All incubations were performed for 30 minutes at room temperature
and followed by a wash in three changes of PBS for 5 minutes. For all immunohistochemical stainings, 3-amino-9-ethylcarbazol (AEC) in 0.01% H2O2 was used as substrate-chromogen. The sections were counterstained with hematoxylin. Negative controls consisted of omission of the primary antibody and were consistently negative. To ensure uniform handling of samples, all sections were processed simultaneously. All immunohistochemically stained slides were evaluated for staining Ibrutinib cost patterns and intensities by four observers (T.R., Y.V., J.W., and L.L.). Histological changes, i.e., portal inflammation; hepatocellular, canalicular, and ductular bilirubinostasis; ductular reaction; steatosis and centrolobular necrosis were graded using a semiquantitative scoring system. BA transporter expression was semiquantitatively graded as compared with what was deemed normal by the pathologist. For the assessment of NRs, intensity of nuclear localized staining was scored. Statistical analysis was performed using Statview 5.0.1 (SAS Institute, Cary, NC). All quantitative
values were assessed for normality. Values with normal distribution, and those that were normalized after logarithmic transformation, are represented as mean ± standard error of the mean (SEM) and were compared using the unpaired Student’s t test. The nonnormally distributed data were represented as medians and interquartile range (IQR) (1st-3rd) and compared by the nonparametric Mann-Whitney U test. Nominal JNK inhibitors high throughput screening and ordinal variables (expressed as numbers and percentages) were compared by Fisher’s exact test. Correlations between variables were calculated using either Pearson’s or Spearman’s rank correlation test. For all comparisons P < 0.05 was deemed significant.
Baseline characteristics of ICU (n = 130) and control (n = 20) patients are described in Table 1. The total ICU population, as well as the subset used for immunohistochemical analysis, was matched with control patients for gender, age, and body mass index (Supporting Data Table 1). Serum total bilirubin on the last day of ICU stay was 8-fold higher in ICU patients than in controls (Table 1) and the hyperbilirubinemia was predominantly conjugated. Compared click here with controls, serum ALP and GGT levels in ICU patients were 1.6- and 3-fold higher, respectively (Table 1). In parallel, serum total BAs were 11-fold higher (P < 0.0001) in ICU patients (Table 1), this increase being mainly attributable to conjugated BAs (Table 2). There was no effect of tight glycemic control on circulating bilirubin or BA levels. There was an increase in conjugation percentage for the primary BA cholic acid (CA) (98.3% in patients versus 55.6% in controls) and chenodeoxycholic acid (CDCA) (95.9% in patients versus 37.