The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Prognostic Significance of Myocardial Fibrosis in Hypertrophic Cardiomyopathy

ObjectivesWe investigated the significance of fibrosis detected by late gadolinium enhancement cardiovascular magnetic resonance for the prediction of major clinical events in hypertrophic cardiomyopathy (HCM).BackgroundThe role of myocardial fibrosis in the prediction of sudden death and heart failure in HCM is unclear with a lack of prospective data.MethodsWe assessed the presence and amount of myocardial fibrosis in HCM patients and prospectively followed them for the development of morbidity and mortality in patients over 3.1 ± 1.7 years.ResultsOf 217 consecutive HCM patients, 136 (63%) showed fibrosis. Thirty-four of the 136 patients (25%) in the fibrosis group but only 6 of 81 (7.4%) patients without fibrosis reached the combined primary end point of cardiovascular death, unplanned cardiovascular admission, sustained ventricular tachycardia or ventricular fibrillation, or appropriate implantable cardioverter-defibrillator discharge (hazard ratio [HR]: 3.4, p = 0.006). In the fibrosis group, overall risk increased with the extent of fibrosis (HR: 1.18/5% increase, p = 0.008). The risk of unplanned heart failure admissions, deterioration to New York Heart Association functional class III or IV, or heart failure-related death was greater in the fibrosis group (HR: 2.5, p = 0.021), and this risk increased as the extent of fibrosis increased (HR: 1.16/5% increase, p = 0.017). All relationships remained significant after multivariate analysis. The extent of fibrosis and nonsustained ventricular tachycardia were univariate predictors for arrhythmic end points (sustained ventricular tachycardia or ventricular fibrillation, appropriate implantable cardioverter-defibrillator discharge, sudden cardiac death) (HR: 1.30, p = 0.014). Nonsustained ventricular tachycardia remained an independent predictor of arrhythmic end points after multivariate analysis, but the extent of fibrosis did not.ConclusionsIn patients with HCM, myocardial fibrosis as measured by late gadolinium enhancement cardiovascular magnetic resonance is an independent predictor of adverse outcome. (The Prognostic Significance of Fibrosis Detection in Cardiomyopathy; NCT00930735

Interaction of tea polyphenols and food constituents with model gut epithelia: The protective role of the mucus gel layer

The luminal surface of the gastrointestinal tract is covered by a mucus gel layer that acts to protect gut epithelial cells from the harsh luminal environment. This study investigated the use of two human colonic adenocarcinoma cell lines, HT29-MTX-E12 and HT29, as a model to mimic gut epithelium with and without a mucus gel layer. The effect of adding the tea polyphenols epigallocatechin gallate (EGCG) and epicatechin (EC) to the cells with subsequent examination of cell morphology and viability was assessed. EGCG, at the concentrations tested, was very toxic to the HT29 cells, but less toxic to the HT29-MTX-E12 cells, suggesting that the mucus gel layer on the HT29-MTX-E12 cells can protect the cells against EGCG toxicity. In contrast, EC had no effect on the viability of either the HT29 or HT29-MTX-E12 cells, suggesting that proteins within the mucus gel layer on the apical surface of gut epithelial cells may bind to the galloyl ring of EGCG. The effect of adding food-related ingredients with the ability to complex with EGCG, β-casein and maltodextrin, on cell viability was also examined. The presence of β-casein was very effective in protecting the cells against the toxicity effect of EGCG, but maltodextrin, at the concentration tested, was less effective in protecting against this toxicity. In conclusion, the results demonstrate that the mucus gel layer on HT29 human colonic adenocarcinoma cells may protect these cells against EGCG toxicity. In addition, the data showing reduced toxicity of EC compared to that of EGCG suggest that the cytotoxic effects of high polyphenol levels may be associated with the ability of polyphenols to interact with cellular proteins and mucins

IHC analysis of nevi and breast skin samples.

<p>Serial sections of a nevi skin sample (A) and a breast skin sample (B) stained immunohistochemically with the F-12 (left-hand column), JC8 (middle column) and E6H4 (right hand column) antibodies to enable field to field comparison. The E6H4 and JC8 antibodies, but not the F-12 antibody, gave qualitatively very similar patterns of staining. The E6H4 and JC8 detected numerous nevus melanocytes (brown staining) in the dermis, whereas only a fraction of these were weakly detected by F-12 (A). Whilst the JC8 and E6H4 antibodies detected fibroblasts (arrows) in a breast sample (B) the F-12 antibody gave cytoplasmic staining in round mononuclear cells which were undetected with the JC8 antibody (arrowheads). The breast and nevi experiments were carried out in independent laboratories using different antibody lots/batches. Magnification bar  = 100 µm.</p

IHC analysis of cervical samples.

<p>Serial sections of three cervical SCC samples stained immunohistochemically with the F-12 (left-hand column), JC8 (middle column) and E6H4 (right hand column) antibodies to enable field to field comparisons. F-12 failed to stain neoplastic structures clearly detected by JC8 and E6H4 in SCC (A) and keratinising SCC (B) samples, and gave a different pattern of cellular staining in tissue peripheral to the neoplastic mass in an adenocarcinoma cervical sample (compare arrows and arrowheads in C). Magnification bar  = 100 µm. For a comparative negative control sections, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053313#pone.0053313.s002" target="_blank">Figure S2</a>.</p

P16INK4A siRNA knockdown.

<p>HeLa cells were transfected with targeting siRNA duplexes 403 and 817, and non-targeting siRNA control duplex AllStars or Lipofectamine 2000 alone. 72 hours post-knockdown cells were fixed and double-labelled with the anti-p16INK4a antibodies (green) H-156, JC8, F-12 and with the TGN46 antibody (red) along with the appropriate secondary AlexaFluor antibodies. Images were separately recorded in the red and green by immunofluorecsence confocal microscopy and merged. Magnification bar  = 20 µm.</p

P16INK4A antibody validation.

<p>P16INK4A subcellular localization by immunofluorescence in HeLa cells using anti-p16INK4a antibodies F-12 (<b>A</b>), H-156 (<b>B</b>), JC8 (<b>C</b>), and E6H4 (<b>D</b>); magnification bar  = 20 µm. <b>E</b>. Comparison of anti-p16INK4a antibodies in Western Blot analysis using p16INK4a positive Hek293 and HeLa whole cell lysates and p16INK4a negative PC3 cell extract (each loaded 20 µg whole protein per well). <b>F</b>. Immunoprecipitation of p16INK4a using F-12 and JC8 mouse monoclonal antibodies, the FLAG mouse monoclonal antibody was used alongside as a negative control.</p

Childlessness and Economic Development: A Survey

This paper provides an introduction to the analysis of childlessness, first by describing the stylized facts and the relevant literature, and then by proposing a theoretical framework. We show that both poverty-driven childlessness and opportunity-driven childlessness matter and are essential to a thorough understanding of childlessness as a socioeconomic phenomenon