Comparison of Efficiency and Function of Vascular Endothelial Growth Factor Adenovirus Vectors in Endothelial Cells for Gene Therapy of Placental Insufficiency.

Severe fetal growth restriction (FGR) affects 1:500 pregnancies, is untreatable and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) and lack of bioavailable VEGF due to placental insufficiency is a major cause. Transduction of uterine arteries in normal or FGR sheep and guinea pigs using an adenovirus (Ad) encoding VEGF isoforms A (Ad.VEGF-A165) and a FLAG-tagged pre-processed short form D (DΔNΔC, Ad.VEGF-DΔNΔC-FLAG) increases endothelial nitric oxide expression, enhances relaxation and reduces constriction of the uterine arteries and their branches. UBF and angiogenesis are increased long term, improving fetal growth in utero. For clinical trial development we compared Ad.VEGF vector transduction efficiency and function in endothelial cells (ECs) derived from different species. We aimed to compare the transduction efficiency and function of the pre-clinical study Ad. constructs (Ad.VEGF-A165, Ad.VEGF-DΔNΔC-FLAG) with the intended clinical trial construct (Ad.VEGF-DΔNΔC) where the FLAG tag is removed. We infected ECs from human umbilical vein, pregnant sheep uterine artery, pregnant guinea pig aorta and non-pregnant rabbit aorta, with increasing multiplicity of infection (MOI) for 24 or 48 hours of three Ad.VEGF vectors, compared to control Ad. containing the LacZ gene (Ad.LacZ). VEGF supernatant expression was analysed by ELISA. Functional assessment used tube formation assay and Erk-Akt phosphorylation by ELISA. VEGF expression was higher after Ad.VEGF-DΔNΔC-FLAG and Ad.VEGF-DΔNΔC transduction compared to Ad.VEGF-A165 in all EC types (*p < 0.001). Tube formation was higher in ECs transduced with Ad.VEGF-DΔNΔC in all species compared to other constructs (***p < 0.001, *p < 0.05 with rabbit aortic ECs). Phospho-Erk and phospho-Akt assays displayed no differences between the three vector constructs, whose effect was, as in other experiments, higher than Ad.LacZ (***p < 0.001). In conclusion, we observed high transduction efficiency and functional effects of Ad.VEGF-DΔNΔC vector with comparability in major pathway activation to constructs used in pre-clinical studies, supporting its use in a clinical trial.

In Vitro Human Placental Studies to Support Adenovirus-Mediated VEGF-DΔNΔC Maternal Gene Therapy for the Treatment of Severe Early-Onset Fetal Growth Restriction.

Severe fetal growth restriction (FGR) affects 1 in 500 pregnancies, is untreatable, and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) is one cause. Transduction of uterine arteries in normal and FGR animal models using an adenovirus (Ad) encoding VEGF isoforms increases UBF and improves fetal growth in utero. Understanding potential adverse consequences of this therapy before first-in-woman clinical application is essential. The aims of this study were to determine whether Ad.VEGF-DΔNΔC (1) transfers across the human placental barrier and (2) affects human placental morphology, permeability and primary indicators of placental function, and trophoblast integrity. Villous explants from normal term human placentas were treated with Ad.VEGF-DΔNΔC (5 × 107-10 virus particles [vp]/mL), or virus formulation buffer (FB). Villous structural integrity (hematoxylin and eosin staining) and tissue accessibility (LacZ immunostaining) were determined. Markers of endocrine function (human chorionic gonadotropin [hCG] secretion) and cell death (lactate dehydrogenase [LDH] release) were assayed. Lobules from normal and FGR pregnancies underwent ex vivo dual perfusion with exposure to 5 × 1010 vp/mL Ad.VEGF-DΔNΔC or FB. Perfusion resistance, para-cellular permeability, hCG, alkaline phosphatase, and LDH release were measured. Ad.VEGF-DΔNΔC transfer across the placental barrier was assessed by quantitative polymerase chain reaction in DNA extracted from fetal-side venous perfusate, and by immunohistochemistry in fixed tissue. Villous explant structural integrity and hCG secretion was maintained at all Ad.VEGF-DΔNΔC doses. Ad.VEGF-DΔNΔC perfusion revealed no effect on placental permeability, fetoplacental vascular resistance, hCG secretion, or alkaline phosphatase release, but there was a minor elevation in maternal-side LDH release. Viral vector tissue access in both explant and perfused models was minimal, and the vector was rarely detected in the fetal venous perfusate and at low titer. Ad.VEGF-DΔNΔC did not markedly affect human placental integrity and function in vitro. There was limited tissue access and transfer of vector across the placental barrier. Except for a minor elevation in LDH release, these test data did not reveal any toxic effects of Ad.VEGF-DΔNΔC on the human placenta.

Colorectal cancer screening practices in Saskatchewan: Survey of family physicians.

OBJECTIVE: To evaluate current colorectal cancer (CRC) screening practices in Saskatchewan and identify barriers to screening with the goal of improving current practice. DESIGN: Survey of family physicians. SETTING: Saskatchewan. PARTICIPANTS: A total of 773 family physicians were surveyed. MAIN OUTCOME MEASURES: Demographic characteristics, individual screening practices, and perceived barriers to screening. RESULTS: The response rate to the survey was 44.5%. When asked what method they used for fecal occult blood testing, almost 40% of respondents were either unsure or did not answer the question. Of those who did respond, 35.8% employed hemoccult testing following digital rectal examination, a practice not recommended for CRC screening. Screening guidelines for average-risk patients were generally well adhered to, with 79.9% of respondents recommending screening beginning at age 50. For screening patients at increased risk of CRC owing to family history, only 64.2% of respondents began screening 10 years before the age of the index patient at diagnosis. Physicians who were more likely to follow guidelines were female, in practice fewer than 10 years, trained in Canada, and practising in urban areas. More than 90% of family physicians agreed that a standard provincewide screening program would be beneficial. CONCLUSION: We have identified considerable knowledge gaps with regard to CRC screening. There is confusion about which fecal occult blood tests are recommended for screening. Also, screening guidelines for patients with a family history of CRC are poorly understood. These findings suggest that better physician education about CRC screening is required. Introduction of a provincewide screening program should improve overall screening success.

A defect in Marco expression contributes to systemic lupus erythematosus development via failure to clear apoptotic cells.

Systemic lupus erythematosus is a multisystem autoimmune disease characterized by the production of numerous antinuclear autoantibodies and inflammatory mediators. The BXSB mouse strain is an excellent model of the disease. Previous work has determined a number of important disease susceptibility intervals that have been isolated in separate congenic strains. Here, we have combined expression data from those strains with functional analyses to demonstrate that reduced expression of the innate scavenger receptor Marco (macrophage receptor with collagenous structure) is a primary event in BXSB mice, that reduced mRNA expression is mirrored at the protein level, and that this results in a significant alteration in function. We have confirmed a role for Marco in the clearance of apoptotic cells and a generalized defect in both endocytosis and phagocytosis. The failure to clear apoptotic cells has previously been linked to the development of systemic lupus erythematosus. However, the use of congenic mice with limited phenotypes in this study has enabled us to propose that in the case of Marco at least, disease results from the production of anti-dsDNA Abs.

Noninvasive imaging of cell proliferation following mitogenic extracellular kinase inhibition by PD0325901.

The mitogenic extracellular kinase 1/2 (MEK1/2) inhibitor, PD0325901, has potent activity in a number of cancer cell types in vitro. In SKMEL-28 human melanoma cells (BRAF mutant), the drug rapidly decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein levels. We investigated if 3'-deoxy-3'-[18F]fluorothymidine-positron emission tomography ([18F]FLT-PET) could be used to image changes in cell proliferation following MEK1/2 inhibition in vivo. Mice bearing SKMEL-28 and human colon cancer HCT116 (K-RAS mutant) xenografts were treated daily with PD0325901 at 25 mg/kg and imaged by dynamic [18F]FLT-PET after 1 and 10 days of initiating treatment. The drug decreased tumor [18F]FLT uptake after 1 and 10 days of treatment compared with control animals. The normalized (maximal) [18F]FLT uptake in SKMEL-28 xenografts (at 60 minutes; NUVmax) after 1 day of vehicle or PD0325901 therapy was 1.81 +/- 0.18 versus 1.23 +/- 0.10, respectively (P = 0.03). In this model, NUVmax after 10 days was 2.07 +/- 0.40 versus 1.08 +/- 0.14, respectively (P = 0.03). The corresponding values for HCT116 tumors were 2.30 +/- 0.84 versus 1.88 +/- 0.36 (P = 0.045) after 1 day, and 1.97 +/- 0.13 versus 1.00 +/- 0.03 (P = 0.03) after 10 days. Similar changes were found for other [18F]FLT retention variables. The drug decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein. Tumor [18F]FLT-PET variables correlated with proliferation as measured by Ki67 labeling index (r > or = 0.6; P > or = 0.003). In summary, [18F]FLT-PET is a sensitive imaging biomarker for detecting the antiproliferative effect of MEK1/2 inhibition by PD0325901.