Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.

The genotyping of human papillomaviruses (HPV) is essential for the surveillance of HPV vaccines. We describe and validate a low-cost PGMY-based PCR assay (PGMY-CHUV) for the genotyping of 31 HPV by reverse blotting hybridization (RBH). Genotype-specific detection limits were 50 to 500 genome equivalents per reaction. RBH was 100% specific and 98.61% sensitive using DNA sequencing as the gold standard (n ‫؍‬ 1,024 samples). PGMY-CHUV was compared to the validated and commercially available linear array (Roche) on 200 samples. Both assays identified the same positive (n ‫؍‬ 182) and negative samples (n ‫؍‬ 18). Seventy-six percent of the positives were fully concordant after restricting the comparison to the 28 genotypes shared by both assays. At the genotypic level, agreement was 83% (285/344 genotype-sample combinations; of 0.987 for single infections and 0.853 for multiple infections). Fifty-seven of the 59 discordant cases were associated with multiple infections and with the weakest genotypes within each sample (P < 0.0001). PGMY-CHUV was significantly more sensitive for HPV56 (P ‫؍‬ 0.0026) and could unambiguously identify HPV52 in mixed infections. PGMY-CHUV was reproducible on repeat testing (n ‫؍‬ 275 samples; 392 genotype-sample combinations; of 0.933) involving different reagents lots and different technicians. Discordant results (n ‫؍‬ 47) were significantly associated with the weakest genotypes in samples with multiple infections (P < 0.0001). Successful participation in proficiency testing also supported the robustness of this assay. The PGMY-CHUV reagent costs were estimated at $2.40 per sample using the least expensive yet proficient genotyping algorithm that also included quality control. This assay may be used in low-resource laboratories that have sufficient manpower and PCR expertise.

The serpin ␣ 2 -antiplasmin ( ␣ 2 -AP) is the major circulating inhibitor of plasmin; it plays a determining role in the regulation of intravascular fibrinolysis. In addition to blood plasma, plasmin formation occurs in various organs where it is thought to fulfill a spectrum of functions not restricted to clot lysis. ␣ 2 -AP is synthesized by hepatocytes, but other possible sites of production have not been investigated. To explore the potential extravascular contribution of ␣ 2 -AP in the regulation of proteolysis, we have isolated the murine ␣ 2 -AP cDNA and determined its mRNA distribution in adult tissues.In

In LLC-PK, cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit P-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.

Our objective was (i) to assess if a self-collected test for human papillomavirus (HPV) may serve as a primary cervical cancer screening method in a low-resource setting, (ii) to evaluate its implication in a screen and treat approach and (iii) to identify the most eligible age group in a screening program. Women were recruited through a cervical cancer screening campaign conducted in Cameroon. Written and oral instructions were given to participants by a health-care professional to carry out an unsupervised self-collected HPV-test (Self-HPV), followed by a physician-collected cervical sample for HPV testing (Physician-HPV) and cytology. Differences in performance between Self-HPV versus Physician-HPV and their ability to detect abnormal cytology results (ASC-US1) were evaluated. Descriptive analyses were used to examine the correlation between HPV positivity and cervical abnormalities by age. A sample of 789 women was prospectively enrolled. HPV prevalence was 14.6% and 12.7% for Self-HPV and Physician-HPV, respectively (Cohen's kappa 5 0.74). HPV positivity by cytological diagnosis for ASC-US1 was similar with the two tests. positive predictive value of the Self-HPV for ASC-US1 was 20.4; odds ratio and number needed to treat were 6.5 (3.2-13.4) and 6 (4.2-10.9), respectively. We observed a trend of increasing cytological abnormalities in 30-49 year-old women and a concomitant trend of decreasing HPV prevalence supporting that this age group might be the most eligible group for screening. In conclusion, Self-HPV can be used as a primary screening test but needs to be followed by a triaging test that would identify the subset of women affected by clinically significant precancer or cancer.