Moderate consumption of red wine (cabernet sauvignon) improves ischemia-induced neovascularization in ApoE-deficient mice: effect on endothelial progenitor cells and nitric oxide.

Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hindlimb ischemia in hypercholesterolemic ApoE-deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine-drinking mice. The wine used in our study is a cabernet sauvignon from Languedoc-Roussillon, France, which contains a relatively high concentration (4-6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia-induced neovascularization in high-cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt-eNOS-NO pathway.

High level of correlation of human papillomavirus-16 DNA viral load estimates generated by three real-time PCR assays applied on genital specimens.

Human papillomavirus-16 (HPV-16) viral load could be a biomarker predictive of the presence of high-grade cervical lesions. Recently, several real-time PCR assays have been developed to accurately measure HPV-16 viral load. However, results from various reports using these assays cannot be compared because interassay test correlation has not been documented. The variability of HPV-16 DNA quantitation was assessed by comparing three real-time PCR assays (HPV-16 L1, HPV-16 E6, and HPV-16 E6 PG) applied on 144 genital samples (125 cervicovaginal lavages and 19 specimens collected using vaginal tampons) obtained from 84 women (66 HIV seropositive and 18 HIV seronegative). Correlation was greater between the HPV-16 E6 assays [correlation coefficient (rho) = 0.92] than between each E6 assay and HPV-16 L1 assay (rho = 0.83 and 0.84, respectively). The median HPV-16 copies measured by HPV-16 E6 PG (14,609 HPV-16 copies/2 muL sample) and HPV-16 E6 (18,846 HPV-16 copies/2 muL) were similar (P = 0.27) but were both greater than the median HPV-16 copies measured with the L1 assay (4,124 HPV-16 copies/2 muL; P < 0.001). Correlations between HPV-16 E6 assays were similar for samples containing non-European (rho = 0.93) or European (rho = 0.95) variants. However, the correlation between HPV-16 L1 and HPV-16 E6 PG or HPV-16 E6 was lower for specimens containing non-European variants (rho = 0.80 and 0.76, respectively) compared with specimens containing European variants (rho > 0.85). HPV-16 DNA quantity estimated with the three assays was comparable although lower with the HPV-16 L1 assay. The level of correlation depended on viral polymorphism, viral load, and cervical disease status.

High levels of HPV-16 DNA are associated with high-grade cervical lesions in women at risk or infected with HIV.

OBJECTIVE: To examine associations between levels of episomal and integrated human papillomavirus (HPV) 16 DNA and the grade of cervical disease. DESIGN: Cross-sectional data were obtained from a cohort of women with and without HIV infection and with high-risk sexual behaviour. METHODS: Episomal and integrated HPV-16 DNA loads were measured in cervicovaginal lavages collected from 75 women (58 HIV seropositive, 17 HIV seronegative) using real-time polymerase chain reaction assays, controlling for cell content and the presence of inhibitors. RESULTS: HPV-16 viral loads were significantly higher in women with high-grade squamous intraepithelial lesions (n = 6) than in women with normal cytology (n = 44), whether total (10(8.28) versus 10(5.10) HPV-16 DNA copies/microg DNA), episomal (10(7.99) versus 10(4.61)) or integrated (10(7.95) versus 10(4.77)) HPV-16 viral loads were measured (P < 0.02 for each comparison). Thirty-nine women had colposcopy [11 normal cervix, 16 cervical intraepithelial neoplasia (CIN) 1, six CIN 2, six CIN 3] and 24 additional women had three consecutive normal cytology smears. Controlling for age, race, CD4 cell count and HIV status, total (OR 3.5, 95% CI 1.2-10.4; P = 0.02), episomal (OR 2.9, 95% CI 1.2-7.4; P = 0.02,) and integrated (OR 1.6, 95% CI 1-2.6; P = 0.05) HPV-16 DNA loads were significantly associated with CIN 2,3, but the differences between CIN 1 and CIN 2,3 were not significant (P > 0.06). A greater amount of cellular DNA was collected from women with CIN 2,3 (P = 0.007). CONCLUSION: Higher HPV-16 DNA loads are associated with cervical lesions detected by either histology or cytology. No additional information is gained by measuring integrated or episomal over total HPV-16 DNA loads.

Human papillomavirus type 16 viral load is higher in human immunodeficiency virus-seropositive women with high-grade squamous intraepithelial lesions than in those with normal cytology smears.

Human papillomavirus type 16 (HPV-16) viral load in cervicovaginal lavage samples collected from 66 human immunodeficiency virus-seropositive women was inversely correlated with blood CD4 count (P = 0.002). HPV-16 viral load was 81-fold higher in women with cervical smears suggestive of high-grade lesions (median, 4,425,883 copies/ micro g of DNA) than in women with normal smears (median, 54,576), controlling for age (P = 0.006).

Prevalence of selective inhibition of HPV-16 DNA amplification in cervicovaginal lavages.

HPV-16 viral load has been assessed with real-time PCR assays by measuring HPV-16 DNA and a human gene in genital samples. HPV-16 viral load measurements are thus based on the inference that inhibitors contained in samples will equally impede amplification of DNA sequences from HPV-16 and human DNA. We have previously shown that sample lysates can inhibit amplification of HPV-16 but not beta-globin DNA. In the current study, cervicovaginal lavages lysates considered adequate for PCR analysis by a qualitative beta-globin PCR test, were screened for the presence of inhibitors using internal controls (IC) for HPV-16 DNA and beta-globin in real-time PCR assays. Of 150 lysates screened with both ICs, 12 (8%) contained inhibitors. Inhibition of amplification of both ICs was demonstrated in four of these specimens. In eight lysates, amplification of HPV-16 IC only was impeded. Six (50%) of these 12 lysates tested positive for HPV-16 DNA despite the presence of PCR inhibitors. The HPV-16 viral load increased significantly after dilution of 11 of 12 lysates, demonstrating the presence of inhibitors in the undiluted lysate. Nine (90%) of 10 samples with inhibitors that were tested after dilution did not demonstrate inhibitory activity. The use of internal controls in real-time PCR is clearly essential to determine HPV viral loads since the effect of inhibitors on primer-driven genomic amplification is variable.

Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-globin DNA in cervicovaginal lavages.

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.