Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice

The MDR1 (multidrug resistance) gene, transferred to hematopoietic cells, is expected to protect them from anticancer chemotherapy and may serve as a selectable marker, restoring gene expression in vivo. Appropriate selection strategies, however, need to be established. To investigate whether preselection ex vivo affects chemoresistance, murine bone marrow cells were retrovirally transduced with high-titer or, as a model for suboptimal gene expression, low-titer retroviruses and exposed to daunomycin or colchicine for 48-96 h. Selection significantly increased chemoresistance of clonogenic progenitor cells. In tissue culture, the entire target population was rendered highly drug resistant after MDR1 transfer with high-titer viruses. If transduction was performed under suboptimal conditions, drug selection increased the frequency of chemoresistant colonies up to 40% over the number of unselected cells. Colchicine and daunomycin were equally efficient in increasing drug resistance ex vivo, but colchicine-preselected cells rescued lethally irradiated mice under conditions where daunomycin-selected bone marrow cells failed to do so. Hence, while hematopoietic cells can be protected by MDR1, the selection strategy is critical for repopulation of bone marrow with transduced cells. Preselection in culture before transplantation significantly increased P-gp expression and chemoresistance in vivo in mice reconstituted with transduced bone marrow cells. This study may help to facilitate the use of MDR1 as a selectable marker in gene therapy of the hematopoietic system. Gene Therapy (2000) 7, 348-358.     

Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources.

Tumor contamination of bone marrow (BM) and peripheral blood (PB) may affect the outcome of patients receiving high dose chemotherapy with autologous transplantation of hematopoietic stem cell products. In this report, we demonstrate that replication defective adenoviral vectors containing the cytomegalovirus (CMV) or DF3/MUC1 carcinoma-selective promoter can be used to selectively transduce contaminating carcinoma cells. Adenoviral-mediated reporter gene expression in breast cancer cells was five orders of magnitude higher than that found in BM, PB, and CD34+ cells. Our results demonstrate that CD34+ cells have low to undetectable levels of integrins responsible for adenoviral internalization. We show that adenoviral-mediated transduction of a reporter gene can detect one breast cancer cell in 5 x 10(5) BM or PB cells with a vector containing the DF3/MUC1 promoter. We also show that transduction of the HSV-tk gene for selective killing by ganciclovir can be exploited for purging cancer cells from hematopoietic stem cell populations. The selective expression of TK followed by ganciclovir treatment resulted in the elimination of 6-logs of contaminating cancer cells. By contrast, there was little effect on CFU-GM and BFU-E formulation or on long term culture initiating cells. These results indicate that adenoviral vectors with a tumor-selective promoter provide a highly efficient and effective approach for the detection and purging of carcinoma cells in hematopoietic stem cell preparations.     

Intrathecal injection of helper-dependent adenoviral vectors results in long-term transgene expression in neuroependymal cells and neurons

Helper-dependent adenoviral (HDAd) vectors are devoid of all viral genes and result in long-term transgene expression in the absence of chronic toxicity. Because of their ability to infect post-mitotic cells, including cells of the central nervous system, HDAd vectors are particularly attractive for brain-directed gene therapy. In this study, we show that intrathecal injection of HDAd results in extensive transduction of ependymal cells and sustained expression of the transgene up to 1 year post-administration. We also demonstrate, for the first time, the ability of HDAd injected by this route of delivery to transduce neuronal cells. The transduced neuroepithelial cells can be potentially used to secrete therapeutic proteins into the cerebrospinal fluid and provide them via cross-correction to nontransduced cells. Targeting of neuronal cells and long-term transgene expression make this approach attractive for the treatment of several neurologic diseases.     

Effect of scaffold attachment region on transgene expression in retrovirus vector-transduced primary T cells and macrophages.

The scaffold attachment region of the human interferon beta gene (IFN-SAR) inserted into a retroviral vector improved transgene expression in human primary CD4+ and CD8+ T cells, and in primary monocytemacrophages. In T cells, expression of the Maloney murine leukemia virus (Mo-MuLV)-based retroviral vectors was high in activated cells but low in resting cells. Addition of the IFN-SAR sequence enhanced vector expression 2- to 10-fold, and the effect was particularly pronounced in resting T cells. In CD33+CD14+CD4+ monocyte-macrophages derived from transduced hematopoietic stem/progenitor cells (HSPCs) in vitro, the IFN-SAR enhanced vector expression three- to sixfold. We have used the IFN-SAR-containing vectors to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) rev gene. Compared with a standard retroviral vector, the IFN-SAR-containing vector was significantly (p < 0.01) more potent at inhibiting HIV-1 replication in infected CD4+ peripheral blood lymphocytes. In monocytes, however, addition of the IFN-SAR did not significantly improve antiviral efficacy. To understand better the reason for the strong effect of the SAR on antiviral efficacy in T cells we have studied the expression of HIV, Mo-MuLV, and Mo-MuLV + SAR vectors in resting and activated cells. While the expression of all three vectors was lower in resting compared with activated cells, the kinetics of the decrease in expression were fastest for the Mo-MuLV vector, followed by the HIV vector and then the Mo-MuLV + SAR vector. Thus, higher level expression of the Mo-MuLV + SAR vector relative to wild-type HIV at all stages of T cell activation is the most likely explanation for the strong antiviral efficacy. Overall, this study demonstrates the utility of the IFN-SAR sequence for achieving high-level retroviral vector expression in lymphoid and myeloid hematopoietic cells.     

Increased functional expression of transgene in primary human lymphocytes using retroviral vectors modified with IRES and splicing motifs

Genetic modification of human lymphocytes is being employed in strategies to correct enzyme deficiencies, encode cytokines and to redirect lymphocytes to antigenic targets other than those encoded by their endogenous T cell receptor. However, expression of transgenes in primary lymphocytes is generally low. Reasoning that vector modification may lead to increased transgene expression and subsequent increases in function, we have performed two retroviral vector modifications and report their effect on the functional expression in primary lymphocytes. A chimeric receptor specific for the colon carcinoma-associated antigen, EGP40, was initially incorporated into the retroviral vector LXSN. In this vector, receptor expression is driven by the Moloney murine leukemia virus LTR, and neomycin phosphotransferase expression driven by the SV40 promoter. Replacement of SV40 with an internal ribosomal entry site (IRES) increased the transgene activity of a mouse T cell line and human PBL as judged by increased cytokine release in response to antigen positive target cells. A further increase in transgene function was generated by the additional incorporation of a splice acceptor motif into the construct. Human PBL transduced with vector incorporating both IRES and intron were consistently more effective at lysing antigen positive colorectal carcinoma cells.     

Plasmid delivery in vivo from porous tissue-engineering scaffolds: transgene expression and cellular transfection.

Tissue engineering scaffolds capable of sustained plasmid release can promote gene transfer locally and stimulate new tissue formation. We have investigated the scaffold design parameters that influence the extent and duration of transgene expression and have characterized the distribution of transfected cells. Porous scaffolds with encapsulated plasmid were fabricated from poly(lactide-co-glycolide) with a gas foaming procedure, with wet granulation employed to mix the components homogeneously prior to foaming. Wet granulation enhanced plasmid incorporation relative to standard procedures and also enhanced in vivo transgene expression, possibly through the increased loading and maintenance of the scaffold pore structure. The plasmid loading regulated the quantity and duration of transgene expression, with expression for 105 days achieved at the highest dosage. Expression was localized to the implantation site, though the distribution of transfected cells varied with time. Transfected cells were initially observed at the scaffold periphery (day 3), then within the pores and adjacent to the polymer (day 17), and finally throughout the scaffold interior (day 126). Delivery of a plasmid encoding VEGF increased the blood vessel density relative to control. Correlating scaffold design with gene transfer efficiency and tissue formation will facilitate application of plasmid-releasing scaffolds to multiple tissues.     

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted expression from parvovirus B19p6 promoter in primary human hematopoietic progenitor cells

Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.     

bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells

Immunization with T cell-dependent antigens generates long-lived memory B cells and antibody-forming cells (AFCs). Both populations originate in germinal centers and, predominantly, produce antibodies with high affinity for antigen. The means by which germinal center B cells are recruited into these populations remains unclear. We have examined affinity maturation of antigen-specific B cells in mice expressing the cell death inhibitor bcl-2 as a transgene. Such mice had reduced apoptosis in germinal centers and an excessive number of memory B cells with a low frequency of V gene somatic mutation, including those mutations encoding amino acid exchanges known to enhance affinity. Despite the frequency of AFCs being increased in bcl-2-transgenic mice, the fraction secreting high-affinity antibody in the bone marrow at day 42 remained unchanged compared with controls. The inability of BCL-2 to alter selection of bone marrow AFCs is consistent with these cells being selected within the germinal center on the basis of their affinity being above some threshold rather than their survival being due to a selective competition for an antigen-based signal. Continuous competition for antigen does, however, explain formation of the memory compartment.     

Retroviral-mediated expression of the P140A, but not P140A/G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment.

O(6)-Benzylguanine (6-BG) inactivates mammalian O(6)-methylguanine DNA methyltransferase (MGMT), an important DNA repair protein that protects cells against chloroethylnitrosourea (CENU) cytotoxicity. 6-BG is being tested as an approach to treat CENU-resistant tumors that overexpress endogenous MGMT. However, in addition to restoring CENU tumor cell sensitivity, 6-BG also increases the cytotoxic effects of CENUs on hematopoietic cells. Several 6-BG-resistant human MGMT mutants have been characterized in Escherichia coli and are predicted to protect mammalian cells against the combination of 6-BG and CENU treatment in vivo. Two mutants, P140A and P140A/G156A, demonstrated 20- and 1200-fold more resistance to 6-BG depletion of MGMT activity compared with wild-type MGMT (WTMGMT). Here, we analyzed retroviral vectors that express either WTMGMT, the P140A or P140A/G156A mutant forms of MGMT. Retroviral-infected L1210 hematopoietic cells demonstrated similar levels of RNA in all transduced clones. However, the amount of MGMT protein and DNA repair activity was reduced in clones expressing the P140A/G156A mutant compared with those expressing WTMGMT or P140A. Expression of P140A was associated with a 4- to 8-fold increase in resistance to 6-BG depletion of MGMT in transduced L1210 clones and a 1, 3-bis(2-chloroethyl)-1-nitrosourea IC(50) of 50 microM (compared with 27.5 microM for WTMGMT) in primary murine hematopoietic cells. These results demonstrate the utility of screening 6-BG-resistant MGMT proteins in hematopoietic cells and provide evidence that the P140A mutant form of MGMT generates 6-BG- and CENU-resistant hematopoietic cells. Retrovirus vectors expressing this mutant may be useful in future human gene therapy trials.     

从ICU继发感染者不同标本中分离鲍曼不动杆菌耐药性分析

目的分析从ICU重症患者不同标本类型中分离出的鲍曼不动杆菌的耐药性差异,为有效预防和控制ICU内鲍曼不动杆菌感染提供依据。方法回顾性分析本院2010年1月至2014年12月住院ICU患者送检标本中分离的568株鲍曼不动杆菌临床资料。结果从ICU感染患者中分离的鲍曼不动杆菌主要来源为痰液(60.0%);药敏试验表明鲍曼不动杆菌对常用抗菌药物的耐药率呈普遍多重耐药,对替加环素、头孢哌酮/舒巴坦最敏感,耐药率小于40.0%;从痰液和无菌体液中分离的鲍曼不动杆菌对亚胺培南、美罗培南、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、环丙沙星、左氧氟沙星、阿米卡星的耐药率普遍高于血液和尿液,差异有统计学意义(P0.05)。结论鲍曼不动杆菌对常用抗菌药物多重耐药,在ICU中不同标本类型的耐药性存在差异,临床必须加强耐药性监测,治疗该菌感染时首选替加环素、头孢哌酮/舒巴坦。     

不同发育阶段拟胚体对小鼠胚胎干细胞造血分化的影响

目的 通过对拟胚体(Embryoid body, EB)在造血发育过程中三个不同发育阶段的初步探讨,以了解它们对胚胎干细胞(Embryonic stem cells, ESCs)造血分化的影响.方法 根据不同阶段拟胚体的发育特点,将拟胚体分为三个阶段:EB 3-5天、EB 6-8天以及EB 9-13天.通过形态学、流式细胞仪分析、Realtime PCR以及造血集落形成试验来分析不同阶段拟胚体在造血分化过程的特点.结果 EB培养至第5天、第7天、第10天时,每6000个ES细胞形成EB的数量分别是97、100、88个;流式细胞仪检测CD34+/Sca-1+细胞,结果分别是8.91%、9.23%、12.73%;Realtime PCR结果分别显示EB培养到第6天左右时中胚层发育最为活跃,第10天时造血发育比前两个阶段要活跃;造血集落形成试验显示EB发育至第5天、第7天以及第10天时,每1×105个EB细胞形成的集落数分别是21、26、32.结论 EB 3-5天处于分化早期,各种检测参数数值都相对比较低,适合于次级拟胚体以及爆发集落的培养;EB 6-8天中胚层发育最为活跃;EB 9-13天造血发育相对比较成熟.根据不同阶段拟胚体发育的特点,可以选择适当的拟胚体作为体外造血分化模型来满足不同研究目的 需要.     

Comparison of the expression of a mutant dihydrofolate reductase under control of different internal promoters in retroviral vectors.

To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.     

Management of lipids in primary and secondary prevention of cardiovascular diseases

Although the frequency of cardiovascular disease is declining, it remains a major present and future threat to health in the United States. The deleterious effects of abnormal blood lipid concentrations have long been recognized, but the benefit of corrective intervention in this process has only recently been demonstrated. We review the major lipid abnormalities and the available clinical therapeutic interventions. In addition, we discuss data that address the premise that reducing low-density lipoprotein cholesterol or raising high-density lipoprotein cholesterol should decrease the progression of coronary atherosclerosis, and we summarize drug trials in which clofibrate, niacin, cholestyramine, and gemfibrozil decreased coronary heart disease events. Studies that used cholestyramine and the combination of colestipol and niacin resulted in decreased progression of coronary artery disease. On the basis of early experience with lovastatin, inhibitors of hydroxymethylglutaryl-coenzyme A reductase are likely to be effective in the treatment of hypercholesterolemia. The available information on the association of low cholesterol levels and cancer suggests that low total cholesterol is a consequence rather than a cause of carcinoma. Current data strongly support the concept of vigorous intervention directed at management of lipids, both with non-pharmacologic treatment and with drug therapy, for the primary and secondary prevention of coronary atherosclerosis.     

阴道光滑念珠菌耐药性及氟康唑耐药相关基因的表达

目的 研究阴道中光滑念珠菌耐药性及氟康唑耐药相关基因的表达.方法 分离、鉴定阴道分泌物中的光滑念珠菌,并进行药敏实验.抽提光滑念珠菌总RNA,通过RT-PCR检测耐药基因CDR1、CDR2和ERG11的相对表达量.结果 光滑念珠菌对制霉菌素、两性霉素B的药物敏感性最好,5-氟胞嘧啶次之,对唑类药物的耐药率(41.00%～67.00%)较高;对氟康唑的耐药率为44.00%.RT-PCR结果显示,光滑念珠菌氟康唑耐药组CDR1、CDR2和ERG11基因高表达.结论 光滑念珠菌对常用抗真菌药物的耐药率呈上升趋势.光滑念珠菌耐药基因CDR1、CDR2和ERG11的高表达,与临床光滑念珠菌对氟康唑耐药有关.     

不同 AFP 浓度下 AFP-L3与 GPC-3在原发性肝癌中的表达分析

目的：分析在不同甲胎蛋白(AFP)浓度下,甲胎蛋白异质体3(AFP-L3)、磷脂酰蛋白聚糖-3(GPC-3)在原发性肝癌(PHC)诊断中的表达,为 PHC 的诊断提供参考。方法以240例门诊、住院患者及体检者为研究对象,检测其血清 AFP、AFP-L3及 GPC-3水平;根据血清 AFP 水平分为阴性组(0≤AFP<20 ng/mL)、低浓度组(20≤AFP<400 ng/mL)和高浓度组(AFP≥400 ng/mL),比较3组 AFP-L3和 GPC-3水平,以及 AFP-L3、GPC-3单独与联合检测对 PHC 的诊断效能。结果低浓度组、高浓度组血清 AFP-L3与 GPC-3水平均高于阴性组,且高浓度组血清 AFP-L3与 GPC-3水平高于低浓度组,差异均有统计学意义(P <0.05)。AFP-L3与 GPC-3联合检测诊断 PHC 的灵敏度、特异度和准确度分别为84.4%、95.9%、93.8%,均高于 AFP-L3、GPC-3单独检测。结论联合检测 AFP-L3和 GPC-3可提高对 PHC 的诊断灵敏度、特异度和准确度,对 PHC 诊断具有重要的临床意义。