[Expression of circ-KEL in acute myeloid leukemia and its regulatory mechanisms in leukemic cells].

Objective: To explore the expression of circ-KEL in patients with acute myeloid leukemia (AML) and the effect and mechanism of circ-KEL on leukemic cells. Methods: The expression of circ-KEL was detected by quantitative real-time polymerase chain reaction in bone marrow mononuclear cells collected from 116 patients with AML and 40 healthy donors. The correlation of circ-KEL expression with the clinical characteristics of patients with AML was further systematically analyzed. The modulations among circ-KEL, miR-335-5p, and LRG1 were predicted through bioinformatics analysis and validated by dual luciferase assay. Cell proliferation and apoptosis were detected using CCK8 and flow cytometry. Results: The expression of circ-KEL was significantly elevated in patients with AML compared with the healthy controls (Relative expression level, -Δct, AML: -7.117±1.831; control: -8.669±1.771, P<0.001) . Moreover, patients with high circ-KEL expression have significantly worse overall survival. The level of circ-KEL in patients with AML was downregulated after chemo-treatment. In addition, circ-KEL could serve as the sponge of miR-335-5p and regulate LRG1. Bioinformatics analysis showed that miR-335-5p correlates with good prognosis and was negatively associated with LRG1. LRG1 could promote cell proliferation and inhibit cell apoptosis. Our results also exhibited the higher expression of LRG1 in patients with AML. Moreover, circ-KEL exerted functional effects via sponging miR-335-5p and regulating LRG1. Conclusion: circ-KEL expresses highly in patients with AML and correlates with poor prognosis, suggesting its important role in the genesis and progress of AML.

Prognostic and predictive significance of plasma hepatocyte growth factor and carcinoembryonic antigen in non-small lung cancer after surgery.

OBJECTIVES: Scatter factor, also known as hepatocyte growth factor (SF/HGF), is a polypeptide growth factor with a number of biologic activities, including cell scattering, stimulation of cell motility, mitogenesis, morphogenesis, angiogenesis, and cellular invasiveness, it is thought to be important in the growth and spread of several carcinomas. We assessed whether preoperative plasma levels of HGF and carcinoembryonic antigen (CEA) can enhance the accuracy of standard models for predicting pathologic features and clinical outcomes. PATIENTS AND METHODS: The study comprised 45 consecutive patients treated with surgery for clinically localized non-small-cell lung cancer. HGF and CEA were measured using the commercially available immunoassay. Multivariate logistic regression was used to assess the relationship between plasma HGF/CEA and pathologic features. Multivariate Cox regression was used to predict disease recurrence. RESULTS: Patients with lung squamous cell cancer (SCC) more frequently had higher plasma HGF, whereas CEA levels were significantly elevated in patients with non-SCC histology. Preoperative plasma HGF and CEA levels were not the independent predictors of overall survival. CONCLUSIONS: Preoperative plasma levels of HGF and CEA are not the independent predictors of non-small lung cancer disease recurrence and metastasis after surgery; HGF is a predictor of lung squamous cell cancer. Use of HGF may help in therapeutic decision-making and estimate the histological type of NSCLC.

Validation of Spilornis cheela hoya TaqMan probes for potential gender identification of many Accipitridae species.

The objective of this study was to test the hypothesis that genders of Accipitridae species, with the same or similar sequences to our previously proposed Spilornis cheela hoya (S. c. hoya) chromo-helicase-DNA binding protein (CHD)-W-specific and CHD-ZW-common TaqMan probes, can be successfully determined. Eight species of Accipitridae with known genders were collected. After PCR, TA cloning, sequencing, and alignment analyses, sequence length differences of Griffiths P2/P8 PCR amplicons between CHD-Z and CHD-W genes ranged from 2 to 19 bp for these Accipitridae species, and they were unsolved in 3% agarose gel. Using our previous proposed S. c. hoya TaqMan probes, the genders of Circaetus gallicus, completely homologous to the sequences for these CHD probes, were successfully identified. With one nucleotide difference to S. c. hoya CHD-W-specific probe, gender identification of Accipiter gularis, Accipiter soloensis, Accipiter trivirgatus, Accipiter virgatus, and Butastur indicus were validated. With two nucleotide differences in the CHD-W-specific probe and one nucleotide difference in the CHD-ZW-common probe, Pernis ptilorhyncus also performed well for gender identification. In conclusion, the S. c. hoyaCHD probes, coupled with the Griffiths P2/P8 primers, were validated to provide accurate and high-throughput gender identification for many Accipitridae species.

High-throughput gender identification of Accipitridae eagles with real-time PCR using TaqMan probes.

The objective was to develop high-throughput gender identification of eagles. Based on BLAST and alignment analyses, the CHD-Z and CHD-W sequences of nine species of eagles were highly homologous with Spilornis cheela hoya (S. c. hoya); therefore, TaqMan probes were designed to target their CHD-ZW-common and CHD-W-specific regions. In S. c. hoya, genders were identified using TaqMan-based, real-time PCR (amplified by P2/P8 primers); this method was validated with anatomically confirmed controls (one of each gender). Both genders had high intensities of the HEX-labeled (CHD-ZW-common) probe, whereas only females had high intensity of the FAM-labeled (CHD-W-specific) probe. The sequence of the CHD-W-specific probe designed for S. c. hoya was completely homologous with the CHD-W-specific region in Circaetus gallicus, Gyps indicus, and Gyps bengalensis, and was only one nucleotide different from those of Accipiter nisus, Spizaetus nipalensis, Aquila chrysaetos, Circus spilonotus, and Milvus migrans. For the CHD-ZW-common probe, all species listed were completely conserved. Using real-time PCR software, we established auto-calling of the genders of 15 individuals of S. c. hoya. In conclusion, this method provided accurate, high-throughput gender identification for S. c. hoya, and has considerable potential for identifying the gender of several related species of eagles.

FGF2-Targeted adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation.

Gene therapy has yet to achieve reproducible clinical efficacy, due to inadequate gene delivery, inadequate gene expression, or dose-limiting toxicity. We have developed a gene therapy technology for tissue repair and regeneration that employs a structural matrix for DNA delivery. The matrix holds the DNA vector at the treatment site and provides a scaffolding for in-growth and accumulation of repair cells and efficient DNA transfection. We now report, for the first time, matrix-mediated delivery of targeted DNA vectors for soft tissue repair. A collagen matrix was used to deliver an adenoviral vector encoding platelet-derived growth factor-B (AdPDGF-B), resulting in efficient transgene expression in vitro and in vivo. Increases in the overall levels of expression and in the relative amounts of secreted PDGF-BB were achieved when AdPDGF-B was conjugated to fibroblast growth factor (FGF2) such that the virus was targeted for cellular uptake via FGF receptors. Matrix-mediated delivery of AdPDGF-B enhanced wound healing responses in vivo, and FGF2 targeting generated effects comparable to nontargeted vectors at significantly lower doses. Therefore, matrix-mediated delivery in combination with FGF2 targeting overcomes some of the safety and efficacy limitations of current gene therapy strategies and is an attractive therapeutic approach for tissue repair and regeneration.

Fibroblast growth factor 2-retargeted adenoviral vectors exhibit a modified biolocalization pattern and display reduced toxicity relative to native adenoviral vectors.

Targeted vectors provide a number of advantages for systemic and local gene delivery strategies. Several groups have investigated the utility of using various ligands to alter the tropism of adenovirus (Ad) vectors. We have previously demonstrated that fibroblast growth factor (FGF) ligands can specifically target DNA transfection and Ad transduction through high-affinity FGF receptors (FGFRs). FGFRs are overexpressed in abnormally proliferating tissues, such as malignancies. The present studies explore the effects of retargeting with FGF2 on the tissue localization pattern and the systemic toxicity of Ad in mice. Results of semiquantitative PCR analyses indicate that the distribution of FGF2-Ad vector genome sequences after intravenous administration in mice is altered. Markedly lower amounts (10- to 20-fold) of FGF2-Ad localize to the liver when compared with native Ad. This decrease in liver deposition translates into a significant reduction in subsequent toxicity as measured by serum transaminases and histopathology in mice injected with FGF2-AdHSV-thymidine kinase with and without ganciclovir administration. In an intraperitoneal model of ovarian cancer, FGF2-Ad generates increased transgene expression in tumor tissue when compared with Ad. Taken together, these results indicate that the retargeting of Ad with FGF2 results in a more efficient vector system for systemic and regional gene therapy applications, with concomitant lower levels of systemic toxicity.

Matrix-enabled gene transfer for cutaneous wound repair.

Several growth factor proteins have been evaluated as therapeutic agents for the treatment of chronic dermal wounds. Unfortunately, most have failed to produce significant improvements in wound healing, in part due to ineffective delivery and poor retention in the wound defect. It has been proposed that gene therapy might overcome the limitations of protein therapy via ongoing transcription and translation, thus prolonging the availability of the therapeutic protein. Reasoning that it would be of further benefit to ensure retention of the DNA vector as well as the therapeutic protein within the wound defect, we have evaluated matrix-enabled gene transfer for cutaneous wound repair (Gene Activated Matrix). Formulations consisting of bovine type I collagen mixed with adenoviral or plasmid gene vectors have been evaluated in 3 in vivo models. The therapeutic transgenes employed encode human platelet-derived growth factor-A or -B, proteins key to each phase of normal wound repair. Increased granulation tissue formation, vascularization, and reepithelialization have been shown compared to controls treated with collagen alone or collagen containing a reporter gene vector. Further enhancements of the tissue repair response have been achieved by combining matrix-enabled gene transfer with molecular targeting, in which the DNA vector is conjugated to a growth factor ligand (basic fibroblast growth factor). These promising results support the clinical evaluation of gene activated matrices for the treatment of chronic dermal wounds.

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.

FGF2-targeted adenoviral vectors for systemic and local disease.

Adenoviral vectors have proven useful for transducing a variety of cell types. However, both adenoviral resistant cell types and vector-related toxicities (due to non-specific tropism), limit their widespread clinical utility. These limitations can be greatly reduced by targeting adenoviral vectors to alternative cell surface receptors. One ligand family, highly effective at targeting adenoviral vectors, is the family of fibroblast growth factors (FGFs). The FGFs allow a high degree of targeting specificity due to their cognate high affinity FGF receptors, which are expressed on cells undergoing repair and regeneration. Recent publications, reviewed herein, demonstrate that FGF-targeted adenoviruses result in enhanced potency and reduced toxicity, and thus substantially increase the therapeutic index in vivo. As a result, vector delivery through FGF receptors provides the targeting specificity required for successful local and systemic clinical applications of gene therapy.

Prediction of rectal temperature by the Questemp II personal heat strain monitor under low and moderate heat stress.

This study assessed the use of aural canal temperature measured with the Questemp II personal heat strain monitor (Tq) relative to rectal temperature (Tre) during simulated industrial work in three different wet bulb globe temperatures (WBGT). Sixteen subjects performed walking and arm curl exercise at a rate of 300 kcal/hour for 4 hours while wearing Saranex protective coveralls in 18, 23, and 27 degrees C WBGT environments and wearing the Questemp II. Correlations were determined between Tre and Tq for the three conditions and for all conditions combined. Pearson r values were 0.48 (18 degrees C WBGT), 0.42 (23 degrees C WBGT), 0.38 (27 degrees WBGT), and 0.50 (all trials). Because a major concern is safe maximum core body temperature, means and standard deviations for differences between Tre and Tq were assessed at peak temperatures to determine the predictability of Tre from Tq solely at these points. Large standard deviations in delta values relative to a small overall tolerable temperature range ruled out the use of Tq in this manner. Based on the current data, aural canal temperature as measured with the Questemp II did not provide an accurate reflection of Tre across time nor at peak core temperatures during low to moderate heat strain.