Real-time PCR quantitation of glucocorticoid receptor alpha isoform

Abstract Background The expression of glucocorticoid-receptor (GR) seems to be a key mechanism in the regulation of glucocorticoid (GC) sensitivity and is potentially involved in cases of GC resistance or hypersensitivity. The aim of this study is to describe a method for quantitation of GR alpha isoform (GRα) expression using real-time PCR (qrt-PCR) with analytical capabilities to monitor patients, offering standard-curve reproducibility as well as intra- and inter-assay precision. Results Standard-curves were constructed by employing standardized Jurkat cell culture procedures, both for GRα and BCR (breakpoint cluster region), as a normalizing gene. We evaluated standard-curves using five different sets of cell culture passages, RNA extraction, reverse transcription, and qrt-PCR quantification. Intra-assay precision was evaluated using 12 replicates of each gene, for 2 patients, in a single experiment. Inter-assay precision was evaluated on 8 experiments, using duplicate tests of each gene for two patients. Standard-curves were reproducible, with CV (coefficient of variation) of less than 11%, and Pearson correlation coefficients above 0,990 for most comparisons. Intra-assay and inter-assay were 2% and 7%, respectively. Conclusion This is the first method for quantitation of GRα expression with technical characteristics that permit patient monitoring, in a fast, simple and robust way.

A New Method to Predict the Epidemiology of Fungal Keratitis by Monitoring the Sales Distribution of Antifungal Eye Drops in Brazil

Purpose: Fungi are a major cause of keratitis, although few medications are licensed for their treatment. The aim of this study is to observe the variation in commercialisation of antifungal eye drops, and to predict the seasonal distribution of fungal keratitis in Brazil. Methods: Data from a retrospective study of antifungal eye drops sales from the only pharmaceutical ophthalmologic laboratory, authorized to dispense them in Brazil (Opthalmos) were gathered. These data were correlated with geographic and seasonal distribution of fungal keratitis in Brazil between July 2002 and June 2008. Results: A total of 26,087 antifungal eye drop units were sold, with a mean of 2.3 per patient. There was significant variation in antifungal sales during the year (p < 0.01). A linear regression model displayed a significant association between reduced relative humidity and antifungal drug sales (R-2 = 0.17, p < 0.01). Conclusions: Antifungal eye drops sales suggest that there is a seasonal distribution of fungal keratitis. A possible interpretation is that the third quarter of the year (a period when the climate is drier), when agricultural activity is more intense in Brazil, suggests a correlation with a higher incidence of fungal keratitis. A similar model could be applied to other diseases, that are managed with unique, or few, and monitorable medications to predict epidemiological aspects.

Goal-directed fluid management based on pulse pressure variation monitoring during high-risk surgery: a pilot randomized controlled trial

Abstract Introduction Several studies have shown that maximizing stroke volume (or increasing it until a plateau is reached) by volume loading during high-risk surgery may improve post-operative outcome. This goal could be achieved simply by minimizing the variation in arterial pulse pressure (ΔPP) induced by mechanical ventilation. We tested this hypothesis in a prospective, randomized, single-centre study. The primary endpoint was the length of postoperative stay in hospital. Methods Thirty-three patients undergoing high-risk surgery were randomized either to a control group (group C, n = 16) or to an intervention group (group I, n = 17). In group I, ΔPP was continuously monitored during surgery by a multiparameter bedside monitor and minimized to 10% or less by volume loading. Results Both groups were comparable in terms of demographic data, American Society of Anesthesiology score, type, and duration of surgery. During surgery, group I received more fluid than group C (4,618 ± 1,557 versus 1,694 ± 705 ml (mean ± SD), P < 0.0001), and ΔPP decreased from 22 ± 75 to 9 ± 1% (P < 0.05) in group I. The median duration of postoperative stay in hospital (7 versus 17 days, P < 0.01) was lower in group I than in group C. The number of postoperative complications per patient (1.4 ± 2.1 versus 3.9 ± 2.8, P < 0.05), as well as the median duration of mechanical ventilation (1 versus 5 days, P < 0.05) and stay in the intensive care unit (3 versus 9 days, P < 0.01) was also lower in group I. Conclusion Monitoring and minimizing ΔPP by volume loading during high-risk surgery improves postoperative outcome and decreases the length of stay in hospital. Trial registration NCT00479011