Determination of bile acids from human gallbladder by 1 H-MRS-Protocol optimization and estimation of reproducibility.

Bile exerts multiple functions in the liver and gut and is involved in multiple disease processes. It is secreted continuously from the liver and stored in the gallbladder until needed, and closely reflects the available bile acid pool. The study objective was therefore to develop a reliable MRS protocol and to assess variability of bile acid determination in human gallbladder. MRS measurements were performed on a 3 T MR scanner with 20 subjects to optimize protocols (26 measurements) and conduct a prospective reproducibility study (18 measurements). Measurements were carried out with subjects lying in either supine (23 scans) or prone positions (21 scans) to compare results from the two positions. For reproducibility determination, six of the 20 volunteers (three males, three females, age = 34.9 ± 10.9 years, BMI = 23.4 ± 2.1 kg/m2 ) were measured three times: back to back to assess technical variability and once again after three weeks to assess total variability, including additional physiological variability. A single voxel was measured in the gallbladder with respiratory triggering. For quantification, apparent T2 times were determined and a non-water-suppressed spectrum was acquired. Total bile acids, glycine and taurine conjugated bile acids, and lipids including choline-containing phospholipids were determined. Higher quality and reliability of gallbladder spectra were obtained with subjects measured in prone compared with supine position. All measurements of the reproducibility sub-study were of sufficient quality to be included in the analysis. Average coefficients of variation within subjects for the main compounds were 37% for total variation (including physiological and technical variation) and 24% for technical variation alone. These values were much smaller than those between subjects, which were >54% for both back-to-back and three weeks separated measurements. These results suggest diagnostic applicability of the method, especially for longitudinal studies aiming at non-invasive characterization of bile composition in humans with various diseases and/or interventional maneuvers

Kaplan-Meier survival curves for autophagy markers in post-treatment tumor tissue of a neo-adjuvant EAC cohort.

<p>(A) LC3B dot-like staining patterns, (B) p62 dot-like staining patterns (C) groupings of LC3B dot-like/p62 dot-like-cytoplasmic expression: Low LC3B/low p62 (LL), low LC3B/high p62 (LH), high LC3B/low p62 (HL) and high LC3B/high p62 (HH); and (D) LC3B dot-like/p62 dot-like-cytoplasmic expression LH versus remainder of all other cases. For each curve the p-value is displayed on the bottom right-hand corner.</p

Comparison of expression of autophagy markers LC3B and p62 in a primary resected EAC cohort and a subcohort of neo-adjuvant chemotherapy (nCTX) treated EAC cases with paclitaxel containing regimens.

<p>Significance was set to 0.05. Statistically significant p-values are shown in bold.</p

Examples of immunohistochemical stainings.

<p>(a) High LC3B dot like staining (score 2). (b) Low LC3B dot like staining (score 1), note a small nerve serving as internal positive control. (c) High p62 cytoplasmic staining (score 3), while negative nuclear staining. (d) Low p62 cytoplasmic/dot-like staining (scores 0), positive nuclear staining. (e) High cytoplasmic (score 2) and low dot-like (score 1) p62 staining. (f) Low cytoplasmic (score 1) and high dot like (score 2) p62 staining. 40x magnification for all images. Error bars indicate 20µm.</p

Cytoplasmic expression of p62 results in decreased responsiveness of EAC cells to paclitaxel.

<p>(a) OE19 p62 knockdown cells were transiently transfected with either a cytoplasmic or nuclear GFP-tagged p62 expression plasmid. GFP (GFP-p62 fusion proteins) and nuclear DAPI staining as analyzed by confocal microscopy are shown. (b) Annexin V/DAPI fluorescence-activated cell sorting (FACS) analysis of OE19 cells expressing cytoplasmic or nuclear p62 after 48 h of paclitaxel treatment. Bars represent four experimental replicates.</p

EAC cell lines exhibit differential response to paclitaxel treatment.

<p>Relative cell viability upon treatment with paclitaxel in increasing concentrations (0, 2.5, 5, 10, 20 and 40nM) was assessed using the Alamar Blue assay in OE19, FLO-1, OE33 and SK-GT-4 after 24hr (a) and 48hr (b). Error bars indicate the standard deviation of three independent experiments. The DMSO equivalent of the highest final concentration was added to the untreated condition as vehicle control and relative toxicity values were normalized to the untreated controls which were set to 100%.</p

No statistically significant independent prognostic factor was identified in a neo-adjuvant chemotherapy treated EAC cohort.

<p>HR–Hazard Ratio.</p

Comparison of expression of autophagy markers LC3B and p62 in primary resected and neo-adjuvant chemotherapy (nCTX) treated EAC cohorts.

<p>Significance was set to 0.05. Statistically significant p-values are shown in bold.</p

Differential response to paclitaxel is not associated with differential autophagy regulation.

<p>OE19, FLO-1, OE33 and SK-GT-4 were treated with paclitaxel, in a final concentration of 20nM, for 24hr with or without the addition of the late stage autophagy inhibitor BafA (200nM) for the last 2hr of the 24hr paclitaxel treatment. LC3B was visualized using Western blotting; total protein was used as loading control. (a) Representative blots of LC3B in all four cell lines, the LC3B-I isoform is not equally visible in all cell lines at the given exposures. (b) Quantification of the LC3B-II normalized to the total protein. Error bars indicate the standard deviation of three independent experiments. Statistical significance was not reached when conditions where compared to one another. (c) WIP1 and LC3B mRNA was assessed via qPCR upon treatment with paclitaxel at 20nM and 40nM for 24hr in OE19 and OE33. Nutrient starvation, achieved with 6hr incubation with EBSS, was included in the experimental setup as a positive control for a known autophagy inducer. Fold change was normalized to mRNA levels of housekeeping gene HBSS. The DMSO equivalent of the highest final concentration of paclitaxel was added to the untreated condition as vehicle control and relative values were normalized to the untreated controls which were set to 1. Error bars indicate the standard deviation of three independent experiments.</p