Bile Duct Cells Research Articles

MULTIPLE LIVER AND SPLENIC ABSCESSES AS AN EARLY COMPLICATION AFTER COVID-19 INFECTION: CASE REPORT AND REVIEW OF LITERATURE

COVID-19 is a major health problem affects millions of people worldwide. The effect of COVID-19 infection is not only related to the cardiopulmonary insult, but rather affecting every system in the body. Many publications have reported common hepatosplenic insult during COVID-19 infection. Here we describe a case ofmultiple liver and splenic abscesses post corona virus infection in a healthy 13-years old girl, highlighting the possible explanations of her condition in the context of literature review. Background:Coronavirus disease 2019 (COVID-19) has been considered overwhelming heath crisis with reported death over 6 million people till now and over half billion confirmed cases according to WHO dashboard (1). This raises a concern that this virus might have the capability of attacking not only the respiratory system but also almost every single organ system. Patients with the virus have presented with features of acute liver injury, gastrointestinal symptoms, endocrine problems, neurological damage, and cardiovascular disease, most of them recover completely with no sequalae, and others develop short- and long-termSide effects(2,3). Recent research on COVID-19 have shown a high incidence of liver injury ranging from 14.8 to 53%, mainly indicated by elevated liver enzymes levels accompanied by slightly elevated bilirubin levels. The proportion of patients who develop liver injury in severe COVID-19 seems to be higher than in mild cases(4). SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as its entry receptor, which is expressed in liver and bile duct cells (5,6), suggesting direct liver injury in patients with COVID-19.In contrary, isolated splenic affection usually passed unnoticed unless splenicthrombosis or infarction with superadded abscess formation occurred (7). Here we describe apostcovid multiple hepatic and splenic abscesses without previous abnormalities shortly after COVID-19 infection in otherwise healthy 13 -years girl.

Human liver organoid derived intra-hepatic bile duct cells support SARS-CoV-2 infection and replication.

Although the main route of infection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory tract, liver injury is also commonly seen in many patients, as evidenced by deranged parenchymal liver enzymes. Furthermore, the severity of liver damage has been shown to correlate with higher mortality. Overall, the mechanism behind the liver injury remains unclear. We showed in this study that intra-hepatic bile duct cells could be grown using a human liver organoid platform. The cholangiocytes were not only susceptible to SARS-CoV-2 infection, they also supported efficient viral replication. We also showed that SARS-CoV-2 replication was much higher than SARS-CoV. Our findings suggested direct cytopathic viral damage being a mechanism for SARS-CoV-2 liver injury.

DHEA Protects Human Cholangiocytes and Hepatocytes against Apoptosis and Oxidative Stress.

Primary biliary cholangitis (PBC) is a rare chronic cholestatic and immune-mediated liver disease of unknown aetiology that targets intrahepatic bile duct cells (cholangiocytes) and primarily affects postmenopausal women, when their estrogen levels sharply decrease. An impaired cholangiocyte response to estrogen characterizes the terminal stage of the disease, as this is when an inefficiency of cholangiocyte proliferation, in balancing the loss of intrahepatic bile ducts, is observed. Here, we report that the estrogen precursor dehydroepiandrosterone (DHEA) and its sulfate metabolites, DHEA-S and 17 β-estradiol, enhance the proliferation of cholangiocytes and hepatocytes in vitro. Flow cytometry analysis showed that DHEA and DHEA-S decreased glyco-chenodeoxycholic acid (GCDC)-driven apoptosis in cholangiocytes. Cell viability assay (MTT) indicated that ER-α, -β, and the G-protein-coupled estrogen receptor, are involved in the protection of DHEA against oxidative stress in cholangiocytes. Finally, immunoblot analysis showed an elevated level of steroid sulfatase and a reduced level of sulfotransferase 1E1 enzymes, involved in the desulfation/sulfation process of estrogens in cirrhotic PBC, and primary sclerosis cholangitis (PSC) liver tissues, another type of chronic cholestatic and immune-mediated liver disease. Taken together, these results suggest that DHEA can prevent the deleterious effects of certain potentially toxic bile acids and reactive oxygen species, delaying the onset of liver disease.

Correlation of LGR5 expression and clinicopathological features in intrahepatic cholangiocarcinoma

Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a known cancer stem cell marker. However, there are no reported analyses of LGR5 mRNA expression in normal liver and liver cancer tissues. Here, we evaluated LGR5 expression by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 25 samples of intrahepatic cholangiocarcinoma (ICC) selected from the medical archives at our hospital. LGR5 expression levels were divided into high and low expression groups by the five-grade scoring system, and clinicopathological features were analyzed. Low LGR5 expression was identified in some normal hepatocytes and bile duct cells. In addition, LGR5 expression was identified in all bile duct cancer samples except one case. Well-differentiated to moderately-differentiated adenocarcinoma tended to show higher LGR5 expression than poorly-differentiated adenocarcinoma (P = 0.0561), and the large duct type showed significantly higher LGR5 expression levels than the small duct type (P = 0.0225). Patients in the high LGR5 expression group tended to have good overall survival (OS) (P = 0.0623). The Cox proportional hazard regression model revealed that the high LGR5 expression group showed independently better OS for ICC (P = 0.0285). High LGR5 expression is possibly a good prognosis factor in ICC. However, the detailed mechanism of LGR5 in this disease remains unclear, and further analysis is warranted.

Management of Simple Hepatic Cyst.

Simple hepatic cysts are typically saccular, thin-walled masses with fluid-filled epithelial lined cavities. They arise from aberrant bile duct cells that develop during embryonic development. With the development of diagnostic modalities such as ultrasonography (US), CT, and MRI, simple hepatic cysts are frequently detected in clinical examinations. US is the most useful and noninvasive tool for diagnosis of simple hepatic cysts and can usually differentiate simple hepatic cysts from abscesses, hemangiomas, and malignancies. Cysts with irregular walls, septations, calcifications, or daughter cysts on US should be evaluated with enhanced CT or MRI, to differentiate simple hepatic cysts from cystic neoplasms or hydatid cysts. Growth and compression of hepatic cysts cause abdominal discomfort, pain, distension, and dietary symptoms such as nausea, vomiting, a feeling of fullness, and early satiety. Complications of simple hepatic cysts include infection, spontaneous hemorrhage, rupture, and external compression of biliary tree or major vessels. Asymptomatic simple hepatic cysts do not require treatment. Treatment for symptomatic simple hepatic cysts includes percutaneous aspiration, aspiration followed by sclerotherapy, and surgery. The American College of Gastroenterology clinical guidelines recommend laparoscopic fenestration because of its high success rate and low invasiveness. Percutaneous procedures for treatment of simple hepatic cysts are particularly effective for immediate palliation of patient symptoms; however, they are not generally recommended because of the high rate of recurrence. Management of simple hepatic cysts requires correct differentiation from neoplasms and infections, and selection of a reliable treatment.

Liver progenitor cells may construct cysts having heterogeneous gene expression of liver-enriched transcription factors in mice with conditional knockout of the Hhex gene

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb-Cre/HhexloxP/loxP) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases.

Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle

Liver regeneration is a complicated biological process orchestrated by various liver resident cells. Hepatic cell proliferation and reconstruction of the hepatic architecture involve multiple signaling pathways. It has been reported that the Hh signal is involved in liver regeneration. However, the signal transduction pathways and cell types involved are ill studied. This study aimed to investigate hedgehog signal response cell types and the specific molecular mechanism involved in the process of liver regeneration. Partial hepatectomy (PH) of 70% was performed on ICR (Institute of Cancer Research) mice to study the process of liver regeneration. We found that the hedgehog signal was activated significantly after PH, including hedgehog ligands, receptors and intracellular signaling molecules. Ligand signals were mainly expressed in bile duct cells and non-parenchymal hepatic cells, while receptors were expressed in hepatocytes and some non-parenchymal cells. Inhibition of the hedgehog signal treated with vismodegib reduced the liver regeneration rate after partial hepatectomy, including inhibition of hepatic cell proliferation by decreasing Cyclin D expression and disturbing the cell cycle through the accumulation of Cyclin B. The current study reveals the important role of the hedgehog signal and its participation in the regulation of hepatic cell proliferation and the cell cycle during liver regeneration. It provides new insight into the recovery of the liver after liver resection.

An Insight into Cholangiocarcinoma and Recent Advances in its Treatment.

Cholangiocarcinoma (CCA) is a malignant disease of the epithelial cells of the intrahepatic andextrahepatic bile ducts. This review focuses on various aspects of cholangiocarcinoma such as its associatedcauses, treatment criteria, and more. Although it remains a rare malignancy and is the second most common primary malignancy of the liver,the incidence is increasing, especially the incidence of intrahepatic CCA. Several studies suggested that surgery isnot only solution; recently, reported targeted drugs may have the potential to become an alternative option. This review provides an overview of the current scenario of targeted therapies for CCA, which weretabulated with their current status and it also included its associated causes and its treatment criteria. Because of its rarity and complexity, surgery remains the preferred treatment in resectable patients.Howerver, the studies suggested that the recently reported drugs may have the potential to be an alternative optionfor the treatment of CCA and related complications. In addition, this review will certainly benefit the community andresearcher for further investigation.

Hesperidin delays cell cycle progression into the G0/G1 phase via suspension of MAPK signaling pathway in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) derived from epithelial cells of bile ducts is highly aggressive tumor. Hesperidin extracted from citrus fruits is a promising antitumor compound. The purpose of this study is to explore molecular mechanism by which hesperidin affects cholangiocarcinoma progression. Cellular functional experiments were performed and subcutaneous transplant xenograft model was established. Our findings indicated that hesperidin suppressed iCCA cell proliferation in time- and concentration-dependent manners. Hesperidin treatment induced cell cycle arrest at G0/G1 phase, whereas it has no effect on cell apoptosis. Further, data revealed that hesperidin attenuated MEK5 and ERK5 phosphorylation and inhibited ERK5 nuclear localization by reducing MEKK2 activity in MAPK signaling pathway. It could cause alterations in expression of the downstream genes, including CDK4, CDK6 (cell cycle protein kinases), Cyclin D1 (a G1/S checkpoint), P21, and P27 (two G1-checkpoint CDK inhibitors), thereby arresting cell cycle distribution of iCCA cells in the G0/G1 phase. BIX02189 treatment, a specific inhibitor of MEK5, in combination with hesperidin displayed synergistic inhibitory effects on cell cycle arrest and gene expressions. Furthermore, hesperidin administration alone or in combination with MEK5 inhibitor BIX02189 restrained iCCA tumor growth in vivo. Taken together, these results confirmed that hesperidin regulated the expression of cell cycle-related genes by inhibiting the activation of MEKK2/MEK5/ERK5 signaling pathway, inducing iCCA cell cycle arrest at the G0/G1 phase. Our study provides a theoretical foundation and experimental basis for further development of hesperidin as a therapeutic agent for iCCA treatment.

Long non-coding RNA ACTA2-AS1 promotes ductular reaction by interacting with the p300/ELK1 complex

Biliary disease is associated with a proliferative/fibrogenic ductular reaction (DR). p300 is an epigenetic regulator that acetylates lysine 27 on histone 3 (H3K27ac) and is activated during fibrosis. Long non-coding RNAs (lncRNAs) are aberrantly expressed in cholangiopathies, but little is known about how they recruit epigenetic complexes and regulate DR. We investigated epigenetic complexes, including transcription factors (TFs) and lncRNAs, contributing to p300-mediated transcription during fibrosis. We evaluated p300 invivo using tamoxifen-inducible, cholangiocyte-selective, p300 knockout (KO) coupled with bile duct ligation (BDL) and Mdr KO mice treated with SGC-CBP30. Primary cholangiocytes and liver tissue were analyzed for expression of Acta2-as1 lncRNA by qPCR and RNA in situ hybridization. Invitro, we performed RNA-sequencing in human cholangiocytes with a p300 inhibitor. Cholangiocytes were exposed to lipopolysaccharide (LPS) as an injury model. We confirmed formation of a p300/ELK1 complex by immunoprecipitation (IP). RNA IP was used to examine interactions between ACTA2-AS1 and p300. Chromatin IP assays were used to evaluate p300/ELK1 occupancy and p300-mediated H3K27ac. Organoids were generated from ACTA2-AS1-depleted cholangiocytes. BDL-induced DR and fibrosis were reduced in Krt19-CreERT/p300fl/fl mice. Similarly, Mdr KO mice were protected from DR and fibrosis after SGC-CBP30 treatment. Invitro, depletion of ACTA2-AS1 reduced expression of proliferative/fibrogenic markers, reduced LPS-induced cholangiocyte proliferation, and impaired organoid formation. ACTA2-AS1 regulated transcription by facilitating p300/ELK1 binding to the PDGFB promoter after LPS exposure. Correspondingly, LPS-induced H3K27ac was mediated by p300/ELK1 and was reduced in ACTA2-AS1-depleted cholangiocytes. Cholangiocyte-selective p300 KO or p300 inhibition attenuate DR/fibrosis in mice. ACTA2-AS1 influences recruitment of p300/ELK1 to specific promoters to drive H3K27ac and epigenetic activation of proliferative/fibrogenic genes. This suggests that cooperation between epigenetic co-activators and lncRNAs facilitates DR/fibrosis in biliary diseases. We identified a three-part complex containing an RNA molecule, a transcription factor, and an epigenetic enzyme. The complex is active in injured bile duct cells and contributes to activation of genes involved in proliferation and fibrosis.

Progress and Current Limitations of Materials for Artificial Bile Duct Engineering.

Bile duct injury (BDI) and bile tract diseases are regarded as prominent challenges in hepatobiliary surgery due to the risk of severe complications. Hepatobiliary, pancreatic, and gastrointestinal surgery can inadvertently cause iatrogenic BDI. The commonly utilized clinical treatment of BDI is biliary-enteric anastomosis. However, removal of the Oddi sphincter, which serves as a valve control over the unidirectional flow of bile to the intestine, can result in complications such as reflux cholangitis, restenosis of the bile duct, and cholangiocarcinoma. Tissue engineering and biomaterials offer alternative approaches for BDI treatment. Reconstruction of mechanically functional and biomimetic structures to replace bile ducts aims to promote the ingrowth of bile duct cells and realize tissue regeneration of bile ducts. Current research on artificial bile ducts has remained within preclinical animal model experiments. As more research shows artificial bile duct replacements achieving effective mechanical and functional prevention of biliary peritonitis caused by bile leakage or obstructive jaundice after bile duct reconstruction, clinical translation of tissue-engineered bile ducts has become a theoretical possibility. This literature review provides a comprehensive collection of published works in relation to three tissue engineering approaches for biomimetic bile duct construction: mechanical support from scaffold materials, cell seeding methods, and the incorporation of biologically active factors to identify the advancements and current limitations of materials and methods for the development of effective artificial bile ducts that promote tissue regeneration.

Generation of functional ciliated cholangiocytes from human pluripotent stem cells

The derivation of mature functional cholangiocytes from human pluripotent stem cells (hPSCs) provides a model for studying the pathogenesis of cholangiopathies and for developing therapies to treat them. Current differentiation protocols are not efficient and give rise to cholangiocytes that are not fully mature, limiting their therapeutic applications. Here, we generate functional hPSC-derived cholangiocytes that display many characteristics of mature bile duct cells including high levels of cystic fibrosis transmembrane conductance regulator (CFTR) and the presence of primary cilia capable of sensing flow. With this level of maturation, these cholangiocytes are amenable for testing the efficacy of cystic fibrosis drugs and for studying the role of cilia in cholangiocyte development and function. Transplantation studies show that the mature cholangiocytes generate ductal structures in the liver of immunocompromised mice indicating that it may be possible to develop cell-based therapies to restore bile duct function in patients with biliary disease.

Histopathological evidence of intrahepatic cholangiocarcinoma occurring in ductal plate malformation: A clinicopathologic study of 5 cases

Ductal plate malformations (DPM) arise from abnormal remodeling of the embryologic ductal plate of the liver. Malignant transformation of DPMs to intrahepatic cholangiocarcinoma (iCCA) has been reported in very rare instances but is viewed with some skepticism. We report the clinicopathological findings in five cases of iCCA, occurring in liver with DPM-like features. All tumors were less than 5 cm, often presented as stage T1a tumors. Histologically, a typical tumor showed a vague multinodular architecture with larger, irregular, tortuous glandular structures with microcystic dilation, intraluminal fibroepithelial projection, and bridge/island formation. The tumor cells were relatively small, bland, and without obvious pleomorphism. Interestingly, DPM presented as a histopathological transition sequence of definitively benign to biliary intraepithelial neoplasia (bilIN), then finally to iCCA. A complete pushing border, with entrapped portal tracts at the edge of the main tumor, suggested a replacing growth pattern. There was gradually increased expression of Ki-67 and p53 in these transition phases from benign to bilIN then to iCCA with DPM-like features. The neoplastic epithelium exhibited immunoreactivity in EpCAM, MUC1, NCAM, and CK19. KRAS mutation was found in 2 of the 5 iCCA cases with DPM-like features. Multifocal DPMs or VMCs with bilIN were dispersed in the non-tumor liver parenchyma in 3 of the 5 cases. The neoplasm was interpreted as iCCA arising in DPM, which may have originated from small bile duct or hepatic precursor cells. More studies are needed to verify this scarce entity and its premalignant properties.

CK19 Predicts Recurrence and Prognosis of HBV Positive HCC

BackgroundCytokeratin is associated with the recurrence and metastasis of some cancers and tends to increase the malignancy of the disease. It is getting more and more attention in cancer research. Abnormal expression of cytokeratin 19 (CK19) has been reported as an important prognostic factor in cancers. CK19 is a marker of bile duct cells, liver progenitor cells (HPCs), and early hepatoblasts, and its expression is associated with poor prognosis in patients diagnosed with hepatocellular carcinoma (HCC). The purpose of this study was to evaluate the predictive value of CK19 for tumor recurrence after radical resection in patients with hepatitis B virus (HBV) positive HCC. MethodsThis study was a retrospective study conducted in two institutions. A total of 674 patients with HBV positive HCC who underwent radical HCC resection from January 2010 to May 2020 were included in this study. Chi-square test or Fisher’s exact test was used to compare the classification variables and continuous variables were compared by t-test or Wilcoxon rank sum test. Cox regression model was used for univariate and multi-variable survival analyses. Based on the results of the multi-variable analyses of Cox regression, the nomogram of 2-year recurrence-free survival (RFS) was plotted. The model was validated internally in the Hangzhou cohort (training set) and then externally in the Lanzhou cohort (test set) and the effectiveness of the model was tested. ResultsFor all 674 patients, 223 cases (33.1%) were positive and 451 cases (66.9%) were negative for CK19. The 2-year RFS rate was higher in patients with CK19 negative than in patients with CK19 positive. In the training set, correlation analysis showed that CK19 expression was correlated with preoperative potassium (P value(P) = 0.030), satellite nodules (P < 0.001) and microvascular invasion (P = 0.020). In the test set, CK19 expression was correlated with postoperative platelet (P = 0.038), satellite nodules (P = 0.003), microvascular invasion (P = 0.011), and maximum tumor size (P = 0.039). Univariate Cox regression correlation analyses showed that CK19 expression was correlated with preoperative potassium (P value(P) = 0.030), satellite nodules (P < 0.001), and microvascular invasion (P = 0.020). Training and test sets showed that postoperative platelet (> 300/L), CK19, satellite nodules in the training set, microvascular invasion, maximum tumor size, and tumor boundary were adverse factors for predicting RFS. Multi-variable analyses showed that in the training set, postoperative platelet > 300/L (hazard ratios (HR) = 2.753, 95% confidence interval (95%CI):1.234–6.142, P = 0.013), CK19 (HR = 1.410, 95%CI:1.006–1.976, P = 0.046), satellite nodule (HR = 1.476, 95%CI:1.026–2.120, P = 0.036), microvascular invasion (HR = 2.927, 95%CI:2.006–4.146, P < 0.001), incomplete tumor capsule (HR = 1.539, 95%CI:1.012–2.341, P = 0.044) were independent prognostic indicator of poor RFS. In the test set, postoperative platelet > 300/L (HR = 2.816, 95%CI:1.043–7.603, P = 0.041), CK19 (HR = 1.586, 95%CI:1.016–2.475, P = 0.042), satellite nodule (HR = 1.706, 95%CI:1.067–2.728, P = 0.026), microvascular invasion (HR = 1.611, 95%CI:1.034–2.510, P = 0.035), and tumor without capsule (HR = 1.870, 95%CI:1.120–3.120, P = 0.017) were independent prognostic indicators of poor RFS. The C-index for the nomogram was 0.698 (95%CI: 0.654–0.742) and the C-index for the test set was 0.670 (95%CI: 0.616–0.724). Both internal and external verification showed good results in identification and calibration. ConclusionCK19 plays a key role in tumor malignancy through overexpression and the expression of CK19 is an independent adverse factor affecting recurrence; therefore, CK19 can be used as a potential biomarker to predict adverse prognosis after surgery and adjuvant therapy in HCC patients.

Proteomic analysis identifies deregulated metabolic and oxidative-associated proteins in Italian intrahepatic cholangiocarcinoma patients

BackgroundCholangiocarcinoma (CCA) is an aggressive disease with poor prognosis. A molecular classification based on mutational, methylation and transcriptomic features could allow identifying tailored therapies to improve CCA patient outcome. Proteomic remains partially unexplored; here, we analyzed the proteomic profile of five intrahepatic cholangiocarcinoma (ICC) derived from Italian patients undergone surgery and one normal bile duct cell line.MethodsProteome profile was investigated by using 2D electrophoresis followed by Mass Spectrometry (MS). To validate proteomic data, the expression of four overexpressed proteins (CAT, SOD, PRDX6, DBI/ACBP) was evaluated by immunohistochemistry in an independent cohort of formalin fixed, paraffin-embedded (FFPE) ICC tissues. We also compared proteomic data with those obtained by transcriptomic profile evaluated by microarray analysis of the same tissues.ResultsWe identified 19 differentially expressed protein spots, which were further characterized by MS; 13 of them were up- and 6 were down-regulated in ICC. These proteins are mainly involved in redox processes (CAT, SODM, PRDX2, PRDX6), in metabolism (ACBP, ACY1, UCRI, FTCD, HCMS2), and cell structure and organization (TUB2, ACTB). CAT is overexpressed in 86% of patients, PRDX6 in 73%, SODM in 100%, and DBI/ACBP in 81% compared to normal adjacent tissues. A concordance of 50% between proteomic and transcriptomic data was observed.ConclusionsThis study pointed out that the impairment of the metabolic and antioxidant systems, with a subsequent accumulation of free radicals, might be a key step in CCA development and progression.

Ultrastructural Profile Combined with Immunohistochemistry of a Hepatic Progenitor Cell Line in Pediatric Autoimmune Hepatitis: New Insights into the Morphological Pattern of the Disease.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I—the most undifferentiated progenitor cells; III—intermediate hepatocyte-like cells; II—intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare—those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Abnormal Liver Biochemistry Tests and Acute Liver Injury in COVID-19 Patients: Current Evidence and Potential Pathogenesis.

Globally, millions of persons have contracted the coronavirus disease 2019 (COVID-19) over the past several months, resulting in significant mortality. Health care systems are negatively impacted including the care of individuals with cancers and other chronic diseases such as chronic active hepatitis, cirrhosis and hepatocellular carcinoma. There are various probable pathogenic mechanisms that have been presented to account for liver injury in COVID-19 patients such as hepatotoxicity cause by therapeutic drugs, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the bile duct cells and hepatocytes, hypoxia and systemic inflammatory response. Liver biochemistry tests such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) are deranged in COVID-19 patients with liver injury. Hepatocellular damage results in the elevation of serum AST and ALT levels in early onset disease while a cholestatic pattern that develops as the disease progress causes higher levels of ALP, GGT, direct and total bilirubin. These liver biochemistry tests are prognostic markers of disease severity and should be carefully monitored in COVID-19 patients. We conducted a systematic review of abnormal liver biochemistry tests in COVID-19 and the possible pathogenesis involved. Significant findings regarding the severity, hepatocellular pattern, incidence and related clinical outcomes in COVID-19 patients are highlighted.

Abstract 62: Development of dual-targeted fine-tuned immune restoring (DFIR) CAR T cell therapy for clear cell renal cell carcinoma (ccRCC)

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. Treatment of RCC has improved dramatically over the last decade, however, a curative treatment for advanced RCC remains rare. Chimeric Antigen Receptor (CAR) T cell therapy is a new type of “living drug”. The FDA approval of CAR-T cell therapies have ushered this new type of cellular immunotherapy into mainstream cancer therapy for hematologic malignancies. To date, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the immunosuppressive tumor microenvironment (TME), and on-target off-tumor toxicities due to shared antigens on normal tissues. Carbonic anhydrase IX (CAIX) is a therapeutic target against ccRCC that is under the control of HIF1α and becomes overexpressed on the surface of ccRCC cells because of VHL inactivation. The first anti-CAIX CAR-T study using the 1st generation G250-CD3 CAR-T cells plus IL-2 to treat patients with metastatic ccRCC was terminated due to hepatitis that developed after CAR-T cell infusions. The on-target off-tumor side effects were attributed to low expression of CAIX on healthy bile duct cells. To translate CAR-T cell therapy to ccRCC, we designed dual-targeted fine-tuned immune restoring (DFIR) CAR-T cells. The DFIR-CAR T cells secreted immune checkpoint inhibitor (ICI) monoclonal antibodies at the tumor site and exhibited superior efficacy and safety profiling. Increased efficacy is achieved through anti-CD70/CAIX dual-targeting CAR which allows the CAR-T cell activation in response to either antigen to mitigate solid tumor heterogeneity. Elevated safety is addressed through fine-tuned CARs which have the affinities of the scFv targeting moieties tailored so that they are activated only by high density tumor associated antigens (TAAs) but not the same antigens expressed at physiologic levels on normal tissues. The ICI payloads act globally on the TME, not only to prevent CAR-T cell exhaustion but also to restore anti-tumor activity of the educated tumor infiltrated lymphocytes (TILs) that have accumulated in the TME. In summary, our DFIR CAR-T cell therapy holds the promise to achieve cures of ccRCC by killing heterogenous ccRCC cells, mitigating against on-target off-tumor toxicity, reversing TME immunosuppression and restoring host anti-tumor immunity. We believe that DFIR-CAR T cells are ready to be translated to the clinic upon completion of pre-IND studies. Citation Format: Yufei Wang, Alicia Buck, Marion Grimaud, Sreekumar Kodangattil, Cecile Razimbaud, Atef Fayed, Matthew Chang, Aedin Culhane, David A. Braun, Toni K. Choueiri, Catherine J. Wu, Kevin S. Wei, Leo L. Chan, Brandon P. Piel, Elena V. Ivanova, Cloud P. Paweletz, David A. Barbie, Rebecca Jennings, Miriam Ficial, Maura Aliezah Sticco-Ivins, Sabina Signoretti, Gordon J. Freeman, Quan K. Zhu, Wayne A. Marasco. Development of dual-targeted fine-tuned immune restoring (DFIR) CAR T cell therapy for clear cell renal cell carcinoma (ccRCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 62.

SARS-CoV-2-related injury of biliary tract

Severe Acute Respiratory Syndrome Coronavirus‑2 (SARS‑CoV‑2), which causes coronavirus disease‑2019 (COVID‑19), affects several organs and systems. The ways of the virus penetration into tissues have been studied. Liver is affected in 15 — 53 % of cases. Data on the RNA‑seq sequencing in the Human Protein Atlas database confirm the expression of ACE2 (angiotensin 2 converting enzyme receptor) in the liver and epithelium of the bile ducts and gallbladder. At the same time, a high frequency of ACE2 expression is observed only in cholangiocytes, but not in hepatocytes, Kupffer cells or endothelial cells. The amount of ACE2 in bile duct cells is significantly higher than in hepatocytes, and is comparable to the level in type 2 alveolar cells in the lungs, which makes the biliary tract a potential target for the virus.Binding of SARS‑CoV‑2 to gallbladder epithelial cells can lead to mucosal inflammation. A systematic review describes the formation of bile clots in small bile ducts. Presence of COVID‑19 in the wall of the gallbladder ­(qrt‑RCR) has been revealed. With this, acute cholecystitis develops clinically, and radiological investigations show thickening of the gallbladder wall, biliary sludge and perivesicular fluid. Onset of acute gangrenous cholecystitis as a late complication of COVID‑19 infection is described. Histologically, inflammatory infiltrates are found in the gallbladder wall, which diffusely affect medium‑sized arteries with obliteration of their lumen, ischemia of the bladder wall; the perineural inflammation take place. These features indicate vasculitis with thrombosis.Thus, despite the theoretical information about the biliary tract injury by the COVID‑19 virus, this aspect of the infection has not been clinically studied, and the published papers are limited to the description of single clinical cases. In our opinion, a deeper and long‑term study of the biliary tract pathology in COVID‑19 infection is needed to provide rationale for the treatment with ursodeoxycholic acid.

Single-cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree.

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.