. RESULTS: Out of the 712 physicians contacted, 179 (25%) completed the questionnaire; 174 (97%) of them routinely prescribed intravenous vancomycin: 95 (55%) by continuous infusion only, 12 (7%) by intermittent infusion, while 67 (38%) used the two modalities. Among continuous administration users, 157 (97%) applied a loading dose of 15 mg/kg or less (n = 80, 49%), 20-25 mg/kg (n = 33, 20%), or 30 mg/kg or more (n = 45, 28%); 143 (88%) used a maintenance dosage of 30 mg/kg/day and 157 (97%) carried out drug monitoring. CONCLUSION: In France, infectious disease specialists favor continuous administration of vancomycin using a loading dose, with systematic monitoring of vancomycin serum concentrations.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease.

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction.

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies.

Precursors of CD3+CD4+CD8+ cells in the human thymus are defined by expression of CD34. Delineation of early events in human thymic development.

Studies of the most immature T cell progenitors in the human thymus have been hampered by the lack of markers and assays that define these cells. In this report we used a novel human fetal thymic organ culture system to determine the potential of T cell precursors isolated from human postnatal thymus, to differentiate into CD3+ thymocytes, and to investigate early stages of human T cell development. It was found that thymocytes that lack the markers CD3, CD4, and CD8 (triple negative [TN]) can differentiate in an allogeneic organotypic thymic culture. The capacity of TN thymocytes to differentiate was exclusively confined to the CD34+ population. CD34- TN thymocytes failed to differentiate in this system. In contrast, cloned lines of CD3- thymocytes could only be established from CD34- TN thymocytes. Five subsets of CD3- thymocytes were found with the following phenotype: CD1-TN, CD1+TN, CD1+CD4+CD8-, CD1+CD4+CD8 alpha+ beta-, and CD1+CD4+CD8 alpha beta+. These subpopulations expressed decreasing levels of CD34. The CD1-CD3- population expressed the highest levels of CD34 supporting the notion that this population is the most immature T cell precursor in the thymus, whereas the CD1+CD4+CD8 alpha+ beta+ which did not express CD34 seems to be the most mature of these CD3- populations. This notion is supported by the observations that CD34+ cells isolated from fetal liver, which differentiated into T cells in a FTOC, developed into CD3+ cells via CD1- and CD4+CD8- intermediates. Based on these data, we present a model of early stages in human intrathymic development.

Lymphoid and myeloid differentiation of fetal liver CD34+lineage- cells in human thymic organ culture.

In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott-Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.

The formation of chondrules at high gas pressures in the solar nebula.

High-precision magnesium isotope measurements of whole chondrules from the Allende carbonaceous chondrite meteorite show that some aluminum-rich Allende chondrules formed at or near the time of formation of calcium-aluminum-rich inclusions and that some others formed later and incorporated precursors previously enriched in magnesium-26. Chondrule magnesium-25/magnesium-24 correlates with [magnesium]/[aluminum] and size, the aluminum-rich, smaller chondrules being the most enriched in the heavy isotopes of magnesium. These relations imply that high gas pressures prevailed during chondrule formation in the solar nebula.

[The plague: An overview and hot topics]. / La peste : mise au point et actualités.

Plague is a bacterial zoonosis caused by Yersinia pestis, usually found in fleas and small rodents that constitute the reservoir of the disease. It is transmitted to humans by flea bite, contact with rodents or inhalation of infected droplets. There are three clinical forms: bubonic plague, pulmonary plague and septicemic plague. The usual presentation is a flu-like syndrome possibly accompanied by an inflammatory lymphadenopathy which appears after 1 to 7days of incubation. Bubonic plague has a case fatality rate of about 50% while other forms of plague are almost always fatal without treatment. Diagnosis can be confirmed by usual bacteriological techniques (Gram examination, culture) but also by serological examination, use of rapid diagnostic tests or PCR. Although aminoglycosides are traditionally regarded as the most effective treatment, fluoroquinolones or cyclins are currently recommended in France. Plague is one of the re-emerging diseases according to the WHO and Madagascar suffered in 2017 the most important plague epidemic of the 21st century with more than 2000 cases and 200 deaths. Peru and the Democratic Republic of Congo are also considered endemic areas. Public health measures and a relentless fight against poverty are the cornerstone of the control of the disease. Vaccine improvement in endemic areas may also play an important role.

Murine Ksr interacts with MEK and inhibits Ras-induced transformation.

BACKGROUND: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway. RESULTS: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity. This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation. Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. CONCLUSIONS: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.

Hemogen is a novel nuclear factor specifically expressed in mouse hematopoietic development and its human homologue EDAG maps to chromosome 9q22, a region containing breakpoints of hematological neoplasms.

We cloned a novel murine gene, designated Hemogen (hemopoietic gene), which was sequentially expressed in active hematopoietic sites and downregulated in the process of blood cell differentiation. Hemogen transcripts were specifically detected in blood islands, primitive blood cells and fetal liver during embryogenesis, and then remained in bone marrow and spleen in adult mice. Immunostaining demonstrated that Hemogen was a nuclear protein. We also identified a human homologue of Hemogen, named EDAG, which was mapped to chromosome 9q22, a leukemia breakpoint. Like Hemogen, EDAG exhibited specific expression in hematopoietic tissues and cells. Taken together, these data are consistent with Hemogen and EDAG playing an important role in hematopoietic development and neoplasms.

Cytokine-mobilized peripheral blood CD34+Thy-1+Lin- human hematopoietic stem cells as target cells for transplantation-based gene therapy.

Gene-therapy of blood-borne disorders may be best achieved using hematopoietic stem cells (HSC) which have extensive self renewal potential as well as multilineage repopulating potential as a cellular target. The human HSC, which is CD34+Thy-1+Lin- has been isolated from fetal, adult bone marrow and cytokine-mobilized peripheral blood (MPB) (1-3). Results presented in this study show that the degree of mobilization of HSC into peripheral blood of cancer patients is highly variable and that the combined use of high dose chemotherapy and GM-CSF as a mobilization strategy is superior to the use of G-CSF with regard to the mobilization of true HSC. A multistep cell isolation procedure has been developed which utilizes high speed flow-cytometric cell sorting and allows the isolation of sufficient numbers of HSC from MPB to permit their use as an hematopoietic graft for clinical transplantation. Hematopoietic stem cells isolated from MPB are capable of self-renewal and differentiation into multiple hematopoietic lineages as shown by their behavior in both in vitro and in vivo assays. Mobilized PB mononuclear cells isolated from cancer patients are frequently contaminated with tumor cells. Using this cell isolation procedure, HSC preparations from patients with multiple myeloma have been created with greatly reduced tumor cell burdens. These CD34+Thy-1+Lin- cells are capable of being stably transduced at high efficiency (32-75%) by co-culture on a cell line producing recombinant retroviruses containing the neomycin-resistant gene. These HSC cell populations are likely ideal targets for hematopoietic cell-based gene therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of L-selectin expression by a dominant negative Ikaros protein.

Ikaros family members play critical roles in hematopoietic development, yet molecules regulated by Ikaros proteins remain incompletely characterized. To determine the requirements for functional Ikaros proteins, we overexpressed Ik7, a dominant negative Ikaros protein, in human cell lines and hematopoietic progenitor cells. Ik7 is known to block the normal function of other Ikaros family members in human and mouse cells. Retroviral-mediated overexpression of Ik7 affected two distinct, migratory properties of the CEM T-cell line. Ik7 down-regulated L-selectin cell-surface expression, an effect not a result of increased shedding but of a decrease in L-selectin mRNA levels. Ik7 also reduced the spontaneous migration of CEM T cells in 3-D collagen gels. A reduction in L-selectin, cell-surface expression was also induced by Ik7 in CD34+ hematopoietic progenitor cells. In contrast, the Reh B cell line showed an up-regulation of L-selectin, cell-surface levels when expressing Ik7. For the first time, this study defines an effect of Ikaros proteins in the control of migration-related properties and shows that intact Ikaros proteins are important in a cell type-specific manner for the normal regulation of L-selectin expression.

Equal distribution of competitive long-term repopulating stem cells in the CD34+ and CD34- fractions of Thy-1lowLin-/lowSca-1+ bone marrow cells.

CD34 antigen is present on most, if not all, human hematopoietic stem cells (HSCs). Consistent with this pattern of expression, we recently reported that primitive murine HSCs defined as competitive long-term repopulating units (CRUs) are highly enriched among CD34+ bone marrow (BM) cells (one CRU/2500 cells). However, in agreement with one recent report that some murine HSCs do not express CD34 (Science 273:242), we observed that 15% of phenotypically defined Thy-1lowLin-/lowSca-1+ (TLS) stem cells were CD34- by fluorescence-activated cell sorting. To examine further the nature of CD34 expression on murine hematopoietic cells, we separated TLS cells into CD34+ (0.022% of BM cells) and CD34- (0.005% of BM cells) fractions, confirmed their phenotype by reverse transcriptase-polymerase chain reaction analysis of CD34 transcripts, and evaluated them in a variety of in vitro and in vivo assays. The CD34+ TLS population contained most (93-95%) of the day 12 spleen colony-forming units (CFU-S) and in vitro colony-forming cells (CFCs). Cobblestone area-forming cells (CAFCs) able to proliferate on a murine bone marrow stromal cell line (SyS-1) represented one of every 5 CD34+ TLS and one of every 31 CD34 TLS cells. When lethally irradiated mice were injected with 100 CD34+ TLS cells, all animals survived and began to recover circulating leukocytes, platelets, and erythrocytes by 15 days. In contrast, only 40% of mice injected with 100 CD34- TLS cells were radioprotected, and hematopoietic reconstitution in surviving mice was not apparent until 21 days. The frequency of CRUs in CD34+ and CD34 TLS cells was determined by injecting limiting numbers of cells into lethally irradiated Ly-5 congenic hosts together with 10(5) "compromised" BM cells to provide radioprotection. CRUs able to regenerate and maintain lymphoid and myeloid cells for at least 6 months in primary and 5 months in secondary hosts represented one of every 156 CD34+ TLS and one of every 35 CD34- TLS cells. However, when normalized for the proportion of TLS cells that were CD34+ or CD34-, it was determined that the recovery of CRU among CD34+ and CD34- TLS cells was equivalent (46% and 54%, respectively). These data are consistent with the previous description of repopulating HSCs among CD34-c-kit+Sca-1+Lin- cells (Science 273:242, 1996) and provide additional evidence that TLS cells are functionally heterogeneous and can be further fractionated on the basis of CD34 expression. Overall, approximately 95% of CFCs, CFU-S, and CAFCs in the TLS population were found to be CD34+, whereas more primitive CRU were distributed equally among CD34+ and CD34 TLS cells. These results should enable better characterization of the most primitive stem cells in murine BM.

High-level expression of a novel epitope of CD59 identifies a subset of CD34+ bone marrow cells highly enriched for pluripotent stem cells.

To further define the hierarchy of human hematopoietic progenitor cells, we have attempted to identify antibodies to cell-surface molecules expressed on CD34+ progenitor cell subsets. Herein we describe the utility of a new monoclonal antibody, HCC-1, which binds to a novel epitope of CD59 differentially expressed among CD34+ progenitor cells. HCC-1 subdivides the adult marrow CD34+ population into HCC-1high and HCC-1low/- fractions of approximately equal size. Cobblestone area-forming cells (CAFC) in long-term bone marrow culture were enriched 10-30-fold in CD34+HCC-1high cells compared with CD34+HCC1-low/- cells and two-fold compared with CD34+ cells. When injected into fetal human bone fragments implanted in SCID mice, the CD34+HCC-1high population showed potent engrafting activity leading to the production of myeloid, lymphoid, and erythroid elements, as well as the retention of progenitor cell phenotype. These studies demonstrate that the CD34+HCC-1high population contains primitive pluripotent hematopoietic stem cells. No hematopoietic engrafting activity was detected in the CD34+HCC-1low/- population. Consistent with this finding, simultaneous five-color flow cytometric analysis revealed that HCC-1high cells include virtually all CD34+Thy-1+Lin- cells, a cell population previously characterized as highly enriched for primitive pluripotent hematopoietic stem cells. The ability of CD34+ cells divided into subsets by HCC-1 to produce T cells was assessed by transplantation of sorted cells into human fetal thymus implanted into SCID mice. A higher frequency of thymus-engrafting activity was observed in the CD34+HCC-1high than in the CD34+HCC-1low/- population. Consistent with the limited ability to engraft in the SCID-hu thymus model, the CD34+HCC-1low/- population was shown to contain a low frequency of CD34+CD10+ lymphoid progenitor cells. We conclude that the HCC-1 epitope is expressed at high levels on a subset of CD34+ cells that contain virtually all primitive pluripotent hematopoietic stem cells and that the population of CD59 molecules expressed on CD34+ cells is not homogeneous.