NFAT regulates the expression of AIF-1 and IRT-1: Yin and yang splice variants of neointima formation and atherosclerosis.

Aims Alternative transcription and splicing of the allograft inflammatory factor-1 (AIF-1) gene results in the expression of two different proteins: AIF-1 and interferon responsive transcript-1 (IRT-1). Here we explore the impact of AIF-1 and IRT-1 on vascular smooth muscle cell (VSMC) activation and neointima formation, the mechanisms underlying their alternative splicing, and associations of AIF-1 and IRT-1 mRNA with parameters defining human atherosclerotic plaque phenotype.Methods and results Translation of AIF-1 and IRT-1 results in different products with contrasting cellular distribution and functions. Overexpression of AIF-1 stimulates migration and proliferation of human VSMCs, whereas IRT-1 exerts opposite effects. Adenoviral infection of angioplasty-injured rat carotid arteries with AdAIF-1 exacerbates intima hyperplasia, whereas infection with AdIRT-1 reduces neointima. Expression of these variants is modulated by changes in nuclear factor of activated T-cells (NFAT) activity. Pharmacological inhibition of NFAT or targeting of NFATc3 with siRNA lowers the AIF-1/IRT-1 ratio and favors an anti-proliferative outcome. NFAT acts as a repressor on the IRT-1 transcriptional start site, which is also sensitive to interferon-γ stimulation. Expression of AIF-1 mRNA in human carotid plaques associates with less extracellular matrix and a more pro-inflammatory plaque and plasma profile, features that may predispose to plaque rupture. In contrast, expression of IRT-1 mRNA associates with a less aggressive phenotype and less VSMCs at the most stenotic region of the plaque.Conclusions Inhibition of NFAT signaling, by shifting the AIF-1/IRT-1 ratio, may be an attractive target to regulate the VSMC response to injury and manipulate plaque stability in atherosclerosis

Quantifying bovine insulin: conversion of units

Xunta de Galicia (10MRU261004PR); Ministerio de Educación, Cultura y Deporte (Beca FPU AP2010-0013

Filter Paper Blood Spot Enzyme Linked Immunoassay for Insulin and Application in the Evaluation of Determinants of Child Insulin Resistance

In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial.) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry.Mean intra-assay (n = 157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at −80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose.Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin

Bland-Altman plot for insulin values from fresh minus frozen samples.

<p>The difference in fasted bloodspot insulin from fresh sample minus the same sample frozen for 8 weeks at −80°C.</p

Bland-Altman plot for fasted bloodspot versus serum insulin assay in 50 paired samples.

<p>Bland-Altman plot for fasted bloodspot versus serum insulin assay in 50 paired samples.</p

Geometric mean (log standard deviation) insulin levels in boys and girls and association with demographic and clinical characteristics, N = 12,812.

<p>SD = standard deviation; CI = confidence interval.</p>a<p>Linear regression coefficient and 95% CI (age-adjusted).</p>b<p>Normal weight, overweight and obese as defined by the International Obesity Task Force, IOTF BMI cut-offs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046752#pone.0046752-Cole1" target="_blank">[40]</a>.</p

Stability of insulin concentrations measured from dried blood spots at +30°C for 1 weeks.

<p>Bloodspot fasting insulin concentrations from three samples analysed at baseline (time = 0), and after 1 hour, 12 hours, 24 hours, 48 hours and 1 week at +30°C.</p

Stability of fasting insulin stored at −80°C for 26 months.

<p>Fasting insulin concentrations of three bloodspot samples analysed prior to freezing (time 0) and then at regular intervals over 26 months. For sample 1, the middle red line is the insulin concentration at time 0 and the upper and lower red lines are the 95% reference range (calculated from the standard deviation of 30 replicates of the time 0 value). For samples 2 and 3, the lines are coloured green and blue, respectively.</p

Comparison of fasting insulin measured from 50 paired serum and whole bloodspot samples.

<p>Comparison of fasting insulin measured from 50 paired serum and whole bloodspot samples.</p