Hydrazone-Functionalized trans-A2B-Corroles: Effective Synergy in Photodynamic Therapy of Lung Cancer.

A synthetic route to trans-A2B-corroles combining the macrocyclic core with a hydrazone moiety, based on the reactivity of azoalkenes toward dipyrromethanes, has been established with the aim of developing a new class of photosensitizers for photodynamic therapy of lung cancer. The study of the photophysical properties of the novel macrocycles allowed the identification of photosensitizers with absorption within the phototherapeutic window and high singlet oxygen quantum yield. Relevant structure-photodynamic activity correlations were established by studying the new corroles-based photodynamic therapy (PDT) in human lung cancer cell lines (A549 and H1299). The methyl-hydrazone corroles were more active than phenyl-hydrazone corroles, with the N-Boc and N-Ts groups being key structural features to ensure high activity. The lead photosensitizers, with IC50 values below 100 nM and no cytotoxicity per se, were significantly more active than 5,10,15-triphenylcorrole, showing that the presence of the hydrazone functional group has a strong influence on PDT activity.

Artificial Intelligence in Coloproctology: A Review of Emerging Technologies and Clinical Applications.

Artificial intelligence (AI) has emerged as a transformative tool across several specialties, namely gastroenterology, where it has the potential to optimize both diagnosis and treatment as well as enhance patient care. Coloproctology, due to its highly prevalent pathologies and tremendous potential to cause significant mortality and morbidity, has drawn a lot of attention regarding AI applications. In fact, its application has yielded impressive outcomes in various domains, colonoscopy being one prominent example, where it aids in the detection of polyps and early signs of colorectal cancer with high accuracy and efficiency. With a less explored path but equivalent promise, AI-powered capsule endoscopy ensures accurate and time-efficient video readings, already detecting a wide spectrum of anomalies. High-resolution anoscopy is an area that has been growing in interest in recent years, with efforts being made to integrate AI. There are other areas, such as functional studies, that are currently in the early stages, but evidence is expected to emerge soon. According to the current state of research, AI is anticipated to empower gastroenterologists in the decision-making process, paving the way for a more precise approach to diagnosing and treating patients. This review aims to provide the state-of-the-art use of AI in coloproctology while also reflecting on future directions and perspectives.

Deep learning and automatic differentiation of pancreatic lesions in endoscopic ultrasound - a transatlantic study.

Endoscopic ultrasound (EUS) allows characterization and biopsy of pancreatic lesions. Pancreatic cystic neoplasms (PCN) include in mucinous (M-PCN) and non-mucinous lesions (NM-PCN). Pancreatic ductal adenocarcinoma (P-DAC) is the commonest pancreatic solid lesion (PSL), followed by pancreatic neuroendocrine tumor (P-NET). While EUS is preferred for pancreatic lesion evaluation, its diagnostic accuracy is suboptimal. This multicentric study aims to develop a convolutional neural network (CNN) for detecting and distinguishing PCN (namely M-PCN and NM-PCN) and PSL (particularly P-DAC and P-NET). A CNN was developed with 378 EUS exams from 4 international reference centers (Centro Hospitalar Universitário São João, Hospital Universitario Puerta de Hierro Majadahonda, New York University Hospitals, Hospital das Clínicas FMUSP). 126.000 images were obtained - 19.528 M-PCN, 8.175 NM-PCN, 64.286 P-DAC, 29.153 P-NET and 4.858 normal pancreas images. A trinary CNN differentiated normal pancreas tissue from M-PCN and NM-PCN. A binary CNN distinguished P-DAC from P-NET. The total dataset was divided in a training and testing dataset (used for model's evaluation) in a 90/10% ratio. The model was evaluated through its sensitivity, specificity, positive and negative predictive values and accuracy. The CNN had 99.1% accuracy for identifying normal pancreatic tissue, 99.0% and 99.8% for M-PCN and NM-PCN, respectively. P-DAC and P-NET were distinguished with 94.0% accuracy. Our group developed the first worldwide CNN capable of detecting and differentiating the commonest PCN and PSL in EUS images, using exams from 4 centers in two continents, minimizing the impact of the demographic bias. Larger multicentric studies are needed for technology implementation.

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration.

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth.

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

Higher frequency of gastric neoplasia in advanced chronic liver disease patients: Impact of screening endoscopy in an intermediate-high risk country.

BACKGROUND: The Baveno VII guidelines were proposed to identify which patients could safely avoid screening esophagogastroduodenoscopy (EGD) for gastroesophageal varices. We aimed to evaluate the frequency of gastric neoplasia in compensated advanced chronic liver disease (cACLD) patients who underwent EGD for screening of gastroesophageal varices (GOEV) compared to a healthy population. METHODS: Retrospective study that enrolled all cACLD patients who underwent EGD for GOEV screening (January 2008-June 2018) in a tertiary reference center. cACLD patients were compared with asymptomatic healthy individuals who underwent EGD in a private hospital setting (April 2017-March 2018). RESULTS: We evaluated 1845 patients (481 cACLD patients, 1364 healthy individuals). A significantly higher frequency of gastric neoplasia was observed in patients with cACLD compared to healthy individuals (4.0% vs. 1.0 %; p < 0.001). Rare histopathological subtypes (WHO Classification) accounted for 28.7 % of gastric carcinoma cases in the cACLD cohort. Seven cases of gastric neoplasia (36.8 % of gastric neoplasia cases in the cACLD patients) were diagnosed in patients who, according to the Baveno VII criteria, would have not been submitted to EGD. CONCLUSION: We found an increased frequency of gastric neoplasia in patients with cACLD in comparison with healthy individuals. In countries with intermediate-high risk for GC, continuing to perform EGD could be beneficial.

Performance of Intracystic Glucose Measurement for the Characterization of Pancreatic Cystic Lesions.

BACKGROUND AND AIMS: Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) is essential for the classification of pancreatic cystic lesions (PCLs). Recently, intracystic glucose has been suggested as an alternative to carcinoembryonic antigen (CEA) level as a predictor of mucinous cystic lesions (M-PCLs). This study aims to evaluate the diagnostic performance of intra-cystic glucose in distinguishing between M-PCLs and non M-PCLs (NM-PCLs) and to analyze the possibility of on-site glucose measurement with a standard glucometer. METHODS: Patients with PCLs submitted to EUS-FNA with simultaneous intracystic glucose measurement between 2017 and 2022 were included. The diagnostic performance of glucose versus CEA for the differentiation between M-PCLs and NM-PCLs was compared to a final diagnosis based on the analysis of surgical specimen, intracystic biopsy or, if this data was unavailable, multidisciplinary evaluation. A cut-off of <50 mg/dL was used for the diagnosis of MCLs. Additionally, the agreement between on-site glucose determination with a standard glucometer and laboratory glucose measurement was assessed. RESULTS: Mucinous lesions accounted for 56% of all PCLs. The median values of glucose and CEA for M-PCLs were 18 mg/dL and 286 ng/mL, respectively. Intracystic glucose had a sensitivity and specificity of 93.2% and 76.5%, respectively, for the diagnosis of MCLs (versus 55.6% and 87.5%, respectively, for CEA). The area under the curve was 0.870 for on-site glucose (versus 0.806 for CEA). An excellent correlation was observed between on-site and laboratory glucose measurement (ρ=0.919). CONCLUSIONS: The measurement of intracystic glucose showed superior performance compared with CEA in distinguishing between M-PCLs and NM-PCLs, with excellent correlation between on-site and conventional lab glucose measurement. Thus, on-site intracystic glucose appears to be an excellent biomarker for the characterization of PCLs due to its low cost, high availability, and the need for a minimal cyst fluid volume for its determination.

Histological analysis of (antral) follicle density in ovarian cortex tissue attached to stripped endometriomas.

PURPOSE: When resecting endometriomas with the stripping technique, in the majority of cases, a thin line of adjacent ovarian cortex is attached to the endometrioma. In this study, we performed histological analysis to determine (antral) follicle density in the ovarian cortex tissue attached to stripped endometriomas and assessed patient- and surgical characteristics that could affect this. METHODS: Histological slides of previously removed endometriomas were assessed. Follicles in the attached ovarian tissue were classified according to maturation, and follicular density was determined. Immunofluorescent staining of antral follicles in a subset of endometriomas was also performed. RESULTS: In 90 out of 96 included endometriomas (93.7%), ovarian tissue attached to the cyst wall was observed. One thousand nine hundred forty-four follicles at different maturation stages were identified (3 follicles/mm3). Follicle density was negatively associated with age (p < 0.001). Antral follicles (< 7-mm diameter) were present in the ovarian tissue attached to 35 endometriomas (36.5%) derived from younger patients compared to endometriomas where none were detected (30 versus 35 years, p = 0.003). Antral follicle density was 1 follicle/mm3. Based on immunofluorescence, healthy antral follicles were identified in two out of four examined endometriomas. CONCLUSIONS: Ovarian tissue attached to stripped endometriomas holds potential as a non-invasive source for antral follicles. In theory, application of IVM could be an interesting alternative FP option in young patients with endometriomas who undergo cystectomy in order to transform the surgical collateral damage to a potential oocyte source. Our results encourage future research with fresh tissue to further assess the quality and potential of these follicles. TRIAL REGISTRATION: Clinical Trials.gov Identifier: B21.055 (METC LDD), date of registration 12-08-2021, retrospectively registered.

Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

Wilson's Disease: A Prevalence Study in a Portuguese Population.

Introduction Wilson's disease (WD) is a rare and underdiagnosed genetic disorder caused by anomalous tissue copper deposition, and for which epidemiological studies, specifically in Portugal, are scarce. Objectives This study aimed to evaluate the prevalence and incidence of WD and provide a description of its main clinical and laboratory features. Methods A retrospective study was carried out, with a search between 1995 and 2015, of all patients with a minimum follow-up of three months and birth confirmed in the northern region of Portugal, with an estimated population of 3,689,682 inhabitants. Database collection was based on the Portuguese National Health Service's clinical coding system, relying on clinical data from 13 northern Portuguese hospitals, liver biopsy histology results, and hospital prescription records. Clinical and biochemical correlations were statistically assessed using chi-square, Mann-Whitney U, Friedman, and Wilcoxon tests. Results Over the 20-year period, a prevalence of 1:37.000 and an incidence of one per million person-year was found. A total of 94 patients were analyzed, with a slight male predominance (53%), the majority with the onset of clinical manifestations in pediatric age (56%), with a median age at diagnosis of 16.6 years (interquartile range of 12.3-20,.8 years). Most patients presented with predominant liver disease (54.8%), with more than a third with cirrhosis; mixed hepatic and neurological manifestations in 17.9%; and mainly neurological symptoms in 10.7% of the patients. Neurological impairment was strongly associated with delayed development of the manifestations of the disease (p = 0.001) and also a higher detection of Kayser-Fleischer rings (p < 0.001), present in 27.0% of the patients. Regarding therapy, penicillamine has been the most widely used, with adverse reactions reported in 24.8%. At six and 12 months after initiation of therapy, a significant decrease in liver enzymes was found (ALT: p = 0.002; AST: p = 0.002, respectively), but no significant reduction was observed in urinary copper excretion. Conclusion This was one of the first studies regarding WD prevalence in a Portuguese population, contributing to a better understanding of the epidemiology, diagnosis, and management of WD in the northern region of Portugal. WD should be considered in any individual with unexplained hepatic or neurological manifestations, and initial symptoms may manifest at an early age, even in children less than five years old. A high percentage of patients were identified in the early stages of the disease by asymptomatic elevation of transaminases. Following copper chelation therapy, cytolysis markers appear to be more sensitive indicators of treatment response.

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids.

Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show efficacy of cytosine and adenine base editors in modeling CTNNB1 hot-spot mutations in hepatocyte organoids. Next, we use C > T base editors to insert nonsense mutations in PTEN in endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug sensitivity assays on organoids harboring either PTEN or PTEN and PIK3CA mutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C > T and A > G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

Modelling Human Post-Implantation Development via Extra-Embryonic Niche Engineering.

Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis formation, and gastrulation events. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons. Additionally, human stem cell models of early post-implantation development with both embryonic and extra-embryonic tissue morphogenesis are lacking. Here, we present iDiscoid, produced from human induced pluripotent stem cells via an engineered a synthetic gene circuit. iDiscoids exhibit reciprocal co-development of human embryonic tissue and engineered extra-embryonic niche in a model of human post-implantation. They exhibit unanticipated self-organization and tissue boundary formation that recapitulates yolk sac-like tissue specification with extra-embryonic mesoderm and hematopoietic characteristics, the formation of bilaminar disc-like embryonic morphology, the development of an amniotic-like cavity, and acquisition of an anterior-like hypoblast pole and posterior-like axis. iDiscoids offer an easy-to-use, high-throughput, reproducible, and scalable platform to probe multifaceted aspects of human early post-implantation development. Thus, they have the potential to provide a tractable human model for drug testing, developmental toxicology, and disease modeling.

Bioengineered 3D Ovarian Models as Paramount Technology for Female Health Management and Reproduction.

Ovarian dysfunction poses significant threats to the health of female individuals. Ovarian failure can lead to infertility due to the lack or inefficient production of fertilizable eggs. In addition, the ovary produces hormones, such as estrogen and progesterone, that play crucial roles not only during pregnancy, but also in maintaining cardiovascular, bone, and cognitive health. Decline in estrogen and progesterone production due to ovarian dysfunction can result in menopausal-associated syndromes and lead to conditions, such as osteoporosis, cardiovascular disease, and Alzheimer's disease. Recent advances in the design of bioengineered three-dimensional (3D) ovarian models, such as ovarian organoids or artificial ovaries, have made it possible to mimic aspects of the cellular heterogeneity and functional characteristics of the ovary in vitro. These novel technologies are emerging as valuable tools for studying ovarian physiology and pathology and may provide alternatives for fertility preservation. Moreover, they may have the potential to restore aspects of ovarian function, improving the quality of life of the (aging) female population. This review focuses on the state of the art of 3D ovarian platforms, including the latest advances modeling female reproduction, female physiology, ovarian cancer, and drug screening.

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss.

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

Autoimmune hepatitis and eosinophilia: A rare case report.

BACKGROUND: Autoimmune hepatitis consists of a chronic liver disease whose etiology is unknown. It is comprised of relevant immunological aspects and of immune-mediated liver injury. Eosinophilia may be a considerable feature, particularly happening in male patients. CASE SUMMARY: We report here a Crohn´s disease patient presenting with de novo hypergammaglobulinemia, circulating autoantibodies and elevated transaminase levels. He also had significant peripheral eosinophilia and elevated immunoglobulin E levels at diagnosis. The pathology findings from liver biopsy were compatible with autoimmune hepatitis with eosinophilic infiltration. CONCLUSION: This is the first report of autoimmune hepatitis with exuberant eosinophilic infiltration in the liver and bone marrow, described in a patient with Crohn's disease.

An unexpected cause of common bile duct obstruction diagnosed by endoscopic ultrasound-guided fine-needle biopsy.

A 43-years-old woman with previous cholecystectomy presented as an outpatient with abnormal liver tests. A diagnosis of well-differentiated neuroendocrine tumor (NET) was made and a 68Ga-DOTA-somatostatin analogue positron emission tomography revealed no apparent metastatic disease.

Liver biopsy in inflammatory bowel disease patients with sustained abnormal liver function tests: a retrospective single-center study.

Background: Inflammatory bowel disease (IBD) may be associated with a wide range of hepatobiliary manifestations. This study aimed to characterize the spectrum of hepatobiliary disorders in patients with IBD who underwent liver biopsy for sustained abnormal liver function tests (LFT). Method: A retrospective study was performed of all patients with IBD who underwent liver biopsy between January 2010 and December 2020 for sustained abnormal LFT (at least 6-month duration). Results: A total of 101 patients were included, mostly male (62.4%), with a mean age of 44.4±13.3 years. The most common IBD type was Crohn's disease (61.4%). Median time interval between abnormal LFT and biopsy was 14 (7-36) months. Abnormal LFT was predominantly hepatocellular in 40 patients (39.6%), cholestatic in 26 (25.7%) and mixed in 35 (34.7%). The most frequent diseases were nonalcoholic fatty liver disease (NAFLD) in 33 patients (32.7%), drug-induced liver disease (DILI) in 30 (29.7%), autoimmune hepatitis (AIH) in 13 (12.9%) and primary sclerosing cholangitis (PSC) in 13 (12.9%). Three patients had primary biliary cholangitis. Remarkably, 70 patients (69.3%) already had fibrosis by the time of liver biopsy and in 6 (5.9%) liver disease was already detected in the stage of cirrhosis. Conclusions: Abnormal LFT in IBD patients had a wide range of etiologies and histology was often essential for reaching a correct diagnosis. NAFLD, DILI, AIH and PSC were the most common diagnoses and patients often presented in cirrhotic stage. Therefore, liver biopsy must be considered early in IBD patients with unexplained sustained abnormal LFT.