Association of Gut Microbiota and Metabolites With Disease Progression in Children With Biliary Atresia.

Background and Aims: Biliary atresia is the most common cause of liver disease and liver transplantation in children. The accumulation of bile acids in hepatocytes and the stimulation of the intestinal microbiome can aggravate the disease progression. This study investigated changes in the composition of the gut microbiota and its metabolites in biliary atresia and the possible effects of these changes on disease progression. Methods: Stool samples of biliary atresia at different disease stages and matched control individuals were collected (early stage: 16 patients, 16 controls; later stage: 16 patients, 10 controls). Metagenomic sequencing was performed to evaluate the gut microbiota structure. Untargeted metabolomics was performed to detect and analyze the metabolites and bile acid composition. Results: A disturbed gut microbiota structure occurred in the early and later stages of biliary atresia. Klebsiella, Streptococcus, Veillonella, and Enterococcus have always been dominant. The abundance of V. atypica displayed significant changes between the early and later stages of biliary atresia. Combined with clinical indicators, Spearman's analysis showed that Klebsiella and Veillonella atypica strongly correlated with liver enzymes. Enterococcus faecium had an enormously positive relationship with lithocholic acid derivatives. Metabolites involved in tryptophan metabolism were changed in the patients with biliary atresia, which had a significant association with stool V. atypica and blood total bilirubin (p < 0.05). Conclusions: The liver damage of biliary atresia was directly or indirectly exacerbated by the interaction of enriched Klebsiella (K. pneumoniae), Veillonella (V. atypica), and Enterococcus (E. faecium) with dysmetabolism of tryptophan and bile acid.

Effects of cytomegalovirus infection on the differential diagnosis between biliary atresia and intrahepatic cholestasis in a Chinese large cohort study.

INTRODUCTION AND OBJECTIVES: Differentiating biliary atresia from other causes of neonatal cholestasis is challenging, particularly when cytomegalovirus (CMV) and biliary atresia occur simultaneously. We aimed to elucidate whether CMV infection would affect the differential diagnosis of biliary atresia and intrahepatic cholestasis. PATIENTS AND METHODS: This retrospective study was conducted among patients with neonatal cholestasis admitted to three tertiary hospitals between January 2010 and August 2019. The clinical characteristics, laboratory, and imaging findings were recorded. On the basis of the CMV serology results, the infants were classified into CMV-IgM (+) and CMV-IgM (-) groups. The clinical differences and diagnostic performances of routine predictors between biliary atresia and intrahepatic cholestasis were analyzed in each group. Finally, we compared the diagnostic performances of various tests in the two groups. RESULTS: A total of 705 patients with neonatal cholestasis were enrolled: 215 (30.5%) patients were positive for CMV-IgM, among whom 97 had biliary atresia and 118 had CMV hepatitis; 490 infants were CMV-IgM (-), among whom 240 had biliary atresia and 250 had intrahepatic cholestasis. The diagnostic performances of stool color, direct bilirubin level, γ-glutamyl transpeptidase level, abnormal gallbladder, triangular cord sign, and hepatobiliary scintigraphy between CMV hepatitis and CMV-IgM (+) biliary atresia were similar to those between CMV-IgM (-) biliary atresia and CMV-IgM (-) intrahepatic cholestasis groups. CONCLUSIONS: Our large-scale study showed a high prevalence of CMV infection in patients with neonatal cholestasis in China. The presence of CMV infection did not affect the routine predictors to discriminate biliary atresia and intrahepatic cholestasis.

Gut microbial profile in biliary atresia: a case-control study.

BACKGROUND AND AIM: Biliary atresia (BA) is a progressive fibro-inflammatory cholangiopathy with an unclear etiology. Various liver disorders are associated with an altered microbiome. However, gut microbiome in BA remains unknown. Here, we performed a case-control study to investigate the gut microbiota in BA. METHODS: A cross-sectional analysis was first conducted for 34 BA patients and 34 healthy controls. Then we investigated the shift in gut microbiota 2 weeks after the Kasai procedure in 16 BA patients. Gut microbiome was initially analyzed using 16S ribosome RNA gene sequencing and further validated by metagenomic sequencing. Fecal bile acids were determined using ultra-high performance liquid chromatography. RESULTS: Compared with healthy controls, BA showed lower diversity and significant structural segregation in the microbiome. At phylum level, Proteobacteria numbers increased, whereas those of Bacteroidetes decreased in BA. At genus level, several potential pathogens such as Streptococcus and Klebsiella thrived in BA, while numbers for Bifidobacterium and several butyrate-producing bacteria declined. The microbiome was also disturbed after the Kasai procedure. Operational taxonomic units responding to BA showed significant correlation with liver function. Furthermore, the abundance ratio of Streptococcus/Bacteroides showed great promise in distinguishing BA from healthy controls. Intestinal bile acids were dramatically decreased in BA, and Clostridium XIVa positively correlated with the ratio of primary/secondary bile acids. CONCLUSIONS: Gut microbial dysbiosis, may be caused by decreased bile acids, was associated with liver function and had a good diagnostic potential for BA. Therefore, further exploration of gut microbiota may provide important insights into their potential diagnostic and therapeutic benefits.

Cxcr2 signaling and the microbiome suppress inflammation, bile duct injury, and the phenotype of experimental biliary atresia.

Biliary atresia is progressive fibro-inflammatory cholangiopathy of young children. Central to pathogenic mechanisms of injury is the tissue targeting by the innate and adaptive immune cells. Among these cells, neutrophils and the IL-8/Cxcl-8 signaling via its Cxcr2 receptor have been linked to bile duct injury. Here, we aimed to investigate whether the intestinal microbiome modulates Cxcr2-dependent bile duct injury and obstruction. Adult wild-type (WT) and Cxcr2-/- mice were fed a diet supplemented with sulfamethoxazole/trimethoprim (SMZ/TMP) during pregnancy and lactation, and their pups were injected intraperitoneally with rhesus rotavirus (RRV) within 24 hours of life to induce experimental biliary atresia. The maternal exposure to SMZ/TMP significantly lowered the incidence of jaundice and bile duct obstruction and resulted in improved survival, especially in Cxcr2-/- mice. Analyses of the microbiome by deep sequencing of 16S rRNA of the neonatal colon showed a delay in bacterial colonization of WT mice induced by SMZ/TMP, with a notable switch from Proteobacteria to Firmicutes. Interestingly, the genetic inactivation of Cxcr2 alone produced a similar bacterial shift. When treated with SMZ/TMP, Cxcr2-/- mice infected with RRV to induce experimental biliary atresia showed further enrichment of Corynebacterium, Anaerococcus and Streptococcus. Among these, Anaerococcus lactolyticus was significantly associated with a suppression of biliary injury, cholestasis, and survivability. These results suggest that the postnatal development of the intestinal microbiota is an important susceptibility factor for experimental biliary atresia.

Unraveling the pathogenesis and etiology of biliary atresia.

Biliary atresia (BA) is the most common and important neonatal hepatobiliary disorder. Because current treatment is inadequate, there is an urgent need to better understand the etiology and pathogenesis of BA. Two major forms of BA are recognized: an embryonic form associated with other congenital anomalies and a perinatal form in which bile ducts were presumably formed normally but underwent fibro-obliteration in the perinatal period. There are currently several proposed pathogenic pathways leading to the phenotype of BA, including an immune or autoimmune response to a perinatal insult (e.g. cholangiotropic viral infection) and dysregulated embryonic development of the extra- or intrahepatic biliary system. Recent advances in developmental biology, genomics and genetics, and cell immunology and biology, coupled with the development of appropriate animal models, have provided support for these postulated mechanisms. Future investigations combining animal model work and evaluation of clinical specimens holds the promise of identifying the etiology of BA and providing a scientific basis for treatment and preventative interventions.

Association of the reovirus S1 gene with serotype 3-induced biliary atresia in mice.

A panel of serotype 3 (T3) reovirus strains was screened to determine their relative capacities to cause lethal infection and hepatobiliary disease following peroral inoculation in newborn mice. A wide range of 50% lethal doses (LD50s) was apparent after peroral inoculation of the different virus strains. Two of the strains, T3 Abney and T3 clone 31, caused mice to develop the oily fur syndrome associated with biliary atresia. The capacity to cause biliary atresia was not related to the capacity to cause lethal infection, however, because the LD50s of T3 Abney and T3 clone 31 were grossly disparate. Examination of liver and bile duct tissues revealed histopathologic evidence of biliary atresia and hepatic necrosis in T3 Abney-infected mice but not in mice inoculated with a T3 strain of similar virulence or with the hepatotropic T1 Lang strain. The consistency with which T3 Abney-infected mice developed biliary atresia-associated oily fur syndrome permitted us to determine the viral genetic basis of reovirus-induced biliary atresia. Analysis of reassortant viruses isolated from an in vitro coinfection with T3 Abney and T1 Lang indicated a strong association of the hepatobiliary disease-producing phenotype with the T3 Abney S1 gene, which encodes the viral cell attachment protein, sigma 1. Amino acid residues within the sigma 1 protein that were unique to disease-producing T3 strains were identified by comparative sequence analysis. Specific changes exist within two regions of the protein, one of which is thought to be involved in binding to host cell receptors. We hypothesize that changes within this region of the protein are important in determining the tropism of this virus for bile-ductular epithelium.

Minimal role of hepatitis C virus infection in childhood liver diseases in an area hyperendemic for hepatitis B infection.

To investigate the role of hepatitis C virus (HCV) in childhood liver disease in Taiwan, an area hyperendemic for hepatitis B, we studied antibody to HCV (anti-HCV) with a second generation enzyme immunoassay in 195 infants and children, including 96 hepatitis B surface antigen (HBsAg) positive children (66 with chronic hepatitis B, 23 children with hepatocellular carcinoma, and 7 with fulminant hepatitis B), 6 children with fulminant non-A, non-B hepatitis, 42 infants with neonatal hepatitis, 11 with biliary atresia, and 40 prospectively followed blood recipients. For comparison, another 748 apparently healthy children (from neonates to 12 years) were also screened for anti-HCV. The positive rate of anti-HCV was low in both apparently healthy children (0.13%) and patients with various liver disorders (0 to 4.4%) except fulminant hepatitis. The seropositive rate in 6 cases of non-A, non-B fulminant hepatitis was higher (16.7%) although the case number was too small. We conclude that HCV is generally not a major etiologic factor in the liver diseases of Taiwanese children.

The pathogenesis of biliary atresia in Japan: immunohistochemical study of HBV-associated antigen.

An immunohistochemical study of HBV-associated antigen in the liver of 16 Japanese infants with biliary atresia revealed positive findings in 13 of the cases for HBc and/or HBs antigens. The positive cells were mainly small liver cells distributed in the peripheral zone of the lobule, and a few lymphocytes were observed in contact with or around the positive liver cells for HBV-associated antigen. Again, HBc antigen was demonstrated almost exclusively in the cytoplasm of positive liver cells. As these findings suggest the possibility of destruction and drop-out of cells constituting the interlobular bile duct in the junctional area by an immunological mechanism, the probability of HBV infection being an important factor in causing and accelerating biliary atresia cannot be denied. Positive findings for HBV-associated antigen similar to those found in biliary atresia are also seen in neonatal hepatitis and choledochal cysts. These conditions are therefore presumed to belong to the same category.

Experimental reovirus type 3-induced murine biliary tract disease.

The aims of this experiment were: (1) to establish a reovirus type 3-induced murine model of biliary atresia/neonatal hepatitis that as far as possible corresponds to the human disease; (2) to demonstrate that the disease is histologically similar to the human disease, and to investigate the natural history of reovirus type 3 infection in this model; (3) to study the host-virus interrelationships at a molecular level; and (4) to develop sensitive assays that could be translated to the human disease. In this study we were unable to produce an exact model for extrahepatic biliary atresia (EHBA) in the laboratory mouse following a perinatal reovirus type 3 infection. However, the ability of reovirus type 3 to persist in the murine liver and the effects produced in the offspring of infected pregnant mice indicate that this preparation may provide the basis for the eventual development of the experimental model of EHBA.

Isolation of respiratory syncytial virus from liver tissue and extrahepatic biliary atresia material.

Respiratory syncytial virus (RSV) was isolated from liver tissue in a 7-month-old infant with extrahepatic biliary atresia. To our knowledge this is the first report of RSV isolation from human liver tissue.

Fecal flora of infants with biliary atresia: effects of the absence of bile on fecal flora.

The fecal flora of nine bottle-fed infants with biliary atresia were examined to determine the effects of the absence of bile on the intestinal flora. The numbers of the following fecal flora were significantly reduced compared with healthy infants: bifidobacteria, lecithinase-negative clostridia, streptococci, and staphylococci. Bifidobacteria were reduced to the greatest extent, constituting 75% of total flora in healthy comparison infants but only 2.4% in infants with biliary atresia. The most prevalent bacteria in infants with biliary atresia were enterobacteria.