One-point population analysis and effect of osmolarity on detection of hetero-vancomycin-resistant Staphylococcus aureus

The original screening test and one-point population analysis (OPPA) were compared for the detection of hetero-vancomycin-resistant Staphylococcus aureus, and the influence of osmolarity on those tests was evaluated. The positivity rates and the reproducibilities were similar for both tests. The addition of NaCl increased the MIC as well as the positivity rates for both tests and the reproducibility of OPPA.     

Efficacy of vancomycin-beta-lactam combinations against heterogeneously vancomycin-resistant Staphylococcus aureus (hetero-VRSA)

There have been conflicting data about the interactions between vancomycin and beta-lactam agents against Staphylococcus aureus strains with heterogeneous resistance to vancomycin. We evaluated the efficacy of these combinations against Mu 3 and heterogeneously vancomycin-resistant S. aureus (hetero-VRSA) strains which were isolated from Korean patients using a population analysis method. Antagonistic effects were observed when less than 1 g/mL of beta-lactam antibiotics was combined with vancomycin, whereas synergistic effects were noticed with more than 4 microgram/mL of beta-lactam antibiotics. The antagonistic effects at low concentrations of beta-lactams were most prominent at 2 microgram/mL of vancomycin, which were the vancomycin MICs of tested hetero-VRSA strains. This study showed the variable effects of vancomycin- beta-lactam combinations depending on the concentrations of beta-lactam antibiotics and this property could be used to develop screening methods for hetero-VRSA strains.     

Impact of booster vaccination on the control of COVID-19 Delta wave in the context of waning immunity: application to France in the winter 2021/22

Europe has experienced a large COVID-19 wave caused by the Delta variant in winter 2021/22. Using mathematical models applied to Metropolitan France, we find that boosters administered to ≥ 65, ≥ 50 or ≥ 18 year-olds may reduce the hospitalisation peak by 25%, 36% and 43% respectively, with a delay of 5 months between second and third dose. A 10% reduction in transmission rates might further reduce it by 41%, indicating that even small increases in protective behaviours may be critical to mitigate the wave.     

Improved gene transfer into canine hematopoietic repopulating cells using CD34-enriched marrow cells in combination with a gibbon ape leukemia virus-pseudotype retroviral vector.

We have used dogs to study gene transfer into hematopoietic stem cells, because of the applicability of results in dogs to human transplantation and the availability of canine disease models that mimic human diseases. Previously we reported successful gene transfer into canine marrow repopulating cells, however, gene transfer efficiency was low, usually below 0.1% (Kiem et al, Hum Gene Ther 1996; 7: 89). In this study we have used CD34-enriched marrow cells to study different retroviral pseudotypes for their ability to transduce canine hematopoietic repopulating cells. Cells were divided into two equal fractions that were cocultivated for 72 h with irradiated packaging cells producing vector with different retroviral pseudotypes (GALV, amphotropic or 10A1). The vectors used contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into four dogs after a myeloablative dose of 920 cGy total body irradiation. Polymerase chain reaction (PCR) analysis of DNA from peripheral blood and marrow after transplant showed that the highest gene transfer rates (up to 10%) were obtained with the GALV-pseudotype vector. Gene transfer levels have remained stable now for more than 18 months. Southern blot analysis confirmed the high gene transfer rate. Interference studies on canine D17 cells revealed that 10A1 virus behaved like an amphotropic virus and was not able to use the GALV receptor. In summary, our results show improved gene transfer into canine hematopoietic repopulating cells when CD34-enriched cells are transduced by cocultivation on a GALV-pseudotype packaging cell line in combination with a GALV-pseudotype vector. Furthermore, these results demonstrate that the monoclonal antibody to canine CD34 used in this study is able to enrich for hematopoietic repopulating cells.     

Highly efficient gene transfer into preterm CD34 hematopoietic progenitor cells

OBJECTIVE: Retrovirus-mediated gene transfer has been shown to transduce CD34(+) cells from term gestation umbilical cord blood with relatively high efficiency. The purpose of this study was to compare the efficiencies of retrovirus-mediated gene transfer into early (23-28 weeks' gestation) and term (37-41 weeks' gestation) umbilical cord blood CD34(+) hematopoietic progenitor cells. STUDY DESIGN: CD34(+) cells were purified from cyropreserved early (23-28 weeks' gestation) and term (37-40 weeks' gestation) umbilical cord blood specimens with fluorescence-activated cell sorting. The CD34(+) cells were then transduced in virus-containing medium (gibbon ape leukemia virus pseudotype vector LAPSN [PG13]) in wells coated with the recombinant human fibronectin fragment CH-296 and in the presence of multiple hematopoietic growth factors (interleukin 6, stem cell factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and megakaryocyte growth and development factor) and protamine sulfate. The LAPSN (PG13) virus-containing medium was changed every 12 hours for 48 hours, after which time colony-forming cells were assayed in soft agar. The gibbon ape leukemia virus pseudotype vector LAPSN (PG13) contains the human placental alkaline phosphatase and neomycin phosphotransferase (neo ) genes. The efficiency of gene transfer was assessed by histochemical staining of colony-forming cells in agar for expression of heat-stable alkaline phosphatase. RESULTS: Gene transfers, as assessed by alkaline phosphatase staining of colony-forming cells (granulocyte-macrophage colony-forming units and erythroid burst-forming units), were similar for CD34(+) hematopoietic progenitor cells from early (58.4% +/- 11.8%) and term (63.2% +/- 12.5%) gestation fetal umbilical cord blood. CONCLUSION: CD34(+) hematopoietic progenitor cells from midgestation fetal blood can be transduced with high efficiency using techniques optimized for postnatal samples with a gibbon ape leukemia virus pseudotype vector. The early fetus may be a preferable target for gene therapy because of the higher number of circulating CD34(+) and CD38(-) cells relative to term cord blood, their greater proliferative capacity, and the rapid expansion of the fetal hematopoietic system that occurs from the second trimester to delivery. Because in vitro studies of gene transfer into hematopoietic progenitor cells and long-term culture-initiation cells have not been predictive of the efficiency of gene transfer into marrow-repopulating cells in vivo, studies that examine clinically applicable approaches to in utero gene therapy in appropriate animal models are still needed.     

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol

The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.     

Direct comparison of steady-state marrow, primed marrow, and mobilized peripheral blood for transduction of hematopoietic stem cells in dogs

The optimal stem cell source for stem cell gene therapy has not been defined. Most gene transfer studies have used peripheral blood or marrow repopulating cells collected after administration of granulocyte colony-stimulating factor and stem cell factor (G-CSF/SCF). For clinical applications, however, growth factor administration may not be feasible. Thus, in the current study we used a competitive repopulation assay in the dog to directly compare transduction efficiency of steady-state marrow, G-CSF/SCF-primed marrow, and G-CSF/SCF-mobilized peripheral blood. Cells from all three sources were transduced, cryopreserved, and thawed together before infusion into myeloablated dogs. Gene marking in hematopoietic repopulating cells was assessed by polymerase chain reaction. While primed marrow resulted in the highest long-term marking levels, steady-state marrow was transduced at least as efficiently as mobilized peripheral blood in all three dogs. These results suggest that steady-state marrow may be an appropriate source for genetic modification of hematopoietic stem cells.     

Overexpression of glutathione-S-transferase, MGSTII, confers resistance to busulfan and melphalan

A major obstacle to hematopoietic gene therapy is the lack of appropriate in vivo selection protocols that can raise the presently low numbers of gene-altered stem cells to therapeutically useful levels. Overexpression of glutathione-S-transferases (GST), in combination with busulfan treatment, may provide an exploitable selection mechanism for hematopoietic gene therapy strategies. GST provides a major route of detoxification of a variety of xenobiotics, including alkylating agents used for myeloablative chemotherapy. The only known route of clearance of busulfan is by GST-mediated conjugation. Using a fibroblast cell line as a model, we have tested the effects of overexpression of three human GST (GSTA1, GSTP1, and MGSTII) on cell survival under a busulfan or melphalan challenge. In two separate assay formats using chronic exposure to busulfan, MGSTII conferred a reproducible twofold selective advantage. GSTA1 and GSTP1 had no effect on busulfan resistance, and melphalan resistance was not affected by expression of any of the GSTs in these assays. In an acute (24-hour) melphalan exposure assay, MGSTII conferred about a twofold selective advantage. Busulfan was not toxic in this assay. RTPCR analysis of human bone marrow CD34+ cells showed that MGSTII is not highly expressed in this stem/early progenitor population. These data indicate that MGSTII may be a useful selective agent in hematopoietic gene therapy.