The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5'-flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75-fold higher LacZ expression levels upon plasmid transfection into GFAP-expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell lines were infected with replication-deficient adenoviral vectors, the enhanced promoters gave LacZ expression levels that were approximately 10-fold higher than those with the parental gfa2 promoter, while retaining specificity for GFAP-expressing cells. Injection of the adenoviral vectors carrying the enhanced promoters into nude mouse brain showed that LacZ expression was limited to GFAP-positive cells. We conclude that gfa2 enhanced promoters are useful for production of short-term, glia-specific, high expression levels of genes in an adenoviral context. Adenoviral vectors containing these enhanced promoters may be useful in glioma gene therapy.
Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.
Glial fibrillary acidic protein (GFAP) is an intermediate filament protein abundantly expressed in malignant gliomas. We have constructed a novel oncolytic adenovirus, Ad5-gfa2(B)3-E1, for treatment of these tumors. In this construct, the E1 region is under control of the tissue-specific GFAP promoter (gfa2) with three additional copies of the glial specific 'B' enhancer. Infection of a GFAP-positive cell line with Ad5-gfa2(B)3-E1 resulted in E1A and E1B expression at 75% and 30% of the levels obtained after wtAd5 infection. Q-PCR showed that Ad5-gfa2(B)3-E1 replicated 4.5 times more efficiently in the GFAP-positive than in the GFAP-negative cell lines. Cell viability assays showed efficient elimination of GFAP-positive cells by Ad5-gfa2(B)3-E1, in some cell lines as efficiently as wtAd5, while the elimination was attenuated in GFAP-negative cell lines. When tested in human tumor xenografts in nude mice, Ad5-gfa2(B)3-E1 effectively suppressed the growth of GFAP-positive SNB-19 glial tumors but not of GFAP-negative A549 lung tumors. In Ad5-gfa2(B)3-E1, the E3 region was deleted to create space for future insertion of heterologous therapeutic genes. Experiments with dl7001, an E3-deleted variant of wtAd5, confirmed that the specificity of Ad5-gfa2(B)3-E1 replication was based on the promoter driving E1 and not on the E3 deletion. Strategies to further improve the efficacy of Ad5-gfa2(B)3-E1 for the treatment of malignant gliomas include the insertion of therapeutic genes in E3 or retargeting to receptors that are more abundantly expressed on primary glioma cells than CAR.
BackgroundA relatively new uncalibrated arterial pressure waveform cardiac output (CO) measurement technique is the Pulsioflex-ProAQT® system. Aim of this study was to validate this system in cardiac surgery patients with a specific focus on the evaluation of a difference in the radial versus the femoral arterial access, the value of the auto-calibration modus and the ability to show fluid-induced changes.MethodsIn twenty-five patients scheduled for ascending aorta, aortic arch replacement, or both we measured CO simultaneously by transpulmonary thermodilution (COtd) and by using the ProAQT® system connected to the radial (COpR), as well as the femoral artery catheter (COpF). Hemodynamic data were assessed at predefined time points; from incision until 16 h after ICU admission.ResultsIn total 175 (radial) and 179 (femoral) pairs of CO measurement were collected. The accuracy of COpR/COpF was evaluated showing a mean bias of −0.31 L/min (±2.9 L/min) and -0.57 L/min (± 2.8 L/min) with percentage errors of 49 and 46% respectively. Trending ability of the ProAQT® device was evaluated; the four quadrant concordance rates in the radial and femoral artery were 74 and 75% and improved to 77 and 85% after auto-calibration. The mean angular biases in the radial and femoral artery were 6.4° and 6.0° and improved to 5° and 3.3° after auto-calibration. The polar concordance rates in the radial and femoral artery were 65 and 70% and improved to 76 and 84% after auto-calibration. Considering the fluid-induced changes in stroke volume(SV), the coefficient of correlation between the changes in SVtd and SVp was 0.57 (p < 0.01) in the radial artery and 0.60 (p < 0.01) in the femoral artery.ConclusionsThe ProAQT® system can be of additional value if the clinician wants to determine fluid responsiveness in cardiac surgery patients. However, the ProAQT® system provided inaccurate CO measurements compared to transpulmonary thermodilution. The trending ability was poor for COpR but moderate for COpF. Auto-calibration of the system did not improve accuracy of CO measurements nor did it improve the prediction of fluid responsiveness. However, the trending ability was improved by auto-calibration, possibly by correcting a drift over a longer time period.
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