Progress in Biomarkers Related to Biliary Atresia.

Biliary atresia (BA) is a congenital cholestatic disease that can seriously damage children's liver function. It is one of the main reasons for liver transplantation in children. Early diagnosis of BA is crucial to the prognosis of patients, but there is still a lack of reliable non-invasive diagnostic methods. Additionally, as some children are in urgent need of liver transplantation, evaluating the stage of liver fibrosis and postoperative native liver survival in children with BA using a straightforward, efficient, and less traumatic method is a major focus of doctors. In recent years, an increasing number of BA-related biomarkers have been identified and have shown great potential in the following three aspects of clinical practice: diagnosis, evaluation of the stage of liver fibrosis, and prediction of native liver survival. This review focuses on the pathophysiological function and clinical application of three novel BA-related biomarkers, namely MMP-7, FGF-19, and M2BPGi. Furthermore, progress in well-known biomarkers of BA such as gamma-glutamyltransferase, circulating cytokines, and other potential biomarkers is discussed, aiming to provide a reference for clinical practice.

NLRP3 inflammasome activation promotes liver inflammation and fibrosis in experimental biliary atresia.

BACKGROUND: Biliary atresia (BA) is characterized by a progressive fibroinflammatory cholangiopathy in early infants with unknown etiology. Although innate immune disorder is involved in its mechanism, role of NLRP3 inflammasome in BA remains largely undefined. AIM: To explore the role of NLRP3 inflammasome in BA. METHODS: The expressions of NLRP3 inflammasome-related genes were determined in BA patients. Role of NLRP3 inflammasome was evaluated using MCC950 in experimental BA. Furthermore, gadolinium chloride, a macrophage scavenger, was applied to validate the inflammasome's cellular localization. Finally, the effects of NLRP3 inflammasome activation on liver fibrosis were explored in vivo and vitro in experimental BA. RESULTS: The components of NLRP3 inflammasome were up-regulated in BA patients. Inflammasome-related genes showed positively correlated with liver inflammation and fibrosis in BA patients. In experimental BA, inflammasome-related genes were up-regulated, and their expressions were inhibited by MCC950, which promoted mice growth, protected liver function, alleviated obstructive jaundice, inhibited liver inflammation, and reduced serum IL-1ß level. NLRP3 inflammasome was expressed in macrophages, and macrophage elimination exerted the same protective roles as MCC950 did in BA. Additionally, NLRP3 inflammasome activation promoted liver fibrosis in experimental BA. CONCLUSIONS: NLRP3 inflammasome activation in macrophages promoted liver inflammation and fibrosis in experimental BA.

Single-cell RNA sequencing of solid pseudopapillary neoplasms of the pancreas in children.

Solid pseudopapillary neoplasm (SPN) of the pancreas is a rare pancreatic tumor in children. Its origin remains elusive, along with its pathogenesis. Heterogeneity within SPN has not been previously described. In addition, low malignant but recurrent cases have occasionally been reported. To comprehensively unravel these profiles, single-cell RNA sequencing was performed using surgical specimens. We identified the cell types and suggested the origin of pancreatic endocrine progenitors. The Wnt/ß-catenin pathway may be involved in tumorigenesis, while the epithelial-to-mesenchymal transition may be responsible for SPN recurrence. Furthermore, NOV, DCN were nominated as primary and S100A10, MGP as recurrent SPN marker genes, respectively. Our results provide insight into the pathogenesis of SPN.

Intestinal fibrosis in aganglionic segment of Hirschsprung's disease revealed by single-cell RNA sequencing.

BACKGROUND: Hirschsprung's disease (HSCR) is a relatively common congenital disability. Accumulating extracellular matrix (ECM) prompts intestinal fibrosis remodelling in the aganglionic segments of HSCR. The contributions of various cellular subsets in the fibrogenesis of HSCR segments are poorly understood. METHODS: Single-cell transcriptomics from 8 aganglionic segments and 5 normal segments of 7 HSCR subjects and 26 healthy segments of seven healthy donors were analysed. Fibrotic phenotype and alterations were explored using differential expression analysis and single-cell trajectory analysis. Fibrosis-related transcription factors were inferred through single-cell regulatory network inference. Bulk transcriptomic data, proteomic data, immunohistochemistry (IHC) and real-time polymerase chain reaction were used to validate the alterations in the HSCR intestine. RESULTS: Various collagen, fibronectin and laminin protein-coding genes expression were up-regulated in the stromal and glial cells of the HSCR intestine. The number of fibroblasts and myofibroblasts in the aganglionic segments increased, and more myofibroblasts were activated at an earlier stage in HSCR segments, which infers that there is an intestinal fibrosis phenotype in HSCR segments. The fibrotic regulators POSTN, ANXA1 and HSP70 were highly expressed in the ECM-related cellular subsets in the transitional segments and aganglionic segments. The transcription factor regulatory network revealed that fibrosis-related and megacolon-related NR2F1 in the fibroblasts and glial subsets was up-regulated in the aganglionic segment. CONCLUSIONS: This work identifies intestinal fibrosis and related regulators in aganglionic segments of HSCR; hence, anti-fibrotic therapy may be considered to prevent HSCR-associated enterocolitis (HAEC), relieve intestinal stricture and improve cell therapy.

Integrative analysis implicates the significance of m6A in the liver fibrosis of biliary atresia by regulating THY1.

Whether N6-methyladenosine (m6A) is involved in biliary atresia (BA) remains undefined. Herein, we comprehensively evaluated the m6A profile in BA. When compared with normal controls, BA had an elevated m6A level with upregulated m6A writers. The m6A level was correlated with liver function, stage of fibrosis and jaundice clearance in BA. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) demonstrated an altered m6A topology in BA. MeRIP-seq and RNA sequencing filtered out 130 m6A-modified genes, which were enriched in fibrogenetic pathways. MeRIP-qPCR in vivo and interventions of LX-2 and primary HSCs in vitro validated the regulatory role of m6A on COL1A1 and THY1. THY1+ myofibroblasts expanded in portal area of BA, and highly expressed profibrogenic genes (COL1A1, MMP2, PDGFRA, and DCN). THY1 was correlated with liver fibrosis and jaundice clearance in BA. Bulk array (GSE46960, GSE15235), single-cell RNA sequencing (GSE136103), primary HSC interventions, and co-immunoprecipitation revealed that THY1 was correlated with extracellular matrix organization, promoted HSC activation, showed higher interactions with integrins on myeloid cells in cholestatic fibrosis, and was correlated with native liver survival in BA. Our study highlights the significance of m6A in BA-induced liver fibrogenesis by regulating THY1, shedding new light on the novel therapies to alleviate liver fibrosis by targeting m6A/THY1 axis in BA.

Preferred diagnostic methods of pyriform sinus fistula in different situations: A systematic review.

PURPOSE: Pyriform sinus fistula (PSF) diagnosis is often easily delayed and incorrect. Diagnostic values of modalities vary in different situations. The aim of this study was to recommend optimal schemes for diagnosing PSF at different ages and infection stages. METHODS: A search of PubMed, Embase, Cochrane Library, and CBM databases was conducted to identify articles written in Chinese and English concerning PSF diagnosis using keywords: "pyriform sinus fistula", "diagnosis", and relevant synonymous terms. Quality assessment was performed using the Joanna Briggs Institute (JBI) levels of evidence and critical appraisal checklist tool. RESULTS: 111 studies describing 3692 patients were included. The highest true positive rate (TPR) of ultrasonography was 66.67 % in adult cases. Computed tomography (CT) yielded a good TPR (approximately 73 %) in both neonatal and adult patients, and contrast-enhanced CT (84.21 %) was better in adult patients. Most children cases could be accurately diagnosed by barium swallow (BS) examination which was significantly different in acute and non-infection stages (AIS, NIS). Magnetic resonance imaging (MRI) produced a nice TPR in fetal cases (69.23 %) and neonatal cases (54.44 %). Laryngoscopy was also affected by infection stages. TPR of gastroscopy (GS) was the highest in children (86.36 %) and adult cases (87.50 %). CONCLUSION: For fetal cases suspected of PSF, an MRI is recommended. MRI or CT is preferred for neonatal cases regardless of infection stages. Children and adult patients are advised to undergo GS during NIS or AIS, while BS is suggested for NIS. Contrast-enhanced CT can also diagnose adults with PSF in AIS.

VEGFA rs3025039 and biliary atresia susceptibility in Chinese population: a systematic review and meta-analysis.

Background: Previous studies have suggested an association between vascular endothelial growth factor A (VEGFA) rs3025039 polymorphism and biliary atresia (BA). However, this conclusion is controversial and there is no published pooled evidence of this association. Methods: This study was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. The protocol was registered with PROSPERO (International Prospective Register of Systematic Reviews). A thorough search was performed on databases including PubMed, Embase, and Chinese Biomedical Database up to August 2020. This study included 846 cases of BA and 2821 controls concerning VEGFA rs3025039 polymorphism. We selected relevant studies based on the following inclusion criteria: (1) the study design was case-control and cohort and (2) the patients carried standard clinical diagnoses of BA, etc. The exclusion criteria were as follows: (1) patients with other related diseases, (2) lack of requisite information and (3) duplicate data. The OR (odd ratio) and the corresponding 95% CI (confidence interval) were calculated to estimate the association. Results: This study on VEGFA rs3025039 polymorphism in the Chinese population included 846 cases and 2821 controls. The results showed that there was no significant association between rs3025039 and susceptibility to BA under four genetic models. The results of the subgroup analysis were similar to the overall results. Conclusions: This meta-analysis shows that rs3025039 was not associated with susceptibility to BA in the Chinese population. Further validation may entail additional research. PROSPERO registration number: CRD42020203812.

Single-cell and spatial transcriptomics reveal the fibrosis-related immune landscape of biliary atresia.

BACKGROUND: Biliary atresia (BA) is a devastating inflammatory and fibrosing cholangiopathy of neonates with unknown aetiology. We aim to investigate the relationship between these two main characteristics. METHODS: Single-cell RNA sequencing and spatial transcriptomics were performed on liver samples from a cohort of 14 objects (BA: n = 6; control: n = 8). We conducted data integration and cell-type annotation based on gene expression profiling. Furthermore, we identified fibrosis-related immune cells according to their spatial locations, GO and KEGG analysis. Finally, SPOTlight and CIBERSORTx were used to deconvolute ST data and microarray data of the GSE46960 cohorts, respectively. RESULTS: Immune subpopulations inhabiting the 'fibrotic niche' (areas of scarring), comprising 'intermediate' CD14++ CD16+ monocytes, scar-associated macrophages, natural killer T cells, transitional B cells and FCN3+ neutrophils were identified. GO and KEGG analyses showed that pathways including 'positive regulation of smooth muscle cell/fibroblast proliferation' and 'positive regulation of/response to VEGFR/VEGF/EGFR/FGF' were enriched in these cell types. Interactions analysis showed that communication among 'FGF_FGFR', 'RPS19-C5AR1', 'CD74_COPA/MIF/APP' and 'TNFRSF1A/B_GRN' was extensive. Finally, the results of deconvolution for ST data and microarray data validated that the proportions of certain identified fibrosis-related cell types we identified were increased in BA. DISCUSSION: Fibrosis is an important feature of BA, in which the immune system plays an important role. Our work reveals the subpopulations of immune cells enriched in the fibrotic niche of BA liver, as well as key related pathways and molecules; some are highlighted for the first time in liver fibrosis. These newly identified interactions might partly explain why the rate of liver fibrosis occurs much faster in BA than in other liver diseases. CONCLUSION: Our study revealed the molecular, cellular and spatial immune microenvironment of the fibrotic niche of BA.

The Cellular and Molecular Landscape of Synchronous Pediatric Sialoblastoma and Hepatoblastoma.

Sialoblastoma (SBL) is an infrequent embryonal malignant tumor originating from the salivary gland, resembling primitive salivary gland anlage, whereas hepatoblastoma (HB) is the most common pediatric liver malignancy. The simultaneous occurrence of both tumors is extremely rare. Here we reported a case of a 6-month-old infant diagnosed with synchronous SBL and HB. The patient received neoadjuvant chemotherapy followed by surgical resection. Fresh tissues of both tumors were collected before and after chemotherapy, which were further profiled by whole exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq). WES analysis revealed potential somatic driver mutation PIK3CA p.Glu454Lys for SBL and canonical mutation CTNNB1 p.Ser45Pro for HB. No shared somatic variants or common copy number alterations were found between SBL and HB primary tumor samples. Though scRNA-seq, single-cell atlases were constructed for both tumors. SBL may recapitulate a pre-acinar stage in the development of salivary gland, including basaloid, duct-like, myoepithelial-like, and cycling phenotypes. In the meantime, HB was composed of tumor cells resembling different stages of the liver, including hepatocyte-like, hepatic progenitor-like, and hepatoblast-like cells. After chemotherapy, both tumors were induced into a more mature phenotype. In terms of transcriptional signatures, SBL and HB showed enhanced expression of epithelial markers KRT8, KRT18, and essential embryo development genes SDC1, MDK, indicating the disruption of normal embryo epithelium development. Finally, heterozygous deleterious germline mutation BLM and FANCI were identified which could predispose the patient to higher cancer risk. It partially explained the reason for the co-occurrence of SBL and HB. Taken together, we provided valuable resources for deciphering cellular heterogeneity and adaptive change of tumor cells after chemotherapy for synchronous SBL and HB, providing insights into the mechanisms leading to synchronous pediatric tumors.

MYBL2 accelerates epithelial-mesenchymal transition and hepatoblastoma metastasis via the Smad/SNAI1 pathway.

Hepatoblastoma (HB) accounts for the majority of hepatic malignancies in children. Although the prognosis of patients with HB has improved in past decades, metastasis is an indicator of poor overall survival. Herein, we applied single-cell RNA sequencing to explore the transcriptomic profiling of 25,264 metastatic cells isolated from the lungs of two patients with HB. The transcriptomes uncovered the heterogeneity of malignant cells after metastatic lung colonization, and these cells had varied expression signatures associated with the cell cycle, epithelial-mesenchymal plasticity, and hepatic differentiation. Single-cell regulatory network inference and clustering (SCENIC) was utilized to identify the co-expressed transcriptional factors which regulated and represented the different cell states. We further screened the key factor by bioinformatics analysis and found that MYBL2 upregulation was significantly associated with metastasis and poor prognosis. The relationship between ectopic MYBL2 and metastasis was subsequently proved by immunohistochemistry (IHC) of HB tissues, and the functions of MYBL2 in promoting proliferation, migration, and epithelial-to-mesenchymal transition (EMT) were verified by in vitro and in vivo assays. Importantly, the levels of Smad2/3 phosphorylation and SNAI1 expression were increased in MYBL2-transfected cells. Consequently, these results indicated that the MYBL2-controlled Smad/SNAI1 pathway induced EMT and promoted HB tumorigenesis and metastasis.

Characteristics of the Gut Microbiome and IL-13/TGF-ß1 Mediated Fibrosis in Post-Kasai Cholangitis of Biliary Atresia.

Aims: Cholangitis in biliary atresia (BA), which accelerates liver fibrosis progression, is among the most common serious complications after Kasai surgery; however, its etiology remains elusive. Gut microbiome migration may contribute to post-Kasai cholangitis. Further, there is no appropriate model of BA post-Kasai cholangitis for use in investigation of its pathogenesis. Methods: We explored the characteristics of gut microbiome in patients with BA before and after Kasai procedure based on 16S rDNA sequencing. We isolated the dominant strain from patient stool samples and established an in vitro model by infecting patient-derived liver organoids. Bulk RNA-seq was performed, and we conducted qPCR, ELISA, and western blot to explore the mechanism of fibrosis. Results: Gut microbiome diversity was lower in patients after, relative to before, Kasai procedure, while the relative abundance of Klebsiella was higher. Patients who developed cholangitis within 1 month after discharge tended to have simpler gut microbiome composition, dominated by Klebsiella. Klebsiella pneumoniae (KPN) was isolated and used for modeling. RNA-seq showed that BA liver organoids expressed markers of hepatic progenitor cells (KRT19, KRT7, EPCAM, etc.) and that organoids were more stable and less heterogeneous among individuals than liver tissues. After infection with KPN, gene expression patterns in BA liver organoids were enriched in pathways related to infection, apoptosis, and fibrosis. Preliminary experiments indicated the presence of IL-13/TGF-ß1-mediated fibrosis in post-Kasai cholangitis. Conclusions: Our findings using a newly-developed model, demonstrate a key role for Klebsiella, and a potential mechanism underlying fibrosis in post-Kasai cholangitis, mediated by the IL-13/TGF-ß1 pathway.