Rapid in vivo evaluation system for cholestasis-related genes in mice with humanized bile acid profiles.

BACKGROUND: Pediatric cholestatic liver diseases (Ped-CLD) comprise many ultrarare disorders with a genetic basis. Pharmacologic therapy for severe cases of Ped-CLD has not been established. Species differences in bile acid (BA) metabolism between humans and rodents contribute to the lack of phenocopy of patients with Ped-CLD in rodents and hinder the development of therapeutic strategies. We aimed to establish an efficient in vivo system to understand BA-related pathogenesis, such as Ped-CLD. METHODS: We generated mice that express spCas9 specifically in the liver (L-Cas9Tg/Tg [liver-specific Cas9Tg/Tg] mice) and designed recombinant adeno-associated virus serotype 8 encoding small-guide RNA (AAV8 sgRNA) targeting Abcc2, Abcb11, and Cyp2c70. In humans, ABCC2 and ABCB11 deficiencies cause constitutional hyperbilirubinemia and most severe Ped-CLD, respectively. Cyp2c70 encodes an enzyme responsible for the rodent-specific BA profile. Six-week-old L-Cas9Tg/Tg mice were injected with this AAV8 sgRNA and subjected to biochemical and histological analysis. RESULTS: Fourteen days after the injection with AAV8 sgRNA targeting Abcc2, L-Cas9Tg/Tg mice exhibited jaundice and phenocopied patients with ABCC2 deficiency. L-Cas9Tg/Tg mice injected with AAV8 sgRNA targeting Abcb11 showed hepatomegaly and cholestasis without histological evidence of liver injury. Compared to Abcb11 alone, simultaneous injection of AAV8 sgRNA for Abcb11 and Cyp2c70 humanized the BA profile and caused higher transaminase levels and parenchymal necrosis, resembling phenotypes with ABCB11 deficiency. CONCLUSIONS: This study provides proof of concept for efficient in vivo assessment of cholestasis-related genes in humanized bile acid profiles. Our platform offers a more time- and cost-effective alternative to conventional genetically engineered mice, increasing our understanding of BA-related pathogenesis such as Ped-CLD and expanding the potential for translational research.

Induced pluripotent stem cell (iPSC) modeling validates reduced GBE1 enzyme activity due to a novel variant, p.Ile694Asn, found in a patient with suspected glycogen storage disease IV.

Background: Glycogen Storage disease type 4 (GSD4), a rare disease caused by glycogen branching enzyme 1 (GBE1) deficiency, affects multiple organ systems including the muscles, liver, heart, and central nervous system. Here we report a GSD4 patient, who presented with severe hepatosplenomegaly and cardiac ventricular hypertrophy. GBE1 sequencing identified two variants: a known pathogenic missense variant, c.1544G>A (p.Arg515His), and a missense variant of unknown significance (VUS), c.2081T>A (p. Ile694Asn). As a liver transplant alone can exacerbate heart dysfunction in GSD4 patients, a precise diagnosis is essential for liver transplant indication. To characterize the disease-causing variant, we modeled patient-specific GBE1 deficiency using CRISPR/Cas9 genome-edited induced pluripotent stem cells (iPSCs). Methods: iPSCs from a healthy donor (iPSC-WT) were genome-edited by CRISPR/Cas9 to induce homozygous p.Ile694Asn in GBE1 (iPSC-GBE1-I694N) and differentiated into hepatocytes (iHep) or cardiomyocytes (iCM). GBE1 enzyme activity was measured, and PAS-D staining was performed to analyze polyglucosan deposition in these cells. Results: iPSCGBE1-I694N differentiated into iHep and iCM exhibited reduced GBE1 protein level and enzyme activity in both cell types compared to iPSCwt. Both iHepGBE1-I694N and iCMGBE1-I694N showed polyglucosan deposits correlating to the histologic patterns of the patient's biopsies. Conclusions: iPSC-based disease modeling supported a loss of function effect of p.Ile694Asn in GBE1. The modeling of GBE1 enzyme deficiency in iHep and iCM cell lines had multi-organ findings, demonstrating iPSC-based modeling usefulness in elucidating the effects of novel VUS in ultra-rare diseases.

Clinicopathological and molecular features of renal cell carcinomas with haemangioblastoma-like features distinct from clear cell renal cell carcinoma.

AIMS: Haemangioblastomas arise in the central nervous system. Rarely, haemangioblastomas may develop in extra-neural sites, such as the kidneys. A few reported cases of renal cell carcinomas (RCCs) with haemangioblastoma-like features have exhibited both clear cell renal cell carcinoma (CCRCC)- and haemangioblastoma-like components. The clinicopathological and molecular characteristics of RCCs with haemangioblastoma-like features were analysed, focusing on VHL alterations, in comparison with CCRCCs partially resembling haemangioblastoma. METHODS AND RESULTS: Four RCCs with haemangioblastoma-like features and five CCRCCs partially resembling haemangioblastoma were included. The RCCs with haemangioblastoma-like features were indolent and lacked adverse prognostic factors. All RCCs with haemangioblastoma-like features had a well-circumscribed appearance and a thick fibromuscular capsule, with fibromuscular bundles extending into the tumour to varying degrees in the three tumours. Each RCC with haemangioblastoma-like features exhibited CCRCC-like areas with indistinct tubular structures and foci of haemangioblastoma-like areas, in which vessels and short spindle cells overwhelmed tumour cells. Whereas haemangioblastoma-like areas in the CCRCCs partially resembling haemangioblastoma exhibited sparse vessels and spindle cells and distinct clear cells. The RCCs with haemangioblastoma-like features exhibited a unique immunohistochemical profile, with positive staining for inhibin-α, S100, carbonic-anhydrase-9, keratin7, and high molecular weight keratin and negative staining for (alpha-methylacyl-CoA racemase) AMACR. RCC with haemangioblastoma-like features did not display any VHL alterations, including VHL mutation, 3p LOH, and methylation of the VHL promoter region, and the two tumours harboured a likely oncogenic missense variant of MTOR (c.7280T>G). CONCLUSION: The histopathological, immunohistochemical, and molecular findings suggest that RCC with haemangioblastoma-like features is a distinct entity from CCRCC.

Neonatal cholestasis in children with Alpha-1-AT deficiency is a risk for earlier severe liver disease with male predominance.

BACKGROUND: Our objective was to better understand the natural history and disease modifiers of Alpha-1-antitrypsin deficiency (AATD), a common genetic liver disease causing hepatitis and cirrhosis in adults and children. The clinical course is highly variable. Some infants present with neonatal cholestasis, which can resolve spontaneously or progress to cirrhosis; others are well in infancy, only to develop portal hypertension later in childhood. METHODS: The Childhood Liver Disease Research Network has been enrolling AATD participants into longitudinal, observational studies at North American tertiary centers since 2004. We examined the clinical courses of 2 subgroups of participants from the several hundred enrolled; first, those presenting with neonatal cholestasis captured by a unique study, enrolled because of neonatal cholestasis but before specific diagnosis, then followed longitudinally (n=46); second, separately, all participants who progressed to liver transplant (n=119). RESULTS: We found male predominance for neonatal cholestasis in AATD (65% male, p=0.04), an association of neonatal gamma-glutamyl transpeptidase elevation to more severe disease, and a higher rate of neonatal cholestasis progression to portal hypertension than previously reported (41%) occurring at median age of 5 months. Participants with and without preceding neonatal cholestasis were at risk of progression to transplant. Participants who progressed to liver transplant following neonatal cholestasis were significantly younger at transplant than those without neonatal cholestasis (4.1 vs. 7.8 years, p=0.04, overall range 0.3-17 years). Neonatal cholestasis had a negative impact on growth parameters. Coagulopathy and varices were common before transplant, but gastrointestinal bleeding was not. CONCLUSIONS: Patients with AATD and neonatal cholestasis are at risk of early progression to severe liver disease, but the risk of severe disease extends throughout childhood. Careful attention to nutrition and growth is needed.

Recent Increase in Incidence of Severe Acute Hepatitis of Unknown Etiology in Children is Associated with Infection with Adenovirus and Other Nonhepatotropic Viruses.

OBJECTIVE: To evaluate whether the nature and severity of non-A-E severe acute hepatitis in children noted by the World Health Organization from late 2021 through early 2022 was indeed increased in 2021-2022 compared with prior years. STUDY DESIGN: We performed a single-center, retrospective study to track the etiology and outcomes of children with non-A-E severe acute hepatitis in 2021-2022 compared with the prior 3-year periods (2018-2019, 2019-2020, and 2020-2021). We queried electronic medical records of children ≤16 years of age with alanine or aspartate aminotransferase levels of >500 IU. Data were analyzed for the periods of October 1, 2021, to May 1, 2022, and compared with the same time periods in 2018-2021. RESULTS: Of 107 children meeting entry criteria, 82 cases occurred from October to May of 2018-2022. The average annual case number was 16.3 in 2018-2021 compared with a 2-fold increase (to 33) in 2021-2022 (P = .0054). Analyses of etiologies showed that this increase was associated with a higher number of children who tested positive for viruses (n = 16) when compared with the average of 3.7 for 2018-2021 (P = .018). Adenovirus (26.1%) and severe acute respiratory syndrome coronavirus-2 (10.3%) were the most frequently detected viruses in 2021-2022. Despite evidence of acute liver failure in 37.8% of children in the entire cohort and in 47% of those with viral infection, the overall survival rate was high at 91.4% and 88.9%, respectively. CONCLUSIONS: The number of children with severe acute hepatitis in our center increased from 2021 to May 2022, with a greater frequency of cases associated with adenovirus, yet transplant-free survival remains high.

Diagnosis and management of glycogen storage disease type IV, including adult polyglucosan body disease: A clinical practice resource.

Glycogen storage disease type IV (GSD IV) is an ultra-rare autosomal recessive disorder caused by pathogenic variants in GBE1 which results in reduced or deficient glycogen branching enzyme activity. Consequently, glycogen synthesis is impaired and leads to accumulation of poorly branched glycogen known as polyglucosan. GSD IV is characterized by a remarkable degree of phenotypic heterogeneity with presentations in utero, during infancy, early childhood, adolescence, or middle to late adulthood. The clinical continuum encompasses hepatic, cardiac, muscular, and neurologic manifestations that range in severity. The adult-onset form of GSD IV, referred to as adult polyglucosan body disease (APBD), is a neurodegenerative disease characterized by neurogenic bladder, spastic paraparesis, and peripheral neuropathy. There are currently no consensus guidelines for the diagnosis and management of these patients, resulting in high rates of misdiagnosis, delayed diagnosis, and lack of standardized clinical care. To address this, a group of experts from the United States developed a set of recommendations for the diagnosis and management of all clinical phenotypes of GSD IV, including APBD, to support clinicians and caregivers who provide long-term care for individuals with GSD IV. The educational resource includes practical steps to confirm a GSD IV diagnosis and best practices for medical management, including (a) imaging of the liver, heart, skeletal muscle, brain, and spine, (b) functional and neuromusculoskeletal assessments, (c) laboratory investigations, (d) liver and heart transplantation, and (e) long-term follow-up care. Remaining knowledge gaps are detailed to emphasize areas for improvement and future research.

Customized Postoperative Therapy Improves Bile Drainage in Biliary Atresia: A Single Center Preliminary Report.

BACKGROUND: Controversies in management of biliary atresia (BA) after hepatoportoenterostomy (HPE) lead to variable treatment protocols. We implemented standardized medical management after HPE, customizing the use of antibiotics and corticosteroids based on patient-specific factors. METHODS: In this retrospective analysis, 20 consecutive infants underwent HPE for BA and were compared to a historical cohort. Analysis of successful biliary drainage 3 months after HPE (defined as serum total bilirubin <2 mg/dL) was the primary endpoint; survival with native liver at 2 years was the secondary endpoint. RESULTS: Sixteen of 20 (80%) infants had successful bile drainage, compared to 8 of 20 (40%) infants in the historical cohort (P = 0.0225). Sixteen of 20 patients in the new protocol have reached 2 years of age or required liver transplantation. Among the sixteen, 11 (68.8%) are alive with native livers versus 10 of 20 (50%) in the historical cohort (P = 0.0970). CONCLUSION: This preliminary report suggests the potential benefit of tailored use of postoperative antibiotics and corticosteroids in improving biliary drainage after HPE. LEVEL OF EVIDENCE: III.

Bacillus Calmette-Guerin intravesical instillation therapy-induced urothelial necrosis of the bladder and upper urinary tract.

Bacillus Calmette-Guerin (BCG) intravesical instillation therapy is the most effective adjuvant therapy for bladder cancer after transurethral resection of bladder tumor (TURBT). We present the first case to our knowledge with bladder and ureteral necrosis as a severe local side effect of it.

The Outcome of Patients With Cystic Biliary Atresia With Intact Proximal Hepatic Ducts Following Hepatic-Cyst-Jejunostomy.

OBJECTIVES: To determine the outcomes of patients with cystic biliary atresia by correlating the anatomy of the hepatic ducts with the choice of biliary reconstruction surgery. BACKGROUND: The Kasai hepatoportoenterostomy (Kasai) is the initial surgical procedure offered to most patients with biliary atresia. In contrast, a hepatic-cyst-jejunostomy has been reported to be effective in patients with the cystic form of biliary atresia. METHODS AND RESULTS: We performed an international multicenter retrospective review. Two hundred eighty-seven patients were included, and 33 cases of cystic biliary atresia were identified. Outcomes were the serum total bilirubin level 3 months post-surgery and native liver survival at 2 years of age and were compared between cases who received the Kasai versus hepatic-cyst-jejunostomy in correlation to the anatomy of proximal hepatic ducts. The patients were categorized into 3 anatomical groups: patent intact hepatic ducts (n = 10), patent hypoplastic hepatic ducts (n = 13), and obliterated hepatic ducts (n = 10). All 10 patients with patent intact hepatic duct group underwent hepatic-cyst-jejunostomy, and 9 experienced bile drainage and native liver survival. Among the 13 patients with hypoplastic hepatic ducts, 11 underwent the Kasai procedure, and 9 had bile drainage, whereas 2 underwent hepatic-cyst-jejunostomy, and one survived with the native liver. All of the patients with obliterated hepatic ducts underwent the Kasai procedure; 5 established biliary drainage and survived with the native liver. Of 5 who did not drain, 3 underwent liver transplantation. CONCLUSIONS: In patients with cystic biliary atresia, the subset with a connection between cyst and intrahepatic bile ducts via intact proximal hepatic ducts had favorable clinical outcomes following hepatic-cyst-jejunostomy.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Oral administration of E-type prostanoid (EP) 1 receptor antagonist suppresses carcinogenesis and development of prostate cancer via upregulation of apoptosis in an animal model.

Prostaglandin E2 plays an important role in carcinogenesis and malignant potential of prostate cancer (PC) cells by binding to its specific receptors, E-type prostanoid (EP) receptors. However, anti-carcinogenic effects of the EP receptor antagonist are unclear. In this study, we used a mouse model of PC. The mice were provided standard feed (control) or feed containing the EP1 receptor antagonist and were sacrificed at 10, 15, 30, and 52 weeks of age. Apoptosis was evaluated by immunohistochemical analysis using a cleaved caspase-3 assay. The incidence of cancer in the experimental group was significantly lower than that in the control group at 15, 30, and 52 weeks of age. The percentage of poorly differentiated PC cells was significantly lower in the experimental group than in the control group at 30 and 52 weeks of age. The percentage of apoptotic cells in the experimental group was significantly higher than that in the control group at 15, 30, and 52 weeks of age. These findings indicate that feeding with the addition of EP1 receptor antagonist delayed PC progression via the upregulation of apoptosis. We suggest that the EP1 receptor antagonist may be a novel chemopreventive agent for PC.

[A Case of Glans Penile Necrosis Due to Hyaluronic Acid into the Penis for Male Genital Augmentation].

A 65-year-old man visited us with a painful penis after receiving an injection of hyaluronic acid into the penis for male genital augmentation. On admission, physical examination revealed black necrotic lesions and ulcerations on his glans penis. We performed partial penectomy to remove the necrotic tissues. Histopathological examination showed necrosis with severe inflammatory infiltration in the dermis and subcutis. Glans penile necrosis due to hyaluronic acid injected into the glans penis for male genital augmentation is exceedingly rare. This case is reported herein along with a review of the Japanese literature.

Multiscale Computational Model Reveals Nerve Response in a Mouse Model for Temporal Interference Brain Stimulation.

There has been a growing interest in the non-invasive stimulation of specific brain tissues, while reducing unintended stimulation in surrounding regions, for the medical treatment of brain disorders. Traditional methods for non-invasive brain stimulation, such as transcranial direct current stimulation (tDCS) or transcranial magnetic stimulation (TMS), can stimulate brain regions, but they also simultaneously stimulate the brain and non-brain regions that lie between the target and the stimulation site of the source. Temporal interference (TI) stimulation has been suggested to selectively stimulate brain regions by superposing two alternating currents with slightly different frequencies injected through electrodes attached to the scalp. Previous studies have reported promising results for TI applied to the motor area in mice, but the mechanisms are yet to be clarified. As computational techniques can help reveal different aspects of TI, in this study, we computationally investigated TI stimulation using a multiscale model that computes the generated interference current pattern effects in a neural cortical model of a mouse head. The results indicated that the threshold increased with the carrier frequency and that the beat frequency did not influence the threshold. It was also found that the intensity ratio between the alternating currents changed the location of the responding nerve, which is in agreement with previous experiments. Moreover, particular characteristics of the envelope were investigated to predict the stimulation region intuitively. It was found that regions with high modulation depth (| maximum| - | minimum| values of the envelope) and low minimum envelope (near zero) corresponded with the activation region obtained via neural computation.

Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.

The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.

High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell-Derived Organoids.

BACKGROUND & AIMS: Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution. METHODS: We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform. RESULTS: HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis. CONCLUSIONS: Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.

Acute liver dysfunction with delayed peak of serum aminotransferase levels as a presentation of ornithine transcarbamylase deficiency in females.

We describe 10 females with ornithine transcarbamylase (OTC) deficiency and liver dysfunction, revealing a unique pattern of hepatocyte injury in which initial hyperammonemia and coagulopathy is followed by a delayed peak in aminotransferase levels. None of the patients required urgent liver transplantation, though five eventually underwent transplant for recurrent metabolic crises. We intend that this novel observation will initiate further investigations into the pathophysiology of liver dysfunction in OTC-deficient patients, and ultimately lead to the development of therapies and prevent the need for liver transplant.

Organoid Medicine in Hepatology.

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