Biliary hCGβ Is a Potential Novel Marker for Prediction of Biliary Neoplasia in Primary Sclerosing Cholangitis Patients

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease, which is associated with an increased risk of cholangiocarcinoma (CCA). Novel markers, to complement or replace CA19-9, are urgently needed for the screening of PSC-associated biliary neoplasia. Previous studies have suggested that serum trypsinogen-2 and human chorionic gonadotropin β-subunit (hCGβ) may serve as such markers. Using highly specific in-house immunoassays, we studied trypsin(ogen)-2 and -3, SPINK1 and hCGβ in bile samples of 214 patients, referred for endoscopic retrograde cholangiography. We found that biliary trypsinogen-2 was decreased (p = 0.027) and hCGβ was elevated (p < 0.001) in PSC patients who were diagnosed 1.6 years (median, range 0.1–8.8 years) later with CCA or in whom biliary dysplasia was observed at least twice in brush cytology (n = 11) as compared to PSC patients without CCA or repeated dysplasia (n = 171). The other studied markers did not show significant differences between these groups. Our results warrant further evaluation of hCGβ as a predictive marker for PSC-associated biliary neoplasia.

Application of DNA Barcoding for the Identification of a Traditional Chinese Medicine Shedan

Background Shedan has a long history of application in Traditional Chinese medicine (TCM), however, Shedan from different original source has been indiscriminately used. So far, there is still a lack of an effective tool to differentiate the original source of Shedan medicinal materials, which brings great risk to the safety and effectiveness of clinical applications. Hence, it is imperative to develop a practicable approach to identify Shedan medicinal materials. Methods The specificity of two pairs of primers, including Folmer’s universal primers and a pair of originally designed primers COISNFF/COISNFR, was tested to screen the more specific primers for further origin identification of Shedan. A total of 253 fresh snake gallbladder samples from 31 morphologically identified snake species were collected and authenticated. Moreover, 51 fresh snake bile samples and 17 fresh bile samples from five other common domestic poultry and livestock (cattle, chicken, duck, pig and sheep) were collected and distinguished using the more specific primers. Additionally, a total of 195 market Shedan samples randomly selected from 18 batches of Shedan medicinal materials were investigated. Sequence definition was executed by querying sequence similarities in GenBank and the Barcode of Life Data System (BOLD), respectively. Results It turned out that the standard COI barcode obtained by COISNFF/COISNFR primers, rather than Folmer’s universal primers, can distinguish all the testing samples from each other in fresh Shedan samples, and COISNFF/COISNFR primers were also specific to snake species and the other four animal species except duck. In terms of market Shedan, 84.6% (165/195) samples can be attributed to 13 snake species from four families and 4.6% (9/195) can be attributed to adulterated chicken species. Conclusion The COI-based DNA barcoding was practicable for species identification of Shedan used in traditional Chinese medicine. The original source of current market Shedan, including adulterated species, has been preliminarily clarified, which provides a foundation for quality control of Shedan medicinal materials.

Corrective effects of benzodiazepine derivative – diazepinone on purine and lipid metabolism in the liver of rats with Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The cause of PD is not fully understood, and effective treatments still do not exist. It is believed that oxidative stress, mitochondrial dysfunction, and impaired lipid metabolism may underlie the pathogenesis of PD. Bile contains the breakdown products of various compounds that form in hepatocytes. This study aimed to evaluate the effect of a new benzodiazepine derivative - diazepinone (DP) on purine and lipid metabolism in the liver of rats with PD caused by rotenone (ROT) by studying the composition of bile. The concentration of ATP, ADP, AMP, xanthine, hypoxanthine, phospholipids (PL), cholesterol (CHOL), cholesterol esters (ECHOL), free fatty acids (FFA), and triglycerides (TG) was quantified in bile samples by thin-layer chromatography. Our findings suggested that the ratio of AMP/ ATP in bile increased almost threefold under the influence of ROT, and with DP, it exceeded the norm by only 1.6 times. ROT also increased the content of xanthine and hypoxanthine by 28.6% and 66.7%, respectively. DP did not affect the increased xanthine content relative to control but significantly reduced the level of hypoxanthine (up to 22.2%, above normal). In addition, ROT reduced the content of bile PL, CHOL, ECHOL, TG by 23.9%, 38.6%, 47.5%, 39.2 %, respectively. Under the influence of the DP, all the above indicators returned to the level of control. Thus, diazepinone improves both the metabolism of purines and lipids in the liver of rats with ROT-simulated PD. This drug may become a therapeutic agent for treating PD and possibly other neurodegenerative diseases in the future.

Prevalence of Hepatitis E Virus Infection among Laboratory Rabbits in China

Hepatitis E virus (HEV) is zoonotic and the leading cause of acute viral hepatitis worldwide. Rabbit HEV can infect humans and is prevalent globally. It is reported that laboratory rabbits are also naturally infected with HEV. Therefore, it is important to investigate in a large scale the prevalence of HEV in laboratory rabbits. Serum samples were collected from 649 laboratory rabbits of 13 different commercial vendors in Beijing, China, from 2017 to 2019, and anti-HEV and HEV antigen (Ag) were tested. Fecal samples were collected from 50 laboratory rabbits from one of the vendors for HEV RNA detection. Six laboratory rabbits with natural HEV infection were euthanized and their liver, kidney, bile and urine samples were collected for HEV RNA quantification. Liver tissues were subjected to histopathology analysis. The overall positive rates of anti-HEV antibodies and HEV-Ag are 2.6% (15/588) and 7.9% (51/649), respectively. HEV RNA was detected in 12.0% (6/50) of the rabbits. High viral load of HEV RNA was detected in liver and bile samples. Liver inflammation was observed. HEV is circulating in laboratory rabbit population in China. Strict screening is crucial to ensure experimental accuracy and prevent zoonotic transmission to research personnel.

Evaluation of high-resolution melt curve analysis for rapid differentiation of Campylobacter hepaticus from other species in birds

Spotty liver disease (SLD) is a bacterial disease of chicken, causing mortalities and reduction in egg production, hence, contributing to economic loss in the poultry industry. The causative agent of SLD has only recently been identified as a novel Campylobacter species, Campylobacter hepaticus. Specific primers were designed from the hsp60 gene of Campylobacter hepaticus and PCR followed by high-resolution melt curve analysis was optimised to detect and differentiate three species of Campylobacter (Campylobacter coli, Campylobacter jejuni and Campylobacter hepaticus). The three Campylobacter species produced a distinct curve profile and was differentiated using HRM curve analysis. The potential of the PCR-HRM curve analysis was shown in the genotyping of 37 Campylobacter isolates from clinical specimens from poultry farms. PCR-HRM curve analysis of DNA extracts from bile samples or cultures from bile samples, were identified as Campylobacter hepaticus and confirmed by DNA sequencing. The DNA sequence analysis of selected samples from each of the three HRM distinctive curves patterns showed that each DNA sequence was associated with a unique melt profile. The potential of the PCR-HRM curve analysis in genotyping of Campylobacter species was also evaluated using faecal specimens from 100 wild birds. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Campylobacter species using either bacterial cultures or clinical specimens.

Bile Microbiota in Liver Transplantation: Proof of Concept Using Gene Amplification in a Heterogeneous Clinical Scenario

Objective: Historically, bile in the biliary tract has been considered sterile. Most of the series are based on patients with biliary tract diseases or the bile has been obtained with procedures susceptible to contamination.Methods: We evaluated the bile in a heterogeneous cohort of liver donors and recipient patients, with samples obtained in a sterile way, directly from the gallbladder and the common bile duct.Results: We assessed the bile microbiota in six liver donors and in six liver recipients after whole or split liver procedures in adult or pediatric recipients. Bile samples were studied using PCR sequencing of the 16S ribosomal RNA gene amplification (rDNA).Conclusions: We demonstrated that the bile is sterile, thereby ruling this out as a source of contamination following transplant.

Biliary Microbial Structure of Gallstone Patients With a History of Endoscopic Sphincterotomy Surgery

The biliary microbiota is related to the pathogenesis of human bile duct stones. However, the extent to which a history of invasive endoscopic sphincterotomy (EST) affects the biliary bacterial community remains largely unknown. We collected bile samples from the common bile duct of 100 choledocholithiasis patients. We performed 16S rRNA sequencing to investigate and compare the biliary microbial community. The patients without antibiotic treatment (AT) were grouped into three clusters based on their biliary microbial compositions. The patients with a history of EST were significantly enriched in one cluster mainly consisting of gastrointestinal bacteria compared with the other two clusters consisting of oral and environmental bacteria. The β-diversities of patients with and without EST were also significantly different, whereas the α-diversities were comparable. The only significantly enriched bacterial genus associated with a history of EST was Pyramidobacter, while eight other genera were significantly decreased. For patients with AT, seven of these genera maintained their association with EST, including Pyramidobacter. However, after AT, the difference in β-diversities was diminished. EST induced a marked shift in the biliary microbial composition. A cluster of biliary bacteria was associated with a history of EST, and Pyramidobacter was specific to EST.

Microbiomic profiles of bile in patients with benign and malignant pancreaticobiliary disease.

417 Background: In pancreaticobiliary (PB) cancers, there is a paucity of data on predictive and pathophysiologic role of the biliary microbiome. We analyzed bile collected from patients with benign and malignant PB diseases to identify microbiomic signatures associated with malignancy. Methods: We collected bile samples from consenting patients during routine endoscopic retrograde cholangiopancreatography at the Cleveland Clinic, approved by the Institutional Review Board. DNA was extracted from bile specimens using PowerViral RNA/DNA Isolation kit. Bacterial 16S rRNA gene amplification and library construction were performed according to the 16S Metagenomic Sequencing Library Preparation guide from Illumina. Post-sequencing analysis was done using QIIME (Quantitative Insights Into Microbial Ecology), Bioconductor phyloseq, microbiomeSeq and mixMC packages. Results: Of 46 enrolled patients, 32 had PB cancers including pancreatic (N = 25), cholangiocarcinoma (N = 6), and gallbladder (N = 1). The rest (N = 14) had benign PB diseases including acute and chronic pancreatitis, and gallstones. Using multivariate approach in mixMC to classify Operational Taxonomic Units (OTUs), we found a predominance of genera Dicekeya (p = 0.0002), [ Eubacterium] hallii group (p = 0.0007) , Bacteroides (p = 0.00099) , Faecalibacterium (p = 0.007) , Facklamia (p = 0.013) , Peptococcus (p = 0.013) , Bergeyella (p = 0.0024) , Lachnospira (p = 0.026) , and Lactobacillus (p = 0.025) in bile samples from PB cancers as compared to benign PB diseases. Furthermore, bile samples from patients with pancreatic cancer showed an increased abundance of genera Enterobacter, Parabacteroides, Atopobium, Alloprevotella, Prevotella 7, Acinetobacter, Bergeyella, Clostridium sensu stricto, Lactobacillus, and Rothia; and a decreased abundance of genera Tannerella, Peptococcus, Colinsella, Capnocytophaga, Achromobacter, Ruminococcus 2, Bacteroides, Alistipes, Barnesiella,, Lachnoclostridium, Lautropia, Akkermansia, and Christensenellaceae R-7 group as compared to bile samples from patients with cholangiocarcinoma. Conclusions: Distinct microbiome signatures are associated with benign and malignant PB diseases. There is a difference in the relative abundance of OTUs in bile samples between patients with benign PB diseases vs PB cancers, and between pancreatic cancer vs cholangiocarcinoma. Our findings raise the possibility that either these OTUs have a role in carcinogenesis, or that changes in the microenvironment of benign PB diseases differ from PB cancers leading to distinct separation of the OTU clusters. Further studies to explore and validate our findings are needed.

Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: Study protocol

Background The performance of the microbiota is observed in several digestive tract diseases. Therefore, reaching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, tests and therapies in hepatobiliopancreatic diseases. Methods Bile samples will be collected in endoscopic retrograde cholangiopancreatography patients (case group) and living liver transplantation donors (control group). We will characterize the microbiome based on two types of sequence data: the V3/V4 regions of the 16S ribosomal RNA (rRNA) gene and total shotgun DNA. For 16S sequencing data a standard 16S processing pipeline based on the Amplicon Sequence Variant concept and the qiime2 software package will be employed; for shotgun data, for each sample we will assemble the reads and obtain and analyze metagenome-assembled genomes. Results The primary expected results of the study is to characterize the specific composition of the biliary microbiota in situations of disease and health. In addition, it seeks to demonstrate the existence of changes in the case of illness and also possible disease biomarkers, diagnosis, interventions and therapies in hepatobiliopancreatic diseases. Trial registration NCT04391426. Registered 18 May 2020, https://clinicaltrials.gov/ct2/show/NCT04391426.