Effects of sex hormones on inflammatory response in male and female vascular endothelial cells.

PURPOSE: Gender-related differences in sex hormones might have a key role in the development of atherosclerosis though direct vascular effects of sex hormones are not yet well understood. Thus, the main purpose of this study was to compare the effects of sex hormones on inflammatory response in Human Umbilical Vein Endothelial Cells (HUVECs) obtained from both male and female donors. METHODS: We analyzed the expression of receptors and enzymes relevant to the action of androgens (AR, 5α-reductase 1 and 5α-reductase 2) and estrogens (ERα, ERß, and aromatase) in male and female HUVECs. Furthermore, we analyzed the effect of testosterone (T), 17ß-estradiol (E2), dihydrotestosterone (DHT), and several androgenic-anabolic steroids (AAS) on VCAM-1, ICAM-1, and E-selectin gene expression and on adhesion of U937 cells to TNF-α-stimulated male and female HUVECs. RESULTS: Our results reveal that in HUVECs, regardless of gender, the components involved in the androgen action pathway are predominant as compared to those of estrogen action pathway. In both HUVEC genders, the inflammatory effect of TNF-α was amplified by co-administration of T or DHT and several AAS frequently used in doping, while E2 had no effect. CONCLUSIONS: This is the first study analyzing, under identical culture conditions, the key components of sex hormone response in male and female HUVECs and the possible role of sex hormones in regulating the endothelial inflammatory response. The data obtained in our experimental system showed a pro-inflammatory effect of androgens, while conclusively excluding any protective effect for all the tested hormones.

Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.

The expression analysis of mouse interleukin-6 splice variants argued against their biological relevance.

Alternative splicing generates several interleukin-6 (IL-6) isoforms; for them an antagonistic activity to the wild-type IL-6 has been proposed. In this study we quantified the relative abundance of IL-6 mRNA isoforms in a panel of mouse tissues and in C2C12 cells during myoblast differentiation or after treatment with the Ca(2+) ionophore A23187, the AMP-mimetic AICAR and TNF-α. The two mouse IL-6 isoforms identified, IL-6δ5 (deletion of the first 58 bp of exon 5) and IL-6δ3 (lacking exon 3), were not conserved in rat and human, did not exhibit tissue specific regulation, were expressed at low levels and their abundance closely correlated to that of full-length IL-6. Species-specific features of the IL-6 sequence, such as the presence of competitive 3' acceptor site in exon 5 and insertion of retrotransposable elements in intron 3, could explain the production of IL-6δ5 and IL-6δ3. Our results argued against biological significance for mouse IL-6 isoforms.

Shape based kinetic outlier detection in real-time PCR.

BACKGROUND: Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. RESULTS: Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point) and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. CONCLUSION: Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.