OBJECTIVETo study the specific binding of the artificial clonal aryl hydrocarbon receptor translocator (ARNT) with the natural aryl hydrocarbon receptor (AhR) and the recolonization by polyclonal antibody. The dose-response relationship with tetrachlo-rodibenzo-dioxin (TCDD) was also studied to develop TCDD detection method and the binding degree related to dose response.

METHODS(1) The target genes including AhR-PAS, AhR-C and ARNT-PAS were amplified by RT-PCR by using the total RNA purified from the liver cells of C57BL/6J mice as templates to construct pGEX-5X1 recombinants. The recombinant plasmids were expressed in E. coli. (2) The rabbits were immuned by the clonal fusion proteins: AhR-PAS, AhR-C to prepare the polyclonal antibody. (3) The natural AhR from the hepatic cytosol of C57BL/6J mice was extracted. The artificial cloning expressed fusion protein:GST-ARNT-PAS and the natural AhR were incubated in different dose of TCDD. The quantity of the heterodimer through affinity adsorption and Western blots were measured.

RESULTS(1) The target proteins including AhR-PAS, AhR-C and ARNT-PAS were successfully cloned and expressed in E. coli. (2) The detection limit of polyclonal antibody AhR-PAS and AhR-C were 5 ng and 1 ng, respectively. (3) The total protein concentration prepared from the liver cells was 60.5 mg/ml. The artificial clonal protein ARNT-PAS could specifically bind to the natural AhR complex with the existence of TCDD. The detection limit of TCDD was 0.25 pmol which was 80 pg approximately.

CONCLUSIONA TCDD detection method based on the aryl hydrocarbon receptor system was established and the detection limit might reach pg grade.

Animals ; Cells, Cultured ; Limit of Detection ; Liver Extracts ; chemistry ; Mice ; Mice, Inbred C57BL ; Polychlorinated Dibenzodioxins ; analysis ; Rabbits ; Receptors, Aryl Hydrocarbon ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: Liver biopsy for the diagnosis of non-alcoholic steatohepatitis (NASH) is limited by its inherent invasiveness and possible sampling errors. Some studies have shown that cytokeratin-18 (CK-18) concentrations may be useful in diagnosing NASH, but results across studies have been inconsistent. We aimed to identify the utility of CK-18 M30 concentrations as an alternative to liver biopsy for non-invasive identification of NASH. METHODS: Individual data were collected from 14 registry centers on patients with biopsy-proven non-alcoholic fatty liver disease (NAFLD), and in all patients, circulating CK-18 M30 levels were measured. Individuals with a NAFLD activity score (NAS) ≥5 with a score of ≥1 for each of steatosis, ballooning, and lobular inflammation were diagnosed as having definite NASH; individuals with a NAS ≤2 and no fibrosis were diagnosed as having non-alcoholic fatty liver (NAFL). RESULTS: A total of 2571 participants were screened, and 1008 (153 with NAFL and 855 with NASH) were finally enrolled. Median CK-18 M30 levels were higher in patients with NASH than in those with NAFL (mean difference 177 U/L; standardized mean difference [SMD]: 0.87 [0.69-1.04]). There was an interaction between CK-18 M30 levels and serum alanine aminotransferase, body mass index (BMI), and hypertension ( P  < 0.001, P  = 0.026 and P  = 0.049, respectively). CK-18 M30 levels were positively associated with histological NAS in most centers. The area under the receiver operating characteristics (AUROC) for NASH was 0.750 (95% confidence intervals: 0.714-0.787), and CK-18 M30 at Youden's index maximum was 275.7 U/L. Both sensitivity (55% [52%-59%]) and positive predictive value (59%) were not ideal. CONCLUSION This large multicenter registry study shows that CK-18 M30 measurement in isolation is of limited value for non-invasively diagnosing NASH.
Humans ; Non-alcoholic Fatty Liver Disease/diagnosis* ; Keratin-18 ; Biomarkers ; Biopsy ; Hepatocytes/pathology* ; Apoptosis ; Liver/pathology*