Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral ...Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Morphine is used to treat pain in several medical conditions including cancer. Here we show that morphine, in a concentration typical of that observed in patients' blood, stimulates human ...microvascular endothelial cell proliferation and angiogenesis in vitro and in vivo. It does so by activating mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation via Gi/Go-coupled G protein receptors and nitric oxide in these microvascular endothelial cells. Other contributing effects of morphine include activation of the survival signal PKB/Akt, inhibition of apoptosis, and promotion of cell cycle progression by increasing cyclin D1. Consistent with these effects, morphine in clinically relevant doses promotes tumor neovascularization in a human breast tumor xenograft model in mice leading to increased tumor progression. These results indicate that clinical use of morphine could potentially be harmful in patients with angiogenesis-dependent cancers.
An association of the dopamine receptor D4 (DRD4) gene located on chromosome 11p15.5 and attention deficit/hyperactivity disorder (ADHD) has been demonstrated and replicated by multiple ...investigators. A specific allele the 7-repeat of a 48-bp variable number of tandem repeats (VNTR) in exon 3 has been proposed as an etiological factor in attentional deficits manifested in some children diagnosed with this disorder. In the current study, we evaluated ADHD subgroups defined by the presence or absence of the 7-repeat allele of the DRD4 gene, using neuropsychological tests with reaction time measures designed to probe attentional networks with neuroanatomical foci in D4-rich brain regions. Despite the same severity of symptoms on parent and teacher ratings for the ADHD subgroups, the average reaction times of the 7-present subgroup showed normal speed and variability of response whereas the average reaction times of the 7-absent subgroup showed the expected abnormalities (slow and variable responses). This was opposite the primary prediction of the study. The 7-present subgroup seemed to be free of some of the neuropsychological abnormalities thought to characterize ADHD.
Associations have been reported of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both attention-deficit/hyperactivity disorder and the personality trait of novelty ...seeking. This polymorphism occurs in a 48-bp tandem repeat in the coding region of DRD4, with the most common allele containing four repeats (4R) and rarer variants containing 2-11. Here we show by DNA resequencing/haplotyping of 600 DRD4 alleles, representing a worldwide population sample, that the origin of 2R-6R alleles can be explained by simple one-step recombination/mutation events. In contrast, the 7R allele is not simply related to the other common alleles, differing by greater than six recombinations/mutations. Strong linkage disequilibrium was found between the 7R allele and surrounding DRD4 polymorphisms, suggesting that this allele is at least 5-10-fold "younger" than the common 4R allele. Based on an observed bias toward nonsynonymous amino acid changes, the unusual DNA sequence organization, and the strong linkage disequilibrium surrounding the DRD4 7R allele, we propose that this allele originated as a rare mutational event that nevertheless increased to high frequency in human populations by positive selection.
The survival of cervix cancer patients is associated with their hemoglobin (Hgb) level during radiotherapy. The Southwest Oncology Group (SWOG) conducted a phase II trial to determine whether ...recombinant human erythropoietin (rHuEPO) safely corrects anemia during chemoradiotherapy for cervix cancer.
Patients had stage IIB–IVA cervix cancer and a Hgb between 8.0 and 12.5 g/dl. All patients received rHuEPO thrice weekly and oral iron starting 10–15 days before their 5-week course of whole pelvic irradiation and weekly cisplatin followed by intracavitary brachytherapy.
Fifty-three patients from 26 institutions received the protocol treatment. The mean Hgb was 10.4 ± 1.3 g/dl on the first day of rHuEPO administration (baseline), 11.0 ± 1.6 g/dl on the first day of chemoradiotherapy, 11.6 ± 1.9 g/dl at the midpoint of chemoradiotherapy, and 11.8 ± 2.2 g/dl at the end of chemoradiotherapy. The target Hgb level of 12.5 g/dl was achieved in 40% of patients (95% CI 26–56%) by the midpoint of Chemoradiotheraphy. Change in Hgb was associated with baseline serum iron (P = 0.008) and transferrin saturation (P = 0.05) levels, but not with baseline Hgb or serum ferritin, or patient age. Seven patients developed deep vein thrombosis. Two-year progression-free survival (PFS) was 43% and overall survival (OS) was 51%. Survival was significantly associated with Hgb level at the end of chemoradiotherapy, but not with the baseline Hgb level.
rHuEPO and iron gradually increased Hgb levels in anemic women with local advanced cervix cancer during chemoradiotherapy. There was a higher than expected incidence of deep vein thrombosis. The progression-free and overall survival rates were lower than reported for women with normal Hgb levels.
Age-related macular degeneration or age-related maculopathy (ARM) is a major public health issue, as it is the leading cause of irreversible vision loss in the elderly in the Western world. Using ...three diagnostic models, we have genotyped markers in 16 plausible candidate regions and have carried out a genome-wide screen for ARM susceptibility loci. A panel of 225 ARM families comprising up to 212 affected sib pairs was genotyped for 386 markers. Under our most stringent diagnostic model, the regions with the strongest evidence of linkage were on chromosome 9 near D9S301 and on 10 near D10S1230, with peak multipoint heterogeneity LOD scores (HLOD) of 1.87 and 1. 42 and peak GeneHunter-Plus non-parametric LOD scores (GHP LOD) of 1. 69 and 1.83. After expanding our initial set of families to 364 ARM families with up to 329 affected sib pairs, the linkage signal on chromosome 9 vanished, while the chromosome 10 signal decreased to a GHP LOD of about 1.0, with a SimIBD P -value of 0.008 under the broadest diagnostic model with marker D10S1236. After error filtration, the GHP LOD increased to 1.27 under our most stringent model and 1.42 under our broadest model, peaking near D10S1236. This peak was seen consistently across all three diagnostic models. Our analyses also excluded up to nine different candidate regions and identified a few other regions of potential linkage, suitable for further studies. Of particular interest was the region on chromosome 5 near D5S1480, where a reasonable candidate gene, glutathione peroxidase 3, resides.
Purpose: Selective cyclooxygenase-2 (COX-2) inhibitors may suppress carcinogenesis by both COX-2-dependent and COX-2-independent mechanisms.
The primary purpose of this study was to evaluate whether ...celecoxib or rofecoxib, two widely used selective COX-2 inhibitors,
possess COX-2-independent antitumor activity.
Experimental Design: PC3 and LNCaP human prostate cancer cell lines were used to investigate the growth inhibitory effects of selective COX-2
inhibitors in vitro . To complement these studies, we evaluated the effect of celecoxib on the growth of PC3 xenografts.
Results: COX-1 but not COX-2 was detected in PC3 and LNCaP cells. Clinically achievable concentrations (2.5-5.0 μmol/L) of celecoxib
inhibited the growth of both cell lines in vitro , whereas rofecoxib had no effect over the same concentration range. Celecoxib inhibited cell growth by inducing a G 1 cell cycle block and reducing DNA synthesis. Treatment with celecoxib also led to dose-dependent inhibition of PC3 xenograft
growth without causing a reduction in intratumor prostaglandin E 2 . Inhibition of tumor growth occurred at concentrations (2.37-5.70 μmol/L) of celecoxib in plasma that were comparable with
the concentrations required to inhibit cell growth in vitro . The highest dose of celecoxib led to a 52% reduction in tumor volume and an ∼50% decrease in both cell proliferation and
microvessel density. Treatment with celecoxib caused a marked decrease in amounts of cyclin D1 both in vitro and in vivo .
Conclusions: Two clinically available selective COX-2 inhibitors possess different COX-2-independent anticancer properties. The anticancer
activity of celecoxib may reflect COX-2-independent in addition to COX-2-dependent effects.
Background & Aims:
Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. The aim of this study was to investigate the role of CTGF in ...progression of colorectal cancer (CRC).
Methods:
Immunohistochemical staining of specimens from 119 patients with CRC was performed. Liposome-mediated transfection was used to introduce a CTGF expression vector into CRC cell lines. Transfectants were tested in invasive ability and experimental hepatic metastasis in BALB/c mice. Furthermore, a FOPflash/TOPflash reporter assay was performed to investigate CTGF on the β-catenin/T-cell factor signaling pathway.
Results:
Patients with stage II and stage III CRC whose tumors displayed high CTGF expression had a significantly higher overall survival and a disease-free advantage over patients with CRC with low CTGF expression. Alterations in the CTGF level in CRC cell lines modulated their invasive ability with an inverse correlation. In addition, a reduction in the CTGF level of CT26 cells after stable transfection with antisense CTGF resulted in increased liver metastasis in BALB/c mice. The activity of the β-catenin/T-cell factor signaling pathway and its downstream effector gene matrix metalloproteinase 7 in these CTGF-transfected cells was strongly attenuated. Blockage of matrix metalloproteinase 7 with its neutralizing antibodies inhibited increased invasiveness in antisense CTGF-transfected CT26 cells.
Conclusions:
Our results implicate CTGF as a key regulator of CRC invasion and metastasis, and it appears to be a useful and better prognosis factor for patients with stage II and stage III CRC.
The inducible prostaglandin synthase cyclooxygenase-2 (Cox-2) is overexpressed in approximately 40% of human breast cancers and at higher frequencies in preinvasive ductal carcinoma in situ (DCIS). ...Cox-2 expression is particularly associated with overexpression of human epidermal growth factor receptor 2 (HER2/neu). To definitively interrogate the role of Cox-2 in mammary neoplasia, we have used a genetic approach, crossing Cox-2-deficient mice with a HER2/neu transgenic strain, MMTV/NDL. At 20 weeks of age, mammary glands from virgin MMTV/NDL females contained multiple focal tumors, or mammary intraepithelial neoplasias, which histologically resembled human DCIS. Mammary tumor multiplicity and prostaglandin E2 (PGE2) levels were significantly decreased in Cox-2 heterozygous and knockout animals relative to Cox-2 wild-type controls. Notably, the proportion of larger tumors was decreased in Cox-2-deficient mice. HER2/neu-induced mammary hyperplasia was also substantially reduced in Cox-2 null mice. Additionally, mammary glands from Cox-2 knockout mice exhibited a striking reduction in vascularization, and expression of proangiogenic genes was correspondingly reduced. Decreased vascularization was observed both in dysplastic and normal-appearing regions of Cox-2-null mammary glands. Our data provide the first genetic evidence that Cox-2 contributes to HER2/neu-induced mammary tumorigenesis. This finding may help to explain the reduced risk of breast cancer associated with regular use of nonsteroidal anti-inflammatory drugs.
The aim of this study was to report the 2-year results of the SURTAVI (Surgical Replacement and Transcatheter Aortic Valve Implantation) trial and confirm the interim Bayesian analysis.
Transcatheter ...aortic valve replacement (TAVR) with a self-expanding valve was noninferior to surgery in patients with severe aortic stenosis and intermediate operative risk using Bayesian statistical methods. Novel Bayesian designs have been used to shorten the time to primary endpoint analysis in randomized clinical trials, although the predictive value of Bayesian analysis compared with frequentist approaches remains debated.
The SURTAVI trial randomized 1,660 patients. An interim analysis was performed 1 year after the 1,400th patient was treated to estimate the primary 2-year endpoint of all-cause mortality or disabling strokes for all patients.
The Kaplan-Meier rate for the complete 2-year primary endpoint was 12.7% in the TAVR group and 12.6% in the surgery group (0.0% difference; 95% confidence interval: −3.4% to 3.5%), compared with 12.6% with TAVR and 14.0% with surgery (−1.4% difference; Bayesian credible interval: −5.2% to 2.3%) in the interim Bayesian analysis. A comparison of individual clinical, hemodynamic, and quality-of-life endpoints using Bayesian and frequentist methods found no significant differences.
The complete analysis of all patients with aortic stenosis at intermediate risk for surgery in the SURTAVI trial confirmed the noninferiority, with respect to the frequency of all-cause mortality or disabling stroke, of TAVR to surgery, as determined in the interim Bayesian analysis. Follow-up will extend out to 10 years.
Display omitted
PDF
