Triage of Women with Minor Cervical Lesions: Data Suggesting a “Test and Treat” Approach for HPV E6/E7 mRNA Testing

Background

Human papillomavirus (HPV) testing is included in the cervical cancer screening program in the triage of women with equivocal (ASC-US) or low-grade (LSIL) cytological lesions. These women have an increased risk for developing high grade dysplasia and cancer (CIN2+) compared to women with normal cytology. However, in order to avoid unnecessary follow-up, as well as overtreatment, a high positive predictive value (PPV) of the triage test is important.

Methodology/Principal Findings

The HPV test PreTect HPV-Proofer, detecting E6/E7 mRNA from the HPV types 16, 18, 31, 33 and 45, is used as triage test together with repeat cytology. PPV data for HPV E6/E7 mRNA testing during the period from January 2006 up to June 2009 are reported. In total, 406 of 2099 women (19.3%) had a positive HPV test result. Of the women with a positive test result and with a histological diagnosis (n = 347), 243 women had histological high-grade dysplasia or cancer (CIN2+), giving a PPV of 70.0% (95% confidence interval [CI], 65.2%–74.8%). For HPV 16 or HPV 33 positive women above 40 years of age, the PPV was 83.7% (95% CI, 73.3%–94.0%) and 84.6% (95% CI, 65.0%–100.0%) respectively. The PPV of test positive women with HSIL cytology was 94.2% (95% CI, 88.7%–99.7%).

Conclusions

When the result in triage is HPV mRNA positive, our data suggest direct treatment for women above 40 years of age or for women with a concurrent cytological HSIL diagnosis, contributing to better clinical safety for these women. In addition, by decreasing the time to treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved, in turn reducing follow-up costs as well as unnecessary psychological stress among patients.     

Analysis of the pattern of gene expression during human adipogenesis by DNA microarray

High density DNA microarrays containing over 5000 cDNA clones were used to carry out a comprehensive investigation of gene expression during adipogenesis. Complex probes synthesized from total RNA were hybridized to the arrays to determine the level of mRNA expression of each arrayed gene. Thirty three genes (29 known and 4 ESTs with no identified homologies) have been found to alter their level of expression more than 2.5-fold after differentiation. The quantitative measurement by DNA array was in good agreement with conventional Northern blot analysis of selected genes. Our results demonstrate that utilization of a DNA array is a speedy, efficient and quantitative approach to profile the expression of a large number of genes.     

Telomerase activity is sufficient to allow transformed cells to escape from crisis

The introduction of simian virus 40 large T antigen (SVLT) into human primary cells enables them to proliferate beyond their normal replicative life span. In most cases, this temporary escape from senescence eventually ends in a second proliferative block known as "crisis," during which the cells cease growing or die. Rare immortalization events in which cells escape crisis are frequently correlated with the presence of telomerase activity. We tested the hypothesis that telomerase activation is the critical step in the immortalization process by studying the effects of telomerase activity in two mortal SVLT-Rasval12-transformed human pancreatic cell lines, TRM-6 and betalox5. The telomerase catalytic subunit, hTRT, was introduced into late-passage cells via retroviral gene transfer. Telomerase activity was successfully induced in infected cells, as demonstrated by a telomerase repeat amplification protocol assay. In each of nine independent infections, telomerase-positive cells formed rapidly dividing cell lines while control cells entered crisis. Telomere lengths initially increased, but telomeres were then maintained at their new lengths for at least 20 population doublings. These results demonstrate that telomerase activity is sufficient to enable transformed cells to escape crisis and that telomere elongation in these cells occurs in a tightly regulated manner.     

The role of parasites in the dynamics of a reindeer population

Even though theoretical models show that parasites may regulate host population densities, few empirical studies have given support to this hypothesis. We present experimental and observational evidence for a host-parasite interaction where the parasite has sufficient impact on host population dynamics for regulation to occur. During a six year study of the Svalbard reindeer and its parasitic gastrointestinal nematode Ostertagia gruehneri we found that anthelminthic treatment in April-May increased the probability of a reindeer having a calf in the next year, compared with untreated controls. However, treatment did not influence the over-winter survival of the reindeer. The annual variation in the degree to which parasites depressed fecundity was positively related to the abundance of O. gruehneri infection the previous October, which in turn was related to host density two years earlier. In addition to the treatment effect, there was a strong negative effect of winter precipitation on the probability of female reindeer having a calf. A simple matrix model was parameterized using estimates from our experimental and observational data. This model shows that the parasite-mediated effect on fecundity was sufficient to regulate reindeer densities around observed host densities.     

Photosensitizing effect of protoporphyrin IX in pigmented melanoma of mice

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.     

Canonical Correspondence Analysis with variation partitioning: some comments and an application

Abstract. This study presents an alternative treatment of data from a comprehensive vegetation study in which the main gradient structure of boreal coniferous forest vegetation in southern Norway was investigated by ordination techniques. The data sets include vegetation samples of different plot sizes, supplied with measurements of 33 environmental explanatory variables (classified in four groups) and nine spatial explanatory variables derived from geographical coordinates. Partitioning the variation of the species-sample plot matrices on different sets of explanatory variables is performed by use of (partial) Canonical Correspondence Analysis. Several aspects of vegetation-environment relationships in the investigation area are discussed on the basis of results obtained by the new method. Generally, ca. 35% of the variation in species abundances are explained by environmental and spatial variables. The results indicate support for the hypothesis of macro-scale topographic control over the differentiation of the vegetation, more strongly so in pine than in spruce forest where soil nutrients play a major role. Towards finer scales, the primary topographical and topographically dependent factors lose importance, and vegetational differentiation is more strongly affected by the accumulated effects of the vegetation (including the tree stand) on soils, shading, litter fall, etc. The fraction of variation in species abundance explained by significant environmental variables was found to be ca. twice as large as the fraction explained by spatial variables. The fraction of variation explained by the supplied variables differed between data sets; it was lower for cryptogams than for vascular plants, and lower for smaller than for larger sample plots. Possible reasons for these patterns are discussed. Some methodological aspects of CCA with variation partitioning are discussed: improvements, necessary precautions, and the advantages over alternative methods.     

Investigation of the metabolic pattern in maple syrup urine disease by means of glass capillary gas chromatography and mass spectrometry

Urine and serum from patients with maple syrup urine disease (MSUD) have been examined quantitatively and qualitatively using glass capillary gas chromatography in combination with mass spectrometry. During clinical episodes, patients with this disease were found to excrete increased amounts of the following metabolites in addition to the previously recognized branched-chain 2-keto and 2-hydroxy acids, lactate and 3-hydroxybutyrate; 2-hydroxybutyrate, 2-hydroxyisobutyrate, 3-hydroxyisovalerate, 3-hydroxylsobutyrate and 2-methyl-3-hydroxybutyrate. Most of the latter compounds seem to accompany ketoacidosis and lactic acidosis. The capillary column also separated the d- and l-forms of 2-keto-3-methylvalerate, and both isomers were, in contrast to earlier assumptions, present in the MSUD patients. The results clearly demonstrate that new information on the metabolic situation in well known disorders may be obtained by exploiting the high resolving power of capillary columns.     

A method for determining minimal sets of markers for the estimation of center of mass,linear and angular momentum

A new method is proposed for finding small sets of points on the body giving sufficient information for estimating the whole body center of mass (CoM), as well as the linear momenta (LM) and angular momenta (AM). In the underlying model each point (whose trajectory is tracked by a marker) is a point mass: Hence the body is represented by a simple system of point masses. The first step is to determine the appropriate set of points and the mass of each point, which is assumed to be specific for the movement performed. The distribution of the mass to each marker is determined from training data for which the true (or reference) trajectories of the CoM, LM or AM are known. This leads to a quadratic optimization problem with inequality constraints. The use of the method is demonstrated on data from discus throw. Results indicate reasonably small errors, considering the magnitude of other error sources, in CoM position (average magnitude of estimation error 1–2 cm), and moderate errors in AM (13–20% of peak value).     

The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxin

Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin-receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.     

Variations in transfection efficiency of VEGF165 and VEGF121-cDNA

Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF₁₂₁ and VEGF₁₆₅ isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51–0.95 pg/mL/μg protein in umbilical artery, through 2.32–2.39 pg/mL/μg protein in coronary artery, to 5.45–7.52 pg/mL/μg protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 μg VEGF₁₂₁-cDNA/in the wells)—an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.