IMBALANCED IMMUNOBIOLOGICAL REACTIVITY, INFECTION AND MUTAGENESIS FACTORS IN THE HEPATOBILIARY SYSTEM AFFECTED BY A NATURAL FOCAL HABITAT FACTOR – A TREMATODE INVASION BY OPISTHORCHIS FELINEUS

An interaction between decreasing host anti-infective defense due to long-term invasion with Opisthorchis felineus in the hepatobiliary system, duct bile colonization by microflora and revealing the endogenous mutagenesis (carcinogenesis) factor - secondary bile acids - in bile is considered in the article. The role of organism genotype in the pathogen-related immune response to Opisthorchis felineus trematode and helminth development in the hepatobiliary system has been shown. The role of dysregulated mechanisms of tissue homeostasis in induction of compensatory chronic homeostatic proliferation and somatic cell oncogenesis is discussed. The study results evidence that disturbed functioning of the regulatory T-cells, inhibition of the NK cell effector function and very high functional activity of the memory B-cells are of great importance in imbalanced host immunobiological reactivity, caused by chronic opistorchosis invasion. Decreased host anti-infective protection causes intrahepatic bile duct infection with different bacterial species. Presence of secondary bile acids in hepatobiliary system was associated with biliary bacterial strains inhabiting intestinal tract: Proteus vulgaris*, Proteus mirabilis*, Citrobacter freundii*, Bacteroides alcaligues faecalis*, Clostridium*, Streptococcus faecalis*, Еscherichia coli* (*gut microflora). Participation of microbiota in bile acid biotransformation immediately in the duct bile has been confirmed in in vitro experiments. Experimental methods on Drosophila melanogaster and Salmonella tiphimurium strains: TA 100, TA 98 allowed to find out that bile from chronic opistorchosis patients exerts higher mutagenic activity compared with control groups. Mutational events in somatic and bacterial cells depend on the presence of secondary bile acids (deoxycholic, lithocholic) in duct bile, as well as the level of total bile acid concentration. The study data confirm the concept by Professor A.A. Shain about presence of endogenous risk factor for developing primary cholangiocellular liver cancer such as secondary bile acids in the bile of chronic opistorchosis patients. A concept of cholangiocarcinogenesis, based on mutational events, is added up with disturbance of generative cycle in tissue cells and their differentiation due to decreased kylon factor activity, as well as sensitivity threshold to it. Level of investigation and understanding of mechanisms underlying cholangiocarcinogenesis during chronic opisthorchiasis invasion will allow to develop pathogenetic approaches to correct homeostasis regulation and prevention of cholangiocarcinomas.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS

Objectives Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. Methods Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. Results The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) “Guideline on bioanalytical method validation”. To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. Conclusions Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

1017. Gut Microbiota Diversity and Beneficial Metabolite Production is Reduced in Liver Transplant Recipients and Associated with Post Operative Infection

Background Liver transplant (LT) recipients have abnormal microbiota before and after transplantation. (1,2) Associations between fecal microbiota, microbial metabolites, and clinical outcomes in liver transplantation are not well established. We correlated fecal microbiota composition and metabolite concentrations with early LT outcomes, including infection. Methods In a prospective observational study, we collected peri-transplant fecal samples and determined microbiota composition by 16S ribosomal RNA gene sequencing in LT recipients. Fecal short chain fatty acid (SCFA) and bile acid concentrations were measured by targeted GC- and LC-MS analyses, respectively. Inverse Simpson index was used to determine microbiota alpha-diversity in subjects and healthy controls. Clinical outcomes including length of stay, ICU admission, liver function, antibiotic use, immunosuppressive requirement and post-operative infection were correlated with microbiota composition. Results 69 patients were enrolled, 70 liver transplants were performed and 307 peri-transplant fecal samples were collected and analyzed. Compared to healthy controls, the fecal microbiota of LT recipients had reduced alpha-diversity (p< 0.001). [Fig1] Bacteroidetes, Ruminococcaceae, and Lachnospiraceae, three taxa associated with a health-promoting microbiota, and their metabolites, SCFA and secondary bile acids, were markedly diminished 55% of LT patients.(3) Intestinal domination ( >30% frequency) by Enterococcus or Proteobacteria species was common and occurred in 36% of LT recipients. 76 post-operative infections occurred in 40 LT recipients, with Enterococci causing 52% and Proteobacteria 41% of bacterial infections. In subjects with fecal samples collected within 5 days of infection, 9/17 infections were caused by the organism dominating the microbiota. [Fig2] Microbiota Composition and Metabolite Production 16s gene sequencing color coded by taxonomy. Each bar represents one stool sample nearest to LT compared to healthy controls. Alpha diversity measured by inverse simpson index. Absolute values of microbial metabolites and ratio of primary to secondary bile acids. Comparison of Microbiota Composition and Post Operative Infection All bacterial infections captured with a microbiota sample within 5 days of infection. Conclusion Microbiota diversity and microbially derived metabolites are markedly reduced in >50% of LT recipients. Intestinal domination and post-operative infections caused by antibiotic-resistant Enterococcus and Proteobacteria correlate with loss of Bacteroidetes, Ruminococcaceae, and Lachnospiraceae species, suggesting a potential role for microbiota reconstitution therapy in LT patients. Disclosures Eric G. Pamer, MD;FIDSA, Nothing to disclose

Comparative Genomic and Physiological Analysis against Clostridium scindens Reveals Eubacterium sp. c-25 as an Atypical Deoxycholic Acid Producer of the Human Gut Microbiota

The human gut houses bile acid 7α-dehydroxylating bacteria that produce secondary bile acids such as deoxycholic acid (DCA) from host-derived bile acids through enzymes encoded by the bai operon. While recent metagenomic studies suggest that these bacteria are highly diverse and abundant, very few DCA producers have been identified. Here, we investigated the physiology and determined the complete genome sequence of Eubacterium sp. c-25, a DCA producer that was isolated from human feces in the 1980s. Culture experiments showed a preference for neutral to slightly alkaline pH in both growth and DCA production. Genomic analyses revealed that c-25 is phylogenetically distinct from known DCA producers and possesses a multi-cluster arrangement of predicted bile-acid inducible (bai) genes that is considerably different from the typical bai operon structure. This arrangement is also found in other intestinal bacterial species, possibly indicative of unconfirmed 7α-dehydroxylation capabilities. Functionality of the predicted bai genes was supported by the induced expression of baiB, baiCD, and baiH in the presence of cholic acid substrate. Taken together, Eubacterium sp. c-25 is an atypical DCA producer with a novel bai gene cluster structure that may represent an unexplored genotype of DCA producers in the human gut.

Gut Microbiota Metabolism of Bile Acids Could Contribute to the Bariatric Surgery Improvements in Extreme Obesity

Bariatric surgery is the only procedure to obtain and maintain weight loss in the long term, although the mechanisms driving these benefits are not completely understood. In the last years, gut microbiota has emerged as one of the drivers through its metabolites, especially secondary bile acids. In the current study, we have compared the gut microbiota and the bile acid pool, as well as anthropometric and biochemical parameters, of patient with morbid obesity who underwent bariatric surgery by two different techniques, namely Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Gut microbiota populations differed after the respective procedures, particularly with respect to the Enterobacteriaceae family. Both techniques resulted in changes in the bile acids pool, but RYGB was the procedure which suffered the greatest changes, with a reduction in most of their levels. Blautia and Veillonella were the two genera that more relationships showed with secondary bile acids, indicating a possible role in their formation and inhibition, respectively. Correlations with the anthropometric and biochemical variables showed that secondary bile acids could have a role in the amelioration of the glucose and HDL-cholesterol levels. Thus, we have observed a possible relationship between the interaction of the bile acids pool metabolized by the gut microbiota in the metabolic improvements obtained by bariatric surgery in the frame of morbid obesity, deserving further investigation in greater cohorts to decipher the role of each bile acid in the homeostasis of the host for their possible use in the development of microbiota-based therapeutics, such as new drugs, postbiotics or probiotics.

The intestinal circadian clock drives microbial rhythmicity to maintain gastrointestinal homeostasis

Diurnal (i.e., 24-hour) oscillations of the gut microbiome have been described in various species including mice and humans. However, the driving force behind these rhythms remains less clear. In this study, we differentiate between endogenous and exogenous time cues driving microbial rhythms. Our results demonstrate that fecal microbial oscillations are maintained in mice kept in the absence of light, supporting a role of the hosts circadian system rather than representing a diurnal response to environmental changes. Intestinal epithelial cell-specific ablation of the core clock gene Bmal1 disrupts rhythmicity of microbiota. Targeted metabolomics functionally link intestinal clock-controlled bacteria to microbial-derived products, in particular branched-chain fatty acids and secondary bile acids. Microbiota transfer from intestinal clock-deficient mice into germ-free mice altered intestinal gene expression, enhanced lymphoid organ weights and suppressed immune cell recruitment. These results highlight the importance of functional intestinal clocks for circadian microbiota composition and function, which is required to balance the hosts gastrointestinal homeostasis.

Abnormal Inflammatory Status Promotes Lymphatic Metastasis of Gallbladder Cancer Cells Through the CACUL1/NRF2/ITGBL1/ITGA9/VEGF-C Pathway

Background: Gallbladder cancer (GBC) is a highly fatal disease with poor prognosis, but the aetiology is poorly understood. Long standing gallstone and chronic inflammation had been observed to be risk factors for GBC. Gallstone and chronic infection may lead to increased turnover of primary bile acids to secondary bile acids and inflammatory cytokines, which were known tumor promoters and might be responsible for GBC. But the exact molecular mechanism was not fully understood. Results: Our results showed that gallstones and chronic infection could increase the levels of inflammatory cytokines, and promoted the expression of CACUL1 and ITGBL1. Our study furtherly identified the highly expressed patients of CACUL1 and ITGBL1 had a poor prognosis, and them therefore might serve as a potential prognostic biomarker for GBC patients. CACUL1 promoted the expression of ITGBL1 through NRF2. Furthermore, ITGBL1 promoted the migration and invasion of GBC cells. In addition, ITGBL1 was found to induce lymphangiogenesis through the ITGA9/VEGF-C pathway and promote lymphatic metastasis. Conclusions: Our study provided new insight into GBC pathogenesis that chronic bacterial infection and gallstones leading to the production of carcinogenic precursors and inflammatory cytokines promote lymphangiogenesis and lymph node metastasis of cancer cells through CACUL1/NRF2/ITGBL1/ ITGA9/VEGF-C pathway.

A prevaccination validated network that drives the breadth of the protective neutralizing antibody response following Dengue Vaccine TV003 immunization

Development of fully protective dengue virus (DV) vaccines has been problematic as infection with DV requires a broad antibody immune response that targets all 4 possible serotypes. Herein, we used an integrated systems vaccinology approach to identify prevaccination features that allow the development of fully protective DV-specific antibody responses. This approach allowed us to identify a transcription network in a subset of monocytes defined by the expression of CD68 and downstream of specific pro- and anti-inflammatory cytokines. Moreover, we identified metabolites as drivers of an immune response that induced neutralizing antibodies to the 4 DV serotypes. Specifically, PC/PE drove the production of TGF-B in CD68 low monocytes, which was a positive correlate of the protective antibody response. In contrast, primary and secondary bile acids triggered a proinflammatory response downstream of TGR5 signaling and inflammasome activation in CD68 high monocytes, which was associated to a non-protective antibody response. These features were validated in vitro in primary myeloid cells. Our results highlight the role of cell and systemic metabolism as regulators of protective immune responses to vaccination, and that systems vaccinology is a key tool to identify such mechanisms.

Disease-Associated Gut Microbiota Reduces the Profile of Secondary Bile Acids in Pediatric Nonalcoholic Fatty Liver Disease

Children with nonalcoholic fatty liver disease (NAFLD) display an altered gut microbiota compared with healthy children. However, little is known about the fecal bile acid profiles and their association with gut microbiota dysbiosis in pediatric NAFLD. A total of 68 children were enrolled in this study, including 32 NAFLD patients and 36 healthy children. Fecal samples were collected and analyzed by metagenomic sequencing to determine the changes in the gut microbiota of children with NAFLD, and an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system was used to quantify the concentrations of primary and secondary bile acids. The associations between the gut microbiota and concentrations of primary and secondary bile acids in the fecal samples were then analyzed. We found that children with NAFLD exhibited reduced levels of secondary bile acids and alterations in bile acid biotransforming-related bacteria in the feces. Notably, the decrease in Eubacterium and Ruminococcaceae bacteria, which express bile salt hydrolase and 7α-dehydroxylase, was significantly positively correlated with the level of fecal lithocholic acid (LCA). However, the level of fecal LCA was negatively associated with the abundance of the potential pathogen Escherichia coli that was enriched in children with NAFLD. Pediatric NAFLD is characterized by an altered profile of gut microbiota and fecal bile acids. This study demonstrates that the disease-associated gut microbiota is linked with decreased concentrations of secondary bile acids in the feces. The disease-associated gut microbiota likely inhibits the conversion of primary to secondary bile acids.