Vivaria housing conditions expose sex differences in brain oxidation, microglial activation, and immune system states in aged hAPOE4 mice.

Apolipoprotein E ε4 allele (APOE4) is the predominant genetic risk factor for late-onset Alzheimer's disease (AD). APOE4 mouse models have provided advances in the understanding of disease pathogenesis, but unaccounted variables like rodent housing status may hinder translational outcomes. Non-sterile aspects like food and bedding can be major sources of changes in rodent microflora. Alterations in intestinal microbial ecology can cause mucosal barrier impairment and increase pro-inflammatory signals. The present study examined the role of sterile and non-sterile food and housing on redox indicators and the immune status of humanized-APOE4 knock-in mice (hAPOe4). hAPOE4 mice were housed under sterile conditions until 22 months of age, followed by the transfer of a cohort of mice to non-sterile housing for 2 months. At 24 months of age, the redox/immunologic status was evaluated by flow cytometry/ELISA. hAPOE4 females housed under non-sterile conditions exhibited: (1) higher neuronal and microglial oxygen radical production and (2) lower CD68+ microglia (brain) and CD8+ T cells (periphery) compared to sterile-housed mice. In contrast, hAPOE4 males in non-sterile housing exhibited: (1) higher MHCII+ microglia and CD11b+CD4+ T cells (brain) and (2) higher CD11b+CD4+ T cells and levels of lipopolysaccharide-binding protein and inflammatory cytokines in the periphery relative to sterile-housed mice. This study demonstrated that sterile vs. non-sterile housing conditions are associated with the activation of redox and immune responses in the brain and periphery in a sex-dependent manner. Therefore, housing status may contribute to variable outcomes in both the brain and periphery.

Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination.

Genetically modified dendritic cell (DC) vaccines expressing tumor-associated antigens are currently used for cancer immunotherapy. Peripheral blood (PB) monocyte precursors are a relatively convenient source of DCs for use in clinical studies, but are often contaminated by lymphocytes. The current study was conducted to examine the impact of T-lymphocyte contamination on genetically modified DC product. PB monocyte-derived DCs were efficiently transduced (75-95%) with an HIV-1-based self-inactivating lentiviral vector encoding a model antigen, the enhanced green fluorescent protein (eGFP). The lymphocyte-free DC culture transduced with Lenti-eGFP showed stable expression of eGFP without measurable decline in viability. In contrast, the eGFP-positive DCs disappeared rapidly in transduced DC cultures containing lymphocyte contaminants, concurrent with detectable activation and expansion of T-lymphocytes. Upon antigen recall, these T cells elicited major histocompatability complex-restricted antigen-specific cytotoxicity against eGFP-positive autologous DCs and mitogen-stimulated T lymphoblasts, mainly through the perforin-mediated pathway. In summary, this study demonstrate that the relative purity of DC cultures could determine the persistence of gene-modified DC, which may affect the induction of effective immune responses by DC vaccination strategies.

Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors.

Dendritic cells (DCs) are potent antigen-presenting cells and are capable of activating naive T cells. Gene transfer of tumor antigen and cytokine genes into DCs could be an important strategy for immunotherapeutic applications. Dendritic cells derived from peripheral blood monocytes do not divide and are therefore poor candidates for gene transfer by Moloney murine leukemia virus (Mo-MuLV)-based retroviral vectors. Lentiviral vectors are emerging as a powerful tool for gene delivery into dividing and nondividing cells. A three-plasmid expression system pseudotyped with the envelope from vesicular stomatitis virus (VSV-G) was used to generate lentiviral vector particles expressing enhanced green fluorescent protein (EGFP). Peripheral blood monocyte-derived DCs were cultured in the presence of GM-CSF and IL-4 and transduced with lentiviral or Mo-MuLV-based vectors expressing EGFP. FACS analysis of lentiviral vector-transduced DCs derived either from normal healthy volunteers or from melanoma patients demonstrated transduction efficiency ranging from 70 to 90% compared with 2-8% using Mo-MuLV-based vectors pseudotyped with VSV-G. Comparison of lentiviral vectors expressing EGFP driven by CMV or human PGK promoters showed similar levels of transgene expression. Lentiviral vector preparations produced in the absence of HIV accessory proteins transduced DCs at efficiencies equal to vectors produced with accessory proteins. Alu-HIV-1 LTR PCR demonstrated the genomic integration of the lentiviral vector in the transduced DCs. Transduced cells showed characteristic dendritic cell phenotype and strong allostimulatory capacity and maintained the ability to respond to activation signals such as CD40 ligand and lipopolysaccharide. These results provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in monocyte-derived DCs that could be useful for immunotherapeutic applications.

Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins.

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)-based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1-based vectors requires the presence of viral accessory proteins. (Blood. 2000;96:1309-1316)

Enhancing hemopoietic drug resistance: a rationale for reconsidering the clinical use of mitozolomide.

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.

Protection of committed murine haemopoietic progenitors against BCNU toxicity does not predict protection of primitive, multipotent spleen colony-forming cells - implications for chemoprotective gene therapy.

The effect of expression of an O6-benzylguanine (O6-beG)-resistant mutant (hATPA/GA) of human O6-alkylguanine-DNA alkyltransferase (ATase) on the in vivo toxicity and clastogenicity of the anti-tumour agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) to murine bone marrow has been investigated. When compared with control animals, the bipotent granulocyte-macrophage colony-forming (GM-CFC) progenitor population of the hATPA/GA transduced mice were somewhat more resistant to BCNU (1.4-fold, P = 0.047) and this effect was more significant in the presence of the ATase inactivator O6-beG (3. 5-fold, P = 0.001). The polychromatic erythrocytes were also significantly protected against BCNU-induced clastogenicity both in the presence (P < 0.001) and absence of O6-beG (P < 0.05). The primitive, multipotent spleen colony-forming cells (CFU-S) in these animals also showed moderate (1.6-fold, P = 0.034) protection in the absence of O6-beG but in the presence of the inactivator they remained as sensitive to BCNU toxicity as those in the control animals (P = 0.133). This result contrasts with previous findings demonstrating significant hATPA/GA-mediated, O6-beG-resistant protection against the toxicity and clastogenicity of a number of O6-alkylating agents, including temozolomide, fotemustine and chlorozotocin. The possibility that our strategy for protective gene therapy may be highly agent and cell-type specific is unexpected and has possible implications for clinical trials of this approach using BCNU or related agents.

A novel dual function retrovirus expressing multidrug resistance 1 and O6-alkylguanine-DNA-alkyltransferase for engineering resistance of haemopoietic progenitor cells to multiple chemotherapeutic agents.

Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future.